大鼠肝纤维化的MR功能成像及磁粒子标记BMSCs移植修复肝损伤的MR示踪实验研究
摘要
第一篇MR功能成像对大鼠肝纤维化早期诊断、定量分析的实验研究
肝脏弥漫性病变中,肝纤维化和早期肝硬化因形态学改变不明显而成为影像学早期诊断面临的难题,传统的以反映解剖结构的影像学方法对此类病变难于提供有价值的诊断信息。目前,临床上应用的非创性的肝纤维化诊断方法敏感性和特异性较差;肝组织活检被认为是诊断肝纤维化的“金标准”,但作为一种侵入性的诊断方法,其临床应用具有许多限度。肝纤维化的早期检测、早期干预有助于阻止病变的进一步发展。因此,迫切需要开发无创性、对慢性肝病出现肝纤维化能进行早期预测、病变程度评价的新的方法。
本研究通过CCI4诱导建立大鼠肝纤维化模型,采用磁共振功能成像(弥散加权成像、波谱成像、灌注成像和肝细胞特异性对比剂应用)技术,并与病理组织学对照,对肝纤维化进展过程中,分子弥散、能量代谢、血流灌注和肝细胞功能等多因素病理生理改变进行动态、系统研究,国内外类似研究尚未见报道。本研究目的在于探讨肝纤维化的MR功能成像参数及最佳成像序列;分析不同程度、不同分期肝纤维化的MR功能成像表现,探索肝纤维化MR功能成像的量化诊断指标;评价MR功能成像在肝纤维化、早期肝硬化诊断中的价值。
第一部分大鼠肝纤维化模型的建立
1.目的:采用SD大鼠皮下注射四氯化碳法,建立肝纤维化动物模型,为肝纤维化的MR功能成像和骨髓基质细胞肝移植MR示踪研究提供不同分期的肝纤维化动物模型。
2.方法:
实验组大鼠腹部皮下注射40%CCl4油溶液,剂量为3ml/kg体重,2次/周,首次剂量为5ml/kg体重;10%乙醇溶液作为唯一饮用水。对照组大鼠腹部皮下注射生理盐水,剂量及用法同实验组,纯净水作为饮用水。注药后第2周开始,每周随机抽取实验组大鼠4只、对照组大鼠1只,肝脏磁共振功能成像(DWI、MRS、PWI)后4小时内处死大鼠,肝脏取材行HE染色、Masson三色染色、网状纤维染色及透射电镜检查,光镜下判定肝纤维化分期。
3.结果:
模型组共有94只大鼠完成实验,死亡46只,死亡率33 %。模型组大鼠均出现不同程度的营养不良、慢性肝病症状。肝脏病理组织学检查见炎症细胞浸润,肝细胞坏死,胶原及网状纤维增生,肝窦毛细血管化等改变。对照组20只全部存活,无相应症状。肝纤维化病理分期:0期28只、1期19只、2期27只、3期25只、4期15只。
4.结论:
复合因素(CCl4+酒精)能成功地诱导大鼠肝纤维化,并且具有成模率高,造模周期短优点,并有较明显的阶段性变化;应用复合因素可建立不同病理分期的肝纤维化模型,为肝纤维化研究提供理想的实验模型。
第二部分大鼠肝纤维化的MR弥散加权成像(DWI)
1.目的:
通过分析大鼠不同程度肝纤维化的DWI信号强度、ADC值和EADC值变化,以探讨应用非创性DWI检测方法对肝脏纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,进行MR弥散成像: SE-EPI序列,梯度因子b分别取:0s/mm2、300 s/mm2、600 s/mm2、800 s/mm2、1000 s/mm2。根据不同b值拟合得到ADC图、EADC图,测定不同b值弥散成像的信号强度,计算ADC值和EADC值,并与病理分期对照。
3.结果:
(1)实验组大鼠随着肝纤维化分期的增加,DWI信号强度呈增加趋势,各叶纤维化进展不近相同,表现为DWI信号强度不均匀。(2)b=300、600、800、1000 s/mm2时,SNR分别为(x±S):36.30±23.25、28.11±12.48、25.71±11.82和15.23±6.54,图像质量呈下降趋势,b=600、800 s/mm2时优于b=1000 s/mm2(P﹤0.05)。(3)ADC值分析:对照组与1期、2期、3期、4期肝纤维化平均ADC值分别为[(x±S)×10-3]:(1.542±0.299)×10-3、(1.334±0.268)×10-3、(1.108±0.198)×10-3、(0.978±0.169)×10-3、(0.680±0.260)×10-3 ,呈下降趋势。对照组与1期、2期、3期、4期比较有显著性差异;1期与2期、3期、4期比较,2期与4期比较,3期与4期比较均有显著性差异;2期与3期比较差异无统计学意义。ADC值与肝纤维化分期的相关性分析:r=-0.766(p﹤0.001)。(4)EADC值分析:对照组与1期、2期、3期、4期肝纤维化平均EADC值分别为[(x±S)×10-3]:(0.315±0.068)×10-3、(0.345±0.081)×10-3、(0.411±0.074)×10-3、(0.465±0.056)×10-3、(0.595±0.106)×10-3,呈增高趋势。对照组与2期、3期、4期比较有显著性差异,1期与2期、3期、4期比较,2期与4期比较,3期与4期比较均有显著性差异。对照组与1期比较,2期与3期比较差异无统计学意义。EADC值与肝纤维化分期的相关性分析:r=0.753 (p﹤0.001)。
4.结论:
(1)DWI信号强度随着肝纤维化分期的增加而增高,各叶纤维化进展不近相同;
(2)梯度因子b取600 s/mm2或800 s/mm2时,即可避免灌注对弥散的影响(低b值),又具有较高的信噪比,为肝脏DWI成像较理想的b值取值;(3)ADC值(1-4期)和EADC值(2-4期)均能对肝纤维化进行分期,且均具有较好的相关性。
第三部分大鼠肝纤维化的MR波谱成像(1H-MRS)
1.目的:
通过分析大鼠不同程度肝纤维化的MRS代谢物波峰峰高、波峰下面积及其相互比值的变化,以探讨应用MRS检测方法对肝纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,采用3D PRESS多体素1H-MRS序列进行MR波谱成像,手动标记波谱图像中不同代谢物,软件自动生成各代谢物的峰高和波峰下面积,分别计算代谢物与脂质的峰高、波峰下面积的比值(Cho/lip、Glx/lip、Lac/lip、Cr/lip)并与病理分期对照。
3.结果:
对照组大鼠肝脏波谱成像可见5个主要波峰;模型组大鼠脂质峰下降,其余代谢物波峰不同程度增高。代谢物与脂质波峰峰高比值:(1)对照组与1期、2期、3期、
4期肝纤维化Cho/lip比值分别为(x±S):0.052±0.034、0.212±0.225、0.117±0.122、0.403±0.299、0.438±0.295。对照组与3期、4期比较P﹤0.05,对照组与1期、2期比较,1期、2期分别与各组比较P﹥0.05。(2)对照组与1期、2期、3期、4期肝纤维化Glx/lip比值分别为(x—±S):0.150±0.132、0.406±0.650、0.656±0.551、0.750±0.452、0.763±0.517。对照组与2期、3期、4期比较P﹤0.05,与1期比较P﹥0.05,余各组两两比较P﹥0.05。(3)对照组与1期、2期、3期、4期肝纤维化Lac/lip比值分别为(x—±S):0.139±0.128、0.262±0.178、0.251±0.344、0.355±0.446、0.233±0.185,差异无统计学意义(P﹥0.05),但随分期增加有增高趋势。(4)对照组与1期、2期、3期、4期肝纤维化Cr/lip比值分别为(x±S):0.136±0.274、0.767±0.902、0.638±0.960、0.917±0.576、0.778±0.856。对照组与3期比较P﹤0.05,余各组两两比较差异无统计学意义。代谢物与脂质波峰峰高比值与肝纤维化分期的相关性分析:Cho/lip(r=0.503 p﹤0.001)、Glx/lip(r=0.388 p﹤0.05)、Lac/lip(r=0.124 p﹥0.05)、Cr/lip(r=0.235 p﹥0.05)。
肝脏主要代谢物与脂质波峰下面积比值:(1)对照组与1期、2期、3期、4期肝纤维化Cho/lip比值分别为(x±S):0.115±0.133、0.257±0.316、0.167±0.187、0.185±0.328、0.468±0.372。对照组与4期比较P﹤0.05,余两两比较差异无显著性。(2)对照组与1期、2期、3期、4期肝纤维化Glx/lip比值分别为(x±S):0.045±0.039、0.540±0.318、0.448±0.364、0.482±0.402、0.531±0.336。对照组与1期、2期、3期、
4期比较P﹤0.05,余各组之间比较P﹥0.05。(3)对照组与1期、2期、3期、4期肝纤维化Lac /lip比值分别为(x—±S):0.062±0.069、0.258±0.266、0.277±0.320、0.170±0.314、0.274±0.312。差异无统计学意义(P﹥0.05),但随分期增加有增高趋势。(4)对照组与1期、2期、3期、4期肝纤维化Cr/lip比值分别为(x±S):0.109±0.231、0.481±0.614、0.704±0.797、0.465±0.525、0.810±0.706。对照组与4期比较P﹤0.05,与1期、2期、3期比较及余各组之间两两比较P﹥0.05。代谢物与脂质波峰下面积比值与肝纤维化分期的相关分析:Cho/lip(r=0.282 p﹥0.05)、Glx/lip(r=0.313 p﹤0.05)、Lac/lip(r=0.135 p﹥0.05)、Cr/lip(r=0.267 p﹥0.05)。
4.结论:
(1)Cho/lip、Glx/lip、Cr/lip波峰峰高比值对肝纤维化分期(Cho/lip对3-4期、Glx/lip对2-4期、Cr/lip对3期)具有一定的价值;代谢物与脂质波峰峰高比值与肝纤维化分期的相关性以Cho/lip、Glx/lip较高。(2)Cho/lip、Glx/lip、Cr/lip波峰下面积比值对肝纤维化分期(Cho/lip、Cr/lip对4期;Glx/lip对1～4期)具有一定的价值;代谢物与脂质波峰下面积比值与肝纤维化分期的相关性以Glx/lip较高(。3)Lac/lip波峰峰高比值、波峰下面积比值对肝纤维化分期均无意义,但均随分期增加呈增高趋势。
第四部分大鼠肝纤维化的MR灌注成像(PWI)
1.目的:
通过分析不同程度肝纤维化的PWI的灌注参数变化,以探讨应用PWI检测方法对肝脏纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,采用单次激发SE-EPI序列进行MR灌注成像:经大鼠尾静脉快速团注Gd-BOPTA,剂量为0.2mmol/kg体重,流率2ml/s,连续扫40个动态,覆盖整个肝脏。应用Perfusion软件自动生成肝实质信号强度-时间曲线,计算相关参数:(1)最大信号下降百分率(SRRmax),(2)到达峰值时间(TTP),(3)平均通过时间(MTT)。分析PWI灌注参数值并与病理肝纤维化分期对照。
3.结果:
(1)肝实质信号强度-时间曲线:对照组曲线呈快速下降、达峰值后缓慢恢复,恢复幅度较大,恢复时程较短;模型组曲线下降速度减慢,下降幅度减小,达峰值时间延长,达峰值后恢复幅度较小,恢复时程较长,波峰宽大,随着肝纤维化分期的增高这种改变更加明显。(2)肝脏灌注参数与肝纤维化分期的关系:1)对照组与1期、2期、3期、4期肝纤维化SRRmax值分别为[(x±S)×100%]:0.754±0.073、0.674±0.137、0.632±0.154、0.603±0.201、0.535±0.135。对照组与3期、4期比较P﹤0.05,与1期、2期比较差异无显著性,余各组两两比较P﹥0.05。2)对照组与1期、2期、3期、4期肝纤维化TTP值分别为[(x±S)s]:14.175±4.845、18.433±7.293、26.789±3.621、31.755±7.308、35.213±6.322。对照组与2期、3期、4期比较P﹤0.05,与1期比较无显著性差异;1期与2期、3期、4期比较,2期与4期比较P﹤0.05;2期与3期比较,3期与4期比较P﹥0.05。3)对照组与1期、2期、3期、4期肝纤维化MTT值分别为[(x—±S)s]:24.620±5.577、28.945±2.758、32.502±4.268、35.861±4.651、35.203±5.674。对照组与2期、3期、4期比较P﹤0.05, 1期与3期、4期比较P﹤0.05,对照组与1期比较,1期与2期比较,2期与3期、4期比较,3期与4期比较差异均无统计学意义。肝脏灌注参数与肝纤维化分期的相关分析:SRRmax(r=-0.439 p﹤0.05)、TTP(r=0.798 p﹤0.001)、MTT(r=0.647 p﹤0.001)。
4.结论:
(1)肝纤维化大鼠肝实质信号强度-时间曲线呈慢降缓升型,波峰低平宽大;(2)SRRmax、TTP、MTT灌注参数分析有助于肝纤维化分期(SRRmax对3-4期、TTP、MTT对2-4期)。SRRmax、TTP、MTT灌注参数与肝纤维化分期均有较好的相关性,其中以TTP和MTT较高。
第五部分大鼠肝纤维化的肝细胞特异性对比剂MR成像
1.目的:
通过对大鼠肝纤维化模型进行Gd-BOPTA增强动态观测,分析不同程度肝纤维化各延迟时间点的动态增强表现、强化程度,并与病理分期对照,以探讨应用Gd-BOPTA增强延迟扫描方法对肝脏纤维化早期诊断、定量分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,进行Gd-BOPTA增强MR扫描:经大鼠尾静脉快速团注Gd-BOPTA,剂量为0.2mmol/kg体重,以TSE-T1WI序列分别于注射对比剂后60min(RER1)、120 min(RER2)、180 min(RER3)时间点延迟扫描,分别测算各不同时间点的信号强度、肝实质相对强化率;观察不同程度肝纤维化大鼠各不同时间点的胆管、血管信号强度变化特点。
3.结果:
不同时间点肝实质相对强化率与肝纤维化分期的关系:(1)对照组与1期、2期、3期、4期肝纤维化RER1值分别为(x±S):1.436±0.374、1.487±0.477、1.476±0.440、1.489±0.431、1.476±0.436。各组差异无统计学意义(P﹥0.05),但随肝纤维化分期的增加有增高趋势。(2)对照组与1期、2期、3期、4期肝纤维化RER2值分别为(x—±S):1.220±0.370、1.292±0.387、1.344±0.367、1.371±0.388、1.405±0.370。各组差异无统计学意义(P﹥0.05),但随肝纤维化分期的增加有增高趋势。(3)对照组与1期、2期、3期、4期肝纤维化RER3值分别为(x±S):0.844±0.275、0.910±0.380、1.041±0.399、1.209±0.299、1.241±0.398。对照组与3期、4期比较P﹤0.05,与1期、2期比较P﹥0.05,肝纤维化各期之间两两比较P﹥0.05。不同时间点肝实质相对强化率与肝纤维化分期的相关分析:RER1(r=0.039 p﹥0.05)、RER2(r=0.174 p﹥0.05)、RER3(r=0.420 p﹤0.05)。胆管、血管显影情况: MR增强延迟期:肝内血管树T1WI为低信号;胆道树T1WI表现为高信号。实验组大鼠肝脏门静脉树和胆管树分支走行迂曲、粗细变化不规律,胆管树见延迟强化。
4.结论:
(1)对比剂Gd-BOPTA增强后60min、120 min时间点肝实质相对强化率对肝纤维化分期无价值,延迟后180 min时间点肝实质相对强化率对肝纤维化分期(3-4期)具有一定的价值,不能对早期肝纤维化(1-2期)进行分期。(2)肝纤维化大鼠胆管树形态改变、延迟强化,提示肝纤维化时胆系重构、肝细胞功能受损。
