B7-H1沉默联合肿瘤抗原负载的新型树突状细胞疫苗体外抗膀胱癌免疫效应的研究

Anti-bladder cancer effect in vitro of novel dendritic cell vaccine modified with B7-H1 short hairpin RNA and pulsed with tumor antigen

目的 通过慢病毒介导B7-H1沉默与负载肿瘤抗原结合的方法构建新型树突状细胞（DC）疫苗,观察其体外抗膀胱癌的免疫效应.方法 分离健康人外周血单个核细胞,体外诱导培养DC,感染含B7-H1短发卡RNA（shRNA）序列的慢病毒载体.逆转录-聚合酶链反应（RT-PCR）及Western blot法检测感染后DC B7-H1的表达,流式细胞术分析DC表型,混合淋巴细胞反应检测其刺激T淋巴细胞增殖能力,酶联免疫吸附试验（ELISA）检测其分泌白细胞介素（IL）-10和IL-12的水平.然后将DC负载膀胱肿瘤抗原,与T细胞共培养诱导细胞毒性T细胞（CTL）,乳酸脱氢酶（LDH）释放法检测其对膀胱癌细胞的体外杀伤活性.结果 DC慢病毒感染效率约为75％,感染后DC上B7-H1表达受到明显抑制,而DC表型却并未受影响.DC疫苗对T细胞诱导增殖效应及IL-12分泌能力明显增强,而IL-10分泌明显减少,同时负载膀胱肿瘤抗原的DC疫苗诱导产生的CTL对膀胱癌T24细胞的杀伤活性[E/T=5∶1时为（12.80±1.06）％,E/T=10∶1时为（23.44±2.40）％,E/T=20∶1时为（36.27±3.06）％]明显高于对照组（P＜0.05）.结论 慢病毒介导的B7-H1沉默联合肿瘤抗原负载的新型DC疫苗可有效诱导体外特异性抗膀胱癌：免疫效应.

Objective To construct novel dendritic cell vaccine by B7-H1 short hairpin RNA （shRNA） transfection via lentivirus and tumor antigen loading jointly,and to investigate its anti-bladder cancer effect in vitro.Methods Monocytes were isolated from peripheral blood of healthy donors and were induced into dendritic cells （DC） in vitro.These DCs were infected by lentivirus containing B7-H1 shRNA sequence.The B7-H1 expression was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting.Flow cytometry was performed to analyze the phenotype of DCs.The ability of stimulating T cell proliferation and secreting interleukin （IL）-10 and IL-12 was tested by Cell Counting Kit-8 （CCK-8） and enzyme linked immunosorbent assay （ELISA） separately.The infected DCs were pulsed by tumor antigen and then co-cultured with autologous T cells to generate CTLs.Lactate dehydrogenase （LDH） release assay was performed to determine the cytotoxicity of CTLs in vitro.Results The infection efficiency was approximately 75%.RT-PCR and Western blotting results showed evident inhibition of B7-H1 expression.Flow cytometry analysis demonstrated a typical mature phenotype of DC which was not affected by lentiviral infection.The capacity of DC vaccine in stimulating T cell proliferation and secreting IL-12 was significantly enhanced with a decreasing IL-10 secretion.Cytotoxicity targeting T24 bladder cancer cells induced by DC vaccine [（12.80 ± 1.06）%,（23.44 ±2.40）%,（36.27 ± 3.06） % at E/T ratios of 5∶1,10∶1,20∶1] was obviously augmented compared with controls （P 〈 0.05）.Conclusion B7-H1 silencing mediated by lentivirus combined with tumor antigen loading could induce effective antitumor immunoresponse of DC towards bladder cancer in vitro.