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小鼠接种HBV外膜小S蛋白产生的细胞免疫反应

来源:《中华医药杂志》
摘要:【摘要】目的研究接种基因重组乙肝病毒疫苗RHBs产生的细胞免疫反应。方法四组小鼠,腹腔分别接种0、0。4周后分离脾T淋巴细胞,分别检测CTL(cytotoxicTlymphocyte,CTL)及特异性增殖(Tlymphocyteproliferation)反应。661%,均高于0组小鼠CTL释放率31。...

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【摘要】  目的 研究接种基因重组乙肝病毒疫苗RHBs产生的细胞免疫反应。方法 四组小鼠,腹腔分别接种0、0.65、1.25、2.5、5μg RHBs;4周后分离脾T淋巴细胞,分别检测CTL (cytotoxic T lymphocyte, CTL)及特异性增殖(T lymphocyte proliferation)反应。结果 CTL特异性释放率分别为:42.36%±19.32%、91.21%±22.97%、69.25%±24.13%、51.49%±21.661%,均高于0组小鼠CTL释放率31.21%±9.23% (对照),差异均有显著性(P<0.05);0.65、1.25、2.5、5μg组小鼠特异性增殖刺激指数SI(体外刺激实验组cpm值/体外未刺激对照组cpm值分别为1.61、2.05、3.74、3.62;用精确Fisher方法分析每个小鼠CTL反应与T淋巴细胞特异性增殖反应的相关性,χ2=5.6979, P<0.05,明显相关。结论 接种RHBs产生特异性CTL及T淋巴细胞增殖反应。

【关键词】  基因重组乙肝病毒疫苗;小鼠;接种;CTL;淋巴细胞特异性增殖

*基金项目:广州市重点科技项目(编号99M04811G)

    The cellular immune response to hepatitis B virus of small S envelop protein in mice

    TAN Chang-cheng,LOU Lu-jun.Department of Parmacy,The 458 Hospital of PLA,Guangzhou 510602,China 

    【Abstract】  Objective  Aims the induction of hepatitis B virus (HBV) specific cellular immune response was studied in mice immunizated with recombinant HBV vaccine (RHBs). Methods  BALB/c(H-2d) mice were inoculated intraperitoneally(i.p.)with RHBs at the doses ranging from 0~5μg,4 weeks after the boosting immunization, spleen T lymphocyte were restimulated in vitro with specific peptides or RHBs separately,to detect specific cytotoxic T lymphocyte (CTL) response and specific helper T lymphocyte (HTL) proliferative activity. Results  For CTL response, the percentages of specific release of the spleen lymphocyte in group 0.65、1.25、2.5、5 (mean±standard deviation) were 42.36%±19.32%、91.21%±22.97%、69.25%±24.13%、51.49%±21.661%, which were all greater than that in control group(31.21%±9.23%, P<0.05).The HTL proliferative activity is strongly associated with the CTL response in the mice immunization with RHBs (χ2=5.6979, P<0.05 by exact Fisher test). Conclusion  Optimal HBV specific CTL response and HTL proliferative activity were induced for the mice immunized with RHBs. There were strong association between CTL response and HTL proliferative activity.

    【Key words】  gene recombinant HBV vaccine (RHBs);mice;inoculated ;CTL;lymphocyte spectific proliferation

    乙肝病毒(Hepatitis B Virus, HBV)感染是一种危害人类健康的常见病、多发病,全世界HBV感染者有3亿人。接种HBV疫苗产生的特异性体液免疫反应对预防HBV感染有重要意义,而特异性细胞免疫反应在清除体内已有的HBV、治疗HBV感染过程中起着重要作用。

    本文给BALB/c小鼠免疫接种不同剂量的HBV外膜小S蛋白(RHBs)疫苗,研究能否产生特异性细胞免疫反应、哪种剂量产生的最强。

    1  材料与方法

    1.1  材料  BALB/c小鼠(H-2d):6~10周龄、SPF级BALB/c。

    RHBs:基因重组乙肝病毒疫苗, Merck公司生产。

    RHBs CTL(S28-39)表位特异性多肽(IPQSLDSWWTSL ,Saibaisheng公司生产),与BALB/c小鼠有相同的H-2d限制),以50mg/ml浓度溶解在70%乙腈/0.1% DMSO储存液中,临用前以培养液稀释。

    P815细胞(H-2d限制):购自中国细胞培养中心。

    Na251CrO4、3H- 甲基胸腺嘧啶:购自中国同位素公司。

    1.2  方法

    1.2.1  小鼠免疫接种  小鼠按以下分组,每组10只。每个小鼠按分组腹腔内各接种不同剂量的RHBs:0组,接种5μg的PBS;1.25组,接种1.25μg的RHBs;2.5组,接种2.5μg的RHBs;5组,接种5μg的RHBs。

    2周后加强免疫接种1次:最后一次接种后4周,分离培养小鼠脾T淋巴细胞。

    1.2.2  CTL反应  P815靶细胞用10μg/ml特异性多肽37℃处理4h,并用100μl  Na251CrO4、在二氧化碳培养箱37℃标记1h。 效应细胞  用含10% FCS的DMEM培养液悬浮小鼠脾T淋巴细胞,加巯基乙醇~5×10-5 M、小鼠IL-2至5u/ml,二氧化碳培养箱内培养5天后,培养液冲洗2次,按50:1与104标记的靶细胞在96孔细胞培养板上反应4h,每个实验对象检测3次;离心后,取100μl上清用r-记数计检测放射性,用以下公式计算特异性释放率,CTL特异性释放率=(实验组释放的量-自然释放的量)/(最大释放的量-自然释放的量);实验组释放的量是靶细胞与效应细胞作用后靶细胞释放的同位素的量;最大释放的量是靶细胞被5% Triton X-100完全破坏后释放的同位素的量;自然释放的量是靶细胞自身在实验期间释放的同位素的量。

    1.2.3  淋巴细胞特异性增殖实验  用含10% FCS的DMEM培养液悬浮小鼠脾T细胞,加巯基乙醇至5×10-5 M、小鼠IL-2至5u/ml,二氧化碳培养箱内培养5天后,用RHBs体外刺激,10天后小鼠脾T细胞用培养液冲洗2次,再用DMEM培养液悬浮,按2×104/孔加入96孔U型培养板,再用RHBs(终浓度10ug/ml)体外刺激4天,结束前18h,按0.5 uCi/孔加入3H-甲基胸腺嘧啶;将细胞收集在玻璃滤膜上,用液闪记数器检测放射性,每例重复3次。

    1.2.4  T淋巴细胞特异性增殖  各组小鼠脾T淋巴细胞体外未用疫苗刺激的增殖实验cpm值为对照,各组小鼠脾T淋巴细胞体外用疫苗刺激的增殖实验cpm值为实验组小鼠脾T淋巴细胞的增殖实验cpm值,以刺激指数SI=实验组cpm值/对照组cpm值,表示T淋巴细胞特异性增殖。

    1.3  统计学方法  用t检验方法比较各组小鼠脾T淋巴细胞特异性CTL及T淋巴细胞特异性增殖实验;每个小鼠脾T淋巴细胞特异性CTL及T淋巴细胞特异性增殖之间的关系用精确Fisher检验;P<0.05为差异有显著性。

    2  结果

    2.1  特异性CTL反应  0.625、1.25、2.5、5组小鼠CTL特异性释放率分别为:42.36%±19.32%、91.21%±22.97%、69.25%±24.13%、51.49%±21.661%,均高于0组小鼠CTL释放率31.21%±9.23% (对照),差异均有显著性(P<0.05)。

    2.2  T淋巴细胞特异性增殖实验  见表1。表1  T淋巴细胞特异性增殖实验 体外用疫苗刺激的增殖实验cpm值(实验组)注:*P<0.05

    2.3  异性CTL反应与T淋巴细胞特异性增殖实验的关系  见表2。表2   T淋巴细胞特异性增殖实验与CTL反应之间的关系注:χ2=5.6979,  P<0.05

    CTL反应对照组(0组)小鼠CTL释放率的平均数±1.96倍标准差(95%可信区间)55%,以此为标准,小于55%为CTL阴性、大于55%为CTL阳性;以增殖实验对照组(未用疫苗体外刺激)小鼠增殖实验cpm值的平均数±1.96倍标准差(95%可信区间)为标准,大于其的为T淋巴细胞特异性增殖实验阳性,小于其的为T淋巴细胞特异性增殖实验阴性。我们的研究显示,免疫接种RHBs疫苗的小鼠产生的特异性CTL与T淋巴细胞特异性增殖实验有非常密切的内在联系(χ2=5.6979,  P<0.05)。

