结肠癌富伴侣分子疫苗的制备及其抗癌作用研究  

Preparation of anticolon carcinoma vaccine with rich chaperone peptides and study on its anticancer efficacy

作　　者：赵建刚[1] 黄常新[1] 杨关根[1] 金建芳[1] 康云平[1] 夏大静[2] 王青青[2] 

机构地区：[1]浙江中医药大学附属杭州市第三人民医院肛肠科,浙江杭州310009 [2]浙江大学免疫学研究所

出　　处：《中华胃肠外科杂志》2009年第3期290-293,共4页Chinese Journal of Gastrointestinal Surgery

基　　金：国家自然科学基金课题（30872380）;浙江省科技攻关项目（2006C33023）;杭州市科技局课题（2006533Q06）.

摘　　要：目的研究高效结肠癌富伴侣分子疫苗的制备及其抗癌治疗作用。方法结肠癌CT26细胞在0.1mg／L天花粉蛋白的培养基中不同温度下培养.诱导癌细胞伴侣分子的表达和抗原肽的合成。超声裂解并离心，盐析法分离上清蛋白，低温透析去除相对分子质量低于50000和高于300000的蛋白分子。凝胶过滤结合变性聚丙烯酰胺凝胶电泳法（SDS.PAGE）收取相对分子质量70000、90000、95000、110000和170000处峰段蛋白。对所获蛋白定性定量，检测不同蛋白复合物的T细胞增殖、抗结肠癌自然杀伤细胞（NK）和细胞毒性T淋巴细胞（CTL）活性等主要抗癌效应。结果主要伴侣分子（HSP60、HSP70、HSP90、gp96、HSP110和HSP170）的抗原肽复合物被较好提取.制备了浓缩的富伴侣分子抗原肽。体外抗癌实验显示，相对于普通处理的瘤苗，各肠癌瘤苗的细胞增殖、NK和CTL细胞活性等主要抗癌效应均有明显提高（P〈0.01）。结论凝胶过滤结合SDS-PAGE法可以更好地提取制备浓缩的富伴侣分子抗原肽；热应激和天花粉蛋白处理可使抗原肽的表达量增加和免疫原性提高。从而诱导出更强的抗癌免疫，其中42℃热应激加天花粉蛋白组瘤苗的抗癌作用最强。Objective To prepare the anticolon carcinoma vaccine with rich chaperone peptide and to examine its anticancer immunological efficacy. Methods CT26 colon carcinoma ceils were cultured in 1mg/L Trichosanthin 1640 medium at different temperatures to induce the chaperone expression and promote the synthesis of antigen peptides. Groups of these cells treated under the different condition were lysed by the sonic disintegration, and the lysates were centrifuged. The rawpurified proteins were obtained from the supernatants by precipitating with saturated ammonium sulfate and removing the molecules below 50 000 and above 300 000 in molecular weight via dialysis. Furthermore, the proteins with the molecular weights in 70 000, 90 000, 95 000, 110 000 and 170 000 were collected through gel filtration and SDS-PAGE. The purified proteins were analysed by Western blotting, and inspected on the anticancer immunological effects including lymphocyte proliferation and the activities of NK and CTL. Results Major of the chaperone peptides of anticancer effects in CT26 ceils, including antigen peptides joining with HSP70, HSP90, gp96, HSP110 and HSP170, was satisfactorily extracted and condensed, and rich chaperone peptide composites were successfully obtained. The composites prepared under various condition could all enhance lymphocyte proliferation and the activities of CTL and NK （P〈0.01）. Conclusions The rich chaperone peptide composites are successfully prepared via dialysis, salt fractionation and gel filtration combined with SDS-PAGE. Both the heat stress and Tfichosanthin can increase the composites, which treated by 42℃ heat stress and Trichosanthin are found to possess the strongest anticancer efficacy.

关 键 词：结肠肿瘤 抗癌疫苗 伴侣分子 热应激 天花粉蛋白 

分 类 号：R73[医药卫生—肿瘤]