BMP-13在C_3H10T_(1/2)干细胞向心肌样细胞分化过程中的作用

Role of bone morphogenetic proteins-13 in C_3H10T_(1/2) stem cells differentiation to myocardiocytes in vitro

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 determine the effect of bone morphogenetic proteins-13（BMP-13） on mesenchymal stem cells（C3H10T1/2） differentiating into cardiomyocyte-like cells in vitro.Methods C3H10T1/2 cells were transfected with recombinant adenovirus plasmids pAdEasy-BMP-13,pAdEasy-BMP-2（positive control）,or AdEasy-GFP（negative control）.The expressions of cardiac-specific proteins cardiac isoform of tropnin T（cTnT） and connexin 43（Cx43） in the above transfected and non-transfected C3H10T1/2 cells were measured by Western blotting,the expressions of cardiac-specific proteins α-MHC and α-actin were detected with immunofluorescence staining and the expressions of cardiac-specific genes GATA4 and MEF2C were detected by real-time quantitative PCR at 3 week after transfection.The ultrastructure of the cells was observed by electron microscopy in 4 weeks after the transfection.Results After pAdEasy-BMP-13 transfection,C3H10T1/2 cells began to stretch,with obviously enhanced refractivity,and close interconnection of cells in a regular order.Positive expressions of cTnT,Cx43,α-MHC and α-actin were found in pAdEasy-BMP-13 and pAdEasy-BMP-2 plasmids transfection groups in 3 weeks after transfection,but not in 1 and 2 weeks.Neither was found in AdEasy-GFP transfection groups and non-transfected group at 1,2 and 3 weeks.The expressions of GATA4 and MEF2C were detected in all cell groups,but more strong in pAdEasy-BMP-13 and pAdEasy-BMP-2 transfection groups than in AdGFP plasmid transfection and control group at week 3（P0.05）.C3H10T1/2 cells after pAdEasy-BMP-13 transfection displayed myocardial leap dish and myofilament-like structure after 4 weeks＇ transfection,but not in pAdEasy-GFP group and control group at the same time point.Conclusion pAdEasy-BMP-13 probably promote the differentiation of stem cells into cardiomyocytes.