中国全科医学2017，Vol.20Issue(21)：2603-2608,6.DOI:10.3969/j.issn.1007-9572.2017.21.009
脂多糖诱导下慢性阻塞性肺疾病大鼠模型远端肺动脉平滑肌细胞中Toll样受体4表达情况研究
Expression of the Toll-like Receptor-4 in Distal Pulmonary Artery Smooth Muscle Cells of Lipopolysaccharide-induced Chronic Obstructive Pulmonary Disease Model Rats
摘要
Abstract
Background Chronic obstructive pulmonary disease(COPD) is a lung disease that includes two main types,chronic bronchitis and emphysema,characterized by chronic incompletely reversible airflow.It is one of the leading causes of pulmonary hypertension.However,its occurrence and development mechanism is not yet fully clear.Objective To explore the TLR-4 expression in distal pulmonary artery smooth muscle cells(PASMCs) of COPD model rats induced by lipopolysaccharide(LPS),so as to provide a theoretical basis for studying the role of TLR-4 signaling pathway in inflammation and immune response to COPD.Methods This study was conducted from December 2015 to March 2016.Twenty-four SPF grade Wistar rats(half males and half females) aging between 6 and 8 weeks were randomly divided into normal group and model group,12 rats in each after feeding adaptively for 1 week.Rats in model group and normal group were respectively administered with 200 μl LPS(1 μg/ml),and 200 μl sodium chloride(0.9%) solutions,via the trachea on the 1st and the 14th day after intervention,and treated by passive inhaling of cigarette smoke in plexiglass cabin for 1 h once daily for 8 weeks.After the intervention,2 rats respectively randomly selected from both groups were sacrificed and the lung tissues were took out for observing the pathological changes via HE staining,and TLR4 expression in PASMCs determined with immunohistochemical(IHC) staining.Six model rats ranomly selected from remaining rats were sacrificed and the distal PASMCs were isolated and cultured.Cultured cells at passages 3-6(logarithmic phase) were used for experiments.The morphology of PASMCs was observed under the optical microscope(the images were magnified 100 times) and α-SM-actin expression in PASMCs stained by immunohistochemistry was observed under the immunofluorescence microscope(the images were magnified 200 times),then they were randomly divided into control group(no intervention),LPS 12 h group(treatment with 10 μl LPS of 1 μg/ml for 12 h),LPS 24 h group(treatment with 10 μl LPS of 1 μg/ml for 24 h),LPS 48 h group(treatment with 10 μl LPS of 1 μg/ml for 48 h) and LPS 72 h group(treatment with 10 μl LPS of 1 μg/ml for 72 h).The TLR-4 protein expression in PASMCs in these 5 groups were measured with Western blotting.Results The pathological changes in lung tissues found in the model group were rupture of alveolar walls,alveolar fusion,formation of bullae,dramatically thickened PASMCs layer in artery,a large number of inflammatory cell infiltration,all these were consistent with COPD typical features.TLR-4 expression in pulmonary artery smooth muscle layer:under the inverted phase contrast microscope,in model group,the TLR-4 expression in pulmonary artery smooth muscle layer was seen to be positive,the pulmonary artery smooth muscle layer was yellow,and it was much deeper than that in the normal group.PASMCs morphology and their α-SM-actin expression:under optical microscope,PASMCs were spindle shaped and presented irregular growth.With the extension of culture time,the number of layers were increased,leading to the formation of "peak valley";under immunofluorescence microscopy,it could be seen that α-SM-actin expression in PASMCs was positive,the cytoplasm had a radial distribution of filaments to the cell poles,parallel to the long axis of the cell,the morphology was clear.TLR-4 expression in PASMCs of LPS 12 h group,LPS 24 h group,LPS 48 h group and LPS 72 h group was higher than that of control group(P<0.05);TLR4 expression in PASMCs of LPS 24 h group,LPS 48 h group and LPS 72 h group was higher than that of LPS 12 h group(P<0.05);TLR4 expression in PASMCs of LPS 48 h group and LPS 72 h group was higher than that of LPS 24 h group(P<0.05).Conclusion TLR-4 expression level increases in distal PASMCs of COPD model rats induced by LPS;it is speculated that LPS could induce the synthesis and secretion of PASMCs through the TLR4 signaling pathway,which aggravated the inflammatory response and pulmonary vascular remodeling.关键词
肺疾病,慢性阻塞性/脂多糖类/肌细胞,平滑肌/肺动脉/Toll样受体4Key words
Pulmonary disease/chronic obstructive/Lipopolysaccharides/Myocytes/smooth muscle/Pulmonary artery/Toll-like receptor 4分类
医药卫生引用本文复制引用
王鹏雁,蒋明,王昌明,韩旭惠,李璐..脂多糖诱导下慢性阻塞性肺疾病大鼠模型远端肺动脉平滑肌细胞中Toll样受体4表达情况研究[J].中国全科医学,2017,20(21):2603-2608,6.基金项目
国家自然科学基金资助项目(81360010) （81360010）
广西壮族自治区卫生厅重点科研课题(重2012004) （重2012004）
