双歧杆菌完整肽聚糖对重组乙肝表面抗原佐剂效应的实验研究

发布时间：2018-04-02 22:05

本文选题：完整肽聚糖　切入点：重组乙肝表面抗原　出处：《重庆医科大学》2005年硕士论文

【摘要】：双歧杆菌完整肽聚糖有很强的免疫增强活性,但它在机体对弱免疫原重组乙肝表面抗原的免疫应答中的作用尚不清楚。本课题旨在初步探讨双歧杆菌完整肽聚糖对重组乙肝表面抗原的佐剂效应及其相关机制。实验使用两歧双歧杆菌分离提纯完整肽聚糖作为佐剂,采用 PBS 液混合、戊二醛或二磷酸腺苷交联来吸附或结合重组乙肝表面抗原,并检测其对抗原的吸附情况;制备免疫原,免疫 BALB/c 小鼠,观察其对小鼠的毒副作用以及对胸腺和脾脏发育的影响;ELISA 法检测小鼠血清的特异性抗体,LDH 释放法检测脾脏 NK细胞和 CTL 细胞体外杀伤活性,MTT 法检测脾脏淋巴细胞体外增殖能力,ELISA法检测脾脏淋巴细胞体外诱生的细胞因子。结果显示,双歧杆菌完整肽聚糖以 PBS 液混合、戊二醛交联或二磷酸腺苷交联等方式均能吸附重组乙肝表面抗原结果,蛋白质交联剂的促进作用并不明显;免疫 BALB/c 小鼠后,未见毒副反应出现,生长情况正常,体重增长平稳,胸腺和脾脏明显增重;免疫 8 周后,小鼠血清抗-HBs 阳性率提高和抗体滴度均比铝佐剂提高,抗体中 IgG2a 亚型的滴度也明显上升;脾脏淋巴细胞体外增殖能力增强,体外诱生细胞因子的能力得到改善,NK 细胞和抗原特异性 CTL 细胞杀伤活性提高。实验表明,双歧杆菌完整肽聚糖在某些缓冲盐溶液中可以吸附一定量的重组乙肝表面抗原;作为佐剂免疫 BALB/c 小鼠后,不出现毒副作用。双歧杆菌完整肽聚糖能够通过缓解胸腺衰老和增加脾重以维持淋巴器官功能,并通过增强脾脏淋巴细胞体外增殖能力和诱生细胞因子能力调节机体免疫应答中 Th1/Th2 的平衡,增强 NK 细胞和 CTL 细胞杀伤活性以增强机体对病毒感染细胞的清除能力,在获得足够保护性抗体的同时产生适当细胞免疫应答,展现出良好的免疫佐剂品质。
[Abstract]:Bifidobacterium peptidoglycan has a strong immune enhancement activity, but its role in the immune response to weak immunogen recombinant hepatitis B surface antigen is unclear.The aim of this study was to investigate the adjuvant effect of complete peptidoglycan of Bifidobacterium on recombinant hepatitis B surface antigen and its related mechanism.The whole peptidoglycan was isolated and purified by bifidobacterium bifidobacterium as adjuvant. The recombinant hepatitis B surface antigen was adsorbed or combined with glutaraldehyde or adenosine diphosphate crosslinking with PBS solution.BALB/c mice were immunized,To observe its toxicity and side effects on the development of thymus and spleen in mice; to detect the activity of splenic NK cells and CTL cells in vitro by Elisa and to detect the lymphatic fineness of spleen by using Elisa method to detect the release of specific antibody against LDH and to detect the cytotoxicity of splenic NK cells and CTL cells in vitro.In vitro proliferative ability and Elisa were used to detect cytokines induced by splenic lymphocytes in vitro.The results showed that the complete peptidoglycan of Bifidobacterium could adsorb recombinant hepatitis B surface antigen by PBS solution, glutaraldehyde crosslinking or adenosine diphosphate crosslinking, but the effect of protein crosslinking agent was not obvious.There were no side effects, normal growth, steady weight gain, significant weight gain in thymus and spleen, and increased anti-HBs positive rate and antibody titer in serum of mice after 8 weeks of immunization compared with aluminum adjuvant.The titer of IgG2a subtype in antibody also increased obviously, the proliferation ability of splenic lymphocytes in vitro was enhanced, the ability of inducing cytokines in vitro was improved, and the killing activity of NK cells and antigen-specific CTL cells was improved.The results showed that Bifidobacterium peptidoglycan adsorb a certain amount of recombinant hepatitis B surface antigen in some buffer salt solution, and no side effects occurred after immunizing BALB/c mice as adjuvant.Bifidobacterium peptidoglycan can maintain lymphoid organ function by relieving thymus aging and increasing spleen weight, and regulate the balance of Th1/Th2 in immune response by enhancing the proliferation and inducing cytokine ability of splenic lymphocytes in vitro.To enhance the killing activity of NK cells and CTL cells in order to enhance the ability of the body to clear the virus infected cells, and to produce appropriate cellular immune response while obtaining sufficient protective antibodies, showing a good quality of immune adjuvant.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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