跳到主要內容

臺灣博碩士論文加值系統

(3.233.217.106) 您好!臺灣時間:2022/08/17 11:54
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:蕭雅文
研究生(外文):Hsiao Ya wen
論文名稱:建立不同p53基因表現的非小細胞肺癌H1299細胞株
論文名稱(外文):Establishment of Different p53 Gene Status In Original p53-Null H1299 Cells
指導教授:黃海美
指導教授(外文):Huang hai mai
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
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:56
中文關鍵詞:p53非小細胞肺癌H1299
外文關鍵詞:p53 Gene StatusH1299 Cellsnon small cell lung cancer
相關次數:
  • 被引用被引用:0
  • 點閱點閱:1622
  • 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:1
H1299細胞中的p53基因有homozygous partial deletion,因此不會表現p53蛋白。論文中是利用H1299細胞的特性,以DNA轉殖技術建立三種背景相同,但是p53 status不同的細胞株。分別是含有穩定表現野生型p53基因的H1299細胞株、含有穩定表現突變型p53基因的H1299細胞株以及只含有空載體不含任何p53基因的H1299細胞株。為了建立wtp53-H1299細胞株,首先,先建構pcDNA3-p53質體。將wtp53從已經鑑定為含有正確p53基因的pc53-SN質體中以BamHI切下轉接到pcDNA3載體上。利用轉殖技術分別將pcDNA3-p53及EGFP-N1質體轉殖進入H1299細胞建立wtp53/EGFP-H1299細胞株。本研究也轉殖了pcDNA3-mp53及EGFP-N1質體到細胞建立mp53/EGFP-H1299細胞株,以及轉殖了pcDNA3及EGFP-N1質體到細胞建立pcDNA3/EGFP-H1299細胞株。三種有轉殖質體的細胞分別在含G418的培養液中培養三週以上,形成穩定有質體的混合細胞族群(stable mix colonies)之後,以西方點墨法檢查三株細胞中p53及p21蛋白表現的情形,結果是:wtp53和mp53細胞中都可以檢查到p53蛋白的表現,無任何轉殖質體的H1299細胞株則完全無p53蛋白的表現。檢查各細胞株之p53下游基因p21蛋白的表現,則wtp53細胞株與無任何轉殖質體的細胞株相較,有較多的p21蛋白的表現。但是mp53細胞株與無任何轉殖質體細胞株,相較其p21蛋白表現,則沒有顯著性差異。由p53蛋白和p21蛋白的表現結果,可以確定這三種細胞株的建立是成功的。
完成以上細胞株建立後,本研究亦進行細胞株對於亞砷酸鈉的藥物敏感度實驗。三株細胞在不同濃度的亞砷酸納處理24小時之後,再培養五日後用SRB assay所得到的結果為:含有wtp53基因的細胞株,對於SA的IC50為3.9uM。含有mtp53基因的細胞株,對於 SA的IC50為6.6uM。negative control的細胞株,對於SA的IC50為15.4uM。因此,wtp53-H1299細胞株對於SA的敏感程度為原來的H1299細胞的4 倍,而mp53-H1299細胞株對於SA的敏感程度為原來的H1299細胞的2.3倍。換句話說,在H1299細胞中回復了p53蛋白的表現,可以增加細胞對藥物的敏感度。在H1299細胞中表現功能不正常的突變型p53蛋白,也使轉殖細胞對藥物的敏感性受到影響。這三種表現不同p53背景的H1299細胞繼續作為臨床上“ p53對應不同抗癌藥物的敏感度測試 ”,可以為本研究之繼續目標之一。

H1299 cells have a homozygous partial deletion of the p53 gene, and thus lack expression of p53 protein. In this study, three kinds of p53-transfected H1299 cells having different p53 gene status have been established from original p53-null H1299 cells. For establishment of wtp53-H1299 cells, normal p53 gene was be constructed from p53-SN into pCDNA3 plasmid , and named it pcDNA3-p53 plasmid. pCDNA3-p53 and EGFP-N1 plasmids were transfected into H1299 cells to establish wtp53/EGFP-H1299 cells by lipofectamine. Two other cell lines, mp53/EGFP-H1299 and pcDNA/EGFP-H1299 cells were established by transfecting pcDNA3-mp53 and pcDNA3, respectively, along with EGFP-N1 plasmids into H1299 cells.
Stable transfected cells were selected in G418-containing medium. They were characterized by Western blotting to examine the background expression of p53 and p21. P53 protein was detected in both wtp53/EGFP-H1299, and mp53/EGFP-H1299 cells. However, p21 protein was only markedly observed in wtp53-H1299 cells in comparison with those in mp53/EGFP-H1299 and pCDNA3/EGFP-H1299 cells. These indicated that all transfectants were have expected results.
The sensitivity to different concentrations of SA in above transfectants was further examined by SRB assay. The IC50 of SA in wtp53/EGFP-H1299, mtp53/EGFP-H1299, and pCDNA3/EGFP-H1299 cells was 3.9 mM, 6.6 mM, and 15.4 mM, respectively. Therefore, the sensitivity to SA was 4-fold and 2.3-fold in wtp53/EGFP-H1299 and mtp53/EGFP-H1299 cells, respectively, in comparison with that in pCDNA3/EGFP-H1299 cells. In other words, expression of wild type p53 could markedly enhance the sensitivity to SA in p53-transfected H1299 cells. However, expression of mutant type p53 also slightly affected the sensitivity to SA in mp3-transfected H1299 cells. We expected above thee transfected cells are suitable in using to investigate the relationship between p53 status and chemosensitivity to various chemotherapeutic agents.

