TLR4表达水平与COPD大鼠肺动脉平滑肌细胞合成分泌功能的关系

摘要

Objective To investigate the relationship between Toll-like receptor 4 (TLR4) expression level of rat pulmonary artery smooth muscle cells with chronic obstructive pulmonary disease(COPD) and its synthesis and secretion function.Methods The rat model of COPD was established.HE staining was used to determine whether the model was established successfully;Immunohistochemical staining was used to observe TLR4 expression in pulmonary artery smooth muscle layer of the control group and model group;primarily cultured and identified pulmonary artery smooth muscle (PASMCs),lipopolysaccharide (LPS),TAK-242 the specificity inhibitors of TLR4 block TLR4,groups as follows:the control group,LPS stimulation group,TAK-242 group,LPS + TAK-242 group,Western blot was used to detect the expression of TLR4 in PASMCs by LPS,Enzyme-linked immunosorbent assay (ELISA) analysis were used to detect the secretion of interferonr-γ (IFN)-γ,interleukin(IL)-6,platelet derived growth factor (PDGF) of rat distal PASMCs in each group.Results Normal rats lung tissue express TLR4,however TLR4 expression level in model group of COPD rats lung tissue obviously higher than normal group (P ＜0.05).The levels of IFN-γ,IL-6 and PDGF in the PPSMCs of LPS group were significantly higher than those in the control group TLR4 expression level was positively related to the concentration of IFN-γ,IL-6 and PDGF (r =0.91,0.89,0.83,P ＜ 0.05).Conclusion TLR4 may participate in regulating the synthesis and secretion of PASMCs.%目的 探讨Toll样受体4(TLR4)表达水平与慢性阻塞性肺疾病(COPD)大鼠肺动脉平滑肌细胞(PASMCs)合成分泌功能的关系.方法 建立大鼠COPD模型,HE染色判定模型建立,免疫组织化学染色观察对照组与COPD模型组肺组织动脉平滑肌层TLR4的表达.分离培养鉴定原代PASMCs,脂多糖(LPS)、TLR4的特异性抑制剂TAK-242干预细胞,实验分组:空白组、LPS组、TAK-242组、LPS+ TAK-242组,Western blot检测各组PASMCs中TLR4的表达,ELISA法检测各组细胞培养上清液中Th1细胞因子干扰素(IFN)-γ、Th2细胞因子白介素(IL)-6及血小板衍化生长因子(PDGF)的浓度;并将TLR4的表达水平与上清液中炎性因子的分泌水平进行相关性分析.结果 COPD模型组大鼠肺组织动脉平滑肌层TLR4的表达明显高于正常大鼠;与空白组比较,LPS组PASMCs中TLR4的表达明显升高(P＜0.05);LPS组PASMCs中合成分泌IFN-γ、IL-6、PDGF的水平较对照组明显升高,TAK-242阻断TLR4,PASMCs中TLR4的表达明显降低(P＜0.05);细胞培养上清液中IFN-γ、IL-6、PDGF的表达水平较空白组明显降低(P＜0.05);TLR4表达水平与细胞培养上清液IFN-γ、IL-6及PDGF的浓度呈显著正相关性(r =0.95、0.87、0.83,P ＜0.05).结论 TLR4可能参与调控PASMCs的合成分泌功能.