TGF⁃β1调控人肺泡上皮细胞中DNA甲基化酶和端粒酶的机制  

Regulatory mechanism of TGF⁃β1 on DNA methylase and telomerase in human alveolar epithelial cells

作　　者：孙梓越 钱力 李丹 朱瑞芳 韩永康 刘学军 SUN Ziyue;QIAN Li;LI Dan;ZHU Ruifang;HAN Yongkang;LIU Xuejun(Shanxi Medical University,Shanxi 030001 China)

机构地区：[1]山西医科大学,山西030001 [2]山西医科大学第一医院

出　　处：《护理研究》2023年第12期2138-2143,共6页Chinese Nursing Research

基　　金：山西省应用基础研究项目自然科学基金项目,编号:201601D011105;山西省应用基础研究项目青年科技研究基金项目,编号:201701D221271。

摘　　要：目的:通过转化生长因子⁃β1(transforming growth factor⁃β1,TGF⁃β1)诱导的人肺泡上皮细胞A549观察脱氧核糖核酸(DNA)甲基化酶和端粒酶的变化。方法:采用TGF⁃β1诱导A549细胞建立体外肺纤维化模型,分为对照组、TGF⁃β12μmol/L干预组、TGF⁃β15μmol/L干预组和TGF⁃β110μmol/L干预组。采用β⁃gal染色法检测细胞衰老程度;酶联免疫吸附测定(ELISA)法和逆转录荧光定量聚合酶链式反应(RT⁃qPCR)检测炎症因子白细胞介素⁃1β、白细胞介素⁃6表达变化;RT⁃qPCR检测I型胶原蛋白(COL⁃I)、纤维连接蛋白1(FN1)、张力蛋白C(Tensin⁃C)、DNA甲基转移酶(DNM)T3a、DNMT3b、钙黏蛋白(CDH1)及端粒酶逆转录酶信使核糖核酸(mRNA)的表达;蛋白质免疫印迹法(Western Blot)检测端粒酶逆转录酶蛋白表达。结果:对照组几乎未见衰老细胞,干预组细胞衰老程度随TGF⁃β1浓度升高而增加;TGF⁃β1干预组白细胞介素⁃1β、白细胞介素⁃6含量明显升高(P<0.05);对照组A549细胞形态呈典型的上皮细胞样形态,TGF⁃β1干预组出现典型的间质细胞样形态,Masson染色结果显示对照组细胞几乎未见蓝色胶原蛋白,而TGF⁃β1干预组蓝染胶原蛋白表达明显增多,发生纤维化改变;TGF⁃β1干预组与对照组比较,COL⁃I、FN1、Tensin⁃C、DNMT3a、DNMT3b mRNA表达明显升高(P<0.05),端粒酶逆转录酶和CDH1基因mRNA的表达降低(P<0.05),Western Blot进一步验证端粒酶逆转录酶蛋白表达降低(P<0.05)。结论:TGF⁃β1导致A549细胞发生衰老,并促进炎症因子和致纤维化因子高表达发生纤维化改变;TGF⁃β1促进了DNA甲基化酶的表达,抑制了端粒酶逆转录酶的表达。Objective:Changes in DNA methylase and telomerase were observed by TGF⁃β1⁃induced human alveolar epithelial cells A549.Methods:A549 cells were induced by TGF⁃β1 to establish an in vitro pulmonary fibrosis model,which was divided into a control group,TGF⁃β12μmol/L intervention group,TGF⁃β15μmol/L intervention group,and TGF⁃β110μmol/L intervention group.β⁃gal staining to detect the degree of cell senescence;ELISA and RT⁃qPCR detected the expression of inflammatory factors IL⁃1βand IL⁃6.RT⁃qPCR detects the expression of Type I collagen(COL⁃I),Fibronectin 1(FN1),Tensin⁃C,DNMT3a,DNMT3b,CDH1,and TERT mRNA.Western Blot method detected TERT protein expression.Results:There were almost no senescent cells in the control group,and the degree of senescence of cells in the intervention group increased with the increase of TGF⁃β1 concentration;IL⁃1βand IL6 expression were significantly increased in the TGF⁃β1 intervention group(P<0.05);The A549 cell morphology of the control group showed a typical epithelial cell⁃like morphology,and the TGF⁃β1 intervention group had a typical mesenchymal cell⁃like morphology,and the Masson staining results showed that the control group cells hardly saw blue collagen,while the expression of indigen⁃stained collagen in the TGF⁃β1 intervention group was significantly increased;Compared with the control group,the expression of COL⁃I,FN1,Tensin⁃C,DNMT3a,and DNMT3b mRNA was significantly increased(P<0.05),the expression of mRNA of TERT and CDH1 genes was reduced(P<0.05),and the expression of TERT protein was further verified by Western Blot(P<0.05).Conclusion:TGF⁃β1 can induce senescence of A549 cells,and promote the high expression of inflammatory factors and fibrogenic factors,resulting in fibrosis changes.TGF⁃β1 promoted the expression of DNA methylase and inhibited the expression of TERT.

关 键 词：特发性肺纤维化 转化生长因子⁃β1 A549细胞 脱氧核糖核酸甲基化 端粒酶逆转录酶 

分 类 号：R563[医药卫生—呼吸系统]