人肝癌裸鼠NKG2D受体的表达及对NK细胞毒活性的影响

目的 通过研究原发性肝癌中NK细胞的细胞毒活性变化及其活化性受体NKG2D的表达，观察肝脏和脾脏组织病理变化，探讨NK细胞免疫抑制机制，为原发性肝癌的免疫治疗提供理论依据。方法 ①建立人肝癌裸鼠皮下-肝原位移植瘤模型：先用人肝癌细胞株Hep3B接种于裸鼠皮下，形成皮下移植瘤，然后用此移植瘤组织再接种于裸鼠肝内，建立肝原位移植瘤模型（间接肝原位移植瘤模型）；②检测裸鼠原发性肝癌对NK细胞免疫活性的影响：分离裸鼠外周血、肝脏及脾脏组织NK细胞，LDH方法检测NK细胞的细胞毒活性，流式细胞技术检测不同组织NK细胞NKG2D表达百分率，H-E染色观察肝脏移植瘤对肝脏和脾脏淋巴细胞的影响。结果 ①外周血、肝脏及脾脏的NK细胞毒活性及NKG2D受体表达随着肿瘤生长逐渐下降，其中肝脏NK细胞毒活性及NKG2D表达下降明显；②荷瘤裸鼠肝脏癌组织与皮下瘤相同，癌组织异型性与时间呈正相关，脾脏淋巴小结在第4周增生明显，第8周小梁结构增多。结论 原发性肝癌通过下调NKG2D的表达，对NK细胞有免疫抑制作用，这种作用主要发生在肝脏，但对外周血和脾脏也有影响。

Expression of NKG2D receptor and its effects on the cytotoxicity of NK cells in nude mice with transplanted human hepatocellular carcinoma

Objective To investigate the cytotoxicity of NK cells and the expression of active NKG2D receptor in hepatocellular carcinoma (HCC). Then, to observe the pathological changes of liver and spleen. Eventually, to study the immunosuppression mechanisms of hepatocellular carcinomas upon NK cells. Methods ①Establishment of the orthotopic transplantation tumor model from the subcutaneous model of human hepatocellular carcinoma in nude mice: Hep3B cells were subcutaneously transplanted to form subcutaneous transplantation tumors in nude mice. Then, the subcutaneous transplantation tumors were injected into liver to form the orthotopic transplantation tumor model (indirect orthotopic model) in nude mice. ②The effects of the primary hepatocellular carcinoma on the immune activity of NK cells in nude mice: NK cells were isolated from the blood, liver and spleen of the nude mice. Then, the cytotoxicity of NK cells was detected with LDH method. Moreover, the NKG2D receptor expression of NK cells was measured with flow cytometry. Subsequently, the effects of the hepatic transplantation tumor on the lymphocytes of liver and spleen were analyzed by H-E staining. Results ①The decrease of the cytotoxicity of NK cells and NKG2D receptor expression in peripheral blood, liver and spleen was associated with the growth of hepatocellular carcinoma, and this tendency was significantly in the liver. ②Both the subcutaneous and hepatic transplantation tumor was homology. The heteromorphism of hepatocellular carcinoma was positive correlated with the growth time of tumors. The hyperplasia lymph follicle obviously increased in the spleen after 4 weeks, and the spleen trabecula significantly increased after 8 weeks. Conclusion The primary hepatocellular carcinoma has the immunosuppression effects upon NK cells though down-regulating the expression of NKG2D. Such effects are obvious in liver, comparing with peripheral blood or spleen.