The Immunosuppression Effects Of Hepatocellular Carcinomas Upon NK Cells

【Objective】:To establish the orthotopic transplantation tumor model from the subcutaneous model of human hepatocellular carcinomas (HCC) in nude mice and the techniques of isolation hepatic NK cells. Then to investigate the cytotoxicity of NK cells and the expression of active NKG2D receptor. Eventually, to observe the pathological changes of liver and spleen.【Methods】:ⅠEstablishment 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.ⅡThe effects of the primary hepatocellular carcinoma on the immune activity of NK cells in nude mice: Total lymphocytes from the blood , liver and spleen of the nude mice were prepared by using Ficoll-Hypaque, digestive enzymes and mechanical dissection , respectively. Then NK cells were purified by Percoll discontinuous density gradient centrifugation , and the cytotoxicity of NK cells was detected by LDH method . Moreover, the NKG2D receptor expression of NK cells was measured by flow cytometry. Subsequently, the effects of the hepatic transplantation tumor on the lymphocytes of liver and spleen were analyzed by H-E staining.【Results】:ⅠThe indirect orthotopic model can stably be propagated. The survival rates of the transplantation tumor were about 100%.ⅡThe purity of NK cells separated by Percoll discontinuous density gradient centrifugation reached to 68.5%.ⅢThe 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.ⅣBoth 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】:ⅠThis model is an ideal means for study of HCC.ⅡNK cells are successfully purified.ⅢThe 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.