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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 investigate the effects and mechanisms of lentivirus-mediated RNA interference on the secretion of fibronectin (FN) and collagen Ⅳ (Ⅳ) from rat mesangial cells (RMCs) under the condition of high glucose. . Methods Gremlin gene-specific short hairpin double-stranded RNA (shRNA) lentivirus GREM1-RNAi-LV was constructed and was divided into 5.5 mmol / L glucose group, 30 mmol / L glucose group, 30 mmol / L glucose + GREM1-RNAi-LV group and 30 mmol / L glucose + NC-GFP-LV (negative control green fluorescent protein-infected lentivirus) group were treated for 48 h, Gremlin mRNA expression was detected by RT-PCR and Gremlin protein by Western Blot The expression of FN and ColⅣ in the supernatant of the cells were detected by ELISA. Results Compared with 5.5 mmol / L glucose group, the mRNA and protein levels of Gremlin in 30 mmol / L glucose group were significantly increased (all P <0.05), FN and ColⅣ protein in extracellular matrix increased (P <0.05) Interference could downregulate the expression of Gremlin mRNA and protein induced by high glucose (P <0.05), and decrease the secretion of extracellular matrix FN and ColⅣ (P <0.05). Conclusion Lentivirus-mediated RNA interference can significantly inhibit high glucose-induced Gremlin expression, reduce extracellular matrix secretion and improve renal fibrosis, which may be a new potential target for the prevention and treatment of diabetic nephropathy.