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

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

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100μg/只(200μl),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-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)、(1 600.750±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。

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.