| 摘要: | 腹膜透析(Peritoneal dialysis, PD)是終末期腎功能衰竭患者的一種治療選擇。 目前，全球約有 11%的腎衰竭患者使用腹膜透析作為治療，長期進行腹膜透析可 能導致腹膜形態學改變和功能惡化。長期腹膜透析可能引起腹膜炎症和腹膜間皮 -間質轉化(Mesothelial-to-Mesenchymal Transition, MMT)，更嚴重則會轉變為包 覆性腹膜硬化(EPS)。根據先前研究指出，長時間進行腹膜透析治療、透析液 中的葡萄糖濃度與腹膜炎都會是引起 MMT 的主要原因。在臨床上他莫昔芬 (Tamoxifen)已經被用於一些纖維化疾病的治療且能有效減緩病徵，例如由腹 膜透析所產生的包膜性腹膜硬化症。目前，由葡萄糖透析液引起 MMT 的發病機 制仍未完全闡明。為了解決這些問題，先前實驗室的研究結果顯示，有五個候選 miRNA 在 EPS 和非 EPS 患者之間具有顯著的倍數變化。因此，我們使用人類肋 膜間皮細胞(MeT-5A)，在體外環境下檢測這些候選 miRNA 對腹膜纖維化的影 響。
首先，我們觀察到透析液在長時間作用於細胞中會對細胞產生毒性，因此我 們使用 TGF-β1 加入間皮細胞中，誘導促纖維化型態發生。在細胞加入 TGF-β1 處理後，使用 CMFDA 染劑觀察細胞形態的變化和纖維化活性的膠原凝膠收縮 測試。此外，TGF-β1 處理 MeT-5A 細胞後，間充質相關標誌物(mesenchymal marker)如:N-鈣粘蛋白(N-cadherin)和波形蛋白(Vimentin)的表現增加。接 著，我們為了研究候選 miRNA 對腹膜纖維化的影響，因此將候選 miRNA 轉染 至 MeT-5A 並同時加入 TGF-β1 進行共同處理，結果發現當過度表達 miR-17-5p、 miR-202-3p 與 miR-483-5p 時，能改變 TGF-β1 誘導的細胞形態。此外，當過度 表現 miR-483-5p 時，能減弱 TGF-β1 誘導含有間皮細胞的凝膠收縮。在西方墨 點法結果顯示，當過度表達 miR-17-5p 時，能增加 E-cadherin 的表現的同時也能減少 vimentin 的表現。當過表達 miR-202-3p 時，能抑制 TGFβ 上游信號路徑的 p-Smad2/3 的蛋白表現，結果表明候選 miRNA 可能參與抑制間皮細胞-間質轉化 的作用，我們將進一步探討這些候選 miRNA 對腹膜纖維化發展的潛在機制。;Peritoneal dialysis (PD) is a treatment option for patients with end-stage renal failure. Currently, approximately 11% of patients with renal failure worldwide are treated with PD, which causes peritoneal morphologic changes and functional deterioration. Long-term peritoneal dialysis may cause peritoneal inflammation and mesothelial-to-mesenchymal transition (MMT) of the peritoneum, and the worst case will cause encapsulated peritoneal sclerosis (EPS). It has been reported that the duration of PD treatment, the glucose concentration in the dialysate, and the occurrence of peritonitis are the major reasons cause MMT. Of note, tamoxifen has been clinically proven to be effective for a series of fibrotic diseases, such as PD-related encapsulated peritoneal sclerosis. At present, the pathogenesis of MMT caused by glucose dialysate is still not fully elucidated. From our previous study, the five candidate miRNAs showed significant fold changes between the EPS and non-EPS patients. Therefore, we dissected the functions of these candidate miRNA on peritoneal fibrosis by in vitro assays in human pleural mesothelial cells (MeT-5A).
In this study, we observed that dialysate caused time and dose-dependent toxicity in the cells, so we used TGF-β1 to induce a profibrotic phenotype in the mesothelial cells. The cellular morphology detected by cell-tracker green CMFDA dye and the fibrosis activity measured by contraction assay were changed after TGF-β1 treatments. Furthermore, the expressions of mesenchymal-related markers including N-cadherin, and vimentin were increased after TGF-β1 treatment in MeT-5A cells. Next, we investigated the effect of candidate miRNAs on peritoneal fibrosis, so we transfected candidate miRNAs into MeT-5A and added TGF-β1 for co-treatment, the results showed that TGF-β1-induced cell morphology and fibrotic phenotype could be suppressed by transfection of three individual miRNA mimics. Additionally, when miR-483-5p was overexpression, the TGF-β1-induced contraction of the gel containing mesothelial cells was attenuated. The western blot results showed that overexpression of miR-17-5p increased E-cadherin′s expression and decreased vimentin′s expression. When miR-202-3p was overexpressed, it inhibited the p-Smad2/3 protein of the TGF- β upstream signaling pathway. The results suggest that candidate miRNAs may be involved in the inhibition of mesothelial-to-mesenchymal transition, and we will further explore the potential mechanism of these candidate miRNAs in the development of peritoneal fibrosis. |
