医学检验信息网 检验医学 2007-2-11 13:02:21

【摘要】 目的 研究乙型肝炎核酸疫苗肌注小鼠后在体内的表达及小鼠体液免疫应答的规律，同时观察接种局部组织的病理变化。方法 以乙型肝炎核酸疫苗NV-HB/s肌注小鼠，然后定期分批处死，采集肌注局部组织检测HBsAg(ABC免疫组化法)，采集外周血标本检测HBsAg和抗-HBs(ELISA法),并常规病理检查。结果 接种乙型肝炎核酸疫苗后1周，HBsAg的表达即可在部分受检小鼠的肌注局部组织内检出；接种后2周，全部受检小鼠的肌注局部组织HBsAg检测均呈阳性，部分受检小鼠的外周血抗-HBs亦阳转，此后检出率进一步增高。接种后1个月，受检小鼠抗-HBs即全部阳转并持续到接种后半年以上；同期间内，至少75%的受检小鼠其肌注部位组织仍保持HBsAg检出阳性，而其外周血标本内的HBsAg检测则始终呈阴性。病理检查提示：核酸疫苗接种后，仅在肌注局部出现一过性非特异性炎症反应。结论 乙型肝炎核酸疫苗NV-HB/s肌注接种小鼠后，可通过在注射部位组织中表达HBsAg，在其体内诱生特异性抗-HBs。 HBsAg expression, anti-HBs induction and pathological observation in the mice inoculated with DNA vaccine against Hepatitis B ZHAO Liansan, QIN Shan, TANG Hong, et al. Sichuan Provincial Key Laloratory of Mdecular Biology of Infectious Diseases, First Affiliatod Hospital of Xuasi Medical University, Chengdu 610041 【Abstract】 Objective To study if special humoral immunological response can be induced by DNA vaccine against Hepatitis B (NV-HB/s) and its mechanism.Medthods The Balb/C mice inoculated with NV-HB/s by intramuscular injection were detected for HBsAg(the expressed product of NV-HB/s)by ABC immunohistochemistry or ELISA, and for anti-HBs by ELISA and also performed the routine pathological examination.Results After inoculated with NV-HB/s, HBsAg could be detected from mice muscle tissue samples of injected sites one week later and almost kept positive(75% and even more) up to 6^th month, but was undetectable from all the serum samples during this period; anti-HBs could be detected from serum samples of some mice two weeks later and of all the mice detected one month later, it still kept positive at 6^th month while the mice were sacrificed. Under microscope, only non-specific inflammation was found in the muscle tissues of injected sites and could completely recover within 4 weeks, no matter whether the mice were inoculated with NV-HBs, or with traditional HB vaccine derived from blood, or even with PBS as control.Conclusion DNA vaccine against Hepatitis B (NV-HB/s) can successfully express HBsAg in the muscle tissue of mice by intramuscular inoculation and subseguently induce anti-HBs in viro. 【key words】 DNA vaccine Hepatitis B HBsAg Antibody 乙型肝炎病毒(HBV)感染可导致肝硬化、甚至肝癌，危害十分严重。目前尚无满意的抗病毒治疗手段。作者等曾报告用乙型肝炎核酸疫苗NV-HBs免疫小鼠，诱导出了特异性体液免疫应答，使小鼠外周血抗HBs阳转^［1］。为了探讨核酸疫苗的作用机理，我们进一步研究了肌注乙型肝炎核酸疫苗NV-HB/s在小鼠体内表达HBsAg的规律及NV-HB/s肌注部位的病理变化特点。 1 材料和方法

1.1 乙型肝炎核酸疫苗NV-HB/s的制备 将乙型肝炎病毒表面抗原(HBsAg)主蛋白编码基因S插入真核细胞表达质粒pRc/CMV/ATG中的CMV启动子下游，以此重组质粒转染真核细胞，可高效表达HBsAg主蛋白；经常规质粒扩增、提纯后制得重组质粒DNA纯品，以1 mg/ml的浓度溶于PBS(pH7.2)，即为乙型肝炎核酸疫苗NV-HB/s。

1.2 小鼠接种 接种用Balb/C小鼠均为6～8周龄，清洁级，随机分为实验组和对照组。实验组32只小鼠，每只小鼠两侧胫前肌处肌注NV-HB/s，接种剂量为每只100 μg/100 μl，两侧各半量。设3个对照组，每组4只小鼠：空白对照组，用等体积的PBS(pH7.2)代替NV-HB/s肌注小鼠；阴性对照组，用等量空载质粒pRc/CMV/ATG代替NV-HB/s肌注小鼠；阳性对照组，用血源乙型肝炎疫苗(成都生物制品研究所产)肌注小鼠，1 μg/只。

1.3 表达产物的检测 将实验组小鼠随机于接种后第3天、1周、2周、1月、2月、4月、6月分批采集眶静脉血，分离血清置-70℃冻存备检：同时处死小鼠，解剖检查其内脏器官，并分离出接种部位胫前肌标本，经常规福尔马林液固定后、石蜡包埋。待所有标本收集完成后，在同一条件下，用ELISA法检测血清内的抗-HBs及HBsAg(检测试剂盒均购自华美生物工程公司)；用ABC免疫组化法检测石蜡包埋组织切片内的HBsAg(检测试剂盒购自美国VECTOR公司)，操作程序参照文献［2］进行；此外，常规H.E.染色上述组织切片标本，显微镜下观察其肌注局部组织内的病理变化。

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