内毒素作用下淋巴细胞中CD4^+CD25^+调节性T细胞变化及连翘对其的影响

Change of CD4^+CD25^+Tregs among lymphocytes with the function of endotoxin and the influence on them with the addition of Forsythia suspensa

目的探讨不同时间点内毒素(LPS)对CD4+CD25+调节性T细胞(CD4+CD25+regulatory T cell,Treg)的影响及连翘(forsythia suspensa,FS)干预后CD4+CD25+调节性T细胞的变化。方法体外培养Wistar大鼠脾脏淋巴细胞,分为对照组、LPS(10ng/ml)组、LPS(10ng/ml)+多黏菌素B组(10μg/ml)、LPS(10ng/ml)+连翘(12.5、25、50mg/ml)组,分别于3、6、12h收集标本,流式细胞术检测CD4+CD25+调节性T细胞在淋巴细胞中的百分比,荧光定量RT-PCR法检测Foxp3mRNA表达,酶联免疫吸附试验(ELISA)法检测TNF-α、IL-10分泌水平。结果脾脏淋巴细胞中CD4+CD25+Treg的百分比和Foxp3mRNA的表达在12h较3、6h明显升高(P<0.01);TNF-α、IL-10的分泌随时间点的延长均显著升高(P<0.01);随连翘剂量增加,TNF-α、IL-10分泌水平,Foxp3mRNA表达和CD4+CD25+Treg百分比均明显降低(P<0.05)。结论内毒素参与并诱导了CD4+CD25+Treg在淋巴细胞中的变化,其随内毒素作用时间延长呈上升趋势。连翘能显著降低CD4+CD25+Treg在淋巴细胞中的百分比。

Objective To discuss the influence of LPS on CD4＋CD25 ＋Tregs during different time and the variation of CD4＋CD25＋Tregs after forsythia suspensa intervention. Methods Spleen lymphocytes of Wister rats in invitro cultivation were allocated into control group, LPS group, LPS （10ng/ml） ＋PloyB （10ug/ml） group and LPS （10ng/ml） ＋AFS （12.5,25,50mg/ml） group, respectively collected samples after 3,6,12 hours. The proportion of CD4＋CD25＋ Tregs among lymphocytes was detected by Flow Cytometry. RT-PCR method was used to detect the expression of Foxp3mRNA in lymphocytes. The secretion level of TNF-a,IL-10 was defected among lymphocytes by the application of ELISA. Results The proportion of CD4＋CD25＋Tregs and the expression of Foxp3mRNA among Spleen lymphocytes after 12 hours had more obvious improvement than the proportion and the expression after 3,6 hours （P〈0.01）. The secretion level of both TNF-a and IL-10 enhanced observably with the time of lengthy delays （P〈0.01）. The secretion level of TNF-a,IL-10,the expression of Foxp3mRNA and the percentage of CD4＋CD25＋ Tregs all reduced dramatically with the increasing in forsythia suspensa（P〈0.05）. Conclusion The endotoxin has induced the change of CD4＋CD25＋Tregs among lymphocytes which presents the rising trend with the lengthening effection of the endotoxin. Forsythia suspensa can reduce remarkably the proportion of CD4＋CD25＋Tregs among lymphocytes.