肿瘤坏死因子相关凋亡诱导配体基因对肝癌细胞杀伤作用及旁观者效应研究

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因在诱导肝癌细胞凋亡时是否存在旁观者效应，以及旁观者效应的可能机制。方法 构建表达TRAIL基因的双腺病毒载体系统Ad／GT-TRAIL Ad／PGK-GV16，通过293包装细胞产生的病毒上清液将TRAIL基因转入肝癌细胞SMMC7721细胞中，RT-PCR检测TRAIL基因的表达，以MTT法检测细胞生长抑制率，流式细胞仪检测细胞凋亡率；不同比例混合培养SMMC7721／TRAIL和SMMC7721细胞，检测混合细胞生长抑制率来评价TRAIL基因的旁观者效应，并以滤去细胞成分的转染细胞培养液培养未转染细胞，观测可溶性因子在TRAIL基因旁观者效应中的作用。结果 TRAIL基因对SMMC7721细胞的生长抑制率与PBS、LacZ基因和Bax基因比较，差异有显著性(P＜0．05)；SMMC7721细胞的凋亡率，TRAIL基因与PBS、LacZ基因和Bax基因比较，差异有显著性(P＜0．05)；混合细胞的生长抑制率，以SMMC7721／TRAIL占0％时为0，占5％时为15．9％，占25％时为67．0％，占50％时为80．2％，占100％时为87．7％；以PBS对SMMC7721的生长抑制率为0，则滤去细胞成分的转染细胞培养液对SMMC7721的生长抑制率为4％，两者差异无显著性。结论 双腺病毒载体系统介导的TRAIL基因对肝癌细胞有明显的抑制生长和促凋亡作用，并存在旁观者效应，可溶性因子在TRAIL的旁观者效应中不起作用。

Studies of killing and bystander effect of tumor necrosis factor related apoptosis-inducing ligand gene on hepatic carcinoma cell

Objective To study the bystander effect of tumor necrosis factor related apoptosis inducing ligand(TRAIL) gene and it's mechanism. Methods Full length cDNA of human TRAIL was transfected into SMMC7721 with binary adenoviral vectors system. RT PCR was used to determine the expression of TRAIL gene. By MTT assay the effects of the TRAIL gene on the proliferation of SMMC7721 was studied; The apoptosis inducing ability of TRAIL gene on SMMC7721 was tested by fluorescence activated cell sorting (FACS). Bystander effect by testing the proliferation of the mixed cells of SMMC7721/TRAIL and SMMC7721 with different ratios, and the mechanism of bystander effect by testing the proliferation of SMMC7721 cultured with media of SMMC7721/TRAIL without cell components were also studied. Results TRAIL gene expressed by binary adenoviral vector system was able to inhibit proliferation(91.2%) and induce apoptosis(29.07%) of SMMC7721, significant difference between TRAIL gene and the other three controls were observed(PBS, LacZ, Bax)( P <0.05). The proliferation of 15.9% mixed cells was inhibited when SMMC7721/TRAIL occupied 5%, 67.0% was inhibited at 25%,80.2% was inhibited at 50%, 87.7% was inhibited when 100%, 4% of SMMC7721 cultured with media of SMMC7721/TRAIL without cell components was also inhibited, there was no significant difference with PBS.Conclusion TRAIL gene transduced by binary adenoviral vector system can inhibit proliferation and induce apoptosis of SMMC7721 with bystander effect, and the soluble factors have no effect on bystander effect.