一、中文摘要 -------------------------------------- 2
二、英文摘要 -------------------------------------- 4
三、緒 論 -------------------------------------- 6
四、研究動機 -------------------------------------- 11
五、材料與方法-------------------------------------- 13
六、結 果 -------------------------------------- 22
七、討 論 -------------------------------------- 31
八、參考文獻 -------------------------------------- 35
九、圖表及附錄-------------------------------------- 42

Blagosklonny MV, Giannakakou P, Wojtowicz M, Romanova LY, Ain KB, Bates SE, Fojo T (1998): Effects of p53-expressing adenovirus on the chemosensitivity and differentiation of anaplastic thyroid cancer cells. J Clin Endocrinol Metab 83:2516-22.
Baker SJ, Markowitz S, Fearon ER, Willson JK, Vogelstein B (1990): Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912-5.
Chen PL, Chen YM, Bookstein R, Lee WH (1990): Genetic mechanisms of tumor suppression by the human p53 gene. Science 250:1576-80.
Biard DS, Martin M, Rhun YL, Duthu A, Lefaix JL, May E, May P (1994): Concomitant p53 gene mutation and increased radiosensitivity in rat lung embryo epithelial cells during neoplastic development. Cancer Res 54:3361-4.
Clarke AR (1993): Transgenic approaches to cancer biology. Curr Opin Biotechnol 4:699-704.
Dalton WS (1997): Mechanisms of drug resistance in hematologic malignancies. Semin Hematol 34:3-8.
el Rouby S, Thomas A, Costin D, Rosenberg CR, Potmesil M, Silber R, Newcomb EW (1993): p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 82:3452-9.
Eliyahu D, Michalovitz D, Eliyahu S, Pinhasi-Kimhi O, Oren M (1989): Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U S A 86:8763-7.
Fan S, Smith ML, Rivet DJ, 2nd, Duba D, Zhan Q, Kohn KW, Fornace AJ, Jr., O'Connor PM (1995): Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res 55:1649-54.
Fan J, Bertino JR (1999): Modulation of cisplatinum cytotoxicity by p53: effect of p53-mediated apoptosis and DNA repair. Mol Pharmacol 56:966-72.
Gazdar AF (1992): The molecular biology of lung cancer. Tohoku J Exp Med 168:239-45.
Gjerset RA, Turla ST, Sobol RE, Scalise JJ, Mercola D, Collins H, Hopkins PJ (1995): Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53. Mol Carcinog 14:275-85.
Greenblatt MS, Bennett WP, Hollstein M, Harris CC (1994): Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54:4855-78.
Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, Hovig E, Smith-Sorensen B, Montesano R, Harris CC (1994): Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res 22:3551-5.
Inoue A, Narumi K, Matsubara N, Sugawara S, Saijo Y, Satoh K, Nukiwa T (2000): Administration of wild-type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene. Cancer Lett 157:105-12.
Jung M, Notario V, Dritschilo A (1992): Mutations in the p53 gene in radiation-sensitive and -resistant human squamous carcinoma cells. Cancer Res 52:6390-3.
Kawabe S, Munshi A, Zumstein LA, Wilson DR, Roth JA, Meyn RE (2001): Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts. Int J Radiat Biol 77:185-94.
Karasuyama H, Tohyama N, Tada T (1989): Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene. J Exp Med 169:13-25.
Kemp CJ, Donehower LA, Bradley A, Balmain A (1993): Reduction of p53 gene dosage does not increase initiation or promotion but enhances malignant progression of chemically induced skin tumors. Cell 74:813-22.
Kigawa J, Sato S, Shimada M, Kanamori Y, Itamochi H, Terakawa N (2002): Effect of p53 gene transfer and cisplatin in a peritonitis carcinomatosa model with p53-deficient ovarian cancer cells. Gynecol Oncol 84:210-5.
Kondo S, Barnett GH, Hara H, Morimura T, Takeuchi J (1995): MDM2 protein confers the resistance of a human glioblastoma cell line to cisplatin-induced apoptosis. Oncogene 10:2001-6.
Kuball J, Wen SF, Leissner J, Atkins D, Meinhardt P, Quijano E, Engler H, Hutchins B, Maneval DC, Grace MJ, Fritz MA, Storkel S, Thuroff JW, Huber C, Schuler M (2002): Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation. J Clin Oncol 20:957-65.
Lai SL, Perng RP, Hwang J (2000): p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells. J Biomed Sci 7:64-70.
Lane DP (1992): Cancer. p53, guardian of the genome. Nature 358:15-6.
Lane DP, Crawford LV (1979): T antigen is bound to a host protein in SV40-transformed cells. Nature 278:261-3.
Liebermann DA, Hoffman B, Steinman RA (1995): Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways. Oncogene 11:199-210.
Lowe SW, Ruley HE, Jacks T, Housman DE (1993): p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957-67.
Malkin D, Li FP, Strong LC, Fraumeni JF, Jr., Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, et al. (1990): Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233-8.
Miller C, Mohandas T, Wolf D, Prokocimer M, Rotter V, Koeffler HP (1986): Human p53 gene localized to short arm of chromosome 17. Nature 319:783-4.
Milner J (1991): The role of p53 in the normal control of cell proliferation. Curr Opin Cell Biol 3:282-6.
Nemunaitis J, Swisher SG, Timmons T, Connors D, Mack M, Doerksen L, Weill D, Wait J, Lawrence DD, Kemp BL, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB, Richli WR, Shin DM, Walsh GL, et al. (2000): Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol
Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA (1996): Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer. J Thorac Cardiovasc Surg 112:1372-6; discussion 1376-7.
Pearson AS, Spitz FR, Swisher SG, Kataoka M, Sarkiss MG, Meyn RE, McDonnell TJ, Cristiano RJ, Roth JA (2000): Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells. Clin Cancer Res 6:887-90.
Pirollo KF, Xu L, Chang EH (2000): Non-viral gene delivery for p53. Curr Opin Mol Ther 2:168-75.
Roberts JM, Koff A, Polyak K, Firpo E, Collins S, Ohtsubo M, Massague J (1994): Cyclins, Cdks, and cyclin kinase inhibitors. Cold Spring Harb Symp Quant Biol 59:31-8.
Rogel A, Popliker M, Webb CG, Oren M (1985): p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors. Mol Cell Biol 5:2851-5.
Roth JA (1996): Modification of tumor suppressor gene expression and induction of apoptosis in non-small cell lung cancer (NSCLC) with an adenovirus vector expressing wildtype p53 and cisplatin. Hum Gene Ther 7:1013-30.
Rotter V, Aloni-Grinstein R, Schwartz D, Elkind NB, Simons A, Wolkowicz R, Lavigne M, Beserman P, Kapon A, Goldfinger N (1994): Does wild-type p53 play a role in normal cell differentiation? Semin Cancer Biol 5:229-36.
Salazar AM, Ostrosky-Wegman P, Menendez D, Miranda E, Garcia-Carranca A, Rojas E (1997): Induction of p53 protein expression by sodium arsenite. Mutat Res 381:259-65.
Sanchez Y, Elledge SJ (1995): Stopped for repairs. Bioessays 17:545-8.
Sasaki R, Shirakawa T, Zhang ZJ, Tamekane A, Matsumoto A, Sugimura K, Matsuo M, Kamidono S, Gotoh A (2001): Additional gene therapy with Ad5CMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells. Int J Radiat Oncol Biol Phys 51:1336-45.
Shen L, Chen TX, Wang YP, Lin Z, Zhao HJ, Zu YZ, Wu G, Ying DM (2000): As2O3 induces apoptosis of the human B lymphoma cell line MBC-1. J Biol Regul Homeost Agents 14:116-9.
Siles E, Villalobos M, Valenzuela MT, Nunez MI, Gordon A, McMillan TJ, Pedraza V, Ruiz de Almodovar JM (1996): Relationship between p53 status and radiosensitivity in human tumour cell lines. Br J Cancer 73:581-8.
Wang Y, Blandino G, Oren M, Givol D (1998): Induced p53 expression in lung cancer cell line promotes cell senescence and differentially modifies the cytotoxicity of anti-cancer drugs. Oncogene 17:1923-30.
Yih LH, Lee TC (1999): Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts. Mutat Res 440:75-82.
Yih LH, Lee TC (2000): Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res 60:6346-52.
Xu L, Pirollo KF, Chang EH (2001): Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy. J Control Release 74:115-28.
ATCC網址:http://www.atcc.org/

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top