BMP-2诱导骨髓间充质干细胞向心肌细胞分化的实验研究

发布时间：2018-05-01 12:39

本文选题：骨髓间充质干细胞 + 骨形态发生蛋白-2　；参考：《苏州大学》2009年硕士论文

【摘要】： 目的:冠心病在发达国家的发病率和死亡率都居于第一位,通常认为心肌细胞在出生后即不能再生,心肌细胞死亡后大量的心肌细胞由疤痕组织替代,导致局部的心肌收缩紊乱,心功能下降,最后导致充血性心衰。本试验针对心衰的基本病理生理,通过BMP-2诱导后的骨髓间充质干细胞移植入受损的心肌内,改善心室重构,促进梗死区血管新生来增加干细胞修复受损心肌的治疗效果,并进一步探讨干细胞治疗缺血性心肌病的机理。方法:从健康梅山猪骨髓中分离培养MSCs,取第四代骨髓间充质干细胞,用BMP-2诱导24小时,促进其向心肌细胞定向分化,免疫细胞化学检测诱导后不同时间点的心肌特异性蛋白cTnT和CX-43及MHC的表达。健康雄性梅山猪15只随机分为3组,经心导管用明胶海绵栓塞LAD第二对角支建立心梗模型,A组(对照组):梗死四周后通过介入方法在心梗区移植5ml DMEM;B组(干细胞组):梗死四周后在心梗区移植1×107个MSCs;C组(诱导组):梗死四周后在心梗区移植1×107个经BMP-2诱导分化后的MSCs。检测心梗前后及移植后心功能的变化,Ⅷ因子免疫组化染色计数新生毛细血管数量,Western blot检测各组梗死区GATA-4蛋白表达,ECT检测心肌缺血状况,大体标本TTC法检查各组心梗面积的变化。结果:利用Ficoll分离和贴壁培养的方法可以获取稳定分裂的梅山猪骨髓间充质干细胞。用BMP-2诱导后,MSCs的形态和排列发生变化。免疫细胞化学染色显示,cTnT和CX-43从诱导后3周开始表达,4周cTnT表达增强,呈现密集的横纹样结构;CX-43在3周位于细胞膜下,4周呈颗粒状密集分布于肌管的相邻细胞连接处;三周后可见MHC阳性棕黄颗粒表达于胞浆及胞核中。移植前,血流动力学LVDP、±dp/dtmax下降明显(与心梗前比较,P0.05),移植后,随着时间的延长,B、C组的心功能较移植前有明显改善(P0.05),而C组的改善更为明显(与B组比较,P0.05);与A、B两组,C组的心梗面积缩小,新生毛细血管数量增多(P0.05);Western blot检测C组GATA-4蛋白表达明显升高(与A,B组比较,P0.05);ECT同时显示诱导组心肌缺血状况改善明显。结论:BMP-2可以在体外诱导骨髓间充质干细胞分化为心肌样细胞并表达心肌特异性蛋白MHC,cTnT和CX-43。诱导后的骨髓间充质干细胞可以在梗死心肌内存活、增殖并上调GATA-4因子的表达,改善心功能。移植经BMP-2诱导分化后的骨髓间充质干细胞较移植未经诱导分化的骨髓间充质干细胞能更好的改善心功能。
[Abstract]:Objective: coronary heart disease (CHD) is the leading cause of morbidity and mortality in developed countries. It is generally believed that cardiomyocytes cannot regenerate after birth and that a large number of cardiac myocytes are replaced by scar tissue after death. Lead to local myocardial contraction disorder, cardiac function decline, and eventually lead to congestive heart failure. According to the basic pathophysiology of heart failure, bone marrow mesenchymal stem cells (BMSCs) induced by BMP-2 were transplanted into damaged myocardium to improve ventricular remodeling and promote angiogenesis in infarcted area to increase the therapeutic effect of stem cells in repairing damaged myocardium. Furthermore, the mechanism of stem cells in the treatment of ischemic cardiomyopathy was discussed. Methods: MSCs were isolated and cultured from the bone marrow of healthy Meishan pig. The fourth generation of bone marrow mesenchymal stem cells (BMSCs) were induced by BMP-2 for 24 hours to promote the differentiation of MSCs into cardiomyocytes. The expression of myocardial specific proteins (cTnT, CX-43 and MHC) at different time points after induction was detected by immunocytochemistry. Fifteen healthy male Meishan pigs were randomly divided into 3 groups. Embolization of the second diagonal branch of LAD via cardiac catheterization with gelatin sponge to establish myocardial infarction model group A (control group: transplantation of 5ml in myocardial infarction area by interventional method after four weeks of infarction) (stem cell group: transplantation of 1 脳 107 MSCSN in myocardial infarction area after four weeks of infarction) Group A (induction group): 1 脳 107 MSCs induced by BMP-2 were transplanted into myocardial infarction area after four weeks of infarction. The changes of cardiac function before and after myocardial infarction and after transplantation were detected. The number of neocapillaries was counted by immunohistochemical staining. Western blot was used to detect the expression of GATA-4 protein in infarcted area. Gross TTC method was used to examine the changes of myocardial infarction area in each group. Results: bone marrow mesenchymal stem cells of Meishan pig could be obtained by Ficoll isolation and adherent culture. The morphology and arrangement of BMP-2 were changed after induction. Immunocytochemical staining showed that the expression of cTnT and CX-43 increased at the beginning of 4 weeks after induction, and CX-43 was located at the junction of adjacent cells in the myotube at the 4th week under the cell membrane. Three weeks later, MHC positive brown granules were found in cytoplasm and nucleus. Before transplantation, hemodynamic LVDPand 卤dp/dtmax decreased significantly (P 0.05 compared with that before myocardial infarction. As time went on, the cardiac function of group C was significantly improved than that of group C before transplantation, while the improvement of group C was more obvious than that of group B (P 0.05), and the area of myocardial infarction in group C was smaller than that in group B (P 0.05), and that in group C was smaller than that in group B (P < 0.05). The expression of GATA-4 protein in group C was significantly higher than that in group A (P 0.05). ConclusionBMP 2 can induce bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells and express myocardial specific proteins MHCC TnT and CX-43 in vitro. The induced bone marrow mesenchymal stem cells (BMSCs) could survive, proliferate and up-regulate the expression of GATA-4 factor in infarct myocardium and improve cardiac function. Transplantation of bone marrow mesenchymal stem cells induced by BMP-2 can improve cardiac function better than that of uninduced bone marrow mesenchymal stem cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.2

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