摘要
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.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2009年第1期58-61,共4页
Chinese Journal of Surgery
基金
基金项目:国家重点基础研究发展规划资助项目(2005CB522602)
国家自然科学基金资助项目(30672178,30872683)