负载HPV16 E7^(44-62)多肽的脐血DC-CIK细胞抗宫颈癌的免疫效应研究

Study the immunological effects on cervical cancer of cord blood-derived HPV16 E7^(44-62) PePtide-DC-CIK cells in vitro

目的:探讨HPV16 E7^(44-62)多肽对脐血DC-CIK细胞分化和功能调节,以及其对宫颈癌CaSki细胞的体外杀伤效应。方法:采集脐血单个核细胞在体外诱导成DC和CIK细胞,HPV16 E7^(44-62)多肽负载DC与CIK细胞以细胞比1∶5共同培养。流式细胞术(FCM)分析细胞表型。RT-qPCR及Western blot法检测T-bet、GATA-3及Foxp3表达水平。ELISA法检测共培养前后CIK细胞上清液中IFN-γ、IL-12、IL-10水平。CCK-8法检测体外DC-CIK细胞抗宫颈癌Caski细胞的细胞毒杀伤效应。结果:DC及CIK细胞体外诱导成功。共培养6天,HPV16 E7^(44-62)-DC-CIK组的CD3+CD56+、CD3+CD8+、CD3+CD4+细胞比例明显高于DC-CIK组及CIK组(P<0.05),DC-CIK组较CIK组高(P<0.05);HPV16 E7^(44-62)-DC-CIK组的CD4+CD25+(Treg)阳性率明显低于DC-CIK组及CIK组(P<0.05)。HPV16 E7^(44-62)-DC-CIK组的T细胞转录因子T-bet、GATA-3、Foxp3表达与DC-CIK组及CIK组比较,差异有统计学意义(P<0.05),其中T-bet表达最高,而GATA-3和Foxp3表达最低。HPV16 E7^(44-62)-DC-CIK组细胞上清液中IFN-γ、IL-12、IL-10与其余两组比较,差异有统计学意义(P<0.05),IFN-γ、IL-12表达最高,IL-10表达最低。在各效靶比下,HPV16 E7^(44-62)-DC-CIK组对宫颈癌CaSki细胞杀伤率最高(P<0.05)。结论:负载HPV16 E7^(44-62)多肽的DC可增强同源CIK细胞体外抗宫颈癌的免疫作用,本研究为宫颈癌DC-CIK细胞免疫治疗提供新的致敏方法。

Objective: To investigate the effect and mechanism of HPV16 E7^44-62 peptideon cord blood-derived DC-CIK cells,and to study the mechanism and effect of anti-cervical cancer. Methods: Mononuclear cells derived from umbilical cord blood were induced to DC and CIK in vitro,then co-cultured in the proportion of 1∶5.Phenotypes were analyzed by FCM,the expression levels of T-bet,GATA3 and Foxp3 were detected by RT-qPCR and Western blot,the levels of IFN-γ,IL-12 and IL-10 were determined by ELISA.While the cytotoxic effect of effector cells against cervical cancer cells in vitro were measured by CCK-8 test. Results: Both induced DC and CIK cells showed typical morphology.On the day 6 of co-culture,the ratios of CD3+CD56+ CD3+CD8+,CD3+CD4+ cells in HPV16 E7^44-62 -DC-CIKs group were obviously higher than the others ( P <0.05),while the ratio of CD4+CD25+ cells was lower ( P <0.05).The T-bet,GATA-3 and Foxp3 expression levels of HPV16 E7^44-62 -DC-CIKs group had significantly differences with others ( P <0.05),the expression level of T-bet was the highest,GATA-3 and Foxp3 were lowest.The levels of IFN-γ and IL-12 in cultured supernatant of HPV16 E7^44-62 -DC-CIKs group were all higher than the others( P <0.05),the level of IL-10 is the lowest( P <0.05).The cytotoxic effect of HPV16 E7^44-62 -DC-CIKs group was obviously higher than the others( P <0.05). Conclusions: The DC loaded HPV16 E7^44-62 peptide can enhanced the immuno-cytoxicity on cervical cancer cells of CIK cells in vitro.Our study provided a new strategy to produce tumor associated antigen to load DC in DC-CIK cells immunotherapy.