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

摘要

目的 探讨血必净注射液对脓毒症大鼠CD+4CD+25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、假于术组、肓肠结扎穿孔(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CD+4CD25+Treg和CD+4CD25-T细胞.CD+4CD25+Treg培养12 h后检测凋亡率、叉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)表达及门细胞介素(IL)-10的分泌.将Treg与CD+4CD25-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α(slL-2Rα)含量的影响.结果 CLP组CD+4CD25+Treg细胞凋亡率<对照组和假于术组(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CD+4CD25+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,sll-2Rα低于CLP组(P<0.01).结论 脓毒症病理过程中CD+4CD25+Treg细胞对Teff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 CD+4 CD25+ regulatory T cells (Tregs) on proliferation as well as secretory fullction of effector T ceils (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). CD+4 CD25+ Tregs in each group were soperated 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 CD+4 CD25 + Treg with CD+4 CD25 - T cells ( 1 : 1 ) for 68 hours, proliferative activity of Teff was determined by MTT, and IL-2/sIL-2Rα 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 sIL-2Rα levels were significantly decreased, while the secretion of IL-2 was increased in Xuebijing injection group ( P < 0. 01 ). Conclusion CD+4 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.