骨髓间充质干细胞移植对易损斑块稳定性的影响及机制研究
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摘要
背景
当今社会,急性心脑血管事件(急性冠脉综合征与脑卒中)严重威胁着中老年人的健康与生命,是公认的健康头号威胁,有着极高的致死率和致残率。心血管疾病是发达国家致死的主因,同时正在快速成为发展中国家的头号杀手。随着社会的发展,人民生活方式的改变及老龄化社会的到来,每年有两千多万人死于动脉粥样硬化性心血管病,其中主要为冠心病,而且这个数字还在不断的递增中。动脉粥样硬化(Atherosclerosis,AS)是心脑血管疾病的共同病理基础,易损斑块的形成以及随之发生的血栓形成是致残、致死性血管疾病的真正元凶及最根本的始动因素,因此AS疾病的防治重点之一是易损斑块的稳定和修复。易于受损的斑块不只是易于破裂的斑块,所有可能有血栓性并发症和进展迅速的斑块都应视为易损斑块。而且易损斑块并非是造成急性冠脉综合症(Acute Coronary Syndrome,ACS)、心肌梗死和心脏猝死的惟一罪犯因子,易损血液(易于发生血栓)和易损心肌(易于发生致命性心律失常)对预后也起着重要作用。近年来,国际上倾向于用“易损患者”一词代替“易损斑块”,以便更好的评估患者发生急性心脑血管事件的危险性。由于AS是散在的、累及全身动脉血管的慢性疾病,有各种不同的局部和周身表现,临床上常表现为冠状动脉粥样硬化(ACS、心肌梗死)、脑血管硬化(脑梗塞、脑血管破裂)、颈动脉粥样硬化(脑供血不足、脑梗塞)、主动脉病变(主动脉夹层、主动脉瘤)、肾动脉粥样硬化(肾脏疾病、高血压)等。因此,对易损性负荷的评估不能仅限于单个不稳定斑块,而应包括总动脉粥样硬化负荷(主动脉和颈动脉、股动脉等)和冠脉的易损斑块,也要包括对血液和心肌易损性的综合危险评估(即易损指数)。
尽管目前已成功应用多种治疗手段防治或减少冠心病的发生发展,主要包括溶栓、降脂、抑炎等内科治疗措施、介入治疗及外科冠状动脉搭桥手术等。这些手段虽然发挥了一定的作用却颇为有限。对于冠脉内径狭窄>70%的ACS患者,介入手术仍将是治疗冠心病的主要技术之一。但是大量的研究表明,约70%-80%ACS形成是由于轻、中度狭窄的斑块破裂及继发血栓形成所致,并不适用介入治疗,其治疗重点应为稳定易损斑块和修复破裂斑块。由于AS的全身性特点,对局部某一易损斑块展开的治疗过于局部和片面,无法最大程度的避免急性心脑血管事件的发生。而针对急性心脑血管事件的易损患者,当下的治疗／预防趋势已倾向于综合评估总动脉粥样硬化负荷和冠脉的易损斑块,包括对患者易损血液、易损心肌的综合评估,从整体上预防急性心脑血管疾病的发生。因此,药物治疗应该是稳定易损斑块的基础。然而,目前仍然没有稳定易损斑块的有效药物。尽管在动物实验中,他汀类药物可明显的降低低密度脂蛋白胆固醇水平,抑制炎症反应,从而增加斑块纤维帽厚度。然而,他汀类的肝毒性使许多患者不能使用此类药物。
近年来,越来越多的医学科学研究者将目光投向了基于组织工程发展起来的细胞(或组织)替代治疗,这为人类征服大量疑难疾病提供了新的途径和希望。骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,MSCs),来源于中胚层,广泛存在于多种成体组织中,相对其它干细胞具有低免疫原性、多分化性、体外易培养扩增及独特的归巢功能等多方面的优点。MSCs可分化为造血相关细胞,也可分化为多种造血以外的细胞,尤其是中胚层和神经外胚层来源的细胞,近年,MSCs在临床中具有广泛的应用前景。目前已有大量实验将MSCs移植用于多种疾病的治疗,并取得了一定的研究成果。目前,在心肌梗死、角膜损伤、神经系统损伤、糖尿病、急性移植物抗宿主反应、肺部多种疾病、肝脏纤维化,骨关节损伤和克罗恩病等多种疾病的临床试验中,MSCs均表现出较好的治疗效果。研究发现,MSCs具有其特殊的生物学特征,包括：①MSCs作为“种子”细胞可定植于受损的组织并可分化成为组织细胞而发挥组织修复作用。②免疫调节作用(可通过旁/自分泌方式),抑制T淋巴细胞、抑制细胞毒性T淋巴细胞和自然杀伤细胞等而起到减轻病变组织内免疫炎性反应的治疗作用。③造血支持功能。④多种生物活性物质的分泌能力。诸多的临床与实验室研究已经证实,体外培养的间充质干细胞具有免疫调控、造血支持和修复多种组织损伤的能力,而这些功能多依赖于间充质干细胞分泌的生物活性物质。
在近些年国内外对MSCs的研究中,并未涉及MSCs移植对机体AS易损斑块的稳定作用。考虑到易损斑块属于多部位多系统疾病,当下有效的治疗方法不能有效发挥“防患于未然”的作用,也无法最大程度的避免血管事件的发生。综合MSCs参与组织修复的多种机制,在本实验中我们对MSCs对易损斑块的稳定作用进行了研究。
目的
1体外扩增培养兔MSCs并对其进行成骨成脂及特异性表面标志物鉴定,并在慢病毒转染及荧光染色剂染色之间选择合适的标记方式对其进行标记,并初步探讨其在体外对氧化型低密度脂蛋白(Oxidized Low-density lipoprotein, ox-LDL)所致血管内皮细胞凋亡的影响。
2兔AS易损斑块模型基础上,利用MSCs独特的“归巢”功能、多向分化潜能及强大的旁分泌功能,通过骨髓间充质干细胞移植,从组织水平、蛋白水平和基因水平观察骨髓干细胞对AS易损斑块稳定性、血清及斑块炎性因子、抑炎蛋白表达及斑块内细胞凋亡的影响,探索MSCs稳定与修复易损斑块的机制,为临床应用MSCs防治急性心脑血管疾病提供理论和实验依据。
方法与结果
第一部分：方法：采用全骨髓贴壁法从新西兰大白兔骨髓中提取、分离和培养兔MSCs。取P3代细胞分别采用成骨、成脂诱导液分别培养21天、14天使诱导其成骨、成脂,并使用流式细胞仪鉴定MSCs的特异性表面标志物CD29,CD44,CD45。分别采用慢病毒转染及二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein-diacetic succinimidyl ester,CFSE)荧光染料染色的方法对MSCs进行标记,并选取合适的标记方法。另外以ox-LDL体外诱导方式建立人脐静内皮细胞(human umbilical vein endothelial cell,hUVECs)损伤模型,采用细胞共培养的方式共同培养MSCs和hUVECs,采用流式细胞仪检测hUVECs凋亡率,ELISA法检测细胞培养液中肿瘤坏死因子a(Tumor necrosis factor-a,TNF-a)和血管内皮生长因子(vascular endothelial growth factor,VEGF)、rt-PCR方法检测凋亡相关基因bcl-2和Bax的表达。采用SPSS13.0统计学软件分析数据。定量数据以(x±s)表示,两样本比较采用独立样本t检验,多组间比较方差齐时采用one-way ANOVA,方差不齐时采用Welch F方法,重复测量数据采用重复测量数据方差分析,P<0.05为差异有统计学意义。
结果表明：(1)体外培养扩增的MSCs为均一性好,增殖能力强的梭型细胞。成骨诱导21天,经茜素红染色可见矿化结节形成；成脂诱导14天,经油红O染色可见脂滴形成。结果表明体外成功培养和扩增了兔MSCs,在特定条件下能够向成骨细胞分化。经流式细胞仪检测,结果显示第3代MSCs的特异性表面标志物：CD29阳性,阳性率为98.0%,CD44阳性,阳性率为98.5%,CD45阴性,阳性率为0.7%。提示第3代贴壁细胞主要是MSCs,为以后的实验提供了细胞基础。慢病毒转染MSCs效率可达80～90%,但是慢病毒转染对MSCs增殖能力有极大的影响,转染组细胞增殖明显低于对照组(F=2970.659,P<0.001)。而CFSE荧光染色作为一种快速、方便且安全的染色方法,终浓度为10μmol/L+染色10分钟为CFSE标记MSCs的理想条件,其不但不影响MSCs的增殖能力(F=0.693,P=0.425),同时也不影响其转化及分泌VEGF的功能(t=2.070,P=0.065),更适用于大剂量移植细胞时采用。
(2)正常hUVECs形态不一,胞核较圆,细胞质丰富且清亮,细胞周围近胞核处可见明显光晕,形成典型的“铺路石样”结构。100μg/ml的ox-LDL作用24h后hUVECs开始出现明显的形态学变化,细胞明显皱缩,细胞间隙增大,胞浆内见较多颗粒、空泡。流式细胞检测发现三组hUVECs凋亡率存在显著差异(F=105.229,P<0.001),ox-LDL组凋亡率显著高于正常对照组(P=0.001)；而MSCs共培养组能显著抑制ox-LDL对hUVECs的损伤,细胞凋亡率显著性降低(P=0.008)。三组VEGF及TNF-α的含量均存在显著性差异(F=50.957,P<0.001；和F=61.578,P<0.001),ox-LDL组VEGF及TNF-α的分泌量均高于空白对照组,差异有显著性意义(P=0.001,P<0.001)；MSCs共培养组上清液中VEGF显著高于对照组和ox-LDL组(P=0.001,P=0.008),而TNF-α分泌量较ox-LDL组显著下降(P=0.002),但仍高于空白对照组,差异有统计学意义(P=0.005)。三组bcl-2和bax mRNA表达存在显著性差异(F=13.381,P=0.002；和F=14.633,P<0.001)。与对照组比较,ox-LDL组bcl-2mRNA表达显著降低(P=0.041)bax mRNA表达显著上调(P=0.001),MSCs共培养组bcl-2mRNA表达显著增加(P=0.044)。与ox-LDL组比较,MSCs共培养组细胞bcl-2mRNA表达显著上调(P=0.004),bax mRNA表达亦显著降低(P<0.001)。三组Bcl-2/Bax比值也有显著性差异(F=42.433,P<0.001),与对照组比较,ox-LDL组Bcl-2/Bax比值显著性降低(P<0.001),MSCs共培养组无显著性差异(P=0.269)；与ox-LDL组比较,MSCs共培养组Bcl-2/Bax比值增加,差异有显著性意义(P=0.001)
第二部分：
方法：34只健康纯种雄性新西兰白兔,随机分为3组,MSCs移植组(MSC组,14只)和易损斑块模型组(VP组,10只)兔先给予高脂饲料喂养1周,然后实施右颈总动脉液氮冻伤术,术后继续高脂饲料喂养7周,并于第8周末再次实施第2次右颈总动脉内膜液氮冻伤术,术后即刻通过耳缘静脉注射1×107个MSCs(1m1)或1mlPBS,改普通饲料继续喂养4周。稳定斑块模型组(SP组,10只)兔先给予高脂饲料喂养1周,然后实施右颈总动脉液氮冻伤术,术后继续高脂饲料喂养7周。改普通饲料继续喂养4周。分别于实验前及高脂饲料喂养后3d、1w、第2w至第12w每2周采血一次检测检测甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL)水平。分别于细胞移植后6h及24h制作全血涂片于荧光显微镜下观察血液内MSCs变化,移植后3d及2w取MSCs组两只兔子,取右颈总动脉于共聚焦显微镜下观察细胞归巢情况；分别于移植后1d、2d、3d、1w、2w、4w末空腹抽取兔静脉血,离心取上清液,ELISA法测定血清高敏C反应蛋白(hs-CRP)、TNF-α、白介素6(IL-6)、白介素10(IL-10)及VEGF水平。细胞移植后4周末处死兔子取出血管,HE染色及Masson三色染色,并行帽／核比值测量,光镜观察斑块修复情况；应用核因子κB (NF-κB)、基质金属蛋白酶1、2、9(MMP-1、MMP-2、MMP-9)以及基质金属蛋白酶抑制因子1(TIMP-1)的单克隆抗体对实验血管进行免疫组织化学染色,以明确斑块处各炎性因子蛋白表达情况；应用rt-PCR和免疫印迹(Western Blot)方法对实验血管处的TSG-6含量进行检测；应用TUNEL法检测各组斑块内凋亡细胞数量,免疫组化检测caspase-9表达水平。
