脂肪分化相关蛋白真核表达载体的构建及其对H9c2心肌细胞增殖和凋亡的影响

摘要

AIM : To construct an adipose differentiation - related protein( ADRP ) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid( PA ).METHODS: The ADRP gene obtained by the method of RT - PCR was cloned into pEGFP - C1 plasmid.The recombinant plasmid was transformed into E.coli DH5α for amplification.The recorubinant plasmid was extracted from E.coli DH5α and transfected into H9c2cells by LipofectamineTM2000.The stable transformants were selected by G418 screening.Expression of green fluorescent protein was ohserved under fluorescence microscope and the ADRP expression was identified hy RT - qPCR and Western hlotting analysis.The effect of PA on the proliferation of H9c2 cells was detected hy MTT assay.The apoptotic percentage of H9c2 cells caused by PA was deterruined by flow cytometry.RESULTS : The eukaryotic expression vector pEGFP - Cl - ADRP was successfully constructed.Green fluorescent was observed in the cells transfected with pEGFP - Cl or pEGFP - Cl - ADRP under fluorescence microscope.RT - qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP.After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells.CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established.The over - expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2cells.%目的:构建编码脂肪分化相关蛋白(adipose differentiation-related protein, ADRP)基因全长序列的真核表达载体,探讨ADRP过表达对软脂酸诱导的H9c2心肌细胞凋亡有何影响.方法:(1)RT-PCR扩增编码ADRP全长的 DNA 序列,重组入pEGFP-C1质粒表达载体中,酶切、测序鉴定后转化大肠埃希菌DH5α,挑取阳性克隆,扩增后提取质粒并进行鉴定;采用脂质体转染法将鉴定后的重组质粒稳定转染H9c2心肌细胞;荧光显微镜下观察细胞绿色荧光蛋白表达情况;RT-qPCR和Western blotting检测ADRP表达情况;(2)采用MTT比色法和流式细胞术检测不同浓度软脂酸对细胞增殖和凋亡的影响.结果:(1)成功构建了真核质粒表达载体pEGFP-C1-ADRP,转染H9c2细胞后,荧光显微镜下观察发现细胞内有绿色荧光蛋白表达;RT-qPCR和Western blotting显示重组质粒转染H9c2细胞ADRP mRNA和蛋白表达水平明显高于空质粒转染组和正常对照组(P＜0.01);(2)经不同软脂酸刺激后,重组质粒转染H9c2细胞增殖抑制率和凋亡率均明显低于空质粒转染组和正常对照组(P＜0.05).结论:(1)成功获得了稳定表达ADRP的H9c2心肌细胞株;(2)软脂酸可抑制H9c2心肌细胞增殖并可诱导其凋亡,而ADRP高表达可对抗软脂酸的这一作用,表明ADRP对高脂环境中心肌细胞可能具有一定的保护作用.