Effect Of Different Polarized Macrophages On Invasiveness And Metastasis In Hepatocellular Carcinoma

Part 1 Objective Isolate monocytes and polarize to be different activated macrophages, explore the effect of different activated macrophages on hepatocellular carcinoma cell proliferation 、 migration 、 invasiveness and proangiogenesis.Methods In vitro, we use gradient centrifugation method to isolate monocytes, using different agents to polarize monocytes to be different activated macrophages. In wound-healing assay, adding supernatants derived from different activated macrophages to tumor medium, after co-culture 24 hours, detect the contribution of different activated macrophages to the migration of hepatocellular cancer cell lines, we also co-culture hepatocellular cancer cell lines with different activated macrophages, at indicated time, count the cell number which passed through the Matrigel plug.Co-culture hepatocellular cells with the supernatant of different activated macrophages, investigate VEGF-A and MMP-9 expression by using RT-PCR and Western-Blot methods. Result After co-cultivation, we found that hepatocelllular cancer cells co-culture with M2 macrophages have stronger migration ability than cells co-culture with M1 macrophages. In Transwell experiment, results indicated that tumor cells co-culture with M2 macrophages have stronger invasiveness than co-culture with M1 macrophages.Co-culture with M2 macrophages,hepatocellular cancer cells obtained stronger ability of proliferation than co-culture with M1 macrophages. Also, in co-culture system, hepatocellular cancer cells express VEGF-A and MMP-9 more strongly after co-culture with M2 macrophages than M1 macrophages.Conclusion Hepatocellular cancer cells co-culture with M2 macrophages make greater contribution to hepatocellular cancer than co-culture with M1 macrophages. Part 2 Objective The aim of the present study is to explore the effect of different activated states of macrophages on the metastasis and invasiveness of HCC and investigate the relation between the macrophages and the prognosis of HCC patients who have undergone surgical resection.Methods CD68+HLA-DR+, CD68+CD163+immunostaining was evaluated as M1 macrophages and M2 macrophages respectively,use indirect immunofluorescence staining, associated with the clinical pathological parameter, drawing survival curve, analysis the impact of different activated states of macrophages on the patients with HCC.Result The results of immunohistochemistry indicated that M2 macrophage is a dependent prognosis index of patients with HCC, associated with poor prognosis of patients with HCC. Kaplan-Meier survival analysis indicates that lower expression of M2 macrophages associated with longer survival time and favourable prognosis.Conclusion M2 macrophages infiltration is a poor progonosis factor for HCC patients, M2 macrophage makes more contribution to HCC invasiveness than M1 macrophage. Part 3 Objective Explore the relationship between different activated macrophages with MMP-9、VEGF-A、NF-κB p50.Method Explore the expression of MMP-9、VEGF-A、NF-κB p50 in hepatocellular carcinoma by IHC method, analysis the relationship between different activated macrophages.Result There is a discrepancy of distribution of MMP-9、VEGF-A、NF-κB p50 expression, high expression of MMP-9、VEGF is associated with shorter survival time.M2 macrophages are more closely associated with MMP-9、VEGF-A、NF-κB p50 expression. Conclusion M2 macrophages contribute to the MMP-9、VEGF-A、NF-κB p50 expression, facilitate hepatocellular carcinoma metastasis.