结果：(1)高脂饲料喂养3天后TG、TC及LDL均出现明显增高(P=0.037,P=0.041,P=0.048),第1周内升高幅度最大,第2-8w血脂缓慢增高。8周后更换为普通饲料喂养后,兔血脂水平迅速下降。8周后,肉眼可见MSC组及VP组兔右颈总动脉有多处黄白色斑块形成,而左侧颈总动脉未见明显斑块形成；12周后光镜下见,SP组兔右颈总动脉内膜显著增厚并纤维化,血管壁向管腔内突出,粥样硬化斑块形态结构完整,纤维帽较厚,斑块内炎细胞较少,未见斑块破裂。VP组斑块中心可见大量脂核,斑块表面覆盖较薄纤维帽,在斑块肩部可见残存泡沫细胞和大量炎细胞(如巨噬细胞和淋巴细胞等)浸润,部分斑块可见破裂和(或)血栓形成。MSC组形态介于之间。左侧颈总动脉内皮细胞及平滑肌细胞排列整齐,呈长型或椭圆型,血管内膜偶有轻度增生,极少部分内膜可见少量脂质沉积,但无突出斑块形成。荧光显微镜下全血涂片显示移植6h后仍可在血液内发现较多荧光细胞,24h后荧光细胞含量明显减少；激光共聚焦显微镜下显示移植3d后可在斑块内膜表面发现较多荧光细胞聚集,2w时荧光细胞明显减少。而在MSCs组兔子左侧颈总动脉表面,未见荧光细胞聚集。Masson染色可见SP组斑块内含有大量的平滑肌细胞和弹力纤维,MSC组斑块内平滑肌细胞、弹力纤维和胶原纤维含量次之。与MSC组和SP组相比,VP组斑块内平滑肌细胞、弹力纤维量明显减少,胶原纤维排列紊乱。三组帽/核比值间有显著性差异(F=28.298,P<0.001),MSC组及SP组斑块帽/核比值均显著性高于VP组(P<0.001)。
(2)不同时间血清hs-CRP有显著差异(F=21.670,P<0.001)；三组血清hs-CRP水平有显著性差异(F=428.245,P<0.001),且各个时间点均为P<0.001,VP组hs-CRP显著高于MSC、SP组,而MSC显著高于SP组。不同时间与不同组别之间存在交互效应(F=11.857,P<0.001)。不同时间TNF-a有显著差异(F=8.056,P<0.001);三组TNF-a水平有显著性差异(F=245.006,P<0.001),且各个时间点均为P<0.001,VP组TNF-a显著高于MSC、SP组。不同时间与不同组别之间存在交互效应(F=9.362,P<0.001)。不同时间IL-6有显著差异(F=5.852,P<0.001)；三组IL-6有显著性差异(F=49.492,P=0.015),且各个时间点均为P<0.05,VP组IL-6显著高于SP组；D2、D3、W1、W2时VP组显著高于MSC组。不同时间与不同组别之间存在交互效应(F=1.951,P=0.044)。不同时间之间血清IL-10有显著差异(F=28.241,P<0.001)；三组血清IL-10有显著性差异(F=244.606,P<0.001),三组比较除D1时P=0.006外均为P<0.001,MSC组IL-10显著高于VP、SP组,而在D3、W1、W2、W4时VP组显著高于SP组。不同时间与不同组别之间存在交互效应(F=12.684,P<0.001)。不同时间之间血清VEGF有显著差异(F=15.241,P<0.001)；三组之间血清VEGF水平有显著性差异(F=132.641,P<0.001),且各个时间点均有显著性差异,均为P<0.001。除D2时MSC组与VP组无显著差异,其余时间点MSC组VEGF均显著高于VP组及SP组,而在W1、W2、W4,VP组VEGF显著高于SP组。不同时间与不同组别之问存在交互效应(F=10.702,P<0.001)。
(3)三组间NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1含量有显著性差异(F=44.953,P<0.001；F=46.530,P<0.001；F=34.292,P<0.001；F=27.108,P<0.001;F=63.138,P<0.001)。MSC组NF-κB、MMP-1、MMP-2、MMP-9含量显著低于VP组,差异有统计学意义(P<0.001；P<0.001；P<0.001；P<0.001),TIMP-1显著高于VP组(P<0.001)；MSC组NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1表达显著高于SP组(P=0.005；P=0.018；P=0.046；P=0.005；P<0.001)；SP组斑块内NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1含量明显低于VP组,差异有统计学意义(P<0.001；P<0.001；P<0.001；P<0.001；P=0.002)。Real-time PCR检测结果显示,三组TSG6mRNA表达有显著性差异(F=106.082,P<0.001),MSCs组TSG6mRNA表达较VP组和SP显著增加(P=0.017,P=0.007)；而VP组TSG6mRNA表达较SP组显著增加(P<0.001)。WesternBlot检测结果显示,三组之间TSG6蛋白表达有显著性差异(F=41.484,P<0.001),其中MSCs组TSG6mRNA表达较VP组和SP显著增加(P<0.001,P<0.001);而VP组TSG6mRNA表达较SP组显著增加(P=0.008)。
TUNEL法示三组斑块内均可见凋亡细胞,三组AI值有显著性差异(F=96.153,P<0.001),VP组显著高于MSCs组及SP组(P<0.001,P<0.001),MSC组AI也显著高于SP组(P<0.001)。MSC组及VP组凋亡细胞主要分布于脂核区及中膜平滑肌区。SP组主要分布于中膜平滑肌区,脂核区及内膜极少凋亡细胞。免疫组化结果显示三组斑块内均可见caspase-9表达。MSCs组、VP组和SP组caspase-9表达水平有显著性差异(F=14.391,P=0.002)；进一步两两比较发现,VP组斑块内caspase-9表达水平显著高于MSCs组及SP组(P<0.001,P<0.001),MSC组AI与SP组之间差异不明显(P=0.335)。
结论
1通过全骨髓贴壁培养法,我们成功提取并扩增出足够数量且活力较好的MSCs,且经多项体外鉴定,证明提取的该种细胞为MSCs。相较于慢病毒转染,CFSE荧光染色对细胞生物学活性无影响,更适用于大剂量的移植细胞标记。且发现MSCs可通过降低炎性因子水平、增加VEGF分泌,并通过一系列机制调节凋亡相关基因的表达,进而改善ox-LDL所致血管内皮细胞凋亡。
2应用同种异体MSCs静脉移植可稳定和修复易损斑块。MSCs进入机体.后能向易损斑块区“归巢”,并通过分泌抗炎蛋白TSG-6抑制巨噬细胞释放NF-κB,进而抑制该信号通路上一系列炎性因子的释放,进而降低血清及斑块局部炎性反应发挥稳定斑块的作用。此外,MSCs还可以抑制斑块局部的细胞凋亡,这可能与MSCs通过分泌VEGF等生长因子抑制线粒体凋亡途径并促进局部细胞组织增殖修复有关。
Background
At present, acute cardiovascular and cerebrovascular events (acute coronary syndrome and stroke) is a serious threat to the health and lives of the elderly, which has high morbidity and mortality. Cardiovascular disease is the main cause of death in developed countries, and is fast becoming the number one killer in developing countries. With the development of society, people's lifestyles change and the arrival of the aging society, more than two thousand people died of atherosclerotic cardiovascular disease every year, mainly for coronary heart disease, and that number continues to increment in. Atherosclerosis (atherosclerosis, AS) is a common pathological basis of cardiovascular and cerebrovascular disease, and the formation of vulnerable plaque and the ensuing thrombosis is the real culprit and the most fundamental initiating factor of the fatal vascular disease, therefore the prevention and treatment of AS disease should be focused on stabilizing and repairing vulnerable plaque. Vulnerable plaque is not only the plaque easy to rupture, all ones are likely to have thrombotic complications and rapid progress should be considered vulnerable plaque. And vulnerable plaque is not the only crime factor for acute coronary syndrome (ACS), myocardial infarction (MI) and sudden cardiac death, the vulnerable blood (easy occurrence of thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) also play an important role on prognosis of these diseases. In recent years, the international community prefers to use the term "vulnerable patients" instead of "vulnerable plaque" in order to better assess the risk of acute cardiovascular and cerebrovascular events in patients. Atherosclerosis is scattered, systemic arteries involved chronic disease, which have various clinical manifestation, usually presents clinical coronary atherosclerosis (ACS, MI), cerebral arteriosclerosis (cerebral infarction, rupture of cerebral blood vessels), carotid atherosclerosis (cerebral insufficiency, cerebral infarction), aortic lesions (aortic dissection, aortic aneurysm), renal artery atherosclerosis (kidney disease, hypertension) et al. Therefore, the assessment of the vulnerability of the plaque load should not be limited to a single unstable plaque, but should include the total atherosclerotic plaque load (aortic and carotid and femoral artery) and coronary vulnerable plaques, also should include blood and myocardial vulnerability risk assessment (vulnerability index).