第二篇磁粒子标记大鼠BMSCs移植修复肝损伤的MR活体示踪实验研究
肝功能衰竭是各种慢性肝病主要的死亡原因,原位肝移植是目前治疗终末期肝病的最有效的方法,但由于供肝严重缺乏,远无法满足临床需求。肝脏的细胞移植为病损肝脏的细胞重建和衰竭肝脏的功能恢复提供了一种新的治疗策略,使患者有可能利用自身的骨髓干细胞作为种子细胞,来修复自体组织的病变和损伤。干细胞移植示踪研究,对动态监测活体内移植细胞的分布、迁移、分化及评估细胞移植效果具有重要作用,但干细胞移植示踪技术一直是医学科研中的难题。传统的移植细胞的示踪方法必须在离体状态下通过组织学观察来实现。因此,迫切需要建立一种安全无创、敏感有效、适于临床的移植细胞活体示踪技术。MR活体示踪在干细胞的研究中日益受到关注,利用MR敏感的对比剂作为分子探针标记供体移植细胞,移植后对受体进行MRI检测是活体观察移植细胞存活和分布的新技术。
应用MR活体示踪技术研究肝纤维化形成环境中移植的BMSCs分布、迁移规律尚未见报道。本研究通过对大鼠BMSCs的分离、增殖和鉴定,采用Brdu和SPIO双标记大鼠BMSCs,对大鼠肝纤维化模型进行同种异体移植后,行MR活体动态观测,并与病理组织学对照,以探讨大鼠BMSCs的分离、增殖和鉴定技术,和SPIO作为分子探针标记BMSCs MR活体示踪的可行性,以及MR成像的最佳扫描参数及成像序列,并探讨移植细胞在肝脏纤维化环境中分布、迁移特点及其对肝损伤的修复作用,以及相应的MR信号变化规律,探索临床应用型1.5T MR用于干细胞示踪研究的可行性,为干细胞移植MR活体示踪的临床应用奠定基础。
第一部分大鼠骨髓基质细胞的体外分离、培养、鉴定和标记
1.目的:
观察大鼠骨髓基质细胞在体外培养的条件,并利用Brdu和超顺磁性氧化铁颗粒对大鼠BMSCs进行双标记,为应用MR对移植骨髓基质细胞进行活体示踪研究奠定基础。
2.方法:
取60~90g SD大鼠,体外培养骨髓基质细胞,利用倒置相差显微镜观察原代及不同传代细胞生长、形态特点,并应用流式细胞仪鉴定原代及不同传代细胞表型。利用Brdu和PLL介导的超顺磁性氧化铁颗粒对大鼠BMSCs进行双标记。
3.结果:
培养的骨髓基质细胞第3代,骨髓基质细胞的细胞表面标志CD90表达阳性的细胞已占到93.1%。造血干细胞的表面标志CD45表达阳性的细胞已从原代的71.2%降低到3.9%。PLL介导的超顺磁性氧化铁颗粒对大鼠BMSCs标记率达100%。
4.结论:
传代3的BMSCs可作为细胞移植治疗的理想细胞来源。PLL介导的超顺磁性氧化铁颗粒可高效率地标记大鼠BMSCs。
第二部分磁粒子标记大鼠骨髓基质细胞的体外MR成像实验
1.目的:
探讨标记细胞不同细胞数量所引起的MR信号强度变化规律,以及磁粒子标记细胞MR示踪最敏感的成像序列,为标记细胞活体内MR示踪奠定基础,并提供理论依据。
2.方法:
用1%的琼脂糖混悬Brdu和SPIO双标记的BMSCs(分别为2×106、1×106、5×105个)和未标记的BMSCs (2×106个),分别置于不同EP管中。行冠状位和轴位TSE序列T1WI、T2WI和FFE-T2WI扫描,测量相同细胞数量不同成像序列以及相同成像序列不同细胞数量的信号强度,并比较MR信号变化率。
3.结果:
磁粒子标记的各不同数量BMSCs各成像序列MR信号变化率(:1)标记组5×105、1×106、2×106个细胞TSE-T1WI序列MR信号变化率分别为[(x±S)%]:-4.19±0.79、-16.35±1.23、-22.80±1.05。(2))标记组5×105、1×106、2×106个细胞TSE-T2WI序列MR信号变化率分别为[(x—±S)%]:-14.15±1.37、-35.09±1.39、-53.02±1.30。(3)标记组5×105、1×106、2×106个细胞FFE-T2WI序列MR信号变化率分别为[(x±S)%]:-44.98±0.46、-69.38±0.82、-87.24±0.82。同一扫描序列中各标记细胞组之间以及相同标记细胞数量各成像序列之间两两比较P﹤0.001,差异均有统计学意义。
4.结论:
磁粒子标记BMSCs,细胞浓度相同条件下,以FFE-T2WI序列信号下降最为明显,标记的细胞数量越多,信号改变越明显,呈细胞数量依赖性。FFE-T2WI序列为磁粒子标记细胞MR示踪的理想序列。
第三部分大鼠骨髓基质细胞的同种异体肝移植
1.目的:
经门静脉途径和肝内途径进行肝脏的BMSCs移植,探讨两种移植途径的特点,为比较两种移植途径的MR示踪效果及信号变化特点,及了解移植细胞在靶器官的分布、迁移规律奠定基础。
2.方法:
将第一篇、第一部分中建立的SD大鼠肝纤维化模型20只分为4组,1组:生理盐水对照组(注射不含BMSCs的等容生理盐水,其中经门静脉2只,经肝内注射2只);2组:未标BMSCs对照组(移植2×106个未经Brdu和SPIO标记的BMSCs,其中经门静脉2只,经肝内注射2只);3组:经门静脉移植BMSCs标记组(经门静脉移植2×106个经Brdu和SPIO标记的BMSCs,6只);4组:经肝内移植BMSCs标记组(经肝内移植2×106个经Brdu和SPIO标记的BMSCs,6只)。手术暴露门静脉主干和肝脏,以微量注射器抽取相应移植内容,穿刺各组目标,缓慢注入。
3.结果:
经肝内注射组:手术操作简便,细胞移植顺利。经门静脉移植组:有3只大鼠因腹膜、系膜粘连,门静脉主干分离困难,给移植手术带来一定的难度,其余大鼠移植手术顺利。因实验大鼠均出现门静脉主干不同程度增粗,门静脉主干穿刺均获成功。缓慢注入移植细胞或生理盐水后,大鼠未出现异常反应。手术切口愈合良好。
4.结论:
经门静脉途径和经肝内注射途径对肝脏进行BMSCs移植,安全、易行。
第四部分大鼠骨髓基质细胞同种异体肝移植的MR活体示踪研究
1.目的:
探讨经门静脉途径和经肝内途径移植标记细胞的MR活体示踪效果及信号变化特点,从而了解肝纤维化环境中移植细胞在靶器官的分布、迁移规律及其对肝损伤的修复作用,为移植干细胞MR活体示踪的临床应用奠定基础。
2.方法:
第三部分移植术后各组大鼠每组随机抽取2只,分别于移植术后2h、3d、7d、2w行TSE序列轴位T2WI-SPAIR、T1WI和FFE-T2WI扫描,对不同序列图像的移植细胞显影效果进行比较;观察各移植组不同时间点的MR信号强度、分布范围及其变化规律。于MR扫描后随机抽取每组大鼠各一只处死,取肝脏标本,并取部分大鼠肺和脾脏组织,采用HE染色、含铁血黄素染色、Brdu免疫组织化学染色。观察含铁血黄素染色阳性细胞和Brdu免疫组织化学染色阳性细胞在肝、肺、脾脏中的分布。
3.结果:
(1)MR检查:在各扫描序列中,以FFE-T2WI序列成像效果最佳。1组和2组:移植术后2h、3d、7d、2w不同时间点、各序列MR图像均未见异常信号改变。3组:移植术后2h,肝门区见多发结节状低信号灶,并随时间延长向肝内分散,低信号结节逐渐变小。移植术后2w示踪效果较差。4组:移植术后2h,局部见团状低信号灶,随时间延长范围逐渐扩大,边缘逐渐模糊,移植术后2w低信号区仍较明显。
(2)病理组织学检查:1)HE染色:移植术后2h、3d各组肝内坏死、炎症及纤维组织增生情况基本类似;移植术后7d、2w,与1组比较,2～4组大鼠肝组织坏死、炎症细胞浸润情况逐渐好转,以经门静脉移植组明显。2)含铁血黄素染色:1组和2组:移植术后2h、3d、7d、2w,肝脏、肺和脾脏含铁血黄素染色,均未见阳性细胞。3组:移植术后2h含铁血黄素染色阳性细胞主要分布于肝门部门静脉内,移植术后3d阳性细胞主要分布于门静脉小分支、肝窦内、肝小叶中央静脉周围,移植术后7d、2w阳性细胞主要分布于损伤较重的肝实质和纤维间隔;各时间点组织学所见与MR成像信号改变一致。同期的肺和脾脏含铁血黄素染色,其内可见少量阳性细胞散在分布。4组:移植术后2h、3d、7d、2w可见含铁血黄素染色阳性细胞密集分布于注射点,随时间延长细胞向周围肝内迁移。各时间点组织学所见与MR成像信号改变一致。同期肺内未见明显阳性细胞,脾脏内可见少量阳性细胞分布。3)Brdu免疫组织化学染色:结果与含铁血黄素染色一致。
4.结论:
(1)FFE-T2WI序列为肝内移植磁粒子标记细胞MR活体示踪的理想序列;(2)经门静脉途径移植BMSCs细胞随时间逐渐向肝内移行,以损伤较重区和纤维间隔分布明显,具有选择性分布特点;移植的部分BMSCs与肝细胞紧密连接,形成规则的肝细胞索,对肝损伤具有修复作用;经门静脉途径移植BMSCs为细胞移植治疗肝脏弥漫性病变的较理想途径,但MR示踪时间窗较短;经肝内途径移植BMSCs细胞分布局限,MR示踪时间窗较长;(3)MR信号变化规律与组织学证实的移植细胞在靶器官的分布、迁移规律具有较好的一致性;(4)MR信号变化规律在一定程度上反映了移植细胞在肝内的分布、迁移、增殖和分化状态;(5)临床应用型1.5T MR可用于肝脏干细胞移植的活体示踪研究。
结论
1.应用复合因素(CCl4+酒精)能成功地建立不同病理分期的大鼠肝纤维化模型,有较明显的阶段性变化。
2. DWI信号强度随着肝纤维化分期的增加而增高;梯度因子600 s/mm2或800 s/mm2为肝脏DWI成像较理想的b值取值;ADC值和EADC值均能对肝纤维化进行分期(ADC值对1-4期;EADC值对2-4期),具有较好的相关性。
3. Cho/lip、Glx/lip、Cr/lip波峰峰高比值(Cho/lip对3-4期、Glx/lip对2-4期、Cr/lip对3期)及波峰下面积比值(Cho/lip对4期、Glx/lip对1-4期、Cr/lip对4期)对肝纤维化具有一定的分期价值;Lac/lip波峰峰高及波峰下面积比值对肝纤维化分期均无意义。
4.肝纤维化大鼠肝实质时间-信号强度曲线呈慢降缓升型,波峰低平宽大;灌注参数SRRmax(对3-4期)、TTP(对2-4期)、MTT(对2-4期)对肝纤维化分期具有一定的价值。
5. Gd-BOPTA增强后延迟60min、120 min时间点肝实质相对强化率对肝纤维化分期无价值,延迟180 min时间点肝实质相对强化率对肝纤维化分期(3-4期)具有一定的价值,但对较早期肝纤维化分期(1-2期)不敏感。
6.传代3的BMSCs细胞可作为细胞移植治疗的理想细胞来源;SPIO-PLL可高效率标记大鼠BMSCs。
7.在体外,FFE-T2WI序列信号下降程度随标记细胞数量增多而增大,示踪效果呈标记细胞数量依赖性。
8.在活体,FFE-T2WI序列为磁粒子标记BMSCs肝移植MR示踪的理想序列。
9.经门静脉途径肝脏BMSCs细胞移植为细胞移植治疗肝脏弥漫性病变的较理想途径。
10.在肝纤维化环境中,移植的BMSCs随时间逐渐向肝实质和纤维间隔内移行,以损伤较重区和纤维间隔分布明显,具有选择性分布特点;移植的BMSCs能与肝细胞紧密连接,形成规则的肝细胞索,对肝损伤具有修复作用。但经门静脉移植BMSCs MR示踪时间窗较短;经肝内途径移植BMSCs细胞分布局限,MR示踪时间窗较长。
11. MR信号变化规律与组织学证实的移植细胞在靶器官的分布、迁移规律具有较好的一致性;MR信号变化规律在一定程度上反映了移植细胞在肝内的分布、迁移、增殖和分化状态。
12.临床应用型1.5T MR可用于肝脏干细胞移植的活体示踪研究。
Chapter I The Experimental Study of Early Diagnosis and Quantitative Analysis of Hepatic Fibrosis of rats with Magnetic Resonance Functional Imaging
In diffuse liver diseases, the diagnosis of hepatic fibrosis and earlier period of liver cirrhosis is the tough problem of early diagnosis of imageology, because the changes of the diseases in morphology are not obvious, and the valuable diagnosis information cannot be provided by the traditional imaging methods which reflect anatomic structure mainly. At present ,it is low that the sensitivity and specificity of the noninvasive diagnostic methods of hepatic fibrosis which are applied clinically;the biopsy of hepatic tissue is thought to be the“gold standard”to diagnose hepatic fibrosis, but it is an invasive method which is limited in clinical application .And the early prediction and intervention is helpful to prevent the development of the process. Thus ,it is urgent to develop new noninvasive methods which can predict hepatic fibrosis early and estimate the degree of the diseases .This study is to study the progression of hepatic fibrosis dynamically and discuss the parameter and the best imaging sequence of magnetic resonance functional imaging by establishing the rat model with liver cirrhosis and applying magnetic functional imaging which include diffusion-weighted imaging、spectrum imaging、perfusion imaging and the application of hepatocyte-specific contrast media); to analyse the appearance of magnetic functional imaging in different degrees and stages of hepatic fibrosis, explore the quantization diadynamic criteria of magnetic functional imaging of hepatic fibrosis and evaluate the value of magnetic functional imaging in the diagnosis of hepatic fibrosis and the early period of liver cirrhosis.
