• 1.四川大學(xué)華西醫(yī)院普外科（成都610041）;;
  • 2.成都地奧制藥集團(tuán)有限公司新藥部（成都610041）;;
  • 通訊作者： 嚴(yán)律南;

【摘要】目的構(gòu)建攜帶多藥耐藥mdr1全長(zhǎng)基因的真核表達(dá)載體并研究其在人肝癌細(xì)胞HepG2中的表達(dá)。
方法雙酶切質(zhì)粒pHaMDR1，獲取含mdr1全長(zhǎng)cDNA片斷，將此片段定向克隆到真核表達(dá)載體PCI-neo多克隆位點(diǎn)，經(jīng)脂質(zhì)體法轉(zhuǎn)染HepG2細(xì)胞，G418篩選穩(wěn)定的細(xì)胞系HepG2/mdr1，PCR檢測(cè)mdr1特異片段，RT-PCR 檢測(cè)HepG2/mdr1細(xì)胞mdr1 mRNA表達(dá)，流式細(xì)胞儀檢測(cè)HepG2/mdr1細(xì)胞P-gp的含量。
結(jié)果成功構(gòu)建攜帶mdr1全長(zhǎng)基因的真核表達(dá)載體，并在HepG2細(xì)胞中表達(dá)，形成穩(wěn)定的細(xì)胞系HepG2/mdr1，其mdr1 mRNA及P-gp的含量較未轉(zhuǎn)染該載體的HepG2細(xì)胞顯著增加。
結(jié)論用真核表達(dá)載體將mdr1全長(zhǎng)基因?qū)肴烁伟┘?xì)胞HepG2能夠建立高效、穩(wěn)定的多藥耐藥細(xì)胞系，為進(jìn)一步研究多藥耐藥機(jī)理提供理想的細(xì)胞模型。

引用本文： 陳永兵,余少鴻,嚴(yán)律南,茍興華,李德華,趙蘭英,張淑彬. 多藥耐藥真核表達(dá)載體的構(gòu)建及其在人肝癌細(xì)胞HepG2中表達(dá)△. 中國(guó)普外基礎(chǔ)與臨床雜志, 2005, 12(3): 254-257. doi: 復(fù)制

版權(quán)信息： ?四川大學(xué)華西醫(yī)院華西期刊社《中國(guó)普外基礎(chǔ)與臨床雜志》版權(quán)所有，未經(jīng)授權(quán)不得轉(zhuǎn)載、改編

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(zhí)民，郭衛(wèi)平，等. 耐藥人肝癌細(xì)胞模型7721/Adm的建立 [Ｊ]. 第四軍醫(yī)大學(xué)學(xué)報(bào)， 2000； 21（4）∶425.
11. 王寶成，郭軍，狄劍時(shí)，等. 肝癌多藥耐藥細(xì)胞株的建立及其多藥耐藥機(jī)理的研究 [Ｊ]. 腫瘤防治研究， 1997； 24（5）∶263.
12. 樊愛(ài)琳，王執(zhí)民，劉國(guó)鵬，等. 逆轉(zhuǎn)錄病毒轉(zhuǎn)染法建立耐藥性大鼠CRBH7919細(xì)胞系 [Ｊ]. 第二軍醫(yī)大學(xué)學(xué)報(bào)， 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.
  1. 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. 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. 3. Rajagopal A, Simon SM. Subcellular localization and activity of multidrug resistance proteins [Ｊ]. Mol Biol Cell, 2003; 14(8)∶3389.
  4. 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. 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. 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. 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. 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. 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. 10. 張洪新，王執(zhí)民，郭衛(wèi)平，等. 耐藥人肝癌細(xì)胞模型7721/Adm的建立 [Ｊ]. 第四軍醫(yī)大學(xué)學(xué)報(bào)， 2000； 21（4）∶425.
  11. 11. 王寶成，郭軍，狄劍時(shí)，等. 肝癌多藥耐藥細(xì)胞株的建立及其多藥耐藥機(jī)理的研究 [Ｊ]. 腫瘤防治研究， 1997； 24（5）∶263.
  12. 12. 樊愛(ài)琳，王執(zhí)民，劉國(guó)鵬，等. 逆轉(zhuǎn)錄病毒轉(zhuǎn)染法建立耐藥性大鼠CRBH7919細(xì)胞系 [Ｊ]. 第二軍醫(yī)大學(xué)學(xué)報(bào)， 2002； 23（5）∶263.
  13. 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. 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.