    3  讨论

    笔者研究发现:(1)各实验组特异性CTL释放率均显著高于作为对照的0组小鼠CTL释放率(31.21%±9.23%),差异有显著性(P<0.05),而以1.25组的最大,0.65组的最小。(2)各组未用疫苗体外刺激(对照组)的淋巴细胞增殖实验cpm值分别为(平均数±标准差):1500.53±307.76、1420.50±485.31、1699.83±891.45、688.67±244.27; 0.65、1.25、2.5、5组用疫苗体外刺激(实验组)的特异性淋巴细胞增殖实验cpm值分别为(平均数±标准差):3830.24±1496.12、2736.19±1238.06、4100.37±1723.74、1246.18±1088.88,2.5组的最大,5组的最小,均高于相应对照组cpm值的平均数±1.96倍标准差(95%可信区间),差异有显著性(P<0.05)。(3) 以作为CTL反应对照的0组小鼠CTL释放率的平均数±1.96倍标准差(95%可信区间, 55%)为标准,小于55%为特异性CTL反应阴性、大于55%为阳性;以未用疫苗体外刺激的淋巴细胞增殖实验的平均数±1.96倍标准差(95%可信区间)为标准,大于其的为淋巴细胞特异性增殖实验阳性,小于其的为淋巴细胞特异性增殖实验阴性,发现特异性CTL反应与淋巴细胞特异性增殖实验明显相关,差异有显著性(χ2=5.6979,  P<0.05 by exact Fisher test)。

    乙肝病毒(Hepatitis B Virus, HBV)感染是一种危害人类健康的常见病、多发病,全世界HBV感染者有3亿人。接种HBV疫苗产生的特异性体液免疫反应对预防HBV感染有重要意义,而特异性细胞免疫反应在清除体内已有的HBV、治疗HBV感染过程中起着重要作用:人体感染HBV后产生的多种抗HBV抗体,并不能清除HBV,但国外研究发现,感染HBV后呈现急性、自限性的患者,体内存在较强的抗HBV特异性细胞免疫反应,而感染HBV后呈现慢性、迁延性的患者,其抗HBV特异性细胞免疫反应缺失或显著降低,表明特异性细胞免疫反应在清除体内已有的HBV、治疗HBV感染过程中起着非常重要的作用。接种HBV疫苗能够预防HBV感染,但能否治疗已经感染的HBV,许多人心存疑虑。笔者的研究显示,加大接种剂量可以产生明显的细胞免疫反应-特异性CTL反应及T淋巴细胞增殖反应,而这两种细胞免疫反应在机体清除HBV过程中其着极其重要的作用-直接杀伤感染HBV肝脏细胞(融细胞作用)、通过分泌细胞因子调节机体免疫反应清除HBV(非融细胞作用)。最近国外的研究已经确定了RHBs的CTL表位在28~39号氨基酸位置上,而且在不同的HBV病毒株之间高度保守,能够被HLA-A2.1型的急性、自限性HBV患者的CTL及MHC-II-2d小鼠的CTL识别;而-HBsAg 21-40, 136-155, 156-175, 211-226号氨基酸序列为PROLIFERATIVE ASSAY表位,是接种RHBs后产生淋巴细胞增殖反应、CTL反应、体液免疫反应所必需的。CTL通过MHC-I限制的“内原性途径”识别MHC-I沟槽内的靶抗原多肽,或在PROLIFERATIVE ASSAY帮助下通过MHC-II限制的“外原性途径”识别靶抗原,直接杀伤、破坏感染HBV的靶细胞(融细胞作用),或通过分泌细胞因子清除HBV,而不破坏感染HBV的细胞(非融细胞作用)。有效的抗HBV免疫反应需要PROLIFERATIVE ASSAY、T淋巴细胞、B淋巴细胞互相协同[18~32]。

    我们使用的Merck & Co.公司出产的RHBs,是至今为止应用最广泛、免疫原性最强、效果最好的预防HBV感染的疫苗。人与小鼠,接种RHBs疫苗产生的免疫反应均受MHC-I限制,识别的RHBs疫苗的免疫表位位点相似。 HBV慢性肝炎T淋巴细胞致病机制的小鼠模型,国外已构建成功,不论是感染表达HBV表面抗原S疫苗的重组病毒的小鼠,还是感染HBV的人,均产生特异性、MHC-I限制性CTL反应及辅助T淋巴细胞(helper T lymphocyte, PROLIFERATIVE ASSAY)介导的淋巴细胞增殖反应[5~17]。

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作者单位:510602 广东广州,解放军第458医院药剂科

作者: 谭昌成,楼陆军 2008-7-4
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