沉默HIF-1α表达逆转缺氧诱导肝癌免疫抑制的实验研究

目的:探讨缺氧诱导因子-1(HIF-1α)表达在缺氧诱导肝癌免疫抑制中的作用,以期阐明缺氧致肝癌免疫抑 制的分子机制。方法:构建缺氧C57BL/6 小鼠移植瘤模型,缺氧探针HypoxyprobeTM-1 标记缺氧组织,免疫组化检测缺氧组织 HIF-1α表达,qPCR 检测缺氧组织肿瘤免疫因子表达;慢病毒构建稳定敲减HIF-1α的Hepa1鄄6 小鼠肝癌细胞株,并移植于 C57BL/6 小鼠,6 周后检测移植瘤瘤重,Western blot 检测移植瘤HIF鄄1琢表达,流式细胞仪检测移植瘤中肿瘤浸润免疫细胞 CD4+CD25+ Foxp3+调节性T 细胞表达,qPCR 检测缺氧组织肿瘤免疫因子表达。结果:C57BL/6 小鼠缺氧移植瘤模型构建成 功,缺氧组织HIF鄄1琢高表达,肿瘤抑制因子IFN-γ、IL-12b 和CXCL10 低表达,肿瘤促进因子IL-10、IL-1β和IL-17 高表达;敲减 HIF-1α明显抑制了移植瘤生长,降低了CD4+ CD25+ Foxp3+调节性T 细胞浸润,同时抑制了肿瘤促进因子IL-10、IL-1β和IL-17 表达,促进了肿瘤抑制因子IFN鄄酌、IL鄄12b 和CXCL10 表达。结论:缺氧可诱导肝癌组织HIF鄄1琢表达及免疫抑制,相反,沉默 HIF鄄1琢可抑制移植瘤生长并逆转肝癌免疫抑制,揭示了HIF-1α在缺氧致肝癌免疫抑制中的作用,可能为将来肝癌诊治提供 新思路。

Silencing HIF-1αexpression reverses hypoxia-induced immunosuppression in#br# human hepatocellular carcinoma

Objective:To investigate the role of hypoxia inducible factor-1(HIF-1α)expression in hypoxia-induced immunosup- pression in hepatocellular carcinoma in order to clarify the molecular mechanism of hypoxia-induced immunosuppression in hepatocellular carcinoma.Methods: HypoxyprobeTM-1 was used to label the hypoxia tissue,the expression of HIF-1α in hypoxia tissue was detected by immunohistochemistry.The qPCR was used to detect the expression of tumor immune factor in hypoxia tissue.The stable shHIF-1αHepa1-6 mouse hepatocarcinoma cell line was constructed by lentivirus and transplanted into C57BL/6 mice.The tumor weight was measured after 6 weeks and the expression of HIF-1α was detected by Western blot.The tumor infiltrating immune cells CD4+ CD25+ Foxp3+ regulatory T cells in transplanted tumor was detected by flow cytometry and qPCR was used to detect tumor immune factor expression.Results: The C57BL/6 mouse models of hypoxia xenografts were successfully established.The expression of HIF-1α,tumor-promoting factor IL-10,IL1βand IL-17 were increased in hypoxic murine tumour samples,while the tumor suppressors IFN-γ,IL-12b and CXCL10 were decreased in non-hypoxic samples.Knockdown of HIF-1αsignificantly inhibited the growth of xenografts and decreased the infiltration of CD4+ CD25+ Foxp3+ regulatory T cells,meanwhile inhibited the expression of tumor-promoting factors IL-10, IL-1βand IL-17 and promoted the expression of tumor suppressor IFN-γ, IL-12b and CXCL10 expression.Conclusion: Hypoxia could induce HIF-1αexpression and immunosuppression in HCC,on the contrary,silencing HIF-1α could inhibit the growth of transplanted tumor and reverse the immunosuppression of HCC,which revealing that the role of HIF-1α in immunosuppression induced by hypoxia,and provided the experimental basis for futher liver cancer diagnosis and treatment.