肿瘤抗原致敏的树突状细胞诱导特异性抗膀胱癌作用的研究

Study on the specific anti-cancer effects by dendritic cells loaded with bladder-tumor lysate or peptides

目的研究不同肿瘤抗原提取物致敏的树突状细胞(DC)诱导特异性体外杀伤膀胱癌细胞的作用。方法反复冻融法和弱酸洗脱法分别获取膀胱癌细胞株BIU-87肿瘤抗原;联合应用重组人的GM-CSF、IL-4和TNF-α体外诱导健康志愿者DC并分别负载膀胱癌肿瘤抗原,进而激活T淋巴细胞;用MTT法检测其对BIU-87体外杀伤效应。结果用枸橼酸-磷酸盐缓冲液洗脱或反复冻融BIU-87(1×107)后获得的肿瘤抗原蛋白含量分别为(426.0±4.3)μg和(681.0±5.6)μg;外周血单个核细胞可培养出DC;负载两种膀胱癌细胞抗原的DC致敏的CTL对BIU-87有明显的杀伤作用,而且负载酸洗脱肿瘤抗原肽的树突状细胞致敏的特异性CTL有更明显的杀伤作用(P<0.05)。结论弱酸洗脱和反复冻融都可以有效获取膀胱肿瘤抗原;以上法致敏的DC均可以高效活化CTL,后者对膀胱癌细胞株发挥特异性杀伤作用,而且弱酸洗脱组可以更为有效杀伤膀胱癌细胞,为以DC为基础的膀胱癌免疫瘤苗的临床应用提供一定的实验依据。

[Objective] To observe specific cytotoxicity induced by dendritic cells loaded with bladder-tumor lysate or acid eluted tumor antigen peptides against bladder cancer cell line. [Methods] BIU-87 cells were frozen and melt repeatedly for 4 times, then tumor lysate were obtained. Otherwise the antigen peptides of BIU-87 cells were obtained by mild acid eluted technique. Healthy volunteer PBMCs cultured with RPMI1640 contained rh GM- CS±rh IL-4±rh TNF-α in vitro for 9 days, and then DCs loaded with bladder-tumor lysate or peptides. Tumor specific CTLs were stimulated by DCs pulsed with bladder-tumor lysate or peptides and cytotoxieity caused by the CTLs against bladder cancer ceils was measured by MTT technique. [Results] The tumor antigen content obtained by citric acidphosptat buffer was （426.0±4.3） μg, and that by frozen and molten repeatedly was （681.0±5.6） μg. DCs can be harvestd through healthy volunteer PBMCs cultured in vitro. Through lactate dehydrogenase release technique, CTLs activated by both DCs can kill BIU-87 cells significantly while eytotoxicity induced by dendrtic cells pulsed with tumor antigens obtained by citric acid- phosptat buffer was stronger, which had a statistic significance （P 〈0.05）. [Conclusions] Bladder cancer cells were eluted by citric acidphosptat buffer or frozen and molten repeatedly ,then tumor antigens were obtained. DCs loaded bladder tumor antigens can produce remarkably specific CTLs,which can kill tumor cells specifically. And this cytotoxieity induced by citric acid- phosptat buffer was stronger. So we provide definite experiment basis for the clinical application of vaccine based on DCs.