选择性上调pSmad 3C/3L基因对人肝星状细胞增殖及α平滑肌肌动蛋白、纤维酶原激活物抑制剂-1和Ⅰ型胶原蛋白表达的影响  

Effect of selective up-regulation of pSmad 3C/3L on LX-2 cell’s proliferation and α-SMA,PAI-1 and Collagen-I protein expression

作　　者：徐媛媛[1] 闻广华 张冲 杨雁[1] XU Yuanyuan;WEN Guanghua;ZHANG Chong;YANG Yan(Author Affiliation:Department of Pharmacology,Institute of Natural Medicine,Anhui Medical University,Hefei,Anhui 230032,China)

机构地区：[1]安徽医科大学药理学教研室和天然药物研究所

出　　处：《安徽医药》2019年第12期2351-2355,共5页Anhui Medical and Pharmaceutical Journal

基　　金：国家自然科学基金面上项目(81573652,81874354)

摘　　要：目的观察选择性上调pSmad 3C/3L基因对人肝星状细胞(LX 2)增殖及α平滑肌肌动蛋白(α SMA)、纤维酶原激活物抑制剂 1(PAI 1)和Ⅰ型胶原蛋白(Collagen Ⅰ)表达的影响。以进一步探究pSmad 3C/3L在肝纤维化进展中的作用机制。方法采用脂质体转染的技术(FuGENE?HD)向LX 2细胞转染野生型Smad3基因(Smad3 WT)、Smad3连接区磷酸化位点突变的基因(Smad3 EPSM)及Smad3 C末端磷酸化位点突变的基因(Smad33S A)3种质粒,选择性上调LX 2细胞中pSmad 3C/3L的表达水平,以蛋白质印迹法(Western Blot)验证转染效率并检测LX 2细胞中α SMA、PAI 1和Collagen Ⅰ的蛋白表达水平。MTT法检测选择性上调pSmad 3C/3L对LX 2细胞增殖能力的影响。结果转化生长因子 β1(TGF β1)刺激1 h后,与LX 2对照组相比,转染3种质粒组Smad3蛋白表达水平均升高[(0.48±0.02),(0.57±0.03),(0.60±0.02),(0.59±0.03);P<0.05];与Smad3 WT组相比,转染Smad3 EPSM质粒组pSmad3C蛋白表达水平明显升高[(0.33±0.02)比(0.44±0.01),P<0.05],转染Smad33S A质粒组pSmad3L蛋白表达水平明显升高[(0.36±0.01)比(0.42±0.02),P<0.05]。MTT结果显示,转染Smad3 EPSM质粒可抑制LX 2细胞增殖,而转染Smad33S A质粒可促进LX 2细胞的增殖[吸光度值(0.48±0.03)比(0.93±0.05),P<0.05]。TGF β1刺激12 h后,α SMA、PAI 1和Collagen Ⅰ在3种质粒转染组中呈现出差异表达(P<0.05)。结论转染3种质粒成功地选择性上调了LX 2细胞中pSmad 3C/3L的表达,选择性高表达pSmad3L可进一步促进TGF β1诱导的细胞增殖反应、促进α SMA、PAI 1和Collagen Ⅰ的表达,选择性高表达pSmad3C可抑制细胞增殖反应、降低α SMA、PAI 1和Collagen Ⅰ的表达;提示TGF β1诱导的Smad3不同位点磷酸化在LX 2细胞增殖和α SMA,PAI 1,Collagen Ⅰ蛋白表达的调控中发挥重要作用,为肝纤维化的逆转提供了新思路。Objective To observe the effect of selective up regulation of pSmad 3C/3L on LX 2 cell proliferation and the expres sions of alpha smooth muscle actin(α SMA),plasminogen activator inhibitor 1(PAI 1)and Collagen Ⅰ,so as to further investi gate the functional mechanism of pSmad 3C/3L in the progression of hepatic fibrosis.Methods Three plasmids,Smad3 WT(wild type Smad3 gene),Smad3 EPSM(Smad3 link region phosphorylation site mutator gene)and Smad33S A(Smad3 C terminal phos phorylation site mutator gene),were transfected in LX 2 cells by using liposome transfection technique(FuGENE?HD).The pS mad 3C/3L expression was selectively up regulated,and Western blot was used to investigate the transfection efficiency and the ex pressions ofα SMA,PAI 1 Collagen Ⅰin LX 2 cells.MTT assay was used to detect the effect of selectively up regulated pSmad 3C/3L on the proliferation of LX 2 cells.Results Compared with the LX 2 control group,after 1 h of transforming growth factor β1(TGF β1)stimulation,the levels of Smad3 expression were increased in the three plasmid transfected groups[(0.48±0.02),(0.57±0.03),(0.60±0.02),(0.59±0.03)](P<0.05).Compared with the Smad3 WT group,the level of pSmad3C expression was signifi cantly increased in the Smad3 EPSM group[(0.33±0.02)vs.(0.44±0.01),P<0.05],and the level of pSmad3L was significantly increased in the Smad33S A group[(0.36±0.01)vs.(0.42±0.02),P<0.05].The results of MTT showed that the transfected Smad3 EPSM plasmid inhibited the proliferation of LX 2 cells,while the transfected Smad33S A plasmid promoted the proliferation of LX 2 cells[absorbance value:(0.48±0.03)vs.(0.93±0.05),P<0.05].After 12 h of TGF β1 stimulation,α SMA,PAI 1,Collagen Ⅰ showed different expressions in the three plasmid transfection groups(P<0.05).Conclusions We succeeded in selec tively up regulating pSmad 3C/3L expression in LX 2 cells by transfecting three plasmids.The selective up regulation of pSmad3L promoted cell proliferation induced by TGF β1 and the expressions of α SMA,PAI 1 and Co

关 键 词：肝硬化 肝星状细胞 质粒 转染 Smad3蛋白质 转化生长因子Β 脂质体 细胞增殖 印迹法 蛋白质 

分 类 号：R73[医药卫生—肿瘤]