The Study Of Construction Of The Over-Expression Of Annexin A10 Gene Lentivirus And Its Effection On Human Hepatocellular Carcinoma Cell Line HepG2 In Vitro

Objective:1. To construct human annexin-10 (ANXA10) and Enhance green fluorescent protein (EGFP) fuse gene of recombinant lentiviral;2. To examine the effects on cell apoptosis and proliferation of human hepatocellular carcinoma HepG2 cell which transfected by recombinant lentiviruses and effect on the expreession of matrix metalloproteinase-9(MMP-9),vascular endothelial growth factor(VEGF). Initially illuminated the effects of ANXA10 gene on human hepatocellular carcinoma HepG2 cell of cell apoptosis,proliferation and transfer.Methods:1.According to the ANXA10 gene screening has determined and subcloned ANXA10 into the lentiviral vector, pGC-FU, to generate the lentiviral expression vector, pGC-FU-ANXA10. The corrected ANXA10 was confirmed by endoenzym digestion, sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGC-FU-ANXA10, with the packaging plasmids pHelper1.0 and pHelper2.0.2.The resulting recombinant lentiviruses with suitable MOI which carrying ANXA10 were then used to transfect huaman hepatocellular carcinoma HepG2 cell in vitro. divide into lentiviral group (LV-ANXA10), containing ANXA10 gene and green fluorescent protein,the control group were the HepG2 cell transfected with negative vector group(Negative vector group),and the HepG2 cell non-transfected with lentivirus(HepG2 group). ANXA10 expression in huaman hepatocellular carcinoma HepG2 cell were dectected by reverse-transcripted polymerase chain reaction (RT-PCR)and Western blotting(WB) analysis.The transfection efficiency was determined by detecting the GFP expression with the fluorescent microscopy.The expression level of ANXA10 mRNA was examined by RT-PCR.The expression level of ANXAIO protein was finally examined by Western blotting for identifing the inhibitory efficiency of ANXA10.In order to analyze the effects of ANXA10 gene on the expression of MMP-9 and VEGF,as well as cell apoptosis and proliferation,the methods mentioned above was applied to detect the changes in expression of MMP-9 and VEGF on mRNA and/or protein level respectively.For the same purpose,flow cytometry were performed to examine the changes in cell apoptosis and proliferation after lentiviruses which carrying ANXA10 were used to transfect HepG2 cell.Results:1.We got lentiviruses which carrying ANXA10 and their gene sequence were correct.The titer of concentrated virus was 2E+8.2.The recombinant lentiviruses which carrying ANXA10 were used to transfect effectively huaman hepatocellular carcinoma HepG2 cell in vitro.After day 3-4, the expression level of the GFP fluorescent protein was higest, The MOI was determined by detecting the GFP fluorescent protein,and the suitable MOI was 10 for HepG2 cell line, and transfected with lentiviral vectors in the presence of polybrene(5μg/ml).3.After the recombinant lentiviruses which carrying ANXA10 were used to transfect effectively huaman hepatocellular carcinoma HepG2 cell 72h,the inhibited proliferation rate of HepG2 cells suggestion, the LV-ANXA10 group 24.646%, was significantly higher than those of the Negative vector group and HepG2 cell group (p<0.05).And the percentage of the apoptosis cells of the LV-ANXA10 group 51.92±1.41%,those of the Negative vector group and HepG2 cell group were 19.00±1.12%,3.59±0.89%,respectively.The percentage of the apoptosis cells of the LV-ANXA10 group was significantly higher than those of the Negative vector group and HepG2 cell group (p<0.05).4.After transfection with ANXA10,the result suggest that the expression level of ANXA10 mRNA of the LV -ANXA10 group was examined by RT-PCRand the expression level of ANXA10 protein by Western blotting were higher than the other two groups.5.After the recombinant lentiviruses which carrying ANXA10 were used to transfect effectively huaman hepatocellular carcinoma HepG2 cell,down-regulating the expression of both MMP-9 and VEGF gene mRNA and protein levels compared with the Negative vector group and HepG2 cells group.(P<0.05)Conclusions: 1.The over-expression of human ANXA10 gene were inhibited proliferation and promoted apoptosis of the huaman hepatocellular carcinoma HepG2 cell;2. The over-expression of human ANXA10 gene were down-regulating the huaman hepatocellular carcinoma HepG2 cell relevant factors expression of MMP-9 and VEGF gene, and thereby maybe inhibited the transfer of huaman hepatocellular carcinoma HepG2 cell.