| [Object] To investigate the reversal effect of multidrug resistance of human hepatocellular cell (HCC) line of SMMC7721/ADM with antisense RNA of multidrug resistance gene (mdr1) delivered by recombinant adenoviruses. [Methods] The primers were designed to code the 5 region flanking the ATG start codon region sequence of 609 bp. The total RNAs of SMMC7721/ADM cells were collected and from the RNAs the fragment of mdrl gene was enlarged by RT-PCR. The fragment of mdrl gene was coloned into the vector of pMD18-T, directly which was transfected into Escherichia coli Top 10, the expression of mdrl gene was verified by gene sequence examination. The fragment of mdrl gene was obtained from the plasmid of pHaMDR1-1 containing the whole human mdrl cDNA, and was cloned reversely into multiple cloning sites of the shuttle plasmid of adenoviral vector AdEasy. The homologous recombination took place in the Escherichia coli BJ5183 with skeleton plasmid of AdEasy system and the recombinant adenoviral plasmid was generated. The adenoviruses of pAdEasy-GFP-ASmdrl containing antisense mdrl gene and pAdEasy-GFPwithout favoriting gene were packaged and amplificated in the 293 cells. The viral titer and the growth curve of human HCC line SMMC7721/ADM inffected by recombination virus were detected. The HCC cells of SMMC7721/ADM were divided into three groups in vitro. The cells of group A were infected by pAdEasy-GFP-ASmdrl (MOI=100), group B were infected by pAdEasy-GFP (MOI=100), and group C were no treated. The chemosensitivity of all groups to the chemotherapeutic drugs of adriamycin and daunorubicin was verified by MTT assay. The levels of mdrl mRNA and gene-encoded P-glycoprotein (P-gp) expression were analysised by RT-PCR and flow cytometry (FCM) respectively at the different time point after infection. The accumulation of the daunorubicin was determinated by FCM simultaneously. In vivo, after the nude mice model of grafting tumor was established by injecting subcutaneously HCC cells treated by different viruses in axilla. Once the tumor diameter reach to 5mm, adriamycin was injected into peritoneal cave. The size and growth inhibition of tumor were evaluated.[Resultes] The amplification fragment of 609bp extracted from the total RNAs of SMMC7721/ADM by RT-PCR was identified with the fragment of mdrl cDNA by gene sequence examination. The recombinant adenoviruses of pAdEasy-GFP-ASmdrl and pAdEasy-GFP were produced successfully. Its titer was 2.5 X 109efu/ml and 3X109efu/ml respectively. The growth and proliferation of SMMC7721/ADM cells was not affected by recombinant adenoviruses. Compared with group B and C, the group A enhanced the sensitivity of two anticancer drugs, the IC50 to ADM and DNR after being infected by pAdEasy-GFP-ASmdrl was 0.44 u g/ml and 0.20 u g/mlrespectively, with the factors of resistance decreased by 40 and 113.33 respectively. The levels of mdrl mRNA and P-gp expression were down-regulated obviously, and the accumulation of DNR in group A was increased evidently. In the nude mice HCC model, the tumorigeneses of three groups were identified. After adriamycin therapy, the tumor size in group A was smaller than the other groups, the tumor growth was inhibited. Compared with the nude mice in group B and C, the tumor growth was inhibited by 44.14% and 41.59% respectively.[Conclusions] Using AdEasy system, recombinant adenoviruses were obtained simply and quickly. In vitro and in vivo, the antisense RNA could inhibit the expression of mdrl gene in HCC cells and partially reverse the multidrug resistance. It would provide the experimental basis of gene therapy in HCC.
|
PDF Full Text Request