【摘要】 目的体外研究诱导多能干细胞(iPSCs)与心肌细胞的杂交细胞的心肌细胞分化效率。方法聚乙二醇体外介导iPSCs与心肌细胞融合;检测杂交细胞染色体数;RT-PCR检测干细胞及心肌细胞特异性基因在杂交细胞中表达情况;细胞免疫荧光法检测干细胞及心肌细胞特异性蛋白在杂交细胞中表达情况;采用悬浮培养法制备拟胚体(EBs),检测杂交细胞和iPSCs的心肌细胞分化效率。结果杂交细胞具有近似四倍体核型;RT-PCR提示杂交细胞中Oct-4的表达与iPSCs没有统计学差异(P>0.05),P1代杂交细胞中Nanog的表达低于iPSCs(P<0.05),α-MHC和β-MHC在iPSCs、P3、P5、P7代杂交细胞中的表达明显低于心肌细胞(P<0.05);细胞免疫荧光提示Oct-4在杂交细胞中呈阳性表达,在心肌细胞中呈阴性表达,cTnT在心肌细胞中阳性表达,在早期杂交细胞中呈阳性表达,P3代以后的杂交细胞呈阴性表达;杂交细胞和iPSCs均能够形成搏动的EBs;杂交细胞在第20 d、25 d及30 d时的搏动率明显高于iPSCs(P<0.05)。结论聚乙二醇能够在体外介导iPSCs与心肌细胞融合,杂交细胞的心肌细胞分化效率高于iPSCs。更多还原

【Abstract】 Objective To study in vitro the differentiation efficiency of cardiomyocytes in hybrid cells and induced pluripotent stem cells(iPSCs). Methods The fusion of iPSCs and cardiomyocytes was mediated in vitro by using polyethylene glycol. The number of chromosomes of hybrid cells was detected. The expressions of iPSCs and cardiomyocyte specific genes in hybrid cells were detected by using RT-PCR. The expressions of iPSCs and cardiomyocyte specific proteins in hybrid cells were detected by using cellular immunofluorescence. The embryoid bodies(EBs) was prepared by using suspension culture method, and differentiation efficiency of cardiomyocytes in hybrid cells and iPSCs was detected. Results The hybrid cells have an approximate tetraploid karyotype. The results of RT-PCR showed that Oct-4 expression had no statistical difference between hybrid cells and iPSCs(P>0.05).The expression of Nanog in P1 generation hybrid cells was lower than that in iPSCs(P<0.05). The expressions of α-MHC and β-MHC in iPSCs and P3, P5, and P7 generation hybrid cells were significantly lower than those in cardiomyocytes(P<0.05). The results of cellular immunofluorescence showed that Oct-4 positively expressed in hybrid cells and negatively expressed in cardiomyocytes, and cTnT positively expressed in cardiomyocytes,positively expressed in early hybrid cells and negatively expressed in hybrid cells after P3 generation. Both hybrid cells and iPSCs were able to form beating EBs. The beating rates of EBs were significantly higher in hybrid cells than those in iPSCs on the 20 th d, 25 th d and 30 th d(P<0.05). Conclusion Polyethylene glycol can mediate in vitro the fusion of iPSCs with cardiomyocytes, and the differentiation efficiency of cardiomyocytes is higher in hybrid cells than that in iPSCs.更多还原