轻链3A在大鼠心肌细胞缺氧模型中的表达及抗凋亡机制  

Expression of Light Chain 3A and Its Anti-Apoptotic Mechanism in Rat Cardiomyocyte Hypoxia Model

作　　者：杨健康 车轰[1] YANG Jiankang;CHE Hong(Cardiac Great vessels Surgery,The First Affiliated Hospital of Anhui Medical,Hefei 230022,China)

机构地区：[1]安徽医科大学第一附属医院心脏大血管外科,合肥230022

出　　处：《岭南心血管病杂志》2024年第4期426-431,共6页South China Journal of Cardiovascular Diseases

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(CoCl_(2))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 CoCl_(2)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 CoCl_(2)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。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 CoCl_(2)-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 CoCl_(2) 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 CoCl_(2) 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.

关 键 词：线粒体自噬 轻链3A 心肌细胞 细胞凋亡 

分 类 号：R33[医药卫生—人体生理学]