Part I The Establishment of Rat Model with hepatic fibrosis
Objective:To establish animal models with hepatic fibrosis with SD rats by injecting carbon tetrachloride subcutaneously to provide different stages of rat models with hepatic fibrosis for magnetic resonance functional imaging of hepatic fibrosis and magnetic resonance tracer study of cellular transplantation . Methods:The rats of experimental group received subcutaneous injection of 40% carbon tetrachloride oil solution 3ml/kg twice per week and the initial dose is 5ml/kg , and 10% Alcohol is the only drinking water. The rats of control group received subcutaneous injection of physiologic saline , and the dosage and usage is similar with the experimental group, but the drinking water is purified water. Since 2 weeks after injection, 4 rats of experimental group and a rat of control group were collected randomly per week to perform magnetic resonance functional imaging (DWI/MRS/PWI) of liver; after MR imaging but in 4 hours these rats were killed and HE staining、Masson trichrome stain、reticular fiber staining and transmission electron microscope(TEM) examination of liver were performed and the stages of hepatic fibrosis were assessed with light microscope. Results:94 rats of experimental group were successful and 46 were death. The death rate was 33%.The symptoms of different degree of nutritional disturbance and chronic liver disease presented in all the rats of experimental group . The histopathologic examination of liver showed that there was inflammatory cell infiltration, hepatic cell necrosis , collagen and reticular fibre hyperplasia , sinusoidal capillarization ,et al.The pathology stages of hepatic fibrosis was shown as follow: 28 rats with 0 stage、19 rats with 1 stage、27 rats with 2 stage、25 rats with 3 stage and 15 rats with 4 stage. The rats of control group were survival totally and had no corresponding symptoms. Conclusions:The composite factor (CCL4+Alcohol) could induce rats to generate hepatic fibrosis ,and had the advantages of high achievement ratio and short cycle of establishing models .Models of different pathology stages of hepatic fibrosis could be established to provide ideal experimental model for the study of hepatic fibrosis with the use of the composite factor.
Part II The Magnetic Resonance Diffusion-weighted Imaging (DWI) of Hepatic Fibrosis of Rat
Objective:To discuss the value of DWI in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the signal intensity of DWI、the value of ADC and the change of EADC of different degree of hepatic fibrosis . Methods: During establishing rat model with hepatic fibrosis in part I, 4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MR diffusion-weighted imaging. The sequence was SE-EPI and the gradient factor b were 0s/mm2、300s/mm2、600s/mm2、800s/mm2 and 1000s/mm2 respectively .ADC diagram and EADC diagram were achieved according to different value of b, then the signal intensity of DWI of different b values were determined and the value of ADC and EADC were calculated to compare with pathology stages . Results: (1) The signal intensity of DWI of the rats of experimental group were in the tendency of increasing with the increase of staging of hepatic fibrosis .The signal intensity of DWI was uneven because the development of each lobe was different. (2)When b values were 300、600、800 and 1000 s/mm2 ,SNR were(x—±S)36.30±23.25、28.11±12.48、25.71±11.82 and 15.23±6.54 respectively, and the picture quality was in the tendency of decreasing. When b values were 600 or 800 s/mm2, the picture quality were superior to that when b values were 1000 s/mm2,and P<0.05. (3)The analysis of ADC: the value of ADC of control group、grade 1of hepatic fibrosis、grade 2 of hepatic fibrosis、grade 3 of hepatic fibrosis、grade 4 of hepatic fibrosis were[(x—±S)×10-3(]1.542±0.299)×10-3、(1.334±0.268)×10-3、(1.108±0.198)×10-3、(0.978±0.169)×10-3、(0.680±0.260)×10-3 respectively , and there was a trend of decreasing . There were significant differences between control group and other groups、grade 1 of hepatic fibrosis and grade 2、grade 1 and 3、grade 1and 4、grade 2 and 3 ; but there was no significant difference between grade 2 and 3 . The result of correlation analysis of ADC and staging of hepatic fibrosis was : r=-0.766(p﹤0.001).(4) The analysis of EADC: the value of EADC of control group、grade 1、grade 2、grade 3 and grade 4 of hepatic fibrosis were[(x—±S)×10-3](0.315±0.068)×10-3、(0.345±0.081)×10-3、(0.411±0.074)×10-3、(0.465±0.056)×10-3 and(0.595±0.106)×10-3 respectively , and there was a tendency of increasing . There were significant differences between control group and grade 2、control group and grade 3 control group and grade 4、grade 1 and 2、grade 1 and 3、grade 1 and 4、grade 2 and 4、grade 3 and 4 stage ; but there was no significant difference between control group and grade 1、grade 2、grade 3 . The result of correlation analysis of EADC and staging of hepatic fibrosis was : r=0.753(p﹤0.001). Conclusions:(1)The signal intensity of DWI was increasing with the increase of staging of hepatic fibrosis and it is different that the degree of fibrosis of each lobe. (2) The better b values of DWI of liver were 600s/mm2 or 800s/mm2, because it could avoid the influence of perfusion and had higher SNR. (3) The hepatic fibrosis could be staged with ADC and EADC ,and there was better dependability between them .
Part III The Magnetic Resonance Spectrum Imaging (1H-MRS) of Hepatic Fibrosis of Rat
Objective:To discuss the value of MRS in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the metabolites peaks、peak areas and the change of the ratio between them of different degree of hepatic fibrosis. Methods: During establishing rat model with hepatic fibrosis in part I, 4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MRS with 3D PRESS multi-voxel 1H-MRS sequence. Then different metabolites of spectrogram were hand-marked, the peak height and area were generated automatically with the software. Then the peak height of metabolites and lipid and the peak area ratio(Cho/lip、Glx/lip、Lac/lip、Cr/lip)were calculated respectively to compare with pathology staging . Results: There were 5 main peaks of MRS of liver of control group, and the lipid peak of rats of experimental group was lower than that of control group ,but the peaks of other metabolites were higher. The ratio of the peak height of metabolites and lipid were as follows: (1)the ratio of Cho and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.052±0.034、0.212±0.225、0.117±0.122、0.403±0.299 and 0.438±0.295 respectively .There were significant differences between control group and grade 3、control group and grade 4 (P<0.05), but there was no significant difference between control group and grade 1、control group and grade 2、grade 1 and other groups、grade 2 and other groups (P>0.05). (2) the ratio of Glx and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.150±0.132、0.406±0.650、0.656±0.551、0.750±0.452 and 0.763±0.517 respectively . There were significant differences between control group and grade 2、grade3、grade 4 (P<0.05), but there was no significant difference between grade 1 and other groups、grade 2 and 3、grade 2 and 4、grade 3 and 4 (P>0.05). (3) the ratio of Lac and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.139±0.128、0.262±0.178、0.251±0.344、0.355±0.446 and 0.233±0.185 respectively ,and there was no significant difference between these groups (P>0.05), but there was a tendency of increasing with the increase of staging. (4) the ratio of Cr and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.136±0.274、0.767±0.902、0.638±0.960、0.917±0.576 and 0.778±0.856 respectively . There were significant differences between control group and grade 3 (P<0.05), but there was no significant between other groups. The results of correlation analysis between the ratio of the peak height of metabolites and lipid and staging of hepatic fibrosis were as follows : Cho/lip(r=0.503 p﹤0.001)、Glx/lip(r=0.388 p﹤0.05)、Lac/lip(r=0.124 p﹥0.05)、Cr/lip(r=0.235 p﹥0.05). The peak area ratio of main metabolites and lipid of liver were as follows: (1) the ratio of Cho and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis wer(x—±S)e 0.115±0.133、0.257±0.316、0.167±0.187、0.185±0.328 and 0.468±0.372 respectively. There were significant differences between control group and grade 4(P<0.05), but there was no significant difference between other groups (P>0.05). (2) the ratio of Glx and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.045±0.039、0.540±0.318、0.448±0.364、0.482±0.402 and 0.531±0.336 respectively. There were significant differences between control group and other groups (P<0.05), but there was no significant difference between other groups(P>0.05) . (3) the ratio of Lac and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.062±0.069、0.258±0.266、0.277±0.320、0.170±0.314 and 0.274±0.312 respectively. There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of staging. (4) the ratio of Cr and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.109±0.231、0.481±0.614、0.704±0.797、0.465±0.525 and 0.810±0.706 respectively. There were significant differences between control group and grade 4 (P<0.05), but there was no significant difference between other groups(P>0.05) . The results of correlation analysis between the peak area ratio of main metabolites and lipid of liver and staging of hepatic fibrosis were as follows : Cho/lip(r=0.282 p﹥0.05)、Glx/lip(r=0.313 p﹤0.05)、Lac/lip(r=0.135 p﹥0.05)、Cr/lip(r=0.267 p﹥0.05). Conclusions:(1)The ratios of peak height of Cho/lip、Glx/lip、Cr/lip were important for the staging of hepatic fibrosis . The correlation between Cho/lip、Glx/lip and staging of hepatic fibrosis were better than others . (2) The peak area ratios of Cho/lip、Glx/lip、Cr/lip were important for the staging of hepatic fibrosis (Cho/lip、Cr/lip is important for grade 4 and Glx/lip for grade1-4). The correlation between Glx/lip and staging of hepatic fibrosis was better than others .(3)The ratio of peak height and peak area ratio of Lac/lip were not important for the staging of hepatic fibrosis , but there was a tendency of increasing with the increase of staging.
Part IV The Perfusion Imaging (PWI) of Hepatic Fibrosis of Rat
Objective:To discuss the value of PWI in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the changes of perfusion parameters of PWI of different degree of hepatic fibrosis . Methods: During establishing rat model with hepatic fibrosis in part I ,,4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MR perfusion imaging with single shot SE-EPI sequence. After bolus injection of Gd-BOPTA (0.2mmlo/kg , 2ml/s) through vena caudalis of rats, 40 dynamic state were scanning continuously to cover the whole liver . The time-signal intensity curve(TIC)was generated automatically with Perfusion software ,then some correlated parameters were calculated as follows: (1) maximal signal reduction ratio (SRRmax);(2)time to peak (TTP);(3)mean transit time(MTT). The perfusion parameters of PWI were analyzed to compare with pathology staging. Results: (1) The time-signal intensity curve(TIC)of liver parenchyma : the curve of control group descended quickly, then recovered slowly after peak value ,and the recovery extent was larger and recovery course was shorter ; the curve of experimental group descended slowly and the extent was smaller , the time to peak was longer , the recovery extent after peak value was smaller and recovery course was longer, and the peak was lenity. And with the development of hepatic fibrosis ,the changes were more obvious.(2)The relationship of the perfusion parameters of liver and the staging of hepatic fibrosis: 1) the values of SRRmax of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were[(x±S)×100%] 0.754±0.073、0.674±0.137、0.632±0.154、0.603±0.201 and 0.535±0.135 respectively . There were significant differences between control group and grade 3、grade 4 (P<0.05) ; but there was no significant difference between other groups(P>0.05). 2) the values of TPP of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were([x—±S)s]14.175±4.845、18.433±7.293、26.789±3.621、31.755±7.308 and 35.213±6.322 respectively . There were significant differences between control group and grade 2、control group and grade 3、control group and grade 4, grade 1 and 2、grade 1 and 3、grade 1 and 4、grade 2 and 4(P<0.05) ; but there was no significant difference between control group and grade 1、grade 2 and 3、grade 3 and 4 (P>0.05). 3) the values of MTT of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were [(x—±S)s] 24.620±5.577、28.945±2.758、32.502±4.268、35.861±4.651 and 35.203±5.674 respectively . There were significant differences between control group and grade 2、control group and grade 3、control group and grade 4、grade 1 and 3、grade 1 and 4 ; but there was no significant difference between control group and grade 1 , grade 1 and 2、grade 2 and 3、grade 2 and 4、grade 3 and 4 (P>0.05). The results of correlation analysis of the perfusion parameters of liver and staging of hepatic fibrosis were : SRRmax (r=-0.439 p﹤0.05)、TTP(r=0.798 p﹤0.001)、MTT(r=0.647 p﹤0.001) . Conclusions:(1) The time-signal intensity curve(TIC)of liver parenchyma of rats of hepatic fibrosis was characterized by slow- washin and slow-washout, and the peak was low and lenity; (2)The analysis of perfusion parameters of SRRmax、TPP and MTT was helpful to the staging of hepatic fibrosis . The correlation was good between these perfusion parameters and the staging of hepatic fibrosis, especially TTP and MTT .
