咪喹莫特作为佐剂的透皮免疫效果研究

发布时间：2018-04-02 05:02

本文选题：咪喹莫特　切入点：疫苗　出处：《中国协和医科大学》2007年硕士论文

【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。
[Abstract]:Skin is the largest tissue and organ of the human body, and its surface area is close to 2 mm2. The skin is also a tissue and organ with unique immune function and closely related to the systemic immune system. Langerhans P, a German medical student, first discovered and described antigen-presenting cells in the skin in 1868, and was later named Langerhans cells. The immature Langerhans cells had a strong phagocytic ability to process antigens. Along the lymphatic vessels, they migrated to the T cell region of the proximal draining lymph nodes-DLNs and differentiated and matured and had strong antigen-presenting (including cross-presentation) capacity. Transdermal immunity (TCI) is a new vaccination method. It can induce systemic immune response by local application of immune adjuvant and vaccine antigen on skin surface. The vaccine antigen enters the epidermis and is swallowed by the Langerhans cells of the epidermis. With the action of adjuvant, the immune response of the whole body is triggered quickly, and the immune effect is achieved. In 2004, Karande et al discovered the combination of sodium laureth sulfate (sodium laureth sulfate) phenyl piperazine (PPP), which can effectively promote the transdermal penetration of biological macromolecules with molecular weight less than 1~10kD. The combination of SLAPP can promote the transdermal penetration of heparin, luteinizing hormone (luteinizing) hormone and other biological macromolecules in vitro. Experiments on the skin of mice also confirmed that Leuprilline acetate was able to enter the mice under the action of a penetration enhancer. When studying the treatment of human herpes simplex virus (HSV), we found that imiquimod has strong antiviral and antitumor activity. Us 3M Company went on the market in 1997 and made 5% external ointment (Aldaraan.imiquimod) to stimulate monocytes. Macrophages, dendritic cells produce a variety of cytokines represented by IFN- 伪, TNF- 伪, IL-1TNF- 伪, IL-1TNF- 伪 and IL-1. We use the immunostimulatory properties of Imiquimod as transdermal adjuvants to study subunit vaccines. The transdermal immune effect of three kinds of virus vaccine and DNA vaccine. Sodium polyoxyethylene dodecyl ether sulfate and phenylpiperazine were dissolved in the mixture of PBS buffer and anhydrous ethanol to prepare transdermal agent. The transdermal agent was mixed with HBV subunit antigen, DNA vaccine, and inactivated HAV antigen. Apply to the skin surface of the mouse's back with imiquimod as adjuvant. The antibody levels in serum were detected by ELISA method at the week of 20 weeks, respectively. After 2 weeks of transdermal immunization, the antibodies in serum were detected by ELISA method in the HBV subunit antigen group and DNA vaccine group with imiquimod as adjuvant. The antibody titer reached its highest level at 8 weeks, with an average of 1: 105.6 and 1: 105.6, respectively; the adjuvant group did not detect antibodies until 4 weeks. The average titer of antibody was 1: 3.31 and 1: 1.82.Conclusion: Imiquimod can efficiently and rapidly induce the production of HBV subunit antigen and DNA vaccine transdermal antibody.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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