中文摘要：

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。

英文摘要：

Objective To investigate the feasibility of using cytokine-induced killer（CIK） cells as targeted vehicle to deliver hIL-21 gene for hepatocellular carcinoma（HCC） treatment. Methods We constructed pDC759-hIL-21 and pDC759-CopGFP for cotransfection of HEK293 cells with pAd5 and pAd5F35 which can express hIL-21 and CopGFP proteins for viral packaging.We isolated CIK cells and plotted the growth curve.Then Ad5/AAV-CopGFP and Ad5F35/AAV-CopGFP of different MOIs（0,1,5,10,50,100,and 1 000） were used to infect CIK cells;Ad5/AAV-hIL-21 and Ad5F35/AAV-hIL-21 of different MOIs（0,1,5,10,20,and 50） were used to infect SMMC-7721 cells and the hIL-21 protein levels were examined by ELISA assay 48 h after infection. Ad5F35/AAV-hIL-21 of different MOIs（0,1,5,10,50,and 100） was used to infect CIK cells and hIL-21 protein levels were also examined 48 h after infection. SMMC-7721 cells were subcutaneously injected to nude mice for tumor forming,and then the tumor-bearing mice were treated with CIK cells carrying hIL-21 and the therapeutic effects were observed. Results Plasmids for viral packaging were constructed.After cotransfection of HEK293 cells,Ad5 and Ad5F35 which can express hIL-21 and CopGFP proteins were successfully packaged.Ad5 and Ad5F35 which can express CopGFP were used in this study.It was found that Ad5F35 was superior to Ad5 in CIK infection,with the suitable MOI being 50.Ad5/AAV-hIL-21 and Ad5F35/AAV-hIL-21 had similar effect on IL-21 expression in SMMC-7721 cells.Results of animal study showed that mice in group CIK/hIL-21 had a better curative effect. Conclusion hIL-21 and CIK cells have synergistic anti-tumor effect in nude mice bearing HCC.