HPV多肽致敏的树突状细胞体外激发特异性CTL能力的研究  

Research of dendritic cells pulsed with HPV peptide inducing specific CTL in vitro

作　　者：浦红[1] 史央 陆英华[1] 朱晨瑶 毛玉荣[1] 张振宇[1] 时宏珍 

机构地区：[1]苏州大学附属第四人民医院妇科肿瘤综合治疗科,无锡214062 [2]南京市免疫细胞生物工程技术研究中心、南京得康生物技术有限公司,210019

出　　处：《中华微生物学和免疫学杂志》2012年第5期468-472,共5页Chinese Journal of Microbiology and Immunology

基　　金：江苏省“六大人才高峰”第四批高层次人才项目资助（07C003）

摘　　要：目的观察以高危型人乳头瘤病毒16型（HPVl6）E6和E7基因编码的多肽E613-21（KLPDLCTEL）和E786-94（TLGIVCPI）为抗原负载的树突状细胞（DC）体外诱导特异性CTL的能力。方法采集HPV＋HIA-A2＋宫颈癌患者外周血，分离单核细胞（Mo）与外周淋巴细胞（PBL）；将Mo诱导成DC后负载E6和町多肽，用其反复致敏PBL（3次）；ELISA法检测致敏细胞上清细胞因子分泌水平；流式细胞标记法检测致敏细胞中特异性CTL的比例；MTr法检测致敏细胞杀伤靶细胞的能力。结果11例HPVl6＋HLA-A2＋宫颈癌患者DC平均得率为（10．79±0．88）×106（每100ml外周血），CDllc＋HLA-DR＋为（97．15±2．41）％，其中CD80＋为（84．28±5．39）％，CD83＋为（85．17±5．06）％，CD86＋为（97．74±0．87）％。PBLs致敏3次后，平均增殖（15．4±1．5）倍；致敏第21天上清细胞因子水平分别为：IL-2（2551．9±195．3）pg／ml、IL-12（554．9±64．0）pg／ml、IFN-γ（2416．9±281．7）pg／ml，TNF-α（632．4±71．1）pg／ml，明显高于未致敏组（P〈0．05），IL-10（235．1±34．7）pg／ml与对照组比较无显著差异；特异性CTL的平均比例为（6．32±1．54）％，明显高于对照组（P〈0．05），对负载E6、E7多肽的T2细胞株杀伤率明显高于对照组（P〈0．05）。结论HPV E6和E7混合多肽负载的DC体外能有效地诱导特异性CTL，刺激Thl型细胞因子的分泌，为治疗型宫颈癌疫苗的研制提供科学依据。Objective To explore the potential of autologous dendritic cells （DC） pulsed with HLA-A201-binding peptide E613.21 （KLPDLCTEL） and E7s6~ （TLGIVCPI）in inducing specific T ceils response in vitro. Methods Cervical carcinoma patients with positive HLA-A201 were enrolled and their monocytes isolated and induced into dendritic ceils and pulsed with HLA-A201-binding peptide E613_21 and E786-94. PBLs were primed by DCs every week for three times. The cytokine level of supernatant of CTLs was tested by ELISA. The percentage of special CTLs was tested by flow cytometry. The specific killing effect of CTLs was tested by MTT. Results the numbers of DCs of eleven cervical carcinoma patients were （ 10.79 ±0.88）×106（100 rnl peripheral blood）. CDlle±HLA-DR±（97.15±2.41）%, CD80±（84.28±5.39）%, CD83±（85.17±5.06）%, CD86^（97.74±0.87）%. Proliferation index of PBLs primed by DCs three times was 15.4±1.5. Cytokine levels including IL-2, IL-12, IFN-γ and TNF-α were obviously higher than non- priming PBLs [ （2551.9±195.3） pg/ml, （554.9±64.0） pg/ml, （2416.9±281.7） pg/ml, （632.4±71.1） pg/ml, respectively] （P〈0.05）, but IL-10 was no significant difference between priming CTLs and non- priming CTLs. The average percentage of special CTLs was obviously higher than eontrol group [ （ 6.32 ± 1.54） %, P〈0.05 ]. The killing effect of CTLs was obviously higher than control group （P〈0.05）. Condusion Dendritic cells pulsed with peptide E613-21 and E786-94 can induce special CTLs in vitro and stimu- late CTLs secret cytokines. This will provide seienee basis for research of therapeutic HPV vaccine.

关 键 词：人乳头瘤病毒 多肽 树突状细胞 细胞毒性T细胞 

分 类 号：R737.33[医药卫生—肿瘤]