Construction Of The RNAi Adenovirus Vector And Study Of The Transfection Of Hepatocellular Carcinoma Cell Targeting MDR1 Gene

Objective:To construct RNAi adenovirus vector targeting MDR1 and to investigate its reverse effect on hepatoma drug fast cell MDR1 gene and the expressional , inhibitive function on P-glycoprotein(P-gp),after transfecting to hepatoma drug fast cell strain SMMC-7721/ADM using expressional vector system targeting MDR1 gene. Method:1.Through the analysis of PGE-1 and the gene order of pshMDR1 to find the integrated shRNA expression unit which contains U6 promoter and shRNA sequence, besides it contains two enzyme sites Xho and XbaI, coincide with MCS enzyme site of pshuttle. shRNA expression unit was constructed to pshuttle by XhoI and XbaI double enzyme incision,named pshuttleMDR1.2. The constructed pshRNA-MDR1was enzyme cutted into filate plasmid by PmeI enzyme,then dephosphorylated to prevent its recirculation, BJ5183 which contained recombinase was concomitantly transformed by adenovirus skeleton plasmid PAdeasy and it, the recombinant adenovirus plasmid PAd- MDR11 was achieved in the strain。3. The viral particle package was achieved by the culture of AD293 cells and the products produced by plasmid PAd- MDR11 enzyme which was cutted by PacI.4. The hepatoma drug fast cell strain SMMC-7721/R infected by the virus, its expression of superficial cell membrane P-gp and the storage of intracellular Rhdaming123(Rh123)(P-gp function test)was detected by flow cytometry,the distribution of intracellular Rh123 was detected by laser scanning confocal microscopy, P-g quantity was detected by Western Blot.Results:1. All of the electrophoretic analysis demonstrated there was a single relucent strap 600bp at of the relative molecular mass following PCR amplification of recombinant pshRNA-MDR1 and Padeasy- shMDR1 vector, which was consistent with the features of the positive clone.2.The detectional results of the co-culture of Luo Danmin 123 and the cells at 30 days by flow cytometry demonstrated the mean fluorescence intensity of the cells transfected by PAd- MDR11 and PAd- MDR13 was significantly higher than the cells of SMMC-7721/R cell line, the positive rates were 91.5%,90.8% respectively,the positive rate of the latter was 29.7%,which has significant difference (p<0.05).3. The detection of superficial cell membrane P-gp could be achieved by indirect immunofluorescence, the positive rates of each group's cells respectively were :SMMC-7721/S, 26.3%; SMMC-7721/R,85.3%; SMMC-7721/R+ PAd- MDR11 22.9%; SMMC-7721/R+PAd- MDR13, 30.8%; SMMC-7721/R+PGE-1 , 85.8% by flow cytometry.4.P-gp protein quantity detection by Western Blot:the coloration of SMMC-7721/R infected by virus at 170Kd was significantly lower than control SMMC-7721/R,but approximated with SMMC-7721/S.Conclusion: recombinant plasmidpAD-MDR11 has been successfully established,equally, it has efficiently cut down the expressions of hepatoma drug fast cell MDR1 mRNA.