抗肝癌单链双功能抗体在Hut-78细胞系中的表达及体外杀伤活性

Expression and in vitro cytotoxicity of anti-hepatocellular carcinoma single-chain bifunctional antibody in Hut-78 cell line

目的 用携带分泌型抗肝癌单链双功能抗体基因(sFv TNF α)的重组逆转录病毒感染T细胞淋巴瘤Hut 78细胞 ,使其表达并分泌针对人肝癌细胞的sFv TNF α融合蛋白 ,观察转导的T细胞对体外培养肝癌细胞的杀伤作用 .方法 用感染性重组病毒产生细胞C2 2 (PA317/PST)产生的病毒上清转导人T细胞淋巴瘤Hut 78细胞 ,采用PCR、RT PCR、细胞原位杂交及免疫组织化学染色等方法 ,对转导的Hut 78细胞进行DNA、mRNA及蛋白水平的分析 .转导的Hut 78细胞分别与SMMC 772 1、HHCC共培养 ,MTT法检测Hut/PST细胞表达产物对两种肝癌细胞的杀伤作用 .结果 PCR、RT PCR结果显示转导Hut细胞中扩增出外源目的基因对应的电泳条带 .neo基因探针原位杂交显示转导细胞质内有较强的紫蓝色阳性反应信号 ,其阳性率约为 95 % .鼠抗人TNF α多克隆抗体免疫组织化学染色 ,转导细胞质内有明确的棕黄色阳性反应信号 .MTT法检测结果 ,分泌型抗肝癌单链双功能抗体对体外培养的两种肝癌细胞的杀伤率分别为 15 .4 %和2 6 .5 % .结论 分泌型抗肝癌单链双功能抗体基因可以在T细胞中整合并稳定表达 ,其分泌的表达产物对两种肝癌细胞均具有一定的亲合活性 ,并且具有一定的体外杀伤作用 .

AIM To investigate the selective cytotoxicity of bifunctional antibody TNF α fusion proteins in cell lines SMMC 7721 and HHCC. METHODS PCR and RT PCR were used to detect the integration and transcription of the sFv TNF α gene in HUT 78 cells transduced with recombinant retroviral virus. MTT method was used to detect the antitumour activity of the antibody TNF α fusion proteins. RESULTS There was integrated sFv TNF α gene in the genome of transduced HUT 78 cells, and that the transduced HUT 78 cells expressed the fusion antibody TNF α proteins. Cell killing was significant in both kinds of HCC cells co cultivated with HUT sFv TNF α cells, whereas the HUT 78 control cells had no significant cytotoxicity effects on HCC cells. CONCLUSION Results demonstrate that the expression of bifunctional antibody TNF α fusion proteins in HUT 78 cells has cytotoxic effects on HCC cells in vitro .