牙龈卟啉单胞菌脂多糖对CD4^＋CD25^＋调节性T细胞免疫抑制功能的影响

目的探讨牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）对CD4^＋CD25^＋调节性T细胞（regulatory T cells，Tregs）免疫抑制功能的影响。方法采用酚水法提取Pg ATCC 33277株脂多糖（lipopolysaccharide，LPS）。免疫磁珠法分离BALB／c小鼠脾脏CD4^＋CD25^＋Tregs并进行体外培养，同时给予不同剂量（0～500ng／ml）Pg—LPS干预，培养48h后收集细胞及上清液。Real-TimePCR法测定培养细胞Foxp3mRNA的表达，ELISA法分别测定细胞上清液中IL-10、TGF-β水平；采用体外淋巴细胞混合培养法对Pg-LPS干预后的CD4^＋CD25^＋Tregs进行功能抑制试验。结果Pg-LPS干预不影响CD4^＋CD25^＋Tregs分泌IL-10和TGF-β，但是能够显著上调CD4^＋CD25^＋TregsFoxp3mRNA的表达，增强其免疫抑制作用；当Ps—LPS浓度低于300ng／m1时，CD4^＋CD25^＋TregsFoxp3mRNA表达以及免疫抑制作用的增强与Ps—LPS浓度之间呈剂量-效应关系。结论Pg-LPS能够增强CD4^＋CD25^＋Tregs的免疫抑制作用，这种免疫抑制增强效应可能与CD4^＋CD25^＋Tregs Foxp3基因表达的上调有关，并且不具有抑制性细胞因子依赖性。

Effects of Porphyromonas gingivalis lipopolysaccharide on immuno-suppressive function of CD4+CD25+ regulatory T cells

Objective To investigate the effects of P. gingivalis lipopolysaccharide （Pg-LPS） on immuno-supressire function of CD4^＋CD25^＋ regulatory T cells （Tregs）. Method Pg-LPS （ Pg strain, ATCC 33277） was prepared using phenol-water method. Tregs were separated from the BALB/c mice splenic cells in two steps by magnetic cell sorting （MACS） system.CD4^＋CD25^＋ Tregs were treated with different doses of Pg-LPS （0 =500 ng/ml） and cultured for 48 hours. The expression of Foxp3 mRNA in treated CD4^＋CD25^＋ Tregs is determined by real-time quantitative PCR. And the secretion of IL-10 and TGF-β were detected using ELISA method. Furthermore ,the direct suppressive effect of Pg-LPS treated CD4^＋CD25^＋ Tregs on CD4^＋CD25^＋ T lymphocytes proliferation was tested by the mixed lymphocytes reaction and measured by^3 H-Thymidine radioactive assay. Result The secretion of IL-10 and TGF-β by CD4^＋CD25^＋ Tregs were not influenced by Pg-LPS. However, Pg-LPS could significantly up-regulate the expression of Foxp3 mRNA and immuno-suppressive effect of CD4^＋CD25^＋ Tregs. When Pg-LPS was less than 300 ng/ml, there was a dose-effect manner existing between Pg- LPS and the expression of Foxp3 mRNA as wen as the immuno-suppressive effect of the treated CD4^＋CD25^＋ Tregs. Conclusion Pg-LPS could enhance the immm,o-suppressive effect of CD4^＋CD25^＋ ~ Tregs,which may correspond to the up-regulation of Foxp3 but not inhibitory cytokines.