成脂诱导剂通过PP2Ac调控人肝星状细胞活化的体外研究  

In vitro study of lipid inducer regulating the activation of human hepatic stellate cells via PP2Ac

作　　者：刘彧冰 蓝春花 卢娜 蓝利 陈成英 袁江浪 孔星星 陆彩玲 李习艺[3] 唐深[1,2] Liu Yubing;Lan Chunhua;Lu Na;Lan Li;Chen Chengying;Yuan Jianglang;Kong Xingxing;Lu Cailing;Li Xiyi;Tang Shen(School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Key Laboratory of Basic Research on Regional Diseases,Education Department of Guangxi Zhuang Autonomous Region,Guangxi Medical University,Nanning 530021,China;School of Public Health,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院,南宁530021 [2]广西医科大学广西高校区域性疾病基础研究重点实验室,南宁530021 [3]广西医科大学公共卫生学院,南宁530021

出　　处：《广西医科大学学报》2023年第9期1433-1439,共7页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.81860585,No.82160612);广西自然科学基金资助项目(No.2021GXNSFAA196019);国家级大学生创新创业训练计划项目(No.202010598026)。

摘　　要：目的:研究成脂诱导剂MDI对活化的人肝星状细胞的作用及其机制。方法:体外培养人肝星状细胞系LX-2细胞,实验分为4组,即空白对照组、溶剂对照组、重组人转化生长因子β1(TGF-β1)组、TGF-β1+MDI组。油红O染色法检测细胞内脂滴变化,实时荧光定量PCR(RT-qPCR)法检测肝纤维化标志物肌动蛋白α2(ACTA2)、Ⅰ型胶原蛋白α1(COL1A1)、金属蛋白酶组织抑制剂1(TIMP1)基因相对表达量。线粒体特异性荧光探针检测TGF-β1/MDI干预后LX-2细胞内线粒体数量的变化。采用western blotting法检测各组蛋白磷酸酶2A催化性C亚基(PP2Ac)蛋白表达。结果:与空白对照组和溶剂对照组比较,TGF-β1组LX-2细胞胞体增大、呈多边形且胞质增多、未见明显脂滴、线粒体数量增加,肝纤维化标志物ACTA2、COL1A1和TIMP-1mRNA表达水平以及PP2Ac去甲基化蛋白表达水平升高(均P<0.01)。与TGF-β1组比较,TGF-β1+MDI组细胞变小、胞质内小脂滴数量增加、线粒体数量减少,ACTA2、COL1A1和TIMP-1 m RNA表达水平以及PP2Ac去甲基化蛋白表达水平降低(均P<0.05)。结论:TGF-β1和MDI可能通过调控肝星状细胞的PP2Ac去甲基化水平调控人肝星状细胞活化,且与脂质生成以及线粒体数量有关。Objective:To investigate the role and mechanism of the lipid inducer MDI on activated human hepatic stellate cells.Methods:Human hepatic stele cell line LX-2 cells were cultured in vitro and divided into 4 groups:control group,solvent control group,recombinant human transforming growth factorβ1(TGF-β1)group and TGF-β1+MDI group.The changes of intracellular lipid droplets were detected by oil red O staining,and the relative expression levels of liver fibrosis markers Actin alpha 2(ACTA2),collagen typeⅠalpha 1(COLlA1)and tissue inhibitors of metalloproteinase 1(TIMP1)were detected by real-time fluorescence quantitative PCR(RT-qPCR).The changes of mitochondria in LX-2 cells after TGF-β1/MDI intervention were observed by mitochondrial specific fluorescence probe.The expression of protein phosphatase 2A catalyzed C subunit(PP2Ac)in each group was detected by western blotting.Results:Compared with the control group and the solvent control group,LX-2 cells of TGF-β1 exhibited cell enlargement,polygonal shape and increased cytoplasm,no distinct lipid droplets were found,the number of mitochondria increased,and the mRNA expression levels of liver fibrosis markers ACTA2,COL1A1 and TIMP-1 and the PP2Ac demethylated protein ex-pression levels increased(all P<0.01).Compared with the TGF-β1 group,the cells in the TGF-β1+MDI group became smaller,the number of intracellular lipid droplets increased,the number of mitochondria decreased,and the mRNA expression levels of liver fibrosis markers ACTA2,COL1A1 and TIMP-1 and the PP2Ac demethylated protein expession levels decreased(all P<0.05).Conclusion:TGF-β1 and MDI may regulate the activation of human hepatic stellate cells by regulating the PP2Ac demethylation levels,and are related to lipogenesis and number of mitochondria.

关 键 词：成脂诱导剂 蛋白磷酸酶2A催化性C亚基 肝星状细胞 

分 类 号：R575.2[医药卫生—消化系统]