负载膀胱癌酸洗抗原肽对树突状细胞分泌IL-12的影响及意义

The Effect and Significance of Capacity of DCs Loaded with Acid Eluted Bladder-tumor Antigen Peptides Excreting IL-12

目的:研究负载膀胱癌酸洗抗原肽对树突状细胞(DCs)分泌IL-12的影响和意义。方法:用弱酸洗脱法获得膀胱癌细胞株BIU-87肿瘤抗原肽;联合应用重组人GM-CSF、IL-4和TNF-α体外诱导健康志愿者DCs并负载膀胱癌肿瘤抗原肽;DC诱导膀胱癌抗原特异性CTLs;用MTT法检测其对BIU-87体外杀伤效应;用ELISA法检测第3、6、9天培养细胞的上清液,评价DCs分泌IL-12 p70的能力。结果:联合应用重组人GMCSF、IL-4和TNF-α可在体外诱导出DCs;负载膀胱癌抗原肽的DC诱导的特异性CTLs可杀伤高达(78.5±7.0)%的BIU-87细胞,显著高于各对照组(P<0.01);经ELISA方法检测不同时期的DCs分泌IL-12的量不同,而且负载抗原肽DCs较未负载抗原DCs有更强的分泌能力(P<0.05)。结论:IL-12 p70在DCs刺激特异性CTLs的过程中发挥重要作用,其分泌量受DCs的成熟状态及某些刺激信号的影响,膀胱癌酸洗抗原肽是其刺激信号之一。

Objective：To examine the effect and significance of capacity of DCs loaded with acid eluted bladdertumor antigen peptides excreting IL-12. Methods： The antigen peptides of BIU-87 cells are obtained by mild acid eluted technique. Healthy volunteer PBMCs which harvested through Ficoll Hypaque technique culture with RP- MI1640＋ 10%FBS contained rhGM-CS＋ rhIL-4 ＋ rhTNF-ain vitro for 9 days. In the meantime immatured DCs loaded with bladder-tumor antigen peptides. When the ninth day matured DCs are harvested. Next, some matured DCs are checked, others are cultured with T Cells for 4 days , which induced tumor specific CTLs. Specific cytotoxicity caused by the CTLs towards bladder cancer cells can be detected through MTT technique. DCs can excrete IL- 12,so the supernatant are detected on every 3 days by ELISA technique. Results： DCs can be harvested through healthy volunteer PBMCs cultured in vitro. Tumor specific CTLs induced by DCs loaded with bladder-tumor antigen peptides can kill BIU-87 cell line significantly,and there are about （78.5±7.0）% tumor cells killed when the ratio was 1：40,which was significant stronger than control groups（P〈0.01）. The capacity of DCs excreting IL- 12,which sensitized by bladder-tumor antigen peptides,is stronger than that of insensitive DCs, and the quantity are different on the different stages（P〈0.05）. Conclusions：IL-12 p70 plays an important role in the process of DCs stimulating specific CTLs. The secretion of which is affected by full-grown status and some stimulant signals,and bladder-tumor antigen peptides is one of stimulant signals.