跳到主要內容

臺灣博碩士論文加值系統

(44.210.21.70) 您好!臺灣時間:2022/08/15 09:21
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:許馥妃
研究生(外文):Fu-Fei Hsu
論文名稱:使用可誘導式載體過量表現p27Kip1基因對鼻咽癌細胞株病理機制之影響
論文名稱(外文):A Study of p27Kip1 Gene Overexpression on Pathogenicity of Nasopharyngeal Carcinoma Cells by an Inducible Vector
指導教授:卓忠隆
指導教授(外文):Chung-Lung Cho
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:72
中文關鍵詞:過量表現細胞週期鼻咽癌可誘導式載體
外文關鍵詞:inducible vectorcell cycleoverexpressionnasopharyngeal carcinomap27kip1
相關次數:
  • 被引用被引用:0
  • 點閱點閱:197
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0

鼻咽癌為東南亞特別盛行的腫瘤,宿有「中國癌之稱」;雖然多種與鼻咽癌有關的危險因子已被發現,如:種族、遺傳、環境、EB病毒…等,但鼻咽癌形成的分子機制仍不是很清楚。在真核細胞,細胞週期的進行受到cyclins、CDKs、CDKIs這些蛋白質的交互作用被調控著,這些細胞週期調節蛋白對正常細胞與腫瘤細胞的生長扮演相當重要的角色。
p27Kip1為Cip/Kip家族的一名成員,p27Kip1蛋白會抑制cyclin D-CDK4與cyclin E-CDK2,使細胞停留在G1期。雖然在人類腫瘤中,p27Kip1基因很少發生突變,但在幾種癌症中,像:直腸癌、乳癌、食道癌發現p27Kip1蛋白的表現量較低。
先前的實驗研究顯示,在兩株鼻咽細胞其p27Kip1的表現量都明顯低於正常鼻咽上皮細胞(NNE)與293(HEK);因此我們將p27Kip1基因與誘導載體結合後,形成一Doxycycline誘導載體pBIG2r/p27Kip1,且轉染到兩株鼻咽癌細胞,觀察一些細胞週期調節蛋白的表現情形。
實驗發現過量表現p27Kip1在兩株鼻咽癌細胞會使cyclin E與CDK2表現量增加,並使較多鼻咽癌細胞停滯在G1期,細胞倍增時間增加,甚至有部分鼻咽癌細胞發生自然凋亡,因此,過量表現p27Kip1或許可以使鼻咽癌細胞休止於G1期且導致凋亡。


Nasopharyngeal carcinoma is a commonly occuring tumor in Southern China. However, the genetic basis underlying its tumorigenicity is still unclear. In eukaryotic cells, progression of the cell cycle is regulated by interactions of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors (CDKIs). These cell cycle-regulator proteins play important roles in growth of both normal and tumor cells. Many human tumors exhibit deregulation of one or more genes which involved in regulation of cell cycle progression.
p27Kip1, a member of the Cip/Kip family, inhibits both cyclin D-CDK4, and cyclin E-CDK2 complexes and regulates progression of the cell cycle from G1 to S phase. Although p27Kip1 gene mutations are rare in human tumors, low expression of p27Kip1 are observed in several cancers, such as colon, breast and esophagus.
In our previous study, p27Kip1 shown lower expression in two NPC cell lines compared with NNE and 293 (HEK). A doxycycline inducible construct, pBIG2r/p27Kip1, included a full length of human p27Kip1 cDNA was transfected into two NPC cell lines. Expression of several cell cycle-related genes were analyzed.
By increasing p27Kip1 in NPC cell lines, we found that the G1 phase and the doubling time were lengthened. Protein expression of cyclin E and CDK2 were up-regulated. These data suggest that the overexpression of p27Kip1 might be cause NPC cells to arrest at G1 phase and might lead to apoptosis.


