【作者】 任翔；

【导师】 胡云章； 胡凝珠；

【作者基本信息】 中国协和医科大学 ， 生物化学和分子生物学， 2007， 硕士

【摘要】 皮肤是人体的最大的组织和器官，它的表面积接近2m~2。皮肤也是一个具有独特免疫功能并与全身免疫系统密切相关的组织器官。德国的医学学生Langerhans P于1868年首次发现并描述了皮肤中的抗原递呈细胞，后被命名为朗格汉斯细胞(Langerhans cell，LC)。未成熟的朗格汉斯细胞具有较强吞噬处理抗原能力，并沿淋巴管迁移至近侧引流淋巴结(draining lymph nodes，DLN)的T细胞区并分化成熟而具有很强的抗原递呈(包括交叉递呈)能力。透皮免疫(Transcutaneous immunization，TCI)是一种新的疫苗接种方法。它通过在皮肤表面局部应用免疫佐剂和疫苗抗原来使机体产生全身性免疫反应。通过透皮剂等技术手段，使疫苗抗原进入表皮中并被表皮朗格汉斯细胞吞噬，在佐剂的作用下，迅速引发全身性的免疫反应，从而达到免疫效果。2004年Karande等发现了透皮剂聚氧乙烯十二烷基醚硫酸钠(sodium laureth sulfate，SLA)+苯基哌嗪(phenyl piperazine，PP)的组合，该组合能够有效促进分子量小于1～10kD的生物大分子的透皮，并不诱导皮肤损伤。SLA+PP的组合能够在离体的皮肤中最大限度的促进肝素、促黄体(生成)激素-释放激素等生物大分子的透皮；在小鼠皮肤上的实验也证实醋酸亮丙瑞林能够在促透剂的作用下进入小鼠体内。在研究人类单纯疱疹病毒的治疗时发现imiquimod具有很强的抗病毒和抗肿瘤活性，美国3M公司在1997年将其上市，制成5％的外用软膏剂(商品名Aldara)。咪喹莫特可以刺激单核细胞、巨噬细胞、树突状细胞产生以干扰素IFN-α、肿瘤坏死因子TNF-α、白介素IL-1、6、8、10、12为代表的多种细胞因子。我们用咪喹莫特免疫刺激特性作为透皮佐剂，研究亚单位疫苗、病毒疫苗、DNA疫苗三种疫苗的透皮免疫效果。聚氧乙烯十二烷基醚硫酸钠和苯基哌嗪混合之后溶于等体积PBS缓冲液和无水乙醇混合液中，制成透皮剂。将其分别与HBV亚单位抗原、HBsAg DNA疫苗、灭活的HAV抗原混合后，涂抹于小鼠脱毛的背部皮肤表面，同时涂抹咪喹莫特作为佐剂，分别在2、4、6、8、12、20周用ELISA法检测血清中的抗体水平。在透皮免疫2周后即可在用咪喹莫特作为佐剂的HBV亚单位抗原组和DNA疫苗组中用ELISA法检测到血清中的抗体，平均分别为1：19.1和1：30.3；抗体滴度在8周达到最高水平，平均分别为1：105.6和1：105.6；无佐剂组在4周才检测到抗体，抗体滴度平均为1：3.31和1：1.82。结论：咪喹莫特能高效迅速诱导HBV亚单位抗原和DNA疫苗透皮免疫的抗体产生。更多还原

【Abstract】 Human’s skin has a large surface area. It is the largest tissue and organ of person.Skin has been regarded as a organ that has peculiar immune function and keeps closecorrelation with immunity system.Langerhans cells are paradigmatic dendriticcells, described in 1868 by a youngmedical student, Paul Langerhans in Berlin. Langerhans cells are present withepithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge,Langerhans cells migrate into the T cell areas of proximal lymph nodes where they actas professional antigen-presenting cells.Transcutaneous immunization (TCI) is a novel strategy for administering adjuvantand antigen to the skin surface. The adjuvant and antigen applied using TCIapparently target Langerhans cells in the skin, eliciting systemic antibodies, includingrobust mucosal IgG and secretory IgA responses in both mice and humans andprotective immunity against mucosal challenge with toxin or live virus.Karande describe particular mixtures of penetration enhancers that increase skinpermeability to macromolecules (＜1-10 kDa) by up to＜100-fold without inducingskin irritation. In vitro experiments demonstrated that the mixtures deliveredmacromolecular drugs, including heparin, leutinizing hormone releasing hormone(LHRH) and oligonulceotides across the skin. In vivo experiments on hairless ratswith leuprolide acetate confirmed the potency and safety of one such mixture, sodiumlaureth sulfate (SLA) and phenylpiperazine (PP).Imiquimod, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (also knownas R-837 and S-26308), is a nonnucleoside heterocyclic amine that belongs to a classof products known as immune-response modifiers. In vivo studies have demonstratedthat imiquimod is a potent inducer of alpha IFN(IFN-a), tumor necrosis factor alpha(TNF-a), and interleukin-1,6,8,10,12 (IL-1,6,8,10,12) and that it has adjuvantproperties greater than those of Freund’s complete adjuvant. In animal models,imiquimod has been demonstrated to have potent antiviral and antitumor effects.We use the imiquimod as adjuvant to investigate the adjuvantal effect of imiquimod on transcutaneous immunization for subunit antigen, virus antigen and DNA vaccine.SLA: PP (weight fractions,0.7:0.3;total concentration of 0.5%(wt/vol)) dissolves in1:1 phosphate buffered saline(PBS):ethanol. This solution admixes with HBV subunitantigen, HAV antigen and HBsAg DNA vaccine distinctively. The admixtures werebesmeared on the depilated mouse back and then the adjuvant imiquimod wasbesmeared on the back. At 2, 4, 6, 8, 12, 20 weeks post inoculation. The specificHBsAg antibody were detected early at two weeks after transcutaneous immunization.The titers reached the highest level at 8 weeks. The array without imiquimod coulddetect antibody only at 4 weeks post immunization.Conclusion The adjuvant imiquimod can enhance specific antibody levelsignificantly and quickly for HBsAg and HBsAg DNA vaccine.更多还原