RNA干扰下调Gremlin表达对高糖培养的大鼠肾小球系膜细胞分泌FN、ColⅣ的影响  

Effects of RNA interference down-regulating Gremlin on extracellular secretions of FN and Col Ⅳ in rat mesangial cells cultured with high glucose

作　　者：宋玉文[1] 韩文霞[1] 高聆[2] 于春晓[1] 刘丹丹[1] 赵家军[1] 

机构地区：[1]山东大学附属省立医院内分泌科,山东省临床医学研究院内分泌代谢研究所,山东省内分泌代谢病临床医学中心,济南250021 [2]山东大学附属省立医院中心实验室,济南250021

出　　处：《山东大学学报（医学版）》2012年第4期5-9,14,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助项目(30701130);山东省自然科学基金资助项目(03BS023)

摘　　要：目的探讨在高糖培养条件下,应用慢病毒介导的RNA干扰下调Gremlin表达对大鼠肾小球系膜细胞(RMCs)分泌纤维连接蛋白(FN)、Ⅳ型胶原(ColⅣ)的影响及机制。方法构建Gremlin基因特异性的短发夹双链RNA(shRNA)慢病毒GREM1-RNAi-LV,感染RMCs后,分为5.5 mmol/L葡萄糖组、30 mmol/L葡萄糖组、30 mmol/L葡萄糖+GREM1-RNAi-LV组及30 mmol/L葡萄糖+NC-GFP-LV(感染携带绿色荧光蛋白的阴性对照慢病毒)组,分别处理48 h,RT-PCR检测Gremlin mRNA表达,Western Blot检测Gremlin蛋白表达,ELISA法检测细胞上清FN、ColⅣ蛋白表达变化。结果与5.5 mmol/L葡萄糖组相比,30 mmol/L葡萄糖组Gremlin mRNA和蛋白水平显著升高(P均<0.05),细胞外基质分泌FN、ColⅣ蛋白增加(P均<0.05),而RNA干扰能下调高糖诱导的Gremlin mRNA与蛋白高表达(P<0.05),降低细胞外基质FN、ColⅣ蛋白分泌(P<0.05)。结论慢病毒介导的RNA干扰可明显抑制高糖诱导的Gremlin高表达,降低细胞外基质分泌,改善肾脏纤维化,可能是一个预防和治疗糖尿病肾病新型的潜在靶点。Objective To explore the effects and mechanism of RNA interference mediated by lentivirus down-regulating Gremlin on extracellular secretions of fibronectin（FN） and collagen type Ⅳ（Col Ⅳ） in rat mesangial cells（RMCs） cultured with high glucose.Methods The lentivirus vector based on small hairpin RNA（shRNA） targeting Gremlin and GREM1-RNAi-LV,was constructed and delivered to RMCs cultured with high glucose.Then the cells were treated for another 48 h with 5.5 mmol/L glucose,30 mmol/L glucose,30 mmol/L glucose plus GREM1-RNAi-LV or 30 mmol/L glucose plus NC-GFP-LV.Gremlin mRNA expressions were detected by RT-PCR,and Western Blot was used for detecting Gremlin protein.FN and Col Ⅳ in the cell culture fluid were detected by ELISA.Results Compared with those in the 5.5 mmol/L glucose group,expression of Gremlin and the secretion of FN and Col Ⅳ in the cell culture fluid obviously increased in the 30mmol/L glucose group（P0.05）.RNA interference mediated by lentivirus successfully suppressed high glucose inducing over-expression of Gremlin mRNA and protein（P0.05）,and at the same time reduced the extracellular secretion of FN and Col Ⅳ（P0.05）.Conclusions With RNA interference mediated by lentivirus,Gremlin mRNA and protein are efficiently inhibited and the secretion of extracellular matrixes decreased,which can improve renal fibrosis.Gremlin may serve as a new therapeutic target in the prevention and treatment of diabetic nephropathy.

关 键 词：RNA干扰 糖尿病肾病 GREMLIN 纤维连接蛋白 Ⅳ型胶原 

分 类 号：R587.1[医药卫生—内分泌]