Construction of mdr1 Expression Vector and Detection of Its Expression in HepG2 Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

CHEN Yongbing ¹ , YU Shaohong ¹ , YAN Lnan ¹ , GOU Xinghua ² , LI Dehua ² , ZHAO Lanying ² , ZHANG Shubin. ¹

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Keywords：

Multidrug resistance Human hepatocellular carcinoma P-glycoprotein Plasmid

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【Abstract】ObjectiveTo construct an mdr1 expression vector and detect its expression in HepG2 cells in vitro.
MethodsThe 4.5-kb mdr1 cDNA was obtained from the plasmid pHaMDR1 cloned into the PCIneo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated，and the specific fragment of mdr1 cDNA, mRNA and the Pgp in these HepG2 cells were detected by means of PCR, RT-PCR and FCM respectively.
ResultsThe mdr1 expression vector was constructed successfully，and the stable multidrug resistance(MDR) hepatocarcinoma cell line （HepG2/mdr1） was developed as well. The outcome of PCR analysis showed that the specific fragment of mdr1 cDNA could be found in HepG2/mdr1 cells, but not in the nontransfection HepG2 cells. Furthermore,the content of the specific fragment of mdr1 mRNA and the expression of P-gp in HepG2/mdr1 cells were (59.7±7.9)% and (12.5±5.45)% respectively, the corresponding value in HepG2 cells were (16.9±3.2)% and (4.63±2.59)% respectively. The difference was statistically significant (P＜0.05).
ConclusionIt is praticable to develop MDR hepatocarcinoma cell line by transferring mdr1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.

Citation： CHEN Yongbing,YU Shaohong,YAN Lnan,GOU Xinghua,LI Dehua,ZHAO Lanying,ZHANG Shubin.. Construction of mdr1 Expression Vector and Detection of Its Expression in HepG2 Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2005, 12(3): 254-257. doi: Copy

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.	张洪新，王执民，郭卫平，等. 耐药人肝癌细胞模型7721/Adm的建立 [Ｊ]. 第四军医大学学报， 2000； 21（4）∶425.
11.	王宝成，郭军，狄剑时，等. 肝癌多药耐药细胞株的建立及其多药耐药机理的研究 [Ｊ]. 肿瘤防治研究， 1997； 24（5）∶263.
12.	樊爱琳，王执民，刘国鹏，等. 逆转录病毒转染法建立耐药性大鼠CRBH7919细胞系 [Ｊ]. 第二军医大学学报， 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. 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. 张洪新，王执民，郭卫平，等. 耐药人肝癌细胞模型7721/Adm的建立 [Ｊ]. 第四军医大学学报， 2000； 21（4）∶425.
11. 王宝成，郭军，狄剑时，等. 肝癌多药耐药细胞株的建立及其多药耐药机理的研究 [Ｊ]. 肿瘤防治研究， 1997； 24（5）∶263.
12. 樊爱琳，王执民，刘国鹏，等. 逆转录病毒转染法建立耐药性大鼠CRBH7919细胞系 [Ｊ]. 第二军医大学学报， 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of mdr1 Expression Vector and Detection of Its Expression in HepG2 Cells

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