巨细胞病毒核酸疫苗辅以分子佐剂的免疫效果研究

发布时间：2018-03-21 04:02

本文选题：巨细胞病毒　切入点：核酸疫苗　出处：《湖南师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：本论文主要由三个部分组成：(一)巨细胞病毒及其疫苗和佐剂的概述；(二)巨细胞病毒M84核酸疫苗辅以分子佐剂HMGB1与IL-18免疫效力的研究；(三)巨细胞病毒IE1核酸疫苗辅以分子佐剂IL-12在正常小鼠以及糖尿病小鼠中的免疫效力的研究。巨细胞病毒(cytomegalovirus; CMV)普遍存在于自然界,属于p疱疹病毒亚科,并具有严格的种属特异性。人类巨细胞病毒(HCMV)在人群中普遍感染,可在新生儿及免疫缺陷个体中引起严重疾病。到目前为止还没有特效药能够很好的治疗人巨细胞病毒感染,因此接种巨细胞病毒疫苗来防治该病毒感染与传播变得尤为重要。虽然人巨细胞病毒疫苗的临床研究已经进行了近30年,但是至今还没有一种巨细胞病毒疫苗获准上市。美国已在2001年将研发CMV疫苗列为优先发展项目。由于巨细胞病毒具有严格的种属特异性,因此临床前实验阶段的CMV疫苗都是在动物模型上面进行,本实验室选用MCMV小鼠模型来研究巨细胞病毒疫苗及其佐剂的免疫效价。高迁移率族蛋白1(high mobility group box-1 protein, HMGB1)是近几年来发现的一种新型细胞因子,它主要由巨噬细胞,单核细胞,垂体细胞分泌,HMGB1能够介导细胞的迁移和分化,促进树突状细胞表达CD40、CD54、CD58等,这表明HMGB1可以促进免疫反应。HMGB1还能提高IL-2的转录水平；白介素18(interleukin 18)是发现较早的一种细胞因子,它能够诱导细胞产生IFN-γ、GM-CSF, IL-2及TNF-α等其他TH1型细胞因子,从而促进免疫反应；白介素12(interleukin 12)主要和细胞免疫应答有关,是免疫过程中重要的调节因子,重组IL-12已被证明是一种很好的免疫佐剂。本论文首先选取鼠巨细胞病毒M84核酸疫苗作为抗原,以HMGB1和IL-18作为分子佐剂共同免疫小鼠。免疫之后四周用致死剂量的病毒攻击小鼠,通过存活率,体重丢失率以及攻毒5天后小鼠脾脏中的病毒滴度去衡量疫苗和佐剂的免疫效价。实验结果表明,M84辅以分子佐剂IL-18与HMGB1共同免疫小鼠后能够显著的提高疫苗的保护率,而且IL-18的效果好于HMGB1。糖尿病是由各种因素作用于机体导致胰岛功能减退而引发的代谢紊乱综合征。糖尿病人的自身免疫系统会出现不同程度降低因此对各种病毒易感。我们选取鼠巨细胞病毒IEl核酸疫苗作为抗原,以IL-12的真核表达质粒作为分子佐剂,分别免疫健康小鼠与糖尿病小鼠。结果显示,加了佐剂可以增强疫苗的细胞应答水平,而且同剂量的疫苗在健康小鼠中引起的细胞免疫应答水平较糖尿病小鼠要高,说明与健康小鼠相比,糖尿病小鼠的免疫应答能力较弱。用致死剂量病毒攻击小鼠后,结果显示,加了佐剂的免疫组比IE1单独免疫组的保护效果要好,在正常小鼠中加入10μg IL-12就能够起到很好的保护效果,而在糖尿病小鼠中需要100μgIL-12配合疫苗共同免疫才能显著提高保护率,这说明糖尿病小鼠需要较多的佐剂剂量才能达到较好的保护效果。
[Abstract]:This paper consists of three parts: (a) an overview of cytomegalovirus and vaccine and adjuvant; (two) study of cytomegalovirus M84 molecules with DNA vaccine adjuvant HMGB1 and IL-18 immune effect; (three) immune efficacy of cytomegalovirus IE1 molecules with DNA vaccine adjuvant of IL-12 in normal mice and diabetic mice.. cytomegalovirus (cytomegalovirus; CMV) exists widely in nature, belongs to the P subfamily of herpes simplex virus, and has a strict species specificity. Human cytomegalovirus (HCMV) infection in the general population, can cause severe disease in neonates and immunocompromised individuals. So far there is no drug can be a good treatment of people cytomegalovirus infection, so vaccination cytomegalovirus vaccine to prevent the virus infection and spread has become particularly important. Although clinical study on human cytomegalovirus vaccine After nearly 30 years, but still does not have a cytomegalovirus vaccine approved. The United States in 2001 will be CMV vaccine as a priority development projects. Since the virus has strict species specificity, so pre clinical experimental stage CMV vaccine were carried out in the animal model, the titer of MCMV mice the laboratory model is chosen to study the CMV vaccine and adjuvant. High mobility group protein 1 (high mobility group box-1 protein, HMGB1) is a new cytokine found in recent years, which is mainly composed of macrophages, mononuclear cells, secretion, migration and differentiation of HMGB1 can mediate cells, dendritic cells promote the expression of CD40, CD54, CD58 etc., which indicates that HMGB1 can promote the immune response of.HMGB1 can enhance the transcriptional level of IL-2; interleukin 18 (interleukin 18) are found in an earlier Cytokine, which can induce IFN- cells to produce GM-CSF, IL-2 gamma, TNF- alpha and other TH1 cytokines, thereby promoting immune response; interleukin 12 (interleukin 12) is mainly concerned with cellular immune response, is an important factor in immune regulation, recombinant IL-12 has been proved to be a good immune this paper first selected adjuvant. Murine cytomegalovirus DNA vaccine M84 as antigen, HMGB1 and IL-18 as molecular adjuvant to immune mice. Four weeks after immunization with a lethal dose of virus attack by mice survival rate, titers of virus titer and infection rate of weight loss in the spleen 5 days in mice to measure vaccine and adjuvant. The experimental results show that M84 with molecular adjuvant IL-18 and HMGB1 co immunized mice can significantly improve the vaccine protection rate, and the effect of IL-18 is better than HMGB1. by all kinds of factors for diabetes For the metabolic disorder of islet dysfunction caused by the syndrome of diabetes patients. Cause the body's own immune system will be reduced so on various virus susceptibility. We select murine cytomegalovirus DNA vaccine IEl as antigen to IL-12 eukaryotic expression plasmid as a molecular adjuvant, were immunized healthy mice with diabetic mice. The results showed, plus adjuvant can enhance cell vaccine response level, and the same dose of vaccine in healthy mice cellular immune response caused by diabetic mice was higher than that of healthy mice and diabetic mice, the immune response is weak. With a lethal dose of virus attack mice. The results showed that with better protection effect group of immune adjuvant immunized group than IE1 alone, adding 10 in normal mice g IL-12 can play a good protective effect, and in diabetes In mice, 100 micron gIL-12 vaccine combined with vaccine can significantly improve the protection rate. This indicates that diabetic mice need more adjuvant dose to achieve better protection effect.

【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392-33

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