Although there have been a variety of treatment applied successfully to control or reduce the occurrence and development of coronary heart disease, mainly including medical treatment measures such as dissolving blood clots, lipid-lowering, suppression the inflammatory, intervention treatment and surgical coronary artery bypass surgery. The treatment plays a certain role but is extremely limited. Surgical intervention will continue to be one of the main technical therapies for ACS patients with more than70%coronary stenosis. However, a large number of studies showed that the formation of about70%-80%ACS is caused by rupture and secondary thrombosis of mild to moderate stenosis plaque, which is not suitable to adopt PCI, its treatment should focus on stabilize and repair vulnerable plaques. Because of the systemic and multi-distribution characteristics of atherosclerosis and vulnerable plaque, and the vulnerability characteristics of the blood, the treatment for a local vulnerable plaque is too partial and one-sided, and can not avoid acute cardio-cerebral vascular events occurred in a greatest degree. Current treatment/prevention trends for acute cardiovascular and cerebrovascular events tend to consolidated total atherosclerotic load and coronary vulnerable plaques and vulnerable patients, including a comprehensive assessment of the fragile blood and vulnerable myocardial of the patients, to prevent the occurrence of acute cardiovascular and cerebrovascular disease. Thus, the drug treatment should be the basis of stabilization of vulnerable plaque. However, there is still no effective drugs to stabilize vulnerable plaque. In animal experiments, statins can significantly reduce the level of LDL-c, inhibition of the inflammatory response, thereby increasing the thickness of the fibrous cap. However, liver toxicity of statins makes so many patients can not use such drugs.
Cells (or organization) replacement therapy and gene therapy technology developed based on tissue engineering and gene engineering in recent years, and become the research focus and cutting-edge in the field of medicine and even in the entire field of life sciences, and provide a new way and hope for conquering many diseases. Bone marrow mesenchymal stem cells(MSCs), is a kind of the early cells from mesoderm, and widely exist in a variety of adult tissues. MSCs have a wide range of advantages relative to other stem cells, such as low immunogenicity, multi-differentiation characteristic, easy to cultivate and amplification in vitro and unique homing feature. Not only can MSCs differentiate into the hematopoietic and stromal cells, but also can differentiate into a variety of other organization, especially the organizations cells from mesoderm and neuroectoderm. In recent years, MSCs has a broad application prospects in clinical practice. There are a large number of experimental to check the transplantation of MSCs for the treatment of a variety of diseases, and have achieved a certain amount of research. Currently, MSCs showed a better therapeutic effect in a variety of clinical trials of disease, such as myocardial infarction, corneal damage, nervous system damage, diabetes, acute graft-versus-host reaction, some lung dieases, liver fibrosis, bone and joint injuries, and Crohn's disease and other diseases. The study found that the MSCs have special biological characteristics, including:①MSCs as a "seed" cells can be planted in the damaged tissues and differentiate into tissue cells and play a role in tissue repair.②immunomodulatory effects (through the bypass/autocrine manner), play to the therapeutic effect by reducing the inflammatory response in the diseased tissue by inhibition of T lymphocytes and cytotoxic T lymphocytes and natural killer cells.③hematopoietic support function.④secretory ability of a variety of biologically active substances. Many of the clinical and laboratory studies have confirmed that MSCs cultured in vitro has some biological functions including immune regulation, hematopoietic support and the ability to repair a variety of tissue damage, which mostly dependent on the bioactive substances secreted by MSCs.
However, domestic and foreign studies on MSCs in recent years did not involve the stabilizing effect of MSCs transplantation on AS vulnerable plaque. Taking into account vulnerable plaque is a multi-site multi-system disease, and current treatment methods can not effectively play a role in prevention, and can not avoid the occurrence of vascular events. Comprehensive multiple mechanisms that MSCs involved in tissue repair, in this experiment, we verify the stabilizing role of MSCs on the vulnerable plaque.
Purpose
1. To culture and amplified rabbit MSCs in vitro and check the osteogenesis and lipogenesis ability, and specific surface markers. And select a appropriate method to labeled MSCs between lentiviral transfection and fluorescent dye staining. And to preliminary explore the effects of MSCs on ox-LDL-induced vascular endothelial cell apoptosis in vitro.
2. Based on the AS vulnerable plaque model, and MSCs' unique "homing" function, multi-differentiation potential and strong secretory function, make use of MSCs transplantation, to explore the effect and mechanism of MSCs on stability and repair of vulnerable plaque, serum and plaque-inflammatory cytokines and anti-inflammatory protein from the organizational level, the protein level and gene level, and to provide theoretical and experimental evidence for the clinical application of MSCs to combat acute coronary syndrome.
Methods and Results
Part Ⅰ:
Methods:MSCs was extracted from the bone marrow of New Zealand white rabbits, and the whole bone marrow adherence method were used to isolate and culture rabbit MSCs. P3generation cells were induced to osteoblasts and lipoblasts by culturing for21days in osteogenic medium and14days in adipogenic medium. And the specific surface markers of MSCs CD29, CD44, CD45were identified by flow cytometry. Lentiviral transfection and CFSE fluorescence dye staining were used to MSCs labeled, and select the appropriate one. In addition, establish human umbilical vein endothelial cells (hUVECs) injury model by Oxidized Low-density lipoprotein (ox-LDL) in vitro, then co-cultured MSCs and hUVECs, apoptosis rate was detected using the flow cytometry, tumor necrosis factor a (TNF-a) and vascular endothelial growth factor (VEGF) of cell culture medium were measured by ELISA, and rt-PCR was used to detect the expression of apoptosis-related genes bcl-2and Bax. The data was analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.
The results show that:
(1) The MSCs cultured and amplified in vitro is uniformity spindle cells with great proliferation ability. After21days'osteogenic induction, mineralized nodule formation is visible by alizarin red staining; and after14days'adipogenic induction, lipid droplets is visible by oil red O staining. The results show that rabbit MSCs was successfully cultured and amplified in vitro, which can differentiate into osteoblasts and lipoblasts under certain conditions. Flow cytometry results showed the3rd generation cells have specific surface markers of MSCs:above98%of cultured cells express surface markers CD29and CD44, but CD45, which illustrate the3rd generation of adherent cells are MSCs. All of these provide the cellular basis for subsequent experiments. Compared to lentiviral transfection to MSCs can reduce the proliferation of the cells, CFSE staining as a fast, convenient and safe method of dyeing, not only do not affect the proliferation of rabbit MSCs, but also not affecting its function of differentiation and secretion, is more suitable for labeling a large dose of transplanted cells.The efficiency of lentiviral transfected MSCs was up to80～90%, but it had a great impact on the proliferation of MSCs, the proliferation of transfected cells was significantly lower than the control group (F=2970.659, P<0.001). CFSE staining was a fast, convenient, and safe method for dyeing. A final concentration of10μmol/L and10minutes stained was ideal conditions for CFSE labeled MSCs, which not only do not affect the proliferation of MSCs (F=0.693, P=0.425), but aslo not affect its conversion function and secretion of VEGF (t=2.070, P=0.065). It's more suitable for large doses of transplanted cells using.
