【摘要】 目的:探讨GCN5调控sublytic C5b-9刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)诱导Cyclin D1基因表达以及GCN5乙酰化修饰SOX9转录因子的作用。方法:先用Western blot检测Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠的肾组织和sublytic C5b-9刺激的GMC中GCN5、SOX9和Cyclin D1的表达。然后分别将pIRES2-GCN5过表达和shGCN5小干扰质粒或将这些质粒与Cyclin D1启动子质粒行不同组合转染GMC,用荧光素酶报告基因实验和real-time PCR、Western blot检测过表达或沉默GCN5后对Cyclin D1表达的影响。同时用免疫共沉淀(Co-IP)和Western blot检测sublytic C5b-9刺激或分别转染GCN5过表达和酶活性突变质粒(ΔGCN5)的GMC中SOX9的乙酰化修饰。结果:Thy-1N大鼠肾组织和sublytic C5b-9刺激的GMC中GCN5、SOX9、Cyclin D1蛋白水平均明显上调,而过表达GCN5基因能上调Cyclin D1的启动子活性、mRNA和蛋白表达;敲低GCN5表达则得到了相反的结果。此外,sublytic C5b-9刺激和过表达GCN5基因促进了SOX9的乙酰化修饰,而沉默GCN5后则降低了SOX9的乙酰化水平。结论:GCN5可上调GMC中Cyclin D1的表达,并促进SOX9的乙酰化修饰。更多还原

【Abstract】 Objective:This study aims to explore the effects of Cyclin D1 expression regulated by GCN5 and the acetylation modification of SOX9 by GCN5 in the glomerular mesangial cells(GMC)stimulated by sublytic C5b-9. Methods:Firstly,GCN5,SOX9 and Cyclin D1 expression in the renal tissue of Thy-1 nephritis(Thy-1 N)rats(in vivo)and in sublytic C5b-9-treated GMC(in vitro)was examined using Western blot. Then the plasmids of pIRES2-GCN5 and shGCN5 or these plasmids with Cyclin D1 promoter plasmids were respectively transfected rat GMC,and Cyclin D1 gene initiation,transcription and expression were measured by luciferase reporter gene assay,real-time PCR and Western blot. Meanwhile,SOX9 acetylation was detected by co-immunoprecipitation(Co-IP)and Western blot in the GMC exposed to sublytic C5b-9 or transfected GCN5 overexpression or enzyme activity mutation(ΔGCN5)plasmids. Results:The expression level of GCN5,SOX9 and Cyclin D1 was significantly up-regulated both in vivo and in vitro,and GCN5 gene overexpression increased Cyclin D1 promoter activity,mRNA transcription and protein expression;GCN5 gene knockdown made for the opposite results. Besides,SOX9 acetylation was also elevated upon sublytic C5b-9 stimulation and GCN5 gene overexpression;SOX9 acetylation was reduced upon GCN5 knockdown. Conclusion:GCN5 can promote Cyclin D1 expression and acetylate SOX9 in GMC.更多还原