摘要
目的 构建人源自噬微管相关蛋白轻链3A(microtubule-autophagy-related protein 1 light chain 3 A,mRFP-C1-LC3A)过表达质粒,并观察其在氯化钴(CoCl2)诱导的大鼠心肌细胞(H9C2细胞)缺氧模型中表达及抗凋亡机制.方法 利用聚合酶链反应(polymerase chain reaction,PCR)技术以cDNA文库为模板克隆人源轻链3A(light chain 3A,LC3A)基因,构建mRFP-C1-LC3A过表达质粒.通过0.8 mmol/L CoCl2 处理H9C2细胞,构建大鼠心肌细胞缺氧模型.应用Western-blot以及荧光成像技术检测细胞缺氧模型中的线粒体自噬水平的变化.通过Western-blot检测H9C2细胞缺氧模型中线粒体自噬水平不同对凋亡相关蛋白B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl2)、胱天蛋白酶3(caspase-3)表达的影响.结果 质粒酶切、测序结果及Western-blot实验证明mRFP-C1-LC3A真核表达载体构建成功.Western-blot结果表明经 0.8 mmol/L CoCl2 处理H9C2 细胞后,细胞内低氧诱导因子-1(hypoxia inducible factor-1,HIF1α)蛋白表达上调、Hoechst/碘化丙啶(propidium iodide,PI)染色证实心肌细胞凋亡比例增加,缺氧模型构建成功(P<0.05).结合Western-blot以及荧光成像结果显示在H9C2细胞缺氧模型中线粒体自噬水平明显升高(P<0.05).通过巴佛洛霉素A1(BafA1)抑制线粒体自噬后,凋亡相关蛋白Bcl2表达下降、活化caspase-3表达升高,表明抑制线粒体自噬后,细胞凋亡水平进一步升高(P<0.05),Hoechst染色进一步明确结论.结论 成功构建人源LC3相关表达载体,并证实线粒体自噬可以抑制心肌细胞缺氧模型中的细胞凋亡.
Abstract
Objectives To construct a human microtubule-autophagy-related protein 1 light chain 3 A(mRFP-C1-LC3A)overexpression plasmid and observe its expression and anti-apoptotic mechanism in CoCl2-induced hypoxia model of rat cardiomyocytes(H9C2 cells).Methods The human light chain 3 A(LC3A)gene was cloned by cDNA library by polymerase chain reaction(PCR)technology,and mRFP-C1-LC3A overexpression plasmid was constructed.H9C2 cells were treated with 0.8 mmol/L CoCl2 to construct a model of hypoxia in rat cardiomyocytes.Western-blot and fluorescence imaging were used to detect changes in mitophagic levels in cellular hypoxia models.The effect of mitochondrial autophagy on the expressions of apoptosis-related proteins B-cell lymphoma-2(Bcl2)and caspase-3 in the hypoxia model of H9C2 cells were detected by Western-blot.Results Plasmid digestion,sequencing results and Western-blot experiments proved that mRFP-C1-LC3A eukaryotic expression vector was successfully constructed.The Western-blot results showed that after 0.8 mmol/L CoCl2 treatment of H9C2 cells,the expression of intracellular hypoxia inducible factor-1(HIF1α)protein was upregulated,and Hoechst/propidium iodide(PI)staining confirmed that the proportion of apoptosis in cardiomyocytes increased,and the hypoxia model was successfully constructed.Combined with Western-blot and fluorescence imaging,the results showed elevated levels of mitophagy in the hypoxic model of H9C2 cells.After inhibition of mitochondrial autophagy by BafA1,the expression of apoptosis-related protein Bcl2 decreased and active caspase-3 expression increased,indicating that the apoptosis level of cells was further increased after inhibition of mitochondrial autophagy.Conclusions Successfully constructed a human LC3-related expression vector,and confirmed that mitochondrial autophagy can inhibit apoptosis in cardiomyocyte hypoxia model.
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