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研究生:曾思詠
研究生(外文):Sih-Yong Zeng
論文名稱:大鼠腎臟足細胞探討狼瘡腎炎療效評估
論文名稱(外文):Effective Evaluation of Lupus Nephritis in Rat Podocyte Cell Culture
指導教授:董光中董光中引用關係
指導教授(外文):Kwong-Chung Tung
口試委員:詹昆衛
口試日期:2011-07-07
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:46
中文關鍵詞:尿肌酶尿肌酶尿肌酶尿肌酶
外文關鍵詞:podocyteuPARIL-6
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全身性紅斑狼瘡 (systemic lupus erythematosus,SLE) 為一種自體免疫疾病。其中,狼瘡腎炎 (lupus nephritis),LN的患者經常伴隨著蛋白尿,其原因在於絲球體尿液形成結構中的足狀細胞從基底膜 (basement membrane) 上剝離而產生蛋白尿。近幾年研究指出,LN產生蛋白尿機轉在於尿激酶接受器或CD87 (urokinase plasminogen activator receptor,uPAR) 會誘導足狀細胞剝離。然而,LN患者腎病變程度又與細胞介白素-6 (Interleukin-6,IL-6) 增加有關,並且透過IL-6接受器單株抗體 (monoclonal antibody Interleukin-6 receptor,mAb IL-6R) 的給予阻斷IL-6結合作用,會顯著降低蛋白尿程度。由於目前研究中尚無資料證實IL-6和mAb IL-6R與足狀細胞內uPAR的相互作用機制影響蛋白尿程度。本研究目的為證實IL-6刺激uPAR增加以誘導足狀細胞剝離導致蛋白尿,並在mAb IL-6R給予後可以阻斷IL-6結合抑制足細胞移行以降低蛋白尿程度達到治療目的。實驗方法利用大鼠分離出腎絲球於體外進行足狀細胞培養,並以免疫染色標定synaptopodin及podocin確定為足細胞,培養至適當細胞量 (1×103/mL) 進行試驗;實驗分組為:正常對照組,IL-6處理組 (100 ng/mL) 及mAb IL-6R (25 μg/mL) 與IL-6 (100 ng/mL) 併用組。評估指標為:(1) 以反轉錄聚合酶連鎖反應 (qRT-PCR)進行足細胞中uPAR mRNA定量分析,(2) 計算足狀細胞移行數量, (3) 免疫螢光染色分析uPAR及轉錄激活因子3 (signal transducer and activator of transcription 3;STAT3) 蛋白表現量。實驗結果顯示:IL-6組足狀細胞uPAR與p-STAT3蛋白表現量、移行數量及uPAR mRNA定量分別與正常對照組、mAb IL-6R/IL-6兩組比較皆增加。mAb IL-6R/IL-6組足狀細胞uPAR蛋白表現量、uPAR mRNA定量與正常對照組、 IL-6組比較皆下降。透過本研究結果瞭解:IL-6會刺激uPAR表現上升於臨床上可能促使足狀細胞從基底膜上剝離產生蛋白尿,但加入mAb IL-6R可阻斷IL-6作用,減低uPAR表現以穩定足狀細胞防止從基底膜上剝離降低蛋白尿程度,對LN以mAb IL-6R治療提供了作用機制的體外試驗印證,為未來動物及人體試驗提供一參考資料。

Lupus nephritis (LN), an autoimmune disease, is the most common forms in systemic lupus erythematosus (SLE). The patients of LN are usually associated with proteinuria which is caused by podocyte’s foot processes effacement. In recent studies, the induction of urokinase-type plasminogen activator receptor or CD78 (uPAR, CD78) signaling in podocyte leads to foot process effacement. The clinical studies show that Interleukin-6 (IL-6) has been implicated in LN. Blockade of IL-6 by interleukin-6 receptor monoclonal antibody (mAb IL-6R) will reduces proteinuria production in murine lupus. Nevertheless, there are no data concerning the effects of IL-6 and mAb IL-6R on the expression of uPAR of podocytes in LN. In this study, we firstly, cultured podocytes from isolated glomerulli in rat, and the podocyte will be identified by immunofluorescence staining of synaptopodin and podocin Ab. Podocytes (1×103/mL) were experimentally grouped into three groups, followed: normal control group, IL-6 (100 ng/mL) group and mAb IL-6R (25 μg/ml) combine with IL-6 (100 ng/mL) group, estimated their expression at different time point. Evaluation indicators of improved effect in LN will be:(1) analysis of uPAR-mRNA level by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR), (2) counting podocytes migration number by transwell assay, (3) measurement of uPAR and phosphor-STAT3 (p-STAT3) protein expression by immunofluorescence staining. The results showed that IL-6 group up-regulated uPAR, p-STAT3 protein expression, podocyte migration and uPAR mRNA rather than normal control and mAb-IL-6R/IL-6 groups. mAb-IL-6R/IL-6 group down-regulated uPAR protein and uPAR mRNA rather than groups normal control and IL-6 groups. In conclusion that IL-6 was seen to significantly stimulate uPAR expression in vitro that suppose leading to podocyte foot process effacement and reduce proteinuria counterpart with in vivo. However, blocked IL-6 by mAb IL-6R will reduce uPAR expression and stable podocyte in glomerular basement may also decrease proteinuria. This study will provide more thraputic application in lupus nephritis and futurer animal and human trial research.



中文摘要……………………………………………………………………………….i
英文摘要……………………………………………………………………………….iii
目次…………………………………………………………………………………….v
圖表目次……………………………………………………………………………….vi
第一章 緒言……………………………………………………………………………1
第二章 文獻探討………………………………………………………………………3
第一節 全身性紅斑狼瘡病因與診斷………………………………………..3
第二節 狼瘡腎炎……………………………………………………………..9
第三章 材料與方法…………………………………………………………………...13
第一節 實驗材料…………………………………………………………….13
第二節 實驗方法…………………………………………………………….13
第四章 結果……………………………………………………………………….......18
第五章 討論…………………………………………………………………………...21
圖表…………………………………………………………………………………….25
參考文獻……………………………………………………………………………….38


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