人羊膜间充质干细胞移植治疗大鼠脑损伤
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摘要
研究背景:脑损伤是严重威胁人类健康的疾病之一。目前对重度脑损伤导致的神经功能缺失,治疗难以达到满意的效果。近年来干细胞研究的进展给脑损伤的治疗带来了希望,取得了令人鼓舞的治疗效果。干细胞移植治疗脑损伤能够改善宿主的神经功能,可能的机制有:外源性干细胞在宿主体内的内分泌作用和机体自身神经细胞的旁分泌作用,使局部的神经营养因子聚集,改善了神经细胞的生存环境,使外源性干细胞更易向神经细胞转化并与宿主的神经细胞建立突触联系,具体的确切机制仍需进一步研究。目前移植干细胞治疗脑损伤的途径有脑损伤区原位移植、通过脑脊液循环及血液循环进行移植,均能对神经功能有所改善。采用何种移植途径能最大程度改善宿主的神经功能,尚没有明确的结论。对各种移植途径进行治疗效果系统比较的研究报道尚未见到,实验将给与探讨,为干细胞移植治疗脑损伤提供实验依据。干细胞移植治疗脑损伤的研究手段多是采用处死动物后获取组织标本,进行病理学的观察。如何活体观察外源性干细胞在宿主体内的存活情况,是干细胞移植研究的难点和重点。在小鼠胚胎干细胞移植治疗大鼠脑损伤的研究方面,应用1.5T核磁共振仪成功进行了活体观察,取得了满意的观察效果。在人羊膜间充质干细胞移植治疗脑损伤的研究方面,报道较少,有关活体示踪羊膜间充质干细胞在宿主体内存活情况的研究也没有见到报道。本文采用人羊膜间充质干细胞进行体外标记后移植治疗脑损伤大鼠,3.0T核磁共振活体示踪细胞的存活迁移情况。
研究表明,神经生长因子对神经元的生长分化及功能的维持具有重要作用。神经生长因子诱导干细胞向神经元样细胞转化的研究已有多篇报道,联合神经生长因子与外源性干细胞移植治疗脑损伤有人进行了研究,对宿主神经功能的改善有一定作用。在脑损伤区长期维持神经生长因子的有效浓度,是神经生长因子联合干细胞移植治疗脑损伤的关键问题,由于神经生长因子通过血脑屏障能力弱,外周血管给药的途径难以有效进入脑损伤区,另外神经生长因子半衰期短,难以在脑损伤区长久维持有效浓度,因此如何使神经生长因子进入脑损伤区并维持有效浓度仍需进一步研究。本文采用量子点观察神经生长因子纳米化后进入羊膜间充质干细胞的情况,通过羊膜间充质干细胞携带神经生长因子进入脑损伤区,取得了满意的效果。
外源性干细胞移植入机体后,对宿主的影响目前尚无明确结论,仍需要研究人员进行探讨。本文通过采集血液检测血常规及肿瘤标志物,了解干细胞移植对脑损伤大鼠的影响,并通过核磁共振观察有无新生物出现。
综合以上研究情况,我们将在人羊膜间充质干细胞移植治疗脑损伤的移植途径、神经生长因子的给药方法、羊膜间充质干细胞移植治疗脑损伤对机体的影响及活体示踪进行探讨,为脑损伤的临床治疗提供依据。
目的:
第一,探讨体外分离、培养和纯化羊膜间充质干细胞(Mesenchmal stem cells,MSCs)的方法;第二,探讨应用量子点神经生长因子纳米粒和Fe_3O_4纳米颗粒(菲力磁)标记羊膜MSCs的可行性及适合条件,并探讨应用3.0T磁共振活体观察负载Fe_3O_4纳米颗粒的羊膜MSCs在大鼠脑损伤区存活迁移的可行性;第三,探讨羊膜MSCs作为载体携带神经生长因子纳米颗粒移植治疗大鼠脑损伤对大鼠神经功能的改善作用;第四,探讨羊膜间充质干细胞移植治疗脑损伤的安全性。
方法:
第一,羊膜MSCs的分离培养与鉴定无菌条件下取正常足月剖腹产胎盘的羊膜,生理盐水充分冲洗后,用0.25%的胰蛋白酶室温消化30min,然后用含1.0g/L胶原酶Ⅳ的DMEM/F12培养液37℃消化1h,200目滤网过滤,制成单细胞悬液,用含10%胎牛血清(fetal bovine serum,FBS)的DMEM/F12培养基进行培养。添加碱性成纤维生长因子(bFGF)进行细胞培养,培养瓶置于37℃、饱和湿度体积分数为5%CO_2培养箱中培养,3d后全量换液,去除未贴壁细胞,每3～5d全量换液。待细胞长至80%～90%融合,按1:2的比例以1×10~6cells/ml密度接种进行传代培养。取第4～6代羊膜MSCs用流式细胞仪检测MSCs的免疫表型CD29,CD44,HLA-ABC的表达。取第3代的羊膜MSCs,用20μg/L bFGF和30μmol/L全反式维甲酸(Retinoic acid,RA)诱导培养的细胞向神经细胞分化。于诱导前及诱导后8d、13d分别进行神经巢蛋白(neuroepithelial stem cell protein,nestin)神经元特异性烯醇化酶(neuron specific enolase,NSE)和胶质纤维酸性蛋白(glial fibrillary acidicProtein,GFAP)免疫细胞化学染色。
第二,量子点神经生长因子纳米粒(QDs-NGF-NPs)、Fe_3O_4纳米颗粒(菲力磁)标记羊膜MSCs及诱导分化
待第三代羊膜间充质干细胞达到80%～90%融合时用2.5g/L胰酶消化,接种于96孔培养板上,每孔200μl,将制好的小玻片置于培养板,待细胞爬满小玻片后,培养基中分别加入不同浓度Fe_3O_4纳米颗粒及QDs-NGF-NPs,Fe_3O_4纳米颗粒标记组分为对照组及实验组,对照组为不含铁纳米颗粒培养基培养的羊膜MSCs,实验组分为四组,使Fe_3O_4纳米颗粒终浓度分别为20μg/ml、30μg/ml、40μg/ml和80μg/ml。每组均加入转染剂多聚左旋赖氨酸,使其终浓度为1.5μg/ml。细胞培养24h均更换为正常培养基,继续培养3周。于细胞培养12h、36h、1周和3周进行普鲁士蓝染色。QDs-NGF-NPs标记组分为实验组及对照组,对照组分为空白对照(培养基中加入等量生理盐水)及单纯量子点对照(培养基中加入终浓度为20、40和60μg/L的量子点)。实验组为QDs-NGF-NPs组,培养基中加入量子点使其终浓度分别为20、40和60μg/L。
根据以上两组实验结果设计QDs-NGF-NPs及Fe_3O_4纳米颗粒联合标记组。该组分为实验组及对照组。QDs-NGF-NPs及Fe_3O_4纳米颗粒组按每孔量子点的终浓度分设3个浓度亚组(20、40、60μg/L)及相应Fe_3O_4纳米颗粒的终浓度为20μg/ml、30μg/ml和80μg/ml,加入多聚左旋赖氨酸终浓度为1.5μg/ml。空载量子点组(不含神经生长因子纳米颗粒)按上述相应量子点浓度分为三组作为对照,正常对照组不加量子点及Fe_3O_4纳米颗粒。培养24h后,更换培养基为正常培养液,继续培养24h、48h、72h后,每孔加入20ul(5g/L)MTT溶液,继续孵育4h后,弃上清,加入100ulDMSO终止反应。微震荡后,于酶标仪波长490nm处测得吸光度值(A)。正常对照组细胞存活率记为100%,其余各组细胞存活率按公式计算:细胞存活率=(实验组A值/对照组A值)×100%,即吸光值越大,存活率越高。置于荧光显微镜下观察量子点在细胞内的分布。分别与12h、36h、1周和3周普鲁士蓝染色,观察Fe_3O_4纳米颗粒进入细胞情况。
细胞分化鉴定:对QDs-NGF-NPs标记组及QDs-NGF-NPs、Fe_3O_4纳米颗粒共标记组用20μg/L bFGF和30μmol/L全反式维甲酸(Retinic acid,RA)诱导羊膜MSCs向神经细胞分化,诱导后第8d及13d用4%多聚甲醛固定30 min,PBS洗涤,加入过氧化物酶阻断液阻断内源性过氧化物酶,非免疫性动物血清封闭,分别加入小鼠抗人神经元烯醇化酶(NSE)、神经巢蛋白(nestin)和胶质纤维酸性蛋白(GFAP)单克隆抗体(即用型1:100稀释),室温孵育1h,PBS洗涤,加入生物素标记的二抗,室温下孵育10min,洗涤,加入链亲和素-过氧化物酶溶液,孵育10min,洗涤,DAB溶液显色,中性树胶封固。
第三,脑损伤模型的制作及不同移植途径的疗效探讨将80只Wistar大鼠,采用自由落体硬膜外撞击法制作大鼠脑创伤模型,损伤装置由底板、固定支架、垂直导杆、20 g砝码、聚乙烯撞击圆锥组成。撞击圆锥头端直径4mm,高2.5mm。10%水合氯醛腹腔注射(3ml/kg)麻醉大鼠后,将头部固定于立体定向仪上,剪去顶部皮毛,消毒,沿中线切开皮肤及骨膜,左侧前囟后3mm,左旁开2mm,钻一直径为5mm的圆形窗,砝码自30cm高处垂直落下,撞击置于硬膜上的圆锥,致中度脑损伤,缝合骨膜及皮肤。大鼠脑外伤模型随机分为对照组与治疗组,对照组20只,治疗组60只,分三组,每组20只,分别标记为A组尾静脉移植组,B组侧脑室移植组及C组脑损伤区移植组。应用平衡木行走试验进行神经功能评分评估大鼠神经功能损伤程度。各组动物分别于1d、4d、1w、2w、3w时处死4只,进行NSE、nestin和GFAP免疫组织化学荧光染色检测,了解移植后的羊膜间充质干细胞的存活、迁移和分化等。根据实验结果选定合适移植途径。
第四,羊膜间充质干细胞移植、活体示踪、神经行为学评分、免疫组化检测实验分为实验组及对照组,对照组20只脑损伤动物模型,相同浓度的菲力磁生理盐水代替羊膜MSCs植入脑损伤区,实验组分为AB两组,共40只大鼠模型,每组20只。A组由第三代羊膜MSCs原位移植于脑损伤区,B组由第三代羊膜MSCs经QDs-NGF-NPs及菲力磁标记24h后原位移植入脑损伤区,在脑损伤区域及其边缘注射含羊膜间充质干细胞的生理盐水10μl(细胞浓度为2.0×10~7/μl),每点进针3mm,针头在注射部位保留5min,然后拔出针头,骨蜡封闭骨窗,缝合骨膜,皮肤。细胞移植后当天记为0d,在移植后的0d,1d,7d,14d,21d进行3.0T核磁共振仪扫描,观察羊膜MSCs的存活迁移情况。成像条件:MRI采用SIEMEMS 3.0 Trio TimⅠ-class MR扫描仪和眼表面线圈,行横断面及冠状面TSE:T1WI、T2WI,T2*WI及SWI序列扫描,扫描参数为:T1WI:TR/TE=430/12ms,T2WI:TR/TE=5990/98ms,层厚2.0mm,FOV10cm×10cm,矩阵288×224;T2*WI:TR/TE=500/20ms,层厚2.0mm,翻转角20°,矩阵256×256;SWI:TR/TE=29/20ms,层厚0.6mm,FOV6cm×6cm,翻转角15°,矩阵256×256。动物模型建立后定时进行神经功能评分。在模型建立后的7d、14d、21d时分别处死动物,进行普鲁士蓝染色了解铁纳米颗粒的存在情况,免疫组织化学荧光法检测神经巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)表达情况。
第五,安全性检测在动物模型建立前、后2w及3w对各组动物尾静脉埋植细针头累积抽血0.5ml,抽血前静脉注入肝素(15U/kg)全身肝素化,对样本进行血常规及12项肿瘤标志物联合检测,观察评估经QDs-NGF-NPs及菲力磁标记的羊膜间充质干细胞移植治疗脑损伤大鼠的安全性。
结果:
经酶消化法自人羊膜组织中提取到的细胞混悬液,置于培养瓶中体外培养,观察到部分细胞最早于10h左右开始贴壁,继续培养1d～2d时,贴壁细胞逐渐增多,培养至1w时,细胞贴满瓶底,细胞形态大多为长梭形,部分为多角形及不规则形状,总体显示为梭形细胞,类似于成纤维细胞的形态。给于消化全量换液传代后,细胞生长加快,在3～6d内铺满瓶底,细胞部分发生融合,细胞以长梭形为主,呈索条状或螺旋状分布。第3～4代的细胞增殖活性较强,一般传代后3d左右可以铺满瓶底,传代至第6～8代后,细胞形态开始出现老化现象,呈扁平状,增殖缓慢或者停止增殖,培养至第9～10代以后,上述现象更加明显。
取第3～4代细胞进行流式细胞术检测,结果显示自人羊膜组织中提取的细胞表达抗原CD29,抗原CD44及抗原HLA-ABC。与间充质干细胞表面抗原一致,提示所得细胞为间充质干细胞。羊膜间充质干细胞体外培养条件下,给与20μg/L bFGF和30μmol/L全反式维甲酸(Retinoic acid,RA)联合诱导后,可以检测到细胞nestin及NSE的表达,提示人羊膜间充质干细胞转化为神经元样细胞。
应用菲立磁标记的羊膜MSCs普鲁士兰染色结果显示,终浓度为20μg/L的Fe_3O_4纳米颗粒组细胞标记率为60%,30,40,80μg/L的Fe_3O_4纳米颗粒细胞标记率均为100%。Fe_3O_4纳米颗粒浓度大者同期进入细胞的铁颗粒就多,36h时各浓度组细胞内铁颗粒聚集达高峰。1w及3w时细胞内铁颗粒逐渐减少。光镜下可以看到细胞内蓝染的铁颗粒分散在胞浆中。
