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C3G基因对H9C2心肌细胞凋亡和增殖的影响
作者:杨东艳 李刚 张蕾 张志升 扶艳波 胡苏蕾 
单位:400016 重庆 重庆医科大学附属第一医院老年病科(杨东艳、李刚、张蕾、张志升、扶艳波、胡苏蕾) 
关键词:C3G 心肌细胞 凋亡 增殖 P-ERK1/2蛋白 bcl-2相关X蛋白质 
分类号:R541.4
出版年,卷(期):页码:2015,40(8):603-609
摘要:

目的 探讨C3G基因对H9C2心肌细胞凋亡、增殖的影响及其机制。方法 构建RNA干扰慢病毒,购买pCXN2-Flag空质粒和pCXN2-Flag-hC3G过表达人C3G mRNA质粒。分别用空白试剂、阴性慢病毒、C3G siRNA慢病毒、C3G siRNA慢病毒+空质粒、C3G siRNA慢病毒+hC3G质粒随机感染和转染H9C2心肌细胞,实验被随机分为5个组,即空白对照组、阴性对照组、沉默C3G组、沉默C3G+空质粒组和沉默C3G+过表达人C3G组。转染质粒72h后,采用RTPCR法检测目的基因C3G mRNA的表达,流式细胞术检测H9C2心肌细胞的凋亡情况,MTT比色法测定细胞增殖率,Western blotting检测细胞C3Gp-ERK1/2BaxFlag的蛋白表达水平。结果 阴性对照组和沉默C3G组经嘌呤霉素筛选,85%以上细胞被绿色荧光蛋白标记,提示细胞被慢病毒感染。与空白对照组和阴性对照组比较,沉默C3G组和沉默C3G+空质粒组的细胞中Bax蛋白表达水平和细胞凋亡率均明显增加(P<0.01P<0.05),且这两组细胞C3G mRNAC3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率均明显降低(P<0.01P<0.05);与沉默C3G组和沉默C3G+空质粒组比较,沉默C3G+过表达人C3G组的细胞中Bax蛋白表达水平和细胞凋亡率明显降低(P<0.01P<0.05),且细胞中C3G mRNAC3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率则明显增加(P<0.01P<0.05)。结论 沉默C3G基因能有效抑制细胞的增殖,促进H9C2心肌细胞凋亡;过表达C3G基因能逆转沉默C3G基因对H9C2心肌细胞的影响,表现为细胞凋亡减少,增殖明显,其机制可能与C3G基因对p-ERK1/2蛋白和促凋亡分子Bax的调控有关。

Objective To study the effect of C3G gene on apoptosis and proliferation of H9C2 cardiomyocytes and its mechanism. Methods The RNA lentivirus was constructed, and pCXN2-Flag plasmid (empty plasmid) and pCXN2-Flag-hC3G plasmid (over-expressed human C3G mRNA) were purchased. H9C2 cardiomyocytes were respectively infected and transfected with blank reagent, negative lentivirus, C3G siRNA lentivirus, C3G siRNA lentivirus+pCXN2-Flag plasmid and C3G siRNA lentivirus+pCXN2-Flag-hC3G plasmid with stochastic method. Thus the experiments were randomly divided into five groups, namely blank group, negative control group, C3G silence group, C3G silence+empty plasmid group, and C3G silence+C3G overexpression group. Seventy two hours after plasmid transfection, the expression of C3G mRNA was detected by PT-RCR, cell apoptosis was determined by flow cytometry, cell proliferation rate was determined by MTT, and the protein levels of C3G, p-ERK1/2, Bax and Flag were determined by Western blotting. Results As screened by puromycin, it was found that more than 85% of the cells in negative control group and C3G silence group were labeled with green fluorescent protein, showing that more than 85% of the cells were infected with lentivirus. Compared with blank group and negative control group, the expressed Bax protein and cell apoptosis rate were increased significantly (P<0.01, P<0.05), and the expression levels of C3G mRNA, C3G protein and p-ERK1/2 protein and the cell proliferation rate were remarkably decreased in C3G silence group and C3G silence+empty plasmid group (P<0.01, P<0.05); Compared with C3G silence group and C3G silence+empty plasmid group, the expression level of Bax protein and the cell apoptosis rate were decreased obviously (P<0.01, P<0.05), while the expression levels of C3G mRNA, C3G protein and p-ERK1/2 protein and the cell proliferation rate were remarkably increased in C3G silence+C3G overexpression group (P<0.01, P<0.05). Conclusion C3G gene silence can induce apoptosis and inhibit proliferation in H9C2 cardiomyocytes, and overexpression of C3G gene can reverse the e?ects of C3G gene silence a?ecting in H9C2 cardiomyocytes, characterized by a reduction of apoptosis rate and promotion of proliferation, and they may be related to p-ERK1/2 protein and pro-apoptotic molecule Bax.

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