摘要
目的探讨中药血必净注射液对体外脂多糖（LPS）刺激CD4^＋CD25^＋调节性T细胞（Treg）凋亡及其介导效应T细胞（Teff）增殖反应、分泌白细胞介素-2（IL-2）功能的影响。方法40只雄性清洁级Wistar大鼠，实验前禁食12h。免疫磁珠法分选大鼠脾脏CD4^＋CD25^＋Treg，分为对照组（n=8）、抗CD3／CD28组（n=8）、抗CD3／CD28＋12S组（n=8）、抗CD3／CD28＋血必净组（n=8）和抗CD3／CD28＋LPS＋血必净组（n=8）。培养3d后流式细胞术检测CD4^＋CD25^＋Treg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，酶联免疫吸附试验检测IL-10分泌。同时，CD4^＋CD25^＋Treg与CD4^＋CD25^-T细胞1：1培养，刀豆素A刺激68h后分析Treg对Teff增殖的抑制效应，并检测上清中IL-2／可溶性IL-2受体水平。结果抗CD3／CD28诱导Treg凋亡率为（33．70±3．06％），显著高于对照组（12．84±0．84）％；抗CD3／CD28＋LPS＋血必净组45．13±2．70）％凋亡率明显高于抗CD3／CD28＋LPS组[（29．41±1．63）％，P〈0．01]。同时，CD4^＋CD25^＋Treg Foxp3和CTLA-4表达及IL-10分泌随凋亡增加而降低。抗CD3／CD28＋血必净组（31．26％）Teff增殖反应平均抑制率显著低于对照组（54．48％，P〈0．05），抗CD3／CD28＋LPS＋血必净组抑制率与抗CD3／CD28＋LPS组比较亦明显下降（P〈0．01）。此外，抗CD3／CD28＋血必净组和抗CD3／CD28＋LPS＋血必净组IL-2分泌水平均显著高于抗CD3／CD28＋LPS组（P〈0．01）。结论LPS刺激可明显上调大鼠Treg对Teff的抑制功能，血必净注射液能通过促进Treg凋亡而有效改善其对Teff的抑制效应。
Objective To investigate the effect of Xuebijing injectiong on lipopolysaccharide （LPS）-induced apoptosis of CD4^ ＋ CD25^ ＋ regulatory T cells （Tregs） and immune function of effector T cells （Teff） in vitro. Method CD4^＋ CD25^＋ Tregs isolated from rat spleens were divided into the control group, anti-CD3/CD28 group, anti-CD3/CD28 ＋ LPS group, anti-CD3/CD28 ＋ Xuebijing injection group, and anti-CD3/CD28 ＋ LPS ＋ Xuebijing injection group. The apoptosis rate and expression of forkhead/winged helix transcription factor p3 （Foxp3） and eytotoxic T-lymphocyte-associated antigen 4 （CTLA-4） of CD4^＋ CD25^＋ Tregs were detected by flow cytometry （FCM）, and the secretion of IL-10 of Tregs was measured by ELISA on day 3. CD4^＋CD25^- T cells were co-cultured with CD4^＋ CD25^ ＋ Tregs （ 1 ： 1 ） for 68 hours, proliferative activity of Teff was determined by MTT, and interleukin （IL）-2/sIL-2Rα levels were measured by ELISA. Results The apoptosis rate of CD4^＋ CD25^＋ Tregs in anti-CD3/CD28 group was 33.70 ± 3.06%, which was significantly higher than that in control group （ 12.84 ± 0.84% ）. Also, apoptosis rate of CD4^＋ CD25^＋ Tregs in anti-CD3/CD28 ＋ LPS ＋ Xuebijinginjection group （45.13 ± 2.70% ） was much higher than that in anti-CD3/CD28 ＋ LPS group （29.41 ± 1.63%, P 〈0.01）. The expression of Foxp3 as well as CTLA-4, and the secretion of IL-10 were markedly decreased along with increases in the apoptosis rates. Compared with control group （54.48 % ）, the mean inhibitory rate of Teff proliferative activity in response to Con A was significantly decreased in anti-CD3/CD28 ＋ Xuebijing injection group （31.26%, P 〈 0.05）, and it was markedly decreased in anti-CD3/CD28 ＋ LPS ＋ Xuebijing injection group comaped to anti-CD3/CD28 ＋ LPS group （ P 〈 0.01 ）. In addition, IL-2 levels in the supematant of anti- CD3/CD28 ＋ Xuebijing injection group and anti-CD3/CD28 ＋ LPS ＋ Xuebijing injection group were significandy higher than those of anti-CD3/CD28 ＋ LPS group （ P 〈 0.01 ）. Conclusions The inhibitory activity of CD4^＋ CD25^＋ Tregs on Teff appears to be upregulated by LPS stimulation in vitro, and Xuebijing injection could markedly enhance apoptosis of CD4^＋ CD25^＋ Tregs, thereby improving suppressive immune function of Teff.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2009年第9期932-936,共5页
Chinese Journal of Emergency Medicine
基金
国家重点基础研究发展规划项目（2005CB522602）
国家自然科学基金课题（30872683）
天津市现代中药大品种群系统开发基金资助项目（2006）
作者简介
通信作者：姚咏明，Email：c_ff@sina．com
