中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (3): 332-338.doi: 10.12140/j.issn.1000-7423.2020.03.012

• 论著 • 上一篇    下一篇

自然杀伤细胞在小鼠脑型疟中对CD4+ T细胞亚群的免疫调节作用

闫谨1, 李丹妮2, 傅炜昕1,*()   

  1. 1 中国医科大学科学实验中心一部,沈阳 110122
    2 中国医科大学基础医学院免疫学教研室,沈阳 110122
  • 收稿日期:2019-08-19 出版日期:2020-06-30 发布日期:2020-07-07
  • 通讯作者: 傅炜昕
  • 作者简介:闫谨(1996-),女,硕士研究生,从事NK细胞在疟原虫感染中的作用的研究。E-mail: 948238408@qq.com

Immunomodulatory effects of natural killer cells on the CD4+ T cell subset in mice with cerebral malaria

YAN Jin1, LI Dan-ni2, FU Wei-xin1,*()   

  1. 1 Science Experiment Center, China Medical University, Shenyang 110122, China
    2 Department of Immunolgy, Basic Medical College, China Medical University, Shenyang 110122, China
  • Received:2019-08-19 Online:2020-06-30 Published:2020-07-07
  • Contact: Wei-xin FU

摘要:

目的 探讨自然杀伤(NK)细胞在小鼠脑型疟中对CD4+ T细胞亚群的免疫调节作用。 方法 雌性C57BL/6小鼠随机分为健康对照组、感染组与NK消除组,每组14只。感染组与NK消除组小鼠经腹腔内注射伯氏疟原虫ANKA株(PbA)感染红细胞1 × 106个;NK消除组小鼠在感染前1 d和感染后2 d腹腔内注射NK细胞封闭抗体anti-Asialo GM-1,15 μl/只,健康对照组和感染组注射等体积的PBS。感染后3 d起,采集小鼠尾静脉血,血涂片检测原虫血症,记录死亡情况。感染后3 d和5 d,各组剖杀小鼠3只,取脾脏,制备脾细胞悬液,流式细胞术检测脾细胞中Th1型细胞和调节性T细胞(Tregs);体外培养脾细胞,48 h后收集上清,ELISA检测γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)和白细胞介素10(IL-10)的水平。感染后6 d,各组取3只小鼠,用伊文思蓝(EB)染液处理后,取脑,体外孵育脑组织,48 h后收集上清,酶标仪检测通过小鼠血脑屏障的EB含量。 结果 感染组感染后6 d开始出现死亡,并伴有神经症状,8 d全部死亡;NK消除组感染后7 d开始出现死亡,12 d全部死亡。感染后7 d,NK消除组原虫血症为5.2%~10.5%,高于感染组的2.4%~5.0%(P < 0.05)。感染后5 d,感染组Th1型细胞的百分率和绝对计数分别为(2.65 ± 0.06)%和(2.09~2.25)× 106,高于健康对照组[(1.37 ± 0.02)%和(0.41~0.47)× 106](P < 0.01);NK消除组分别为(1.82 ± 0.07)%和(1.48~1.86)× 106,低于感染组(P < 0.01或P < 0.05)。感染后3 d和5 d,感染组Tregs的百分率分别为(1.21 ± 0.06)%和(1.70 ± 0.13)%,高于健康对照组[(0.90 ± 0.01)%和(0.91 ± 0.02)%](P < 0.01);感染组Tregs的绝对计数分别为(0.63~0.73)× 106和(1.39~1.62)× 106,高于健康对照组[(0.27~0.31)× 106和(0.47~0.56)× 106](P < 0.01);NK消除组Tregs百分率分别为(1.86 ± 0.07)%和(2.15 ± 0.04)%,绝对计数分别为(0.77~0.90)× 106和(1.68~2.15)× 106,均高于感染组(P < 0.01或P < 0.05)。感染后3 d和5 d,感染组脾细胞培养上清中的IFN-γ分别为(790.75 ± 84.80)和(989.58 ± 199.59)pg/ml,TNF-α分别为(2 637.47 ± 283.50)和(3 124.58 ± 964.70)pg/ml,均高于健康对照组[(73.69 ± 16.67)和(75.19 ± 15.97)pg/ml、(290.01 ± 187.46)和(290.51 ± 186.76)pg/ml](P < 0.01或P < 0.05);NK消除组培养上清中的IFN-γ分别为(15.83 ± 4.27)和(266.63 ± 108.62)pg/ml,TNF-α分别为(165.89 ± 71.02)和(842.77 ± 311.94)pg/ml,均低于感染组(P < 0.01或P < 0.05)。感染后3 d,NK消除组IL-10为(3 588.20 ± 1 436.38)pg/ml,高于感染组[(1 255.77 ± 190.01)pg/ml](P < 0.05)。感染后5 d,感染组IL-10为(4 991.36 ± 1 030.89)pg/ml,高于健康对照组[(848.50 ± 501.79)pg/ml](P < 0.05);NK消除组为(9 317.95 ± 1 077.89)pg/ml,高于感染组(P < 0.05)。感染后6 d,感染组脑组织EB含量为(4.02 ± 0.10)μg/ml,高于健康对照组[(2.09 ± 0.06)μg/ml](P < 0.05),NK消除组为(3.14 ± 0.02)μg/ml,低于感染组(P < 0.05)。 结论 NK细胞消除可减弱脑型疟小鼠Th1免疫应答,增强Tregs免疫应答,降低血脑屏障通透性,延长小鼠生存时间。