(2) The normal hUVECs have different forms, the round nucleus, rich and clear cytoplasm, near the nucleus of cells surrounding seen clearly Halo, forming a typical "cobblestone structure". After24h'100μg/ml ox-LDL, hUVECs began to appear obvious morphological changes, the cells were shrunken, cell gap increases, more particles and vacuoles were see in intracytoplasmic. Flow cytometry results showed that hUVECs apoptosis rate of the three groups were significantly different (F=105.229, P<0.001), ox-LDL group is significantly higher than that of control group (P=0.001); the MSCs co-culture group can significantly inhibited the ox-LDL-induced hUVECs damage, and apoptosis rate significantly reduced (P=0.008). VEGF and TNF-a content of three groups are significant differences (F=50.957, P<0.001and F=61.578, P<0.001). VEGF and TNF-a levels were significantly increased compared with the blank control group(P=0.001, P<0.001), VEGF in MSCs co-culture group supernatant was significantly higher than the control group and the ox-LDL group(P=0.001, P=0.008), and TNF-a level was significantly reduced compared with ox-LDL group (P=0.002), but still higher than the control group ((P=0.005). Bcl-2and Bax mRNA expression of three groups are significant different (F=13.381, P=0.002and F=14.633, P<0.001). Bcl-2mRNA expression in ox-LDL group had a significant reduction (P=0.041) and bax mRNA expression significant increased compared with the control group (P=0.001). Compared with ox-LDL group, MSCs co-culture group had increased bcl-2mRNA expression (P=0.004) and reduced bax mRNA expression (P<0.001). The Bcl-2/Bax ratios of three groups were significant difference (F=42.433, P<0.001). And comparing with the control group, the ratio of ox-LDL group was significantly decreased (P<0.001), MSCs co-culture group had no significant difference(P=0.269). Compared with ox-LDL group, the Bcl-2/Bax ratio of MSCs co-culture group increased, the difference was statistically significant (P=0.001).
Part II:
Methods:34healthy male New Zealand white rabbits were randomly divided into three groups, the MSC group (14) and (10) VP group rabbit first underwent a high fat diet for one week, and then cold-induced endothelial injury with liquid nitrogen, postoperative continued high fat diet fed for7weeks, and again in the8th week to2nd liquid nitrogen frostbite surgery, immediately following transplantation of1×107MSCs (approximately1ml) or lml PBS via ear vein, continue to change ordinary feed fed for four weeks. SP group (10) rabbit first underwent the high fat diet for one week, and then the liquid nitrogen frostbite surgery, postoperative were fed high-fat diet for7weeks and followed normal diet fed for four weeks. Before the experiment and after the high-fat diet3d,1w,2w to12w once every2weeks, triglyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL) levels of serum were detected. After6h and24h cell transplantation respectively, whole blood film was produced and MSCs changes in blood was observed under fluorescence microscope.3Days and2weeks after transplantation, two rabbit in the MSCs group were taken the right common carotid artery, cells' homing was observed under confocal microscope. The high sensitivity C reactive protein (hs-CRP), tumor necrosis factor a (TNF-a), interleukin6(IL-6), interleukin10(IL-10) and VEGF level in serum was determined respectively at Id,2d,3d,1w,2w,4w end after transplant by ELISA method. Rabbits were sacrificed4weeks after cell transplantation, the blood vessels were removed to make HE and Masson staining, measure cap nuclear ratio, and detected the nuclear factor κB (NF-κB), matrix metalloproteinase1,2,9(MMP-1, MMP-2, MMP-9) and tissue inhibitor of metalloproteinase1(TIMP-1) level in plaque using immunohistochemical study, the number of apoptotic cells in the plaque was measured by TUNEL. Rt-PCR was to test the level TSG6mRNA expression and Western blot was to test the level TSG6protein expression. The data were analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.
Results:
(1) After high fat diet for3days, TG, TC and LDL significantly increased (P=0.037, P=0.041,P=0.048), and increased fastest in one week, the first2to8W, lipids increased slowly. After replaced normal diet, rabbit serum lipid levels declined rapidly. All rabbits in MSC group and VP group had multiple yellow-white plaques in right carotid artery at8weeks, and the left common carotid artery had no obvious and visible plaque. And at12weeks, SP group showed localized thickening and fibrotic intima, and integrity morphology of the atherosclerotic plaque with thick fibrous cap, fewer inflammatory cells in the plaque, no plaque rupture. VP group showed a large lipid core in plaque center and a thinner fibrous cap, and residual foam cells and a large number of inflammatory cells (such as macrophages and lymphocytes) infiltrated in the plaque shoulder. In addition, rupture (or) thrombosis can be seen in some plaques. The MSC group showed a moderate morphology between VP and SP group. The endothelial cells and smooth muscle cells of left common carotid artery have a normal morphology and structure, with occasional mild intimal hyperplasia, a very small part of the intima showed a small amount of lipid deposition, but no prominent plaque formation. We can see more fluorescent cells in whole blood film under a fluorescence microscope after6hours transplantation, and after24hours, the fluorescent cells were significantly reduced. Fluorescence laser scanning confocal microscope showed after3days transplantation, more fluorescent cell aggregation can be found in the plaque intimal surface, but the cells decreased significantly after2weeks transplantation. And no fluorescent cell was found in the surface of left common carotid artery in the MSCs group. The Masson staining showed SP group plaque contains a large number of smooth muscle cells and elastic fibers, and followed by MSC group. Compared with MSC and VP group, the VP group plaque had decreased smooth muscle cells and elastic fibers, and disordered arrangement of collagen fibers. There are significant differences between the three groups of cap/core ratio (F=28.298, P<0.001). The plaque cap/nuclear ratio of MSC and SP group were significantly higher than the VP group (P<0.001).
(2) There were significant differences of serum hs-CRP in different times (F=21.670, P<0.001). The serum hs-CRP level in three groups were significant difference (F=428.245, P<0.001), and at each time point was P<0.001. Hs-CRP in VP group was significantly higher than MSC and SP group, MSC was significantly higher than SP group. Interaction between different times with different groups were significantly (F=11.857, P<0.001). There were significant differences of TNF-a in different times (F=8.056, P<0.001). The TNF-a level in three groups were significant difference (F=245.006, P<0.001), and at each time point was P<0.001. TNF-α in VP group was significantly higher than the MSC and SP group. Interaction between different times with different groups were significantly (F=9.362, P<0.001). There were significant differences of IL-6in different times (F=5.852, P<0.001); The IL-6level in three groups were significant differences (F=49.492, P=0.015), and at each time point was P<0.05. IL-6in VP group was significantly higher than the SP group; the VP group was significantly higher than the MSC group at D2, D3, W1and W2. Interaction between different times with different groups were significantly (F=1.951, P=0.044). There were significant differences of VEGF in different times (F=15.241, P<0.001); The VEGF level in three groups were significant differences (F=132.641, P<0.001), and all were P<0.001. While MSC group had no significant difference with VP group at D2, but in the rest of the time points the VEGF level of MSC group were significantly higher the VP and SP group. And VEGF of VP was significantly higher than SP group at W1, W2and W4. Interaction between different times with different groups were significantly (F=10.702, P<0.001).
(3) The content of NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1among the three groups were significant differences (F=44.953, P<0.001; F=46.530, P<0.001; F=34.292, P<0.001; F=27.108, P<0.001; F=63.138, P<0.001). The content of NF-κB, MMP-1, MMP-2, MMP-9in MSC group were significantly lower than that in the VP group, and the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001), and TIMP-1level was significantly higher than that in VP group(P<0.001). The NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1content in the plaques of SP group were significantly lower than VP group, the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001, P=0.002); the NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1expression in MSC group were also significantly higher than the SP group (P=0.005, P=0.018, P=0.046, P=0.005, P<0.001). There were significant differences of TSG6mRNA and protein expression among three groups (F=106.082, P<0.001; F=41.484, P<0.001), TSG6mRNA and protein expression in MSCs group were significant higher than the VP and SP groups (P=0.017, P=0.007; P<0.001, P<0.001); while TSG6expression in VP group increased significantly than SP group (P<0.001; P=0.008).
TUNEL results showed that apoptotic cells were seen in the plaques of three groups, and AIs of three groups were significant differences (F=96.153, P<0.001).AI of VP group was significantly higher than MSCs and SP group (P<0.001, P<0.001), and AI of MSC group was also significantly higher than SP group (P<0.001). The apoptotic cells were mainly distributed in the lipid nucleus and medial smooth muscle area in the plaques of MSC and VP group, and were mainly distributed in the area of medial smooth muscle of SP group, there were little of apoptotic cells in the minimal lipid core area and intimal of SP group.
The content of caspase-9among the three groups were significant differences (F=14.391, P=0.002), the caspase-9expression of VP group was significantly higher than those of MSCs and SP group (P<0.001, P<0.001). There were not statistically differences between MSC group and SP group(P=0.335).
Conclusion
1We successfully extracted and amplified a sufficient number and vigorous MSCs through whole bone marrow adherent culture method. And through a series of identifications in vitro, we proved the cells extracted were MSCs. Compared to the lentivirus transfection, the CFSE fluorescence staining has no effect on the biological activities, was more applicable to large doses of transplanted cells labeled. And we found that MSCs could reduce the level of inflammatory factors, increased the secretion of VEGF, and through a series of mechanisms to regulate the expression of apoptosis-related genes, thereby improving the ox-LDL-induced vascular endothelial cell apoptosis.
2Transplation of allogeneic bone marrow mesenchymal stem cells (MSCs) in vein can stabilize and repair the vulnerable plaque. MSCs in the body can "homing" to the vulnerable plaques, can reduce inflammatory factors and increase anti-inflammatory protein in serum and local plaque, thereby stabilizing the vulnerable plaque. The specific mechanisms may be that the transplanted MSCs can secrete TSG-6, which can inhibits macrophage releasing NF-κB, thereby inhibiting the release of a series of inflammatory cytokines in the signaling pathway. In addition, MSCs can inhibit local cells apoptosis in plaque, which may be associated with MSCs by suppressing inflammation reduce the damage and secreting growth factors such as VEGF to promote the repair and proliferation of local cells and inhibite mitochondrial apoptotic pathway.
