臺灣博碩士論文加值系統

　　內質網(Endoplasmic reticulum, ER)主要功能是負責蛋白質合成，為控制蛋白質修飾和摺疊重要的胞器，當未摺疊或摺疊錯誤的蛋白質累積在內質網時，細胞則會產生內質網壓力(ER stress)。B型肝炎病毒(HBV)的表面抗原(HBsAg)是由pre-S1、S2和S基因所組成。在可能因感染HBV所引起的肝癌病人中，發現大B肝病毒表面抗原(Large HBsAg, LBHs)中的pre-S1或S2基因有部分缺失的現象，暗示著與腫瘤形成有關，而且pre-S突變的LBHs會累積在內質網內，而導致內質網壓力的產生。原致癌基因c-jun是組成AP-1(activator protein 1)的主要成員，對於肝細胞的發育是很重要的。因此我們想要探討HBV pre-S mutant、ER stress和c-jun的關係。在我們實驗中證實，當細胞處理ER stress inducers, Tunicamycin和Brefeldin A時，造成細胞產生內質網壓力，會促進c-Jun的表現和活化；在表現pre-S wild type和mutant的ML-1細胞，也會增加c-Jun的表現，可能藉此來調控細胞的生理狀況。另外，會受到AP-1調控的基因-MMP-9，在內質網壓力下的表現有隨時間增加的趨勢，c-Jun也有隨時間的進行增加結合在MMP-9 promoter區域的能力。而且在表現pre-S wild type和mutant的ML-1細胞， MMP-9的表現也增多，但只有pre-S2 mutant的細胞株有較強的移動(migration)及侵入(invasion)能力。

　Endoplasmic reticulum(ER) is the first compartment of secretory pathway to modify and fold the proteins. When misfolded or unfolded proteins accumulate in the ER, cells activate a self-protective mechanism, termed the ER stress response. Hepatitis B virus (HBV) is a small, enveloped DNA virus that causes acute and chronic liver diseases. In HBV-associated hepatocarcinoma, the large HBsAg with deletions in pre-S1 or pre-S2 region have been detected. These pre-S mutant HBsAg accumulate in endoplasmic reticulum (ER), resulting in ER stress. Specifically, expressoion of pre-S2�� is associated with potential carcinogenesis. Since c-jun is essential important for hepatocyte development. We wish investigate whether there is a linkage between HBV pre-S mutant, ER stress, and c-jun. Tunicamycin and brefeldin A, two ER stress inducers, increase the expression of c-Jun in ML-1 cells. Expression of pre-S mutant HBsAg also enhance the expression of c-Jun in ML-1 cells. Furthermore, one of the c-Jun regulated gene, MMP-9, was increased under ER stress. MMP-9 is known to play a key role in cancer invasion and metastasis by degradating the extracellular matrix (ECM) and basement membrane barriers. Since AP-1 acts on the promoter of MMP-9 gene, we further demonstrate that enhanced binding of c-Jun on the promoter of MMP-9 under ER stress. ML-1 cells expressing wild type or mutant HBV surface protein is associated with the expression of c-Jun and MMP-9. In addition, higher migration ability was observed in ML-1 cells expressed pre-S2 mutant compared with that of wild type pre-S.

目錄

緒論 1
一、內質網壓力概論 1
二、訊息傳遞與轉錄調節因子– c-Jun 3
三、基質水解酶-9 (matrix metalloproteinese-9 ; MMP9) 4
四、肝癌與B型肝炎病毒概論 6
材料與方法 9
一、細胞培養與藥物處理 (Cell culture and drug treatment) 9
二、基因殖入與細胞株的篩選 (Transfection and selection) 12
三、質體製備 (Plasmid preparation) 13
四、HBsAg ELISA kit assay 16
五、免疫組織化學染色法 (Immunohistochemistry) 17
六、細胞核和細胞質蛋白萃取 (Nuclear and cytoplasmic extraction) 19
七、藥物毒殺細胞存活率計算— MTT assay 20
八、抽取total RNA (Isolation total RNA) 21
九、反轉錄聚合酶連鎖反應 (RT-PCR) 23
十、西方點墨法 (Western blotting) 24
十一、電泳膠體位移分析 (Electophoretic mobility shift assay) 31
十二、染色質免疫沉澱法 (Chromatin immunoprecipitation) 33
十三、細胞移動分析 (Boyden chamber assay) 40
十四、細胞侵入分析 (Transwell invasion assay) 41
結果 44
一、利用Tunicamycin(TM)或Brefeldin A(TM)誘導內質網壓力產生，
造成c-Jun表現量增加 44
二、內質網壓力增加MMP-9的表現 45
三、c-Jun參與調控MMP-9的表現 45
四、建立pre-S wild type和mutant的ML-1細胞株 46
五、pre-S wild type和mutant的ML-1細胞株促進c-Jun和MMP-9 表現 46
六、pre-S wild type和mutant的ML-1細胞株移動(migration)與侵入
(invasion)能力的差異 47
結論 48
討論 49
參考文獻 54
表 59
圖 61
自述 77

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