西咪替丁伍用弓形虫ROP2蛋白免疫小鼠诱导免疫反应的初步研究

中国寄生虫学与寄生虫病杂志 ›› 2013, Vol. 31 ›› Issue (2): 4-99-103.

西咪替丁伍用弓形虫ROP2蛋白免疫小鼠诱导免疫反应的初步研究

陈兴智1,2 *，杨小迪1，杨雯1，陈勇1，刘丽丽2，沈继龙2，孙新1

1 蚌埠医学院病原生物学教研室，安徽省感染与免疫重点实验室，蚌埠 233030；2 安徽病原生物学省级实验室，人兽共患病安徽省重点实验室，安徽医科大学病原生物学教研室，合肥 230032

出版日期:2013-04-30 发布日期:2013-07-02

Study on Immune Response in BALB/c Mice Induced by ROP2 Protein of Toxoplasma gondii with Cimetidine

CHEN Xing-zhi1,2 *, YANG Xiao-di1, YANG Wen1, CHEN Yong1, LIU Li-li2, SHEN Ji-long2, SUN Xin1

1 Department of Microbiology and Parasitology，Bengbu Medical College；Anhui Key Laboratory of Infection and Immunity，Bengbu 233030，China；2 Provincial Laboratory of Microbiology & Parasitology and Key Laboratory of Zoonoses；Department of Parasitology, Anhui Medical University, Hefei 230032, China

Online:2013-04-30 Published:2013-07-02

摘要/Abstract

摘要： 目的观察西咪替丁伍用弓形虫棒状体蛋白2（ROP2）免疫小鼠诱导的免疫反应。方法 80只BALB/c小鼠随机分为A组、B组、C组和D组，各组分别经皮下注射PBS、弓形虫ROP2蛋白+PBS、弓形虫ROP2蛋白+福氏佐剂和弓形虫ROP2蛋白+西咪替丁[200 mg/(kg·d）]免疫BALB/c小鼠，每次ROP2蛋白免疫剂量为100 μg/只（200 μl），共免疫3次，每次间隔2周。于首次免疫前和每次免疫后2周，采血分离血清。末次免疫后2周，每组小鼠随机处死8只，无菌分离脾细胞，CCK-8法测定淋巴细胞增殖活性。流式细胞术检测小鼠全血T细胞亚群。ELISA法检测小鼠血清IgG抗体和γ干扰素(IFN?鄄γ)水平。各组剩余12只小鼠经腹腔感染弓形虫RH株速殖子（5×104个/鼠），观察攻击感染后小鼠生存时间。结果末次免疫后2周，A、B、C和D组小鼠的血清IFN-γ水平分别为（659.750±239.962）、（872.750±197.011）、（1600.750±480.680）和（1 494.375±451.655）pg/ml，血清特异性IgG抗体水平分别为0.504±0.078、0.592±0.160、1.068±0.111和1.046±0.147，脾细胞增殖活性分别为0.504±0.078、0.592±0.160、0.831±0.130和0.762±0.089，外周血CD4+/CD8+比值分别为0.504±0.078、0.592±0.160、0.831±0.130和0.762±0.089，C和D组均显著高于A和B组（前3个指标，均P<0.01；后者，均P<0.05），D组与C组的差异均无统计学意义（P＞0.05）。各免疫组弓形虫攻击感染后，A、B、C 和D 组小鼠的生存时间中位数分别为96、108、132和132 h，C组和D组小鼠平均存活时间显著长于A组和B组（P<0.05），D组与C组的相比，差异无统计学意义（P＞0.05）。结论西咪替丁可增强ROP2蛋白诱导的细胞免疫和体液免疫反应。

关键词: 刚地弓形虫, 西咪替丁, 棒状体蛋白2, 免疫反应, 蛋白疫苗

Abstract: Objective To observe the immune response induced by ROP2 protein of Toxoplasma gonddi with cimetidine in mice. Methods Eighty BALB/c mice were randomly divided into 4 groups: PBS（group A）, ROP2 protein （group B）, ROP2 protein-Freund’s adjuvant （group C） and ROP2 protein-cimetidine（group D）. Mice were immunized with PBS, ROP2 protein, ROP2 protein and Freund’s adjuvant, ROP2 protein and cimetidine[200 mg/(kg·d）] by subcutaneous injection three times at an interval of 2 weeks, respectively. Experimental mice were immunized with 100 μg ROP2 proteins, which were diluted to a final volume of 200 μl in PBS. On day 13, 27 and 41 after immunization, mice sera were collected for determination of antibody IgG and cytokine IFN-γ by ELISA. Two weeks after the final immuni-zation, T cells subpopulation was detected by flow cytometry and splenocyte proliferation activity was determined with CCK-8. Another 12 immunized mice in each group were intraperitoneally challenged with 5×104 tachyzoites of T. gondii and the survival time was observed. Results Two weeks after final immunization, compared with groups A［（659.750±239.962） pg/ml］ and B ［（872.750±197.011） pg/ml］, the level of IFN-γ significantly increased in groups C ［（1 600.750±480.680） pg/ml］ and D ［（1 494.375±451.655） pg/ml］（P<0.01）. Similarly, compared with groups A （0.636±0.108） and B （0.871±0.089）, the level of IgG was also higher in groups C（1.068±0.111） and D（1.046±0.147）（P<0.01）. The proliferation of splenocytes rose in group C（0.831±0.130） after immunization, and similarly in group D （0.762±0.089）, and were both significantly higher than that of groups A （0.504±0.078） and B （0.592±0.160）（P<0.01）. Moreover, ratio of CD4+/CD8+ in groups C （0.831±0.130） and D （0.762±0.089） were higher than that of groups A（0.504±0.078） and B（0.592±0.160）（P<0.05）. After challenge with violent virulence strain of tachyzoites, the median survival time of mice in groups A, B, C, and D were 96, 108, 132, and 132 h, respectively. The mean survival time of mice in groups C and D were longer than that of groups A and B（P<0.05）. There was no significant difference in 5 parameters between C and D: the level of IFN-γ and IgG, CD4+/CD8+ ratio, splenocyte proliferation, and survival time of mice（P>0.05）. Conclusion Cimetidine can enhance the humoral and cellular immune response induced by ROP2 protein.

Key words: Toxoplasma gondii；Cimetidine, Rhpotry protein 2；Immune response；Protein vaccine

陈兴智1,2 *，杨小迪1，杨雯1，陈勇1，刘丽丽2，沈继龙2，孙新1. 西咪替丁伍用弓形虫ROP2蛋白免疫小鼠诱导免疫反应的初步研究[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(2): 4-99-103.

CHEN Xing-zhi1,2 *, YANG Xiao-di1, YANG Wen1, CHEN Yong1, LIU Li-li2, SHEN Ji-long2, SUN Xin1. Study on Immune Response in BALB/c Mice Induced by ROP2 Protein of Toxoplasma gondii with Cimetidine[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2013, 31(2): 4-99-103.

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