Rogdi 是一個尚未知其功能的基因。依據 NCBI基因資料庫，大鼠此基因位於其染色體10q12上，遺傳編碼區864 bp，轉譯出287個胺基酸。本實驗室之前研究指出人類ROGDI會影響 HeLa、Hep 3B 與 NIH3T3細胞的增生速度。後來發現肝纖維化的組織中，Rogdi蛋白質有增加表現的現象。肝臟纖維化過程中，肝細胞受到損傷，造成發炎反應，刺激肝星狀細胞從靜默態轉型成活化態，表現出α-平滑肌肌動蛋白(α-SMA)、加速細胞增生與沈積大量膠原蛋白， 造成肝纖維化。本研究主要以分離培養的大鼠肝星狀細胞，首先發現活化態肝星狀細胞的Rogdi蛋白質表現量也較多。接著藉由轉染質體改變初代肝星狀細胞培養中 Rogdi基因表現，顯示Rogdi會影響細胞增生、也影響肝星狀細胞活化的標記蛋白α-SMA的表現，以及造成肝纖維化主要的蛋白質collagen type I的表現。並且也發現經過 PDGF刺激的肝星狀細胞，其Rogdi的表現量明顯增加。更深入研究顯示Rogdi參與MAPK與PI3K/Akt訊號傳遞路徑。最後我們發現 Rogdi基因表現會影響細胞移動的能力。

Rogdi was a novel gene with unknown function. According to GeneBank database, the gene is located on chromsome 10q12 and the length of coding regeion is 864 bp that encods 287 animo acids. Earlier studies in our laboratory showed that human ROGDI influenced rate of cell proliferaion in HeLa, Hep3B and NIH3T3 cells. In addition, we found Rogdi protien was up-regulated in fibrotic livers. Following various types of injury to liver, quiescent hepatic stellate cells (HSCs) transform to activated phenotype, leading to exprssion of α-SMA, increasing rate of cell proliferation and depositing of extracellular matrix. In this study, we found that Rogdi protein was up-regulated in activated HSCs isolated and cultured from rat livers. By either overexpression or RNA interference of Rogdi, we found that Rogdi affected rate of HSCs proliferation, and expressions of α-SMA and collagen type I. Expression of Rogdi protein was induced after PDGF treatment of rat HSCs. Additionally, we found that Rogdi was involved in MAPK and PI3K/Akt pathways. Furthermore, using wound healing assay and migration assay, Rogdi was found to regulate migration of activated HSCs.

中文摘要
英文摘要
頁次
前言 --------------------------------------------------------------------1
ROGDI -----------------------------------------------------------------------1
肝纖維化與肝星狀細胞 --------------------------------------------------1
肝纖維化模式的應用 -----------------------------------------------------2
肝星狀細胞的異質性與可塑性 -----------------------------------------5
研究星狀細胞的模式與方法 --------------------------------------------6
生長因子與細胞激素對肝星狀細胞的影響 --------------------------9
實驗目的 -------------------------------------------------------------11
材料與方法 ----------------------------------------------------------12
Total RNA的萃取 --------------------------------------------------------12
反轉錄反應 ----------------------------------------------------------------12
聚合酶連鎖反應 ----------------------------------------------------------13
建構表達蛋白質質體 ----------------------------------------------------13
轉型作用 -------------------------------------------------------------------14
製備小量質體 -------------------------------------------------------------15
表現與萃取大量蛋白質 -------------------------------------------------15
純化His-ROGDI融合蛋白 ---------------------------------------------16
ROGDI多株抗體製備及純化 ------------------------------------------16
西方墨點法 ----------------------------------------------------------------17
誘導動物肝纖維化 -------------------------------------------------------18
分離與培養肝星狀細胞 -------------------------------------------------18
細胞轉染 -------------------------------------------------------------------20
免疫細胞化學染色 -------------------------------------------------------20
免疫螢光染色 -------------------------------------------------------------20
即時聚合酵素連鎖反應 -------------------------------------------------23
細胞增生分析 -------------------------------------------------------------23
細胞移行能力分析 -------------------------------------------------------25
數據統計分析 -------------------------------------------------------------26
結果 -------------------------------------------------------------------28
大量表現His-ROGDI融合蛋白 --------------------------------------28
His-ROGDI融合蛋白的純化 ------------------------------------------29
多株抗體anti-ROGDI的純化 -----------------------------------------29
多株抗體anti-ROGDI的確認 -----------------------------------------29
動物肝纖維化模式檢測Rogdi的變化 -------------------------------30
確認大鼠肝星狀細胞與純度 -------------------------------------------31
Rogdi在大鼠肝星狀細胞的表現 --------------------------------------32
LPS處理對大鼠肝星狀細胞內Rogdi蛋白質的影響 -------------33
細胞激素處理對大鼠肝星狀細胞內Rogdi蛋白質的影響 -------34
Rogdi對大鼠肝星狀細胞增生的影響 --------------------------------34
Rogdi對大鼠肝星狀細胞其他基因mRNA表現的影響 ----------38
Rogdi對大鼠肝星狀細胞活化的影響與訊號傳遞路徑的參與 --39
Rogdi對大鼠肝星狀細胞Wound healing assay的影響 -----------42
Rogdi對大鼠肝星狀細胞侵入能力的影響 --------------------------43
討論 -------------------------------------------------------------------44
參考文獻 -------------------------------------------------------------47
圖 ----------------------------------------------------------------------55
表 ----------------------------------------------------------------------72
附錄 -------------------------------------------------------------------73

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