壹、緖論
一、前言---------------------------------------------------------------------1
二、細胞週期---------------------------------------------------------------2
三、細胞週期調節基因p27Kip1------------------------------------------5
四、鼻咽癌------------------------------------------------------------------6
貳、研究目的---------------------------------------------------------------------11
參、材料與方法
一、鼻咽癌細胞與正常鼻咽上皮細胞之培養(Cell Lines)--------12
二、生長曲線(Growth Curve)-------------------------------------------13
三、反轉錄聚合酵素連鎖反應(RT-PCR)----------------------------13
四、勝任細胞(Competent Cell)的製備---------------------------------15
五、pUCmT與可誘導式載體pBIG2r的製備----------------------16
六、具有p27Kip1基因重組質體的製備-------------------------------17
(1)pUCmT/p27Kip1質體的製備
(2) pBIG2r /p27Kip1質體的製備
七、轉染細胞(Transfection)---------------------------------------------21
八、西方墨點法(Western Blot Analysis)------------------------------22
九、多重探針核糖核酸分解酵素保護分析(RPA)------------------26
十、流式細胞儀分析細胞週期之變化(Flow Cytometry)----------30
肆、結果與討論------------------------------------------------------------------32
伍、結論---------------------------------------------------------------------------37
陸、參考文獻---------------------------------------------------------------------68


