【作者】 周循；

【导师】 刘伏友；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 腹膜透析(peritoneal dialysis, PD)是终末期肾脏疾病(end stage renal disease, ESRD)患者最主要的替代治疗之一,但是长期腹膜透析引起腹膜组织纤维化(peritoneal fibrosis, PF)而导致超滤衰竭(ultrafiltration failure, UFF),限制了腹膜透析的应用。腹膜间皮细胞转分化(epithelial-mesenchymal transition, EMT)是腹膜纤维化的起始和可逆环节,细胞外基质(extracellular matrix, ECM)沉积是腹膜纤维化的主要组织学改变。研究表明转化生长因子-β(transforming growth factor-β, TGF-p)在腹膜透析病人腹膜EMT及ECM沉积的过程中起到了关键的作用,并受到多个因素的调控。microRNA是存在于动植物基因组中的功能性非编码小RNA,大多为21-25个核苷酸,可与靶mRNA 3’端非翻译区(untranslated region, UTR)碱基互补配对结合,从而调控相关基因的表达。microRNA广泛参与到生长发育、细胞分化、增殖、凋亡、肿瘤发生等过程中,且与许多疾病,包括EMT相关。研究显示部分microRNA在TGF-β诱导的EMT中调控相关的转录抑制因子如Smad作用蛋白-1 (Smad interacting protein 1, SIP1),或TGF-β的下游结缔组织生长因子(connective tissue growth factor, CTGF)等的表达,从而发挥对EMT的调节作用。基于以上分析,我们认为,特定的microRNA参与了调控腹膜间皮细胞EMT及纤维化。为此,本研究开展如下：目的检测腹膜透析患者腹膜透析流出液细胞中1microRNA的表达及EMT发生情况,初步探讨nicroRNA-129-5p与腹膜间皮细胞EMT的关系。方法随机选取新开管持续不卧床腹膜透析(continuous ambulatory peritoneal dialysis, CAPD)患者10人,腹透半年以上者12人,分离培养并鉴定流出液细胞；通过micro array得出二者microRNA差异表达谱并用realtime PCR Taqman探针法验证microRNA-129-5p的表达；采取realtime PCR、western blot观察流出液细胞中E-cadherin、claudin1、vimentin、FN的表达。结果腹透流出液细胞为腹膜间皮细胞,不同透龄患者流出液细胞存在形态学改变。透析半年以上组较新开管组33个microRNA表达上调,58个下调,其中microRNA-129-5p在半年以上组中明显下调。半年以上组流出液细胞E-钙黏素(E-cadherin)、claudin 1 mRNA及蛋白表达明显低于新开管组,而波形蛋白(vimentin)及纤维连接蛋白(fibronectin, FN) mRNA及蛋白表达明显高于新开管组。结论腹膜透析患者腹透流出液细胞经证实为HPMC,并表达多个EMT相关的microRNA;随腹透时间延长,microRNA-129-5p表达显著降低同时伴有EMT及ECM沉积,提示microRNA-129-5p可能与腹膜纤维化有关。目的阐明microRNA-129-5p在TGF-β1诱导的HMrSV5细胞EMT及ECM沉积中的作用,以寻找逆转腹膜间皮细胞纤维化的方法方法用5ng／ml TGF-β1刺激HMrSV5细胞,通过realtime PCR及western blot检测细胞中E-cadherin、claudin1、vimentin、FN及microRNA-129-5p的表达；转染microRNA-129-5p的前体(pre-mir-129-5p)及阴性对照pre-miR negative control,再予TGF-p1刺激,通过细胞免疫荧光、realtime PCR及western blot检测HMrSV5细胞E-cadherin、claudin1、vimentin、FN的表达变化。结果5ng／ml TGF-β1刺激后,HMrSV5细胞E-cadherin、claudin1 mRNA及蛋白表达水平呈时间依赖性降低,vimentin、FN mRNA及蛋白表达水平呈时间依赖性升高,microRNA-129-5p呈时间依赖性降低。与TGF-β1刺激组比较,过表达microRNA-129-5p使E-cadherin、claudin1上调,vimentin、FN下调；但转染pre-miR negative control对TGF-β1诱导的HMrSV5细胞E-cadherin、claudin1、vimentin、FN的表达变化无明显影响。结论TGF-β1诱导HMrSV5细胞EMT及ECM沉积；TGF-β1诱导microRNA-129-5p表达下调；上调microRNA-129-5p可逆转HMrSV5细胞EMT,抑制ECM沉积。目的阐明microRNA-129-5p参与调控TGF-β1诱导的HMrSV5细胞EMT和ECM沉积的可能机制,为靶向microRNA-129-5p防治腹膜纤维化提供理论依据。方法用5ng/ml TGF-β1刺激HMrSV5细胞,通过realtime PCR及western blot检测细胞中SIP1的表达；转染pre-mir-129-5p及阴性对照pre-miR negative control,再予TGF-β1刺激,通过细胞免疫荧光、realtime PCR及western blot检测HMrSV5细胞SIP1的表达变化。结果5ng/ml TGF-β1刺激后,HMrSV5细胞SIP1 mRNA及蛋白表达水平呈时间依赖性上调。与TGF-β1刺激组比较,过表达microRNA-129-5p使SIP1蛋白水平下调,而mRNA水平无变化；转染pre-miR negative control对TGF-β1诱导的HMrSV5细胞SIP1的表达变化无明显影响。结论TGF-β1诱导HMrSV5细胞SIP1表达上调；miRNA-129-5p通过转录后水平调节SIP1,影响E-cadherin、claudin1的表达,从而参与TGF-β1诱导的腹膜间皮细胞EMT及ECM的沉积。更多还原

【Abstract】 Background Peritoneal dialysis (PD) is an established alternative for the replacement therapy of end stage renal disease (ESRD). Unfortunately, the limitation of long-term PD is peritoneal fibrosis (PF), resulting in ultrafiltration failure (UFF). Epithelial-mesenchymal transition (EMT) is the initiate and reversible stage of PF and extracellular matrix (ECM) accumulation is the key histological change of PF. It is established that transforming growth factor-P (TGF-P) plays a key role in these processes, and is regulated by multi-factors.microRNAs are 21～25 nucleotide small non-coding RNAs that participate in gene regulation, and exist in species ranging from plants to humans genome. They regulate gene expression by base-pairing to 3’ untranslated region of target mRNAs. microRNAs have been implicated in various biological processes such as developing, differentiation, proliferation, apoptosis, tumorigenesis, et al. They are also related to pathological processes such as EMT. It is reported that EMT-related protein like Smad interacting protein 1 (SIP1) or connective tissue growth factor (CTGF) can be regulated by some microRNAs.Therefore, novel microRNAs may play a key role in the process of EMT and ECM accumulation