当今社会,急性心脑血管事件(急性冠脉综合征与脑卒中)严重威胁着中老年人的健康与生命,是公认的健康头号威胁,有着极高的致死率和致残率。心血管疾病是发达国家致死的主因,同时正在快速成为发展中国家的头号杀手。随着社会的发展,人民生活方式的改变及老龄化社会的到来,每年有两千多万人死于动脉粥样硬化性心血管病,其中主要为冠心病,而且这个数字还在不断的递增中。动脉粥样硬化(Atherosclerosis,AS)是心脑血管疾病的共同病理基础,易损斑块的形成以及随之发生的血栓形成是致残、致死性血管疾病的真正元凶及最根本的始动因素,因此AS疾病的防治重点之一是易损斑块的稳定和修复。易于受损的斑块不只是易于破裂的斑块,所有可能有血栓性并发症和进展迅速的斑块都应视为易损斑块。而且易损斑块并非是造成急性冠脉综合症(Acute Coronary Syndrome,ACS)、心肌梗死和心脏猝死的惟一罪犯因子,易损血液(易于发生血栓)和易损心肌(易于发生致命性心律失常)对预后也起着重要作用。近年来,国际上倾向于用“易损患者”一词代替“易损斑块”,以便更好的评估患者发生急性心脑血管事件的危险性。由于AS是散在的、累及全身动脉血管的慢性疾病,有各种不同的局部和周身表现,临床上常表现为冠状动脉粥样硬化(ACS、心肌梗死)、脑血管硬化(脑梗塞、脑血管破裂)、颈动脉粥样硬化(脑供血不足、脑梗塞)、主动脉病变(主动脉夹层、主动脉瘤)、肾动脉粥样硬化(肾脏疾病、高血压)等。因此,对易损性负荷的评估不能仅限于单个不稳定斑块,而应包括总动脉粥样硬化负荷(主动脉和颈动脉、股动脉等)和冠脉的易损斑块,也要包括对血液和心肌易损性的综合危险评估(即易损指数)。
尽管目前已成功应用多种治疗手段防治或减少冠心病的发生发展,主要包括溶栓、降脂、抑炎等内科治疗措施、介入治疗及外科冠状动脉搭桥手术等。这些手段虽然发挥了一定的作用却颇为有限。对于冠脉内径狭窄>70%的ACS患者,介入手术仍将是治疗冠心病的主要技术之一。但是大量的研究表明,约70%-80%ACS形成是由于轻、中度狭窄的斑块破裂及继发血栓形成所致,并不适用介入治疗,其治疗重点应为稳定易损斑块和修复破裂斑块。由于AS的全身性特点,对局部某一易损斑块展开的治疗过于局部和片面,无法最大程度的避免急性心脑血管事件的发生。而针对急性心脑血管事件的易损患者,当下的治疗／预防趋势已倾向于综合评估总动脉粥样硬化负荷和冠脉的易损斑块,包括对患者易损血液、易损心肌的综合评估,从整体上预防急性心脑血管疾病的发生。因此,药物治疗应该是稳定易损斑块的基础。然而,目前仍然没有稳定易损斑块的有效药物。尽管在动物实验中,他汀类药物可明显的降低低密度脂蛋白胆固醇水平,抑制炎症反应,从而增加斑块纤维帽厚度。然而,他汀类的肝毒性使许多患者不能使用此类药物。
近年来,越来越多的医学科学研究者将目光投向了基于组织工程发展起来的细胞(或组织)替代治疗,这为人类征服大量疑难疾病提供了新的途径和希望。骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,MSCs),来源于中胚层,广泛存在于多种成体组织中,相对其它干细胞具有低免疫原性、多分化性、体外易培养扩增及独特的归巢功能等多方面的优点。MSCs可分化为造血相关细胞,也可分化为多种造血以外的细胞,尤其是中胚层和神经外胚层来源的细胞,近年,MSCs在临床中具有广泛的应用前景。目前已有大量实验将MSCs移植用于多种疾病的治疗,并取得了一定的研究成果。目前,在心肌梗死、角膜损伤、神经系统损伤、糖尿病、急性移植物抗宿主反应、肺部多种疾病、肝脏纤维化,骨关节损伤和克罗恩病等多种疾病的临床试验中,MSCs均表现出较好的治疗效果。研究发现,MSCs具有其特殊的生物学特征,包括：①MSCs作为“种子”细胞可定植于受损的组织并可分化成为组织细胞而发挥组织修复作用。②免疫调节作用(可通过旁/自分泌方式),抑制T淋巴细胞、抑制细胞毒性T淋巴细胞和自然杀伤细胞等而起到减轻病变组织内免疫炎性反应的治疗作用。③造血支持功能。④多种生物活性物质的分泌能力。诸多的临床与实验室研究已经证实,体外培养的间充质干细胞具有免疫调控、造血支持和修复多种组织损伤的能力,而这些功能多依赖于间充质干细胞分泌的生物活性物质。
在近些年国内外对MSCs的研究中,并未涉及MSCs移植对机体AS易损斑块的稳定作用。考虑到易损斑块属于多部位多系统疾病,当下有效的治疗方法不能有效发挥“防患于未然”的作用,也无法最大程度的避免血管事件的发生。综合MSCs参与组织修复的多种机制,在本实验中我们对MSCs对易损斑块的稳定作用进行了研究。
目的
1体外扩增培养兔MSCs并对其进行成骨成脂及特异性表面标志物鉴定,并在慢病毒转染及荧光染色剂染色之间选择合适的标记方式对其进行标记,并初步探讨其在体外对氧化型低密度脂蛋白(Oxidized Low-density lipoprotein, ox-LDL)所致血管内皮细胞凋亡的影响。
2兔AS易损斑块模型基础上,利用MSCs独特的“归巢”功能、多向分化潜能及强大的旁分泌功能,通过骨髓间充质干细胞移植,从组织水平、蛋白水平和基因水平观察骨髓干细胞对AS易损斑块稳定性、血清及斑块炎性因子、抑炎蛋白表达及斑块内细胞凋亡的影响,探索MSCs稳定与修复易损斑块的机制,为临床应用MSCs防治急性心脑血管疾病提供理论和实验依据。
方法与结果
第一部分：方法：采用全骨髓贴壁法从新西兰大白兔骨髓中提取、分离和培养兔MSCs。取P3代细胞分别采用成骨、成脂诱导液分别培养21天、14天使诱导其成骨、成脂,并使用流式细胞仪鉴定MSCs的特异性表面标志物CD29,CD44,CD45。分别采用慢病毒转染及二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein-diacetic succinimidyl ester,CFSE)荧光染料染色的方法对MSCs进行标记,并选取合适的标记方法。另外以ox-LDL体外诱导方式建立人脐静内皮细胞(human umbilical vein endothelial cell,hUVECs)损伤模型,采用细胞共培养的方式共同培养MSCs和hUVECs,采用流式细胞仪检测hUVECs凋亡率,ELISA法检测细胞培养液中肿瘤坏死因子a(Tumor necrosis factor-a,TNF-a)和血管内皮生长因子(vascular endothelial growth factor,VEGF)、rt-PCR方法检测凋亡相关基因bcl-2和Bax的表达。采用SPSS13.0统计学软件分析数据。定量数据以(x±s)表示,两样本比较采用独立样本t检验,多组间比较方差齐时采用one-way ANOVA,方差不齐时采用Welch F方法,重复测量数据采用重复测量数据方差分析,P<0.05为差异有统计学意义。
结果表明：(1)体外培养扩增的MSCs为均一性好,增殖能力强的梭型细胞。成骨诱导21天,经茜素红染色可见矿化结节形成；成脂诱导14天,经油红O染色可见脂滴形成。结果表明体外成功培养和扩增了兔MSCs,在特定条件下能够向成骨细胞分化。经流式细胞仪检测,结果显示第3代MSCs的特异性表面标志物：CD29阳性,阳性率为98.0%,CD44阳性,阳性率为98.5%,CD45阴性,阳性率为0.7%。提示第3代贴壁细胞主要是MSCs,为以后的实验提供了细胞基础。慢病毒转染MSCs效率可达80～90%,但是慢病毒转染对MSCs增殖能力有极大的影响,转染组细胞增殖明显低于对照组(F=2970.659,P<0.001)。而CFSE荧光染色作为一种快速、方便且安全的染色方法,终浓度为10μmol/L+染色10分钟为CFSE标记MSCs的理想条件,其不但不影响MSCs的增殖能力(F=0.693,P=0.425),同时也不影响其转化及分泌VEGF的功能(t=2.070,P=0.065),更适用于大剂量移植细胞时采用。
(2)正常hUVECs形态不一,胞核较圆,细胞质丰富且清亮,细胞周围近胞核处可见明显光晕,形成典型的“铺路石样”结构。100μg/ml的ox-LDL作用24h后hUVECs开始出现明显的形态学变化,细胞明显皱缩,细胞间隙增大,胞浆内见较多颗粒、空泡。流式细胞检测发现三组hUVECs凋亡率存在显著差异(F=105.229,P<0.001),ox-LDL组凋亡率显著高于正常对照组(P=0.001)；而MSCs共培养组能显著抑制ox-LDL对hUVECs的损伤,细胞凋亡率显著性降低(P=0.008)。三组VEGF及TNF-α的含量均存在显著性差异(F=50.957,P<0.001；和F=61.578,P<0.001),ox-LDL组VEGF及TNF-α的分泌量均高于空白对照组,差异有显著性意义(P=0.001,P<0.001)；MSCs共培养组上清液中VEGF显著高于对照组和ox-LDL组(P=0.001,P=0.008),而TNF-α分泌量较ox-LDL组显著下降(P=0.002),但仍高于空白对照组,差异有统计学意义(P=0.005)。三组bcl-2和bax mRNA表达存在显著性差异(F=13.381,P=0.002；和F=14.633,P<0.001)。与对照组比较,ox-LDL组bcl-2mRNA表达显著降低(P=0.041)bax mRNA表达显著上调(P=0.001),MSCs共培养组bcl-2mRNA表达显著增加(P=0.044)。与ox-LDL组比较,MSCs共培养组细胞bcl-2mRNA表达显著上调(P=0.004),bax mRNA表达亦显著降低(P<0.001)。三组Bcl-2/Bax比值也有显著性差异(F=42.433,P<0.001),与对照组比较,ox-LDL组Bcl-2/Bax比值显著性降低(P<0.001),MSCs共培养组无显著性差异(P=0.269)；与ox-LDL组比较,MSCs共培养组Bcl-2/Bax比值增加,差异有显著性意义(P=0.001)
第二部分：
方法：34只健康纯种雄性新西兰白兔,随机分为3组,MSCs移植组(MSC组,14只)和易损斑块模型组(VP组,10只)兔先给予高脂饲料喂养1周,然后实施右颈总动脉液氮冻伤术,术后继续高脂饲料喂养7周,并于第8周末再次实施第2次右颈总动脉内膜液氮冻伤术,术后即刻通过耳缘静脉注射1×107个MSCs(1m1)或1mlPBS,改普通饲料继续喂养4周。稳定斑块模型组(SP组,10只)兔先给予高脂饲料喂养1周,然后实施右颈总动脉液氮冻伤术,术后继续高脂饲料喂养7周。改普通饲料继续喂养4周。分别于实验前及高脂饲料喂养后3d、1w、第2w至第12w每2周采血一次检测检测甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL)水平。分别于细胞移植后6h及24h制作全血涂片于荧光显微镜下观察血液内MSCs变化,移植后3d及2w取MSCs组两只兔子,取右颈总动脉于共聚焦显微镜下观察细胞归巢情况；分别于移植后1d、2d、3d、1w、2w、4w末空腹抽取兔静脉血,离心取上清液,ELISA法测定血清高敏C反应蛋白(hs-CRP)、TNF-α、白介素6(IL-6)、白介素10(IL-10)及VEGF水平。细胞移植后4周末处死兔子取出血管,HE染色及Masson三色染色,并行帽／核比值测量,光镜观察斑块修复情况；应用核因子κB (NF-κB)、基质金属蛋白酶1、2、9(MMP-1、MMP-2、MMP-9)以及基质金属蛋白酶抑制因子1(TIMP-1)的单克隆抗体对实验血管进行免疫组织化学染色,以明确斑块处各炎性因子蛋白表达情况；应用rt-PCR和免疫印迹(Western Blot)方法对实验血管处的TSG-6含量进行检测；应用TUNEL法检测各组斑块内凋亡细胞数量,免疫组化检测caspase-9表达水平。
