胰高血糖素样肽-1受体敲除H9c2细胞株建立及其抗凋亡作用初探

Establishment of Glucagon-Like Peptide-1 Receptor Knockout H9c2 Cell Line and Its Anti-Apoptotic Effect

目的通过CRISPR/Cas9技术构建大鼠H9c2心肌细胞胰高血糖素样肽-1受体(GLP-1R^(-/-))基因敲除的稳转株,探讨甲基乙二醛对其凋亡的影响。方法利用CRISPR/Cas9技术敲除H9c2心肌细胞的GLP-1R^(-/-)基因建立GLP-1R^(-/-)H9c2细胞系,单克隆后通过测序和免疫印迹进行鉴定。H9c2细胞为野生型(WT)组,GLP-1R^(-/-)H9c2细胞为GLP-1R^(-/-)组。采用免疫荧光对两组细胞进行染色,电子显微镜观察细胞形态;采用甲基乙二醛对两组细胞进行干预,利用CCK-8检测细胞活力,JC-1检测线粒体膜电位,实时荧光定量PCR检测GLO1、Bax、Bcl-2和caspase-3 mRNA的表达。结果基因测序及免疫印迹结果证实,GLP-1R^(-/-)H9c2细胞系构建成功;免疫荧光染色显示GLP-1R^(-/-)组细胞形态较WT组显著缩小(P<0.01);CCK-8检测结果显示,GLP-1R^(-/-)组细胞活力较WT组显著降低(P<0.01);JC-1检测结果显示,GLP-1R^(-/-)组细胞的线粒体膜电位损伤显著高于WT组(P<0.01);实时荧光定量PCR检测结果显示,GLP-1R^(-/-)组细胞的Bax mRNA表达显著上调(P<0.01),Bcl-2 mRNA表达显著下调(P<0.01)。而caspase-3 mRNA和GLO1 mRNA的表达在两组细胞间无显著差异(P>0.05)。结论在GLP-1R^(-/-)基因敲除的H9c2细胞系中,GLP-1R^(-/-)蛋白缺失会影响H9c2细胞的细胞形态,降低细胞活力,损伤线粒体膜电位,增强甲基乙二醛诱导的凋亡,从而证明GLP-1R^(-/-)蛋白在H9c2细胞中的保护作用。

Objective To investigate the effect of methylglyoxal(MGO) on the apoptosis of stable strains of glucagon-like peptide-1 receptor(GLP-1R^(-/-)) gene knockout H9c2 cells which was constructed by CRISPR/Cas9 technology.Methods GLP-1R^(-/-)H9c2 cell line was established by knocking out the GLP-1R^(-/-) gene of H9c2 cells by CRISPR/Cas9 technology, and identification was carried out by sequencing and Western blot after monoclonalization.H9c2 cells are wild-type(WT) group and GLP-1R^(-/-)H9c2 cells are GLP-1R^(-/-)group.Two groups of cells were stained with immunofluorescence, and cell morphology was observed by electron microscopy.MGO was used to intervene in both groups of cells, cell viability was detected by CCK-8,mitochondrial membrane potential(MMP) was detected by JC-1,and real-time PCR was used to detect the expression of GLO1,Bax, Bcl-2 and caspase-3 mRNA.Results Gene sequencing and Western blot results confirmed that the GLP-1R^(-/-)H9c2 cell line was successfully constructed.Immunofluorescence staining showed that the cell morphology of GLP-1R^(-/-)group was significantly smaller than that of WT group(P<0.01).The CCK-8 results showed that the cell viability of GLP-1R^(-/-)group was significantly reduced compared with that of WT group(P<0.01).The JC-1 results showed that the MMP damage of GLP-1R^(-/-)group cells was significantly higher than that of WT group(P<0.01).The real-time PCR results showed that the Bax mRNA expression of GLP-1R^(-/-)group cells was significantly upregulated(P<0.01),and Bcl-2 mRNA expression was significantly downregulated(P<0.01),while there was no significant difference in caspase-3 mRNA and GLO1 mRNA expression between the two groups(P>0.05).Conclusion In the H9c2 cell line with GLP-1R^(-/-)gene knockout, the deletion of GLP-1R^(-/-) protein affects the cell morphology of H9c2 cells, reduces cell viability, damages the MMP and enhances MGO-induced apoptosis, thereby demonstrating the protective role of GLP-1R^(-/-) protein in H9c2 cells.