多藥耐藥真核表達載體的構建及其在人肝癌細胞HepG2中表達△_《中國普外基礎與臨床雜志》

作者：

陳永兵 ¹ , 余少鴻 ¹ , 嚴律南 ¹ , 茍興華 ² , 李德華 ² , 趙蘭英 ² , 張淑彬 ¹

1.四川大學華西醫院普外科（成都610041）;;
2.成都地奧制藥集團有限公司新藥部（成都610041）;;
通訊作者：嚴律南;

關鍵詞：

多藥耐藥人肝癌細胞 P-糖蛋白質粒

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【摘要】目的構建攜帶多藥耐藥mdr1全長基因的真核表達載體并研究其在人肝癌細胞HepG2中的表達。
方法雙酶切質粒pHaMDR1，獲取含mdr1全長cDNA片斷，將此片段定向克隆到真核表達載體PCI-neo多克隆位點，經脂質體法轉染HepG2細胞，G418篩選穩定的細胞系HepG2/mdr1，PCR檢測mdr1特異片段，RT-PCR 檢測HepG2/mdr1細胞mdr1 mRNA表達，流式細胞儀檢測HepG2/mdr1細胞P-gp的含量。
結果成功構建攜帶mdr1全長基因的真核表達載體，并在HepG2細胞中表達，形成穩定的細胞系HepG2/mdr1，其mdr1 mRNA及P-gp的含量較未轉染該載體的HepG2細胞顯著增加。
結論用真核表達載體將mdr1全長基因導入人肝癌細胞HepG2能夠建立高效、穩定的多藥耐藥細胞系，為進一步研究多藥耐藥機理提供理想的細胞模型。

引用本文： 陳永兵,余少鴻,嚴律南,茍興華,李德華,趙蘭英,張淑彬. 多藥耐藥真核表達載體的構建及其在人肝癌細胞HepG2中表達△. 中國普外基礎與臨床雜志, 2005, 12(3): 254-257. doi: 復制

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1. Chang G, Roth CB. Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters [Ｊ]. Science， 2001; 293(5536)∶1793.
2. Siddiqui A, Kerb R, Weale ME, et al. Association of multidrug resistance in epilepsy with a polymorphism in the drugtransporter gene ABCB1 [Ｊ]. N Engl J Med, 2003; 348(15)∶1442.
3. Rajagopal A, Simon SM. Subcellular localization and activity of multidrug resistance proteins [Ｊ]. Mol Biol Cell, 2003; 14(8)∶3389.
4. Ueda K, Cardarelli C, Gottesman MM, et al. Expression of a fulllength cDNA for the human “MDR1” gene confers resistance to colchicine, doxorubicin, and vinblastine [Ｊ]. Proc Natl Acad Sci USA, 1987; 84(9)∶3004.
5. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants [Ｊ]. Biochim Biophys Acta, 1976; 455(1)∶152.
6. Callen DF, Baker E, Simmers RN, et al. Localization of the human multiple drug resistance gene, MDR1, to 7q21.1 [Ｊ]. Hum Genet, 1987; 77(2)∶142.
7. Scanlon KJ, Ishida H, KashaniSabet M. Ribozymemediated reversal of the multidrugresistant phenotype [Ｊ]. Proc Natl Acad Sci USA, 1994; 91(23)∶11123.
8. Yang LY, Trujillo JM. Biological characterization of multidrugresistant human colon carcinoma sublines induced/selected by two methods [Ｊ]. Cancer Res, 1990; 50(11)∶3218.
9. Wang FS, Kobayashi H, Liang KW, et al. Retrovirusmediated transfer of antiMDR1 ribozymes fully restores chemosensitivity of Pglycoproteinexpressing human lymphoma cells [Ｊ]. Hum Gene Ther, 1999; 10(7)∶1185.
10. 張洪新，王執民，郭衛平，等. 耐藥人肝癌細胞模型7721/Adm的建立 [Ｊ]. 第四軍醫大學學報， 2000； 21（4）∶425.
11. 王寶成，郭軍，狄劍時，等. 肝癌多藥耐藥細胞株的建立及其多藥耐藥機理的研究 [Ｊ]. 腫瘤防治研究， 1997； 24（5）∶263.
12. 樊愛琳，王執民，劉國鵬，等. 逆轉錄病毒轉染法建立耐藥性大鼠CRBH7919細胞系 [Ｊ]. 第二軍醫大學學報， 2002； 23（5）∶263.
13. Ledoux S, Yang R, Friedlander G, et al. Glucose depletion enhances Pglycoprotein expression in hepatoma cells: role of endoplasmic reticulum stress response [Ｊ]. Cancer Res, 2003; 63(21)∶7284.
14. Nakajima T, Takayama T, Miyanishi K, et al. Reversal of multiple drug resistance in cholangiocarcinoma by the glutathione Stransferasepispecific inhibitor O1hexadecylgammaglutamylSbenzylcysteinylDphenylglycine ethylester [Ｊ]. J Pharmacol Exp Ther, 2003; 306(3)∶861.

《中國普外基礎與臨床雜志》

多藥耐藥真核表達載體的構建及其在人肝癌細胞HepG2中表達△

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《中國普外基礎與臨床雜志》

多藥耐藥真核表達載體的構建及其在人肝癌細胞HepG2中表達△

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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