Stefin B调控巨噬细胞极化对肝癌细胞增殖、迁移、侵袭及上皮间质转化的影响

Effects of polarized macrophages regulated by stefin B on proliferation,migration,invasion and epithelial-mesenchymal transition of hepatoma carcinoma cells

目的探讨stefin B(CSTB)调控巨噬细胞极化对肝癌细胞增殖迁移侵袭及上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法诱导人单核细胞系THP-1极化成未分化巨噬细胞(M0)、经典活化的巨噬细胞(M1)、替代性活化的巨噬细胞(M2)和肿瘤相关巨噬细胞(tumor associated macrophage,TAM);构建慢病毒stefin B(LV-CSTB)及对照空载体病毒(LV-NULL),感染各亚型巨噬细胞,ELISA法检测IL-10和IL-12分泌;病毒感染各亚型巨噬细胞后的上清分别制备成条件培养液用于培养MHCC97H细胞,CCK-8法、划痕法和小室法分别检测细胞的增殖、迁移和侵袭能力,RT PCR和Western blotting法分别检测细胞E-cadherin、N-cadherin、β-catenin及Twist的mRNA和蛋白表达。结果LV-CSTB感染M0、M2、TAM亚型细胞的上清中IL-10明显增加,感染M1后上清中IL-12明显增加;LV-CSTB感染M0、M2、TAM亚型的条件培养液作用时,MHCC97H细胞的增殖、迁移和侵袭能力均明显增强,E-cadherin的mRNA和蛋白表达明显下降,N-cadherin、β-catenin(M0除外)及Twist的mRNA和蛋白表达明显上升。结论Stefin B调控巨噬细胞M0向M2亚型极化,stefin B介导M0、M2、TAM促进肝癌细胞增殖、迁移、侵袭及EMT。

Objective To investigate the effects of polarized macrophages regulated by stefin B(CSTB)on proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of hepatoma carcinoma cells.Methods Human monocyte THP-1 was induced to differentiate into macrophages(M0),classically activated macrophages(M1),alternatively activated macrophages(M2)and tumor associated macrophages(TAM),respectively.The recombinant lentiviral vectors containing human CSTB gene and a control lentivirus(LV-CSTB and LV-NULL,respectively)were constructed.M0,M1,M2 and TAM were transfected by LV-CSTB and LV-NULL,respectively.The IL-10 and IL-12 secretion of macrophages was detected by ELISA.MHCC97H cells were incubated with conditioned medium of macrophages transfected by LV-CSTB and LV-NULL,respectively.Proliferation,migration and invasion of MHCC97H cells were determined using Cell-Counting Kit-8(CCK-8),wound healing and Transwell assay,respectively.The mRNA and protein expressions of E-cadherin,N-cadherin,β-catenin and Twist were assessed by quantitative RT-PCR and Western blotting,respectively.Results The IL-10 secretion of M0,M2,TAM and IL-12 of M1 transfected with LV-CSTB was significantly increased.Proliferation,migration and invasion of MHCC97H cells were significantly raised by the conditioned medium of M0,M2 and TAM transfected with LV-CSTB.The conditioned medium of M0,M2 and TAM also promoted mRNA and protein expressionsof N-cadherin,β-catenin(except for M0)and Twist in MHCC97H cells,while the expression of E-cadherin was downregulated.Conclusion CSTB can regulate the macrophages'polarization from M0 to M2,thus promoting the proliferation,migration,invasion and epithelial-mesenchymal transition of hepatoma carcinoma cells.