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研究生:蔣濶如
研究生(外文):Kuo-Ru
論文名稱:研究Peripheral myelin protein 22基因對人類肺癌細胞株生長的影響
論文名稱(外文):Study the effects of peripheral myelin protein 22 gene on cell growth in human lung cancer cell lines
指導教授:蔡菁華
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:75
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PMP22 (Peripheral myelin protein22)基因也就是Growth arrest – specific-3 (GAS3)基因,位於染色體17p11.2,包含6個exons,為周邊神經系統髓鞘的結構蛋白。PMP22在細胞的增生、分化、細胞與細胞之間和細胞與基質之間的交互作用,扮演一個很重要的角色。PMP22 mRNA在肺癌病人之正常肺組織的表現量較肺腫瘤組織高約2~45倍不等(附圖一)。在CL1系列的細胞株中,CL1-0之PMP22的表現量最低。因此我們在CL1-0肺癌細胞中過度表現PMP22基因,觀察PMP22蛋白對細胞生長、死亡、細胞群落的形成、細胞週期的分佈及細胞週期調控蛋白的表現有什麼影響。由實驗結果發現,當PMP22基因過度表現時,會使CL1-0細胞有多核的現象產生。由MTT assay和細胞計數結果得知,過度表現PMP22會抑制CL1-0細胞的生長。由流氏細胞儀結果顯示,過度表現PMP22會使CL1-0細胞比CL1-0 parental及vector control的G1、G2/M期減少,S phase增加。進而利用西方點墨法偵測細胞週期調控蛋白的表現量,結果發現過度表現PMP22會使細胞調控蛋白CDKs (cyclin-dependent kinases)表現量減少,其中CDK1的表現量會隨著PMP22表現量的增加而降低。暗示PMP22會直接或間接調控CDK1的表現。接著利用RT-real time PCR得知PMP22對CDK1 mRNA的表現並無影響,因此PMP22對CDK1的影響並不是在mRNA level。此外,也發現過度表現PMP22時,會使細胞p21、p53的表現量增加及pRb的表現量減少。當過度表現PMP22時,會使得CL1-0細胞形成群落的初期比CL1-0 parental及vector control鬆散,不貼附生長( anchorage-independent growth )的能力降低,並可由西方點墨法發現β- catenin表現量的下降。可見PMP22會影響細胞的cell-cell interaction或cell-cell junction,而其作用機制還需要進一步探討。由以上的結果推論,PMP22基因在肺癌細胞CL1-0中可能扮演一個抑癌(tumor supreesor)的角色,其詳細的作用機轉及影響路徑仍有待探討。之後,可以從過度表現PMP22對CDK1後轉譯修飾的影響,更進一步研究之後的作用機轉及影響路徑來觀察PMP22對肺癌的影響。

Peripheral myelin protein 22 (PMP22), also known as Growth arrest-specific-3 (GAS3) gene, is located at chromosome 17p11.2. PMP22 is composed of six exons and is a structural protein of the myelin sheath in the peripheral nervous system (PNS). The PMP22 gene plays an important role in cell proliferation, differentiate, cell-cell interactions and cell-matrix interaction. The mRNA level of PMP22 was 2 to 45 fold higher in normal lung than in lung cancer. The PMP22 expression level of CL1-0 is the lowest in CL1 series cell lines. We overexpressed PMP22 in CL1-0 to observe the effect of PMP22 on cell growth, apoptosis, colony formation, cell cycle distribution and expression of cell cycle regulators. When PMP22 is overexpressed in CL1-0 cells, 500μg/ml of G418 resistant cells developed mutinuclei, while this phenomenon disappeared in stable clones. MTT assay and cell counting data showed that overexpression of PMP22 decreased cell growth. Flow cytometry analysis showed that overexpression of PMP22 decreased the percentage of G1 and G2/M phase cells while increased S phase cells. We used Western blot analyses to examine the expression level of cell cycle regulators (CDKs). Overexpression of PMP22 decreased protein levels of CDKs. Among them, the expression of CDK1 decreased with the increasing expression level of exogenous PMP22, suggesting that PMP22 reduces CDK1 expression directly or indirectly. To assess whether PMP22 overexpression would decrease CDK1 by decreasing its mRNA level, RT-real time PCR was performed and showed that overexpression of PMP22 did not affect the mRNA level of CDK1. In addition, overexpression of PMP22 increased the protein level of p21, p53 and decreased the level of phosporylated-Rb. When PMP22 was overexpressed, CL1-0 cells formed losser colonies, and decreased the anchorage-independent growth. By Western blot analysis, the expression of β-catenin was decreased in cells overexpressed PMP22, suggesting that PMP22 may affect cell-cell interaction or cell-cell junction. Together, these results indicated that PMP22 might acts as a tumor suppressor in lung cancer. The detail mechanisms and pathways involved in tumor suppressor activity of PMP22 are required for further investigation.

