桥粒芯胶蛋白-2基因沉默对P19细胞向心肌细胞诱导分化的影响

The effects of silencing desmocollin-2 gene on the cardiomyocyte-like cells differentiated from P19 cell lines

目的探讨在P19细胞诱导分化成心肌细胞过程中,桥粒芯胶蛋白-2(DSC2)基因沉默对其的影响。方法设计并合成针对DSC2基因编码区的干扰序列,构建真核细胞表达质粒并转染P19细胞。荧光定量聚合酶链式反应(RT-PCR)和蛋白免疫印迹(Western blot)技术检测DSC2在mRNA和蛋白水平表达变化,筛选出沉默效率最佳的细胞株。二甲基亚矾诱导分化为心肌细胞,观察其超微结构和细胞凋亡等改变,以及对纤维化与脂肪化相关基因mRNA水平表达的影响。结果成功构建了5种ShDSC2重组质粒,转染P19细胞并获得稳定转染细胞株,筛选出对DSC2基因mRNA水平(69.47%vs 0,P<0.01)和蛋白水平表达(65.62%vs 0,P<0.01)的抑制效率最显著的ShDSC2-613组,并成功诱导分化为心肌样细胞后,电镜扫描显示后者细胞出现脂滴、空泡样变性、线粒体肿胀及嵴消失,流式细胞仪检测提示细胞凋亡显著增加,RT-PCR示纤维化相关基因(Collal、Colla2、Col3a1)与脂肪化相关基因(Adiponectin、PPAR-γ、C/EBP-α)的mRNA表达均显著升高。结论建立能有效抑制DSC2表达的P19细胞株并分化为心肌样细胞,表现出与致心律失常型右室心肌病(ARVC)患者病理和分子生物学特点相似的表型特征,提示其可作为深入研究ARVC致病机制的前体细胞。

Objective To investigate the effects of down-regulation of desmocollin-2 （ DSC2 ） gene expression on cardiac-like myocyte differentiated from P19 ceils. Methods The DSC2 gene specific siRNAs and control siRNA （siNC） were synthesized and cloned into pGPU6/GFP/Neo vector. The recombinant plasmids （ShDSC2 and ShNC） were transfected into P19 cells, and the stable cell lines were selected with G418. The efficiency of DSC2 knock down at the levels of mRNA and protein expression were determined by RT-PCR and Western blot, respectively. The selected P19 cells lines were then induced and differentiated into cardiomyocyte-like cells by DMSO. The apoptosis process and ultrastructure chan- ges of cardiomyocyte-like cell derived from P19 cells were evaluated by flow cytometry and transmission electron microscope （ TEM）, respectively. RT-PCR was used to measure the expression of adipogenesis and fibrogenesis related genes at mRNA level. Results Five recombinant ShDSC/ plasmids were constructed successfully and transfected into P19 cells, after G418 selection, positive clones were established by RNA interference. Compared with the ShNC control group, the expression levels of mRNA and protein in ShDSC2 group were decreased by 69.47% （P〈0.01） and 65.62% （P〈0.01）, respectively. In ShDSC2 group, TEM showed that fat droplet, cellular vacuoles, swollen mitochondria and disappeared cristae, flow cytometry demonstrated that the apoptosis was also increased. A marked increase in expression levels of three major regulators of adipogenesis, namely PPAR-γ C/EBP-α and Adiponectin, and their target gene adiponectin, were also observed in ShDSC2 group. The expression levels of procollagen genes Collal, Col1a2 and Col3a1 were increased remarkably. Conclusions We established DSC2 gene silenced P19 cell line and further differentiated it into cardiomyocyte-like cells. The later exhibited increased apoptosis, adipogenesis and fibrogenesis, thus recapitulating the phenotype of human arrhythmogenic right ventricular cardiomyopathy. This cellular model could provide the opportunity to explore the pathogenesis and novel tool for therapeutic studies of ARVC in vitro.