Part V The Magnetic Resonance Imaging of Hepatic Fibrosis of Rat with Hepatocyte-specific Contrast Media
Objective:To discuss the value of the method of delayed - contrast enhanced scanning with Gd-BOPTA in early diagnosis、quantization analysis and staging of hepatic fibrosis by observing rat models with hepatic fibrosis dynamically after the injection of Gd-BOPTA and analyzing the characteristics of dynamic contrast-enhanced MR imaging and enhancement pattern to compare with pathology staging . Methods: During establishing rat model with hepatic fibrosis in part I , ,4 rats of experimental group and 1 rat of control group were selected randomly per week to perform contrast-enhanced MR imaging with hepatocyte-specific contrast media . After bolus injection of Gd-BOPTA (0.2mmlo/kg) through vena caudalis of rats, delayed scans were performed with TSE-T1WI-TRA sequence after 60min、120min and 180min after contrast media were injected . Then the signal intensity and relative enhancement ratio of liver parenchyma of different time were measured respectively ; and the changes of signal intensity of bile duct and blood vessel of different time of different stages of hepatic fibrosis were observed . Results: The relationship of relative enhancement ratio of liver parenchyma in different time and the staging of hepatic fibrosis were as follows : (1)the value of RER1 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 1.436±0.374、1.487±0.477、1.476±0.440、1.489±0.431 and 1.476±0.436 respectively .There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of the staging of hepatic fibrosis .(2) the value of RER2 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S)1.220±0.370、1.292±0.387、1.344±0.367、1.371±0.388 and 1.405±0.370 respectively .There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of the staging of hepatic fibrosis . (3) the value of RER3 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.844±0.275、0.910±0.380、1.041±0.399、1.209±0.299 and 1.241±0.398 respectively. There were significant differences between control group and grade 3、grade 4 (P<0.05) ; but there was no significant difference between other groups(P>0.05). The results of correlation analysis of relative enhancement ratio of liver parenchyma in different time and staging of hepatic fibrosis were : RER1(r=0.039 p﹥0.05)、RER2(r=0.174 p﹥0.05)、RER3(r=0.420 p﹤0.05) . The appearance of the visualization of bile duct and blood vessel were as follows: the delayed phase of contrast enhanced MRI showed that the tree-like vessels of liver were low signal while the bile duct was high signal in T1WI. The courser of portal vein and bile duct of liver of rats of experimental group were winding and the changes of caliber were not regular, and the bile duct was enhanced in the delayed phase. Conclusions:(1)It was helpless that measuring the relative enhancement ratio of liver parenchyma when 60 min or 120min after injection of Gd-BOPTA to the staging of hepatic fibrosis . It was helpful that measuring the relative enhancement ratio of liver parenchyma when 180 min after injection of Gd-BOPTA to the staging of hepatic fibrosis (especially grade3 and 4), but it was helpless to grade 1 and 2.(2) The morphological changes and enhancement in delayed phase of bile duct of rat model with hepatic fibrosis were a cue of reconstitution of biliary tree and damage of hepatocyte function .
Chapter II The Magnetic Resonance Tracing Experiment in vivo about the Repair of Hepatic Injury by Transplanting BMSCs Labeled with Magnetic Particles
Liver function failure is the main cause of death of various chronic liver diseases. Orthotopic liver transplantation is the most effective method to treat terminal stage of liver diseases at present , but the donor liver are too famine to satisfy clinical requirement . The cellular transplant provides a new therapy strategy for cell reconstitution of the lesion liver and functional recovery of failure liver to make it possible to make use of self-bone marrow stem cell as seed to repair the affection and injury of autologous tissue. The stem cellular transplant tracer study is very important to monitor the disposition、immigration and differentiation of transplanted cells in vivo dynamically and evaluate the effective of cellular transplant ,but this tracer technique is always a puzzle in medical scientific research and traditional tracer technique of transplanted cells must be carry out with histology observation ex vivo . Therefore, it is urgent to establish a transplanted cell tracer technique in vivo which is safe、sensitive、effective and fit to clinic . Close attention was paid to magnetic resonance tracer technique in vivo in the study of stem cell increasingly, and it is a new technique of observing the survival and distribution of transplanted cells in vivo to perform MRI examination after cellular transplant while the cell is labled with contrast media as molecular probe which is sensitive to MR. The MRI tracer technique of transplanted cell which is applied in the study of cardiac muscle and central nervous system is more , but that applied in the study of bone marrow stem cell in hepatic fibrosis is seldom . This study was focused on: (1)Separation、proliferation and identification of BMSCs from rats.(2) Establishing rat model with hepatic fibrosis. (3) Transplanting BMSCs labeled with SPIO and bromodeoxyuridine (BrdU) to treat the rats with hepatic fibrosis . (4) Monitoring the labeled cells after transplantation into the hepatic fibrosis rats with MR scanning dynamically in vivo and contrasting with pathohistology . This study was to discuss the feasibility of the technique of separation、proliferation and identification of BMSCs、MR tracer technique of BMSCs in vivo which was labeled with SPIO as molecular probe、the optimum scanning parameters and imaging sequence、the feature of distribution and immigration of transplanted cells in liver and the rule of the MR signal intensity changes , and to explore the feasibility of the application of 1.5T MR in stem cell tracer study to establish the foundation of clinical application of stem cell transplant MR tracer study in vivo .
Part I :In Vitro Culture of BMSCs from SD Rats and label of BMSCs with Brdu and SPIO
Objective To develop the in-vitro culture condition of BMSCs from SD rats and to establish the foundation of clinical application of stem cell transplant with MR tracer study in vivo. Methods SD rats (weight 60~90g) were sacrificed. BMSCs were isolated from their bone marrows. Cultured BMSCs were observed daily under phase-contrast microscope and the cell purity were further confirmed by FACS analysis. Results 93.1% of the passage 3 cells were CD90 (one of the BMSCs’surface markers) positive. On the other hand, CD45 + cells were dropped from 71.2% in primary culture to 3.9% in the passage 3. 100% of BMSCs were labeled with SPIO- PLL.Conclusion The passage 3 cells could be used to cell transplantation because of its high purity of BMSCs. It was effective to label BMSCs with SPIO- PLL.
PartⅡ:MRI Study of Bone Marrow Stromal Cells of Rats Labeled with Magnetic Particle in Vitro
Objective:To discuss the changes of MR signal intensity of different numbers of labeled BMSCs and the most sensitive sequences of MR tracing after stromal cells were labeled with magnetic particle to establish the foundation for MR tracing in vivo and provide theoretical evidence . Methods:BMSCs which were labeled with Brdu and SPIO and that which were not labeled were suspended with 1% agarose solutions . The subjects were divided into four groups and put into different EP ducts, including 2×106labeled cells、1.0×106 labeled cells、5×105 labeled cells and 2×106 unlabeled cells .MR scanning sequences included coronal and axial TSE-T1WI、TSE- T2WI and FFE-T2WI.Signal intensity of different sequences of the same object were measured and the MR signal intensity changing rate were compared . Results : The MR signal intensity changing rate of BMSCs which were labeled and not labeled were as follows : (1)the MR signal intensity changing rate of TSE-T1WI of 5×105 labeled cells、1×106 labeled cells and 2×106labeled cells were [(x±S)%] -4.19±0.788、-16.35±1.228、-22.80±1.053 respectively . (2) the MR signal intensity changing rate of TSE-T2WI of 5×105 labeled cells、1×106 labeled cells and 2×106 labeled cells were([x±S)%] -14.15±1.366、-35.09±1.391、-53.02±1.299 respectively . (3)the MR signal intensity changing rate of FFE-T2WI of 5×105 labeled cells、1×106 labeled cells and 2×106 labeled cells were[(x±S)%] -44.98±0.4562、-69.38±0.820、-87.24±0.818 respectively . There were significant differences among labeled cells groups(P<0.001) and significant differences among MRI sequences(P<0.001) .Conclusion : The signal intensity changes of FFE-T2WI sequence is the most apparent when the BMSCs which were labeled with magnetic particle were in the same concentration , and with the number of the labeled cells increasing ,the signal intensity changes were more visible. FFE-T2WI sequence was the ideal sequence for MR tracing of cells labeled with magnetic particle.
PartⅢ:The Homogeneous Transplantation of BMSCs of Rat into Liver
Objective:To transplant BMSCs into liver with two ways -direct injection and intravenous injection via portal vein . To discuss the feature of the two transplant ways to establish the foundation of comparing the effective of MR tracing and feature of signal intensity changes of them and understanding the rule of distribution and immigration of transplanted cells in the target organ .Methods:20 rat models with hepatic fibrosis which were established in part I of chapter I were selected and divided into four groups , including 1 group :control group (normal sodium which contained no BMSCs were transplanted , and in the group , the number of intra-portal-venous injection and direct intra-liver injection was 2 and 2 respectively); 2 group: BMSCs control group (2×106 BMSCs which were not labeled with Brdu and SPIO were transplanted , and in the group , the number of intra-portal-venous injection and direct intra-liver injection was 2 and 2 respectively ) ; 3 group : labeled cells transplanted via portal vein (2×106 BMSCs which were labeled with Brdu and SPIO were transplanted via portal vein .n=6) ; 4 group : labeled cells transplanted into liver by direct injection (2×106 BMSCs which were labeled with Brdu and SPIO were transplanted into liver by direct injection.n=6).The transplanted cells were taken suction with microinjector and injected into the main portal vein or liver slowly. Results :In the process of transplanted cells into liver by direct injection , the operative procedure was convenient and the cellular transplant was successful . In the process of transplanted cells via portal vein , the transplant operation of 3 rats were difficult because the separation of main portal vein was difficult which was resulted from adherence of abdominal membrane and mesenterium in abdominal cavity , and the operation of other rats were successful . Because the main portal vein of experimental rats was thickening in different degree , the puncturation of main portal vein was successful . After cells or normal saline were transplanted into liver slowly, there was no paradoxical reaction occurred in the rats. The healing of operative incision was well. Conclusion: Transplant stromal cells into liver via portal vein and direct injection into liver were safe and effective. And the choice of transplant way was determined according to clinical or investigative purpose.The difficulty of the separation of main portal vein because of adherence of abdominal contents of subjects should be considered.
