携带溶瘤病毒的CIK细胞载体构建及其抗肝癌初步研究  

Construction of CIK cell vehicles carrying oncolytic virus and preliminary study on its antihepatoma ability

作　　者：李沛[1] 王铮[1] 徐勤鸿[1] 段万星[1] 雷建军[1] 马清涌[1] 仵正[1] 

机构地区：[1]西安交通大学医学院第一附属医院肝胆外科,陕西西安710061

出　　处：《肿瘤》2013年第11期959-965,共7页Tumor

基　　金：国家自然科学基金资助项目(编号:81071877)

摘　　要：目的:构建含肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)基因的重组腺病毒的细胞因子诱导杀伤(cytokine-induced killer,CIK)细胞载体,并初步观察其抗肝癌能力。方法:分离外周血淋巴细胞,进行CIK细胞培养及表型鉴定;构建携带CD40L启动子的慢病毒载体pLentihCD40L-E1AB及重组腺病毒载体pAd5/F35-TRAIL,两步法感染CIK细胞;应用ELISA、MTT、小管形成实验及软琼脂克隆形成实验观察双病毒感染后CIK细胞的分泌功能、体外增殖能力变化以及对血管生成能力和对肝癌细胞集落形成能力的影响。结果:CIK细胞生长旺盛,双染色结果显示CD3、CD56、CD11a和CD226为阳性,CD8和CD305为阴性。成功获得了具有hCD40L启动子活性及腺病毒E 1基因的慢病毒pLenti-hCD40LE1AB,同时成功构建了重组腺病毒载体pAd5/35-TRAIL。功能学检测结果显示,感染Ad5/35-TRAIL和pLentihCD40L-E1AB两种病毒的CIK细胞中干扰素γ表达水平升高(P<0.05),血管生成及肝癌细胞集落形成能力受到明显抑制,但对CIK细胞自身增殖能力的影响较小。结论:成功构建了携带腺病毒并表达TRAIL基因的CIK细胞,并初步观察到该CIK细胞对肝癌细胞生长具有一定的抑制作用。Objective： To construct cytokine-induced killer （CIK） cell vehicles carrying recombinant adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） gene, and preliminarily observe its anti-hepatoma ability. Methods： Lymphocytes were isolated from peripheral blood to culture CIK cells. The phenotypic identification of CIK cells was performed by flow cytometry （FCM）. Then, the lentiviral pLenti-hCD40L-E1AB containing CD40L promoter and the recombined adenovirus vector pAd5/35-TRAIL were constructed, respectively. The two viruses were infected into CIK cells by two-step method. After that, the secretory function and proliferative capacity of CIK cells as well as the effect on angiogenesis and the ablility of colony-formation of hepatoma cells were assessed by ELISA, MTT method, Tubule formation assay and soft agarose assay, respectively. Results： The CIK lymphocytes grew vigorously, in which the expressions of CD3, CD56, CD11a and CD226 were positive, while the expressions of CD8 and CD305 were negative. The lentiviral pLenti-hCD40L-E1AB containing active hCD40L promoter and adenovirus E1 gene was successfully constructed, and the recombined adenovirus vector pAd5/35-TRAIL containing human TRAIL gene was also constructed. In CIK cells infected with Ad5/F35-TRAIL and pLenti-hCD40L-E1AB, the expression level of interferon-γ was significanly increased （P 〈 0.05）, and the angiogenesis and the colony-formation rate of hepatoma cells were inhibited, but the proliferative capacity of CIK cells was less affected. Conclusion： CIK cell vehicles carrying adenovirus and expressing TRAIL gene are successfully constructed. The growth inhibition of hepatoma cells may be induced by CIK cells.

关 键 词：肝肿瘤 实验性 溶瘤病毒疗法 杀伤细胞 淋巴因子激活 TNF相关凋亡诱导配体 新生血管化  病理性 集落形成单位测定 

分 类 号：R730.51[医药卫生—肿瘤]