应用QDs-NGF-NPs(浓度小于40μg/L)标记组及QDs-NGF-NPs(浓度小于40μg/L)、菲力磁(浓度小于30μg/ml)联合标记组的培养基培养羊膜MSCs6h后,荧光显微镜下均可见羊膜MSCs的胞膜及胞浆中有红色荧光发出,随时间延长进入细胞的量子点逐渐增多,于32h荧光强度达到高峰,2周内量子点荧光强度未见减弱。普鲁士兰染色见QDs-NGF-NPs(浓度小于40μg/L)、菲力磁(浓度小于30μg/ml)联合标记组羊膜MSCs的胞浆中有大量蓝染的铁颗粒存在,经诱导后两组细胞均有部分细胞表达神经元特异性烯醇化酶(NSE),部分细胞表达神经巢蛋白(nestin),未检测到胶质纤维酸性蛋白(GFAP)的表达。
在移植后第1d开始,钉板平衡木行走测试,评分在对照组5.42±0.98、A组(尾静脉移植)5.02±1.98、B组(侧脑室移植)5.54±1.34和C组(脑损伤区移植)5.12±1.04,各组相比无统计学差异(P＞0.05)。移植后第7d评分对照组5.11±1.62、A组5.15±1.02、B组5.51±2.04和C组4.55±1.02,C组与对照组、A组及B组比较统计学有差异(P＜0.05)。其它各组之间统计学差异不明显(P＞0.05)。在术后13d进行评分显示,对照组5.11±1.92、A组5.01±0.92、B组4.86±0.87和C组2.66±1.72,C组与其它各组比较有统计学差异(P＜0.05),B组与A组及对照组相比有统计学意义(P＜0.05)。A组与对照组相比无统计学差异(P＞0.05)。饲养至第3周时各组评分均未见进一步变化。侧脑室注射羊膜间充质干细胞组显示损伤区表达NSE和nestin的细胞较少,侧脑室壁显示有少量NSE和nestin表达。大鼠脑损伤区定点注射羊膜间充质干细胞组,可见损伤区附近有较多细胞表达NSE和nestin,提示植入的羊膜间充质干细胞部分种植、存活。大鼠尾静脉注射组,在脑损伤区局部组织中未检测到NSE和nestin表达。
菲力磁标记的MSCs脑内移植后对照组及实验组B组在0d扫描时显示T1、T2、T2*GRE扫描在移植区可见低信号区。其中T2*GRE扫描最敏感。在1d,7d,14d,21dMR扫描时,对照组不显影,实验组显示T1、T2、T2*GRE扫描在移植区可见低信号区。其中T2*GRE扫描最敏感。在14d及21d扫描显示在细胞植入区仍能清晰观察到低信号区,向周围弥散现象不明显。A组在MR扫描时不显影。神经功能评分显示细胞移植后的前4d,各组评分统计学没有明显差异,在细胞移植第4d后A组B组评分开始低于对照组,统计学有意义,B组与A组相比统计学有差异,至14d左右,评分没有更进一步变化。普鲁士兰染色结果显示,在移植针道附近可见蓝染的铁颗粒存在,随时间延长,蓝染程度下降,在针道附近蓝色有弥散趋势。免疫组织化学荧光染色显示对照组没有检测到神经巢蛋白(nestin)神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达,实验组两组均有不同数量的细胞表达神经巢蛋白(nestin)及神经元特异性烯醇化酶(NSE),未检测到胶质纤维酸性蛋白(GFAP)的表达。B组神经巢蛋白(nestin)及神经元特异性烯醇化酶(NSE)表达明显多于A组(p＜0.05)。
在动物模型建立前及后2w、3w时抽血,进行血常规及12项肿瘤标志物联合检测,结果显示血常规及12项肿瘤标志物未见异常。
结论:
酶消化法能够从人羊膜组织中分离提取出MSCs,经bFGF和全反式维甲酸体外诱导,能够分化为神经元样细胞,证实人羊膜MSCs具有多分化潜能。
量子点神经生长因子纳米颗粒(QDs-NGF-NPs)及菲力磁可以在体外成功标记羊膜MSCs,浓度小于40μg/L的QDs-NGF-NPs及浓度小于30μg/ml的菲力磁对羊膜MSCs的生长分化没有影响。
脑损伤区移植羊膜间充质干细胞较尾静脉及侧脑室移植能更大程度改善大鼠的神经功能。
神经生长因子纳米化颗粒能有效进入羊膜MSCs,经脑损伤区原位移植能有效改善脑损伤大鼠的神经功能。
羊膜MSCs经菲立磁标记移植大鼠后,能够在脑损伤区存活分化,借助3.0T核磁共振仪能有效观察到羊膜MSCs移植后的存活情况。
经QDs-NGF-NPs及菲力磁标记的羊膜MSCs移植治疗脑损伤大鼠,观察3w检测大鼠血常规及各项肿瘤标志物均未见异常,提示该方法安全。
Background:Brain damage is one of the diseases that pose great threat to people's health.Presently no satisfying effect has been seen in treating the loss of nerve function brought by the suffering of severe brain damage.Yet studies on stem cells bring hope for such patients.Treating brain damage by transplanting stem cells can improve the nerve function of the host.The possible meachanism is that the endocrine effect of exogenous stem cells in hosts and the paracrine effect of nerve cells in the organism itself can bring local neurotrophic factors together,improve the living environment of nerve cells and make it easier to transform from exogenous stem cells into nerve cells and have synaptic connections with them.While the possibility is expected to do more research.Currently there are three ways to transplant stem cells which are different in improving nerve function,namely, tranplanting through the original brain injured area,circulation of blood-cerebrospinal fluid,as well as blood circulation,all of which are relatively effective in improving the nerve function yet no definite conclusion can show which way is the best.Also, no relevant systemic report has ever been seen in comparing their exact treating effect. Thus the author will make further discussion about this and offer some experimental references for treating brain damage.In general,the traditional practice for studying stem cell transplantation in treating brain damage is to sacrifice animals and make them as samples for histopathologic observation.Therefore,how to make observation in vivo about the survival of exogenous stem cells is the difficulty as well as priority for doing research on stem cell transplantation.Through 1.5Tmagnetic resonance, some researchers successfully made observation in vivo about the effect of transplanting labelled mouse embryonic stem cell in vitro in treating brain injured rats, while few reports have been released to study the application of transplantation of human amniotic membrane-derived stem cells(human AD-MSCs) as well as tracking them in vivo for observing their survival in hosts.In this essay,the author treats injured rats by lablelling human AD-MSCs in vitro and the survival as well as immigration of labelled cells are traced through 3.0T nuclear magnetic resonance Previous research shows that NGF play a key role in nerve cells growth differentiation as well as the maintenance of their function.A number of studies have been done to prove that NGF can induce stem cells to transform into neuron-like cells and brain damage can be treated through transplanting NGF and exogenous stem cells which can relatively improve the nerve function of the hosts.However,how to ensure long maintenance of effective concentration of NGF in brain injured area is critical to treating brain damage by combining NGF with stem cells since NGF is weak to pass blood-brain barrier,the administration way of medicine in peripheral vascular is difficult to enter the brain injured area and NGF is hard to maitain effective concentration for a long time due to short half-life.The author will observe the situation of NGF nanoparticles by means of quantum dots(QDs) after entering the brain injured area.The treatment effect turns out to be quite positive.
After tranplanting exogenous stem cells into the organism,further study need to be done about the effect to host.This essay successfully discovers the effect of transplanting stem cells to brain injured rats and finds no neoplasm with the assistance of muclear magnetic resonance after blood routine and tumor marker examination from collected blood.
Based on the above study,this essay is going to explore in such matters as tracking in vitro of human for treating brain damage,the inserting ways of NGF as well as the safety issue so as to provide evidence for clinical treatment of brain damage.
Objective:Ⅰ.To investigates the method of in vitro isolation,culture and purification of MSCs.Ⅱ.To explore the feasibility and necessary conditions for labelling AD-MSCs with QDs-NGF-NPsas well as Fe_3O_4 nanoparticles and also explores the feasibility of the survival and migration of human AD-MSCs into brain injured rats by 3.0T magnetic resonance.Ⅲ.To analyse improving effect of rats' ethology and nerve function by transplanting AD-MSCs with QDs-NGF.Ⅳ.To explore the safety of treating brain damage through transplanting MSCs.
Methods:
Ⅰ.Isolated culture and identification of AD-MSCs
Under the sterile condition,AD-MSCs were obtained from normal placenta after abdominal delivery.Firstly,after being rinsed with physiological saline,they were digested with 0.25%parenzyme at ordinary temperature for 30 minutes and digested with DMEM/F12 solution that contained 1.0g/L collagenase for one hour at 37℃.Then they were filtered with 150 wells filter,processed into unicell suspension cultured in DMEM/F 12 nutritive medium that contained 10%fetal bovine serum (FBS).After adding basic fibroblast growth factors(bFGF),cell culture was undertaken in incubators at 37℃with volume fraction of 5%CO2 and saturation humidity.Three days later,nutritive medium change was done and the unattached cells were removed.In the following research,the removal was done every three to five days.When the cells reached 80%to 90%confluence,serial subcultivation was undertaken in the proportion of 1:2 with the concentration of 1×10~6cells/ml.The expression of immunophenotype for MSCs from the fourth to the sixth generation such as CD29,CD44,HLA-ABC were detected by flow cytometer.The third generation AD-MSCs were chosen,and the cultured cells were induced to differentiate into cellula nervosa with the assistance of 20μg/L bFGF and 30μmol/L Retinoic acid(RA).In eight days and thirteen days before and after the induction, immunocytochemical staining was undertaken to nestin,neuronspecific enolase(NSE) and glial fibrillary acidic protein(GFAP)
Ⅱ.The induction and differentiation of AD-MSCs labelled by QDs-NGF-NPs and Fe_3O_4 nanoparticles
With DMEM/F12 as their culture solution that contained 10%FBS and 20μg/L When the cells reached 80%confluence,the cells were inoculated into 96-hole culture plate for 200μl per hole.Glass slide were put onto the plates.When the slides were coated with cells,QDs-NGF-NPs and Fe_3O_4 nanoparticles at different concentration were added into the nutrient medium which were divided into experimental and control groups.Groups only with Fe_3O_4 nanoparticles were classified into experimental group and control group.The control group had no Fe_3O_4 nanoparticles nutrient medium while experimental group were further divided into four sub-groups at final concentration of 20、30、40μg/L,80μg/ml respectively.Then we added poly-1-lysine at concentration of 1.5μg/ml in each group.After culturing for 24 hours,the nutrient medium was changed into medium without NGF and NPs and continue to culture for 3w and were given Prussian blue staining in the.corresponding time,The labelled groups were divided into blank control group(equal amount of physiological saline in it and pure QDs,in which the final concentration of QDs were 20、40 and 60μg/L;The experimental group was group with QDs-NGF-NPs which was added woth QDs at concentration of 20,40,and 60μg/L respectively.
According to the experimental results,a combined labelled group between QDs-NGF -NPs and Fe_3O_4 nanoparticles was made which was further divided into experimental group and control group.Group with QDs-NGF-NPs were divided into three sub-groups(with concentration of 20、40、60μg/L),and group with Fe_3O_4 nanoparticles were divided into 3 sub-groups(with concentration of 20μg/ml、30μg/ml and 80μg/ml).Then they were added into Poly L lysine with the final concentration of 1.5μg/ml.Group without QDs-NGF-NPs at the same conresponding final concentration were divided into control groups for comparision.Normal control group without no QDs-NGF-NPs and Fe_3O_4 nanoparticles were changed nutrient medium for nutrient solution after being cultured for 24h.Being culturing for another 24h、48h、72h,each hole was added 20ul(5g/L)MTT solution and continued to incubate for 4 hours.At last,the supernatant was removed and 100ulDMSO was used to terminate reaction.After shaking slightly,the absorbance value(A) were measured at the wavelength of 490mm.The cell survival in normal control groups were 100% while survival in other groups was to be calculated according to the following formula: cell survival=(experimental groups A/control groups A)×100%.That is to say,the larger the A value was,the better survival rate was.The distribution of QDs among the cells was observed under fluorescent microscope.Then Prussian blue staining was applied at 12h、36h、1w and 3w so that how much Fe_3O_4 nanoparticles had entered into cells could be observed.
Identification of cell differentiation:20μg/L bFGF and 30μmol/L RA were used to induce AD-MSCs to differentiate into cellula nervosa in both labelled group with QDs-NGF-NPs and combined group with QDs-NGF-NPs and Fe_3O_4 nanoparticles. Then after 8days and 13 days,they were fixed by 4%paraformaldehyde for 30 minutes and washed by PBS.Peroxidase blocking solution was used to block endogenous peroxidase and nonimmune animal serum.Then neuron-specific enolase (NSE),nestin,monoclonal antibody(1:100dilution) of glial fibrillary acidic protein (GFAP) were added to the cells which were incubated for 1 hour at ordinary temperature and washed with PBS.After that,cells were incubated for 10 minutes and washed.At last they were incubated for another 10 minutes and washed while adding streptavidin-peroxydase solution which were developed color with DAB solution and fixed with neutral gum.
Ⅲ.Study on therapeutic effect of different transplantation methods and establishment of brain injured models
About 80 wistar rats were chosen to establish brain injured models by the Feeney's free falling weight method.The necessary injury devices were made up of backboard,fixed bracket,vertical lead-screw,20g counterweight and polythene knocking conus with the diameter of 4mm and 2.5mm high.After being undertaken peritoneal injection with 10%chloral hydrate(3ml/kg),their heads were fixed in stereotaxic apparatus,the skin and hair of their heads were cut and sterilized Then the part of skin and periosteum were cut a round shape along the centerline with diameter of 5 mm,3mm behind anterior fontanel and 2mm on the left side.Then,the weight was vertically dropped the from 30 mm high,making it stroke the conus so that the rats could be damaged moderately.At last,the cut was sutured.
Those brain injured models were divided into control group(n=20)and treatment group(n=60)which was further divided into 3 groups(n=20),that is group A(vein transplantation),groupB(ventricle transplantation) and group C(brain injured area transplantation).The degree of neural injury was evaluated with pegged beam walking test method.4 rats from each group were killed 1d、4d、1d、2w、3w later separately and NSE as well as nestin and GFAP immunohistochemistry staining were done to those dead rats in order to observe the survival,migration as well as differentiation of AD-MSCs.We would choose the appropriate way to transplant.
Ⅳ.The transplantation,in vivo labelling,evaluation of neuroethology,and immunehistochemical detection of AD-MSCs
The rats were divided into control group and experimental group.In control group,all of the 20 models were transplanted with Feridex physiologic saline at the same concentration that replaced AD-MSCs.40 models were divided into two experimental groups(n=20).In groupA,the third generation AD-MSCs was transplanted into the original brain injured area.In group B,the third generation AD-MSCs labelled with QDs-NGF-NPs and Feridex for 24 hours.Then 10μl physiologic saline that contained AD-MSCs(with concentration of 2.0×107/μl)was injected into and around the injured area.Each injection should be 3mm deep and remained in the injected place for about 5min.After this,bone wax was used to seal bone window incision off and periost were sutured.SIEMEMS 3.0 Trio Tim I-class MR scanner was used to performT1WI、T2WI、T2~* Wias well as SWI sequence scanning.Scan parameters were T1WI:TR/TE = 430/12ms,T2WI:TR/TE= 5990/98ms with thickness of 2.0mm,FOV10cm×10cm and matrix 288×224 and so on.The neurological scores was graded in fixed time after establishing the models. Then those models were sacrificed to observe the existence of iron nano-particles with Prussian staining 7days,14days and 21 days later respecttively.The expression of nestin and NSE and GFAP were observed with immune histochmistry method.
Ⅴ.Safety detection
Before establishing the models and After that for 2w and 3w,0.5ml blood was drawn from vena caudalis.But heparin(15U/kg) should be injected before hemospasia to ensure whole body heparinization.Blood routine examination and 12 tumor markers should be detected together in order to observe and evaluate the availability and safety of tranplanting AD-MSCs labelled by QDs-NGF-NPs and Feridex.
Results:The cells suspension solution extracted by enzymic digestion from AD-MSCs was cultured in vitro.It was soon found that some cells became adhering to the wall as early as 10 hours later.Being cultured for another day or two days,the adhering cells increased steadily and filled the bottom in various forms.Most of them were long fusiform while some of them were polygon or in irregular forms.In general, they appeared to be spindle cells,similar to fibroblast cells.After cell passage through total change of digestion solution,cells growth accelerated and spreaded the flasks bottom all over 3 to 6 days later with some cell fusion.Most of them were long fusiform cells,distributing in stripy and spiral shape.Cells in the third generation proliferated actively and could cover the bottom of the flasks during that stage yet cells in the sixth to eighth generation became aging in flat shape proliferatng slowly or even ceasing to do so.When it comes to the 9th or 10th generation cells,this phenomenon became even more obvious.
According to flow cytometry,cells in the third and fourth generation turned out that: cells extracted from AD-MSCs could express antigens CD29,CD44 and HLA-ABC which were the same with those of MSCs,indicating those were MSCs.Under the cultivation in vitro,AD-MSCs jointly induced by 20μg/L bFGF and 30μmol/ LRetinoic acid(RA) could express nestin and NSE,indicating that AD-MSCs could differentiate into neuron-like cells
Resultl from Prussian staining toAD-MSCs lablelled by Feridex indicated as follows:cell labeling rate in group with Fe_3O_4 nanoparticles at the final concentration of 20μg/L was 60%,yet cell labelling rate in group with Fe_3O_4 nanoparticles at the final concentration of 30,40,80μg/L was 100%.That is to say,the higher the concentration was,the more Fe_3O_4 particles entered.And Fe_3O_4 particles from each groups reached the highest amount 36h later and they decreased when it was 1w and 3w later.We could see those Fe_3O_4 particles stained by Prussian method scattered in kytoplasm.
Labelled group with QDs-NGF-NPs(concentration was less than40μg/L) and combined group between QDs-NGF-NPs(concentration was less than40μg/L) as well as Feridex(concentration was less than30μg/ml) were cultured for 6h.Under fluorescent microscope,red fluorescent light could be observed radiating from cell membrane kytoplasm.Meanwhile,the amount of QDs increased as time went by.The light reached its peak 32h later and it didn't fade away 2w later.After being stained,a lot of blue-stained Fe_3O_4 particles could be detected in the Fe_3O_4 particles of the combined group.In the two groups,some cells expressed as NSE or nestin.No cells were detected to express GFAP.
Balance beam walking test was adopted to those groups one day after transplantation.But there was not significant differences between control group 5.42±0.98、group A 5.02±1.98、group B 5.54±1.34 and group C 5.12±1.04(P＞0.05). There was statistic significance between control group 5.11±1.62、group A 5.15±1.02、group B 5.51±2.04 and group C 4.55±1.02,between group C and control group,beteen group A and group B(P＜0.05).The rest of other groups were not seen any significant difference(P＞0.05).Scores were graded 13 days after the operation,there was significant diffrences between control group 5.11±1.92、groupA 5.01±0.92、groupB 4.86±0.87 and group C 2.66±1.72,between group C and other groups,between group B,group A and the control group(P＜0.05).But there was no significant differences between group A and the control group(P＞0.05).No change in the above results after raising those rats for 3 weeks.In group B with intracerebroventricular injection indicated that few cells in the injured region expressed as NSE and GFAP,though some cells in the ventricular walls expressed as NSE and GFAP.While in group C,a lot of cells expressed as NSE and GFAP indicating that most of the transplanted AD-MSCs managed to grow and survive in that area.As for group A.nothing was observed to express as NSE and GFAP.
Shortly after tranaplantating AD-MSCs labelled with feridex,the control group and group B were undertaken T1、T2、T2~*G scaning,and low signal could be found in the transplanted area in which T2~*GRE was the most sensitive one.In the MR scanning 1d,7d,14d and 21d later,nothing could be found in the control group while low signal could be observed in group B.Particularly,the signal of feridex could be detected in the transplanted area 14d and 21d later while group A appeared to be nonvisulized under MR scaning.According to the score of nerve function,no significant differences in statistics could be found in the first 7 days after transplantation.Yet 8 days later,the scores in group A and group B became lower than that in control group,thus there was statistic significance while group B had differences compared with group A and it remained so for 14 days.The Prussian staining showed that some blue stained iron praticles existed along the injection point and the color became lighter and lighter as time went by and eventually spread to surrounding part.Immunohistochemical staining results showed that there was no expression of nestin,NSE and GFAP in the control group while there were such apparent expression in the two experimental groups with more expression in group A (p＜0.05).Before establishing the models and after that for 2w and 3w,0.5ml blood was drawn from vena caudalis.And blood routine examination and 12 tumor markers showed that everything was normal.
Conclusion:MSCs can be obtained from human amniotic membranes by enzyme digestion.Induced by bFGF and RA,they can differentiate into neuron-like cells,fully demonstrating that human amniotic-derived MSCs posess multi-differentiating potential
Human AD- MSCs can be successfully labelled in vitro by QDs-NGF-NPs and Feridex.QDs-NGF-NPs with concentration of less than 40μg/L and Feridex with concentration of less than 30μg/ml have no impact to the growth and differentiation of MSCs.
Transplanting AD-MSCs from the injured area is the best way to treat brain injured rats which shows better improvement of the nerve function of rats compared with those of transplanting via tail veins or via intracerebroventricular.
