高糖促进小鼠肾小管上皮细胞DNMT3B表达并通过激活β-catenin信号通路促进纤维化相关蛋白分泌  

High glucose promotes the expression of DNA methyltransferase 3B(DNMT3B)in cultured mouse renal tubular epithelial cells and promotes the secretion of fibrosis associated proteins by activating the Wnt/β-catenin signaling pathway

作　　者：屈玲玲 邹琴 李清璇 李晓颖 刘忠强[1] 周宇霞 郭兵 石明隽[1,3] QU Lingling;ZOU Qin;LI Qingxuan;LI Xiaoying;LIU Zhongqiang;ZHOU Yuxia;GUO Bing;SHI Mingjun(Department of Pathology and Pathophysiology,Guizhou Medical University,Guiyang 550025;Department of Nephrology,Guiyang First People’s Hospital,Guiyang 550002;Guizhou Provincial Key Laboratory of Pathogenesis and Drug Control of Major Diseases,Guiyang 550025,China)

机构地区：[1]贵州医科大学病理学与病理生理学教研室,贵州贵阳550025 [2]贵州省贵阳市第一人民医院肾内科,贵州贵阳550002 [3]贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳550025

出　　处：《细胞与分子免疫学杂志》2021年第7期608-615,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：贵州省科技计划项目(黔科合LH字[2016]7353号,黔科合基础[2016]1117号);贵阳市科技局基金项目(筑科合同[2017]30-23号)。

摘　　要：目的观察高糖条件下小鼠肾小管上皮细胞(RTEC)中DNA甲基转移酶3B(DNMT3B)对分泌型卷曲相关蛋白1(SFRP1)表达的影响及对Wnt/β联蛋白(β-catenin)信号通路的调控作用。方法将体外培养的小鼠RTEC分为正常糖(NG)组和高糖(HG)组。RTEC分别转染DNMT3B短发夹RNA(sh-DNMT3B)及DNMT3B过表达(DNMT3B-OE)质粒后,反转录PCR检测RTEC的DNMT3B、 SFRP1、Ⅳ型胶原蛋白(Col4)、纤连蛋白(FN)mRNA表达,Western blot法检测DNMT3B、 SFRP1、糖原合成酶3β(GSK3β)、磷酸化的GSK3β(p-GSK3β)、β-catenin、 Col4、 FN的蛋白表达。免疫荧光细胞化学染色检测DNMT3B和SFRP1在RTEC的表达和定位。结果与NG组相比,HG组RTEC中DNMT3B、β-catenin、 p-GSK3β、 Col4、 FN蛋白表达增加,SFRP1表达降低。与空载体组比较,敲低DNMT3B后,HG条件下RTEC的SFRP1 mRNA及蛋白表达均增加,β-catenin、 p-GSK3β、 Col4蛋白表达降低,FN的mRNA和蛋白表达均降低,而β-catenin mRNA表达无明显改变;过表达DNMT3B后,各指标表达情况与上述结果相反。DNMT3B及SFRP1在RTEC的细胞核及细胞质中均有表达,与NG组相比,HG组的细胞可见DNMT3B在细胞核内聚集,且与SFRP1的共定位增加。结论 HG刺激增加RTEC的DNMT3B表达,下调SFRP1表达,激活Wnt/β-catenin信号通路促进细胞外基质形成。Objective To observe the effects of DNA methyltransferase 3B(DNMT3B) on the expression of secreted frizzled-related protein 1(SFRP1) and regulation of Wnt/β-catenin signaling pathway in renal tubular epithelial cells(RTECs) of mice under high glucose conditions. Methods in vitro cultured mouse RTECs were divided into normal glucose(NG) group and high glucose(HG) group. After DNMT3B short-hairclip RNA(sh-DNMT3B) and DNMT3B over-expression(DNMT3B-OE) plasmids were transfected separately into RTECs, mRNA expression of DNMT3B, SFRP1, collagen Ⅳ(Col4) and fibronectin(FN) were detected by reverse-transcription PCR. Protein expression of DNMT3B, SFRP1, glycogen synthase kinase 3β(GSK3β), phosphorylated glycogen synthase kinase 3β(p-GSK3β), β-catenin, Col4 and FN weredetected by Western blotting. The localization of DNMT3B and SFRP1 in RTECs was observed by immunofluorescence cytochemistry combined with confocal microscopy. Results Compared with the NG group, the protein expression of DNMT3B, β-catenin, p-GSK3β, Col4 and FN increased in the HG group, while SFRP1 protein expression was reduced in the HG group. Compared with the sh-vector group, SFRP1 mRNA and protein expression increased in the sh-DNMT3B group, while the expression of β-catenin, p-GSK3β and Col4 proteins decreased. FN mRNA and protein expression dropped in the sh-DNMT3B group, however, the expression of β-catenin mRNA did not change significantly. Visually, DNMT3B over-expression reversed the above changes. Both DNMT3B and SFRP1 were expressed in the nucleus and cytoplasm of RTECs, and DNMT3B was aggregated in the nuclei of the cells in the HG group and the co-localization between DNMT3B and SFRP1 was also promoted in the HG group. Conclusion The expression of DNMT3B increases and the expression of SFRP1 decreases when the mouse RTECs were stimulated by HG. This subsequently leads to the activation of the Wnt/β-catenin signaling pathway and promotes the formation of extracellular matrix.

关 键 词：肾脏纤维化 小鼠肾小管上皮细胞(RTEC) DNA甲基转移酶3B(DNMT3B) 分泌型卷曲相关蛋白1(SFRP1) Wnt β联蛋白(β-catenin) 

分 类 号：R692[医药卫生—泌尿科学] R392-33[医药卫生—外科学]