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目的探讨Wnt3a单独以及联合骨形态发生蛋白9(BMP9)诱导C3H10T1/2细胞向心肌细胞样细胞分化的作用。方法利用HEK293细胞扩增重组腺病毒AdGFP、AdBMP9、AdWnt3a;分别转染C3H10T1/2细胞3周后,倒置显微镜和荧光显微镜观察细胞形态及绿色荧光蛋白表达;流式细胞术检测细胞转染效率;Westernblot法、免疫荧光技术及实时定量PCR(qRT-PCR)分别检测缝隙连接蛋白43(Cx43)、肌钙蛋白T(cTnT)、GATA结合蛋白4(GATA4)、心肌细胞增强因子2C(MEF2C)的表达。结果经HEK293细胞扩增可得到高滴度的重组腺病毒;转染C3H10T1/2细胞3周后,Wnt3a组Cx43、cTnT、GATA4、MEF2C的表达量与空白组比较无明显差异;Wnt3a联合BMP9组的Cx43、cTnT、GATA4、MEF2C基因的表达量显著高于空白组、GFP组和Wnt3a组,与单独BMP9组相比无显著性差异。结论Wnt3a不能单独诱导C3H10T1/2细胞向心肌细胞样细胞分化;Wnt3a联合BMP9可诱导C3H10T1/2细胞向心肌细胞样细胞分化。
Objective To investigate the effect of Wnt3a alone and in combination with BMP9 on the differentiation of C3H10T1 / 2 cells into cardiomyocyte-like cells. Methods The recombinant adenovirus AdGFP, AdBMP9 and AdWnt3a were amplified by HEK293 cells. After transfection of C3H10T1 / 2 cells for 3 weeks respectively, the cell morphology and green fluorescent protein expression were observed by inverted microscope and fluorescence microscope. The transfection efficiency was detected by flow cytometry. The expressions of Cx43, cTnT, GATA4 and MEF2C were detected by Western blot, immunofluorescence and real-time quantitative PCR (qRT-PCR) expression. Results The recombinant adenovirus with high titer was obtained by amplification of HEK293 cells. The expression of Cx43, cTnT, GATA4 and MEF2C in Wnt3a group was not significantly different from that in blank group after 3 weeks of C3H10T1 / 2 transfection. Wnt3a combined with BMP9 group The expression of Cx43, cTnT, GATA4 and MEF2C was significantly higher than that of blank group, GFP group and Wnt3a group, but no significant difference compared with BMP9 alone group. Conclusion Wnt3a can not induce the differentiation of C3H10T1 / 2 cells into cardiomyocyte-like cells alone. Wnt3a and BMP9 can induce the differentiation of C3H10T1 / 2 cells into cardiomyocyte-like cells.