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目的研究骨形态形成蛋白-13(bone morphogenetic proteins-13,BMP-13)促骨髓间充质干细胞C3H10T1/2向心肌样细胞分化过程中的作用。方法骨髓间充质干细胞C3H10T1/2经pAdEasy-BMP-13重组腺病毒质粒感染后,利用Western blot以及免疫荧光技术于1、2、3周时分别检测C3H10T1/2干细胞心肌特异性蛋白cTnT、Cx43、α-MHC、α-actin的表达变化;同时以实时荧光定量PCR技术进行心肌分化特异性基因GATA4、MEF2C的检测,4周后应用电子显微镜观察干细胞超微结构变化。结果 C3H10T1/2干细胞经pAdEasy-BMP-13感染诱导后,细胞开始伸展,折光性增强,细胞间连接较紧密且细胞排列趋向一致;pAdEasy-BMP-13和pAdEasy-BMP-2感染后细胞于3周时,Western blot技术检测出心肌特异性肌钙蛋白(cardiac isoform of tropnin T,cTnT)、连接蛋白Cx43(connexin 43,Cx43)(P<0.05),免疫荧光检测有蛋白α-MHC、α-actin表达,而于1周和2周时未检测出。同时pAdEasy-GFP感染后细胞和未感染组细胞1、2周和3周均未发现有心肌特异性蛋白cTnT、Cx43、α-MHC、α-actin的表达。3周对各组均出现心脏早期发育特异性基因GATA4、MEF2C的表达,但pAdEasy-BMP-13和pAdEasy-BMP-2感染后细胞表达量明显高于pAdEasy-GFP感染后细胞和未感染组细胞(P<0.05);pAdEasy-BMP-13感染后细胞于4周时透射电镜下观察出现闰盘及肌丝样细胞超微结构,而pAdEasy-GFP感染后细胞和未感染组细胞没有出现类似结构。结论 pAdEasy-BMP-13可以诱导C3H10T1/2干细胞向心肌样细胞分化。
Objective To investigate the role of bone morphogenetic proteins-13 (BMP-13) in the differentiation of cardiomyocyte-like cells from C3H10T1 / 2 cells. Methods C3H10T1 / 2 was transfected into C3H10T1 / 2 cells by pAdEasy-BMP-13 recombinant adenovirus plasmid. Western blot and immunofluorescence were used to detect the expression of cTnT, Cx43 , Α-MHC and α-actin. Real-time quantitative PCR was used to detect myocardial differentiation-specific genes GATA4 and MEF2C. Four weeks later, ultrastructural changes of stem cells were observed by electron microscopy. RESULTS: After induced by pAdEasy-BMP-13, C3H10T1 / 2 cells began to stretch, and the refolding of C3H10T1 / 2 stem cells was stronger than that of pAdEasy-BMP-13 cells. Western blot was used to detect cardiac-specific troponin T (cTnT) and connexin 43 (Cx43) (P <0.05) actin expression, but not detected at 1 week and 2 weeks. At the same time, the expression of cardiac specific protein cTnT, Cx43, α-MHC and α-actin were not found in the cells of 1 and 2 weeks and 3 weeks after infection with pAdEasy-GFP. The expression of GATA4 and MEF2C in early stage of cardiac development appeared in each group after 3 weeks, but the expression of pAdEasy-BMP-13 and pAdEasy-BMP-2 was significantly higher than those in pAdEasy-GFP infected and uninfected (P <0.05). After transfection with pAdEasy-BMP-13, the ultrastructures of intercalated disc and myofibroblasts were observed by transmission electron microscopy at 4 weeks. However, no similar structure was found between cells transfected with pAdEasy-BMP-13 and non-infected cells . Conclusion pAdEasy-BMP-13 can induce the differentiation of C3H10T1 / 2 stem cells into cardiomyocyte-like cells.