NGF-NPs can effectively enter AD-MSCs and improve the nerve function of brain injured rats.
It is feasible and convenient to observe the survival and differentiation of transplanted AD-MSCs with 3.0T nuclear magnetic resonance analyser and Feridex as labelled marker.
After treating brain injured rats through transplanting AD-MSCs labelled with QDs-NGF-NPs and Feridex the detection to blood routine examination and 12 tumor markers turn out to be normal,all of which indicate that this treatment was safe.
研究表明,神经生长因子对神经元的生长分化及功能的维持具有重要作用。神经生长因子诱导干细胞向神经元样细胞转化的研究已有多篇报道,联合神经生长因子与外源性干细胞移植治疗脑损伤有人进行了研究,对宿主神经功能的改善有一定作用。在脑损伤区长期维持神经生长因子的有效浓度,是神经生长因子联合干细胞移植治疗脑损伤的关键问题,由于神经生长因子通过血脑屏障能力弱,外周血管给药的途径难以有效进入脑损伤区,另外神经生长因子半衰期短,难以在脑损伤区长久维持有效浓度,因此如何使神经生长因子进入脑损伤区并维持有效浓度仍需进一步研究。本文采用量子点观察神经生长因子纳米化后进入羊膜间充质干细胞的情况,通过羊膜间充质干细胞携带神经生长因子进入脑损伤区,取得了满意的效果。
外源性干细胞移植入机体后,对宿主的影响目前尚无明确结论,仍需要研究人员进行探讨。本文通过采集血液检测血常规及肿瘤标志物,了解干细胞移植对脑损伤大鼠的影响,并通过核磁共振观察有无新生物出现。
综合以上研究情况,我们将在人羊膜间充质干细胞移植治疗脑损伤的移植途径、神经生长因子的给药方法、羊膜间充质干细胞移植治疗脑损伤对机体的影响及活体示踪进行探讨,为脑损伤的临床治疗提供依据。
目的:
第一,探讨体外分离、培养和纯化羊膜间充质干细胞(Mesenchmal stem cells,MSCs)的方法;第二,探讨应用量子点神经生长因子纳米粒和Fe_3O_4纳米颗粒(菲力磁)标记羊膜MSCs的可行性及适合条件,并探讨应用3.0T磁共振活体观察负载Fe_3O_4纳米颗粒的羊膜MSCs在大鼠脑损伤区存活迁移的可行性;第三,探讨羊膜MSCs作为载体携带神经生长因子纳米颗粒移植治疗大鼠脑损伤对大鼠神经功能的改善作用;第四,探讨羊膜间充质干细胞移植治疗脑损伤的安全性。
方法:
第一,羊膜MSCs的分离培养与鉴定无菌条件下取正常足月剖腹产胎盘的羊膜,生理盐水充分冲洗后,用0.25%的胰蛋白酶室温消化30min,然后用含1.0g/L胶原酶Ⅳ的DMEM/F12培养液37℃消化1h,200目滤网过滤,制成单细胞悬液,用含10%胎牛血清(fetal bovine serum,FBS)的DMEM/F12培养基进行培养。添加碱性成纤维生长因子(bFGF)进行细胞培养,培养瓶置于37℃、饱和湿度体积分数为5%CO_2培养箱中培养,3d后全量换液,去除未贴壁细胞,每3～5d全量换液。待细胞长至80%～90%融合,按1:2的比例以1×10~6cells/ml密度接种进行传代培养。取第4～6代羊膜MSCs用流式细胞仪检测MSCs的免疫表型CD29,CD44,HLA-ABC的表达。取第3代的羊膜MSCs,用20μg/L bFGF和30μmol/L全反式维甲酸(Retinoic acid,RA)诱导培养的细胞向神经细胞分化。于诱导前及诱导后8d、13d分别进行神经巢蛋白(neuroepithelial stem cell protein,nestin)神经元特异性烯醇化酶(neuron specific enolase,NSE)和胶质纤维酸性蛋白(glial fibrillary acidicProtein,GFAP)免疫细胞化学染色。
第二,量子点神经生长因子纳米粒(QDs-NGF-NPs)、Fe_3O_4纳米颗粒(菲力磁)标记羊膜MSCs及诱导分化
待第三代羊膜间充质干细胞达到80%～90%融合时用2.5g/L胰酶消化,接种于96孔培养板上,每孔200μl,将制好的小玻片置于培养板,待细胞爬满小玻片后,培养基中分别加入不同浓度Fe_3O_4纳米颗粒及QDs-NGF-NPs,Fe_3O_4纳米颗粒标记组分为对照组及实验组,对照组为不含铁纳米颗粒培养基培养的羊膜MSCs,实验组分为四组,使Fe_3O_4纳米颗粒终浓度分别为20μg/ml、30μg/ml、40μg/ml和80μg/ml。每组均加入转染剂多聚左旋赖氨酸,使其终浓度为1.5μg/ml。细胞培养24h均更换为正常培养基,继续培养3周。于细胞培养12h、36h、1周和3周进行普鲁士蓝染色。QDs-NGF-NPs标记组分为实验组及对照组,对照组分为空白对照(培养基中加入等量生理盐水)及单纯量子点对照(培养基中加入终浓度为20、40和60μg/L的量子点)。实验组为QDs-NGF-NPs组,培养基中加入量子点使其终浓度分别为20、40和60μg/L。
根据以上两组实验结果设计QDs-NGF-NPs及Fe_3O_4纳米颗粒联合标记组。该组分为实验组及对照组。QDs-NGF-NPs及Fe_3O_4纳米颗粒组按每孔量子点的终浓度分设3个浓度亚组(20、40、60μg/L)及相应Fe_3O_4纳米颗粒的终浓度为20μg/ml、30μg/ml和80μg/ml,加入多聚左旋赖氨酸终浓度为1.5μg/ml。空载量子点组(不含神经生长因子纳米颗粒)按上述相应量子点浓度分为三组作为对照,正常对照组不加量子点及Fe_3O_4纳米颗粒。培养24h后,更换培养基为正常培养液,继续培养24h、48h、72h后,每孔加入20ul(5g/L)MTT溶液,继续孵育4h后,弃上清,加入100ulDMSO终止反应。微震荡后,于酶标仪波长490nm处测得吸光度值(A)。正常对照组细胞存活率记为100%,其余各组细胞存活率按公式计算:细胞存活率=(实验组A值/对照组A值)×100%,即吸光值越大,存活率越高。置于荧光显微镜下观察量子点在细胞内的分布。分别与12h、36h、1周和3周普鲁士蓝染色,观察Fe_3O_4纳米颗粒进入细胞情况。
细胞分化鉴定:对QDs-NGF-NPs标记组及QDs-NGF-NPs、Fe_3O_4纳米颗粒共标记组用20μg/L bFGF和30μmol/L全反式维甲酸(Retinic acid,RA)诱导羊膜MSCs向神经细胞分化,诱导后第8d及13d用4%多聚甲醛固定30 min,PBS洗涤,加入过氧化物酶阻断液阻断内源性过氧化物酶,非免疫性动物血清封闭,分别加入小鼠抗人神经元烯醇化酶(NSE)、神经巢蛋白(nestin)和胶质纤维酸性蛋白(GFAP)单克隆抗体(即用型1:100稀释),室温孵育1h,PBS洗涤,加入生物素标记的二抗,室温下孵育10min,洗涤,加入链亲和素-过氧化物酶溶液,孵育10min,洗涤,DAB溶液显色,中性树胶封固。
第三,脑损伤模型的制作及不同移植途径的疗效探讨将80只Wistar大鼠,采用自由落体硬膜外撞击法制作大鼠脑创伤模型,损伤装置由底板、固定支架、垂直导杆、20 g砝码、聚乙烯撞击圆锥组成。撞击圆锥头端直径4mm,高2.5mm。10%水合氯醛腹腔注射(3ml/kg)麻醉大鼠后,将头部固定于立体定向仪上,剪去顶部皮毛,消毒,沿中线切开皮肤及骨膜,左侧前囟后3mm,左旁开2mm,钻一直径为5mm的圆形窗,砝码自30cm高处垂直落下,撞击置于硬膜上的圆锥,致中度脑损伤,缝合骨膜及皮肤。大鼠脑外伤模型随机分为对照组与治疗组,对照组20只,治疗组60只,分三组,每组20只,分别标记为A组尾静脉移植组,B组侧脑室移植组及C组脑损伤区移植组。应用平衡木行走试验进行神经功能评分评估大鼠神经功能损伤程度。各组动物分别于1d、4d、1w、2w、3w时处死4只,进行NSE、nestin和GFAP免疫组织化学荧光染色检测,了解移植后的羊膜间充质干细胞的存活、迁移和分化等。根据实验结果选定合适移植途径。
第四,羊膜间充质干细胞移植、活体示踪、神经行为学评分、免疫组化检测实验分为实验组及对照组,对照组20只脑损伤动物模型,相同浓度的菲力磁生理盐水代替羊膜MSCs植入脑损伤区,实验组分为AB两组,共40只大鼠模型,每组20只。A组由第三代羊膜MSCs原位移植于脑损伤区,B组由第三代羊膜MSCs经QDs-NGF-NPs及菲力磁标记24h后原位移植入脑损伤区,在脑损伤区域及其边缘注射含羊膜间充质干细胞的生理盐水10μl(细胞浓度为2.0×10~7/μl),每点进针3mm,针头在注射部位保留5min,然后拔出针头,骨蜡封闭骨窗,缝合骨膜,皮肤。细胞移植后当天记为0d,在移植后的0d,1d,7d,14d,21d进行3.0T核磁共振仪扫描,观察羊膜MSCs的存活迁移情况。成像条件:MRI采用SIEMEMS 3.0 Trio TimⅠ-class MR扫描仪和眼表面线圈,行横断面及冠状面TSE:T1WI、T2WI,T2*WI及SWI序列扫描,扫描参数为:T1WI:TR/TE=430/12ms,T2WI:TR/TE=5990/98ms,层厚2.0mm,FOV10cm×10cm,矩阵288×224;T2*WI:TR/TE=500/20ms,层厚2.0mm,翻转角20°,矩阵256×256;SWI:TR/TE=29/20ms,层厚0.6mm,FOV6cm×6cm,翻转角15°,矩阵256×256。动物模型建立后定时进行神经功能评分。在模型建立后的7d、14d、21d时分别处死动物,进行普鲁士蓝染色了解铁纳米颗粒的存在情况,免疫组织化学荧光法检测神经巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)表达情况。
第五,安全性检测在动物模型建立前、后2w及3w对各组动物尾静脉埋植细针头累积抽血0.5ml,抽血前静脉注入肝素(15U/kg)全身肝素化,对样本进行血常规及12项肿瘤标志物联合检测,观察评估经QDs-NGF-NPs及菲力磁标记的羊膜间充质干细胞移植治疗脑损伤大鼠的安全性。
结果:
经酶消化法自人羊膜组织中提取到的细胞混悬液,置于培养瓶中体外培养,观察到部分细胞最早于10h左右开始贴壁,继续培养1d～2d时,贴壁细胞逐渐增多,培养至1w时,细胞贴满瓶底,细胞形态大多为长梭形,部分为多角形及不规则形状,总体显示为梭形细胞,类似于成纤维细胞的形态。给于消化全量换液传代后,细胞生长加快,在3～6d内铺满瓶底,细胞部分发生融合,细胞以长梭形为主,呈索条状或螺旋状分布。第3～4代的细胞增殖活性较强,一般传代后3d左右可以铺满瓶底,传代至第6～8代后,细胞形态开始出现老化现象,呈扁平状,增殖缓慢或者停止增殖,培养至第9～10代以后,上述现象更加明显。
取第3～4代细胞进行流式细胞术检测,结果显示自人羊膜组织中提取的细胞表达抗原CD29,抗原CD44及抗原HLA-ABC。与间充质干细胞表面抗原一致,提示所得细胞为间充质干细胞。羊膜间充质干细胞体外培养条件下,给与20μg/L bFGF和30μmol/L全反式维甲酸(Retinoic acid,RA)联合诱导后,可以检测到细胞nestin及NSE的表达,提示人羊膜间充质干细胞转化为神经元样细胞。
应用菲立磁标记的羊膜MSCs普鲁士兰染色结果显示,终浓度为20μg/L的Fe_3O_4纳米颗粒组细胞标记率为60%,30,40,80μg/L的Fe_3O_4纳米颗粒细胞标记率均为100%。Fe_3O_4纳米颗粒浓度大者同期进入细胞的铁颗粒就多,36h时各浓度组细胞内铁颗粒聚集达高峰。1w及3w时细胞内铁颗粒逐渐减少。光镜下可以看到细胞内蓝染的铁颗粒分散在胞浆中。
