進階搜尋


 
系統識別號 U0026-0812200911343421
論文名稱(中文) 探討在內質網壓力下肝細胞中的c-jun所扮演的可能角色
論文名稱(英文) The potential role of c-Jun in hepatocytes under ER stress
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 93
學期 2
出版年 94
研究生(中文) 趙詠梅
研究生(英文) Yung-Mei Chao
學號 s1692116
學位類別 碩士
語文別 中文
論文頁數 77頁
口試委員 指導教授-賴明德
口試委員-劉校生
口試委員-張文粲
中文關鍵字 B型肝炎病毒  內質網壓力  肝癌 
英文關鍵字 ER tress  pre-S  c-jun  Hepatopa  HBV  MMP-9 
學科別分類
中文摘要   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。



英文摘要  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.



論文目次 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


參考文獻 Arii S., Mise M., Harada T., Furutani M., Ishigami SI. and Niwano M. Overexpression of matrix metalloproteinase 9 gene in hepatocellular carcinoma with invasive potential. Hepatology 24, 316-322, 1996.

Atkinson JJ. and Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am. J.Ewapie. Cell Mol. Biol. 28, 12-24, 2003.

Back SH., Schröder M., Lee K., Zhang K. and Kaufman RJ. ER stress signaling by regulated splicing: IRE1/HAC1/XBP1. Methods 35, 395-416, 2005.

Boyce M., Bryant KF., Jousse C., Long K., Harding HP., Scheuner D., Kaufman RJ., Ma D., Coen DM., Ron D. and Yuan J. A selective inhibitor of eIF2a dephosphorylation protects cells from ER stress. Science 307, 935-939, 2005.

Bruix J. and Llovet JM. Hepatitis B virus and hepatocellular carcinoma. J. Hepatol. 39, S59-S63, 2003.

Caselmann WH., Meyer M., Kekule AS., Lauer U., Hofschneider PH. and Koshy R. A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA. Proc. Natl. Acad. Sci. U.S.A. 87, 2970-2974, 1990.

Eferl R., Sibilia M., Hilberg F., Fuchsbichler A., Kufferath I., Guertl B., Zenz R., Wagner EF., and Zatloukal K. Functions of c-Jun in liver and heart development. J. Cell Biol. 145, 1049-1061, 1999.

Eferl R. and Wagner EF. AP-1: a double-edged sword in tumorignesis. Nat. Rev. Cancer 3, 859-868, 2003.

Entschladen F., Drell IV TL., Lang K., Joseph J. and Zaenker KS. Lancet Oncol. 5, 254-258, 2004.

Fan YF., Lu CC., Chang YC., Chang TT., Lin PW., Lei HY. and Su IJ. Identification of a pre-S2 mutant in hepatocytes expression a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J. Gastroenterol. Hepatol. 15, 519-528, 2000.

Gum R., Lengyel E., Juarez J., Chen JH., Sato H., Seiki M. and Boyd D. Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen- activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. J. Cell Biol. 271, 10672-10680, 1996.

Gum R., Wang H., Lengyel E., Juarez J. and Boyd D. Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 14, 1481-1493, 1997.

Hayasaka A., Suzuki N., Fusimoto N., Iwama S., Fukuyama E. and Kanda Y. Elevated plasma levels of matrix metalloproteinase-9 (92-kd type IV collagenase/gelatinase B) in hepatocellular carcinoma. Hepatology 24, 1058-1062, 1996.

Hsieh YH., Su IJ., Wang HC., Chang WW., Lei HY., Lai MD., Chang WT. and Huang W. Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis 25, 2023-2032, 2004.

Hilberg F., Agguzi A., Howells N. and Wagner EF. c-Jun is essential for normal mouse development and hepatogenesis. Nature 365, 179-181, 1993.

Hildt E. and Hofschneider P. The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways. Recent Results Cancer Res. 154, 315-329, 1998.

Hildt E., Hofschneider P. and Urban S. The role of hepatitis B virus(HBV) in the development of hepatocellular carcinoma. Virology 7, 333-347, 1996.

