摘要
目的探索姥鲛烷诱导的系统性红斑狼疮(SLE)模型小鼠肾小球系膜细胞过表达AMP激活蛋白激酶(AMPK)对系膜细胞增生、基质合成和炎性因子分泌的影响。方法小鼠分为对照组和模型组,姥鲛烷诱导建立小鼠SLE模型。ELISA检测血清自身抗体水平;分离培养对照组和模型组小鼠原代肾小球系膜细胞,模型组小鼠原代肾小球系膜细胞分为模型组、空载体组和过表达组。pEGFP-C1-vector和pEGFP-C1-AMPK分别转染空载组和过表达组细胞。Western blot法检测AMPK证明其过表达情况。AMPK过表达后,采用流式细胞术检测系膜细胞的凋亡情况,Western blot法检测细胞周期蛋白D1(cyclin D1)、增殖细胞核抗原(PCNA)、P21、P27、纤连蛋白(FN)和Ⅳ型胶原蛋白(Col4)、白细胞介素6(IL-6)、IL-1β蛋白水平。结果模型组小鼠血清抗dsDNA IgG、抗dsDNA IgM、抗Sm IgG、抗Sm IgM水平明显增加,说明建模成功。在SLE模型小鼠肾小球系膜细胞过表达AMPK后,系膜细胞凋亡率明显增加、cyclin D1、PCNA和P21蛋白水平降低、P27蛋白水平增加。FN和Col4及IL-6和IL-1β蛋白水平明显降低。结论过表达AMPK抑制姥鲛烷诱导的狼疮鼠中肾小球系膜细胞增生,基质合成和炎症反应。
Objective To explore the effects of AMP-activated protein kinase(AMPK) over-expression on the proliferation,matrix synthesis and inflammatory cytokine production in glomerular mesangial cells from pristane-induced systemic lupus erythematosus( SLE) mice. Methods C57 BL/6 mice were randomly divided into control group and model group. The SLE mouse model was established by the treatment with pristane. The levels of autoantibodies were detected by ELISA. The primary glomerular mesangial cells were isolated from the mice of the control and model groups. The primary glomerular mesangial cells of the model mice were divided into model subgroup,empty vector subgroup and over-expression subgroup.The pEGFP-C1-vector and pEGFP-C1-MPK were transferred into the cells of the empty vector and over-expression subgroups, respectively. The expression of AMPK was tested by Western blotting. After AMPK was successfully over-expressed,cell apoptosis was detected by flow cytometry. The expressions of AMPK,cyclin D1,proliferating cell nuclear antigen( PCNA),P21,P27,fibronectin( FN),collagen Ⅳ( Col4),interleukin 6( IL-6) and interleukin 1 β( IL-1 β)were measured by Western blotting. Results The levels of serum dsDNA IgG,dsDNA IgM,Sm IgG,Sm IgM significantly increased in the model group compared with the control group,indicating that the mouse model used in this study successfully imitated the pathological process of SLE. In addition,over-expression of AMPK promoted the apoptosis of mesangial cells from the SLE mice. Moreover,the expressions of cyclin D1,PCNA,P21,FN,Col4,IL-6 and IL-1β were markedly depressed,whereas the level of P27 protein was raised in these cells after AMPK over-expression. Conclusion AMPK over-expression can inhibit the cell proliferation,matrix synthesis and inflammatory response of mesangial cells in pristane-induced SLE mice.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第11期1479-1485,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
2010年河南省医学科技攻关计划(201003089)