M2型肿瘤相关巨噬细胞通过p53途径下调肝癌化疗敏感性的机制研究

摘要

Objective To study the mechanism of tumor associated macrophages (TAM) down-regulating the p53 expression in chemotherapy HCC cells,so as to broad the measures of establishing the chemoresistance.Methods The mouse liver tumor models were established and the liver cancer tissue was isolated,grinded and extracted.Then the expression of CD206 and CD163 which existing on the surface of TAM were detected by RT-PCR.The human monocyte macrophages (THP-1) were cultured and induced differectiation of TAM with PMA and IL-4.Then TAM was co-cultured with liver cancer cells(HepG2,SMMC7721 and Hep 3B),and oxaliplatin(20ug / mL) was added for 24 hours.The expression of Caspase 3,anti Bcl-2 and p53 were detected by western blot.The proliferation of hepatocellular carcinoma cells was detected by MTT.Results The mouse HCC model was successfully constructed.And the expression of CD206 and CD163 existing on the surface of macrophage was significantly increased in tumor tissue.The expression level of CD206 and CD163 which exist on the surface of Human mononuclear cells (THP-1) induced with PMA (100ng/mL) and IL-4(100ng/mL) also significantly increased.After the effect of oxaliplatin,the expression of Bcl-2 in HCC cells increased significantly in tumor cells co-cultured with TAM.And the expression of Caspase 3 and p53 decreased significantly.Moreover,the proliferation inhibition rate of tumor cells was significantly decreased.Howover,in Hep 3B co-cultured with TAM caspase 3 levels were decreased obviously.Conclusion Regulation of p53 expression in hepatocellular carcinoma cells by tumor associated macrophages could inhibit the sensitivity of the liver cancer chemotherapy.%目的 通过研究M2肿瘤相关巨噬细胞调控p53表达来下调肝癌化疗敏感性的机制,以此拓宽建立抗肿瘤耐药的措施.方法 构建小鼠肝癌模型及分离肝癌组织,研磨组织提取巨噬细胞,通过RT-PCR方法检测巨噬细胞表面CD206及CD163分子表达情况.培养人单核巨噬细胞(THP-1),PMA (100ng/mL)和IL-4 (100ng/mL)作用诱导分化成肿瘤相关巨噬细胞后,与肝癌细胞(HepG2、SMMC7721,Hep 3B)共培养,加入奥沙利铂(20μ g/mL)作用24小时,通过Western blot方法检测凋亡相关蛋白Caspase 3、抗凋亡蛋白Bcl-2及p53的表达,通过MTT方法检测化疗对肝癌细胞增殖情况.结果 成功构建了肝癌模型,分离出了肝癌组织巨噬细胞,RT-PCR检测CD206和CD163的表达显著升高;人单核细胞(THP-1)经加入PMA(100ng/mL)和IL-4 (100ng/mL)分化诱导48h后其细胞的表面CD206和CD163的表达明显高于THP1分化诱导的细胞表面表达量;肝癌细胞SMMC7721和HepG2与M2型肿瘤相关巨噬细胞共培养24h,经奥沙利铂作用后,肿瘤细胞Bcl-2表达明显升高,Caspase 3和p53的表达显著降低;肿瘤细胞的增殖抑制率明显降低,而Hep 3B细胞的凋亡则明显降低.结论 M2型肿瘤相关巨噬细胞调控肝癌细胞中p53的表达,进而抑制了肝癌化疗的敏感性.