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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中和抗体组(14.96±0.83)%(P<0.05);但Tg Ctwh3株ESA加抗IFN-γ中和抗体与否诱导CD4~+CD25~+调节性T细胞的早期凋亡率变化不大(P>0.05)。RH株ESA诱导IFN-γ基因敲除小鼠的CD4~+CD25~+调节性T细胞凋亡率为(10.64±0.55)%,明显低于野生型小鼠的(15.21±1.11)%(P<0.01);Tg Ctwh3株ESA诱导两组小鼠的CD4~+CD25~+调节性T细胞凋亡率的差异无统计学意义(P>0.05)。结论刚地弓形虫RH株ESA在体外诱导CD4~+CD25~+调节性T细胞凋亡及促IFN-γ分泌强于Tg Ctwh3株ESA。弓形虫ESA诱导机体产生IFN-γ可通过介导调节性T细胞凋亡来促进弓形虫感染早期抗感染免疫力的形成。
Objective To investigate the difference of CD4 ~ + CD25 ~ + regulatory T cell apoptosis and IFN-γ secretion induced by Toxoplasma gondii RH strain and Tg Ctwh3 strain excretory-secretory antigen (ESA) in vitro. Methods ESA of T. gondii RH strain and Tg Ctwh3 strain were prepared respectively. Spleen mononuclear cells from wild-type C57BL / 6 mice were randomly divided into 3 groups (2 × 10 ~ 6 cells per well), and were respectively added with ES strain of RH strain, ESA of Tg Ctwh3 strain (both 10 μg / ml) (OVA). Flow cytometry was used to detect the early apoptosis of CD4 ~ + CD25 ~ + regulatory T cells induced by stimulation for 48 h and 72 h in each group. The level of IFN-γ secretion in the supernatant. In addition, isolated wild-type C57BL / 6 mouse splenic mononuclear cells were randomly divided into two groups (2 × 10 ~ 6 cells per well). One group was divided into two groups: ESA (10μg / ml) While anti-IFN-γ neutralizing antibody (10μg / ml) was added to induce stimulation for 72 hours. Flow cytometry was used to detect the early apoptosis of CD4 + CD25 + regulatory T cells in each group. The splenic mononuclear cells (2 × 10 ~ 6 cells per well) of IFN-γ knockout and wild-type C57BL / 6 mice were stimulated with ESA (10μg / ml) Cytometry was used to detect the early apoptosis of CD4 ~ + CD25 ~ + regulatory T cells in each group. Results The protein concentrations of ESA antigen of T. gondii RH strain and Tg Ctwh3 strain were 0.54 mg / ml and 2.14 mg / ml, respectively. The results of flow cytometry showed that the early apoptotic rates of CD4 ~ + CD25 ~ + regulatory T cells were (12.90 ± 1.26)% and (9.71 ± 1.04), respectively, in RH and Tg Ctwh3 ESA groups for 48 h ) Were significantly higher than those in OVA control group (4.48 ± 0.48)% (P <0.01). After induction for 72 h, the apoptosis rate of CD4 + CD25 + T cells in ESA group was (15.21 ± 1.11)%, which was still significantly higher than that of ESA group (11.02 ± 0.92)% (P <0.05) and OVA control group (10.10 ± 1.49)% (P <0.01) of Tg Ctwh3 strain. The results of ELISA showed that the levels of IFN-γ in the culture supernatant induced by stimulation of splenic mononuclear cells by RH strain and Tg Ctwh3 ESA group were (4764.0 ± 118.7) pg / ml and (3 629.0 ± 33.6) pg / ml (P <0.01), which was significantly higher than that of OVA control group (679.4 ± 30.6) pg / ml (P <0.01). The results of flow cytometry showed that the early apoptosis rate of CD4 ~ + CD25 ~ + regulatory T cells induced by ESA plus anti-IFN-γ neutralizing antibody was (10.44 ± 1.44)%, which was significantly lower than that of non-anti-IFN- (14.96 ± 0.83)% (P <0.05), but no significant difference was found between the ESCC plus anti-IFN-γ neutralizing antibody and Tg Ctwh3 antibody induced early CD4 ~ + CD25 ~ + regulatory T cells apoptosis Little change (P> 0.05). The apoptosis rate of CD4 ~ + CD25 ~ + regulatory T cells in RH strain ESA-induced IFN-γ knockout mice was (10.64 ± 0.55)%, which was significantly lower than that in wild-type mice (15.21 ± 1.11)% <0.01). The apoptosis rate of CD4 ~ + CD25 ~ + regulatory T cells induced by Tg Ctwh3 ESA in both groups had no statistical significance (P> 0.05). Conclusion ESA of T. gondii RH strain induced CD4 ~ + CD25 ~ + regulatory T cells apoptosis and enhanced IFN-γ secretion in vitro than that of Tg Ctwh3 ESA. Toxoplasma gondii ESA-induced IFN-γ production can promote the formation of Toxoplasma gondii infection-resistant early anti-infective immunity by mediating apoptosis of regulatory T cells.