天津医药

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新型树突状细胞疫苗的构建及功能的初步鉴定

霍佳   

  1. 天津医科大学附属肿瘤医院免疫室
  • 收稿日期:2010-10-13 修回日期:2010-11-19 出版日期:2011-04-15 发布日期:2011-04-15
  • 通讯作者: 霍佳

Construction and Identification of a Novel DC Vaccine

  • Received:2010-10-13 Revised:2010-11-19 Published:2011-04-15 Online:2011-04-15

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摘要： 摘要 目的：构建可同时实现沉默细胞因子信号抑制因子1（SOCS1）、高迁移率族蛋白B1（HMGB1）刺激和肿瘤相关抗原负载的新型树突状细胞(DC)疫苗。方法：构建沉默SOCS1并共表达HMGB1及肿瘤相关抗原NY-ESO-1的质粒（p/shS1-NY-IRES-sig- HMGB1）。体外诱导单核细胞分化为DC，将p/shS1-NY-IRES-sig-HMGB1转染入DC细胞。同时设立未转染组、shNC/GFP转染组和p/shS1-NY-IRES-sig-HMGB1转染组。PCR和Western blot验证基因表达后，ELISA法检测DC细胞分泌细胞因子TNF-α、IL-12、IL-1及IL-6的变化。结果：经测序证实所构建质粒序列与预期序列一致，酶切和PCR验证质粒结构正确。成功将p/shS1-NY-IRES-sig-HMGB1转染入DC细胞，并在基因和蛋白水平验证NY-ESO-1的正确表达，HMGB1可在胞浆内表达。SOCS1 shRNA质粒shS可沉默SOCS1表达。p/shS1-NY-IRES-sig-HMGB1转染组DC TNF-α、IL-12、IL-1及IL-6分泌量较未转染组和shNC/GFP转染组升高（p＜0.05）。结论：成功构建可同时实现沉默SOCS1、HMGB1刺激和肿瘤相关抗原负载的新型DC疫苗。

关键词: 细胞因子信号抑制因子1, 高迁移率族蛋白B1, 质粒, DC疫苗

Abstract: [Abstract]: Objective: To construct a novel DC vaccine which can coexpressing suppressor of cytokine signaling 1 (SOCS1) inhibitor, high mobility group proteins B1 (HMGB1), and tumor-associated antigens. Method: First construct the plasmid （p/shS1-NY-IRES-sig-HMG B1）that coexpress SOCS1 inhibitor, HMGB1, and tumor associated antigens. Then p/shS1-NY-IRES-sig-HMGB1 was transfected into DC. PCR and western blot was used to verify the expression of the p/shS1-NY-IRES-sig-HMGB1. ELISA was used to analyze the variation of TNF-α、IL-12、IL-1 and IL-6. Result: The sequence of the plasmid coincided with the sequence that expected. NY-ESO-1 can be expressed correctly. HMGB1 can be express in the cytoplasm. SOCS1 can be silenced by SOCS1 shRNA plasmid shS. TNF-α、IL-12、IL-1 and IL-6 was up-regulated in the culture medium（p＜0.05）. Conclusion: Successfully construct a novel DC vaccine which can coexpressing SOCS1 inhibitor, HMGB1, and tumor-associated antigens.

Key words: SOCS1, HMGB1, plasmid, DC vaccine