体外不同诱导条件对骨髓间充质干细胞分化为心肌细胞的实验研究

发布时间：2018-04-14 04:22

本文选题：骨髓间充质干细胞 + 心肌细胞　；参考：《广西医科大学》2010年硕士论文

【摘要】： 目的探讨体外大鼠骨髓间充质干细胞(marrow mesenchyma stem cells, MSCs)和心肌细胞(cardiomyocytes, CM)的分离、培养及5-氮胞苷(5-Aza)和共培养两种方法对MSCs诱导分化为CM的作用；不同方法诱导MSCs分化后,MSCs在不同时间段分化为CM的能力及表达心肌细胞特异性标记物缝隙连接蛋白43(Cx43)、肌钙蛋白T(cTnT)的差别,为MSCs移植治疗急性心肌梗死(acute myocardial infarction,AMI)提供一定的理论基础。方法在无菌条件下分离Wistar大鼠股骨骨髓,采用密度梯度离心与贴壁培养法相结合进行MSCs培养、纯化和扩增,并行透射电镜鉴定。在无菌条件下分离同种乳鼠心脏,采用差速贴壁法进行CM培养、纯化并行Cx43、cTnT的免疫细胞化学检测。在获得稳定的细胞系后,选取生长良好的第三代MSCs用4',6-二脒基-2-苯基吲哚(DAPI)标记,分三组：①正常对照组：DAPI-MSCs在普通培养基中生长；②5-Aza诱导组：DAPI-MSCs加入5-Aza诱导,用不同浓度5-Aza分别作用不同时长,以观察最佳浓度和作用时间并行免疫组化鉴定cTnT、Cx43;③共培养组：与培养第3天的CM共培养。培养后1w、2w、3w、4w在倒置相差显微镜下观察各组细胞形态结构变化及免疫细胞化学染色鉴定cTnT、Cx43,并计算其诱导阳性率。结果Wistar大鼠MSCs经过分离、贴壁、纯化能在体外进行增殖并保持良好的未分化潜能。同种乳鼠CM分离、贴壁后1天即能出现自发性搏动,培养2-3d可长成单层并形成细胞簇,并出现成簇细胞的同步搏动。经10μmol/L 5-Aza持续作用15天的MSCs,4w时呈现出心肌细胞的典型改变,并有部分细胞出现自发搏动,频率为15～20次／分。MSCs传至第3代时用DAPI标记,标记效率为100%,DAPI-MSCs在正常培养基中未见有细胞收缩,cTnT、Cx43表达阴性；在诱导组和共培养组,培养至4w时细胞排列方向(?)致,成肌性排列,部分细胞呈现搏动,诱导1w时cTnT、Cx43表达阴性,诱导2w时cTnT阳性率为(9.98±1.67)%,Cx43阳性率为(13.38±2.15)%,培养至4w时阳性率升高,而且与3w,2w相比(P0.01)；共培养组第5天cTnT、Cx43就开始表达了,随着培养时间延长,阳性率逐渐升高,到第4w时cTnT、Cx43阳性率分别为(88.3±1.33)%,(90.38±1.87)%,与3w,2w,1w相比(P0.01)；相同周数内比较,共培养组的阳性率均高于诱导组(P0.01)。结论MSCs在体外有良好的增殖能力并保持其未分化潜能特性,CM经差速贴壁法分离纯化,细胞纯度可达95%以上。MSCs经适宜浓度5-Aza诱导和与CM共培养,可转化为心肌细胞并表达心肌细胞特异性标记物cTnT、Cx43,且共培养MSCs分化为心肌细胞的能力比5-Aza诱导强。
[Abstract]:Objective To investigate the effects of two methods on the induction and differentiation of bone marrow mesenchymal stem cells ( MSCs ) and cardiomyocytes ( MSCs ) and myocardial cells ( CM ) in vitro .
After inducing MSCs to differentiate , MSCs differentiated into CM at different time intervals and expressed myocardial cell specific marker gap junction protein 43 ( Cx43 ) and troponin T , which provided a theoretical basis for MSCs transplantation for acute myocardial infarction ( AMI ) .

Methods The bone marrow of Wistar rats was isolated under aseptic conditions . MSCs were cultured , purified and amplified by density gradient centrifugation and adherent culture method .
( 2 ) 5 - Aza - induced group : DAPI - MSCs were induced by 5 - Aza , and different concentrations of 5 - Aza were used to observe the optimal concentration and the time of action .

Results MSCs were isolated , adherent and purified in Wistar rats to proliferate and maintain good undifferentiated potential in vitro .
The positive rate was ( 9.98 卤 1.67 ) % , the positive rate of Cx43 was ( 13.38 卤 2.15 ) % , and the positive rate of Cx43 was ( 13.38 卤 2.15 ) % , and the positive rate of Cx43 was higher than that of 3w and 2w ( P0.01 ) .
At the 5th day of the coculture , Cx43 began to express , and the positive rate gradually increased with the increase of culture time . The positive rate was ( 88.3 卤 1.33 ) % and ( 90.38 卤 1 . 87 ) % , respectively , compared with 3w , 2w and 1w ( P0.01 ) .
The positive rate of co - culture group was higher than that in the induction group ( P0.01 ) .

Conclusion MSCs can proliferate in vitro and maintain their undifferentiation potential . The purity of MSCs can reach more than 95 % . MSCs can be transformed into cardiomyocytes and express cardiac muscle cell - specific markers for cardiac muscle cells . MSCs can be transformed into cardiomyocytes and express cardiac muscle cell - specific markers . The ability of MSCs to differentiate into cardiomyocytes is stronger than that of 5 - Aza .

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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