目 錄
壹、 中文摘要 1
貳、 英文摘要 2
參、 前言
一、肺癌流行病學及危險因子 4
二、肺癌的分類及成因 5
三、鑑定參與肺癌形成的基因 5
四、人類PMP22基因 6
五、細胞週期調控蛋白 8
肆、 實驗材料
1.藥品 11
2.商業套組 11
3.試藥 11
4.抗體 11
5.人類肺癌細胞株來源 12
6.載體來源 12
7.儀器 12
伍、 實驗方法
1.質體DNA的萃取(Plasmid extract) 14
2.細胞染色體DNA的萃取 15
3.細胞RNA的萃取 16
4.利用去氧核醣核酸酶除去total RNA裡的染色體DNA 16
5. 反轉錄反應(Reverse Transciption;RT) 17
6.定量PCR(Real time PCR) 17
7.構築具有表現PMP22(overexpression constract)的載體18
8.肺癌細胞培養(cell culture) 22
9.轉染作用(transfection) 23
10.Stable clones的篩選(selection)步驟 24
11.西方墨點法(western blot) 25
12.3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium
bromide分析法 27
13.細胞計數生長試驗(Counting cell numbers) 28
14.群落形成試驗(Colony formation) 28
15.軟培養基非附著性細胞生長試驗
(Anchorage-independent growth assay) 28
16.細胞轉移試驗(Modified Boyden chamber) 29
17.流氏細胞分析(Flow cytometry) 29
18.Hematoxylin & Eosin stain(H&E stain) 30
19.統計分析 30
陸、 實驗結果
一、建立穩定過量表現PMP22的細胞株 31
二、利用RT-real time PCR和Western blot定量PMP22在
stable clone的表現量 32
三、利用GFP綠色螢光蛋白觀察PMP22-GFP融合蛋白於CL1-0細
胞株表現的位置 33
四、利用MTT assay和細胞計數觀察PMP22的表現對於人類肺
癌細胞株生長能力的影響 33
五、利用細胞群落形成試驗(colony formation assay)觀察
PMP22對細胞群落形成的影響 34
六、觀察過度表現PMP22是否影響anchorage–independent
growth 34
七、利用流式細胞儀觀察PMP22的表現對細胞週期的影響 35
八、觀察PMP22的表現對於人類肺癌細胞之細胞週期調控蛋白
影響 36
九、利用RT-real time PCR觀察過度表現PMP22對CDK1、
p21、p53和Rb之mRNA表現量的影響 36
十、利用H&E stain觀察過度表現PMP22對CL1-0產生多核的現
象 37
柒、 討論 38
捌、 圖表說明
圖一、分析PMP22基因於載體控制組(vector control)裡的表
現量 44
圖二、分析過度表現PMP22之細胞株PMP22 mRNA的表現量 45
圖三、利用西方點墨法偵測PMP22/CL1-0 stable clones中
PMP22-Flag融合蛋白(fusion protein)的表現量 46
圖四、利用GFP綠色螢光蛋白觀察PMP22於CL1-0細胞株 47
圖五、利用MTT assay分析PMP22 overexpression對CL1-0細
胞生長的影響 48
圖六、利用細胞計數分析過度表現PMP22對CL1-0細胞生長的
影響 49
圖七、觀察過度表現PMP22對於CL1-0細胞珠之PCNA及β-
catenin的表現量 50
圖八、利用Colony formation assay觀察PMP22
overexpression對細胞群落形成的影響 51
圖九、利用anchorage-independent growth assay觀察過度
表現PMP22 對細胞轉型能力的影響 52
圖十、以流式細胞儀分析PMP22 overexpression對細胞週期
分佈影響 55
圖十一、觀察過度表現PMP22對於CL1-0細胞株之細胞週期調
控蛋白的影響 57
圖十二、觀察過度表現PMP22對於CL1-0細胞株產生之多核現
象 60
表一、過度表現PMP22對CDK1、p21、p53及Rb mRNA表現影響 62
玖、 附圖
附圖一、肺癌病人之正常及腫瘤組織之PMP22相對於18srRNA
或GAPDH的差異表現量 63
附圖二、2006年主要癌症死亡率 64
附圖三、PMP22的結構圖 65
附圖四、細胞週期參考圖 66
附圖五、在人類肺癌細胞株中以RT-real time PCR定量
PMP22基因的表現情形 67
附圖六、C-Terminal p3xFLAG-CMV之map (Sigma) 68
附圖七、pEGFP-N1之map (BD Biosciences Clontech) 69
拾、 附表
附表一、在不同肺癌細胞株中PMP22基因相對於18s rRNA表現量 70
附表二、細胞株的種類 71
附表三、引子序列表 72
拾壹 文獻探討 73




Allan, L.A. and Clarke, P.R. (2007) Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis. Mol Cell, 26, 301-310.
Amici, S.A., Dunn, W.A., Jr., Murphy, A.J., Adams, N.C., Gale, N.W., Valenzuela, D.M., Yancopoulos, G.D. and Notterpek, L. (2006) Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina. J Neurosci, 26, 1179-1189.
