摘要
目的评价肠道病毒71型(enterovirus 71,EV71)灭活疫苗诱导小鼠的细胞免疫效果。方法分别以不同剂量(1、2.5、5、10μg/ml,均含铝佐剂1 mg/ml)、不同剂型(含铝佐剂1 mg/ml或不含铝佐剂)、不同免疫剂次(单次免疫或加强免疫)EV71灭活疫苗经腹腔免疫小鼠,0.5 ml/只,均设铝佐剂对照组(仅注射铝佐剂1 mg/ml)。采用经典小鼠树突状细胞(dendritic cell,DC)培养方法制备正常小鼠DC,瑞氏染色法检测细胞形态,流式细胞术分析其表型及纯度;免疫磁珠分选法(magnetic activated cell sorting,MACS)分离小鼠脾脏CD4+、CD8+T淋巴细胞,流式细胞术检测细胞纯度。ELISPOT法检测各组免疫小鼠CD4+、CD8+T淋巴细胞分泌IL-2、IL-4、IFNγ的水平;细胞因子试剂盒检测小鼠淋巴细胞培养上清及小鼠血清中细胞因子的水平。结果 DC形态不规则,表面树枝状突起形态不同,细胞核较大且不规则;DC纯度为(81.39±9.24)%,可高水平表达MHⅡ(I-A/I-E)类分子,中度表达CD86和CD40。CD4+、CD8+T细胞纯度分别为95.27%和94.08%。随着疫苗免疫剂量的增加,CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC显著增加,5μg/ml疫苗组达最大值,且明显高于其他组(P均<0.05);5μg/ml疫苗组淋巴细胞培养上清中IFNγ、IL-2、IL-4、IL-6、IL-10、IL-13、IL-5、TNF-α含量明显高于其他组(P<0.05);1、2.5、5μg/ml疫苗组血清中IL-10含量明显高于铝佐剂对照组(P<0.05),1μg/ml疫苗组明显低于2.5及5μg/ml疫苗组(P<0.05)。含铝佐剂疫苗组CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC、淋巴细胞培养上清中IL-2、IFNγ、IL-4、IL-5、IL-6、IL-10、IL-13、TNF-α水平及血清中IL-10含量均明显高于无铝佐剂疫苗组(P均<0.05)。加强免疫组CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC、淋巴细胞培养上清中IL-2、IFNγ、IL-4、IL-5、IL-6、IL-10、IL-13、TNF-α水平、血清中IL-10含量均明显高于单次免疫组(P均<0.05)。结论 EV71灭活疫苗可诱导小鼠产生特异性细胞免疫反应,本实验为其进一步人体临床试验研究奠定了基础。
Objective To evaluate the cellular immune effect of inactivated enterovirus 71 （EV71 ） vaccine in mice. Methods BALB/c mice were immunized i.p. with inactivated EV71 vaccine at various dosages （1,2. 5,5 and 10 μg/ml, containing 1 mg/ ml aluminum adjuvant）, of various formula （with and without aluminum adjuvant） by various schedules （a single dose and two doses）,0. 5 ml for each,using 1 mg/ml aluminum adjuvant as control. DCs were extracted from mouse bone marrow precursor cells by typical culture and observed for morphology by Wright staining,and analyzed for pheno- type and purity by flow cytometry. CD4＋ and CD8＋T lymphocytes were separated from mouse spleen by magnetic activated cell sorting（MACS） and determined for purity by flow cytometry, by which the secretion level of IL-2, IL-4 and IFNγ, were determined by ELISPOT. The cytokine contents in lymphocyte culture supematants and sera of mice were deter- mined by cytokine detection kit. Results DCs showed irregular morphology,expressed as processes of irregular shapes and large irregular nuclei. The DCs reached a purity of （81.39 ± 9. 24）%,in which MH II （ I -A/ I -E） molecules were highly and CD86 and CD40 were moderately expressed. The purifies of CD4＋ and CD8＋ T lymphocytes were 95.27% and 97. 08% respectively. The spot-forming cells （SFCs） of CD4＋ T lymphocytes secreting IL-2,IL-4 and IFNγ and CD8＋ lymphocytes secreting IFNγ increased significantly with the increasing dosage of vaccine, which were significantly higher in the mice immunized with 5 μg/ml vaccine than in those with vaccine at other dosages （each P 〈 0. 05）. The IFNγIL-2,IL-4, IL-6,IL-IO,IL-13 and IL-5 contents in culture supernatant of lymphocytes of mice immunized with 5 μg/ml vaccine were significantly higher than those in other groups （P 〈 O. 05）. The IL-10 content was significantly higher in sera of mice immunized with 1,2. 5 and 5 μg vaccine than with aluminum adjuvant （P 〈 0. 05）,while significantly lower in those with 1 Ixg/ml than those with 2. 5 and 5 μg/ml （P 〈 0. 05）. The SFCs of CD4＋ T lymphocytes secreting IL-2,IL-4 and IFNγ and CD8＋ lymphocytes secreting IFNγ, as well as IL-2, IFNγ, IL-4, IL-5, IL-6, IL-10, IL-13 and TNF-a contents in culture supernatant of lymphocytes and IL-10 content in sera of mice immunized with aluminum adjuvant-containing vaccine were significantly higher than those with aluminum adjuvant-free vaccine （each P 〈 O. 05）. However, the above- mentioned indexes after booster immunization were significantly higher than those after primary immunization. Conclusion Inactivated EV71 vaccine induce specific cellular immune response in mice, which laid a foundation of further clinical tri- al of the vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第1期1-8,共8页
Chinese Journal of Biologicals
基金
国家重大新药创制项目(2011ZX2009401-403-11)
863计划项目(2012AA02A401)
重大新药创制项目(2012ZX09101319)
关键词
肠道病毒71型
灭活疫苗
细胞免疫
Enterovirus 71 （EV71 ）
Inactivated vaccine
Cellular immunity