结果：(1)高脂饲料喂养3天后TG、TC及LDL均出现明显增高(P=0.037,P=0.041,P=0.048),第1周内升高幅度最大,第2-8w血脂缓慢增高。8周后更换为普通饲料喂养后,兔血脂水平迅速下降。8周后,肉眼可见MSC组及VP组兔右颈总动脉有多处黄白色斑块形成,而左侧颈总动脉未见明显斑块形成；12周后光镜下见,SP组兔右颈总动脉内膜显著增厚并纤维化,血管壁向管腔内突出,粥样硬化斑块形态结构完整,纤维帽较厚,斑块内炎细胞较少,未见斑块破裂。VP组斑块中心可见大量脂核,斑块表面覆盖较薄纤维帽,在斑块肩部可见残存泡沫细胞和大量炎细胞(如巨噬细胞和淋巴细胞等)浸润,部分斑块可见破裂和(或)血栓形成。MSC组形态介于之间。左侧颈总动脉内皮细胞及平滑肌细胞排列整齐,呈长型或椭圆型,血管内膜偶有轻度增生,极少部分内膜可见少量脂质沉积,但无突出斑块形成。荧光显微镜下全血涂片显示移植6h后仍可在血液内发现较多荧光细胞,24h后荧光细胞含量明显减少；激光共聚焦显微镜下显示移植3d后可在斑块内膜表面发现较多荧光细胞聚集,2w时荧光细胞明显减少。而在MSCs组兔子左侧颈总动脉表面,未见荧光细胞聚集。Masson染色可见SP组斑块内含有大量的平滑肌细胞和弹力纤维,MSC组斑块内平滑肌细胞、弹力纤维和胶原纤维含量次之。与MSC组和SP组相比,VP组斑块内平滑肌细胞、弹力纤维量明显减少,胶原纤维排列紊乱。三组帽/核比值间有显著性差异(F=28.298,P<0.001),MSC组及SP组斑块帽/核比值均显著性高于VP组(P<0.001)。
(2)不同时间血清hs-CRP有显著差异(F=21.670,P<0.001)；三组血清hs-CRP水平有显著性差异(F=428.245,P<0.001),且各个时间点均为P<0.001,VP组hs-CRP显著高于MSC、SP组,而MSC显著高于SP组。不同时间与不同组别之间存在交互效应(F=11.857,P<0.001)。不同时间TNF-a有显著差异(F=8.056,P<0.001);三组TNF-a水平有显著性差异(F=245.006,P<0.001),且各个时间点均为P<0.001,VP组TNF-a显著高于MSC、SP组。不同时间与不同组别之间存在交互效应(F=9.362,P<0.001)。不同时间IL-6有显著差异(F=5.852,P<0.001)；三组IL-6有显著性差异(F=49.492,P=0.015),且各个时间点均为P<0.05,VP组IL-6显著高于SP组；D2、D3、W1、W2时VP组显著高于MSC组。不同时间与不同组别之间存在交互效应(F=1.951,P=0.044)。不同时间之间血清IL-10有显著差异(F=28.241,P<0.001)；三组血清IL-10有显著性差异(F=244.606,P<0.001),三组比较除D1时P=0.006外均为P<0.001,MSC组IL-10显著高于VP、SP组,而在D3、W1、W2、W4时VP组显著高于SP组。不同时间与不同组别之间存在交互效应(F=12.684,P<0.001)。不同时间之间血清VEGF有显著差异(F=15.241,P<0.001)；三组之间血清VEGF水平有显著性差异(F=132.641,P<0.001),且各个时间点均有显著性差异,均为P<0.001。除D2时MSC组与VP组无显著差异,其余时间点MSC组VEGF均显著高于VP组及SP组,而在W1、W2、W4,VP组VEGF显著高于SP组。不同时间与不同组别之问存在交互效应(F=10.702,P<0.001)。
(3)三组间NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1含量有显著性差异(F=44.953,P<0.001；F=46.530,P<0.001；F=34.292,P<0.001；F=27.108,P<0.001;F=63.138,P<0.001)。MSC组NF-κB、MMP-1、MMP-2、MMP-9含量显著低于VP组,差异有统计学意义(P<0.001；P<0.001；P<0.001；P<0.001),TIMP-1显著高于VP组(P<0.001)；MSC组NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1表达显著高于SP组(P=0.005；P=0.018；P=0.046；P=0.005；P<0.001)；SP组斑块内NF-κB、MMP-1、MMP-2、MMP-9及TIMP-1含量明显低于VP组,差异有统计学意义(P<0.001；P<0.001；P<0.001；P<0.001；P=0.002)。Real-time PCR检测结果显示,三组TSG6mRNA表达有显著性差异(F=106.082,P<0.001),MSCs组TSG6mRNA表达较VP组和SP显著增加(P=0.017,P=0.007)；而VP组TSG6mRNA表达较SP组显著增加(P<0.001)。WesternBlot检测结果显示,三组之间TSG6蛋白表达有显著性差异(F=41.484,P<0.001),其中MSCs组TSG6mRNA表达较VP组和SP显著增加(P<0.001,P<0.001);而VP组TSG6mRNA表达较SP组显著增加(P=0.008)。
TUNEL法示三组斑块内均可见凋亡细胞,三组AI值有显著性差异(F=96.153,P<0.001),VP组显著高于MSCs组及SP组(P<0.001,P<0.001),MSC组AI也显著高于SP组(P<0.001)。MSC组及VP组凋亡细胞主要分布于脂核区及中膜平滑肌区。SP组主要分布于中膜平滑肌区,脂核区及内膜极少凋亡细胞。免疫组化结果显示三组斑块内均可见caspase-9表达。MSCs组、VP组和SP组caspase-9表达水平有显著性差异(F=14.391,P=0.002)；进一步两两比较发现,VP组斑块内caspase-9表达水平显著高于MSCs组及SP组(P<0.001,P<0.001),MSC组AI与SP组之间差异不明显(P=0.335)。
结论
1通过全骨髓贴壁培养法,我们成功提取并扩增出足够数量且活力较好的MSCs,且经多项体外鉴定,证明提取的该种细胞为MSCs。相较于慢病毒转染,CFSE荧光染色对细胞生物学活性无影响,更适用于大剂量的移植细胞标记。且发现MSCs可通过降低炎性因子水平、增加VEGF分泌,并通过一系列机制调节凋亡相关基因的表达,进而改善ox-LDL所致血管内皮细胞凋亡。
2应用同种异体MSCs静脉移植可稳定和修复易损斑块。MSCs进入机体.后能向易损斑块区“归巢”,并通过分泌抗炎蛋白TSG-6抑制巨噬细胞释放NF-κB,进而抑制该信号通路上一系列炎性因子的释放,进而降低血清及斑块局部炎性反应发挥稳定斑块的作用。此外,MSCs还可以抑制斑块局部的细胞凋亡,这可能与MSCs通过分泌VEGF等生长因子抑制线粒体凋亡途径并促进局部细胞组织增殖修复有关。
Background
At present, acute cardiovascular and cerebrovascular events (acute coronary syndrome and stroke) is a serious threat to the health and lives of the elderly, which has high morbidity and mortality. Cardiovascular disease is the main cause of death in developed countries, and is fast becoming the number one killer in developing countries. With the development of society, people's lifestyles change and the arrival of the aging society, more than two thousand people died of atherosclerotic cardiovascular disease every year, mainly for coronary heart disease, and that number continues to increment in. Atherosclerosis (atherosclerosis, AS) is a common pathological basis of cardiovascular and cerebrovascular disease, and the formation of vulnerable plaque and the ensuing thrombosis is the real culprit and the most fundamental initiating factor of the fatal vascular disease, therefore the prevention and treatment of AS disease should be focused on stabilizing and repairing vulnerable plaque. Vulnerable plaque is not only the plaque easy to rupture, all ones are likely to have thrombotic complications and rapid progress should be considered vulnerable plaque. And vulnerable plaque is not the only crime factor for acute coronary syndrome (ACS), myocardial infarction (MI) and sudden cardiac death, the vulnerable blood (easy occurrence of thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) also play an important role on prognosis of these diseases. In recent years, the international community prefers to use the term "vulnerable patients" instead of "vulnerable plaque" in order to better assess the risk of acute cardiovascular and cerebrovascular events in patients. Atherosclerosis is scattered, systemic arteries involved chronic disease, which have various clinical manifestation, usually presents clinical coronary atherosclerosis (ACS, MI), cerebral arteriosclerosis (cerebral infarction, rupture of cerebral blood vessels), carotid atherosclerosis (cerebral insufficiency, cerebral infarction), aortic lesions (aortic dissection, aortic aneurysm), renal artery atherosclerosis (kidney disease, hypertension) et al. Therefore, the assessment of the vulnerability of the plaque load should not be limited to a single unstable plaque, but should include the total atherosclerotic plaque load (aortic and carotid and femoral artery) and coronary vulnerable plaques, also should include blood and myocardial vulnerability risk assessment (vulnerability index).
