The Study Of Molecular Mechanism About Vasculogenic Mimicry In Hepatocellular Carcinoma

Objective (1) This experiment is to study if Vasculogenic Mimicry exists in Hepatocellular carcinoma (HCC) by constructing tumor cell HepG₂ tri-dimension culture system and comparing it with tri-dimension cell culture of normal hepatocyte. We detect the expression of COX-2 and MMP-2 protein in tumor cells to reveal its effect to Vasculogenic Mimicry in tri-dimension culture system.(2) To observe Vasculogenic Mimicry in Hepatocelllar carcinoma, and detect the expression of COX-2 and MMP-2 in human Hepatocellular carcinoma for investigating its effect to Vasculogenic Mimicry and tumor differentiated degree.Methods (1) This experiment constructs tumor cell HepG₂ tri-dimension culture system. We cultured HepG₂ cells and normal hepatocytes for comparing the capacity of them to form Vasculogenic Mimicry. COX-2 and MMP-2 antibodies were added in HepG₂ tri-dimension culture system for study of antibody inhibition experiment, stained with PAS and immunohistochemistry for observing the capacity of HepG₂ cells to form VasculogenicMimicry.(2) 31 samples of Hepatocellular carcinoma were collected. All cases were observed byserial section and HE staining, detected the expression of Ferritin and CD31 proteins in the cases by immnohistochemistrial and PAS staining, and detected the expression of COX-2 and MMP-2 proteins by immnohistochemistrial staining.Results (1) Hepatocellular carcinoma cells (HepG₂) formed distinctive loops or networks after 2 days of three-dimensional cultures in collogen matrix, and the cells linked each other to form wreath and net work structures at the seventh day. But normal hepatocytes stretched out leptos tubers. At the third day, the tumor cells began to atrophy. The cells turned round and small, and the nuclei pycnosys. The normal hepatocytes could not form wreath and net work structures.(2) In antibody inhibition experiment, culture dishes which had been added COX-2 antibody were fixed and stained with PAS after cultured for three days. The tumor cells could be observed to stretch out tubers, and had the tendency to form net work structures. But the network structures were less than those tumor cells which not added antibody. Culture dishes which had been added MMP-2 antibody had a kind of tendency to form network structure only in a few part of cells, more cells were dispersal. The culture dishes which have been added COX-2 and MMP-2 antibodies could not form network structure. The tumor cells grew scattered.(3) The expression of COX-2 proteins was stronger than MMP-2 proteins in immunohistochemical staining. The expression of MMP-2 proteins was weaker in the cells added MMP-2 antibody than not added MMP-2 antibody. Added COX-2 proteins could inhibit the expression of MMP-2.(4) Vasculogenic Mimicry could be observed in 22 simples of Hepatocellular carcinoma.(5) COX-2 proteins were detected in all 31 cases of Hepatocellular carcinoma. The expression of COX-2 proteins in Hepatocellular carcinoma with VM was significantly higher than that group without VM (P<0.05).(6) MMP-2 proteins were detected in 30 cases of hepatocellular carcinoma. The expression of MMP-2 proteins in hepatocellular carcinoma with VM was significantly higher than that group without VM (P<0.05).(7) The expression of COX-2 and MMP-2 proteins showed differentiated in different depth groups of differentiation (P<0.05).(8) The expression of COX-2 and MMP-2 proteins was positive correlation in hepatocellular carcinoma.Conclusions (1) Hepatocellular carcinoma cells can form vasculogenic mimicry in vitro, but normal hepatocytes can not.(2) HepG₂ can produce COX-2 proteins. It is very important for the formation of Vasculogenic Mimicry. VM can be abrogated by using inhibitors of COX-2 proteins in tri-dimension cell culture system.(3) COX-2 proteins can inhibit the formation of Vasculogenic Mimicry by mediating the up-regulation of MMP-2.(4) Vasculogenic Mimicry exists in human Hepatocellular carcinoma.(5) The expression of COX-2 and MMP-2 proteins all can be detected in Hepatocellular carcinoma. Expression of COX-2 proteins is related with VM in Hepatocellular carcinoma and the expression of COX-2 and MMP-2 proteins is positive correlation.(6) As the differentiated degree degrades, the expression of COX-2 and MMP-2 proteins is positive correlation.