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为探索临床头颈肿瘤生物治疗新途径,对CD3AK、LAK细胞单独应用及其联合p53基因重组腺病毒体外抗喉癌的效应进行比较研究.重组腺病毒载体的Anderson CancerCenter提供,含巨细胞病毒(CMV)启动子、野生型p53基因cDNA和SV4D多聚腺苷信号,为E1区缺失的复制缺陷型病毒.病毒在293内大量繁殖,CSC1密度梯度离心法纯化,空斑形成法测定纯化病毒滴度.(3)于5%FCS的RPMI1640中传代培养靶细胞-喉鳞癌Hep-2细胞.杀伤活性检测前24-48h传代Hep-2,实验当日消化成单个细胞.从健康献血者新鲜外周静脉血,常规法分离单个核细胞,体外诱导LAK细胞和CD3AK细胞.(5)杀伤活性的检测:于细胞活性高峰
In order to explore a new approach for the biological treatment of clinical head and neck tumors, the effects of CD3AK and LAK cells alone and combined with p53 gene recombinant adenovirus against laryngeal cancer were compared in vitro. Anderson CancerCenter of the recombinant adenovirus vector provided with cytomegalovirus (CMV ) Promoter, wild-type p53 gene cDNA and SV4D polyadenylation signal, were replication deficient viruses deleted in E1 region.The virus multiplied in 293, purified by CSC1 density gradient centrifugation, and purified virus titer was determined by plaque formation (3) Hep-2 cells were subcultured in 5% FCS RPMI 1640. Hep-2 cells were passaged 24-48 hours before cytotoxicity test and digested into single cells on the day of experiment.From the fresh peripheral vein of healthy donors Blood, conventional method of isolation of mononuclear cells, in vitro induction of LAK cells and CD3AK cells. (5) detection of cytotoxic activity: peak in cell activity