Huang C., Jacobson K. and Schaller MD. A role for JNK-paxillin signaling in cell migration. Cell Cycle 3, 4-6, 2004.

Hung JH., Su IJ., Lei HY., Wang HC., Lin WC., Chang WT., Huang W., Chang WC., Chang YS., Chen CC. and Lai MD. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase. J. Biol. Chem. 279, 46384-46392, 2004.

Jochum W., Passegué E. and Wagner EF. AP-1 in mouse development and tumorigensis. Oncogene 20, 2401-2412, 2001.

Kadowaki H., Nishitoh H. and Ichijo H. Survival and apoptosis signals in ER stress: the role of protein kinases. J. Chem. Neuroanat. 28, 93-100, 2004.

Karin M., Liu Z. and Zandi E. AP-1 function and regulation. Curr. Opin. Cell Biol. 9, 240-246, 1997.

Kidd-Ljunggren K., Miyakawa Y. and Kidd AH. Genetic variability in hepatitis B viruses. J. Gen. Virol. 83, 1267-1280, 2002.

Kuang E., Wan Q., Li X., Xu H., Liu Q. and Qi Y. ER Ca(2+) depletion triggers apoptotic signals for endoplasmic reticulum (ER) overload response induced by overexpressed reticulon 3 (RTN3/HAP). J. Cell. Physiol. 204, 549-559, 2005.

Larman M. and Lucy J. The endoplasmic reticulum: structure and function. Mol. Membr. Biol. 16, 313-316, 1990.

Leppa S. and Bohmann D. Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene 18, 6158-6162, 1999.

Liotta LA. and Stetler-Stevenson WG. Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res. 51, 5054s-5059s, 1991.

Luo S., Baumeister P., Yang S., Abcouwer SF. and Lee AS. Induction of Grp78/Bip by translational block. J. boil. Chem. 278, 37375-37385, 2003.

Mercurio F. and Manning AM. NF-B as a primary regulator of the stress response. Oncogene 18, 6163-6171, 1999.

Mitra SK., Hanson DA. and Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat. Rev. Mol. Cell Biol. 6, 56-68, 2005.

Nakabeppu Y., Ryder K., and Nathans D. DNA binding activities of thress murine Jun proteins: stimulation by Fos. Cell 55, 907-915, 1988.

Papavassiliou AG., Treier M. and Bohmann D. Intramolecular signal transduction in c-Jun. EMBO J. 14, 2014-2019, 1995.

Sato H., Kita M. and Seiki M. v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements. J. Biol. Chem. 268, 23460-23468, 1993.

Schröder M. and Kaufman RJ. ER stress and the unfolded protein response. Mutat. Res. 569, 29-63, 2005.

Shaulian E. and Karin M. AP-1 in cell proliferation and survival. Oncogene 20, 2390-2400, 2001.

Shen J. and Prywes R. ER stress and signaling by regulated proteolysis of ATF6. Methods 35, 382-389, 2005.

Szczesna-Skorupa E., Chen CD., Liu H. and Kemper B. Gene expression changes associated with the endoplasmic reticulum stress response induced by microsomal cytochrome p450 overproduction. J. Biol. Chem. 279, 13953-13961, 2004.

Wang HC., Chang WT., Chang WW., Wu HC., Huang W., Lei HY., Lai MD., Fausto N. and Su IJ. Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes. Hepatology 41, 761-70, 2005.

Wang HC., Wu HC., Chen CF., Fausto N., Lei HY. and Su IJ. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am. J. Pathol. 163, 2441-2449, 2003.

Wilhelm SM., Collier IE., Marmer BL., Eisen AZ., Grant GA. and Goldberg GI. SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J.Biol. Chem. 264, 17213-17221, 1989.

Vogt PK. Jun, the oncoprotein. Oncogene 20, 2365-2377, 2001.

Zhang B., Liu S., Perpetua MD., Walker WH. and Harbrecht BG. Cytokines increase CRE binding but decrease CRE-mediated reporter activity in rat heptocytes by increasing c-Jun. Hepatology 39, 1343-1352, 2004.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2008-07-25起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2008-07-25起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw