泛素特异性修饰酶2-69对大鼠系膜细胞内饰胶蛋白聚糖表达和泛素化降解的调节  

Effect of USP2-69 on regulating the expression and ubiquitous degradation of intracellular decorin in mesangial cells in rats

作　　者：骆伟丽 茅幸[1,3] 孙建勇 赵仲华[1] 张志刚[1,2,3] 吴慧娟[1,2,3] 

机构地区：[1]复旦大学基础医学院病理系,上海200032 [2]复旦大学附属中山医院上海市肾病与透析研究所,上海200032 [3]复旦大学基础医学院教育部分子医学重点实验室,上海200032 [4]中科院上海生命科学研究所、健康科学研究所,上海200032

出　　处：《中国循证儿科杂志》2015年第4期297-301,共5页Chinese Journal of Evidence Based Pediatrics

基　　金：国家自然科学基金:30900683

摘　　要：目的研究泛素特异性修饰酶2-69(USP2-69)对系膜细胞(MC)内饰胶蛋白聚糖(DCN)表达和泛素化的调节作用。方法 1培养MC细胞,采用Western blot法检测大鼠肾MC内USP2-69的表达;2采用免疫共沉淀、激光共聚焦和免疫荧光法,检测MC内USP2-69是否与DCN结合,并观察两者在MC内的定位;3将pRK5-USP2-69-HA质粒瞬时转染MC后,用Western blot法检测HA、USP2-69和DCN的蛋白表达,用免疫共沉淀法检测DCN的泛素化水平;4采用USP2-69siRNA处理MC,用PCR检测USP2-69和DCN的mRNA表达,用time-course Western blot法检测DCN的半衰期,Western blot法检测DCN的下游效应分子(TGF-β1和ColⅣ)的蛋白表达。结果 1 USP2-69在MC、肝细胞(BRL-3A)和足细胞(GEC)内均有表达,其在MC内的基础蛋白表达水平显著高于BRL-3A和GEC[MCs:(0.27±0.05),BRL-3A:(0.035±0.009),GEC:(0.012±0.004),P<0.01]。2 MC总蛋白经DCN抗体免疫沉淀后,可检测到USP2-69的表达;经USP2-69抗体免疫沉淀后,可检测到DCN表达;且在MC胞质内,代表USP2-69蛋白的荧光与代表DCN蛋白的荧光存在共定位。3转染pRK5-USP2-69-HA质粒的MC内可检测到代表外源性USP2-69表达的HA蛋白,USP2-69和DCN蛋白表达的升高,同时DCN的泛素化水平明显降低。4经USP2-69 RNA干扰的MC内DCN的mRNA水平没有明显改变,但其半衰期明显缩短(3 h),且TGF-β1和ColⅣ的蛋白表达均明显升高。结论大鼠肾MC内USP2-69可与DCN相结合,及两者在胞质内共定位,USP2-69能够降低MC内DCN的泛素化水平及提高DCN蛋白总量并促进其功能。Objective To investigate the effects of ubiquitin-specific processing protease 2-69（ USP2-69） on regulating the expression and ubiquitous degradation of decorin（ DCN） in mesangial cells（ MC）. Methods Western blot was used to examine the protein expression of USP2-69 in rat MC. Co-IP,confocal and immunofluorescence were used to analyze the interaction between USP2-69 and DCN,and their location in MC. After pRK5-USP2-69-HA eukaryon expression plasmid was transfected into MC,Western blot was used to examine protein expression of HA,USP2-69 and DCN. Co-IP was used to analyze the ubiquitination of DCN. After USP2-69 was knocked down by USP2-69 RNA interference,PCR was used to analyze the mRNA expression of USP2-69 and DCN,time-course Western blot was used to analyze the half-life of DCN and Western blot was used to examine the protein levels of TGF-β1 and Col Ⅳ,two downstream effectors of DCN. Results 1 USP2-69 was expressed in MC,BRL-3A and GEC,the basal protein expression was higher in MC than in hepatocytes and podocytes [MCs：（ 0. 27 ± 0. 05） vs BRL-3A：（ 0. 035 ± 0. 009）,GEC：（ 0. 012 ± 0. 004）,P〈0. 01]. 2 After the total protein of MC was immunoprecipitated by anti-DCN antibody,USP2-69 could be detected. Conversely,DCN could be detected after the total protein of MC was immunoprecipitated by anti-USP2-69 antibody. Moreover,there was a co-localization between fluorescence of USP2-69 and DCN in cytoplasm of MC. 3 Protein expressions of HA which represented extrinsic USP2-69,elevated protein level of USP2-69 and DCN,and an obvious decrease of ubiquitinated DCN was detected in MCs transfected with pRK5-USP2-69-HA plasmid. 4 RNA interference of USP2-69 in MC didn＇t change the mRNA level of DCN,but markedly shortened the half-life of DCN from 6 h to 3 h and increased the protein expression of TGF-β1 and Col Ⅳ. Conclusion In cultured rat MC,a physical interaction and a co-localization in cytoplasm existed between USP2-69 and DCN. USP2-69 could decrease the ubiquitinated form of D

关 键 词：饰胶蛋白聚糖 泛素特异性修饰酶2-69 系膜细胞 

分 类 号：R726.9[医药卫生—儿科]