应用QDs-NGF-NPs(浓度小于40μg/L)标记组及QDs-NGF-NPs(浓度小于40μg/L)、菲力磁(浓度小于30μg/ml)联合标记组的培养基培养羊膜MSCs6h后,荧光显微镜下均可见羊膜MSCs的胞膜及胞浆中有红色荧光发出,随时间延长进入细胞的量子点逐渐增多,于32h荧光强度达到高峰,2周内量子点荧光强度未见减弱。普鲁士兰染色见QDs-NGF-NPs(浓度小于40μg/L)、菲力磁(浓度小于30μg/ml)联合标记组羊膜MSCs的胞浆中有大量蓝染的铁颗粒存在,经诱导后两组细胞均有部分细胞表达神经元特异性烯醇化酶(NSE),部分细胞表达神经巢蛋白(nestin),未检测到胶质纤维酸性蛋白(GFAP)的表达。
在移植后第1d开始,钉板平衡木行走测试,评分在对照组5.42±0.98、A组(尾静脉移植)5.02±1.98、B组(侧脑室移植)5.54±1.34和C组(脑损伤区移植)5.12±1.04,各组相比无统计学差异(P＞0.05)。移植后第7d评分对照组5.11±1.62、A组5.15±1.02、B组5.51±2.04和C组4.55±1.02,C组与对照组、A组及B组比较统计学有差异(P＜0.05)。其它各组之间统计学差异不明显(P＞0.05)。在术后13d进行评分显示,对照组5.11±1.92、A组5.01±0.92、B组4.86±0.87和C组2.66±1.72,C组与其它各组比较有统计学差异(P＜0.05),B组与A组及对照组相比有统计学意义(P＜0.05)。A组与对照组相比无统计学差异(P＞0.05)。饲养至第3周时各组评分均未见进一步变化。侧脑室注射羊膜间充质干细胞组显示损伤区表达NSE和nestin的细胞较少,侧脑室壁显示有少量NSE和nestin表达。大鼠脑损伤区定点注射羊膜间充质干细胞组,可见损伤区附近有较多细胞表达NSE和nestin,提示植入的羊膜间充质干细胞部分种植、存活。大鼠尾静脉注射组,在脑损伤区局部组织中未检测到NSE和nestin表达。
菲力磁标记的MSCs脑内移植后对照组及实验组B组在0d扫描时显示T1、T2、T2*GRE扫描在移植区可见低信号区。其中T2*GRE扫描最敏感。在1d,7d,14d,21dMR扫描时,对照组不显影,实验组显示T1、T2、T2*GRE扫描在移植区可见低信号区。其中T2*GRE扫描最敏感。在14d及21d扫描显示在细胞植入区仍能清晰观察到低信号区,向周围弥散现象不明显。A组在MR扫描时不显影。神经功能评分显示细胞移植后的前4d,各组评分统计学没有明显差异,在细胞移植第4d后A组B组评分开始低于对照组,统计学有意义,B组与A组相比统计学有差异,至14d左右,评分没有更进一步变化。普鲁士兰染色结果显示,在移植针道附近可见蓝染的铁颗粒存在,随时间延长,蓝染程度下降,在针道附近蓝色有弥散趋势。免疫组织化学荧光染色显示对照组没有检测到神经巢蛋白(nestin)神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达,实验组两组均有不同数量的细胞表达神经巢蛋白(nestin)及神经元特异性烯醇化酶(NSE),未检测到胶质纤维酸性蛋白(GFAP)的表达。B组神经巢蛋白(nestin)及神经元特异性烯醇化酶(NSE)表达明显多于A组(p＜0.05)。
在动物模型建立前及后2w、3w时抽血,进行血常规及12项肿瘤标志物联合检测,结果显示血常规及12项肿瘤标志物未见异常。
结论:
酶消化法能够从人羊膜组织中分离提取出MSCs,经bFGF和全反式维甲酸体外诱导,能够分化为神经元样细胞,证实人羊膜MSCs具有多分化潜能。
量子点神经生长因子纳米颗粒(QDs-NGF-NPs)及菲力磁可以在体外成功标记羊膜MSCs,浓度小于40μg/L的QDs-NGF-NPs及浓度小于30μg/ml的菲力磁对羊膜MSCs的生长分化没有影响。
脑损伤区移植羊膜间充质干细胞较尾静脉及侧脑室移植能更大程度改善大鼠的神经功能。
神经生长因子纳米化颗粒能有效进入羊膜MSCs,经脑损伤区原位移植能有效改善脑损伤大鼠的神经功能。
羊膜MSCs经菲立磁标记移植大鼠后,能够在脑损伤区存活分化,借助3.0T核磁共振仪能有效观察到羊膜MSCs移植后的存活情况。
经QDs-NGF-NPs及菲力磁标记的羊膜MSCs移植治疗脑损伤大鼠,观察3w检测大鼠血常规及各项肿瘤标志物均未见异常,提示该方法安全。
Background:Brain damage is one of the diseases that pose great threat to people's health.Presently no satisfying effect has been seen in treating the loss of nerve function brought by the suffering of severe brain damage.Yet studies on stem cells bring hope for such patients.Treating brain damage by transplanting stem cells can improve the nerve function of the host.The possible meachanism is that the endocrine effect of exogenous stem cells in hosts and the paracrine effect of nerve cells in the organism itself can bring local neurotrophic factors together,improve the living environment of nerve cells and make it easier to transform from exogenous stem cells into nerve cells and have synaptic connections with them.While the possibility is expected to do more research.Currently there are three ways to transplant stem cells which are different in improving nerve function,namely, tranplanting through the original brain injured area,circulation of blood-cerebrospinal fluid,as well as blood circulation,all of which are relatively effective in improving the nerve function yet no definite conclusion can show which way is the best.Also, no relevant systemic report has ever been seen in comparing their exact treating effect. Thus the author will make further discussion about this and offer some experimental references for treating brain damage.In general,the traditional practice for studying stem cell transplantation in treating brain damage is to sacrifice animals and make them as samples for histopathologic observation.Therefore,how to make observation in vivo about the survival of exogenous stem cells is the difficulty as well as priority for doing research on stem cell transplantation.Through 1.5Tmagnetic resonance, some researchers successfully made observation in vivo about the effect of transplanting labelled mouse embryonic stem cell in vitro in treating brain injured rats, while few reports have been released to study the application of transplantation of human amniotic membrane-derived stem cells(human AD-MSCs) as well as tracking them in vivo for observing their survival in hosts.In this essay,the author treats injured rats by lablelling human AD-MSCs in vitro and the survival as well as immigration of labelled cells are traced through 3.0T nuclear magnetic resonance Previous research shows that NGF play a key role in nerve cells growth differentiation as well as the maintenance of their function.A number of studies have been done to prove that NGF can induce stem cells to transform into neuron-like cells and brain damage can be treated through transplanting NGF and exogenous stem cells which can relatively improve the nerve function of the hosts.However,how to ensure long maintenance of effective concentration of NGF in brain injured area is critical to treating brain damage by combining NGF with stem cells since NGF is weak to pass blood-brain barrier,the administration way of medicine in peripheral vascular is difficult to enter the brain injured area and NGF is hard to maitain effective concentration for a long time due to short half-life.The author will observe the situation of NGF nanoparticles by means of quantum dots(QDs) after entering the brain injured area.The treatment effect turns out to be quite positive.