Baechner, D., Liehr, T., Hameister, H., Altenberger, H., Grehl, H., Suter, U. and Rautenstrauss, B. (1995) Widespread expression of the peripheral myelin protein-22 gene (PMP22) in neural and non-neural tissues during murine development. J Neurosci Res, 42, 733-741.
Brancolini, C., Marzinotto, S., Edomi, P., Agostoni, E., Fiorentini, C., Muller, H.W. and Schneider, C. (1999) Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22. Mol Biol Cell, 10, 2441-2459.
Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R. and Wu, C.W. (1997) Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol, 17, 353-360.
Cook, D., Fry, M.J., Hughes, K., Sumathipala, R., Woodgett, J.R. and Dale, T.C. (1996) Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. Embo J, 15, 4526-4536.
Cukier, I.H., Li, Y. and Lee, J.M. (2007) Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin. FEBS Lett, 581, 1661-1672.
Fabbretti, E., Edomi, P., Brancolini, C. and Schneider, C. (1995) Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev, 9, 1846-1856.
Farid, S.S., Washbrook, J. and Titchener-Hooker, N.J. (2005) Combining multiple quantitative and qualitative goals when assessing biomanufacturing strategies under uncertainty. Biotechnol Prog, 21, 1183-1191.
Feng, Y. and Walsh, C.A. (2004) The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat Cell Biol, 6, 1034-1038.
Frank, C.J., Hyde, M. and Greider, C.W. (2006) Regulation of telomere elongation by the cyclin-dependent kinase CDK1. Mol Cell, 24, 423-432.
Gachet, Y., Tournier, S., Millar, J.B. and Hyams, J.S. (2001) A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast. Nature, 412, 352-355.
Jeffrey, P.D., Russo, A.A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J. and Pavletich, N.P. (1995) Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature, 376, 313-320.
Karlsson, C., Afrakhte, M., Westermark, B. and Paulsson, Y. (1999) Elevated level of gas3 gene expression is correlated with G0 growth arrest in human fibroblasts. Cell Biol Int, 23, 351-358.
Knudsen, E.S. and Wang, J.Y. (1997) Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation. Mol Cell Biol, 17, 5771-5783.
Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A. and Jacks, T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature, 362, 847-849.
Notterpek, L., Roux, K.J., Amici, S.A., Yazdanpour, A., Rahner, C. and Fletcher, B.S. (2001) Peripheral myelin protein 22 is a constituent of intercellular junctions in epithelia. Proc Natl Acad Sci U S A, 98, 14404-14409.