关键词: 脑型疟, 自然杀伤细胞, Th1免疫应答, 调节性T细胞

Abstract:

Objective To explore the immunomodulatory effects of natural killer (NK) cells on the CD4+T cell subset in mice with cerebral malaria. Methods Female C57BL/6 mice were randomly divided into the health control group, infection group and NK elimination group, 14 mice in each. The mice in the infection group and NK elimination group were injected intraperitoneally with 1 × 106 Plasmodium berghei ANKA (PbA)-infected red cells. The mice in NK elimination group were intraperitoneally injected with anti-Asialo GM-1(15 μl), a blocking antibody against NK cells, on day 1 before and day 2 after infection, while the mice in the health control and infection groups were injected with equal volumes of PBS. From day 3 on post-infection, blood was collected from the tail vein to make blood smears for examining parasitemia, and death of mine was recorded. On days 3 and 5 after infection, 3 mice were sacrificed in each group. The spleens were taken to make single cell suspensions for assaying Th1 type cells and regulatory T cells (Tregs) by flow cytometry. The spleen cells were cultured in vitro for 48 h, then the culture supernatant was collected to assay the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10) by ELISA. On day 6 after infection, 3 mice each group received Evans blue (EB) intravenously, brain tissues were incubated in vitro for 48 h, supernatant was collected and the EB content passing through the blood-brain barrier of the mice was detected by microplate reader. Results In the infection group, mice deaths began to occur on day 6 after infection, accompanied by neurological symptoms, and all died on day 8. In the NK elimination group, deaths began to occur on day 7 after infection, and all mice died on day 12. On day 7 after infection, mice in the NK elimination group had significantly higher parasitemia (5.2%-10.5%) than those in the infection group (2.4%-5.0%) (P < 0.05). On day 5 after infection, the percentage and absolute counts of Th1 type cells were (2.65 ± 0.06)% and (2.09-2.25) × 106in the infection group, which were significantly higher than those in the health control group (1.37 ± 0.02)% and (0.41-0.47) × 106 (P < 0.01). and those in the NK elimination group [(1.82 ± 0.07)% and (1.48-1.86) × 106] were significantly lower than those in the infection group (P < 0.01 or P < 0.05). The percentages of Tregs on days 3 and 5 in the infection group were(1.21 ± 0.06)% and (1.70 ± 0.13)%, respectively, which were significantly higher than those in the health control group[(0.90 ± 0.01)% and (0.91 ± 0.02)%] (P < 0.01). The absolute counts of Tregs in the infection group on day 3 and 5 post-infection were (0.63-0.73) × 106 and (1.39-1.62) × 106, respectively, which were higher than those in the health control group[(0.27-0.31) × 106 and (0.47-0.56) × 106] (P < 0.01); the percentages of Tregs in the NK elimination group were (1.86 ± 0.07)% and (2.15 ± 0.04)%, and the absolute counts were (0.77-0.90) × 106 and (1.68-2.15) × 106, respectively, all significantly higher than those in the infection group (P < 0.01 or P < 0.05). On days 3 and 5 after infection, the concentrations of IFN-γ in the cultural supernatant of splenocytes in the infection group were (790.75 ± 84.80) and (989.58 ± 199.59) pg/ml, respectively, and the TNF-α concentrations were (2 637.47 ± 283.50) and (3 124.58 ± 964.70) pg/ml, respectively, which were higher than those in the health control group [(73.69 ± 16.67) and (75.19 ± 15.97) pg/ml, (290.01 ± 187.46) and (290.51 ± 186.76) pg/ml respectively] (P < 0.01 or P < 0.05). In the NK elimination group, the IFN-γ concentrations were (15.83 ± 4.27) and (266.63 ± 108.62) pg/ml, and the TNF-α concentrations were (165.89 ± 71.02) and (842.77 ± 311.94) pg/ml, respectively, which were lower than those in the infection group (P < 0.01 or P < 0.05). On day 3 after infection, the IL-10 concentration in the NK elimination group was (3 588.20 ± 1 436.38) pg/ml, which was higher than that in the infection group [(1 255.77 ± 190.01) pg/ml] (P < 0.05). On day 5 after infection, the IL-10 concentration in the infection group was (4 991.36 ± 1 030.89) pg/ml, which was higher than that in the health control group as (848.50 ± 501.79) pg/ml (P < 0.05). In the NK elimination group, the IL-10 concentration was (9 317.95 ± 1 077.89) pg/ml, which was higher than that in the infection group (P < 0.05). On day 6 after infection, the EB content in the brain tissue of the infection group was (4.02 ± 0.10) μg/ml, which was higher than that in the health control group [(2.09 ± 0.06) μg/ml] (P < 0.05), and the EB content in the NK elimination group was (3.14 ± 0.02) μg/ml, which was lower than that in the infection group (P < 0.05). Conclusion The elimination of NK cells weakens the Th1 immune response, enhances the Tregs immune response, reduces the blood-brain barrier permeability and prolongs the survival time of mice with cerebral malaria.

Key words: Cerebral malaria, Natural killer cells, Th1 immune response, Regulatory T cells

中图分类号: