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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。
OBJECTIVE: To construct an eukaryotic expression vector encoding the full length of ADRP gene and investigate the effect of ADRP overexpression on palmitate-induced H9 c2 cardiomyocyte apoptosis. Methods: (1) PCR method was used to amplify the full-length cDNA of ADRP from the cDNA of adult SD rat myocardium, and then recombined into pEGFP-C1 plasmids. After digestion and sequencing, the recombinant plasmid was transformed into Escherichia coli DH5α, the positive clones were picked out, and the plasmids were amplified after amplification for enzyme digestion and sequencing; (2) The identified recombinant plasmids were transfected into H9 c2 cardiomyocytes by lipofection, and screened by G418 to obtain resistance The expression of green fluorescent protein (EGFP) reporter gene was observed by fluorescence microscope. The expression of ADRP mRNA and protein in transfected and normal cells was detected by RT-qPCR and Western blot. (3) Flow cytometry was used to detect the expression of green fluorescent protein Effect of palmitate on the apoptosis rate of the above cells. Results: (1) Enzyme-linked digestion and DNA sequence analysis confirmed that the target gene was inserted into the expression plasmid pEGFP-C1. (2) The expression of green fluorescent protein in cells was observed by fluorescence microscope and disappeared after more than 20 passages; RT-qPCR and Western The results showed that the expression of ADRP mRNA and protein in H9 c2 cells transfected with recombinant plasmids was significantly higher than that in empty plasmid transfected and normal control groups (P <0.01). (3) After stimulation with different palmitate, the recombinant plasmids were transfected into H9 c2 The apoptosis rate was significantly lower than that of the empty plasmid transfected group and the normal H9 c2 cell group (P <0.05). Conclusions: (1) H9 c2 cell eukaryotic plasmid expression vector pEGFP-C1-ADRP was successfully constructed and H9 c2 cardiomyocyte strain stably expressing ADRP gene was obtained. (2) ADRP could inhibit palmitate-induced H9 c2 cardiomyocytes Apoptosis, indicating that ADRP may have some protective effect on cardiomyocytes in high fat environment.