一种体外评价佐剂诱导人急性白血病单核细胞THP-1炎症因子方法的建立  

Developmemt of a method for evaluation in vitro of inflammatory factor in human acute leukemia monocyte THP-1induced by adjuvant

作　　者：魏多前 单璞 张转 郝少杰 王志标 李树香 徐静 WEI Duo-qian;SHAN Pu;ZHANG Zhuan;HAO Shao-jie;WANG Zhi-biao;LI Shu-xiang;XU Jing(The Third Laboratory of National Vaccine and Serum Institute,Beijing 101lll,China)

机构地区：[1]国药中生生物技术研究院有限公司第三研究室,北京101111

出　　处：《中国生物制品学杂志》2022年第12期1482-1487,1494,共7页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2017ZX10202201)。

摘　　要：目的以人急性白血病单核细胞THP-1为模型,建立一种体外评价佐剂诱导人源化细胞炎症因子水平的方法。方法以细菌内毒素标准品为阳性对照,与佛波酯(phorbol myristate acetate,PMA)共刺激THP-1细胞,检测炎症因子IL-8、干扰素诱导蛋白-10(interferon-induced protein,IP-10)、IL-1β及单核细胞趋化蛋白(monocyte chemotactic protein,MCP-1)的分泌水平,分别对PMA终浓度(25、5、1、0.1 ng/mL)、刺激时间(4、16、24 h)和细胞密度(1×10^(5)、5×10^(5)、1×10^(6)个/mL)进行优化,确定最佳条件。用不同佐剂[(Toll样受体(toll-like receptors,TLR)激动剂类佐剂Pam2CSK4和TLR4以及乳剂类佐剂MF59和AS03]与PMA刺激THP-1细胞,考察不同佐剂刺激产生炎症因子的水平。结果炎症因子IL-8、IP-10、IL-1β、MCP-1的最佳诱导条件分别为:PMA终浓度0.1、1、5、1 ng/mL,培养时间16、16、4、24 h,细胞密度均为1×10^(6)个/mL。不同佐剂诱导产生的炎症因子水平不同,其中Pam2CSK4和LPS-B5大于0.001μg/mL即可诱导产生较高水平的炎症因子,而MF59和AS03诱导产生的炎症因子水平较低。结论成功建立了一种体外检测方法,可用于评价佐剂诱导产生的炎症因子IL-8、IP-10、IL-1β、MCP-1水平,为佐剂反应原性的研究提供了参考。Objective To develop a method for evaluation in vitro of the levels of human inflammatory cytokines induced by adjuvant using human acute leukemia monocyte THP-1 as a model.Methods THP-1 cells were co-stimulated with phorbol myristate acetate(PMA)and bacterial endotoxin standard which was used as positive control,and determined for the secretion of inflammatory cytokine IL-8,interferon-induced protein(IP-10),IL-1βand monocyte chemotactic protein(MCP-1).PMA concentration(25,5,1 and 0.1 ng/mL),incubation time(4,16 and 24 h)and cell density(1×10^(5),5×10^(5)and 1×10^(6)cells/mL were optimized to determine the optimal conditions.THP-1 cells were co-stimulated with PMA and various adjuvants[toll-like receptor(TLR),agonist type adjuvants Pam2CSK4 and TLR4 and emulsion type adjuvants MF59 and AS03]to determine the levels of inflammatory cytokines stimulated by different adjuvants.Results The optimal induction conditions of IL-8,IP-10,IL-1βand MCP-1 were as follows:final concentrations of PMA were 0.1,1,5 and 1 ng/mL,incubation times were 16,16,4 and 24 h,respectively,and each cell density was 1×10^(6)cells/mL.Various adjuvants induced different levels of inflammatory cytokines,of which Pam2CSK4 and LPS-B5 at a concentration of more than 0.001μg/mL induced high levels of inflammatory cytokines,while MF59 and AS03 induced low levels.Conclusion An in vitro method to evaluate the levels of inflammatory cytokines IL-8,IP-10,IL-1βand MCP-1 induced by adjuvants was developed successfully,which provided a reference for the study of adjuvant reactivity.

关 键 词：佐剂 THP-1细胞 炎症因子 

分 类 号：R392-33[医药卫生—免疫学]