Patel, P.I., Roa, B.B., Welcher, A.A., Schoener-Scott, R., Trask, B.J., Pentao, L., Snipes, G.J., Garcia, C.A., Francke, U., Shooter, E.M., Lupski, J.R. and Suter, U. (1992) The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nat Genet, 1, 159-165.
Porter, L.A. and Donoghue, D.J. (2003) Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res, 5, 335-347.
Puri, A., Hug, P., Jernigan, K., Rose, P. and Blumenthal, R. (1999) Role of glycosphingolipids in HIV-1 entry: requirement of globotriosylceramide (Gb3) in CD4/CXCR4-dependent fusion. Biosci Rep, 19, 317-325.
Re, F.C., Manenti, G., Borrello, M.G., Colombo, M.P., Fisher, J.H., Pierotti, M.A., Della Porta, G. and Dragani, T.A. (1992) Multiple molecular alterations in mouse lung tumors. Mol Carcinog, 5, 155-160.
Roux, K.J., Amici, S.A., Fletcher, B.S. and Notterpek, L. (2005) Modulation of epithelial morphology, monolayer permeability, and cell migration by growth arrest specific 3/peripheral myelin protein 22. Mol Biol Cell, 16, 1142-1151.
Roux, K.J., Amici, S.A. and Notterpek, L. (2004) The temporospatial expression of peripheral myelin protein 22 at the developing blood-nerve and blood-brain barriers. J Comp Neurol, 474, 578-588.
Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C., Munemitsu, S. and Polakis, P. (1996) Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science, 272, 1023-1026.
Snipes, G.J. and Suter, U. (1995) Molecular anatomy and genetics of myelin proteins in the peripheral nervous system. J Anat, 186 (Pt 3), 483-494.
Snipes, G.J., Suter, U., Welcher, A.A. and Shooter, E.M. (1992) Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13). J Cell Biol, 117, 225-238.
Suter, U. and Nave, K.A. (1999) Transgenic mouse models of CMT1A and HNPP. Ann N Y Acad Sci, 883, 247-253.
Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y. and Nakamura, Y. (2000) A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature, 404, 42-49.
van Dartel, M. and Hulsebos, T.J. (2004) Characterization of PMP22 expression in osteosarcoma. Cancer Genet Cytogenet, 152, 113-118.
Vodenicharov, M.D. and Wellinger, R.J. (2006) DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase. Mol Cell, 24, 127-137.
Wang, L.H. (2004) Molecular signaling regulating anchorage-independent growth of cancer cells. Mt Sinai J Med, 71, 361-367.
Wulf, P. and Suter, U. (1999) Embryonic expression of epithelial membrane protein 1 in early neurons. Brain Res Dev Brain Res, 116, 169-180.
Xiong, Y. (1996) Why are there so many CDK inhibitors? Biochim Biophys Acta, 1288, 01-05.
Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R. and Beach, D. (1993) p21 is a universal inhibitor of cyclin kinases. Nature, 366, 701-704.
Yamashiro, S. and Matsumura, F. (1991) Mitosis-specific phosphorylation of caldesmon: possible molecular mechanism of cell rounding during mitosis. Bioessays, 13, 563-568.
Yutaka, H., Mariko, Y., Shinichiro, O., Kunihiko, M., Yusuke, T. and Yasuo, I. (2006) Thalidomide for the treatment of leptomeningeal multiple myeloma. Eur J Haematol, 76, 358-359.
Zephir, H., Stojkovic, T., Latour, P., Hurtevent, J.F., Blankaert, F. and Vermersch, P. (2005) A family with a novel frameshift mutation in the PMP22 gene (c.433_434insC) causing a phenotype of hereditary neuropathy with liability to pressure palsies. Neuromuscul Disord, 15, 493-497.
Zhou, B.B. and Elledge, S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature, 408, 433-439.
Zoidl, G., Blass-Kampmann, S., D''Urso, D., Schmalenbach, C. and Muller, H.W. (1995) Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth. Embo J, 14, 1122-1128.
Zoidl, G., D''Urso, D., Blass-Kampmann, S., Schmalenbach, C., Kuhn, R. and Muller, H.W. (1997) Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts. Cell Tissue Res, 287, 459-470.



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