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目的研究4种耐药蛋白:多药耐药蛋白(multi-drug resistance protein 1,MDR1),多药耐药相关蛋白1(multi-drug resistance related protein 1,MRP1),肺耐药蛋白(lung resistance protein, LRP),乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)在肝癌多药耐药中的作用。方法通过培养液中阿霉素(adriamycin,ADM)浓度递增诱导法筛选培养,建立HePG2／ADM肝癌多药耐药细胞株;采用real time荧光定量PCR检测四种耐药蛋白mRNA在正常肝细胞系L02、肝癌细胞系HePG2及HePG2／ADM中的表达差异;Western blot检测3种细胞系中4种耐药蛋白的表达。结果(1)建立HePG2／ADM细胞系。MTT法检测阿霉素对HePG2／ADM细胞IC50为亲本细胞的282倍(P<0．05)。(2)HePG2／ADM中MDR1和BCRP mRNA表达分别是HePG2的400倍(F=87．49, P<0．05)和9倍(F=1006,P<0．05)。MRP1和LRP mRNA表达差异无统计学意义(FMRP1=3．43, FLRP=2．44,Pall>0．05)。(3)L02和HePG2中4种耐药蛋白mRNA的表达均无差异(FMDR1=1006, FBCRP=87．49,FMRP1=3．43,FLRP=2．44,Pall>0．05)。(4)Western blot检测发现HePG2／ADM细胞中MDR1,BCRP,LRP蛋白表达显著高于HePG2和L02细胞(FMDR1=28．68,FBCRP=18．60,FLRP= 6．28,Pall<0．05),MRPI蛋白表达不增高(FMRP1=0．70,P>0．05)。(5)4种耐药蛋白表达在HePG2和L02细胞差异均无统计学意义(FMDR1=28．68,FBCRP=18．60,FMRP1=0．70,FLRP=6．28, Pall>0．05)。结论MDR1和BCRP在HePG2／ADM多药耐药中起重要作用,HePG2／ADM多药耐药与MRP1和LRP可能无关。
Objective To investigate the expression of four multidrug resistance proteins (MDR1), multi-drug resistance related protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) in multidrug resistance of liver cancer. Methods HepG2 / ADM multidrug-resistant hepatocarcinoma cell lines were established by increasing the concentration of adriamycin (ADM) in culture medium. Real-time fluorescent quantitative PCR was used to detect the expression of four multidrug resistance protein mRNAs in normal liver cell line L02, HepG2 and HepG2 / ADM cell lines. Western blot was used to detect the expression of four drug resistance proteins in three cell lines. Results (1) HePG2 / ADM cell line was established. The IC50 of Adriamycin on HepG2 / ADM cells was 282-fold higher than that of the parental cells by MTT assay (P <0.05). (2) The mRNA expressions of MDR1 and BCRP in HepG2 / ADM were 400 times (F = 87.49, P <0.05) and 9 times (HepG2, F = 1006, P <0.05) respectively. There was no significant difference in MRP1 and LRP mRNA expression (FMRP1 = 3.43, FLRP = 2.44, Pall> 0.05). (3) There was no difference in the expression of four kinds of resistance protein mRNA in L02 and HePG2 (FMDR1 = 1006, FBCRP = 87.49, FMRP1 = 3.43, FLRP = 2.44, Pall> 0.05). (4) Western blot showed that the expression of MDR1, BCRP and LRP in HePG2 / ADM cells was significantly higher than that in HePG2 and L02 cells (FMDR1 = 28.68, FBCRP = 18.60, FLRP = 6.28, Pall ), MRPI protein expression was not increased (FMRP1 = 0.70, P> 0.05). (5) There was no significant difference in the expression of four kinds of resistance proteins between HepG2 and L02 cells (FMDR1 = 28.68, FBCRP = 18.60, FMRP1 = 0.70, FLRP = 6.28, Pall> 0.05 ). Conclusion MDR1 and BCRP play an important role in multidrug resistance of HePG2 / ADM. Multidrug resistance of HePG2 / ADM may not be related to MRP1 and LRP.