TLR9信号通路参与急性肺损伤的作用机制研究

摘要

Objective To study the function mechanism of Toll-like receptor 9 in acute lung injury (ALI),providing a theoretical basis for clinical diagnosis and treatment.Methods 40 cases of ALI with isolated peripheral blood mononuclear cells,using RT-PCR detection of Toll-like receptor-9 mRNA expression in peripheral blood mononuclear cells (PBMC) after unmethylated cytosine-phosphate-guanine (CpG) sequence oligodeoxynucleotide (CpG ODN) and didn' t contain unmethylated CpG dinucleotide sequence oligodeoxynucleotide (non-CpG ODN),three forms of blank culturing for 72 h.Using 3H-TdR incorporation assay PBMC to get value changes,flow cytometry was used to detect the proportion of population using T cells,T cells and CD8 surface expression of CD69 molecules change IFN-γ and IL-4+T cells within.ELISA method was used to detect the IFN-α concentration in the supernatant of PBMC,and evaluate their degree of response to chloroquine and the inhibitory ODN.Results Patients with ALI PBMC expressed TLR9 mRNA expression level was (0.75 ± 0.08),and the healthy group (0.76 ± 0.09) with no significant difference (P ＞ 0.05); CpG ODN induced PBMC proliferation in patients with ALI (P ＜ 0.05),increased expression of CDTT cell surface molecule CD69 (P ＜ 0.05); enhancing CD8+T cells to produce IFN-γ in (P ＜ 0.05); enhanced D4+T/CD8+T ratio (P ＜ 0.05); CpG ODN promotes IFN-α secretion (P ＜ 0.05); suppressive ODN and chloroquine inhibit CpG ODN produced IFN-α.Conclusion TLR9 ALI patients involved in immune regulation,includes the promotion of its activation effect of PMBC,activating PBMC increase in the proportion of CD4+T,and the proliferation induced by IFN-α production,promoting CD8+T secreted IFN-γ.%目的 研究Toll样受体-9在急性肺损伤中的作用机制,为临床诊疗提供理论依据.方法 分离40例急性肺损伤患者的外周血单个核细胞,采用RT-PCR检测Toll样受体-9 mRNA表达,外周血单个核细胞(PBMC)经过非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸(CpG ODN)和不含非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸(non-CpG ODN)、空白培养三种形式培养72 h.采用3H-TdR掺入法测定PBMC增值变化,采用流式细胞仪检测T细胞亚群比例、T细胞表面CD69分子变化及CD8+T细胞内IFN-γ和IL-4的表达.采用ELISA法对PBMC上清液IFN-α的浓度,并评价其对氯喹和抑制性ODN的反应程度.结果 急性肺损伤患者PBMC表达TLR9 mRNA的表达强度为(0.75±0.08),与健康组(0.76±0.09)比较差异无统计学意义(P＞0.05);CpG ODN诱导急性肺损伤患者PBMC增殖(P＜0.05),增加CD3+T细胞表面CD69分子的表达(P＜0.05);增强CD8+T细胞产生IFN-γ的能力(P＜0.05);增强CD4+T/CD8+T比值(P＜0.05);CpG ODN可促进IFN-α的分泌(P＜0.05);氯喹和抑制性ODN会抑制CpG ODN产生IFN-α.结论 TLR9参与急性肺损伤患者的免疫调节,其激活效应包括促进PMBC活化、增加PBMC中CD4+T的比例,诱导并增殖IFN-α产生,促进CD8+T分泌IFN-γ.