抗原致敏IL-27基因修饰的树突状细胞活化的CTL对食管癌细胞线粒体膜电位及凋亡的影响

Effects of specific CTL which are activated by DC modified with IL-27 gene and esophageal tumor lysate on esophageal carcinoma cell mitochondria membrane potential and cell apoptosis

目的研究IL-27基因转染的树突状细胞(dendritic cell,DC)活化CTL体内诱导食管癌细胞凋亡的影响。方法通过在裸鼠的移植瘤周注射食管癌细胞抗原致敏、IL-27基因修饰DC(DC_(IL-27+Ag))活化的特异CTL,采用流式细胞术检测细胞凋亡水平,荧光染料罗丹明123染色检测细胞线粒体膜电位水平并检测caspase-3蛋白表达水平。结果 5组之间凋亡率、膜电位水平、caspase-3表达差异均有统计学意义(P<0.05):DC_(IL-27+Ag)、DCIL-27、DCnaive、Tnaive组凋亡率、caspase-3表达均高于PBS组,膜电位水平低于PBS组;DC_(IL-27+Ag)、DCIL-27、DCnaive组凋亡率、caspase-3表达均高于Tnaive组,膜电位水平低于Tnaive组;DC_(IL-27+Ag)、DC_(IL-27)组凋亡率、caspase-3表达均高于DCnaive组,膜电位水平低于DCnaive组;DC_(IL-27+Ag)组凋亡率、caspase-3表达均高于DCIL-27组,膜电位水平低于DCIL-27组。结论在荷瘤小鼠体内,经食管癌细胞抗原致敏、IL-27基因修饰的DC可活化特异性CTL产生较强的细胞毒作用,其机制可能是CTL通过降低食管癌细胞线粒体膜电位,启动内源性线粒体凋亡途径,激活caspase-3蛋白,从而诱导细胞凋亡。

Objective To explore the effect of specific cytotoxic lymphocyte（CTL）which are activated by dendritic cell（DC）modified with IL-27 gene and esophageal tumor lysate on esophageal carcinoma cell apoptosis.Methods After making the nude mice models with esophageal cancer cell successfully,the mice were immunized with specific CTL which are activated by IL-27 gene modified DC loaded with esophageal tumor lysate（DC_（IL-27＋Ag））.The cell apoptosis rate was analyzed by Annexin V/PI staining.The mitochondrial membrane potential was measured by Rhodamine 123 staining and the expression level of caspase-3protein was detected by flow cytometry.Results The ratio of apoptosis,membrane potential of mitochondria and expression of caspase-3of 5groups showed significant difference（P0.05）.The ratio of apoptosis and caspase-3protein expression in DCIL-27＋Ag group,DCIL-27 group and DCnaive group were higher than those in PBS group（control group）.The membrane potential of mitochondria in DCIL-27＋Aggroup,DCIL-27 group and DCnaivegroup was higher than that in PBS group.The ratio of apoptosis and caspase-3protein expression in DCIL-27＋Ag,group,DCIL-27 group and,DCnaivegroup were higher than those in Tnaive group,but the membrane potential of mitochondria in DCIL-27＋Ag group,DCIL-27 group and DCnaivegroup was lower than that in Tnaive group.The ratio of apoptosis and caspase-3protein expression in DCIL-27＋Ag group,DCIL-27 group were higher than those in DCnaivegroup,but the membrane potential of mitochondria in DCIL-27＋Aggroup,DCIL-27 group was lower than that in DCnaive group.The ratio of apoptosis and caspase-3 protein expression in DCIL-27＋Ag group,were higher than those in DCIL-27 group,but the membrane potential of mitochondria in DCIL-27＋Aggroup was lower than that in DCIL-27 group.Conclusion Specific CTL,activated by DC modified with IL-27 gene and esophageal tumor lysate,efficiently play cytotoxic effect on tumor-bearing mice in vivo through reducing the mitochondrial membrane potential,initiating the intrinsic mitochondrial apoptotic pathways and increasing caspase-3 protein expression.