增强型绿色荧光蛋白标记骨髓间充质干细胞体外诱导分化成心肌样细胞研究
发布时间：2019-05-26 20:18
【摘要】： 目的 探讨增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转染骨髓间充质干细胞的可行性,明确经蛋白标记后的间充质干细胞(mesenchymal stem cell,MSC)是否可以稳定生长并被诱导成为心肌细胞以及心肌微环境对转染MSC在体外向心肌样细胞分化的影响,为进一步做标记的MSC体内移植研究奠定基础。 方法 1、提取、鉴定pEGFP质粒; 2、大鼠骨髓间充质干细胞的的分离和培养; 3、新生乳鼠心肌细胞的分离和培养; 4、制备心肌细胞裂解液; 5、EGFP基因转染骨髓间充质干细胞; 6、筛选阳性转染细胞后在荧光显微镜观察,然后对比建系,并体外诱导其向心肌样细胞转化; 7、把诱导的细胞做免疫组化检测心脏特异性肌钙蛋白T(cTnT)、α-肌动蛋白、连接蛋白43及CD31的表达情况,通过计算机分析系统测定其阳性率。 结果 原代MSC多呈纺锤形或梭形,有聚集生长的倾向,3～5个细胞成为一个集落。传代后,细胞变为形态均一的排列有序的成纤维细胞样,长梭形,胞浆突起较少,胞体轮廓不甚清晰,胞体也相对较大;在脂质体介导下将EGFP基因导入MSC,在荧光显微镜下观察,细胞的生长和增殖不受影响。增强型绿色荧光蛋白在基因转染12h后开始表达,48～72h达高峰。在1周内有较强的表达。1周后表达绿色荧光的细胞逐渐减少,到4周时仍可见少量散在的荧光。瞬时转染率达20%～30%。质粒和脂质体的浓度比例在1:2到1:3转染效率最高。 结论 绿色荧光蛋白可成功转染骨髓间充质干细胞;转染效率与脂质体和质粒浓度有关;转染后骨髓间充质干细胞生长良好,在体外可以成功诱导成为心肌样细胞。
[Abstract]:Objective to investigate the feasibility of enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP (enhanced green fluorescent protein (mesenchymal stem cell,) transfer into bone marrow mesenchymal stem cells (MSCs). Whether MSC) can grow stably and be induced into cardiomyocytes and the effect of myocardial microenvironment on the differentiation of MSC into cardiomyocytes in vitro lays a foundation for the further study of in vivo transplantation of labeled MSC. Methods 1, pEGFP plasmid was extracted and identified, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes. 4. Cardiomyocyte lysate was prepared. 5, EGFP gene was transferred into bone marrow mesenchymal stem cells. 6. The positive cells were screened and observed by fluorescence microscope, and then the lines were compared and induced to transform into cardiac muscle-like cells in vitro. 7. The expression of cardiac specific troponin T (cTnT), 伪-actin, junction protein 43 and CD31 was detected by immunohistochemistry, and the positive rate of cardiac specific troponin CD31 was measured by computer analysis system. Results most of the primary MSC were fusiform or fusiform, with the tendency of aggregation and growth. 3 / 5 cells became a colony. After passage, the cells became homogeneously arranged fibroblasts, long fusiform, less cytoplasm protrusions, the outline of the cell body was not very clear, and the cell body was relatively large. The introduction of EGFP gene into MSC, mediated by liposomes was observed under fluorescence microscope, and the growth and proliferation of the cells were not affected. The expression of enhanced green fluorescent protein began 12 hours after gene transfer and reached the peak at 48 h and 72 h. There was strong expression within 1 week. After 1 week, the number of cells expressing green fluorescence decreased gradually, and a small amount of scattered fluorescence was still observed at 4 weeks. The instantaneous dye transfer rate was 20% 鈮，
本文编号：2485604
[Abstract]:Objective to investigate the feasibility of enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP (enhanced green fluorescent protein (mesenchymal stem cell,) transfer into bone marrow mesenchymal stem cells (MSCs). Whether MSC) can grow stably and be induced into cardiomyocytes and the effect of myocardial microenvironment on the differentiation of MSC into cardiomyocytes in vitro lays a foundation for the further study of in vivo transplantation of labeled MSC. Methods 1, pEGFP plasmid was extracted and identified, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes. 4. Cardiomyocyte lysate was prepared. 5, EGFP gene was transferred into bone marrow mesenchymal stem cells. 6. The positive cells were screened and observed by fluorescence microscope, and then the lines were compared and induced to transform into cardiac muscle-like cells in vitro. 7. The expression of cardiac specific troponin T (cTnT), 伪-actin, junction protein 43 and CD31 was detected by immunohistochemistry, and the positive rate of cardiac specific troponin CD31 was measured by computer analysis system. Results most of the primary MSC were fusiform or fusiform, with the tendency of aggregation and growth. 3 / 5 cells became a colony. After passage, the cells became homogeneously arranged fibroblasts, long fusiform, less cytoplasm protrusions, the outline of the cell body was not very clear, and the cell body was relatively large. The introduction of EGFP gene into MSC, mediated by liposomes was observed under fluorescence microscope, and the growth and proliferation of the cells were not affected. The expression of enhanced green fluorescent protein began 12 hours after gene transfer and reached the peak at 48 h and 72 h. There was strong expression within 1 week. After 1 week, the number of cells expressing green fluorescence decreased gradually, and a small amount of scattered fluorescence was still observed at 4 weeks. The instantaneous dye transfer rate was 20% 鈮，
本文编号：2485604
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2485604.html
最近更新
教材专著