PartⅣ: MR Tracing of the Homogeneous Transplantation of Bone Marrow Stromal Cells of Rat into Liver in Vivo
Objective:To discuss the effective of MR tracing and the changes of MR signal intensity of the two transplant ways (BMSCs were transplanted into liver which were labeled with Brdu and SPIO via portal vein and direct injection ); to understand the rule of distribution and immigration of transplanted cells in the target organ to establish the foundation of MR tracing in vivo . Methods:2 rats were selected from every group randomly after transplantation in partⅢ, and performed axial TSE-T2WI-SPAIR、TSE-T1WI and FFE-T2WI when 2hours、3days、7days and 2weeks after transplantation .The developing effect of transplanted cells of different sequences were compared. MR signal intensity、distribution range and rule of change of each group in different time were observed . After MR scanning ,1 rat was selected from erery group randomly and killed to obtain liver specimen、fraction of lung and spleen to perform HE staining、hemosiderin staining and Brdu immunohistochemistry staining .The distribution of hemosiderin-positive cells and Brdu immunohistochemistry-positive cells in liver、lung and spleen should be noticed . Results: (1)MR examination showed as followed :the imaging of FFE-T2WI sequence was the best of all sequences .1 group and 2 group showed no signal changes when 2hours、3days、7days and 2weeks after transplant .3 group showed that there were many hypo-intense lesions in hepatic hilar region when 2 hours after transplant , and with the time going on ,the hypo-intense lesions became smaller . The effect of MR tracing was worst when 2 weeks after transplant. 4 group showed that there were mass-like hypo-intense area in the area of cell injection, and with the time going on , the area was becoming larger , the edge was becoming ambiguous , and when 2 weeks after transplant , the hypo-intense area was still obvious .(2) The histopathologic examination showed as follows : 1) HE staining showed that 2 hours and 3 days after transplant , necrosis、inflammation and fibroplasia of liver of all groups were similar, but when 7days and 2 weeks after transplant , the necrosis and inflammatory cell infiltration of 2-4 group were improving and more obvious than control group . 2) Hemosiderin staining showed that 2hours、3days、7days and 2weeks after transplant ,there were no hemosiderin-positive cells in 1-2 group .The hemosiderin-positive cells of 3group were in the portal vein of porta hepatic 2 hours after transplant , in the small branches of portal vein、sinus hepaticus and around central veins of hepatic lobules 3 days after transplant , and in the liver parenchyma (especially in the area of lesion) 7 days and 2weeks after transplant . The histology change in different time was corresponding with the signal intensity changes of MRI. At the same time, there was a few positive cells in the lung and in the spleen . The hemosiderin-positive cells of 4 group were around the injection point, and with the time going on, the cells immigrated into the liver parenchyma nearby. The histology change in different time was corresponding with the signal intensity change of MRI. At the same time, there was no positive cells in the lung ,and a few in the spleen . 3) Brdu immunohistochemistry staining showed that the result was in coincidence with Hemosiderin staining. Conclusion: (1) FFE-T2WI sequence is the ideal sequence of liver MR tracing of transplanted cells labeled with magnetic particle in vivo. (2)The transplanted cells were scattered in the whole liver (especially in the area of lesion) with the time going on . It was helpful to repair the lesion of liver by transplanting BMSCs .But the time window of MR tracing was shorter when BMSCs were transplanted into liver via portal vein ;It is the ideal way to treat diffuse disease of liver when BMSCs were transplanted into liver via portal vein;but the distribution of transplanted cells were localized and the time window of MR tracing was longer when BMSCs were injected into liver directly. (3) The rule of MR signal intensity changes and the rule of distribution and immigration of transplanted cells in target organ were in good concordance.(4) The rule of MR signal intensity changes reflected the condition of distribution、immigration、proliferation and differentiation of transpl
肝脏弥漫性病变中,肝纤维化和早期肝硬化因形态学改变不明显而成为影像学早期诊断面临的难题,传统的以反映解剖结构的影像学方法对此类病变难于提供有价值的诊断信息。目前,临床上应用的非创性的肝纤维化诊断方法敏感性和特异性较差;肝组织活检被认为是诊断肝纤维化的“金标准”,但作为一种侵入性的诊断方法,其临床应用具有许多限度。肝纤维化的早期检测、早期干预有助于阻止病变的进一步发展。因此,迫切需要开发无创性、对慢性肝病出现肝纤维化能进行早期预测、病变程度评价的新的方法。
本研究通过CCI4诱导建立大鼠肝纤维化模型,采用磁共振功能成像(弥散加权成像、波谱成像、灌注成像和肝细胞特异性对比剂应用)技术,并与病理组织学对照,对肝纤维化进展过程中,分子弥散、能量代谢、血流灌注和肝细胞功能等多因素病理生理改变进行动态、系统研究,国内外类似研究尚未见报道。本研究目的在于探讨肝纤维化的MR功能成像参数及最佳成像序列;分析不同程度、不同分期肝纤维化的MR功能成像表现,探索肝纤维化MR功能成像的量化诊断指标;评价MR功能成像在肝纤维化、早期肝硬化诊断中的价值。
第一部分大鼠肝纤维化模型的建立
1.目的:采用SD大鼠皮下注射四氯化碳法,建立肝纤维化动物模型,为肝纤维化的MR功能成像和骨髓基质细胞肝移植MR示踪研究提供不同分期的肝纤维化动物模型。
2.方法:
实验组大鼠腹部皮下注射40%CCl4油溶液,剂量为3ml/kg体重,2次/周,首次剂量为5ml/kg体重;10%乙醇溶液作为唯一饮用水。对照组大鼠腹部皮下注射生理盐水,剂量及用法同实验组,纯净水作为饮用水。注药后第2周开始,每周随机抽取实验组大鼠4只、对照组大鼠1只,肝脏磁共振功能成像(DWI、MRS、PWI)后4小时内处死大鼠,肝脏取材行HE染色、Masson三色染色、网状纤维染色及透射电镜检查,光镜下判定肝纤维化分期。
3.结果:
模型组共有94只大鼠完成实验,死亡46只,死亡率33 %。模型组大鼠均出现不同程度的营养不良、慢性肝病症状。肝脏病理组织学检查见炎症细胞浸润,肝细胞坏死,胶原及网状纤维增生,肝窦毛细血管化等改变。对照组20只全部存活,无相应症状。肝纤维化病理分期:0期28只、1期19只、2期27只、3期25只、4期15只。
4.结论:
复合因素(CCl4+酒精)能成功地诱导大鼠肝纤维化,并且具有成模率高,造模周期短优点,并有较明显的阶段性变化;应用复合因素可建立不同病理分期的肝纤维化模型,为肝纤维化研究提供理想的实验模型。
第二部分大鼠肝纤维化的MR弥散加权成像(DWI)
1.目的:
通过分析大鼠不同程度肝纤维化的DWI信号强度、ADC值和EADC值变化,以探讨应用非创性DWI检测方法对肝脏纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,进行MR弥散成像: SE-EPI序列,梯度因子b分别取:0s/mm2、300 s/mm2、600 s/mm2、800 s/mm2、1000 s/mm2。根据不同b值拟合得到ADC图、EADC图,测定不同b值弥散成像的信号强度,计算ADC值和EADC值,并与病理分期对照。
3.结果:
(1)实验组大鼠随着肝纤维化分期的增加,DWI信号强度呈增加趋势,各叶纤维化进展不近相同,表现为DWI信号强度不均匀。(2)b=300、600、800、1000 s/mm2时,SNR分别为(x±S):36.30±23.25、28.11±12.48、25.71±11.82和15.23±6.54,图像质量呈下降趋势,b=600、800 s/mm2时优于b=1000 s/mm2(P﹤0.05)。(3)ADC值分析:对照组与1期、2期、3期、4期肝纤维化平均ADC值分别为[(x±S)×10-3]:(1.542±0.299)×10-3、(1.334±0.268)×10-3、(1.108±0.198)×10-3、(0.978±0.169)×10-3、(0.680±0.260)×10-3 ,呈下降趋势。对照组与1期、2期、3期、4期比较有显著性差异;1期与2期、3期、4期比较,2期与4期比较,3期与4期比较均有显著性差异;2期与3期比较差异无统计学意义。ADC值与肝纤维化分期的相关性分析:r=-0.766(p﹤0.001)。(4)EADC值分析:对照组与1期、2期、3期、4期肝纤维化平均EADC值分别为[(x±S)×10-3]:(0.315±0.068)×10-3、(0.345±0.081)×10-3、(0.411±0.074)×10-3、(0.465±0.056)×10-3、(0.595±0.106)×10-3,呈增高趋势。对照组与2期、3期、4期比较有显著性差异,1期与2期、3期、4期比较,2期与4期比较,3期与4期比较均有显著性差异。对照组与1期比较,2期与3期比较差异无统计学意义。EADC值与肝纤维化分期的相关性分析:r=0.753 (p﹤0.001)。
4.结论:
(1)DWI信号强度随着肝纤维化分期的增加而增高,各叶纤维化进展不近相同;
(2)梯度因子b取600 s/mm2或800 s/mm2时,即可避免灌注对弥散的影响(低b值),又具有较高的信噪比,为肝脏DWI成像较理想的b值取值;(3)ADC值(1-4期)和EADC值(2-4期)均能对肝纤维化进行分期,且均具有较好的相关性。
第三部分大鼠肝纤维化的MR波谱成像(1H-MRS)
1.目的:
通过分析大鼠不同程度肝纤维化的MRS代谢物波峰峰高、波峰下面积及其相互比值的变化,以探讨应用MRS检测方法对肝纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,采用3D PRESS多体素1H-MRS序列进行MR波谱成像,手动标记波谱图像中不同代谢物,软件自动生成各代谢物的峰高和波峰下面积,分别计算代谢物与脂质的峰高、波峰下面积的比值(Cho/lip、Glx/lip、Lac/lip、Cr/lip)并与病理分期对照。
3.结果:
对照组大鼠肝脏波谱成像可见5个主要波峰;模型组大鼠脂质峰下降,其余代谢物波峰不同程度增高。代谢物与脂质波峰峰高比值:(1)对照组与1期、2期、3期、
4期肝纤维化Cho/lip比值分别为(x±S):0.052±0.034、0.212±0.225、0.117±0.122、0.403±0.299、0.438±0.295。对照组与3期、4期比较P﹤0.05,对照组与1期、2期比较,1期、2期分别与各组比较P﹥0.05。(2)对照组与1期、2期、3期、4期肝纤维化Glx/lip比值分别为(x—±S):0.150±0.132、0.406±0.650、0.656±0.551、0.750±0.452、0.763±0.517。对照组与2期、3期、4期比较P﹤0.05,与1期比较P﹥0.05,余各组两两比较P﹥0.05。(3)对照组与1期、2期、3期、4期肝纤维化Lac/lip比值分别为(x—±S):0.139±0.128、0.262±0.178、0.251±0.344、0.355±0.446、0.233±0.185,差异无统计学意义(P﹥0.05),但随分期增加有增高趋势。(4)对照组与1期、2期、3期、4期肝纤维化Cr/lip比值分别为(x±S):0.136±0.274、0.767±0.902、0.638±0.960、0.917±0.576、0.778±0.856。对照组与3期比较P﹤0.05,余各组两两比较差异无统计学意义。代谢物与脂质波峰峰高比值与肝纤维化分期的相关性分析:Cho/lip(r=0.503 p﹤0.001)、Glx/lip(r=0.388 p﹤0.05)、Lac/lip(r=0.124 p﹥0.05)、Cr/lip(r=0.235 p﹥0.05)。
肝脏主要代谢物与脂质波峰下面积比值:(1)对照组与1期、2期、3期、4期肝纤维化Cho/lip比值分别为(x±S):0.115±0.133、0.257±0.316、0.167±0.187、0.185±0.328、0.468±0.372。对照组与4期比较P﹤0.05,余两两比较差异无显著性。(2)对照组与1期、2期、3期、4期肝纤维化Glx/lip比值分别为(x±S):0.045±0.039、0.540±0.318、0.448±0.364、0.482±0.402、0.531±0.336。对照组与1期、2期、3期、
4期比较P﹤0.05,余各组之间比较P﹥0.05。(3)对照组与1期、2期、3期、4期肝纤维化Lac /lip比值分别为(x—±S):0.062±0.069、0.258±0.266、0.277±0.320、0.170±0.314、0.274±0.312。差异无统计学意义(P﹥0.05),但随分期增加有增高趋势。(4)对照组与1期、2期、3期、4期肝纤维化Cr/lip比值分别为(x±S):0.109±0.231、0.481±0.614、0.704±0.797、0.465±0.525、0.810±0.706。对照组与4期比较P﹤0.05,与1期、2期、3期比较及余各组之间两两比较P﹥0.05。代谢物与脂质波峰下面积比值与肝纤维化分期的相关分析:Cho/lip(r=0.282 p﹥0.05)、Glx/lip(r=0.313 p﹤0.05)、Lac/lip(r=0.135 p﹥0.05)、Cr/lip(r=0.267 p﹥0.05)。
4.结论:
(1)Cho/lip、Glx/lip、Cr/lip波峰峰高比值对肝纤维化分期(Cho/lip对3-4期、Glx/lip对2-4期、Cr/lip对3期)具有一定的价值;代谢物与脂质波峰峰高比值与肝纤维化分期的相关性以Cho/lip、Glx/lip较高。(2)Cho/lip、Glx/lip、Cr/lip波峰下面积比值对肝纤维化分期(Cho/lip、Cr/lip对4期;Glx/lip对1～4期)具有一定的价值;代谢物与脂质波峰下面积比值与肝纤维化分期的相关性以Glx/lip较高(。3)Lac/lip波峰峰高比值、波峰下面积比值对肝纤维化分期均无意义,但均随分期增加呈增高趋势。
第四部分大鼠肝纤维化的MR灌注成像(PWI)
1.目的:
通过分析不同程度肝纤维化的PWI的灌注参数变化,以探讨应用PWI检测方法对肝脏纤维化早期诊断、量化分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,采用单次激发SE-EPI序列进行MR灌注成像:经大鼠尾静脉快速团注Gd-BOPTA,剂量为0.2mmol/kg体重,流率2ml/s,连续扫40个动态,覆盖整个肝脏。应用Perfusion软件自动生成肝实质信号强度-时间曲线,计算相关参数:(1)最大信号下降百分率(SRRmax),(2)到达峰值时间(TTP),(3)平均通过时间(MTT)。分析PWI灌注参数值并与病理肝纤维化分期对照。
3.结果:
(1)肝实质信号强度-时间曲线:对照组曲线呈快速下降、达峰值后缓慢恢复,恢复幅度较大,恢复时程较短;模型组曲线下降速度减慢,下降幅度减小,达峰值时间延长,达峰值后恢复幅度较小,恢复时程较长,波峰宽大,随着肝纤维化分期的增高这种改变更加明显。(2)肝脏灌注参数与肝纤维化分期的关系:1)对照组与1期、2期、3期、4期肝纤维化SRRmax值分别为[(x±S)×100%]:0.754±0.073、0.674±0.137、0.632±0.154、0.603±0.201、0.535±0.135。对照组与3期、4期比较P﹤0.05,与1期、2期比较差异无显著性,余各组两两比较P﹥0.05。2)对照组与1期、2期、3期、4期肝纤维化TTP值分别为[(x±S)s]:14.175±4.845、18.433±7.293、26.789±3.621、31.755±7.308、35.213±6.322。对照组与2期、3期、4期比较P﹤0.05,与1期比较无显著性差异;1期与2期、3期、4期比较,2期与4期比较P﹤0.05;2期与3期比较,3期与4期比较P﹥0.05。3)对照组与1期、2期、3期、4期肝纤维化MTT值分别为[(x—±S)s]:24.620±5.577、28.945±2.758、32.502±4.268、35.861±4.651、35.203±5.674。对照组与2期、3期、4期比较P﹤0.05, 1期与3期、4期比较P﹤0.05,对照组与1期比较,1期与2期比较,2期与3期、4期比较,3期与4期比较差异均无统计学意义。肝脏灌注参数与肝纤维化分期的相关分析:SRRmax(r=-0.439 p﹤0.05)、TTP(r=0.798 p﹤0.001)、MTT(r=0.647 p﹤0.001)。
4.结论:
(1)肝纤维化大鼠肝实质信号强度-时间曲线呈慢降缓升型,波峰低平宽大;(2)SRRmax、TTP、MTT灌注参数分析有助于肝纤维化分期(SRRmax对3-4期、TTP、MTT对2-4期)。SRRmax、TTP、MTT灌注参数与肝纤维化分期均有较好的相关性,其中以TTP和MTT较高。
第五部分大鼠肝纤维化的肝细胞特异性对比剂MR成像
1.目的:
通过对大鼠肝纤维化模型进行Gd-BOPTA增强动态观测,分析不同程度肝纤维化各延迟时间点的动态增强表现、强化程度,并与病理分期对照,以探讨应用Gd-BOPTA增强延迟扫描方法对肝脏纤维化早期诊断、定量分析及其分期的价值。
2.方法:
第一部分在大鼠给药建立肝纤维化模型过程中,每周随机抽取模型组大鼠4只,对照组大鼠1只,进行Gd-BOPTA增强MR扫描:经大鼠尾静脉快速团注Gd-BOPTA,剂量为0.2mmol/kg体重,以TSE-T1WI序列分别于注射对比剂后60min(RER1)、120 min(RER2)、180 min(RER3)时间点延迟扫描,分别测算各不同时间点的信号强度、肝实质相对强化率;观察不同程度肝纤维化大鼠各不同时间点的胆管、血管信号强度变化特点。
3.结果:
不同时间点肝实质相对强化率与肝纤维化分期的关系:(1)对照组与1期、2期、3期、4期肝纤维化RER1值分别为(x±S):1.436±0.374、1.487±0.477、1.476±0.440、1.489±0.431、1.476±0.436。各组差异无统计学意义(P﹥0.05),但随肝纤维化分期的增加有增高趋势。(2)对照组与1期、2期、3期、4期肝纤维化RER2值分别为(x—±S):1.220±0.370、1.292±0.387、1.344±0.367、1.371±0.388、1.405±0.370。各组差异无统计学意义(P﹥0.05),但随肝纤维化分期的增加有增高趋势。(3)对照组与1期、2期、3期、4期肝纤维化RER3值分别为(x±S):0.844±0.275、0.910±0.380、1.041±0.399、1.209±0.299、1.241±0.398。对照组与3期、4期比较P﹤0.05,与1期、2期比较P﹥0.05,肝纤维化各期之间两两比较P﹥0.05。不同时间点肝实质相对强化率与肝纤维化分期的相关分析:RER1(r=0.039 p﹥0.05)、RER2(r=0.174 p﹥0.05)、RER3(r=0.420 p﹤0.05)。胆管、血管显影情况: MR增强延迟期:肝内血管树T1WI为低信号;胆道树T1WI表现为高信号。实验组大鼠肝脏门静脉树和胆管树分支走行迂曲、粗细变化不规律,胆管树见延迟强化。
4.结论:
(1)对比剂Gd-BOPTA增强后60min、120 min时间点肝实质相对强化率对肝纤维化分期无价值,延迟后180 min时间点肝实质相对强化率对肝纤维化分期(3-4期)具有一定的价值,不能对早期肝纤维化(1-2期)进行分期。(2)肝纤维化大鼠胆管树形态改变、延迟强化,提示肝纤维化时胆系重构、肝细胞功能受损。
第二篇磁粒子标记大鼠BMSCs移植修复肝损伤的MR活体示踪实验研究
肝功能衰竭是各种慢性肝病主要的死亡原因,原位肝移植是目前治疗终末期肝病的最有效的方法,但由于供肝严重缺乏,远无法满足临床需求。肝脏的细胞移植为病损肝脏的细胞重建和衰竭肝脏的功能恢复提供了一种新的治疗策略,使患者有可能利用自身的骨髓干细胞作为种子细胞,来修复自体组织的病变和损伤。干细胞移植示踪研究,对动态监测活体内移植细胞的分布、迁移、分化及评估细胞移植效果具有重要作用,但干细胞移植示踪技术一直是医学科研中的难题。传统的移植细胞的示踪方法必须在离体状态下通过组织学观察来实现。因此,迫切需要建立一种安全无创、敏感有效、适于临床的移植细胞活体示踪技术。MR活体示踪在干细胞的研究中日益受到关注,利用MR敏感的对比剂作为分子探针标记供体移植细胞,移植后对受体进行MRI检测是活体观察移植细胞存活和分布的新技术。
应用MR活体示踪技术研究肝纤维化形成环境中移植的BMSCs分布、迁移规律尚未见报道。本研究通过对大鼠BMSCs的分离、增殖和鉴定,采用Brdu和SPIO双标记大鼠BMSCs,对大鼠肝纤维化模型进行同种异体移植后,行MR活体动态观测,并与病理组织学对照,以探讨大鼠BMSCs的分离、增殖和鉴定技术,和SPIO作为分子探针标记BMSCs MR活体示踪的可行性,以及MR成像的最佳扫描参数及成像序列,并探讨移植细胞在肝脏纤维化环境中分布、迁移特点及其对肝损伤的修复作用,以及相应的MR信号变化规律,探索临床应用型1.5T MR用于干细胞示踪研究的可行性,为干细胞移植MR活体示踪的临床应用奠定基础。
第一部分大鼠骨髓基质细胞的体外分离、培养、鉴定和标记
1.目的:
观察大鼠骨髓基质细胞在体外培养的条件,并利用Brdu和超顺磁性氧化铁颗粒对大鼠BMSCs进行双标记,为应用MR对移植骨髓基质细胞进行活体示踪研究奠定基础。
2.方法:
取60~90g SD大鼠,体外培养骨髓基质细胞,利用倒置相差显微镜观察原代及不同传代细胞生长、形态特点,并应用流式细胞仪鉴定原代及不同传代细胞表型。利用Brdu和PLL介导的超顺磁性氧化铁颗粒对大鼠BMSCs进行双标记。
3.结果:
培养的骨髓基质细胞第3代,骨髓基质细胞的细胞表面标志CD90表达阳性的细胞已占到93.1%。造血干细胞的表面标志CD45表达阳性的细胞已从原代的71.2%降低到3.9%。PLL介导的超顺磁性氧化铁颗粒对大鼠BMSCs标记率达100%。
4.结论:
传代3的BMSCs可作为细胞移植治疗的理想细胞来源。PLL介导的超顺磁性氧化铁颗粒可高效率地标记大鼠BMSCs。
第二部分磁粒子标记大鼠骨髓基质细胞的体外MR成像实验
1.目的:
探讨标记细胞不同细胞数量所引起的MR信号强度变化规律,以及磁粒子标记细胞MR示踪最敏感的成像序列,为标记细胞活体内MR示踪奠定基础,并提供理论依据。
2.方法:
用1%的琼脂糖混悬Brdu和SPIO双标记的BMSCs(分别为2×106、1×106、5×105个)和未标记的BMSCs (2×106个),分别置于不同EP管中。行冠状位和轴位TSE序列T1WI、T2WI和FFE-T2WI扫描,测量相同细胞数量不同成像序列以及相同成像序列不同细胞数量的信号强度,并比较MR信号变化率。
3.结果:
磁粒子标记的各不同数量BMSCs各成像序列MR信号变化率(:1)标记组5×105、1×106、2×106个细胞TSE-T1WI序列MR信号变化率分别为[(x±S)%]:-4.19±0.79、-16.35±1.23、-22.80±1.05。(2))标记组5×105、1×106、2×106个细胞TSE-T2WI序列MR信号变化率分别为[(x—±S)%]:-14.15±1.37、-35.09±1.39、-53.02±1.30。(3)标记组5×105、1×106、2×106个细胞FFE-T2WI序列MR信号变化率分别为[(x±S)%]:-44.98±0.46、-69.38±0.82、-87.24±0.82。同一扫描序列中各标记细胞组之间以及相同标记细胞数量各成像序列之间两两比较P﹤0.001,差异均有统计学意义。
4.结论:
磁粒子标记BMSCs,细胞浓度相同条件下,以FFE-T2WI序列信号下降最为明显,标记的细胞数量越多,信号改变越明显,呈细胞数量依赖性。FFE-T2WI序列为磁粒子标记细胞MR示踪的理想序列。
第三部分大鼠骨髓基质细胞的同种异体肝移植
1.目的:
经门静脉途径和肝内途径进行肝脏的BMSCs移植,探讨两种移植途径的特点,为比较两种移植途径的MR示踪效果及信号变化特点,及了解移植细胞在靶器官的分布、迁移规律奠定基础。
2.方法:
将第一篇、第一部分中建立的SD大鼠肝纤维化模型20只分为4组,1组:生理盐水对照组(注射不含BMSCs的等容生理盐水,其中经门静脉2只,经肝内注射2只);2组:未标BMSCs对照组(移植2×106个未经Brdu和SPIO标记的BMSCs,其中经门静脉2只,经肝内注射2只);3组:经门静脉移植BMSCs标记组(经门静脉移植2×106个经Brdu和SPIO标记的BMSCs,6只);4组:经肝内移植BMSCs标记组(经肝内移植2×106个经Brdu和SPIO标记的BMSCs,6只)。手术暴露门静脉主干和肝脏,以微量注射器抽取相应移植内容,穿刺各组目标,缓慢注入。
3.结果:
经肝内注射组:手术操作简便,细胞移植顺利。经门静脉移植组:有3只大鼠因腹膜、系膜粘连,门静脉主干分离困难,给移植手术带来一定的难度,其余大鼠移植手术顺利。因实验大鼠均出现门静脉主干不同程度增粗,门静脉主干穿刺均获成功。缓慢注入移植细胞或生理盐水后,大鼠未出现异常反应。手术切口愈合良好。
4.结论:
经门静脉途径和经肝内注射途径对肝脏进行BMSCs移植,安全、易行。
第四部分大鼠骨髓基质细胞同种异体肝移植的MR活体示踪研究
1.目的:
探讨经门静脉途径和经肝内途径移植标记细胞的MR活体示踪效果及信号变化特点,从而了解肝纤维化环境中移植细胞在靶器官的分布、迁移规律及其对肝损伤的修复作用,为移植干细胞MR活体示踪的临床应用奠定基础。
2.方法:
第三部分移植术后各组大鼠每组随机抽取2只,分别于移植术后2h、3d、7d、2w行TSE序列轴位T2WI-SPAIR、T1WI和FFE-T2WI扫描,对不同序列图像的移植细胞显影效果进行比较;观察各移植组不同时间点的MR信号强度、分布范围及其变化规律。于MR扫描后随机抽取每组大鼠各一只处死,取肝脏标本,并取部分大鼠肺和脾脏组织,采用HE染色、含铁血黄素染色、Brdu免疫组织化学染色。观察含铁血黄素染色阳性细胞和Brdu免疫组织化学染色阳性细胞在肝、肺、脾脏中的分布。
3.结果:
(1)MR检查:在各扫描序列中,以FFE-T2WI序列成像效果最佳。1组和2组:移植术后2h、3d、7d、2w不同时间点、各序列MR图像均未见异常信号改变。3组:移植术后2h,肝门区见多发结节状低信号灶,并随时间延长向肝内分散,低信号结节逐渐变小。移植术后2w示踪效果较差。4组:移植术后2h,局部见团状低信号灶,随时间延长范围逐渐扩大,边缘逐渐模糊,移植术后2w低信号区仍较明显。
(2)病理组织学检查:1)HE染色:移植术后2h、3d各组肝内坏死、炎症及纤维组织增生情况基本类似;移植术后7d、2w,与1组比较,2～4组大鼠肝组织坏死、炎症细胞浸润情况逐渐好转,以经门静脉移植组明显。2)含铁血黄素染色:1组和2组:移植术后2h、3d、7d、2w,肝脏、肺和脾脏含铁血黄素染色,均未见阳性细胞。3组:移植术后2h含铁血黄素染色阳性细胞主要分布于肝门部门静脉内,移植术后3d阳性细胞主要分布于门静脉小分支、肝窦内、肝小叶中央静脉周围,移植术后7d、2w阳性细胞主要分布于损伤较重的肝实质和纤维间隔;各时间点组织学所见与MR成像信号改变一致。同期的肺和脾脏含铁血黄素染色,其内可见少量阳性细胞散在分布。4组:移植术后2h、3d、7d、2w可见含铁血黄素染色阳性细胞密集分布于注射点,随时间延长细胞向周围肝内迁移。各时间点组织学所见与MR成像信号改变一致。同期肺内未见明显阳性细胞,脾脏内可见少量阳性细胞分布。3)Brdu免疫组织化学染色:结果与含铁血黄素染色一致。
4.结论:
(1)FFE-T2WI序列为肝内移植磁粒子标记细胞MR活体示踪的理想序列;(2)经门静脉途径移植BMSCs细胞随时间逐渐向肝内移行,以损伤较重区和纤维间隔分布明显,具有选择性分布特点;移植的部分BMSCs与肝细胞紧密连接,形成规则的肝细胞索,对肝损伤具有修复作用;经门静脉途径移植BMSCs为细胞移植治疗肝脏弥漫性病变的较理想途径,但MR示踪时间窗较短;经肝内途径移植BMSCs细胞分布局限,MR示踪时间窗较长;(3)MR信号变化规律与组织学证实的移植细胞在靶器官的分布、迁移规律具有较好的一致性;(4)MR信号变化规律在一定程度上反映了移植细胞在肝内的分布、迁移、增殖和分化状态;(5)临床应用型1.5T MR可用于肝脏干细胞移植的活体示踪研究。
结论
1.应用复合因素(CCl4+酒精)能成功地建立不同病理分期的大鼠肝纤维化模型,有较明显的阶段性变化。
2. DWI信号强度随着肝纤维化分期的增加而增高;梯度因子600 s/mm2或800 s/mm2为肝脏DWI成像较理想的b值取值;ADC值和EADC值均能对肝纤维化进行分期(ADC值对1-4期;EADC值对2-4期),具有较好的相关性。
3. Cho/lip、Glx/lip、Cr/lip波峰峰高比值(Cho/lip对3-4期、Glx/lip对2-4期、Cr/lip对3期)及波峰下面积比值(Cho/lip对4期、Glx/lip对1-4期、Cr/lip对4期)对肝纤维化具有一定的分期价值;Lac/lip波峰峰高及波峰下面积比值对肝纤维化分期均无意义。
4.肝纤维化大鼠肝实质时间-信号强度曲线呈慢降缓升型,波峰低平宽大;灌注参数SRRmax(对3-4期)、TTP(对2-4期)、MTT(对2-4期)对肝纤维化分期具有一定的价值。
5. Gd-BOPTA增强后延迟60min、120 min时间点肝实质相对强化率对肝纤维化分期无价值,延迟180 min时间点肝实质相对强化率对肝纤维化分期(3-4期)具有一定的价值,但对较早期肝纤维化分期(1-2期)不敏感。
6.传代3的BMSCs细胞可作为细胞移植治疗的理想细胞来源;SPIO-PLL可高效率标记大鼠BMSCs。
7.在体外,FFE-T2WI序列信号下降程度随标记细胞数量增多而增大,示踪效果呈标记细胞数量依赖性。
8.在活体,FFE-T2WI序列为磁粒子标记BMSCs肝移植MR示踪的理想序列。
9.经门静脉途径肝脏BMSCs细胞移植为细胞移植治疗肝脏弥漫性病变的较理想途径。
10.在肝纤维化环境中,移植的BMSCs随时间逐渐向肝实质和纤维间隔内移行,以损伤较重区和纤维间隔分布明显,具有选择性分布特点;移植的BMSCs能与肝细胞紧密连接,形成规则的肝细胞索,对肝损伤具有修复作用。但经门静脉移植BMSCs MR示踪时间窗较短;经肝内途径移植BMSCs细胞分布局限,MR示踪时间窗较长。
11. MR信号变化规律与组织学证实的移植细胞在靶器官的分布、迁移规律具有较好的一致性;MR信号变化规律在一定程度上反映了移植细胞在肝内的分布、迁移、增殖和分化状态。
12.临床应用型1.5T MR可用于肝脏干细胞移植的活体示踪研究。
Chapter I The Experimental Study of Early Diagnosis and Quantitative Analysis of Hepatic Fibrosis of rats with Magnetic Resonance Functional Imaging
In diffuse liver diseases, the diagnosis of hepatic fibrosis and earlier period of liver cirrhosis is the tough problem of early diagnosis of imageology, because the changes of the diseases in morphology are not obvious, and the valuable diagnosis information cannot be provided by the traditional imaging methods which reflect anatomic structure mainly. At present ,it is low that the sensitivity and specificity of the noninvasive diagnostic methods of hepatic fibrosis which are applied clinically;the biopsy of hepatic tissue is thought to be the“gold standard”to diagnose hepatic fibrosis, but it is an invasive method which is limited in clinical application .And the early prediction and intervention is helpful to prevent the development of the process. Thus ,it is urgent to develop new noninvasive methods which can predict hepatic fibrosis early and estimate the degree of the diseases .This study is to study the progression of hepatic fibrosis dynamically and discuss the parameter and the best imaging sequence of magnetic resonance functional imaging by establishing the rat model with liver cirrhosis and applying magnetic functional imaging which include diffusion-weighted imaging、spectrum imaging、perfusion imaging and the application of hepatocyte-specific contrast media); to analyse the appearance of magnetic functional imaging in different degrees and stages of hepatic fibrosis, explore the quantization diadynamic criteria of magnetic functional imaging of hepatic fibrosis and evaluate the value of magnetic functional imaging in the diagnosis of hepatic fibrosis and the early period of liver cirrhosis.