Draetta G.F., Mammalian G1 Cyclins. Current Opinion in Cell Biology, 6:842-846 (1994) Erdamar S., Yang G., Harper J.W., Lu X., Kattan M.W., Thompson T.C. and Wheeler T.M., Level of Expression p27 Kip1 of Protein in Human Prostate Cancer. Modern Pathology, 12:751-755 (1999)Fan C.S., Wong N., Leung S.F., To K.F., Lo K.W., Lee S.W., Mok T., Johnson P.J. and Huang D.P., Frequent c-Myc and Int-2 Overrepresentation in Nasopharyngeal Carcinoma. Human Pathology, 31:169-178 (2000) Fero M.L., Rivkin M., Tasch M., Porter P., Carow C.E., Firpo E., Polyak K., Tsai L.H., Broudy V., Perlmutter R.M., Kaushansky K. and Roberts J.M., A Syndrome of Multiorgan Hyperplasia with Features of Gigantism , Tumorigenesis, and Female Sterility in p27kip1 —Deficient Mice. Cell, 85:733-744 (1996) Fredersdorf S., Burns J., Milne A.M., Packham G., Fails L., Gillett C.E., Royads J.A., Peston D., Hall P.A., Hanby A.M., Barnes D.M., Shousha S., O’Hare M.J. and Lu X., High Level Expression of p27kip1 and Cyclin D1 in some Human Breast Cancer Cells:Inverse Correlation between the Expression of p27kip1 and Degree of Malignancy in Human Breast and Colorectal Cancer. Proceeding of the National Academy of Science of the USA, 94:6380-6385 (1997)Gulley M.L., Nicholls J.M., Schneider B.G., Amin M.B., Ro J.Y. and Geradts J., Nasopharyngeal Carcinoma Frequently Lack the p16/MTS1 Tumor Suppressor Protein but Consistently Express the Retinoblastoma Gene Product. American Journal of Pathology, 152:865-869 (1998)Hartwell L.H. and Kastan M.B., Cell Cycle Control and Cancer. Science, 266:1821-1828 (1994)Hunter T. and Pines J., Cyclin and CancerⅡ:Cyclin D and CDK inhibitors come of age. Cell, 79:573-582 (1994)Katayose Y., Kim M., Rakkar N.S., Li Z., Cowan K.H. and Seth P., Promoting Apoptosis:A Novel Activity Associated with the Cyclin-dependent Kinase Inhibitor p27. Cancer Research, 57:5441-5445 (1997)Kiyokawa H., Kinerman R.D., Manova-Todorova K.O., Soares V.C., Hoffman E.S., Ono M., Khanam D., Hayday A.C., Frohman L.A. and Koff A., Enhanced Growth of Mice Lacking the Cyclin-Dependent Kinase Inhibitor Function of p27 kip1. Cell, 85:721-732 (1996) Lloyd R.V., Erickson L.A., Jin L., Kulig E., Qian X., Cheville J.C. and Scheithauer B.W., p27kip1:A Multifunctional Cyclin-dependent Kinase Inhibitor with Prognostic Significance in Human Cancers. American Journal of Pathology, 154:313-323 (1999) Lin C.T., Chan W.Y., Chen W. and Shew J.Y., Nasopharyngeal Carcinoma and Retinoblastoma Gene Expression. Laboratory Investigation, 67:56-70 (1992)Lin C.T., Chan W.Y., Chen W., Huang H.M., Wu H.C., Hsu M.M., Chuang S.M. and Wang C.C., Characterization of Seven Newly Established Nasopharyngeal Carcinoma Cell Lines. Laboratory Investigation, 68:716-727 (1993)Lo K.W., Cheung S.T., Leung S.F., Hasselt A., Tsang Y.S., Mak K.F., Chung Y.F., Woo K.S., Lee C.K. and Huang D.P., Hypermethylation of the p16 Gene in Nasopharyngeal Carcinoma. Cancer Research, 56:2721-2725 (1996) Luo J., Xiao J., Tao Z. and Li X., Detection of c-Myc Gene Expression in Nasopharyngeal Carcinoma by Nonradioactive in Situ Hybirdization and Immunohistochemistry. Chinease Medical Journal (England) 110:229-232 (1997)Mineta H., Miura K., Suzuki I., Takebayashi S., Misawa K., Ueda Y. and Ichimura K., p27 Expression in Correlates with Prognosis in Patients with Hypopharyngeal Cancer. Anticancer Research, 19:4407-4412 (1999) Murakami Y. and Sekiya T., Accumulation of Genetic Alterations and Their Significance in each Primary Human Cancer and Cell Lines. Mutation Research, 400:421-437 (1998)Naruse I., Hoshino H., Dobashi K., Minato K., Saito R. and Mori M., Overexpression of p27 kip1 Induces Growth Arrest and Apoptosis Mediated by Changes of pRB Expression in Lung Cancer Cell Lines. International Journal of Cancer, 88:377-383 (2000) Nakayama K.M., Ishida M., Shirane A., Inomata T., Inoue N., Shisido L., Horri D.Y., Loh D. and Nakayama K.I., Mice Lacking p27Kip1 Display Increased Body Size, Multiple Organ Hyperplasia, Retinal Dysplasia and Pituitary Tumors. Cell, 85:721-732 (1996) Pance-Cestaneda M.V., Lee M.H., Latres E., Polyak K. and Lacombe L., p27Kip1 :Chromosomal Mapping to 12p12-12p13.1 and Absennce of Mutation in HumanTumors. Cancer Research, 55:1211-1214 (1995) Polyak K., Lee M.H., Erdjument-Bromage H., Koff A., Robert J.M., Tempst P. and Massague J., Cloningof p27Kip1, Cyclin-dependent Kinase Inhibitor and a Potential Mediator of Extracellular Antimitogenic Signals. Cell, 78:59-66 (1994)Porter M.J., Field J.K., Lee J.C., Leung S.F., Lo D. and van Hasselt C.A., Detection of the Tumor Suppressor Gene p53 in Nasopharyngeal Carcinoma in Hong Kong Chinease. Anticancer Research, 14:1359-1360 (1994) Rosenthal E.T., Hunt T. and Ruderman J.V., Selective Translation of mRNA Controls the Pattern of Protein Synthesis During Early Development of the Surfclam, Spisula Solidissima. Cell, 20:487-494 (1980) Sgambato A., Cittadini A., Faraglia B. and Weinstein I.B., Multiple Functions of p27Kip1 and Its Alteration in Tumor cells:A Review. Journal of Cellular Physiology, 183:18-27 (2000) Sherr C.J., Mammalian G1 Cyclins. Cell, 6:1059-1065 (1994)Spirin K.S., Simpson J.F., Takeuchi S., Kawamata N. and Miller C. W., p27/Kip1 Mutation Found in Breast Cancer. Cancer Research, 56:2400-2404 (1996)Strathdee C.A., McLeod M.R. and Hall J.R., Efficient Control of Tetracycline-responsive Gene Expression from an Autoregulated Bi-direction Expression Vector. Gene, 229:21-29 (1999)Wang G.L., Lo K.W., Tsang K.S., Chung N.Y., Tsang Y.S., Cheung S.T., Lee J.C. and Huang D.P., Inhibiting Tumorigenic Potential by Restoration of p16 in Nasopharyngeal Carcinoma. British Journal of Cancer, 81:1122-1126 (1999)Weinberg R.A., How cancer arises? Scientific American, 275:62-67 (1996)

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top