After tranplanting exogenous stem cells into the organism,further study need to be done about the effect to host.This essay successfully discovers the effect of transplanting stem cells to brain injured rats and finds no neoplasm with the assistance of muclear magnetic resonance after blood routine and tumor marker examination from collected blood.
Based on the above study,this essay is going to explore in such matters as tracking in vitro of human for treating brain damage,the inserting ways of NGF as well as the safety issue so as to provide evidence for clinical treatment of brain damage.
Objective:Ⅰ.To investigates the method of in vitro isolation,culture and purification of MSCs.Ⅱ.To explore the feasibility and necessary conditions for labelling AD-MSCs with QDs-NGF-NPsas well as Fe_3O_4 nanoparticles and also explores the feasibility of the survival and migration of human AD-MSCs into brain injured rats by 3.0T magnetic resonance.Ⅲ.To analyse improving effect of rats' ethology and nerve function by transplanting AD-MSCs with QDs-NGF.Ⅳ.To explore the safety of treating brain damage through transplanting MSCs.
Methods:
Ⅰ.Isolated culture and identification of AD-MSCs
Under the sterile condition,AD-MSCs were obtained from normal placenta after abdominal delivery.Firstly,after being rinsed with physiological saline,they were digested with 0.25%parenzyme at ordinary temperature for 30 minutes and digested with DMEM/F12 solution that contained 1.0g/L collagenase for one hour at 37℃.Then they were filtered with 150 wells filter,processed into unicell suspension cultured in DMEM/F 12 nutritive medium that contained 10%fetal bovine serum (FBS).After adding basic fibroblast growth factors(bFGF),cell culture was undertaken in incubators at 37℃with volume fraction of 5%CO2 and saturation humidity.Three days later,nutritive medium change was done and the unattached cells were removed.In the following research,the removal was done every three to five days.When the cells reached 80%to 90%confluence,serial subcultivation was undertaken in the proportion of 1:2 with the concentration of 1×10~6cells/ml.The expression of immunophenotype for MSCs from the fourth to the sixth generation such as CD29,CD44,HLA-ABC were detected by flow cytometer.The third generation AD-MSCs were chosen,and the cultured cells were induced to differentiate into cellula nervosa with the assistance of 20μg/L bFGF and 30μmol/L Retinoic acid(RA).In eight days and thirteen days before and after the induction, immunocytochemical staining was undertaken to nestin,neuronspecific enolase(NSE) and glial fibrillary acidic protein(GFAP)
Ⅱ.The induction and differentiation of AD-MSCs labelled by QDs-NGF-NPs and Fe_3O_4 nanoparticles
With DMEM/F12 as their culture solution that contained 10%FBS and 20μg/L When the cells reached 80%confluence,the cells were inoculated into 96-hole culture plate for 200μl per hole.Glass slide were put onto the plates.When the slides were coated with cells,QDs-NGF-NPs and Fe_3O_4 nanoparticles at different concentration were added into the nutrient medium which were divided into experimental and control groups.Groups only with Fe_3O_4 nanoparticles were classified into experimental group and control group.The control group had no Fe_3O_4 nanoparticles nutrient medium while experimental group were further divided into four sub-groups at final concentration of 20、30、40μg/L,80μg/ml respectively.Then we added poly-1-lysine at concentration of 1.5μg/ml in each group.After culturing for 24 hours,the nutrient medium was changed into medium without NGF and NPs and continue to culture for 3w and were given Prussian blue staining in the.corresponding time,The labelled groups were divided into blank control group(equal amount of physiological saline in it and pure QDs,in which the final concentration of QDs were 20、40 and 60μg/L;The experimental group was group with QDs-NGF-NPs which was added woth QDs at concentration of 20,40,and 60μg/L respectively.
According to the experimental results,a combined labelled group between QDs-NGF -NPs and Fe_3O_4 nanoparticles was made which was further divided into experimental group and control group.Group with QDs-NGF-NPs were divided into three sub-groups(with concentration of 20、40、60μg/L),and group with Fe_3O_4 nanoparticles were divided into 3 sub-groups(with concentration of 20μg/ml、30μg/ml and 80μg/ml).Then they were added into Poly L lysine with the final concentration of 1.5μg/ml.Group without QDs-NGF-NPs at the same conresponding final concentration were divided into control groups for comparision.Normal control group without no QDs-NGF-NPs and Fe_3O_4 nanoparticles were changed nutrient medium for nutrient solution after being cultured for 24h.Being culturing for another 24h、48h、72h,each hole was added 20ul(5g/L)MTT solution and continued to incubate for 4 hours.At last,the supernatant was removed and 100ulDMSO was used to terminate reaction.After shaking slightly,the absorbance value(A) were measured at the wavelength of 490mm.The cell survival in normal control groups were 100% while survival in other groups was to be calculated according to the following formula: cell survival=(experimental groups A/control groups A)×100%.That is to say,the larger the A value was,the better survival rate was.The distribution of QDs among the cells was observed under fluorescent microscope.Then Prussian blue staining was applied at 12h、36h、1w and 3w so that how much Fe_3O_4 nanoparticles had entered into cells could be observed.
Identification of cell differentiation:20μg/L bFGF and 30μmol/L RA were used to induce AD-MSCs to differentiate into cellula nervosa in both labelled group with QDs-NGF-NPs and combined group with QDs-NGF-NPs and Fe_3O_4 nanoparticles. Then after 8days and 13 days,they were fixed by 4%paraformaldehyde for 30 minutes and washed by PBS.Peroxidase blocking solution was used to block endogenous peroxidase and nonimmune animal serum.Then neuron-specific enolase (NSE),nestin,monoclonal antibody(1:100dilution) of glial fibrillary acidic protein (GFAP) were added to the cells which were incubated for 1 hour at ordinary temperature and washed with PBS.After that,cells were incubated for 10 minutes and washed.At last they were incubated for another 10 minutes and washed while adding streptavidin-peroxydase solution which were developed color with DAB solution and fixed with neutral gum.
Ⅲ.Study on therapeutic effect of different transplantation methods and establishment of brain injured models
About 80 wistar rats were chosen to establish brain injured models by the Feeney's free falling weight method.The necessary injury devices were made up of backboard,fixed bracket,vertical lead-screw,20g counterweight and polythene knocking conus with the diameter of 4mm and 2.5mm high.After being undertaken peritoneal injection with 10%chloral hydrate(3ml/kg),their heads were fixed in stereotaxic apparatus,the skin and hair of their heads were cut and sterilized Then the part of skin and periosteum were cut a round shape along the centerline with diameter of 5 mm,3mm behind anterior fontanel and 2mm on the left side.Then,the weight was vertically dropped the from 30 mm high,making it stroke the conus so that the rats could be damaged moderately.At last,the cut was sutured.
