刚地弓形虫排泄-分泌抗原体外诱导的IFN-γ促CD4^+CD25^+调节性T细胞凋亡作用  

Interferon-γ Induced by Toxoplasma gondii Excreted/Secreted Antigens Promotes Apoptosis of CD4^+CD25^+ Regulatory T Cells

作　　者：葛可 仇晓艳 王丽娟[1] 陈慧[3] 张璐彦 邱竞帆[1] 王勇[1] 

机构地区：[1]南京医科大学基础医学院病原生物学系,南京210029 [2]盱眙县人民医院检验科,盱眙211700 [3]南京医科大学第四临床医学院,南京210029

出　　处：《中国寄生虫学与寄生虫病杂志》2016年第3期183-188,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省高校自然科学研究面上项目(No.13KJB310004);江苏省大学生创新创业训练计划项目(No.201510312024Z)~~

摘　　要：目的研究刚地弓形虫(Toxoplasma gondii)RH株和Tg Ctwh3株排泄-分泌抗原(ESA)体外诱导小鼠CD4^+CD25^+调节性T细胞凋亡和IFN-γ分泌方面的差异。方法分别制备刚地弓形虫RH株和Tg Ctwh3株的ESA。将分离的野生型C57BL/6小鼠脾单个核细胞随机分为3组(每孔2×10~6细胞),分别加入RH株ESA、Tg Ctwh3株ESA(均为10μg/ml)和鸡卵清蛋白(OVA)进行诱导刺激,流式细胞术检测各组诱导刺激48 h和72 h的CD4^+CD25^+调节性T细胞的早期凋亡情况,ELISA检测各组诱导刺激72 h细胞培养上清中的γ干扰素(IFN-γ)分泌水平。另外,将分离的野生型C57BL/6小鼠脾单个核细胞随机分为2大组(每孔2×10~6细胞),其中一大组在分别加入两株ESA(10μg/ml)和OVA的同时加入抗IFN-γ中和抗体(10μg/ml)进行诱导刺激72 h,流式细胞术检测各组CD4^+CD25^+调节性T细胞早期凋亡情况。用两虫株ESA(10μg/ml)及OVA分别诱导刺激IFN-γ基因敲除型和野生型C57BL/6小鼠的脾单个核细胞(每孔2×10~6细胞)72 h后,流式细胞术检测各组CD4^+CD25^+调节性T细胞早期凋亡情况。结果制备的刚地弓形虫RH株和Tg Ctwh3株ESA抗原蛋白浓度分别为0.54 mg/ml和2.14 mg/ml。流式细胞术检测结果显示,RH株和Tg Ctwh3株ESA组诱导刺激48 h后,CD4^+CD25^+调节性T细胞的早期凋亡率分别为(12.90±1.26)%和(9.71±1.04)%,均显著高于OVA对照组(4.48±0.48)%(P<0.01);诱导刺激72 h后,RH株ESA组诱导CD4^+CD25^+调节性T细胞的凋亡率为(15.21±1.11)%,仍明显高于Tg Ctwh3株ESA组(11.02±0.92)%(P<0.05)和OVA对照组(10.10±1.49)%(P<0.01)。ELISA检测结果显示,RH株和Tg Ctwh3株ESA组诱导刺激脾单个核细胞72 h的培养上清中分泌的IFN-γ水平分别为(4 764.0±118.7)pg/ml和(3 629.0±33.6)pg/ml(P<0.01),均显著高于OVA对照组的(679.4±30.6)pg/ml(P<0.01)。流式细胞术检测结果显示,RH株ESA加抗IFN-γ中和抗体组诱导CD4^+CD25^+调节性T细胞的早期凋亡率为(10.44±1.44)%,明显低于未加抗IFN-γ中Objective To investigate the effect of excreted/secreted antigens（ESAs） from Toxoplasma gondii RH strain and TgCtwh3 strain on apoptosis of CD4^＋CD25^＋ regulatory T cells and interferon-γ（IFN-γ） secretion. Methods ESAs of Toxoplasma gondii RH strain and TgCtwh3 strain were prepared. Splenic mononuclear cells were isolated from C57BL/6 mice and randomly divided into RH ESA group（2×10^6 cells/well with addition of 10 μg/ml RH ESA）, TgCtwh3 ESA group （2×10^6 cells/well with addition of 10 μg/ml TgCtwh3 ESA） and control group（2×10^6 cells/well with addition of 10 μg/ml ovalbumin）. Flow cytometry was performed to examine the early apoptosis of CD4^＋CD25^＋ regulatory T cells after treatment for 48 h and 72 h. ELISA was conducted to determine the level of IFN-γ in the supernatant after treatment for 72 h. In another experiment, the 3 groups of splenic mononuclear cells were added with 10 μg/ml anti-IFN-γ antibody for 72 h and flow cytometry was performed to examine the early apoptosis of CD4^＋CD25^＋ regulatory T cells. Meanwhile, splenic mononuclear cells from IFN-γ knockout and wild-type C57BL/6 mice were also divided into the above-described 3 groups, and flow cytometry was performed to examine the early apoptosis of CD4^＋CD25^＋ regulatory T cells after treatment for 72 h. Results The concentrations of RH ESA and TgCtwh3 ESA were 0.54 mg/ml and 2.14 mg/ml, respectively. Flow cytometry showed that the early apoptosis rate of CD4^＋CD25^＋ regulatory T cells in the RH ESA group and the TgCtwh3 ESA group after 48 h treatment was （12.90±1.26）% and （9.71±1.04）%, respectively （P〈0.05）, both significantly higher than that in control group （4.48±0.48）% （P〈0.01） . The early apoptosis rate of CD4^＋CD25^＋ regulatory T cells after 72 h in the RH ESA group was（15.21±1.11）%, significantly higher than that in the TgCtwh3 ESA group[（11.02±0.92）%] （P〈0.05） and the control group[（10.10±1.49）%]（P〈0.01）. ELISA showed that the level

关 键 词：刚地弓形虫 排泄-分泌抗原 CD4^+CD25^+调节性T细胞 细胞凋亡 Γ-干扰素 

分 类 号：R382.5[医药卫生—医学寄生虫学]