Study On Intratumoral Injection Of Recombinant Lentivirus Mediated ANNEXIN A10Inhibits The Growth Of HepG2Xenografts In Nude Mice

Objective：1, To construct subcutaneously xenografts tumor models of humanhepatocarcinoma cell line HepG2in nude mice.2,Recombinant lentivirus mediated Annexin A10injecting into xenografts ofnude mice,observe the growth of Subcutaneously transplanted tumor and theexpression of matrix metalloproteinase-9(MMP-9), vascular endothelial growthfactor(VEGF).Initially illuminated the effects of ANXA10gene overexpression inxenografts in nude mice on cell proliferation, apoptosis and the expression of livercancer invasion and metastasis-related cytokines MMP-9,VEGF.Methods：1,Human hepatocellular carcinoma HepG2cell suspension(0.2ml,1×10~7/ml)inject into right subcutaneously in nude mice.24days later, the tumor diameter up to1.7-2.0cm,remove and cut into1mm×1mm×1mm size organizations,wereinoculated into the right subcutaneously of the24female nude mice and measuringtumor size in every3d to calculate the tumor volume.2,7days later, the tumor diameter up to0.3-0.5cm,divided the mice intoexprimental group(LV-ANXA10),negative control group(LV-GFP) and blank controlgroup randomly.LV-ANXA10,LV-GFP and PBS were intratumorally injected with2×10~7TU (100ul) respctively in subcutaneous xenograft model,repeated injection3hlater.Nude mice were sacrificed and tumor samples removed later. The level ofANXA10gene in tumor samples were dectected by reverse-transcripted polymeraseChain reaction(RT-PCR) and The protein expression of ANXA10were dectected byimmunohistochemistry in organizatiional level. In order to analyze the effects ofANXA10gene on the expression of MMP-9and VEGF,as well as cell apoptosis andproliferation,the methods mentioned above was applied to detect the changes inexpression of MMP-9and VEGF on gene and/or organizational level respectively.TUNEL(TdT-mediated biotinylated-dUTP nick end labing method)staining were used to determine apoptosis and apoptosis index were measured.Results：1, construct subcutaneously transplanted tumor models of humanhepatocarcinoma cell line HepG2in nude mice successfully.2, The growth rate of subcutaneous xenograft significantly decreased inexprimental group,the tumor's size and weight of experimental group weresignificantly lesser than those of negative control group and blank control group(forvolume, F=19.13,P＜0.01,;for weight,F=46.91,P＜0.01)3,RT-PCR and Immunohistochemistry：in exprimental group, the expressionlevel of ANXA10mRNA and protein were higher than the other two groups,(P＜0.05). in negative control group and blank control group,the expression level ofVEGF and MMP-9mRNA and protein were higher than the exprimental groups,(P＜0.05),all of the difference were statistically signifiant.4, TUNEL assay apoptosis index:AI of exprimental group was(27.87±1.20)%,there was significant difference among negative control group(3.47±0.72)%and blank control group(3.38±0.77%)%,(P＜0.05).Conclusions：1,LV-ANXA10can dramatically inhibit the growth of human hepatocarcinomacell line HepG2and induce it apoptosis in vivo.2,The overexpression of ANXA10in HepG2xenografts maybe inhibited theexpression of MMP-9and VEGF,which were the cytokines ralated to liver cancerinvasion and metastasis...