细粒棘球蚴重组铁蛋白和重组mMDH的免疫保护性差异机制的研究

发布时间：2018-01-06 08:20

本文关键词：细粒棘球蚴重组铁蛋白和重组mMDH的免疫保护性差异机制的研究　出处：《宁夏医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的通过对具有免疫保护力的重组铁蛋白和不具有免疫保护力的重组mMDH蛋白的比较分析,从而找出小鼠被疫苗免疫后产生免疫保护和抗细粒棘球蚴的机制,为疫苗的广泛应用提供理论依据。方法(1)实验动物分组:选取雌性ICR小鼠随机分为3组:rEg.ferritin免疫组、rEg.mMDH免疫组和对照组;(2)动物免疫:rEg.ferritin和rEg.mMDH免疫组小鼠分别注射10μg重组抗原+佐剂+PBS,100μl,共三次,每次间隔两周,对照组注射100μl PBS;(3)动物攻击感染:第3次免疫后2周,各组均以1500个活的原头蚴/每只小鼠攻击感染;(4)免疫保护力观察:攻击感染20周后剖杀小鼠,统计小鼠腹腔内包囊数量和直径,计算免疫保护力。(5)脾细胞悬液的制备:剖杀小鼠,无菌取脾,分离脾细胞;(6)用分离得到的脾细胞,应用FCM检测CD4~+和CD8~+T细胞亚群的百分比变化。(7)用MTT法检测脾淋巴细胞增殖情况。(8)获得数据后对具有免疫保护力和不具有免疫保护力的两组进行对照比较,分析其内在机制。结果(1)根据Dempster公式计算:rEg.ferritin抗原免疫ICR小鼠可获得86.37%的免疫保护力,而rEg.mMDH抗原免疫ICR小鼠未获得免疫保护力,提示rEg.ferritin具有疫苗的潜质,而rEg.mMDH则无。(2)免疫组与对照组经统计学检验比较:①r Eg.ferritin免疫组小鼠在剖杀前CD4~+T细胞增加(P㩳0.01),CD8~+T细胞无明显变化(P㧐0.05),而rEg.mMDH免疫组小鼠脾淋巴细胞亚群与对照组相比无明显变化,提示rEg.ferritin免疫小鼠的保护机制有CD4~+T细胞的参与,CD8~+T细胞的作用不是很大;②MTT法检测证实rEg.ferritin免疫的小鼠在rEg.ferritin刺激下脾淋巴细胞增殖水平明显高于对照组(P0.01),而rEg.mMDH无明显作用(P㧐0.05),提示rEg.ferritin抗原免疫小鼠的保护机制可能与其刺激脾淋巴细胞增殖有关。结论rEg.ferritin抗原诱导小鼠产生细胞免疫的保护机制可能与激活CD4~+T细胞,刺激脾淋巴细胞增殖有关,而rEg.mMDH抗原无免疫保护的机制可能与未激活CD4~+T细胞,未刺激脾淋巴细胞增殖有关。
[Abstract]:Objective through a comparative analysis on immune protection of the recombinant ferritin and do not have protective immunity of recombinant mMDH protein in mice, so as to find out the mechanism of immune protection caused by vaccine and anti Echinococcus, provide a theoretical basis for the widely used vaccine. Methods (1) experimental animal groups: female ICR mice were randomly selected divided into 3 groups: rEg.ferritin immune group, rEg.mMDH immune group and control group; (2) animal immunity: the rEg.ferritin and rEg.mMDH immunized mice were injected with 10 g recombinant antigen + adjuvant +PBS, 100 L, a total of three times, each time interval of two weeks, the control group was injected with 100 l PBS; (3) animal attack: third infection 2 weeks after immunization, each group are 1500 living protoscolices / per mouse against infection; (4) to observe the protective immunity against infection: 20 weeks after the mice were killed, the number and diameter of statistical intraperitoneal cyst, calculation of protective immunity (5). Spleen cell suspension was prepared, the mice were sacrificed and spleens from spleen cells; (6) with spleen cells isolated and detected by FCM CD4~+ and CD8~+T cell subsets percentage change. (7) MTT was used to detect the proliferation of spleen lymphocytes. (8) to obtain data on immune protection and two groups do not have protective immunity were compared, analysis of its internal mechanism. The results calculated according to Dempster formula (1): rEg.ferritin immunized ICR mice can get protective immunity in 86.37%, and rEg.mMDH antigen immune ICR mice did not get immune protection force, suggesting that rEg.ferritin has the potential of vaccines, and rEg.mMDH No. (2) comparison of immune group and control group were tested by the R Eg.ferritin immunized mice in killing CD4~+T cells before increased (P? 0.01), the CD8~+T cells did not change significantly (P? 0.05), and mouse spleen lymphocyte subsets and immune group of rEg.mMDH There was no significant change in the control group, suggesting that the protective mechanism in mice immunized with rEg.ferritin CD4~+T cells, not CD8~+T cells; detection of MTT showed that the rEg.ferritin immunized mice in spleen lymphocyte proliferation stimulated by rEg.ferritin was significantly higher than the control group (P0.01), and rEg.mMDH had no significant effect (P? 0.05), protection the mechanism may be related to the rEg.ferritin antigen in mice stimulated proliferation of splenic lymphocytes. The protective mechanism of immunity in mice induced by the conclusion of rEg.ferritin antigen and activation of CD4~+T cells, stimulate the proliferation of spleen lymphocytes, and rEg.mMDH antigen immune protection mechanism may not activate CD4~+T cells did not stimulate the proliferation of lymphocytes in spleen.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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