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  

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

作　　者：闫谨 李丹妮 傅炜昕 YAN Jin;LI Dan-ni;FU Wei-xin(Science Experiment Center,China Medical University,Shenyang 110122,China;Department of Immunolgy,Basic Medical College,China Medical University,Shenyang 110122,China)

机构地区：[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

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第3期332-338,共7页Chinese Journal of Parasitology and Parasitic Diseases

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(Pb A)感染红细胞1×10^6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-1,15μl/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)×10^6,高于健康对照组[(1.37±0.02)%和(0.41~0.47)×10^6](P<0.01);NK消除组分别为(1.82±0.07)%和(1.48~1.86)×10^6,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)×10^6和(1.39~1.62)×10^6,高于健康对照组[(0.27~0.31)×10^6和(0.47~0.56)×10^6](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)×10^6和(1.68~2.15)×10^6,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)Objective To explore the immunomodulatory effects of natural killer(NK)cells on the CD4^+T cell subset in mice with cerebral malaria.Methods Female C57 BL/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 1×10^6 Plasmodium berghei ANKA(Pb A)-infected red cells.The mice in NK elimination group were intraperitoneally injected with anti-Asialo GM-1(15μl),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 in-fection,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 day8.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)×10^6 in the infection group,which

关 键 词：脑型疟 自然杀伤细胞 TH1免疫应答 调节性T细胞 

分 类 号：H531.33[语言文字]