抗原致敏IL-27基因修饰的树突细胞活化的细胞毒性T淋巴细胞对食管癌裸鼠移植瘤生长的影响

Effects of specific cytotoxicity T lymphocyte activated by interleukin-27 gene modified DC and loaded with esophageal tumor lysate on growth of transplantation tumor of human esophageal carcinoma in nude mice

目的研究白细胞介素27(IL-27)基因转染树突细胞(DC)体内诱导免疫杀伤食管癌细胞的效能及其机制。方法采用基因转染的方法建立表达IL-27基因的树突细胞(DCIL-27),构建人食管癌裸鼠移植瘤模型,皮下注射食管癌细胞抗原致敏、IL-27基因修饰的DC(DCIL-27+Ag)活化的特异细胞毒性T淋巴细胞(CTL)。30只裸鼠分为PBS组、Tnaive组、DC+Tnaive组、DCIL-27+Tnaive组和DCIL-27+Ag+Tnaive组,每组6只。观察荷瘤裸鼠的肿瘤体积、瘤重和抑瘤率,流式细胞术检测移植瘤细胞的细胞周期、凋亡率及凋亡相关蛋白Fas和caspase-3的表达。结果 RT-PCR结果显示,DCIL-27细胞中有IL-27 p28和EBI3亚基基因表达,提示转染成功。DCIL-27+Ag+Tnaive组裸鼠移植瘤的体积和重量明显下降,抑瘤率为58.28%,明显大于其他各组(P<0.01)。流式细胞术分析显示,DCIL-27+Ag+Tnaive组裸鼠肿瘤组织内细胞增殖指数为23.92±1.60,显著低于其他各组,细胞凋亡率为32.78%±0.83%,明显高于其他各组(P<0.01)。DCIL-27+Ag+Tnaive组Fas和caspase-3蛋白表达水平均明显高于其他各组(P<0.01)。结论食管癌细胞抗原致敏、IL-27基因修饰DC可活化特异性CTL,且在荷瘤小鼠体内对食管癌细胞具有明显的细胞毒作用,其机制可能是通过Fas/FasL途径抑制食管癌细胞的体内增殖并促进其凋亡。

Objective To investigate the effect of specific cytotoxicity T lymphocyte （CTL） activated by IL-27 gene modified DC and loaded with esophageal tumor lysate on the growth of transplantation tumor of human esophageal carcinoma in nude mice, and study the effect of IL-27 on cancer cell apoptosis and its mechanisms. Methods With gene transfection method, human DCIL_Z7 cells secreting IL-27 were successfully obtained. After the nude mouse model of human esophageal carcinoma transplantation tumor was reproduced, the mice were immunized with specific CTL activated by IL-27 gene modified DC loading with esophageal tumor lysate （DCIL-27±Ag）. Thirty nude mice were divided into PBS group, Tnaive group, DC±Tnaive group, DCIL-27±Tnaive group and DCIL.27±Ag±Tnaive group （6 each）. The volume, weight and inhibition rate of transplantation tumors were measured. The cell cycle and apoptosis rate of transplantation tumors at G0/G1, S and G2/M stages were determined by flow cytometry. The expression of Fas and caspase-3 protein were also detected by flow cytometry. Results RT-PCR showed that the subunits of IL-27, namely p28 and EBI3, were expressed in DC1L-27 cells, and it proved that gene transfection was successful. The volume and weight of transplantation tumors in nude mice of DCIL-27±Ag±Tnaive group were significantly smaller than those in other treatment groups and control group. The inhibition rate was 58.28%, which was much higher than that in other therapy groups and control group （P〈0.01）. Flow cytometry showed that the proliferation index of transplantation tumor tissue with specific CTL activated by DCIL.E7＋Ag was 23.92% ± 1.60% ,which was evidently lower than that in other therapy groups and control group （P〈0.01）. The apoptosis rate of cells in the transplantation tumors with specific CTL activated by DCIL-27±Ag was 32.78% ± 0.83%, which was significantly higher than that in other therapy groups and control group （P〈0.01）. Flow cytometry showed also that the expressions of Fas and caspase-3 protein in the transplantation tumor tissue with specific CTL activated by DCIL-27±Ag were significantly higher than those in other groups （P〈0.01）. Conclusions Specific CTL activated by IL-27 gene modified DC loading with esophageal tumor lysate possesses great cytotoxic effects on esophageal carcinoma of tumor-bearing mice in vivo and it is able to inhibit the proliferation and enhance the apoptosis of esophageal carcinoma cells through Fas/FasL pathway in vivo.