Establishment Of Multidrug Resistant Cell Line, HepG2/mdr1, Based On Transferring The Mdr1 CDNA Into Hepatocellular Carcinoma Cell

The incidence rate of hepatocellar cancinoma (HCC) continues to increase and its deaths in the whole world are more than 5 millions every year. Although chemical therapy is one of the most important treatments for HCC, the multidrug resistance(MDR) is still an obsruct. To solve the problem, MDR cell lines are dispensible. Untill now, the popular method to establish MDR cells line is drug selection, which has many shortcomings, i.e. time-expending, MDR-unstability and michanism-complexity. In order to overcome these disadvantages, a creative mothed based on transferring the mdrl cDNA into target cells has been found in human bladder cancer cells line and rat lung cancer cells line, but not yet in HCC. Therefore the HCC MDR cells line by gene transfer will be an ideal model for HCC MDR research.[Object] To construct a mdrl expression vector which can express the transmembular P-gp in HepG2 cells in vitro; To establish multidrug resistant hepatocellular carcinoma cell line, HepG2/mdrl, based on transferring the mdrl cDNA into HepG2 cells; To study the MDR michanism of HepG2/mdrl cells by observing the drug resistance of HepG2/mdrl cells invivo and in vitro as well as the reversal effect of antisense RNA of mdrl. [Methods] The fragment of human mdrl cDNA was obtained from the plasmid pHaMDRl by enzymatic digestion, and was cloned into multiple cloning sites of the PCI-neo mammalian expression vector, which was later transfered to human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and prolifered and exposed to mild concentration of ADM transiently to screen out the multidrug resistant hepatocellular carcinoma cell line, HepG2/mdrl. The total DNAs of HepG2/mdrl cells were collected and the specific fragment of mdrl gene was enlarged by PCR. The growth curve of HepG2/mdrl cells was detected and the chemosensitivity of HepG2/mdrl cells to the chemotherapeutic drugs of ADM and MMC was verified by MTT assay. The levels of mdrl mRNA and gene-encoded P-glycoprotein (P-gp) and MRP expression were analysised by RT-PCR and flow cytometry (FCM) respectively. The accumulation of the daunorubicin was determinated by FCM simultaneously. The HepG2/mdrl cells were infected by the recombinant adenoviruses of pAdEasy-GFP-ASmdrl containing antisense mdrl gene to determind whether it can reverse MDR of HepG2/mdrl cells. 48 h after the infection, the chemosensitivity to ADM, the levels of mdrl mRNA and the P-gp were analysed as above. The nude mice model of grafting tumor was established by injecting subcutaneously HepG2/mdrl cells in axilla. Once the tumor diameter reachs to 5mm, ADM was injected into peritoneal cave. The size and growth inhibition of tumor were evaluated. All through the whole experiment, the same do the parental cells(HepG2 cells)as the control group. [Resultes] The mdrl expression vector was constructed successfully andalso the MDR hepatocarcinoma cell line HepG2/mdrl was developed as well. The outcome of PCR analysis showed that the specific fragment of mdrl cDNA could be found in HepG2/mdrlcells, but not in the control group HepG2 cells. The growth and proliferation of HepG2/mdrl cells was not affected by the mdrl transfer. Compared with the parental cells(HepG2 cells), the sensitivity of cells to ADM and MMC was decreased by 35 times and 125 times respectively, the IC50 was 25 u g/ml and 10 u g/ml consequently.Except for MRP, the levels of mdrl mRNA and P-gp expression were upregulated obviously, and the accumulation of DNR in group A was decreased evidently. After the infection of the recombinant adenoviruses, the sensitivity of HepG2/mdrl cells to ADM was completely restored, the difference of IC50 and the accumulation of DNR as well as the levels of mdrl mRNA and P-gp expression between HepG2/mdrl and HepG2 cells was not significant. In the nude mice HCC model, the tumorigenes of both groups was identified. After ADM therapy, the mean size of HepG2 cell tumors was obviously smaller than HepG2/mdrl cell tumors, Compared to HepG2/mdrl cell tumors, the tumor growth was inhibited by 44.6%.[Conclusions] The transfer of mdrl cDNA with the help of the PCI-neo mammalian expression vector can evidently enhance the expressiong of functional P-gp in HepG2 cells. The approach using the transfer of mdrl cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechnism is lucid and which would provide the experimental basis of MDR research.