The Expression And Drug Inhibition Of CD206 In Tumor Associated Macrophages Of Primary Hepatocellular Carcinoma

PART Ⅰ XPRESSION OF CD206 IN TUMOR ASSOCIATEDMACROPHAGES OF HEPATOCELLULAR CARCINOMAObjective: To study the relationship between the expression of tumor associated macrophage(TAM) marker CD206 and the prognosis of primary hepatocellular carcinoma(HCC) and to analyze the clinical characteristics of HCC patients with CD206 expression by using hepatocellular carcinoma tissue microarray.Method: We have collected the HCC tissue samples and the corresponding clinical data during January 2010 to September 2011 who undergo surgery in our department. The follow-up period was until September 2013, the complete clinical data were followed, including gender, age, liver function and tumor stage. The tumor and the peri-carcinoma tissues were made into 10x18, totally 180 points microarray(One point in the tumor and the other in the peri-carcinoma tissues). Using immunohistochemistry to detect the expression of CD206 in the microarray. The Kaplan Meier method, log- rank test was applied to analyze the relationship of survival rates of patients with liver cancer and the expressions of CD206.COX regression model was applied to analyze the correlation between survival rates of patients and clinical indicators.Results: Tissue microarray results show the level of CD206 is significantly higher in HCC than the pericarcinous tissue(P<0.05).Kaplan Meier method and the log- rank test showed that the overall survival rate of higher CD206 expression group in tumors was significantly lower than that of CD206 lower expression group(p=0.008),while the expression of CD206 in peri-tissues had no significant difference(p=0.772).COX regression analysis suggests pathologic stage(HR=1.449, P=0.043), tumor TNM stagge(HR=2.117, P=0.013), gender(HR=3.581, P= 0.019), and the in-tumor CD206 expression(HR=2.371, P=0.036) were independent prognostic factors associated with the survival rates in HCC patients.Conclusion: Tumor associated macrophage marker CD206 in primary liver cancer tissues were highly expressed than peri-tumor tissues. Higher CD206 expression in the tumor could be an independent prognostic factor in HCC patients.PART Ⅱ ONSTRUCTION AND IDENTIFICATION OFDIFFERENT PHENOTYPES OF MACROPHAGES INVITROObjective: To induce normal murine RAW264.7 macrophage into tumor associated macrophages and identify the phenotype by various assay.Methods: RAW264.7 cell line was derived from murine leukemia cells, which was commonly used for the study of tumor associated macrophages. In our study, we applied 2.5μg/ml lipopolysaccharide and co-cultured for 2 hours to induce RAW264.7 cells transfer into M1 phenotype and 10ng/ml IL-4 for 48 hours to transfer the cell into M2 phenotype. Macrophage associated marker F4/80, CD16/32, CD206 were detected by flow cytometry, Arg-1, i NOS, NF-kb1 were detected by RT-PCR and Western Blot.Results: The LPS induced RAW264.7 cells were transformed into M1 phenotype sucssecefully, with higher expression of NOS2, CD16/32, NF-kb1 while IL-4 induced RAW264.7 cells were transformed into M2 phenotype, the CD206, Arg-1 were highly expressed.Conclusions: We have successfully induced the RAW264.7 cells into different phenotypes, the M2 macrophage, which is considered as TAM have a higher expression level of M2 markers, such as Arg-1 and CD206.PART Ⅲ DCL3 SUPPRESSES THE MALIGNANCY OF HEPATOCELLULAR CARCINOMA BY INHIBITING THEEXPRESSION OF CD206 IN TUMOR-ASSOCIATEDMACROPHAGESObjective: To demonstrate that Gd Cl3 could inhibit the malignant functions of tumor associated macrophages via down regulating the epression of CD206.Methods: Raw264.7 cells were treated with Gd Cl3 solutions in different concentrations, the IC50 and IC10 concentrations were confirmed; The flow cytometry, immunohistochemistry, PCR and Western Blot assay were applied to demonstrate whether Gd Cl3 could inhibit the CD206 expression in Raw264.7 cells. CD206 RNAi was conducted to knock down the CD206 expression in M2 macrophages. The co-culture system of the M2 macrophages, mice Hepa1-6 tumor cells were builded. The level of epithelial-mesenchymal transition(EMT) was analyzed. The diethylnitrosamine(DEN) was administrated to induce hepatocarcinogenesis in BALB/c mice, and Gd Cl3 was injected regularly in this process, the surviving period, mice liver tissue or blood samples were acquired at different time point, the CD206 expression, growth kinetical parameter of mice liver and the mice survival data were analysed.Results: The IC10 concentration of Gd Cl3 on RAW264.7 growth is 0.07ug/ul.The expression of CD206 was significantly reduced under IC10 concentrations of Gd Cl3 treatments. The CD206-suppressed M2 macrophages have given the Hepa1-6 cells a lower proliferation rate, and the EMT property of Hepa1-6 cells was attenuated. DEN can successfully induce mice HCC, the expression of CD206 was inhibited notably under Gd Cl3 long term treatments. Finally, the HCC growth in a lower level and mice survival rate were increased after Gd Cl3 treatment.Conclusions: Gd Cl3 could significantly inhibit the expression of CD206 in tumor-associated macrophages, followed with tumor malignancy decreased. Animal experiments show that Gd Cl3 has no distinct biotoxicity and long term administration is possible.We demonstrated that Gd Cl3 could be a promising HCC therapy agent, further mechanism research is needed.