in petitoneal dialysis. To this end, experimental research is carried out as follow. Objective To investigate the expression of microRNAs and the EMT status in HPMC from effluents, and to observe the relationship of microRNA-129-5p and EMT in peritoneal dialysis.Methods 22 patients undergoning continuous ambulatory peritoneal dialysis (CAPD) were enrolled into this study,10 patients undergoing PD start while 12 patients undergoing PD over 6 months. The isolated cells from effluents in dialysis fluid were cultured and identified by morphology and immunohistochemistry. Scaning the samples through micro array to determine the different microRNAs expression between PD-start group and PD-over-6-month group. Taqman assay was used to determine the expression of microRNA-129-5p. The expressions of E-cadherin, claudin1, vimentin and FN were tested by realtime PCR and western blot.Results Cells isolated from effluents of dialysis fluid were identified as HPMC. The morphology varied according to how long patients undergoing PD. Micro array showed that 33 miRNAs up-regulated and 58 miRNAs down-regulated in cells from patients undergoing PD over 6 months, compared to patients undergoing PD start. The down-regulation of microRNA-129-5p was clarified. Poor expression of E-cadherin and claudin1 and excessive expression of vimentin and fibronectin (FN) both in mRNA and protein level was remarkable in PD-over-6-month group, compared to PD-start group.Conclusion Multiple EMT-related microRNAs can be found in HPMC from effluents. Down-regulation of miRNA-129-5p along with EMT and ECM accumulation during long-term PD indicated that miRNA-129-5p was negatively related to peritoneal fibrosis. Methods HMrSV5 cells were exposed to 5ng/ml TGF-β1. The expressions of E-cadherin, claudin1, vimentin and FN were examined by realtime PCR and western blot, meanwhile realtime PCR for miRNA-129-5p. Furthermore HMrSV5 cells were transfected with miRNA-129-5p precursor (pre-mir-129-5p) or pre-miR negative control before exposing to TGF-β1. Then the expressions of E-cadherin, claudin1, vimentin and FN were detected by immunofluorescence, realtime PCR and western blot.Results Stimulation of HMrSV5 cells with TGF-β1 resulted in a significant decrease of E-cadherin and claudinl, and increase of vimentin and FN, all in time-dependent manner. TGF-β1 also repressed miRNA-129-5p. Compared to TGF-β1 group, overexpression of miRNA-129-5p in HMrSV5 cells upregulated the mRNA and protein expression of E-cadherin and claudinl, and downregulated the mRNA and protein expression of vimentin and FN. However, pre-miR negative control had no significant effect on the expression of E-cadherin, claudin1, vimentin and FN. Conclusion TGF-β1 leads to EMT and ECM accumulation and represses miRNA-129-5p level. Overexpression of miRNA-129-5p can reverse the EMT of HMrSV5 cells and inhibit the ECM accumulation.Ⅸ Objective To investigate the mechanism by which miRNA-129-5p modulates the process of TGF-β1 induced EMT and ECM accumulation in HMrSV5 cell lines.Methods HMrSV5 cells were exposed to 5ng/ml TGF-β1. The expression of SIP1 was examined by realtime PCR and western blot. Furthermore HMrSV5 cells were transfected with pre-mir-129-5p or pre-miR negative control before exposing to TGF-β1. Then the expression of SIP1 was detected by immunofluorescence, realtime PCR and western blot.Results Stimulation of HMrSV5 cells with TGF-β1 resulted in a significant increase of SIP1 in time-dependent manner. Compared to TGF-β1 group, overexpression of miRNA-129-5p in HMrSV5 cells downregulated the protein expression of SIP1, but had no effect on its mRNA expression. However, pre-miR negative control had no significant effect on the expression of SIP 1.Conclusion TGF-β1 leads to upregulation of SIP1. During the process of TGF-β1 induced EMT in HMrSV5 cells, miRNA-129-5p modulates E-cadherin and claudinl expression by posttranscriptional repression of SIP1.更多还原