GPI锚定B7蛋白对原发性肝癌的免疫治疗作用

The effect of tumor cell membranes anchored by glycosyl-phosphatidylinositol-B7-1 protein on the immuno-regulatory activity for hepatocellular carcinoma

目的GPI—B7—1蛋白转移修饰肝癌细胞膜，观察其对肿瘤细胞的免疫诱导效应。方法GPI—B7—1和B7—1蛋白修饰肝癌细胞膜，流式法测定其CD80（B7—1）的表达，分离患者外周血T细胞，检测不同蛋白修饰的肝癌细胞膜对T淋巴细胞的致增殖效应及granzyme B、perforin、IFN-γ mRNA表达的影响。结果GPI—B7-1蛋白修饰肝癌细胞膜表面B7—1表达率为85％。GPI—B7—1蛋白修饰的肝癌细胞膜致T淋巴细胞的增殖程度显著强于对照组和B7—1蛋白修饰组（P〈0．01）；该作用源于其可上调肝癌外周血T淋巴细胞granzyme B、perforin及IFN-γ mRNA表达水平。结论GPI—B7—1蛋白转移修饰的肝癌细胞膜可上调granzyme B、perforin及IFN-γ mRNA表达，激活T细胞增殖，促进杀伤性T细胞的效应。

Objective To use isolated tumor membranes modified by transfer of a glycosyl-phosphatidylinositol （GPI）-anchored form of the eostimulatory molecule,B7-1 （CD80）,as a cell free cancer vaccine for hepatocellular carcinoma （ HCC ） immunity. Methods Isolated tumor cell membranes were prepared from surgically removed HCC tissue. GPI-B7-1 was transferred optimally onto isolated HCC cell membranes at physiological temperature （37 ℃ ）. Flow cytometry was used to directly detect the expression of B7-1 on isolated membranes. T cells were isolated from peripheral blood of patients with HCC. T cell stimulation assays were carried out for 72 h, followed by other 4-h incubation by adding 0.5 g/L MTT （final concentration） per well. The absorbanee （A） was read on an ELISA reader. The expression of granzyme B,perforin and IFN-γ/mRNA was detected by real-time PCR. Results Transfer of GPI-B7-1 to isolated membranes resulted in the stable expression, with strong effect to costimulate T cells （P 〈 0.01 ）. There were high expression levels of granzyme B, perforin and IFN-γ/ mRNA from T cells. Conclusion Transfer of GPI-B7-1 to isolated HCC cell membranes could up-regulate the expression of granzyme B, perfofin and IFN-γ mRNA, activate proliferation of T cells, and promote the effects of killing T cells.