miR-200c在腹膜透析流出液细胞中的表达变化及意义

    Expression and significance of miR-200c in mesothelial cells of the peritoneal dialysate effluent fluid of peritoneal dialysis patients

    • 摘要: 目的 检测腹膜透析(PD)患者PD流出液细胞发生上皮-间充质细胞转换(EMT)的分子标志物蛋白及miR-200c的表达,初步探讨miR-200c与人腹膜间皮细胞(HPMCs)EMT的关系。方法 随机选取PD患者28例,将其分为新开管组(12例)和PD ≥ 6个月组(16例),分离培养并鉴定2组患者PD流出液细胞;通过实时定量荧光(Realtime)PCR检测流出液细胞中miR-200c的表达;利用Realtime PCR及蛋白免疫印迹法(Western blot)分别检测流出液细胞中E-钙黏素(E-cadherin)、波形蛋白(Vimentin)、纤维连接蛋白(FN)、胶原蛋白(Collagen)的mRNA及蛋白表达。结果 PD流出液细胞经鉴定为HPMCs,不同透析龄患者流出液细胞存在形态学改变。新开管组流出液HPMCs中miR-200c相对表达量为(7.55±0.34),PD ≥ 6个月组流出液HPMCs中miR-200c的相对表达量为(1.7±1.1),较新开管组明显下降(P<0.01);PD ≥ 6个月组患者流出液HPMCs中E-cadherin mRNA及蛋白表达均显著低于新开管组(P<0.05),而在PD ≥ 6个月组流出液HPMCs中Vimentin、Col-1及FN mRNA及蛋白表达明显高于新开管组(P<0.05)。结论 PD患者腹透流出液细胞经证实为HPMCs,并表达miR-200c;随PD时间延长,miR-200c表达显著降低并同时伴有EMT发生,提示miR-200c与HPMCs的EMT相关,并可能参与了HPMCs的EMT的调控。

       

      Abstract: Objective To detect the expression of molecular marker protein and miR-200c in the mesothelial cells of the peritoneal dialysate effluent fluid of peritoneal dialysis (PD) patients, and to investigate the association between miR-200c and human peritoneal mesothelial cell (HPMC) epithelial-mesenchymal transition(EMT). Methods Twelve patients recently given continuous ambulatory peritoneal dialysis (initial PD group) and 16 patients undergoing peritoneal dialysis for more than 6 months (PD over 6 months group) were randomly chosen for the isolation, culture and identification of effluent cells. Real-time PCR and Western blotting were used to detect the expression levels of miR-200c, and the levels of four cellular marker proteins:E-cadherin, vimentin, fibronectin (FN) and COL-1, in effluent cells. Results Cells isolated from effluents of dialysis fluid were identified as HPMCs. The morphology of effluent cells varied according to how long patients undergoing PD. The relative expression of miR-200c in the effluents HPMCs of the initial PD group was (7.546±0.341), and the relative expression of miRNA-200c in the effluent HPMCs of the PD over 6 months group was (1.701±1.070)(P<0.01). The level of E-cadherin mRNA and protein expression was significantly lower in the PD over 6 months group than in the initial PD group, while vimentin, FN and COL-1 mRNA and protein expression levels were significantly increased in the PD over 6 months group as compared with those in the PD initial group. Conclusions Cells isolated from effluents of dialysis fluid were identified as HPMCs, and the expression of miRNA-200c in HPMCs were confirmed. With the prolongation of PD time, miR-200c expression was significantly reduced and associated with EMT, suggesting that miR-200c may be associated with peritoneal fibrosis.

       

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