重组乙型肝炎表面抗原(汉逊酵母菌表达)免疫小鼠后不同细胞免疫测定方法的比较研究  

Comparison of two Assays for Detection of Cellular Immune Responses of HBsAg

作　　者：胡忠玉[1] 何鹏[1] 邱少辉[1] 方鑫[1] 梁争论[1] 李河民[1] 庄辉[2] 

机构地区：[1]中国药品生物制品检定所,北京100050 [2]北京大学医学部

出　　处：《中国预防医学杂志》2008年第6期456-459,共4页Chinese Preventive Medicine

基　　金："十五"国家科技攻关课题(2004BA718B02)

摘　　要：目的探讨不同细胞免疫测定方法对小鼠免疫乙型肝炎表面抗原(HBsAg)后细胞免疫应答测定的影响。方法小鼠皮下接种HBsAg,于免疫后4、7、10、14和25d分离脾单个核细胞(MNC),应用酶联斑点法(ELISPOT)测定MNC细胞体外刺激后所产生的细胞因子IFN-γ斑点形成细胞计数(SFC),其中刺激物选择多肽和HBsAg;流式荧光检测技术(Luminex)测定MNC体外经HBsAg刺激后所产生的细胞因子IFN-γ、IL-4、IL-5和IL-10含量;小鼠血清检测抗-HBs。结果小鼠接种HBsAg后,应用ELISPOT法测定,7d和14d小鼠IFN-γ阳转率分别为60%和80%;而应用Luminex方法测定,7d和14d小鼠IFN-γ阳转率均为80%;IL-4、IL-5、IL-10阳转率分别为60%,80%;100%,100%;80%,100%;25d时,IFN-γ与14d比较统计学上差异有统计学意义(P25,14d=0.030,<0.05),而IL-5、IL-10和IL-4与14d比较差异均无统计学意义(P>0.05)。抗-HBs水平随时间而呈上升趋势。结论Elispot和Luminex法测定小鼠IFN-γ应答符合率高,小鼠接种单针HBsAg后细胞免疫应答高峰为免疫后7～14d。25d时IFN-γ分泌水平随抗-HBs抗体水平升高而下降,而IL-4、IL-5、IL-10分泌继续保持较高水平。Objective To evaluate the effect of cellular immune assays through the study on the cellular immune response induced by recombinant hepatitis B surface antigen in mice. Methods At different time, the level of IFN -γ secreted by splenic mononuclear ceils （MNC） of mice were detected by ELISPOT after stimulation in vitro with HBsAg and HBsAg MHC class I peptide S28-39, respectively. The cytokine concentration of IFN -γ, IL -4, IL -5 and IL -10 secreted by MNCs of mice were detected by Luminex after stimulation with HBsAg and the titers of anti - HBs of mice were assayed by ARCHITECH Anti - HBs kits of Abbott. Results The positive rates of IFN - γ by ELISPOT were 60% at day 7 and 80% at day 14 after immunization with HBsAg in mice, respectively, while those by Lumines were both 80%. The positive rates of IL -4, IL -5 and IL - 10 were 60%, 80% ; 100%, 100%, 80%, 100%, respectively. On day 25, the level of IFN -γ was significantly lower than that on day 14 （ P 〈 0. 05） while the levels of IL - 5, IL - 4 and IL - 10 were not significantly different from those on day 14 （P 〉 0. 05 ）. The level of anti - HBs rose with the increase of immunization time. Conclusion The two assays of ELIS- POT and Luminex had the same ability in detection of IFN - γ induced by HBsAg in mice. The response of cellular immune response reached peak on day 7 - 14. On day 25, the level of IFN - γ decreased with the rising of anti - HBs while the level of IL-5, IL-4 and IL- 10 remained high.

关 键 词：乙型肝炎 酶联免疫斑点法 流式荧光检测技术 单个核细胞 斑点形成细胞 细胞因子 

分 类 号：R392.1[医药卫生—免疫学]