Although there have been a variety of treatment applied successfully to control or reduce the occurrence and development of coronary heart disease, mainly including medical treatment measures such as dissolving blood clots, lipid-lowering, suppression the inflammatory, intervention treatment and surgical coronary artery bypass surgery. The treatment plays a certain role but is extremely limited. Surgical intervention will continue to be one of the main technical therapies for ACS patients with more than70%coronary stenosis. However, a large number of studies showed that the formation of about70%-80%ACS is caused by rupture and secondary thrombosis of mild to moderate stenosis plaque, which is not suitable to adopt PCI, its treatment should focus on stabilize and repair vulnerable plaques. Because of the systemic and multi-distribution characteristics of atherosclerosis and vulnerable plaque, and the vulnerability characteristics of the blood, the treatment for a local vulnerable plaque is too partial and one-sided, and can not avoid acute cardio-cerebral vascular events occurred in a greatest degree. Current treatment/prevention trends for acute cardiovascular and cerebrovascular events tend to consolidated total atherosclerotic load and coronary vulnerable plaques and vulnerable patients, including a comprehensive assessment of the fragile blood and vulnerable myocardial of the patients, to prevent the occurrence of acute cardiovascular and cerebrovascular disease. Thus, the drug treatment should be the basis of stabilization of vulnerable plaque. However, there is still no effective drugs to stabilize vulnerable plaque. In animal experiments, statins can significantly reduce the level of LDL-c, inhibition of the inflammatory response, thereby increasing the thickness of the fibrous cap. However, liver toxicity of statins makes so many patients can not use such drugs.
Cells (or organization) replacement therapy and gene therapy technology developed based on tissue engineering and gene engineering in recent years, and become the research focus and cutting-edge in the field of medicine and even in the entire field of life sciences, and provide a new way and hope for conquering many diseases. Bone marrow mesenchymal stem cells(MSCs), is a kind of the early cells from mesoderm, and widely exist in a variety of adult tissues. MSCs have a wide range of advantages relative to other stem cells, such as low immunogenicity, multi-differentiation characteristic, easy to cultivate and amplification in vitro and unique homing feature. Not only can MSCs differentiate into the hematopoietic and stromal cells, but also can differentiate into a variety of other organization, especially the organizations cells from mesoderm and neuroectoderm. In recent years, MSCs has a broad application prospects in clinical practice. There are a large number of experimental to check the transplantation of MSCs for the treatment of a variety of diseases, and have achieved a certain amount of research. Currently, MSCs showed a better therapeutic effect in a variety of clinical trials of disease, such as myocardial infarction, corneal damage, nervous system damage, diabetes, acute graft-versus-host reaction, some lung dieases, liver fibrosis, bone and joint injuries, and Crohn's disease and other diseases. The study found that the MSCs have special biological characteristics, including:①MSCs as a "seed" cells can be planted in the damaged tissues and differentiate into tissue cells and play a role in tissue repair.②immunomodulatory effects (through the bypass/autocrine manner), play to the therapeutic effect by reducing the inflammatory response in the diseased tissue by inhibition of T lymphocytes and cytotoxic T lymphocytes and natural killer cells.③hematopoietic support function.④secretory ability of a variety of biologically active substances. Many of the clinical and laboratory studies have confirmed that MSCs cultured in vitro has some biological functions including immune regulation, hematopoietic support and the ability to repair a variety of tissue damage, which mostly dependent on the bioactive substances secreted by MSCs.
However, domestic and foreign studies on MSCs in recent years did not involve the stabilizing effect of MSCs transplantation on AS vulnerable plaque. Taking into account vulnerable plaque is a multi-site multi-system disease, and current treatment methods can not effectively play a role in prevention, and can not avoid the occurrence of vascular events. Comprehensive multiple mechanisms that MSCs involved in tissue repair, in this experiment, we verify the stabilizing role of MSCs on the vulnerable plaque.
Purpose
1. To culture and amplified rabbit MSCs in vitro and check the osteogenesis and lipogenesis ability, and specific surface markers. And select a appropriate method to labeled MSCs between lentiviral transfection and fluorescent dye staining. And to preliminary explore the effects of MSCs on ox-LDL-induced vascular endothelial cell apoptosis in vitro.
2. Based on the AS vulnerable plaque model, and MSCs' unique "homing" function, multi-differentiation potential and strong secretory function, make use of MSCs transplantation, to explore the effect and mechanism of MSCs on stability and repair of vulnerable plaque, serum and plaque-inflammatory cytokines and anti-inflammatory protein from the organizational level, the protein level and gene level, and to provide theoretical and experimental evidence for the clinical application of MSCs to combat acute coronary syndrome.
Methods and Results
Part Ⅰ:
Methods:MSCs was extracted from the bone marrow of New Zealand white rabbits, and the whole bone marrow adherence method were used to isolate and culture rabbit MSCs. P3generation cells were induced to osteoblasts and lipoblasts by culturing for21days in osteogenic medium and14days in adipogenic medium. And the specific surface markers of MSCs CD29, CD44, CD45were identified by flow cytometry. Lentiviral transfection and CFSE fluorescence dye staining were used to MSCs labeled, and select the appropriate one. In addition, establish human umbilical vein endothelial cells (hUVECs) injury model by Oxidized Low-density lipoprotein (ox-LDL) in vitro, then co-cultured MSCs and hUVECs, apoptosis rate was detected using the flow cytometry, tumor necrosis factor a (TNF-a) and vascular endothelial growth factor (VEGF) of cell culture medium were measured by ELISA, and rt-PCR was used to detect the expression of apoptosis-related genes bcl-2and Bax. The data was analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.
The results show that:
(1) The MSCs cultured and amplified in vitro is uniformity spindle cells with great proliferation ability. After21days'osteogenic induction, mineralized nodule formation is visible by alizarin red staining; and after14days'adipogenic induction, lipid droplets is visible by oil red O staining. The results show that rabbit MSCs was successfully cultured and amplified in vitro, which can differentiate into osteoblasts and lipoblasts under certain conditions. Flow cytometry results showed the3rd generation cells have specific surface markers of MSCs:above98%of cultured cells express surface markers CD29and CD44, but CD45, which illustrate the3rd generation of adherent cells are MSCs. All of these provide the cellular basis for subsequent experiments. Compared to lentiviral transfection to MSCs can reduce the proliferation of the cells, CFSE staining as a fast, convenient and safe method of dyeing, not only do not affect the proliferation of rabbit MSCs, but also not affecting its function of differentiation and secretion, is more suitable for labeling a large dose of transplanted cells.The efficiency of lentiviral transfected MSCs was up to80～90%, but it had a great impact on the proliferation of MSCs, the proliferation of transfected cells was significantly lower than the control group (F=2970.659, P<0.001). CFSE staining was a fast, convenient, and safe method for dyeing. A final concentration of10μmol/L and10minutes stained was ideal conditions for CFSE labeled MSCs, which not only do not affect the proliferation of MSCs (F=0.693, P=0.425), but aslo not affect its conversion function and secretion of VEGF (t=2.070, P=0.065). It's more suitable for large doses of transplanted cells using.
