【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.
【Abstract】ObjectiveTo construct a mrp1 expression vector and investigate its biological characteristics in HepG2 cells in vitro. MethodsThe 6.5 kb multidrug resistanceassociated protein （MRP) cDNA obtained from plasmid pGEM-mrp1 was 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 mrp1 mRNA and MRP in these HepG2 cells were detected by means of RT-PCR and FCM respectively. ResultsThe mrp1 expression vector was established successfully, and the stable MDR hepatocarcinoma cell line (HepG2/mrp1) was developed as well. The content of the specific fragment of mrp1 mRNA was (56.8±6.37)% and MRP was 7.89 in the HepG2/mrp1 cells, the corresponding value in HepG2 cells was (9.67±3.26)% and 0.79 respectively. The difference was statistically significant (P＜0.05). ConclusionIt is practicable to establish MDR hepatocarcinoma cell line by transferring mrp1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
【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 PCIneo 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 Pgp 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.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.