活化的肝星状细胞在肿瘤微环境中的免疫调节作用

Immune modulatory activity of activated hepatic stellate cells in the tumor microenvironment

目的研究活化的肝星状细胞（hepatic stellate cells，HSCs）在小鼠肝细胞癌发生发展中的免疫调节作用及肿瘤微环境的免疫功能变化。方法于体外，用Brdu细胞增殖实验检测HSCs上清对肿瘤增殖的影响，用混合淋巴实验检测HSCs的免疫调节作用。体内实验采用皮下移植瘤模型，分为H22单纯注射组与H22＋HSCs联合注射组。20d后对荷瘤小鼠行瘤组织解剖测量移植瘤大小；用免疫组化法观察两组肿瘤中T细胞亚群浸润变化情况和接种肿瘤PD-L1的表达变化；用流式细胞术分析脾脏中T细胞亚群变化。然后，通过Tunel检测肿瘤组织单核细胞凋亡情况。结果在体外实验，Brdu检测显示HSC条件培养基（HSCs-CM）能明显诱导H22细胞的增殖，而混合淋巴实验则显示HSCs对T细胞增殖有明显的抑制作用。在体内实验，HSCs＋H22组小鼠皮下移植瘤的形成时间与生长速度明显快于单纯接种H22组；肿瘤组织免疫组化检测显示HSCs＋H22组浸润的T细胞及其亚型较单纯H22组有不同程度降低；HSCs＋H22组脾脏亦显示T细胞及其亚型出现不同程度降低。TUNEI。试剂盒检测显示HSCs＋H22组肿瘤组织中单核细胞的凋亡数量增加，免疫组化分析显示HSCs＋H22组肿瘤组织中PD-L1表达增加。结论HSCs可通过免疫抑制作用在肿瘤微环境中促进肿瘤的发牛、发展，这一发现可能为肝癌的治疗提供新的思路。

Objective To determine immune modulatory activity of activated hepatic stellate cells（HSCs） in hepatocellular carcinoma and immune response in tumor microenvironment. Methods Cell proliferation was measured by BrdU incorporation with a microtiter plate reader at 450 nm. The effect of HSCs on T cell proliferation was measured by MLR. Mouse hepatic cancer cell line H22 were implanted on the backs of BALB/c mice to establish the subcutaneous transplanted tumor model. Then the mice were sacrificed after 20 days for anatomical and size determination. Furthermore, Paraffin-embedded tissue was removed by serial tissue sectioning and immunohistochemically examined for expression of T lymphocyte subsets. T lymphocyte subsets in splenocytes were detected by FCM. Apoptotic mononuclear cells were evaluated by FITC-labeled Tunel assay. Results We determined that HSCsCM promoted hepatocellular carcinoma（HCC） cell line proliferation and HSCs inhibit T cell prolifera tion by MLR in vitro. We also examined normal immune mice to assess the immunosuppression of HSCs in the development of HCC. In the co transplantation with HSCs group, T cells and their subtypes decreased obviously in the tumors and the spleen. The results showed that co-transplanted HSCs can induce more PD-L1 expression and more mononuclear cell apoptosis in tumor tissue. Conclusion Our results demonstrated that HSCs promote HCC progression partially because of their immune regulatory activity. Our data supply new information to support an integral role for HSCs in promoting HCC progression and suggest that HSCs may serve as a therapeutic target for HCC.