黏膜佐剂在黏膜耐受治疗大鼠实验性结肠炎中的作用

目的探讨％11样受体（TLR）参与黏膜佐剂增强黏膜耐受和缓解实验性结肠炎的可能机制。方法建立大鼠三硝基苯磺酸实验性结肠炎模型。以卵白蛋白为诱导抗原，脂多糖为佐剂，将已建立的三硝基苯磺酸结肠炎大鼠模型根据不同的干预方法分为结肠炎组（无干预）、口服耐受组、经鼻耐受组、口服加佐剂组、经鼻加佐剂组、佐剂对照组和空白对照组。用免疫组织化学方法检测各组大鼠结肠组织局部TLR2和TLR4表达水平。用三色流式细胞技术检测各组大鼠外周血调节性T细胞CD4^＋CD25^＋亚群和CD8^＋CD28^-亚群表面TLR2和TLR4表达水平。结果在结肠组织局部，结肠炎组的TLR2和TLR4阳性细胞数均显著高于正常对照组（P〈0．05）；佐剂对照组和口服加佐剂组的TLR4表达较结肠炎组显著下降（P〈0．05）。外周血CD4^＋CD25^＋亚群内，口服加佐剂组、经鼻耐受组、经鼻加佐剂组和佐剂对照组的TLR2^＋细胞比例显著低于结肠炎组（P〈0．05，P〈0．01）；口服加佐剂组和经鼻加佐剂组的TLR4^＋细胞比例显著低于结肠炎组（P〈0．05）。外周血CD4^＋CD28^-亚群内，各组的TLR2^＋细胞比例差异均无显著性；口服加佐剂组和经鼻耐受组的TLR4^＋细胞比例显著低于结肠炎组（P〈0．05）。结论多次给予脂多糖佐剂可能通过下调调节性T细胞的TLR2和TLR4表达而增强黏膜局部和循环的调节性T细胞功能（对CD4^＋CD25^＋亚群作用更明显），从而增强免疫耐受的效果。

Effect of Mucosal Adjuvant in Therapeutic Effects of Mucosal Tolerance on Experimental Colitis of Rats

Objective To investigate the mechanisms of Toll-like receptors （TLR） in therapeutic effects of oral tolerance, nasal tolerance and both combined with mucosal adjuvant on experimental colitis of rats. Methods Rat models of experimental colitis were established by enema with 2, 4, 6-trinitrobenzenesulfonic acid. Ovalbumin （OVA） was used as inducing antigen and lipopolysaccharide （LPS） as adjuvant. Rat models of experimental colitis were grouped according to therapies： experimental colitis （no therapy）, OVA oral administration, OVA nasal administration, OVA and LPS oral administration, OVA and LPS nasal administration, and LPS oral administration. PBS was orally or nasally administerd as controls. Colonic expressions of TLR2 and TLR4 were examined by immunohistochemistry. TLR2 and TLR4 expressions onCD4^±CD25^±and CD8± CD28^- regu- latory T ceils in peripheral blood were detected by flow cytometry. Results Colonic expressions of both TLR2 （52.5 ±17.5 vs. 22.0±8.5, P〈0.05） andTLR4 （52.0 ±16.9 vs. 22.5 ±3.5, P〈0.05） in experimental colitis of rats were significantly higher than in normal rats. Colonic expressions of TLR4 decreased signif- icantly after the oral administration of OVA and LPS （34.3 ± 11.5 vs. 52.0 ± 16.9, P 〈0.05） or LPS oral administration （30.0 ± 7.2 vs. 52.0 ± 16.9, P 〈 0.05 ）. TLR2 expressions on CD4^±CD25^± significantly decreased after therapies including OVA and LPS oral administration （61.2 ± 15.5 vs. 83.2 ± 8.9, P 〈0.05 ） , OVA nasal administration （ 66.2 ± 14.6 vs. 83.2 ± 8.9, P 〈 0.05 ） , OVA and LPS nasal administration （58.6±6.5 vs. 83.2±8.9, P〈0.01）, and LPS oral administration （65.3 ±12.2 vs. 83.2 ±8.9, P〈 0.05）. TLR4 expressions on CD4^±CD25^± significantly decreased after OVA and LPS oral administration （59.0 ± 16.1 vs. 74.0 .± 8.8, P 〈 O. 05 ） or OVA and LPS nasal administration （ 54.6 .± 13.0 vs. 74.0 ± 8.8, P 〈 0.05 ）. TLR4 expressions on CD8 ± CD28 - significantly decreased afte