IL-2基因逆转录载体的构建与B7联合表达的抗肿瘤研究

Effect of IL-2 and B7 Co-expression on the Anti-Tumor Response

目的 :IL - 2是一种具有抗肿瘤作用的细胞因子 ,B7是诱导细胞免疫的共刺激分子 ,联合在肿瘤细胞中表达IL - 2或IL - 2 /B7,诱导机体抗肿瘤免疫应答 ,为IL - 2基因治疗进入临床提供依据 .方法 :利用逆转录载体PLXSN构建了插入IL - 2基因的重组逆转录病毒载体 .将重组逆转录病毒载体引入包装细胞CRIP ,测病毒滴毒为 6× 10 5CFU/mL ,用成熟重组逆转录病毒转染小鼠胰腺癌细胞MPC - 35 ,经G4 18筛选得抗性克隆MPC - 35 1,PCR检测证实目的基因已整合在MPC - 35细胞基因组中 ;引入携带B7基因的腺病毒载体转染MPC - 35 1,经流式细胞仪证实B7基因已在MPC - 35 1中获得表达 ,命名为MPC - 35 2 .结果 :采用ELISA方法检测MPC - 35 1、MPC - 35 2 分泌IL - 2水平分别为 95 5U/mL及 92 5U/mL ,与亲代MPC- 35比较 ,MPC - 35 1体外增长率及形态无明显变化 ;MPC - 35 2 无形态的改变 ,但增长速度减缓 ,流式细胞仪测试显示 :MPC - 35本身不表达MHC -classI类分子 ,MPC - 35 1有ICAM - 1的表达增加 ,MPC - 35 2 有classII和ICAM -I的表达增加 ;小鼠体内移瘤实验表明 :与作为对照的MPC - 35细胞相比 ,MPC - 35 1,MPC - 35 2 在小鼠体内的生长明显受抑制 ,出瘤时间晚 (P <0 0 5 ) ,瘤块小 (P <0 0 1) ,生命时间延长

Objective: Interlukin-2 (IL-2) is a cytokine with anti-tumor effect. B7 is a co-stimulate factor .The aim of the study is to induce anti-tumor response by co-expression Il2/B7 in tumor cells. Methods: We constructed an expression vector of retrovirus containing the IL-2 cDNA. The titer of the expression vector was high to 6×10 5 CFU/mL. The vector was used to transfect to MPC-35 cells, a murine carcinoma cells. The cells of expressing the IL-2 were selected to resist to G418 and termed as MPC-35 1. Electrophoresis of PCR product from the IL-2 expressing cells showed a proper band of IL-2 .The IL-2 expressing cells were second transfected to express the B7 by using adenovirous vector containing the B-7 cDNA. The cells expressing both IL-2 and B7 were termed as MPC-35 2. The IL-2 secretion of MPC-35 1 and MPC-35 2 were 95 5?U/mL and 92 5?U/mL respectively. Results: The Biological characters of two modified cells were compared with native MPC-35 cells. We found that the growth speed was similar between MPC-35 and MPC-35 1, but significant difference (P<0 05) between MPC-35 2 and MPC-35/MPC-35 1. Flow cytometry showed that MPC-35 1 expressing ICAM-1 and MPC-35 2 expressing ICAM-1 and Class II increase, compared with the native MPC-35 cells. When these modified cells were injected in back of mouse, the MPC-35 1 (P<0 05) and MPC-35 2 (P<0 01) showed a significant delay for tumor formation, and survive ratio increased in both MPC-35 1 (P<0 01) and MPC-35 2 (P<0 01).