脓毒症大鼠调节性T细胞凋亡对效应T细胞增殖和分泌功能的影响及血必净注射液的干预作用

Effect of apoptosis of CD4＋CD25＋ regulatory T cells on proliferation as well as secretion of effector T cells and interventional activity of Xuebijing injection in septic rats

目的探讨血必净注射液对脓毒症大鼠CD4＋CD25＋调节性T细胞（Treg）凋亡的影响及与效应T细胞（Teff）增殖及分泌功能的关系。方法Wister大鼠随机分为对照组、假手术组、盲肠结扎穿孔（cLP）组和血必净治疗组，每组8只；于第3天采用免疫磁珠法分选大鼠脾脏CD4＋CD25＋Treg和CD4＋CD25＋T细胞。CD4＋CD25＋Treg培养12h后检测凋亡率、又头翼状螺旋转录因子（Foxp3）和T淋巴细胞毒性相关抗原4（CTLA-4）表达及白细胞介素（IL）-10的分泌。将．rreg与cDfcD25-T细胞共培养，并以刀豆素A刺激后观察Treg对Teff增殖活性和细胞培养上清IL-2／可溶性IL-2受体仪（sIL-2Rd）含量的影响。结果CLP组CD4＋CD25＋Treg细胞凋亡率〈对照组和假手术组（P〈0．01），而血必净治疗组明显〉CLP组（P〈0．01）。与CLP组比较，血必净治疗组CD4＋CD25＋Treg细胞FOXp3、CTLA-4表达和IL-10分泌显著下降（P〈0．01）。同时，CLP组Teff增殖反应抑制率明显〉对照组，而血必净组其抑制率〈CLP组和对照组（P〈0．01）。与对照组比较,CLP组IL-2分泌水平明显降低（P〈0．01），而sIL-2Rd水平升高；血必净组IL-2分泌水平显著〉clJP组，sIL-2Rd低于CLP组（P〈0．01）。结论脓毒症病理过程中CD4＋CD25＋Treg细胞对TeⅡ抑制效应明显增强，而血必净注射液能诱导Treg细胞凋亡，减轻Treg对Teff的抑制作用。

Objective To investigate the effect of apoptosis of CD4＋CD25＋regulatory T cells （Tregs） on proliferation as well as secretory fullction of effector T cells （Teff） and potential influence of Xuebijing injection on them in septic rats. Methods A sepsis model was reproduced by cecal ligation puncture （CLP）, and Wister rats were randomly divided into the control group （n = 8 ）, sham-operated group （ n = 8 ）, CLP group （ n = 8 ）, and Xuebijing injection treatment group （ n = 8 ）. CD4＋CD25＋ Tregs in each group were seperated by immunomagnetic beads isolate system on day 3, the apoptosis rate, expression of forkhead/winged helix transcription factor p3 （Foxp3） and cytotoxic T-lymphocyte-associated antigen 4 （CTLA-4） on Tregs were analyzed by flow cytometry, and secretion levels of interleukin （IL） -10 from Tregs were measured by ELISA. Following co-culture of CD4＋CD25＋ Treg with CD4＋CD25＋ T cells （ 1 ： 1 ） for 68 hours, proliferative activity of Teff was determined by MTT, and IL-2/slL-2Rot levels were measured by ELISA. Results The apoptosis rate of Tregs in control group was 12.03% ± 0. 89%, which was not significantly different from sham-operated group 9.48% ± 2. 17%. The apoptosis rate of Tregs in CLP group 5.87% ± 0.44% was lower than that in control group （ P 〈 0. 01 ） , and treatment with Xuebijing injection markedly enhanced the apoptosis of Tregs 27.29% ± 2. 48%. Compared to CLP group, expression of Foxp3, CTLA-4, and the secretion of IL-10 of Treg were significantly lowered in Xuebijing injection group （ all P 〈 0. 01 ）. The Teff proliferative activity in response to ConA, and IL-2 levels of Teff in CLP group were significantly suppressed compared with control group （ P 〈 0. 01 ）, and secretion of slL-2Rα in the supernatants was much higher than that of the control group. In comparison to the CLP group, inhibitory rate of Teff proliferative activity and the slL-2Rα levels were significantly decreased, while the secretion of IL-2 was increased in Xuebijing injection group （ P 〈 0. 01 ）. Conclusion CD4＋CD25＋ Tregs could markedly upregulate the suppressive function on Teff in sepsis, and treatment with Xuebijing injection effectively enhanced apoptosis of Tregs, thereby down-regulating the suppression on Teff.