(2) The normal hUVECs have different forms, the round nucleus, rich and clear cytoplasm, near the nucleus of cells surrounding seen clearly Halo, forming a typical "cobblestone structure". After24h'100μg/ml ox-LDL, hUVECs began to appear obvious morphological changes, the cells were shrunken, cell gap increases, more particles and vacuoles were see in intracytoplasmic. Flow cytometry results showed that hUVECs apoptosis rate of the three groups were significantly different (F=105.229, P<0.001), ox-LDL group is significantly higher than that of control group (P=0.001); the MSCs co-culture group can significantly inhibited the ox-LDL-induced hUVECs damage, and apoptosis rate significantly reduced (P=0.008). VEGF and TNF-a content of three groups are significant differences (F=50.957, P<0.001and F=61.578, P<0.001). VEGF and TNF-a levels were significantly increased compared with the blank control group(P=0.001, P<0.001), VEGF in MSCs co-culture group supernatant was significantly higher than the control group and the ox-LDL group(P=0.001, P=0.008), and TNF-a level was significantly reduced compared with ox-LDL group (P=0.002), but still higher than the control group ((P=0.005). Bcl-2and Bax mRNA expression of three groups are significant different (F=13.381, P=0.002and F=14.633, P<0.001). Bcl-2mRNA expression in ox-LDL group had a significant reduction (P=0.041) and bax mRNA expression significant increased compared with the control group (P=0.001). Compared with ox-LDL group, MSCs co-culture group had increased bcl-2mRNA expression (P=0.004) and reduced bax mRNA expression (P<0.001). The Bcl-2/Bax ratios of three groups were significant difference (F=42.433, P<0.001). And comparing with the control group, the ratio of ox-LDL group was significantly decreased (P<0.001), MSCs co-culture group had no significant difference(P=0.269). Compared with ox-LDL group, the Bcl-2/Bax ratio of MSCs co-culture group increased, the difference was statistically significant (P=0.001).
Part II:
Methods:34healthy male New Zealand white rabbits were randomly divided into three groups, the MSC group (14) and (10) VP group rabbit first underwent a high fat diet for one week, and then cold-induced endothelial injury with liquid nitrogen, postoperative continued high fat diet fed for7weeks, and again in the8th week to2nd liquid nitrogen frostbite surgery, immediately following transplantation of1×107MSCs (approximately1ml) or lml PBS via ear vein, continue to change ordinary feed fed for four weeks. SP group (10) rabbit first underwent the high fat diet for one week, and then the liquid nitrogen frostbite surgery, postoperative were fed high-fat diet for7weeks and followed normal diet fed for four weeks. Before the experiment and after the high-fat diet3d,1w,2w to12w once every2weeks, triglyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL) levels of serum were detected. After6h and24h cell transplantation respectively, whole blood film was produced and MSCs changes in blood was observed under fluorescence microscope.3Days and2weeks after transplantation, two rabbit in the MSCs group were taken the right common carotid artery, cells' homing was observed under confocal microscope. The high sensitivity C reactive protein (hs-CRP), tumor necrosis factor a (TNF-a), interleukin6(IL-6), interleukin10(IL-10) and VEGF level in serum was determined respectively at Id,2d,3d,1w,2w,4w end after transplant by ELISA method. Rabbits were sacrificed4weeks after cell transplantation, the blood vessels were removed to make HE and Masson staining, measure cap nuclear ratio, and detected the nuclear factor κB (NF-κB), matrix metalloproteinase1,2,9(MMP-1, MMP-2, MMP-9) and tissue inhibitor of metalloproteinase1(TIMP-1) level in plaque using immunohistochemical study, the number of apoptotic cells in the plaque was measured by TUNEL. Rt-PCR was to test the level TSG6mRNA expression and Western blot was to test the level TSG6protein expression. The data were analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.
Results:
(1) After high fat diet for3days, TG, TC and LDL significantly increased (P=0.037, P=0.041,P=0.048), and increased fastest in one week, the first2to8W, lipids increased slowly. After replaced normal diet, rabbit serum lipid levels declined rapidly. All rabbits in MSC group and VP group had multiple yellow-white plaques in right carotid artery at8weeks, and the left common carotid artery had no obvious and visible plaque. And at12weeks, SP group showed localized thickening and fibrotic intima, and integrity morphology of the atherosclerotic plaque with thick fibrous cap, fewer inflammatory cells in the plaque, no plaque rupture. VP group showed a large lipid core in plaque center and a thinner fibrous cap, and residual foam cells and a large number of inflammatory cells (such as macrophages and lymphocytes) infiltrated in the plaque shoulder. In addition, rupture (or) thrombosis can be seen in some plaques. The MSC group showed a moderate morphology between VP and SP group. The endothelial cells and smooth muscle cells of left common carotid artery have a normal morphology and structure, with occasional mild intimal hyperplasia, a very small part of the intima showed a small amount of lipid deposition, but no prominent plaque formation. We can see more fluorescent cells in whole blood film under a fluorescence microscope after6hours transplantation, and after24hours, the fluorescent cells were significantly reduced. Fluorescence laser scanning confocal microscope showed after3days transplantation, more fluorescent cell aggregation can be found in the plaque intimal surface, but the cells decreased significantly after2weeks transplantation. And no fluorescent cell was found in the surface of left common carotid artery in the MSCs group. The Masson staining showed SP group plaque contains a large number of smooth muscle cells and elastic fibers, and followed by MSC group. Compared with MSC and VP group, the VP group plaque had decreased smooth muscle cells and elastic fibers, and disordered arrangement of collagen fibers. There are significant differences between the three groups of cap/core ratio (F=28.298, P<0.001). The plaque cap/nuclear ratio of MSC and SP group were significantly higher than the VP group (P<0.001).
(2) There were significant differences of serum hs-CRP in different times (F=21.670, P<0.001). The serum hs-CRP level in three groups were significant difference (F=428.245, P<0.001), and at each time point was P<0.001. Hs-CRP in VP group was significantly higher than MSC and SP group, MSC was significantly higher than SP group. Interaction between different times with different groups were significantly (F=11.857, P<0.001). There were significant differences of TNF-a in different times (F=8.056, P<0.001). The TNF-a level in three groups were significant difference (F=245.006, P<0.001), and at each time point was P<0.001. TNF-α in VP group was significantly higher than the MSC and SP group. Interaction between different times with different groups were significantly (F=9.362, P<0.001). There were significant differences of IL-6in different times (F=5.852, P<0.001); The IL-6level in three groups were significant differences (F=49.492, P=0.015), and at each time point was P<0.05. IL-6in VP group was significantly higher than the SP group; the VP group was significantly higher than the MSC group at D2, D3, W1and W2. Interaction between different times with different groups were significantly (F=1.951, P=0.044). There were significant differences of VEGF in different times (F=15.241, P<0.001); The VEGF level in three groups were significant differences (F=132.641, P<0.001), and all were P<0.001. While MSC group had no significant difference with VP group at D2, but in the rest of the time points the VEGF level of MSC group were significantly higher the VP and SP group. And VEGF of VP was significantly higher than SP group at W1, W2and W4. Interaction between different times with different groups were significantly (F=10.702, P<0.001).
(3) The content of NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1among the three groups were significant differences (F=44.953, P<0.001; F=46.530, P<0.001; F=34.292, P<0.001; F=27.108, P<0.001; F=63.138, P<0.001). The content of NF-κB, MMP-1, MMP-2, MMP-9in MSC group were significantly lower than that in the VP group, and the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001), and TIMP-1level was significantly higher than that in VP group(P<0.001). The NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1content in the plaques of SP group were significantly lower than VP group, the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001, P=0.002); the NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1expression in MSC group were also significantly higher than the SP group (P=0.005, P=0.018, P=0.046, P=0.005, P<0.001). There were significant differences of TSG6mRNA and protein expression among three groups (F=106.082, P<0.001; F=41.484, P<0.001), TSG6mRNA and protein expression in MSCs group were significant higher than the VP and SP groups (P=0.017, P=0.007; P<0.001, P<0.001); while TSG6expression in VP group increased significantly than SP group (P<0.001; P=0.008).
TUNEL results showed that apoptotic cells were seen in the plaques of three groups, and AIs of three groups were significant differences (F=96.153, P<0.001).AI of VP group was significantly higher than MSCs and SP group (P<0.001, P<0.001), and AI of MSC group was also significantly higher than SP group (P<0.001). The apoptotic cells were mainly distributed in the lipid nucleus and medial smooth muscle area in the plaques of MSC and VP group, and were mainly distributed in the area of medial smooth muscle of SP group, there were little of apoptotic cells in the minimal lipid core area and intimal of SP group.
The content of caspase-9among the three groups were significant differences (F=14.391, P=0.002), the caspase-9expression of VP group was significantly higher than those of MSCs and SP group (P<0.001, P<0.001). There were not statistically differences between MSC group and SP group(P=0.335).
Conclusion
1We successfully extracted and amplified a sufficient number and vigorous MSCs through whole bone marrow adherent culture method. And through a series of identifications in vitro, we proved the cells extracted were MSCs. Compared to the lentivirus transfection, the CFSE fluorescence staining has no effect on the biological activities, was more applicable to large doses of transplanted cells labeled. And we found that MSCs could reduce the level of inflammatory factors, increased the secretion of VEGF, and through a series of mechanisms to regulate the expression of apoptosis-related genes, thereby improving the ox-LDL-induced vascular endothelial cell apoptosis.
2Transplation of allogeneic bone marrow mesenchymal stem cells (MSCs) in vein can stabilize and repair the vulnerable plaque. MSCs in the body can "homing" to the vulnerable plaques, can reduce inflammatory factors and increase anti-inflammatory protein in serum and local plaque, thereby stabilizing the vulnerable plaque. The specific mechanisms may be that the transplanted MSCs can secrete TSG-6, which can inhibits macrophage releasing NF-κB, thereby inhibiting the release of a series of inflammatory cytokines in the signaling pathway. In addition, MSCs can inhibit local cells apoptosis in plaque, which may be associated with MSCs by suppressing inflammation reduce the damage and secreting growth factors such as VEGF to promote the repair and proliferation of local cells and inhibite mitochondrial apoptotic pathway.
引文
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