Those brain injured models were divided into control group(n=20)and treatment group(n=60)which was further divided into 3 groups(n=20),that is group A(vein transplantation),groupB(ventricle transplantation) and group C(brain injured area transplantation).The degree of neural injury was evaluated with pegged beam walking test method.4 rats from each group were killed 1d、4d、1d、2w、3w later separately and NSE as well as nestin and GFAP immunohistochemistry staining were done to those dead rats in order to observe the survival,migration as well as differentiation of AD-MSCs.We would choose the appropriate way to transplant.
Ⅳ.The transplantation,in vivo labelling,evaluation of neuroethology,and immunehistochemical detection of AD-MSCs
The rats were divided into control group and experimental group.In control group,all of the 20 models were transplanted with Feridex physiologic saline at the same concentration that replaced AD-MSCs.40 models were divided into two experimental groups(n=20).In groupA,the third generation AD-MSCs was transplanted into the original brain injured area.In group B,the third generation AD-MSCs labelled with QDs-NGF-NPs and Feridex for 24 hours.Then 10μl physiologic saline that contained AD-MSCs(with concentration of 2.0×107/μl)was injected into and around the injured area.Each injection should be 3mm deep and remained in the injected place for about 5min.After this,bone wax was used to seal bone window incision off and periost were sutured.SIEMEMS 3.0 Trio Tim I-class MR scanner was used to performT1WI、T2WI、T2~* Wias well as SWI sequence scanning.Scan parameters were T1WI:TR/TE = 430/12ms,T2WI:TR/TE= 5990/98ms with thickness of 2.0mm,FOV10cm×10cm and matrix 288×224 and so on.The neurological scores was graded in fixed time after establishing the models. Then those models were sacrificed to observe the existence of iron nano-particles with Prussian staining 7days,14days and 21 days later respecttively.The expression of nestin and NSE and GFAP were observed with immune histochmistry method.
Ⅴ.Safety detection
Before establishing the models and After that for 2w and 3w,0.5ml blood was drawn from vena caudalis.But heparin(15U/kg) should be injected before hemospasia to ensure whole body heparinization.Blood routine examination and 12 tumor markers should be detected together in order to observe and evaluate the availability and safety of tranplanting AD-MSCs labelled by QDs-NGF-NPs and Feridex.
Results:The cells suspension solution extracted by enzymic digestion from AD-MSCs was cultured in vitro.It was soon found that some cells became adhering to the wall as early as 10 hours later.Being cultured for another day or two days,the adhering cells increased steadily and filled the bottom in various forms.Most of them were long fusiform while some of them were polygon or in irregular forms.In general, they appeared to be spindle cells,similar to fibroblast cells.After cell passage through total change of digestion solution,cells growth accelerated and spreaded the flasks bottom all over 3 to 6 days later with some cell fusion.Most of them were long fusiform cells,distributing in stripy and spiral shape.Cells in the third generation proliferated actively and could cover the bottom of the flasks during that stage yet cells in the sixth to eighth generation became aging in flat shape proliferatng slowly or even ceasing to do so.When it comes to the 9th or 10th generation cells,this phenomenon became even more obvious.
According to flow cytometry,cells in the third and fourth generation turned out that: cells extracted from AD-MSCs could express antigens CD29,CD44 and HLA-ABC which were the same with those of MSCs,indicating those were MSCs.Under the cultivation in vitro,AD-MSCs jointly induced by 20μg/L bFGF and 30μmol/ LRetinoic acid(RA) could express nestin and NSE,indicating that AD-MSCs could differentiate into neuron-like cells
Resultl from Prussian staining toAD-MSCs lablelled by Feridex indicated as follows:cell labeling rate in group with Fe_3O_4 nanoparticles at the final concentration of 20μg/L was 60%,yet cell labelling rate in group with Fe_3O_4 nanoparticles at the final concentration of 30,40,80μg/L was 100%.That is to say,the higher the concentration was,the more Fe_3O_4 particles entered.And Fe_3O_4 particles from each groups reached the highest amount 36h later and they decreased when it was 1w and 3w later.We could see those Fe_3O_4 particles stained by Prussian method scattered in kytoplasm.
Labelled group with QDs-NGF-NPs(concentration was less than40μg/L) and combined group between QDs-NGF-NPs(concentration was less than40μg/L) as well as Feridex(concentration was less than30μg/ml) were cultured for 6h.Under fluorescent microscope,red fluorescent light could be observed radiating from cell membrane kytoplasm.Meanwhile,the amount of QDs increased as time went by.The light reached its peak 32h later and it didn't fade away 2w later.After being stained,a lot of blue-stained Fe_3O_4 particles could be detected in the Fe_3O_4 particles of the combined group.In the two groups,some cells expressed as NSE or nestin.No cells were detected to express GFAP.
Balance beam walking test was adopted to those groups one day after transplantation.But there was not significant differences between control group 5.42±0.98、group A 5.02±1.98、group B 5.54±1.34 and group C 5.12±1.04(P＞0.05). There was statistic significance between control group 5.11±1.62、group A 5.15±1.02、group B 5.51±2.04 and group C 4.55±1.02,between group C and control group,beteen group A and group B(P＜0.05).The rest of other groups were not seen any significant difference(P＞0.05).Scores were graded 13 days after the operation,there was significant diffrences between control group 5.11±1.92、groupA 5.01±0.92、groupB 4.86±0.87 and group C 2.66±1.72,between group C and other groups,between group B,group A and the control group(P＜0.05).But there was no significant differences between group A and the control group(P＞0.05).No change in the above results after raising those rats for 3 weeks.In group B with intracerebroventricular injection indicated that few cells in the injured region expressed as NSE and GFAP,though some cells in the ventricular walls expressed as NSE and GFAP.While in group C,a lot of cells expressed as NSE and GFAP indicating that most of the transplanted AD-MSCs managed to grow and survive in that area.As for group A.nothing was observed to express as NSE and GFAP.
Shortly after tranaplantating AD-MSCs labelled with feridex,the control group and group B were undertaken T1、T2、T2~*G scaning,and low signal could be found in the transplanted area in which T2~*GRE was the most sensitive one.In the MR scanning 1d,7d,14d and 21d later,nothing could be found in the control group while low signal could be observed in group B.Particularly,the signal of feridex could be detected in the transplanted area 14d and 21d later while group A appeared to be nonvisulized under MR scaning.According to the score of nerve function,no significant differences in statistics could be found in the first 7 days after transplantation.Yet 8 days later,the scores in group A and group B became lower than that in control group,thus there was statistic significance while group B had differences compared with group A and it remained so for 14 days.The Prussian staining showed that some blue stained iron praticles existed along the injection point and the color became lighter and lighter as time went by and eventually spread to surrounding part.Immunohistochemical staining results showed that there was no expression of nestin,NSE and GFAP in the control group while there were such apparent expression in the two experimental groups with more expression in group A (p＜0.05).Before establishing the models and after that for 2w and 3w,0.5ml blood was drawn from vena caudalis.And blood routine examination and 12 tumor markers showed that everything was normal.
Conclusion:MSCs can be obtained from human amniotic membranes by enzyme digestion.Induced by bFGF and RA,they can differentiate into neuron-like cells,fully demonstrating that human amniotic-derived MSCs posess multi-differentiating potential
Human AD- MSCs can be successfully labelled in vitro by QDs-NGF-NPs and Feridex.QDs-NGF-NPs with concentration of less than 40μg/L and Feridex with concentration of less than 30μg/ml have no impact to the growth and differentiation of MSCs.
Transplanting AD-MSCs from the injured area is the best way to treat brain injured rats which shows better improvement of the nerve function of rats compared with those of transplanting via tail veins or via intracerebroventricular.
NGF-NPs can effectively enter AD-MSCs and improve the nerve function of brain injured rats.
It is feasible and convenient to observe the survival and differentiation of transplanted AD-MSCs with 3.0T nuclear magnetic resonance analyser and Feridex as labelled marker.
After treating brain injured rats through transplanting AD-MSCs labelled with QDs-NGF-NPs and Feridex the detection to blood routine examination and 12 tumor markers turn out to be normal,all of which indicate that this treatment was safe.
引文
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75.徐虹,李康生.神经营养因子对颅脑损伤保护作用的研究进展.国外医学,生理、病理科学与临床分册.2001,9(2):147-148.
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94.Marmarou A,Foda MA,van den Brink W,et al.A new model of diffuse brain injury in rats,part 1 and part 2.J Neurosurg,1994,80(4):291-313.
95.王公平,杨波,关方霞等.不同途径羊膜间充质干细胞移植治疗大鼠脑外伤的研究.中南大学学报(医学版),2008,33(10):926-930.
96.Himes N,Min JY,Lee R,et al.Invivo MRI of embryonic stem cells in a mouse model of myocardial infarction.Magn Resort Med,2004,52(9):1214-1219.
97.徐健,郑敏文,宦怡,等.干细胞的磁性标记及肝内活体磁共振示踪第四军医大学学报,2007,28(6):484-488.