miR-200a在腹膜透析相关性腹膜纤维化中的表达  

Expression of miR-200a in peritoneal fibrosis associated with peritoneal dialysis

作　　者：魏昕[1] 郝国军[1] 刘燕丽[1] 史欣甜 陈岩冰[1] 周静[1] 陈钦开[1] 

机构地区：[1]南昌大学第一附属医院肾内科,330006

出　　处：《中华肾脏病杂志》2015年第4期261-268,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81260120）;江西省自然科学基金（20122BAB215004）

摘　　要：目的通过基因芯片技术寻找与腹膜纤维化相关的关键性miRNA，体内外实验验证其表达，确定其与腹膜纤维化的关系。方法30只雄性昆明小鼠被分为对照组和实验组。腹腔注射脂多糖（LPS）＋高糖透析液建立腹膜纤维化小鼠模型。芯片法检测腹膜组织miRNA表达谱，寻找纤维化腹膜组织中差异表达的miRNA。进一步扩大样本量，用实时定量PCR法验证差异miRNA（miR-200a）表达水平。转化生长因子β1（TGF-β1）刺激，建立体外腹膜间皮细胞上皮-间充质转分化（EMT）模型，分别用细胞免疫荧光、Western印迹、RT—PCR法检测人腹膜间皮细胞中E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α—SMA）、I型胶原（Col-I）、纤连蛋白（FN）的表达变化及定位，实时定量PCR、Taqman探针法检测体外间皮细胞EMT模型中miR-200a的表达变化。结果与对照组相比，实验组小鼠腹膜组织有差异表达的miRNA表达谱。差异倍数〉2的miRNA有8个，其中miR-200a表达明显下调（差异倍数3.31，P〈0．05）。实时定量PCR验证了miR-200a在腹膜组织中的表达。体外TGF—β1诱导的腹膜间皮细胞EMT模型中发现：分别用TGF—β1作用1、2、3h后，腹膜间皮细胞的miR-200a表达水平明显下调，作用1h时下调了46％（P〈0．05）。结论miR-200a在纤维化腹膜组织及腹膜间皮细胞EMT过程中的表达明显下调，提示miR-200a与腹膜纤维化过程密切相关。作用机制可能为其影响相关靶基因，参与调控腹膜间皮细胞EMT，影响腹膜纤维化过程。Objective To find the key miRNA that relative to peritoneal fibrosis associated with peritoneal dialysis （PD） by microarray technology, and verify its expression in vitro and in vivo. Methods The peritoneal fibrosis mouse model associated with PD were established by intraperitoneal injection of lipopolysaccharide （LPS）＋ 4.25% peritoneal dialysate. The expression of miRNA was detected by microarray in peritoneal tissues. The expression of miRNA profiles between fibrotic and normal peritoneal tissues was compared. The differentially expressed miRNA （miR-20Oa） was validated by real-time PCR in lager sample size cohorts. The expressions of miR-200a were also detected in the epithelial-mesenchymal transition （EMT） process of human peritoneal mesothelium cells. Results In mice model of PD, peritoneal tissue was markedly thickened and with a massive extracellular matrix accumulation. In contrast with control, the expression level of epithelial marker E- cadherin was significantly decreased, α- SMA, Col- I and FN were remarkably increased （P 〈 0.05）. By miRNA microarray analysis, miR- 200a was significantly downregulated （3.31 folds change, P 〈 0.05） in fibrotic peritoneal tissues. The down-regulated expression level of miR-200a was also validated by real- time PCR in larger cohorts （P 〈 0.05）. Then, the expression level of miR-200a was detected in the EMT process of human peritoneal mesothelium ceils. During the process of TGF-β1 induced EMT, miR -200a was significantly down-regulated compared with the control （P 〈 0.05）. Conclusions Down- regulated expression of miR-200a was observed both during peritoneal fibrosis and TGF-131 induced EMT in vivo and in vitro, suggesting that miR- 200a may be involved in the peritoneum fibrosis by regulating the target genes of EMT.

关 键 词：上皮-间充质转分化 腹膜透析 腹膜纤维化 miR-200a 

分 类 号：R459.5[医药卫生—治疗学]