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
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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﹤0.01),CD8~+T细胞无明显变化(P﹥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(P<0.01),而rEg.mMDH无明显作用(P﹥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。
Objective Through recombinant ferritin on immune protection and do not have protective immunity of recombinant mMDH comparative analysis of proteins, To find out, mechanism of immune protection caused by vaccine immune and mechanism of Ec hinococcus granulosus protoscolex in mice, provide theoretical basis for wider use of vaccines. Methods (1) ICR mice(female)were divided into three groups with random process;(2) All the mice of experimental group were vaccinated subcutaneously on the back with 10μl antigen dissolved in phosphate-buffered saline(PBS)and emulsified with 50ul Freund’s complete adjuvant(FCA)respectively in the first time,two weeks after the first vaccination with the same preparation except that FCA was replaced by Freund’s incomplete adjvant(FIA),all mice were vaccinated thirdly, the same as the second time with an interval week,PBS group did merely with PBS 100ul each time,the process was as before. (3) All the mice were challenged with 1500 protoscolices after vaccinating thirdly about 2 weeks; (4) Attack after 20 weeks of infection after cesarean section to kill mice, statistical number of cysts in mice peritoneal and diameter, calculation of immune protection;(5) Profile control and kill the mice, aseptic preparation of spleen, separation of spleen cells;(6) Spleen cells isolated, Application of FCM for detecting percentage of T-cell CD4~+, and CD8~+ change;(7) MTT assay for detection of splenic lymphocyte proliferation;(8) Obtain data on immune protection and do not have protective immunity of two group comparisons, analysis of inherent mechanism.Result (1)Using Dempster theory: rEg.ferritin can induce 86.37% immune protective efficiency in mice against the challenge infection, However rEg.mMDH Antigen immune ICR mice did not get immune protection,it indicated that rEg.ferritin has the potential of vaccines, rEg.mMDH is not.(2)①Immunity of mice with recombinant ferritin before the generation kill, CD4~+T cell increase(P﹤0.01), no obvious changes in CD8~+T cells(P﹥0.05), spleen lymphocyte subsets of mice with rEg.mMDH compared with control group, no significant change,it indicated that protective mechanism relate with participation of CD4~+T cells, role of CD8~+T cells are not significant;②As demonstrated by MTT assay,the proliferation activity of splenic T lymphocyte in rEg.ferritin group was higher than that in the control group(P﹤0.01), rEg.mMDH is not(P﹥0.05).It suggested that mechanisms of rEg.ferritin-induced mice relate with proliferation of splenic lymphocytes.Conclusions Cell immunity protective mechanism of rEg.ferritin induced mice may be associated with activated CD4~+T cell and stimulated proliferation of splenic lymphocytes.rEg.mMDH antigen non- immune protective mechanism may be associated with non-activated CD4~+T cell and non-stimulated proliferation of splenic lymphocytes.
Objective Through recombinant ferritin on immune protection and do not have protective immunity of recombinant mMDH comparative analysis of proteins, To find out, mechanism of immune protection caused by vaccine immune and mechanism of Ec hinococcus granulosus protoscolex in mice, provide theoretical basis for wider use of vaccines. Methods (1) ICR mice(female)were divided into three groups with random process;(2) All the mice of experimental group were vaccinated subcutaneously on the back with 10μl antigen dissolved in phosphate-buffered saline(PBS)and emulsified with 50ul Freund’s complete adjuvant(FCA)respectively in the first time,two weeks after the first vaccination with the same preparation except that FCA was replaced by Freund’s incomplete adjvant(FIA),all mice were vaccinated thirdly, the same as the second time with an interval week,PBS group did merely with PBS 100ul each time,the process was as before. (3) All the mice were challenged with 1500 protoscolices after vaccinating thirdly about 2 weeks; (4) Attack after 20 weeks of infection after cesarean section to kill mice, statistical number of cysts in mice peritoneal and diameter, calculation of immune protection;(5) Profile control and kill the mice, aseptic preparation of spleen, separation of spleen cells;(6) Spleen cells isolated, Application of FCM for detecting percentage of T-cell CD4~+, and CD8~+ change;(7) MTT assay for detection of splenic lymphocyte proliferation;(8) Obtain data on immune protection and do not have protective immunity of two group comparisons, analysis of inherent mechanism.Result (1)Using Dempster theory: rEg.ferritin can induce 86.37% immune protective efficiency in mice against the challenge infection, However rEg.mMDH Antigen immune ICR mice did not get immune protection,it indicated that rEg.ferritin has the potential of vaccines, rEg.mMDH is not.(2)①Immunity of mice with recombinant ferritin before the generation kill, CD4~+T cell increase(P﹤0.01), no obvious changes in CD8~+T cells(P﹥0.05), spleen lymphocyte subsets of mice with rEg.mMDH compared with control group, no significant change,it indicated that protective mechanism relate with participation of CD4~+T cells, role of CD8~+T cells are not significant;②As demonstrated by MTT assay,the proliferation activity of splenic T lymphocyte in rEg.ferritin group was higher than that in the control group(P﹤0.01), rEg.mMDH is not(P﹥0.05).It suggested that mechanisms of rEg.ferritin-induced mice relate with proliferation of splenic lymphocytes.Conclusions Cell immunity protective mechanism of rEg.ferritin induced mice may be associated with activated CD4~+T cell and stimulated proliferation of splenic lymphocytes.rEg.mMDH antigen non- immune protective mechanism may be associated with non-activated CD4~+T cell and non-stimulated proliferation of splenic lymphocytes.
引文
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