目的研究CD4^+CD25^+调节性T淋巴细胞(Treg)在小鼠肺结核发生中的作用。方法PC61处理组小鼠腹腔注射抗-CD25(PC61),50μg/只,消除体内的Treg,IgG对照组注射相同剂量的IgG同型对照抗体。3d后静脉注射H37Rv0.1mL(含1×10^6CFU活菌),攻击小鼠。流式细胞技术分析PC61对小鼠体内不同组织中Treg的消除效果,体外分析Treg消除对特异性细胞免疫、肺组织病理学和肺组织内结核分枝杆菌生长情况的影响。采用方差齐性F检验和非配对t检验比较实验数据。结果PC61处理组与IgG对照组小鼠脾细胞中Treg占CD4^+T淋巴细胞比例在第10天分别为(21.13±3.58)%和(30.42±4.20)%(t=2.38,P〈0.05);第30天为(16.12±1.26)%和(17.34±1.62)%(t=0.84,P〉0.05),Foxp3^++占CD4^+T淋巴细胞比例在第10天为(32.07±3.95)%和(60.55±5.48)%(t=5.96,P〈0.05).外周血也有类似结果。在PC61处理组及IgG对照组,感染后第10、30和60天时体外培养小鼠脾细胞分泌的卡介苗特异性细胞因子IL-17(ng/L)分别为5.1±0.9比0、43.1±10.0比5.9±2.8、124.8±5.8比102.5±8.1(t=7.90,t=5.10,t=3.19;均P〈0.05);IFN-γ(ng/L)分别为28.4±8.2比4.0±1.3、685.9±128.6比418.7±20.4、310.9±119.7比32.8±7.5(t=4.21,t=8.43,t=3.27;均P〈0.05);TNF—α(ng/L)分别为38.6±5.0比16.34±0、112.9±12.3比71.5±12.6、86.2±8.2比0(t=4.95,t=3.33,t=14.8;均P〈0.05)。PC61处理小鼠在感染早期(第10天)肺内结核菌量低于IgG对照小鼠(t=4.63,P〈0.01),但后期比较差异无统计学意义。PC61处理小鼠感染后期(第120天)肺组织病理改变较IgG对照组为轻。结论结核菌感染后小鼠Treg显著增加;Treg能有效抑制结核菌特异性细胞免疫,进而影响小鼠肺内结?
Objective To investigate the role of CD4^+ CD25^+ regulatory T lymphocytes (Treg) in modulating the cellular immune response and pathogenesis of murine pulmonary tuberculosis. Methods Inactivation of Treg was achieved by intraperitoneal injection anti CD25 (clone PC61, 50 μg/mouse) in PC61 group, and rat-IgG (50 μg/mouse) was injected intraperitoneally in control group. All the mice were inoculated intravenously with H37Rv 0. 1 mL (1 × 10^6 CFU) 3 days after Treg inactivation. The effects of Treg inactivation in different tissues were analyzed by flow cytometry. The cellular immune response, pulmonary histopathology and bacterial load were determined in vitro at different time points. The data were compared using homogeneity of variance F test and non-paired t test. Results In spleen, the percentages of Treg/CD4^+ T lymphocytes in PC61 group and control group were (21. 13 ± 3. 58) % and (30. 42 ±4. 20) %, respectively at day 10 of inoculation (t= 2. 38, P〈0. 05), and those were (16. 12± 1. 26)% and (17. 34± 1. 62)%, respectively at day 30 of inoculation (t = 0. 84, P〉 0. 05). The percentages of Foxp3^+/CD4^+ T lymphocytes in PC61 group and control group were (32. 07 ± 3. 95)% and (60. 55 ± 5. 48)%, respectively at day 10 of inoculation (t= 5. 96, P〈 0. 05). Similar results were achieved in the peripheral blood. Bacillus calmette guerin (BCG)-specific IL-17 (ng/L) secreted by murine spleen cells in PC61 group and control group at day 10, 30 and 60 of inoculation were 5. 1±0.9 vs 0, 43.1± 10.0vs5.9±2. S and 124. 8±5.8 vs102. 5±8.1, respectively (t =7.90, t=5.10, t =3.19; all P〈 0.05) ; those of BCG-specific IFN-γ (ng/L) were 28.4±8.2 vs 4.0±1.3, 685.9±128.6 vs 418.7± 20.4 and 310.9±119. 7 vs 32.8±7. 5, respectively (t=4.21, t=8. 43, t=3. 27; all P〈0.05); those of TNF-α (ng/L) were 38.6±5.0 vs 16.3±4.0, 112.9±12.8 vs 71.5±12.6 and 86.2±8.2 vs 0, respectively (t=4.95, t=3.33, t=14.8; all P?