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目的探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对CD4+CD25+调节性T细胞(regulatory Tcells,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(0～500 ng/ml)Pg-LPS干预,培养48 h后收集细胞及上清液。Real-Time PCR法测定培养细胞Foxp3 mRNA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+Tregs Foxp3 mRNA的表达,增强其免疫抑制作用;当Pg-LPS浓度低于300 ng/ml时,CD4+CD25+Tregs Foxp3 mRNA表达以及免疫抑制作用的增强与Pg-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。
Objective To investigate the effect of Porphyromonas gingivalis (Pg) on the immunosuppressive function of CD4 + CD25 + regulatory T cells (Tregs). Methods Pg ATCC 33277 lipopolysaccharide (LPS) was extracted by phenol water method. CD4 + CD25 + Tregs from spleen of BALB / c mice were isolated by immunomagnetic beads method and cultured in vitro. Different concentrations of Pg-LPS were administrated at different doses (0-500 ng / ml). Cells were cultured for 48 h and supernatants were collected. The expression of Foxp3 mRNA in cultured cells was detected by Real-Time PCR. The levels of IL-10 and TGF-β in the supernatant of the cells were measured by ELISA. The levels of CD4 + CD25 + Tregs Function inhibition test. Results Pg-LPS treatment did not affect the secretion of IL-10 and TGF-β by CD4 + CD25 + Tregs, but significantly up-regulated the Foxp3 mRNA expression in CD4 + CD25 + Tregs and enhanced their immunosuppression. When Pg-LPS was lower than 300 ng / ml, the expression of Foxp3 mRNA and the immunosuppressive effect of CD4 + CD25 + Tregs were dose-effect relationship with the concentration of Pg-LPS. Conclusion Pg-LPS can enhance the immunosuppressive effect of CD4 + CD25 + Tregs, which may be related to the up-regulation of Foxp3 gene expression in CD4 + CD25 + Tregs and not cytokine-dependent.