中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (3): 218-223.

• 论著 • 上一篇    下一篇

四种刺激剂对日本血吸虫感染小鼠脾脏CD8+ T细胞内细胞因子及表面分子CD62L的影响

郑力1,2, 胡媛1, 王燕娟1, 黄希宝2, 沈玉娟1, 徐馀信1, 巩文词1, 蔡顺祥2, 曹建平1,*()   

  1. 1 中国疾病预防控制中心寄生虫病预防控制所,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 210025
    2 湖北省疾病预防控制中心,武汉 430079
  • 收稿日期:2017-01-26 出版日期:2017-03-30 发布日期:2017-09-07
  • 通讯作者: 曹建平
  • 基金资助:
    国家自然科学基金(No. 81371841);上海市第四轮公共卫生三年行动计划(No. 15GWZK0101)

Effects of four common stimulants on intracellular cytokines and CD62L of splenic CD8+ T cells from mice infected with Schistosoma japonicum

Li ZHENG1,2, Yuan HU1, Yan-juan WANG1, Xi-bao HUANG2, Yu-juan SHEN1, Yu-xin XU1, Wen-ci GONG1, Shun-xiang CAI2, Jian-ping CAO1,*()   

  1. 1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; WHO Collaborating Center for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
    2 Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China
  • Received:2017-01-26 Online:2017-03-30 Published:2017-09-07
  • Contact: Jian-ping CAO
  • Supported by:
    Supported by the National Natural Science Foundation of China (No. 81371841) and The Fourth Round of Three-year Action Planning of Public Health in Shanghai (No. 15GWZK0101)

摘要: 目的 探讨4种常用刺激剂佛波酯(phorbol-12-myristate-13-acetate,P)、离子霉素(ionomycin,I)、布雷非德菌素A(brefeldin A,B)和莫能霉素(monensin,M)对日本血吸虫(Schistosoma japonicum)感染小鼠脾脏CD8+ T细胞内细胞因子及表面分子CD62L的影响。 方法 21只C57BL/6雌性小鼠腹部贴片法感染日本血吸虫尾蚴,(20 ± 2)条/鼠。感染后第8周和第12周,分别取小鼠脾脏制备单细胞悬液,用P(50 ng/ml)+ I(1 μmol/L)+ M(2 μmol/L)体外刺激4 h,采用流式细胞术检测分泌γ干扰素(IFN-γ)的CD8+ T细胞比例,同时检测细胞表面CD62L的表达情况。制备感染后4周的小鼠脾脏单细胞悬液,按I组(1 μmol/L)、P组(50 ng/ml)、B组(1 μg/ml)、M组(2 μmol/L)、P + I组、P + I + M组、P + I + B组、P + I + M + B组分组,用对应刺激剂体外刺激4 h后,利用细胞因子微球检测法检测各组培养上清中白细胞介素4(IL-4)、IL-17A、IFN-γ、IL-10、IL-1β和集落刺激因子(G-CSF)等细胞因子水平,同时用流式细胞术检测CD8+ T细胞表面CD62L的表达情况,计算平均荧光强度(MFI)。 结果 流式细胞术检测结果显示,小鼠感染日本血吸虫后第8周和第12周,产生IFN-γ的CD8+ T细胞比例分别降至(1.3 ± 0.8)%和(0.7 ± 0.2)%,均低于未感染对照组的(5.6 ± 0.8)%(P < 0.05),但两者差异无统计学意义(P > 0.05)。健康对照组和感染组小鼠CD8+ T细胞表面均未检测到CD62L阳性群。不同组合刺激剂作用感染后4周,小鼠脾脏细胞培养上清细胞因子的检测结果显示,P + I组和P + I + M组的IL-4水平分别为(177.2 ± 56.0)和(13.7 ± 2.2)pg/ml;P + I组和P + I + M组的IL-17A浓度分别为(361.8 ± 81.3)和(33.7 ± 2.9)pg/ml;P + I组和P + I + M组上清中的IFN-γ浓度分别为(1 534.0 ± 316.6)和(135.3 ± 16.1)pg/ml;P + I组、P组和I组上清中IL-10的浓度分别为(705.5 ± 179.6)、(34.8 ± 13.9)和(43.1 ± 13.9)pg/ml;P组和P + I组中G-CSF的浓度为(44.6 ± 8.0)和(21.7 ± 2.9)pg/ml;P组、I组和P + I + M组中IL-1β的浓度分别为(3.9 ± 1.0)、(6.4 ± 0.2)和(3.7 ± 0.3)pg/ml;上述各组与其对应的对照组比较,差异均有统计学意义(P < 0.05或P < 0.01)。流式细胞术检测结果显示,P + I组、P + I + B组、P + I + M组和P + I + B + M组中CD62L峰较对照组明显左移,MFI分别为2.7 ± 0.1、2.6 ± 0.2、2.5 ± 0.1、2.5 ± 0.1,低于对照组(P < 0.01)。 结论 日本血吸虫感染晚期,小鼠脾脏细胞产生IFN-γ的CD8+ T细胞比例减少,经P + I刺激后,CD62L表达明显下调.

关键词: 日本血吸虫, 感染, 细胞因子, 流式细胞术, CD62L, CD8+ T细胞

Abstract: Objective To investigate the effects of four stimulants Ionomycin (I), Phorbol-12-myristate-13-acetate(P), Brefeldin A(B), and Monensin (M) on intracellular cytokines and CD62L of splenic CD8+ T cells from mice infected with Schistosoma japonicum. Methods Twenty-one C57BL/6 female mice were infected with S. japonicum using the abdominal patch method (20 ± 2 cercaria per mouse). The splenic single cell suspension was prepared at 8 and 12 weeks after infection, and were stimulated in vitro by P (50 ng/ml) + I (1 μmol/L) + M (2 μmol/L) for 4 h. Flow cytometry was performed to determine the proportion of CD8+ T cells secreting IFN-γ and detect surface expression of CD62L. Meanwhile, the splenic single cell suspension at 4 weeks after infection was stimulated by I (1 μmol/L), P (50 ng/ml), B (1 μg/ml), M (2 μmol/L), P + I, P + I + M, P + I + B, or P + I + M + B for 4 h, and the supernatant levels of IL-4, IL-17A, IFN-γ, IL-10, IL-1β and G-CSF were determined with cytometric bead array. CD62L expression on CD8+ T cells was examined by flow cytometry, and the mean fluorescence intensity (MFI) was calculated. Results After infection with S. japonicum, the proportion of IFN-γ/CD8+ T cells was(1.3 ± 0.8)% at 8 weeks, and dropped to(0.7 ± 0.2)% at 12 weeks, which were both significantly lower than(5.6 ± 0.8)% in the uninfected control group(P < 0.05). CD62L was not detected on CD8+ T cell surface in control and infection groups. The supernatant levels of IL-4 in the P + I and P + I + M groups were(177.2 ± 56.0) and (13.7 ± 2.2) pg/ml, respectively. The supernatant levels of IL-17A in the P + I and P + I + M groups were (361.8 ± 81.3) and (33.7 ± 2.9) pg/ml, respectively. The supernatant levels of IFN-γ in the P + I and P + I + M groups were (1534.0 ± 316.6) and (135.3 ± 16.1) pg/ml, respectively. The supernatant levels of IL-10 in the P + I, P and I groups were (705.5 ± 179.6), (34.8 ± 13.9) and (43.1 ± 13.9) pg/ml, respectively. The supernatant levels of G-CSF in the P and P + I groups were (44.6 ± 8.0) and (21.7 ± 2.9) pg/ml, respectively. The supernatant levels of IL-1β in the P, I, and P + I + M groups were (3.9 ± 1.0), (6.4 ± 0.2) and (3.7 ± 0.3) pg/ml, respectively. The above cytokine levels were all significantly different from the corresponding control level (P < 0.01 or P < 0.05). Results of flow cytometry showed a significant leftward shift of CD62L peak in the P + I, P + I + B, P + I + M, and P + I + B + M groups, compared with the control group, and MFIs of CD62L on CD8+ T cells were 2.7 ± 0.1, 2.6 ± 0.2, 2.5 ± 0.1 and 2.5 ± 0.1, all significantly lower than the control (P < 0.01). Conclusion In the late stage of schistosome infection(8-12 weeks), there is a lower percentage of IFN-γ-secreting CD8+ T cells among splenic cells. The P + I stimulation decreases the expression of CD62L.

Key words: Schistosoma japonicum, Infenction, Cytokine, Flow cytometry, CD62L, CD8+ T cells

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