ICOS基因转染对CIK细胞杀伤胆管癌细胞作用的研究

目的 探讨转染可诱导共刺激分子(inducible co-stimulator, ICOS)基因对细胞因子诱导杀伤(cytokine. induced killer, CIK)细胞杀伤胆管癌细胞的作用.方法 构建含ICOS基因的腺病毒载体并转染给CIK细胞(CIK-ICOS细胞组),单纯CIK及CIK-EGFP细胞为对照组,观察3组CIK细胞体外增殖与凋亡情况以及对胆管癌细胞的杀伤作用;ELISA法检测3组CIK细胞上清液中IFN-γ、IL-2及TNF-α的表达.建立胆管癌移植瘤的SCID小鼠模型,按随机数字表法将40只SCID小鼠分为生理盐水对照组、CIK、CIK-EGFP、CIK-ICOS细胞治疗组,观察转染ICOS基因的CIK细胞对小鼠胆管癌细胞的杀伤作用.结果 CIK-ICOS细胞组体外增殖明显强于CIK及CIK-EGFP细胞组;培养第20天CIK-ICOS与CIK细胞凋亡率分别为0.69%和2.90%,第23天分别为0.89%和4.92%;在不同效靶比CIK-ICOS细胞组杀伤效应均显著高于CIK与CIK-EGFP细胞组(F=13.37,6.46,25.51,P<0.05);CIK-ICOS细胞组上清液中IFN-γ的浓度为(49.50±4.73)μg/L,显著高于CIK细胞组(30.53±3.73)μg/L及CIK-EGFP细胞组(30.12±2.64)μg/L(F=38.89,P<0.05).CIK-ICOS细胞治疗组小鼠肿瘤生长速度明显低于CIK与CIK-EGFP细胞治疗组,肿瘤坏死面积明显大于CIK与CIK-EGFP细胞治疗组,瘤内CIK细胞数量最多.结论 CIK细胞在体内外均具有杀伤胆管癌细胞的作用.转染ICOS基因后,CIK细胞体外存活时间延长、增殖能力增强、IFN-γ的表达增多,其在体内外抗胆管癌作用明显增强.

Effects of inducible co-stimulator gene on the cytotoxic activity of cytokine-induced killer cells against cholangiocarcinoma cells

Objective To explore the effects of inducible co-stimulator (ICOS) gene on the cytotoxic activity of cytokine-induced killer (CIK) cells against cholangiocarcinoma cells. Methods CIK-ICOS cells were obtained by stable transfecting ICOS genes into CIK cells through the adenovirus vector whereas untransfected and EGFP-transfected CIK cells were treated as controls. The proliferation and apoptosis of different CIK cells, as well as their cytotoxicity against cholangiocarcinoma cells in the three groups were detected. The expressions of IFN-T, IL-2 and TNF-α in the supernatant of different CIK cells were measured by ELISA. SCID mice with cholangiocarcinoma were randomly divided into CIK group, CIK-EGFP group, CIK-ICOS group and normal saline group. The cytotoxic activity of CIK-ICOS cells against cholangiocarcinoma cells in vivo was observed. Results CIK-ICOS cells displayed better proliferation than CIK cells and CIK-EGFP cells. At day 20 and 23 of culture, the apoptosis rate of CIK-ICOS cells was 0.69% and 0.89%, respectively, while that of the CIK cells was 2.90% and 4.92%. The cytotoxic effect of CIK-ICOS cells at different E: T ratio against cholangiocarcinoma cells was significantly stronger than that of CIK cells and CIK-EGFP cells (F=13.37, 6.46, 25.51, P<0.05). The concentration of IFN-γ in CIK-ICOS cultured supernatant was (49.50±4.73)μg/L, which was significantly higher than that in the cultured supernatant of CIK cells (30.53±3.73)μg/L] and CIK-EGFP cells (30.12±2.64)μg/L](F=38.89, P<0.05). The growth of cholangiocarcinoma was significantly slower in CIK-ICOS group than that in CIK group and CIK-EGFP group, whereas the necrosis area of tumor was larger and the CIK cells in CIK-ICOS group was more than those in the other two groups. Conclusions CIK cells had the function of killing cholangiocarcinoma cells in vitro and in vivo. After ICOS genes were transfected into CIK cells, the survival time of CIK cells in vitro was prolonged and the proliferation of CIK cells was enhanced, as well as the secretion of IFN-γ was increased so that the cytotoxicity of CIK cells against cholangiocarcinoma cells in vitro and in vivo was enhanced.