Part I The Establishment of Rat Model with hepatic fibrosis
Objective:To establish animal models with hepatic fibrosis with SD rats by injecting carbon tetrachloride subcutaneously to provide different stages of rat models with hepatic fibrosis for magnetic resonance functional imaging of hepatic fibrosis and magnetic resonance tracer study of cellular transplantation . Methods:The rats of experimental group received subcutaneous injection of 40% carbon tetrachloride oil solution 3ml/kg twice per week and the initial dose is 5ml/kg , and 10% Alcohol is the only drinking water. The rats of control group received subcutaneous injection of physiologic saline , and the dosage and usage is similar with the experimental group, but the drinking water is purified water. Since 2 weeks after injection, 4 rats of experimental group and a rat of control group were collected randomly per week to perform magnetic resonance functional imaging (DWI/MRS/PWI) of liver; after MR imaging but in 4 hours these rats were killed and HE staining、Masson trichrome stain、reticular fiber staining and transmission electron microscope(TEM) examination of liver were performed and the stages of hepatic fibrosis were assessed with light microscope. Results:94 rats of experimental group were successful and 46 were death. The death rate was 33%.The symptoms of different degree of nutritional disturbance and chronic liver disease presented in all the rats of experimental group . The histopathologic examination of liver showed that there was inflammatory cell infiltration, hepatic cell necrosis , collagen and reticular fibre hyperplasia , sinusoidal capillarization ,et al.The pathology stages of hepatic fibrosis was shown as follow: 28 rats with 0 stage、19 rats with 1 stage、27 rats with 2 stage、25 rats with 3 stage and 15 rats with 4 stage. The rats of control group were survival totally and had no corresponding symptoms. Conclusions:The composite factor (CCL4+Alcohol) could induce rats to generate hepatic fibrosis ,and had the advantages of high achievement ratio and short cycle of establishing models .Models of different pathology stages of hepatic fibrosis could be established to provide ideal experimental model for the study of hepatic fibrosis with the use of the composite factor.
Part II The Magnetic Resonance Diffusion-weighted Imaging (DWI) of Hepatic Fibrosis of Rat
Objective:To discuss the value of DWI in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the signal intensity of DWI、the value of ADC and the change of EADC of different degree of hepatic fibrosis . Methods: During establishing rat model with hepatic fibrosis in part I, 4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MR diffusion-weighted imaging. The sequence was SE-EPI and the gradient factor b were 0s/mm2、300s/mm2、600s/mm2、800s/mm2 and 1000s/mm2 respectively .ADC diagram and EADC diagram were achieved according to different value of b, then the signal intensity of DWI of different b values were determined and the value of ADC and EADC were calculated to compare with pathology stages . Results: (1) The signal intensity of DWI of the rats of experimental group were in the tendency of increasing with the increase of staging of hepatic fibrosis .The signal intensity of DWI was uneven because the development of each lobe was different. (2)When b values were 300、600、800 and 1000 s/mm2 ,SNR were(x—±S)36.30±23.25、28.11±12.48、25.71±11.82 and 15.23±6.54 respectively, and the picture quality was in the tendency of decreasing. When b values were 600 or 800 s/mm2, the picture quality were superior to that when b values were 1000 s/mm2,and P<0.05. (3)The analysis of ADC: the value of ADC of control group、grade 1of hepatic fibrosis、grade 2 of hepatic fibrosis、grade 3 of hepatic fibrosis、grade 4 of hepatic fibrosis were[(x—±S)×10-3(]1.542±0.299)×10-3、(1.334±0.268)×10-3、(1.108±0.198)×10-3、(0.978±0.169)×10-3、(0.680±0.260)×10-3 respectively , and there was a trend of decreasing . There were significant differences between control group and other groups、grade 1 of hepatic fibrosis and grade 2、grade 1 and 3、grade 1and 4、grade 2 and 3 ; but there was no significant difference between grade 2 and 3 . The result of correlation analysis of ADC and staging of hepatic fibrosis was : r=-0.766(p﹤0.001).(4) The analysis of EADC: the value of EADC of control group、grade 1、grade 2、grade 3 and grade 4 of hepatic fibrosis were[(x—±S)×10-3](0.315±0.068)×10-3、(0.345±0.081)×10-3、(0.411±0.074)×10-3、(0.465±0.056)×10-3 and(0.595±0.106)×10-3 respectively , and there was a tendency of increasing . There were significant differences between control group and grade 2、control group and grade 3 control group and grade 4、grade 1 and 2、grade 1 and 3、grade 1 and 4、grade 2 and 4、grade 3 and 4 stage ; but there was no significant difference between control group and grade 1、grade 2、grade 3 . The result of correlation analysis of EADC and staging of hepatic fibrosis was : r=0.753(p﹤0.001). Conclusions:(1)The signal intensity of DWI was increasing with the increase of staging of hepatic fibrosis and it is different that the degree of fibrosis of each lobe. (2) The better b values of DWI of liver were 600s/mm2 or 800s/mm2, because it could avoid the influence of perfusion and had higher SNR. (3) The hepatic fibrosis could be staged with ADC and EADC ,and there was better dependability between them .
Part III The Magnetic Resonance Spectrum Imaging (1H-MRS) of Hepatic Fibrosis of Rat
Objective:To discuss the value of MRS in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the metabolites peaks、peak areas and the change of the ratio between them of different degree of hepatic fibrosis. Methods: During establishing rat model with hepatic fibrosis in part I, 4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MRS with 3D PRESS multi-voxel 1H-MRS sequence. Then different metabolites of spectrogram were hand-marked, the peak height and area were generated automatically with the software. Then the peak height of metabolites and lipid and the peak area ratio(Cho/lip、Glx/lip、Lac/lip、Cr/lip)were calculated respectively to compare with pathology staging . Results: There were 5 main peaks of MRS of liver of control group, and the lipid peak of rats of experimental group was lower than that of control group ,but the peaks of other metabolites were higher. The ratio of the peak height of metabolites and lipid were as follows: (1)the ratio of Cho and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.052±0.034、0.212±0.225、0.117±0.122、0.403±0.299 and 0.438±0.295 respectively .There were significant differences between control group and grade 3、control group and grade 4 (P<0.05), but there was no significant difference between control group and grade 1、control group and grade 2、grade 1 and other groups、grade 2 and other groups (P>0.05). (2) the ratio of Glx and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.150±0.132、0.406±0.650、0.656±0.551、0.750±0.452 and 0.763±0.517 respectively . There were significant differences between control group and grade 2、grade3、grade 4 (P<0.05), but there was no significant difference between grade 1 and other groups、grade 2 and 3、grade 2 and 4、grade 3 and 4 (P>0.05). (3) the ratio of Lac and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.139±0.128、0.262±0.178、0.251±0.344、0.355±0.446 and 0.233±0.185 respectively ,and there was no significant difference between these groups (P>0.05), but there was a tendency of increasing with the increase of staging. (4) the ratio of Cr and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.136±0.274、0.767±0.902、0.638±0.960、0.917±0.576 and 0.778±0.856 respectively . There were significant differences between control group and grade 3 (P<0.05), but there was no significant between other groups. The results of correlation analysis between the ratio of the peak height of metabolites and lipid and staging of hepatic fibrosis were as follows : Cho/lip(r=0.503 p﹤0.001)、Glx/lip(r=0.388 p﹤0.05)、Lac/lip(r=0.124 p﹥0.05)、Cr/lip(r=0.235 p﹥0.05). The peak area ratio of main metabolites and lipid of liver were as follows: (1) the ratio of Cho and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis wer(x—±S)e 0.115±0.133、0.257±0.316、0.167±0.187、0.185±0.328 and 0.468±0.372 respectively. There were significant differences between control group and grade 4(P<0.05), but there was no significant difference between other groups (P>0.05). (2) the ratio of Glx and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.045±0.039、0.540±0.318、0.448±0.364、0.482±0.402 and 0.531±0.336 respectively. There were significant differences between control group and other groups (P<0.05), but there was no significant difference between other groups(P>0.05) . (3) the ratio of Lac and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 0.062±0.069、0.258±0.266、0.277±0.320、0.170±0.314 and 0.274±0.312 respectively. There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of staging. (4) the ratio of Cr and Lip of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.109±0.231、0.481±0.614、0.704±0.797、0.465±0.525 and 0.810±0.706 respectively. There were significant differences between control group and grade 4 (P<0.05), but there was no significant difference between other groups(P>0.05) . The results of correlation analysis between the peak area ratio of main metabolites and lipid of liver and staging of hepatic fibrosis were as follows : Cho/lip(r=0.282 p﹥0.05)、Glx/lip(r=0.313 p﹤0.05)、Lac/lip(r=0.135 p﹥0.05)、Cr/lip(r=0.267 p﹥0.05). Conclusions:(1)The ratios of peak height of Cho/lip、Glx/lip、Cr/lip were important for the staging of hepatic fibrosis . The correlation between Cho/lip、Glx/lip and staging of hepatic fibrosis were better than others . (2) The peak area ratios of Cho/lip、Glx/lip、Cr/lip were important for the staging of hepatic fibrosis (Cho/lip、Cr/lip is important for grade 4 and Glx/lip for grade1-4). The correlation between Glx/lip and staging of hepatic fibrosis was better than others .(3)The ratio of peak height and peak area ratio of Lac/lip were not important for the staging of hepatic fibrosis , but there was a tendency of increasing with the increase of staging.
Part IV The Perfusion Imaging (PWI) of Hepatic Fibrosis of Rat
Objective:To discuss the value of PWI in early diagnosis、quantization analysis and staging of hepatic fibrosis by analyzing the changes of perfusion parameters of PWI of different degree of hepatic fibrosis . Methods: During establishing rat model with hepatic fibrosis in part I ,,4 rats of experimental group and 1 rat of control group were selected randomly per week to perform MR perfusion imaging with single shot SE-EPI sequence. After bolus injection of Gd-BOPTA (0.2mmlo/kg , 2ml/s) through vena caudalis of rats, 40 dynamic state were scanning continuously to cover the whole liver . The time-signal intensity curve(TIC)was generated automatically with Perfusion software ,then some correlated parameters were calculated as follows: (1) maximal signal reduction ratio (SRRmax);(2)time to peak (TTP);(3)mean transit time(MTT). The perfusion parameters of PWI were analyzed to compare with pathology staging. Results: (1) The time-signal intensity curve(TIC)of liver parenchyma : the curve of control group descended quickly, then recovered slowly after peak value ,and the recovery extent was larger and recovery course was shorter ; the curve of experimental group descended slowly and the extent was smaller , the time to peak was longer , the recovery extent after peak value was smaller and recovery course was longer, and the peak was lenity. And with the development of hepatic fibrosis ,the changes were more obvious.(2)The relationship of the perfusion parameters of liver and the staging of hepatic fibrosis: 1) the values of SRRmax of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were[(x±S)×100%] 0.754±0.073、0.674±0.137、0.632±0.154、0.603±0.201 and 0.535±0.135 respectively . There were significant differences between control group and grade 3、grade 4 (P<0.05) ; but there was no significant difference between other groups(P>0.05). 2) the values of TPP of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were([x—±S)s]14.175±4.845、18.433±7.293、26.789±3.621、31.755±7.308 and 35.213±6.322 respectively . There were significant differences between control group and grade 2、control group and grade 3、control group and grade 4, grade 1 and 2、grade 1 and 3、grade 1 and 4、grade 2 and 4(P<0.05) ; but there was no significant difference between control group and grade 1、grade 2 and 3、grade 3 and 4 (P>0.05). 3) the values of MTT of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were [(x—±S)s] 24.620±5.577、28.945±2.758、32.502±4.268、35.861±4.651 and 35.203±5.674 respectively . There were significant differences between control group and grade 2、control group and grade 3、control group and grade 4、grade 1 and 3、grade 1 and 4 ; but there was no significant difference between control group and grade 1 , grade 1 and 2、grade 2 and 3、grade 2 and 4、grade 3 and 4 (P>0.05). The results of correlation analysis of the perfusion parameters of liver and staging of hepatic fibrosis were : SRRmax (r=-0.439 p﹤0.05)、TTP(r=0.798 p﹤0.001)、MTT(r=0.647 p﹤0.001) . Conclusions:(1) The time-signal intensity curve(TIC)of liver parenchyma of rats of hepatic fibrosis was characterized by slow- washin and slow-washout, and the peak was low and lenity; (2)The analysis of perfusion parameters of SRRmax、TPP and MTT was helpful to the staging of hepatic fibrosis . The correlation was good between these perfusion parameters and the staging of hepatic fibrosis, especially TTP and MTT .
