CAJ | 학술논문

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lh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.
목적 통과기인심편기술심조여복막섬유화상관적관건성miRNA,체내외실험험증기표체,학정기여복막섬유화적관계.방법 30지웅성곤명소서피분위대조조화실험조.복강주사지다당(LPS)+고당투석액건립복막섬유화소서모형.심편법검측복막조직miRNA표체보,심조섬유화복막조직중차이표체적miRNA.진일보확대양본량,용실시정량PCR법험증차이miRNA(miR-200a)표체수평.전화생장인자β1(TGF-β1)자격,건입체외복막간피세포상피-간충질전분화(EMT)모형,분별용세포면역형광、Western인적、RT-PCR법검측인복막간피세포중E개점단백(E-cadherin)、α평활기기동단백(α-SMA)、Ⅰ형효원(Col-Ⅰ)、섬련단백(FN)적표체변화급정위,실시정량PCR、Taqman탐침법검측체외간피세포EMT모형중miR-200a적표체변화.결과 여대조조상비,실험조소서복막조직유차이표체적miRNA표체보.차이배수＞2적miRNA유8개,기중miR-200a표체명현하조(차이배수3.31,P＜ 0.05).실시정량PCR험증료miR-200a재복막조직중적표체.체외TGF-β1유도적복막간피세포EMT모형중발현:분별용TGF-β1작용1、2、3h후,복막간피세포적miR-200a표체수평명현하조,작용lh시하조료46％ (P＜ 0.05).결론 miR-200a재섬유화복막조직급복막간피세포EMT과정중적표체명현하조,제시miR-200a여복막섬유화과정밀절상관.작용궤제가능위기영향상관파기인,삼여조공복막간피세포EMT,영향복막섬유화과정.
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-200a) 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 down-regulated (3.31 folds change,P ＜ 0.05) in fibrotic peritoneal tissues.The down-regulated expression level of miR-200a was also validated by realtime PCR in larger cohorts (P ＜ 0.05).Then,the expression level of miR-200a was detected in the EMT process of human peritoneal mesothelium cells.During the process of TGF-β1 induced EMT,miR -200a was significantly down-regulated compared with the control (P ＜ 0.05).Conclusions Downregulated expression of miR-200a was observed both during peritoneal fibrosis and TGF-β 1 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.