体外模拟心肌环境诱导骨髓间充质干细胞向心肌细胞分化的研究

发布时间：2018-07-06 21:01

本文选题：骨髓间充质干细胞 + 心肌细胞　；参考：《山西医科大学》2008年硕士论文

【摘要】： 目的体外模拟心肌环境,诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)向心肌细胞分化,观察其诱导作用,同时与5-氮杂胞苷(5-azacytidine,5-aza)的诱导作用进行比较。方法自成年Wister大鼠的骨髓分离MSCs,新生Wister乳鼠分离心肌细胞并制备心肌细胞冻融液作为培养基体外模拟心肌环境,分四组培养MSCs。A组为对照组即用普通培养基培养细胞;B组为5-aza组;C组为心肌细胞冻融液与5-aza共同培养;D组为心肌细胞冻融液培养。观察细胞形态变化,利用免疫细胞化学技术鉴定心肌特异性蛋白T(Troponin T,cTnT),连接蛋白43(Connexin43),a一横纹肌动蛋白(a—Sarcomeric Actin),CD31的表达情况。在光镜下随机选择8个非重叠视野,图像分析分别计算阳性细胞所占比例。数据采用SPSS13.0统计软件,测量数据以均数±标准差( x±s)表示,作成组比较t检验。P0.05有统计学意义。结果 ①体外模拟心肌微环境情况:培养7d时心肌细胞形成细胞簇或细胞单层,呈放射状排列的同心圆状或峰谷样生长,细胞簇搏动呈明显同步性。传代后的心肌细胞仍具有自主收缩的特性。 ②骨髓间充质干细胞的体外诱导结果:诱导处理1周后,5-氮杂胞苷组多数细胞呈杆状,紧密平行排列生长,细胞体积变大,可见肌丝样结构形成;心肌细胞冻融液组细胞有聚集生长趋势,形成大量的子细胞,可见大量的肌丝样的结构;5-氮杂胞苷+心肌细胞冻融液组脱落或降解的细胞数明显少于5-氮杂胞苷组,也形成肌丝样结构,但部分细胞内有脂肪空泡形成。 ③免疫细胞化学鉴定结果:诱导培养1周后,血清对照组仅表达α-横纹肌肌动蛋白;5-氮杂胞苷组表达心肌特异性蛋白T、α-横纹肌肌动蛋白、连接蛋白43,其阳性率分别为20%,28%,25%,抗CD31染色呈阴性;心肌细胞冻融液组上述蛋白表达阳性率分别为23%,32%,28%,明显高于5-氮杂胞苷组与5-氮杂胞苷+心肌细胞冻融液组(P0.05),同时抗CD31染色呈阳性;5-氮杂胞苷+心肌细胞冻融液组上述蛋白表达阳性率分别为21%,28%,24%,与5-氮杂胞苷组相比无明显差异(P0.05),同时抗CD31染色呈阳性结论 ①以心肌细胞冻融液体外模拟心肌微环境,可高效诱导骨髓间充质干细胞分化为心肌细胞。 ②部分骨髓间充质干细胞表达CD31,即可向血管内皮细胞分化,提示与5-氮杂胞苷单一的肌细胞诱导作用相比,心肌细胞冻融液更能提供一个心肌再生所需的自然条件。
[Abstract]:Objective to induce the differentiation of bone marrow mesenchymal stem cells (mesenchymal stem cells) into cardiomyocytes by simulating myocardial environment in vitro, and to observe the induction effect of 5-azacytidineine 5-aza (5-azacytidineine 5-aza). Methods Myocardial cells were isolated from bone marrow of adult Wister rats and cardiomyocytes were isolated from newborn Wister rats. Cardiomyocyte freeze-thawed solution was used as culture medium to simulate myocardial environment in vitro. MSCs.A group was divided into four groups as control group: normal culture medium group B was 5-aza group C group was cardiomyocyte freezing-thawing solution and 5-aza co-culture group D group was cardiomyocyte freeze-thawing solution culture. The expression of cardiac specific protein T (Troponin TnT) and Connexin 43 (Connexin 43) and a-Sarcomeric Actin (a-sarcomeric Actin) CD31 were detected by immunocytochemistry. Eight unoverlapped visual fields were randomly selected under light microscope and the proportion of positive cells was calculated by image analysis. The data were measured by SPSS 13.0 software and the data were expressed as mean 卤standard deviation (x 卤s). Results 1 simulated myocardial microenvironment in vitro: after 7 days of culture, cardiomyocytes formed cell clusters or monolayers, which grew radially in concentric circles or peaks and valleys, and cell cluster pulsation showed obvious synchronism. In vitro induction of bone marrow mesenchymal stem cells: after one week of induction, most of the cells in the 5-azacytidine group were in the shape of rods and grew in close parallel order. The size of the cells became larger and the myofilm-like structure was formed, and the cells in the cardiomyocyte freezing and thawing fluid group had the tendency of aggregation and growth, forming a large number of daughter cells. It can be seen that a large number of myofilm-like structure, 5-azacytidine cardiomyocyte freeze-thaw solution group, the number of cells dropped or degraded significantly less than 5-azacytidine group, but also formed a myofilament structure. But adipose vacuoles formed in some cells. 3 Immunocytochemical identification results: 1 week after induction, In the serum control group, only 伪 -rhabdomyosin 5 azacytidine expressed myocardial specific protein T, 伪 -rhabdomydomycin 43, the positive rates were 20% and 28% 25%, respectively, and the anti-CD31 staining was negative. The positive rate of the above protein expression in the cardiomyocyte freeze-thaw solution group was 2332 / 28, which was significantly higher than that in the 5-azacytidine group and 5-azacytidine cardiomyocyte freeze-thaw group (P0.05), and the anti-CD31 staining was positive in the 5-azacytidine cardiomyocyte freezing and thawing group (P0.05). The positive rates of these proteins were 21% and 28%, respectively, and there was no significant difference compared with the 5-azacytidine group (P0.05). At the same time, the anti-CD31 staining showed positive conclusion: 1 the myocardial microenvironment was simulated by cardiomyocyte freeze-thawing fluid, and no significant difference was found between 5-azacytidine group and 5-azacytidine group. Bone marrow mesenchymal stem cells can be induced to differentiate into cardiomyocytes. 2 some of bone marrow mesenchymal stem cells express CD31, which can differentiate into vascular endothelial cells, suggesting that compared with 5-azacytidine single myocyte induction, some bone marrow mesenchymal stem cells can differentiate into vascular endothelial cells. Cardiomyocyte freeze-thawing fluid can provide a natural condition for myocardial regeneration.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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