幽门螺杆菌微球疫苗制备及其免疫效应研究

发布时间：2018-03-09 02:04

  本文选题：幽门螺杆菌　切入点：MS　出处：《第三军医大学》2005年博士论文　论文类型：学位论文

【摘要】：幽门螺杆菌(Helicobacter pylori, Hp)是引起人类慢性胃炎、消化性溃疡、胃癌等的主要致病菌,现阶段在全世界人群中的感染率达50%以上,目前临床上常用的三联疗法不足以根治Hp 感染,且费用昂贵、病人依从性差、容易复发及产生耐药菌株。疫苗接种是彻底消除Hp 感染的有效措施之一,根据共同粘膜免疫系统(common mucosal immunity system, CMIS)理论及文献报道,口服途径是其中最简单、直接、安全且有效的给药方式,但必须克服消化道中大量酸性、酶类物质对抗原破坏的问题。 近年来,以微球(microsphere, MS)、微囊、脂质体等为代表的缓释、控释给药制剂研究得到迅猛发展,它不仅能耐受各种复杂生理环境,而且还具有靶向给药的特点。其靶向性决定于制剂本身粒径大小,有文献报道当粒径为lμm 至l0μm 之间的MS经口服后,既可以产生全身免疫,也可以在局部粘膜形成有效的免疫应答。另外,由于MS 具有显著的缓释特性,能够使药物在靶器官形成长时间的有效浓度,因此可以在一定程度上减少疫苗的免疫剂量及免疫次数。 Hp 疫苗免疫保护机理的深入探讨对于Hp 的有效防治及疫苗的研发会起到较大的推定作用。目前尽管对其进行了大量的研究,但其确切机制尚不清楚。本研究以Hp全菌蛋白(HpWCP)为抗原成分,主要从MS 制备工艺及特征分析、免疫效应、免疫保护及保护机理等三个方面进行了研究。 (一) HpWCP-CAMS 的制备及其表征分析 在本研究中,采用生物可降解天然高分子物质-壳聚糖(chitosan, CTS)、海藻酸钠(sodium alginate, AGS)为MS 主要制备材料,通过系统考察制备材料水溶液的流动性、粘稠度、稳定性等结果确定了AGS 乳液浓度为2%、CTS 浓度为1%、植物油与AGS乳液配比为2:8、CaCl2溶液反滴滴定法等MS 制备工艺参数及程序。在此基础上,对影响MS 粒径大小的因素进行均匀设计分析,并以确定的搅拌速率800rpm、药物浓度2mg/ml、制备温度25℃、搅拌时间30min 等参数制备出平均粒径为3.33μm 壳聚糖-海藻酸钠包裹牛血清白蛋白微球(BSA-CAMS),其平均载药量为31.5%,平均药物包封率为61.0%。电镜观察所得MS 表面光整、大小均一。体外释药实验结果显示此MS具有良好的缓释功能,最长释药时间可达到20d 以上。在人工胃液中观察其具有强耐
[Abstract]:Helicobacter pylori (HP) is the main pathogen causing chronic gastritis, peptic ulcer, gastric cancer and so on. At present, the infection rate of Helicobacter pylori is more than 50% in the world population. At present, the triple therapy commonly used in clinical practice is not enough to cure HP infection. And the cost is high, the patient compliance is poor, easy to relapse and to produce drug-resistant strains. Vaccination is one of the effective measures to completely eliminate HP infection, according to the common mucosal immune system, common mucosal immunity system (CMISS) theory and literature report. Oral administration is the most simple, direct, safe and effective way of administration, but it is necessary to overcome the problem that a large number of acidic and enzymatic substances destroy the antigen in the digestive tract. In recent years, the research of microsphere, MSG, microcapsule, liposome and controlled release drug delivery has been developed rapidly, which can not only tolerate various complex physiological environments. Moreover, it has the characteristics of targeted administration. Its targeting depends on the size of the preparation itself. It has been reported that when MS with the particle size of 1 渭 m to 1.0 渭 m is taken orally, it can produce systemic immunity. It can also form an effective immune response in the local mucous membrane. In addition, because MS has a remarkable slow-release property, it can make the effective concentration of the drug in the target organ form for a long time. Therefore, the immunization dose and times of immunization can be reduced to a certain extent. The further study on the mechanism of immunization protection of HP vaccine will play an important role in the effective prevention and treatment of HP and the research and development of the vaccine. Although a great deal of research has been done on it at present, However, the exact mechanism of HpWCPS was not clear. In this study, HpWCPs were used as antigen components, mainly from three aspects: preparation process and characteristic analysis of MS, immune effect, immune protection and protective mechanism. (1) preparation and characterization of HpWCP-CAMS. In this study, the biodegradable natural macromolecules, chitosan chitosan, CTS, alginate sodium alginate, AGSwere used as the main preparation materials of MS. The fluidity and viscosity of aqueous solution of the prepared materials were systematically investigated. The results of stability determined the process parameters and procedures of MS preparation, such as the concentration of AGS emulsion was 2 and CTS concentration was 1, the ratio of vegetable oil to AGS emulsion was 2: 8, CaCl2 solution reverse drop titration, and so on. On the basis of this, the factors affecting the size of MS particle size were designed and analyzed uniformly. The stirring rate was 800rpm, the drug concentration was 2 mg / ml, and the preparation temperature was 25 鈩，

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