摘要
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。
AIM： To investigate the transdifferentiation of bone marrow mesenchymal stem cells （MSCs） into cardiomyocytes and the temporal expression of regulatory genes in the simulated myocardial microen- vironment in which MSCs were co-cultured with cardiomyocytes by indirect contact in vitro and to screen some important genes in this differentiation. METHODS： MSCs isolated from adult rats were cocuhured with CMs obtained from neonatal rat ventricles at a 1 ： 5 ratio in a dual-chamber dish separated by a semipermeable membrane for 2 weeks. During this indirect contact eocuhure procedure, cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining against α-actin and cardiac troponin-T （cTnT） , and reverse transcfiptase-polymerase chain reaction was performed for temporal expres- sion of regulatory genes TGF-fl, Nkx-2. 5, GATA-4, MEF-2C and TEF-1. RESULTS ： After cocuhure with CMs, MSCs showed a stick-like or elliptical morphology. Immunofluorescent stain results revealed that α-actin- and cardiac troponin T （cTnT）-positive cells were found in MSCs at day 5 after coculture with CMs, and 29.63% of MSCs were positive for α-actin and 27.38% for cTnT at 14 days. RT-PCR demon-strated that the expression level of TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes began to increase at day 1 after cocuhure, reached their peak at day 7 and remained relatively high afterwards, even though there was a slight decrease. Only insignificant changes were observed in the expression level of TEF-1 gene in the cocuhure. CONCLUSION ： Indirect contact with CMs （ 1 ： 5 ） is conducive for MSCs to differentiate into CMs in vitro. TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may play a crucial role during transdifferentiation.
出处
《心脏杂志》
CAS
2010年第6期801-806,共6页
Chinese Heart Journal
基金
江西省卫生厅重大招标项目资助(200602)
关键词
骨髓间充质干细胞
间接接触
分化
调控基因
心肌细胞
bone marrow mesenchymal stem cells
indirect contact
differentiation
genes
cardiomyocytes