Part V The Magnetic Resonance Imaging of Hepatic Fibrosis of Rat with Hepatocyte-specific Contrast Media
Objective:To discuss the value of the method of delayed - contrast enhanced scanning with Gd-BOPTA in early diagnosis、quantization analysis and staging of hepatic fibrosis by observing rat models with hepatic fibrosis dynamically after the injection of Gd-BOPTA and analyzing the characteristics of dynamic contrast-enhanced MR imaging and enhancement pattern to compare with pathology staging . Methods: During establishing rat model with hepatic fibrosis in part I , ,4 rats of experimental group and 1 rat of control group were selected randomly per week to perform contrast-enhanced MR imaging with hepatocyte-specific contrast media . After bolus injection of Gd-BOPTA (0.2mmlo/kg) through vena caudalis of rats, delayed scans were performed with TSE-T1WI-TRA sequence after 60min、120min and 180min after contrast media were injected . Then the signal intensity and relative enhancement ratio of liver parenchyma of different time were measured respectively ; and the changes of signal intensity of bile duct and blood vessel of different time of different stages of hepatic fibrosis were observed . Results: The relationship of relative enhancement ratio of liver parenchyma in different time and the staging of hepatic fibrosis were as follows : (1)the value of RER1 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S) 1.436±0.374、1.487±0.477、1.476±0.440、1.489±0.431 and 1.476±0.436 respectively .There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of the staging of hepatic fibrosis .(2) the value of RER2 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x—±S)1.220±0.370、1.292±0.387、1.344±0.367、1.371±0.388 and 1.405±0.370 respectively .There was no significant difference between these groups(P>0.05) ,but there was a tendency of increasing with the increase of the staging of hepatic fibrosis . (3) the value of RER3 of control group、grade1、grade 2、grade 3 and grade 4 of hepatic fibrosis were(x±S) 0.844±0.275、0.910±0.380、1.041±0.399、1.209±0.299 and 1.241±0.398 respectively. There were significant differences between control group and grade 3、grade 4 (P<0.05) ; but there was no significant difference between other groups(P>0.05). The results of correlation analysis of relative enhancement ratio of liver parenchyma in different time and staging of hepatic fibrosis were : RER1(r=0.039 p﹥0.05)、RER2(r=0.174 p﹥0.05)、RER3(r=0.420 p﹤0.05) . The appearance of the visualization of bile duct and blood vessel were as follows: the delayed phase of contrast enhanced MRI showed that the tree-like vessels of liver were low signal while the bile duct was high signal in T1WI. The courser of portal vein and bile duct of liver of rats of experimental group were winding and the changes of caliber were not regular, and the bile duct was enhanced in the delayed phase. Conclusions:(1)It was helpless that measuring the relative enhancement ratio of liver parenchyma when 60 min or 120min after injection of Gd-BOPTA to the staging of hepatic fibrosis . It was helpful that measuring the relative enhancement ratio of liver parenchyma when 180 min after injection of Gd-BOPTA to the staging of hepatic fibrosis (especially grade3 and 4), but it was helpless to grade 1 and 2.(2) The morphological changes and enhancement in delayed phase of bile duct of rat model with hepatic fibrosis were a cue of reconstitution of biliary tree and damage of hepatocyte function .
Chapter II The Magnetic Resonance Tracing Experiment in vivo about the Repair of Hepatic Injury by Transplanting BMSCs Labeled with Magnetic Particles
Liver function failure is the main cause of death of various chronic liver diseases. Orthotopic liver transplantation is the most effective method to treat terminal stage of liver diseases at present , but the donor liver are too famine to satisfy clinical requirement . The cellular transplant provides a new therapy strategy for cell reconstitution of the lesion liver and functional recovery of failure liver to make it possible to make use of self-bone marrow stem cell as seed to repair the affection and injury of autologous tissue. The stem cellular transplant tracer study is very important to monitor the disposition、immigration and differentiation of transplanted cells in vivo dynamically and evaluate the effective of cellular transplant ,but this tracer technique is always a puzzle in medical scientific research and traditional tracer technique of transplanted cells must be carry out with histology observation ex vivo . Therefore, it is urgent to establish a transplanted cell tracer technique in vivo which is safe、sensitive、effective and fit to clinic . Close attention was paid to magnetic resonance tracer technique in vivo in the study of stem cell increasingly, and it is a new technique of observing the survival and distribution of transplanted cells in vivo to perform MRI examination after cellular transplant while the cell is labled with contrast media as molecular probe which is sensitive to MR. The MRI tracer technique of transplanted cell which is applied in the study of cardiac muscle and central nervous system is more , but that applied in the study of bone marrow stem cell in hepatic fibrosis is seldom . This study was focused on: (1)Separation、proliferation and identification of BMSCs from rats.(2) Establishing rat model with hepatic fibrosis. (3) Transplanting BMSCs labeled with SPIO and bromodeoxyuridine (BrdU) to treat the rats with hepatic fibrosis . (4) Monitoring the labeled cells after transplantation into the hepatic fibrosis rats with MR scanning dynamically in vivo and contrasting with pathohistology . This study was to discuss the feasibility of the technique of separation、proliferation and identification of BMSCs、MR tracer technique of BMSCs in vivo which was labeled with SPIO as molecular probe、the optimum scanning parameters and imaging sequence、the feature of distribution and immigration of transplanted cells in liver and the rule of the MR signal intensity changes , and to explore the feasibility of the application of 1.5T MR in stem cell tracer study to establish the foundation of clinical application of stem cell transplant MR tracer study in vivo .
Part I :In Vitro Culture of BMSCs from SD Rats and label of BMSCs with Brdu and SPIO
Objective To develop the in-vitro culture condition of BMSCs from SD rats and to establish the foundation of clinical application of stem cell transplant with MR tracer study in vivo. Methods SD rats (weight 60~90g) were sacrificed. BMSCs were isolated from their bone marrows. Cultured BMSCs were observed daily under phase-contrast microscope and the cell purity were further confirmed by FACS analysis. Results 93.1% of the passage 3 cells were CD90 (one of the BMSCs’surface markers) positive. On the other hand, CD45 + cells were dropped from 71.2% in primary culture to 3.9% in the passage 3. 100% of BMSCs were labeled with SPIO- PLL.Conclusion The passage 3 cells could be used to cell transplantation because of its high purity of BMSCs. It was effective to label BMSCs with SPIO- PLL.
PartⅡ:MRI Study of Bone Marrow Stromal Cells of Rats Labeled with Magnetic Particle in Vitro
Objective:To discuss the changes of MR signal intensity of different numbers of labeled BMSCs and the most sensitive sequences of MR tracing after stromal cells were labeled with magnetic particle to establish the foundation for MR tracing in vivo and provide theoretical evidence . Methods:BMSCs which were labeled with Brdu and SPIO and that which were not labeled were suspended with 1% agarose solutions . The subjects were divided into four groups and put into different EP ducts, including 2×106labeled cells、1.0×106 labeled cells、5×105 labeled cells and 2×106 unlabeled cells .MR scanning sequences included coronal and axial TSE-T1WI、TSE- T2WI and FFE-T2WI.Signal intensity of different sequences of the same object were measured and the MR signal intensity changing rate were compared . Results : The MR signal intensity changing rate of BMSCs which were labeled and not labeled were as follows : (1)the MR signal intensity changing rate of TSE-T1WI of 5×105 labeled cells、1×106 labeled cells and 2×106labeled cells were [(x±S)%] -4.19±0.788、-16.35±1.228、-22.80±1.053 respectively . (2) the MR signal intensity changing rate of TSE-T2WI of 5×105 labeled cells、1×106 labeled cells and 2×106 labeled cells were([x±S)%] -14.15±1.366、-35.09±1.391、-53.02±1.299 respectively . (3)the MR signal intensity changing rate of FFE-T2WI of 5×105 labeled cells、1×106 labeled cells and 2×106 labeled cells were[(x±S)%] -44.98±0.4562、-69.38±0.820、-87.24±0.818 respectively . There were significant differences among labeled cells groups(P<0.001) and significant differences among MRI sequences(P<0.001) .Conclusion : The signal intensity changes of FFE-T2WI sequence is the most apparent when the BMSCs which were labeled with magnetic particle were in the same concentration , and with the number of the labeled cells increasing ,the signal intensity changes were more visible. FFE-T2WI sequence was the ideal sequence for MR tracing of cells labeled with magnetic particle.
PartⅢ:The Homogeneous Transplantation of BMSCs of Rat into Liver
Objective:To transplant BMSCs into liver with two ways -direct injection and intravenous injection via portal vein . To discuss the feature of the two transplant ways to establish the foundation of comparing the effective of MR tracing and feature of signal intensity changes of them and understanding the rule of distribution and immigration of transplanted cells in the target organ .Methods:20 rat models with hepatic fibrosis which were established in part I of chapter I were selected and divided into four groups , including 1 group :control group (normal sodium which contained no BMSCs were transplanted , and in the group , the number of intra-portal-venous injection and direct intra-liver injection was 2 and 2 respectively); 2 group: BMSCs control group (2×106 BMSCs which were not labeled with Brdu and SPIO were transplanted , and in the group , the number of intra-portal-venous injection and direct intra-liver injection was 2 and 2 respectively ) ; 3 group : labeled cells transplanted via portal vein (2×106 BMSCs which were labeled with Brdu and SPIO were transplanted via portal vein .n=6) ; 4 group : labeled cells transplanted into liver by direct injection (2×106 BMSCs which were labeled with Brdu and SPIO were transplanted into liver by direct injection.n=6).The transplanted cells were taken suction with microinjector and injected into the main portal vein or liver slowly. Results :In the process of transplanted cells into liver by direct injection , the operative procedure was convenient and the cellular transplant was successful . In the process of transplanted cells via portal vein , the transplant operation of 3 rats were difficult because the separation of main portal vein was difficult which was resulted from adherence of abdominal membrane and mesenterium in abdominal cavity , and the operation of other rats were successful . Because the main portal vein of experimental rats was thickening in different degree , the puncturation of main portal vein was successful . After cells or normal saline were transplanted into liver slowly, there was no paradoxical reaction occurred in the rats. The healing of operative incision was well. Conclusion: Transplant stromal cells into liver via portal vein and direct injection into liver were safe and effective. And the choice of transplant way was determined according to clinical or investigative purpose.The difficulty of the separation of main portal vein because of adherence of abdominal contents of subjects should be considered.
PartⅣ: MR Tracing of the Homogeneous Transplantation of Bone Marrow Stromal Cells of Rat into Liver in Vivo
Objective:To discuss the effective of MR tracing and the changes of MR signal intensity of the two transplant ways (BMSCs were transplanted into liver which were labeled with Brdu and SPIO via portal vein and direct injection ); to understand the rule of distribution and immigration of transplanted cells in the target organ to establish the foundation of MR tracing in vivo . Methods:2 rats were selected from every group randomly after transplantation in partⅢ, and performed axial TSE-T2WI-SPAIR、TSE-T1WI and FFE-T2WI when 2hours、3days、7days and 2weeks after transplantation .The developing effect of transplanted cells of different sequences were compared. MR signal intensity、distribution range and rule of change of each group in different time were observed . After MR scanning ,1 rat was selected from erery group randomly and killed to obtain liver specimen、fraction of lung and spleen to perform HE staining、hemosiderin staining and Brdu immunohistochemistry staining .The distribution of hemosiderin-positive cells and Brdu immunohistochemistry-positive cells in liver、lung and spleen should be noticed . Results: (1)MR examination showed as followed :the imaging of FFE-T2WI sequence was the best of all sequences .1 group and 2 group showed no signal changes when 2hours、3days、7days and 2weeks after transplant .3 group showed that there were many hypo-intense lesions in hepatic hilar region when 2 hours after transplant , and with the time going on ,the hypo-intense lesions became smaller . The effect of MR tracing was worst when 2 weeks after transplant. 4 group showed that there were mass-like hypo-intense area in the area of cell injection, and with the time going on , the area was becoming larger , the edge was becoming ambiguous , and when 2 weeks after transplant , the hypo-intense area was still obvious .(2) The histopathologic examination showed as follows : 1) HE staining showed that 2 hours and 3 days after transplant , necrosis、inflammation and fibroplasia of liver of all groups were similar, but when 7days and 2 weeks after transplant , the necrosis and inflammatory cell infiltration of 2-4 group were improving and more obvious than control group . 2) Hemosiderin staining showed that 2hours、3days、7days and 2weeks after transplant ,there were no hemosiderin-positive cells in 1-2 group .The hemosiderin-positive cells of 3group were in the portal vein of porta hepatic 2 hours after transplant , in the small branches of portal vein、sinus hepaticus and around central veins of hepatic lobules 3 days after transplant , and in the liver parenchyma (especially in the area of lesion) 7 days and 2weeks after transplant . The histology change in different time was corresponding with the signal intensity changes of MRI. At the same time, there was a few positive cells in the lung and in the spleen . The hemosiderin-positive cells of 4 group were around the injection point, and with the time going on, the cells immigrated into the liver parenchyma nearby. The histology change in different time was corresponding with the signal intensity change of MRI. At the same time, there was no positive cells in the lung ,and a few in the spleen . 3) Brdu immunohistochemistry staining showed that the result was in coincidence with Hemosiderin staining. Conclusion: (1) FFE-T2WI sequence is the ideal sequence of liver MR tracing of transplanted cells labeled with magnetic particle in vivo. (2)The transplanted cells were scattered in the whole liver (especially in the area of lesion) with the time going on . It was helpful to repair the lesion of liver by transplanting BMSCs .But the time window of MR tracing was shorter when BMSCs were transplanted into liver via portal vein ;It is the ideal way to treat diffuse disease of liver when BMSCs were transplanted into liver via portal vein;but the distribution of transplanted cells were localized and the time window of MR tracing was longer when BMSCs were injected into liver directly. (3) The rule of MR signal intensity changes and the rule of distribution and immigration of transplanted cells in target organ were in good concordance.(4) The rule of MR signal intensity changes reflected the condition of distribution、immigration、proliferation and differentiation of transpl
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