脂肪分化相关蛋白对软脂酸诱导的H9c2心肌细胞凋亡的影响  

Effect of adipose differentiation-related protein on the apoptosis of H9c2 cells by palmitic acid

作　　者：鲍苑苑[1] 方舟[2] 周丽诺[1] 胡仁明[1] 丁薇[1] 

机构地区：[1]复旦大学附属华山医院内分泌科,复旦大学附属华山医院内分泌糖尿病研究所 [2]复旦大学生命科学院,上海200040

出　　处：《细胞与分子免疫学杂志》2011年第4期382-385,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30270627)

摘　　要：目的:构建编码脂肪分化相关蛋白(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H9 c2心肌细胞凋亡有何影响。方法:(1)采用PCR的方法,以成年SD大鼠心肌cDNA为模板扩增编码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,进行酶切和测序鉴定;(2)采用脂质体转染法将鉴定后的重组质粒转染H9 c2心肌细胞株,经G418筛选获得抗性细胞克隆后扩大培养;荧光显微镜观察细胞内绿色荧光蛋白报告基因表达情况;RT-qPCR和Western blot检测转染细胞与正常细胞ADRP mRNA和蛋白表达水平;(3)采用流式细胞术检测不同浓度软脂酸对上述细胞细胞凋亡率的影响。结果:(1)酶切图谱分析和DNA序列测定证实目的基因已插入表达质粒pEGFP-C1;(2)荧光显微镜观察细胞内有绿色荧光蛋白表达且传20代以上无消失;RT-qPCR和Western blot证明重组质粒转染H9 c2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);(3)经不同软脂酸刺激后,重组质粒转染H9 c2细胞凋亡率明显低于空质粒转染组和正常H9 c2细胞组(P<0.05)。结论:(1)成功构建了H9 c2细胞真核质粒表达载体pEGFP-C1-ADRP,获得了稳定表达ADRP基因的H9 c2心肌细胞株;(2)ADRP能够抑制软脂酸诱导的H9 c2心肌细胞凋亡,表明ADRP对高脂环境中心肌细胞可能具有一定的保护作用。AIM：To construct a eukaryotic expression vector for expressing adipose differentiation-related protein（ADRP） protein and obtain a stable transfected H9c2 cell line.METHODS：（1）The full length DNA fragment of ADRP gene was amplified by PCR from the myocardium of adult SD rats and was inserted into pEGFP-C1 with T4 ligase.After the identification of digestion and sequencing on the recombinant pEGFP-C1-ADRP,the recombinant was transformed into E.coli strain DH5α.and the positive recombinant plasmid was selected.（2）The selected positive recombinant clones were amplified and transfected into H9c2 cells by lipofectamineTM2000.Cells containing stable transformants were selected by the ability of resistance to G418.The stable transfected cell line expressing high level of ADRP were obtained.Expression of green fluorescent protein gene was observed under fluorescence microscope and the expression of ADRP was identified by RT-qPCR and Western blot analysis.（3）The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry.RESULTS：（1）Restriction map analysis and DNA sequencing analysis revealed that our target gene fragment was inserted into the expressing vector pEGFP-C1 successfully.（2）Green fluorescent was observed by fluorescence microscope on the cells which were transfected with pEGFP-C1 and pEGFP-C1-ADRP and did not disappear even at the twentieth passage of H9c2 cells.RT-qPCR and Western blot analysis showed that recombinant cells exhibit higher levers of mRNA and protein.（3）After treatment with different levels of PA,the apoptosis percentage of recombinant cells was lower than those of the other two cells.CONCLUSION：（1）The eukaryotic expression vector pEGFP-C1-ADRP and stable transfected H9c2 cells were established successfully.（2）The overexpression of ADRP gene could prevent apoptosis of cells caused by PA,which indicated that ADRP might play a protective role in H9c2 cells.

关 键 词：脂肪分化相关蛋白 H9C2心肌细胞 软脂酸 凋亡 

分 类 号：R392.12[医药卫生—免疫学]