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目的探讨细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK细胞)作为人IL-21(hIL-21)基因的载体治疗肝癌的可行性。方法构建携带hIL-21、CopGFP基因的质粒pDC759-hIL-21、pDC759-CopGFP并分别与pAd5、pAd5F35共转染HEK293细胞,包装表达hIL-21、CopGFP蛋白的Ad5、Ad5F35病毒。分离培养CIK细胞并绘制生长曲线,将携带CopGFP基因的Ad5、Ad5F35病毒以不同MOI(0、1、5、10、50、100、1 000)感染CIK细胞;将携带hIL-21基因的Ad5、Ad5F35病毒以不同MOI(0、1、5、10、20、50)感染SMMC-7721细胞48h后检测hIL-21蛋白的表达;将携带hIL-21基因的Ad5F35病毒以不同MOI(0、1、5、10、50、100)感染CIK细胞48h后检测hIL-21蛋白的表达。皮下注射SMMC-7721细胞致裸鼠成瘤,以表达hIL-21的CIK细胞治疗荷瘤裸鼠,评价疗效。结果成功构建出用于Ad5、Ad5F35病毒的质粒;构建的质粒与pAd5、pAd5F35脂质体共转染HEK293细胞,包装出表达hIL-21及CopGFP蛋白的Ad5、Ad5F35病毒。携带CopGFP基因的Ad5、Ad5F35病毒对CIK细胞的感染实验表明Ad5F35腺病毒感染率更高;携带hIL-21基因的Ad5、Ad5F35病毒对SMMC-7721细胞的感染实验表明hIL-21表达量差异无统计学意义;携带hIL-21基因的Ad5F35病毒以50MOI感染CIK细胞后hIL-21的表达量较高;动物实验统计分析说明表达hIL-21的CIK细胞对荷瘤裸鼠疗效较好。结论 hIL-21与CIK细胞在荷瘤小鼠体内联合发挥抗肿瘤作用。
Objective To investigate the feasibility of cytokine-induced killer cell (CIK) as a carrier of human IL-21 (hIL-21) gene in the treatment of liver cancer. METHODS: Plasmids pDC759-hIL-21 and pDC759-CopGFP carrying hIL-21 and CopGFP gene were constructed and co-transfected into HEK293 cells with pAd5 and pAd5F35 respectively to package Ad5 and Ad5F35 viruses expressing hIL-21 and CopGFP protein. CIK cells were isolated and cultured and the growth curve was drawn. The Ad5 and Ad5F35 viruses carrying CopGFP gene were infected with CIK cells at different MOI (0, 1, 5, 10, 50, 100 and 1000). The Ad5, Ad5F35 virus was used to infect SMMC-7721 cells at different MOI (0, 1, 5, 10, 20, 50) for 48 hours to detect the expression of hIL-21 protein. Ad5F35 virus carrying hIL- 5, 10, 50, 100) infected CIK cells 48h after the detection of hIL-21 protein expression. SMMC-7721 cells were injected subcutaneously into nude mice, and the tumor-bearing nude mice were treated with CIK cells expressing hIL-21 to evaluate the curative effect. Results The plasmids for Ad5 and Ad5F35 were successfully constructed. The constructed plasmids were cotransfected into HEK293 cells with pAd5 and pAd5F35 liposomes and packaged the Ad5 and Ad5F35 viruses expressing hIL-21 and CopGFP protein. Ad5F35 adenovirus infection rate of Ad5 and Ad5F35 viruses carrying CopGFP gene was higher than that of Ad5F35 virus. Infection experiments of SMMC-7721 cells with Ad5 and Ad5F35 carrying hIL-21 gene showed no difference in hIL-21 expression Significance of the study; hIL-21 gene carrying Ad5F35 virus in 50MOI infected CIK cells after high hIL-21 expression levels; animal experiments and statistical analysis shows that hIL-21-expressing CIK cells in tumor-bearing nude mice better. Conclusion hIL-21 and CIK cells in tumor-bearing mice combined anti-tumor effect.