CAJ | 학술논문

目的探讨重组乙型肝炎(乙肝)疫苗和汉逊酵母菌表达的乙肝表面抗原(rHBsAg)诱导动物和人体细胞免疫应答的动态变化.方法应用不同剂量rHBsAg免疫小鼠(BALB/c,H-2d),酶联免疫斑点法(ELISPOT)检测小鼠脾单个核细胞(MNC)体外刺激诱生IFN-γ的水平;单剂rHBsAg免疫后不同时间检测MNC、CD8+T淋巴细胞产生IFN-γ水平;同时测定免疫后不同时间细胞毒T淋巴细胞反应(CTL)活性;检测免疫单剂和多剂乙肝疫苗后小鼠MNC分泌IFN-γ、IL-2、IL-5和抗-HBs抗体水平;对乙肝感染标志阴性的4名成年人按0、1、2个月程序接种乙肝疫苗,检测免疫后外周血单个核细胞(PBMC)、CD8+T淋巴细胞分泌IFN-γ、IL-2和IL-4水平以及抗体动态.结果应用ELISPOT法,汉逊rHBsAg免疫小鼠7 d时,可检测到IFN-γ应答,14 d时达到高峰;CTL在免疫后7 d时可检出,峰值位于28 d.在1～8μg免疫剂量范围内,MNCs IFN-γ免疫应答与剂量呈显著正相关(阳转率:Υ=0.951,P=0.049＜0.05;SFC:Υ=0.996,P=0.000＜0.05),在1～4 μg免疫剂量范围内,CD8+T淋巴细胞的IFN-γ与剂量也呈显著正相关(Υ=0.999,P=0.025＜0.05);三剂乙肝疫苗免疫小鼠诱生细胞免疫和体液免疫应答水平均显著高于单剂(P值均＜0.05);成年人接种汉逊乙肝疫苗后不同个体间细胞免疫和体液免疫应答的模式不同,IL-2、IL-4应答与个体抗体滴度有关.结论用ELISPOT法成功测定了汉逊HBsAg免疫小鼠后不同淋巴细胞的细胞免疫剂量效应和CTL时间动态变化,并分析了成年人的细胞免疫应答特点,为规范疫苗细胞免疫评价提供了基础.
목적 탐토중조을형간염(을간)역묘화한손효모균표체적을간표면항원(rHBsAg)유도동물화인체세포면역응답적동태변화.방법 응용불동제량rHBsAg면역소서(BALB/c,H-2d),매련면역반점법(ELISPOT)검측소서비단개핵세포(MNC)체외자격유생IFN-γ적수평;단제rHBsAg면역후불동시간검측MNC、CD8+T림파세포산생IFN-γ수평;동시측정면역후불동시간세포독T림파세포반응(CTL)활성;검측면역단제화다제을간역묘후소서MNC분비IFN-γ、IL-2、IL-5화항-HBs항체수평;대을간감염표지음성적4명성년인안0、1、2개월정서접충을간역묘,검측면역후외주혈단개핵세포(PBMC)、CD8+T림파세포분비IFN-γ、IL-2화IL-4수평이급항체동태.결과 응용ELISPOT법,한손rHBsAg면역소서7 d시,가검측도IFN-γ응답,14 d시체도고봉;CTL재면역후7 d시가검출,봉치위우28 d.재1～8μg면역제량범위내,MNCs IFN-γ면역응답여제량정현저정상관(양전솔:Υ=0.951,P=0.049＜0.05;SFC:Υ=0.996,P=0.000＜0.05),재1～4 μg면역제량범위내,CD8+T림파세포적IFN-γ여제량야정현저정상관(Υ=0.999,P=0.025＜0.05);삼제을간역묘면역소서유생세포면역화체액면역응답수평균현저고우단제(P치균＜0.05);성년인접충한손을간역묘후불동개체간세포면역화체액면역응답적모식불동,IL-2、IL-4응답여개체항체적도유관.결론 용ELISPOT법성공측정료한손HBsAg면역소서후불동림파세포적세포면역제량효응화CTL시간동태변화,병분석료성년인적세포면역응답특점,위규범역묘세포면역평개제공료기출.
Objective To study the kinetics of immune response in mice and human immunized with rHB vaccine or rHBsAg derived from yeast cells(Hansenula polymorpha).Methods With different doses,the level of IFN-γ secreted by spleen mononuclear cells(MNC)including CD8+T cells by MACs of mice were detected by enzyme-linked immunospot(ELISPOT)methods after stimulation in vitro with HBsAg MHC class Ⅰ peptide S28-39,respectively.At serial time points.the immunized mice were detected for IFN-γ by ELISPOT as above and for the lymphocytotoxicity test(CTL)by specific lysis assay.The levels of IFN-γ,IL-2,IL-5 and anti-HBs in mice induced by rHB vaccine were detected after single or three doses.Four adults were vaccinated with rHB vaccine according to 0,1 and 2 month schedule.The peripheral blood mononuclear cells(PBMCs)were collected at the 3,8,21,34 and 65 days after the first dose.The CD8+T cells with high purity obtained by sorting from PBMcs were stimulated with rHBsAg or HBsAg peptides.The SFC of IFN-γ,IL-2 and IL-4 of CD4+ and CD8+ T cells were determined by ELISPOT.Results The cytokine of IFN-γ became detectable on day 7 and its peak value appeared on day 14 by ELISPOT.The CTL was detected on day 7 and the maximum lysis of CTL appeared on day 28.The cellular immune response of IFN-γ of MNCs were significantly correlated with the doses vaccinated from 1 μg to 8 μg(Υpositive rates=0.951,Ppositive rates=0.049＜0.05;rSFC=0.996,PSFC=0.000＜0.05).IFN-γSFC of CD8+T cells were significantly associated with the doses from 1 μg to 4 μg(Υ=0.999,P=0.025＜0.05).The HBsAg specific cellular immune and humoral responses of mice immunized with three doses were significantly higher than that with a single dose(P＜0.05).The characteristics of IFN-γ,IL-2 and IL-4 of CD4+ and CD8+ T cells were variable between individuals immunized with the same rHB vaccine.The level of IL-2 and IL-4 of responders were significantly related to the titer of anti-HBs.Conclusion Data from this study showed the kinesis of cellular immunity in mice and adults vaccinated with rHBsAg or rHB vaccine respectively.and the characteristics of cellular immune response in adults induced by the vaccine.Our data provided the basis of standardizing the analysis of cellular immune response to rHB vaccine.