论文部分内容阅读
目的:研究间接接触共培养条件下骨髓间充质干细胞(mesenchymal stem cells,MSCs)向心肌细胞(myocadium-lkce cells,CMs)的分化及相关调控基因的时序表达;筛选MSCs定向分化为CMs的重要调控基因。方法:将MSCs与CMs按1∶5的比例进行间接接触共培养,连续观察两周,在相差显微镜下观察MSCs的形态变化。采用免疫荧光染色法检测心肌特征性肌动蛋白α(α-actin)和心脏肌钙蛋白T(cTnT)的表达。应用半定量RT-PCR分析TGF-β、Nkx-2.5、GATA-4、MEF-2C及TEF-1等相关调控基因在分化过程中的时序表达。结果:共培养后,MSCs的体积增大,其梭形形态逐渐变短变粗近似棒状或椭圆形,细胞之间形成连接,排列方向趋于一致。共培养两周时,α-actin及cTnT阳性细胞的比例分别为29.63%和27.38%。TGF-β、Nkx-2.5、GATA-4和MEF-2C基因在共培养后1 d表达开始增强,诱导后7 d达高峰,以后虽有所下降但仍维持在较高水平;TEF-1基因在诱导过程中表达无明显变化。结论:间接接触共培养条件下,MSCs可分化为心肌细胞。在此过程中,TGF-β、Nkx-2.5、GATA-4和MEF-2C基因可能是调控MSCs定向分化为心肌样细胞的重要调控基因。
OBJECTIVE: To study the temporal expression of mesenchymal stem cells (MSCs) differentiating into cardiomyocytes (CMs) and the related regulatory genes under indirect contact co-culture conditions. It is important to screen MSCs for differentiation into CMs Regulatory genes. Methods: MSCs and CMs were co-cultured by indirect contact at a ratio of 1: 5 for two weeks. The morphological changes of MSCs were observed under a phase contrast microscope. The expression of α-actin and cardiac troponin T (cTnT) were detected by immunofluorescence staining. Semi-quantitative RT-PCR was used to analyze the temporal expression of TGF-β, Nkx-2.5, GATA-4, MEF-2C and TEF-1 in the process of differentiation. Results: After co-culture, the volume of MSCs increased, the shape of spindle gradually became shorter and became thicker and roughly rod-like or oval, and the MSCs became connected and aligned in the same direction. At two weeks of co-culture, the ratios of α-actin and cTnT positive cells were 29.63% and 27.38%, respectively. The expression of TGF-β, Nkx-2.5, GATA-4 and MEF-2C began to increase on the 1st day after co-culture and reached a peak on the 7th day after induction, No significant changes in expression during induction. Conclusion: MSCs differentiate into cardiomyocytes under indirect contact co-culture conditions. In this process, TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may be important regulatory genes that regulate the directional differentiation of MSCs into cardiomyocyte-like cells.