包覆性腹膜硬化症相關miRNAs在腹膜纖維化之研究

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姓名

吳柏儒(Po-Ju Wu) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

包覆性腹膜硬化症相關miRNAs在腹膜纖維化之研究
(Study of Encapsulating peritoneal sclerosis related miRNAs in peritoneal fibrosis)

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至系統瀏覽論文 (2027-10-6以後開放)

摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 包覆性腹膜硬化症
★ 小分子核糖核酸
★ 腹膜纖維化

關鍵字(英)

★ Encapsulating peritoneal sclerosis
★ MicroRNA
★ Peritoneal fibrosis

論文目次

目錄
中文摘要 ..................................................................................................................i
Abstract .................................................................................................................iii
誌謝 .........................................................................................................................v
圖目錄 ...............................................................................................................viii
表目錄 .................................................................................................................ix
符號說明 ..................................................................................................................x
ㄧ、介紹（Introduction）..........................................................................................1
1. 腹膜透析（Peritoneal dialysis)...........................................................................1
2. 包囊性腹膜硬化症（Encapsulating peritoneal sclerosis)....................................3
2-1 腹膜纖維化的發生（Development of peritoneal fibrosis）..................................3
2-2 包覆性腹膜硬化症的診斷與治療（Diagnosis and Treatment of Encapsulating peritoneal sclerosis） ..........................................................................................4
2-3 miRNA作為診斷EPS的生物標誌物（miRNA as a biomarker for diagnosis of EPS） .................................................................................................................4
3. 轉化生長因子信號通路（TGF-β signaling pathway） .....................................5
3-1 TGF-β透過活化典型Smad信號誘導EMT發生 ....................................................5
3-2 TGF-β透過活化非典型信號途徑誘導EMT發生 ....................................................6
4. 微型核糖核酸（microRNA） ...........................................................................8
4-1 微型RNA的生成（Genesis of miRNA） ............................................................8
4-2 微型RNA的調控機制（Regulatory mechanism of miRNA） ..............................8
4-3 微型RNA在疾病上的應用（miRNA application in disease） ............................10
5. 研究目的（Purpose of the study） ...........................................................11
5-1 候選miRNA在EMT或纖維化中的作用 ...........................................................11
5-2 探討候選miRNA在腹膜間皮-間質轉化中的作用 ...........................................13
二、實驗材料與方法(Materials and methods) ...................................................14
1. 實驗材料（Material） .................................................................................14
1-1.細胞株（Cell lines） .........................................................................................14
1-2. 藥物（Drugs） .........................................................................................14
1-3. 螢光染色試劑（Fluorescent reagent） ...........................................................14
1-4. 核糖核酸模擬物（miRNA mimics） ..................................................................14
1-5. 膠原凝膠收縮試劑（Type I Collagen Solution） ...........................................15
1-6. 抗體（Antibodies） .........................................................................................15
2.實驗方法（Method） .........................................................................................15
2-1.細胞增殖檢測實驗（Cell proliferation assay） ...........................................15
2-2. miRNA模擬物轉染（miRNA mimic transfection） ...................................15
2-3. 螢光染色（Fluorescent staining） ..................................................................16
2-4. 蛋白質萃取製備（Preparation of protein extraction） ....................................16
2-5. 西方墨點法（Western blot） ..........................................................................17
2-6. 膠原凝膠收縮測定（Collagen gel contraction assay） ....................................17
2-7. 即時定量聚合酶連鎖反應（RT-qPCR） ...........................................................18
三、實驗結果（Results） .................................................................................19
1. 葡萄糖透析液對間皮細胞的生長影響 ...........................................................19
2. TGF-β1誘導MeT-5A細胞發生間皮-間質轉化 ...................................................19
3. 候選miRNA在MeT-5A細胞中的表現 ..........................................................20
4. 候選miRNA調控TGF-β1誘導的細胞形態變化 ...................................................21
5. 候選miRNA調控間皮細胞的收縮能力 ..........................................................22
6. miR-17-5p、miR-202-3p與miR-483-5p調控間皮細胞中TGF-β1/Smad2/3信號途徑與間皮-間質轉化 ................................................................................................23
四、結論與討論 ................................................................................................27
1. 探討葡萄糖透析液內容物對間皮細胞-間質轉化的影響 ...................................27
2. 探討候選miRNA對間皮細胞中TGF-β1/Smad2/3信號途徑與間皮-間質轉化的影響 ..............................................................................................................................28
3. 探討候選miRNA對TGF-β1誘導間皮細胞凝膠收縮的影響 ...................................28
4. 探討miR-17-5p在大鼠腹膜纖維化中的表現 ..................................................29
5. 探討候選miRNA在不同疾病中的表現與影響 ..................................................30
6. 未來展望 .......................................................................................................31
五、參考資料與文獻 ........................................................................................32

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指導教授

馬念涵(Nian-Han Ma)

審核日期

2022-10-11

推文