细胞因子诱导杀伤细胞杀伤胃癌的特异性与非特异性研究

目的 观察特异性与非特异性杀伤在细胞因子诱导的杀伤细胞(CIK)体外杀伤胃癌细胞(SGC-7901)中所起的作用.方法 从正常人外周血单个核细胞诱导树突状细胞(DC)、CIK细胞,用人胃癌细胞SGC-7901提取肿瘤抗原致敏DC,用DC诱导CIK生成DC诱导CIK细胞(DC-CIK)和经胃癌抗原致敏的DC诱导CIK细胞(抗原-DC-CIK).培养的第14天,用流式细胞仪检测CIK、DC-CIK、抗原-DC-CIK细胞表型CD3、CD3/CD8和CD3/CD56.以CIK、DC-CIK和抗原-DC-CIK为效应细胞,以经主要组织相容性复合体(MHC)分子阻断的SGC-7901细胞和未经MHC分子阻断的SGC-7901细胞为靶细胞,效靶比分别为5:1、10:1、20:1,用噻唑蓝(MTT)比色法分别检测各组杀伤效应(每组n=3).结果 (1)培养的第14天,抗原-DC-CIK中CD3~+CD56~+和CD3~+CD8~+双阳性细胞的比例较DC-CIK和单独培养CIK明显增高(P均<0.01).(2)效靶比为20:1时,CIK组、DC-CIK组、抗原-DC-CIK组对SGC-7901的杀伤活性分别为:(46.6±2.2)%、(52.6±1.6)%、(72.5±2.1)%(P均<0.01);对经MHC分子阻断的SGC-7901的杀伤活性分别为:(44.3±1.1)%、(49.9±0.9)%、(50.4±1.9)%.抗原-DC-CIK中特异性杀伤占总杀伤效力的比例明显高于DC-CIK和CIK.结论 CIK杀伤胃癌细胞以非特异性杀伤为主,特异性杀伤仅占总杀伤效力的4.9%;未致敏的DC诱导CIK后,总杀伤效力增强,以非特异性杀伤为主,特异性杀伤效力增加不明显,占总杀伤效力的5.1%;抗原致敏的DC诱导CIK后,杀伤仍以非特异性杀伤为主,特异性杀伤效力大幅度增加,占总杀伤效力的30.5%.

The specific and non-specific cytotoxic activity of killer cells against gastric cancer cells induced by cytokines

Objective To observe the roles of specific and non-specific cytotoxic activity in cyto-kine-induced killer cells (CIK) against gastric cancer (SGC-7901 ) cells in vitro. Methods Dendritic cells (DC) and CIK were induced from peripheral blood mononuclear cells (PBMC) from healthy donors. The DC were sensitized with tumor antigen extracted from gastric cancer SGC-7901 cells. The CIK were in-duced with DC to form DC-CIK,or induced with gastric cancer antigen-pulsed DC to form antigen-DC-CIK. On the 14th day of culture,CIK, DC-CIK and antigen-DC-CIK phenotypes (CD3 ,CD3/CD8 and CD3/CD56) ware analyzed by flow cytometry. CIK, DC-CIK and antigen-DC-CIK were used as the effector cells, and the SGC-7901 cells with or without blockade of major histocompatibility complex (MHC) molecule as the target cells. At 5: 1,10:1 and 20:1 effector-target ratio,the cytotoxic activity was detected by MTT. Results On the 14th day of culture,the proportion of CD3~+ CD8~+ and CD3~+ CD56~+ double positive cells in antigen-DC-CIK was significantly higher than that in DC-CIK and pure CIK (P <0.01 ). At 20:1 effector-target ratio in vitro,the cytotoxic activity of CIK, DC-CIK and antigen-DC-CIK against SGC-7901 cells was (46.6± 2.2) %, (52.6 ± 1.6) % and (72.5±2.1 ) % (P < 0.01 ), and after MHC molecule was blocked in SGC-7901 cells, the cytotoxic activity was (44.3 ± 1.1 ) %, (49.9 ±0. 9) % and (50.4 ± 1.9) % respectively. The proportion of specific cytotoxic activity in antigen-DC-CIK was higher than in DC-CIK and CIK. Conclusion The major eytotoxic activity of CIK against gastric cancer cells is non-specific, and specific cytotoxic activity only accounts for 4.9%. After induction of CIK by DC ,the total cytotoxic activity was enhanced and the ma-jor cytotoxic activity is non-specific, and specific cytotoxie activity was enhanced slightly to 5.1%. After in-duction of CIK by antigen-pulsed DC, the major cytotoxic activity is still non-specific, and the specific cyto-toxic activity was largely increased to 30.5%.