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

发布时间：2018-06-01 01:48

本文选题：肺疾病 + 慢性阻塞性　；参考：《中国全科医学》2017年21期

【摘要】：背景慢性阻塞性肺疾病(COPD)是以不完全可逆的气流受限为特征的慢性支气管炎和肺气肿,也是导致肺动脉高压的主要原因之一,但其发生发展机制尚未完全清楚。目的探讨脂多糖(LPS)诱导下COPD大鼠模型远端肺动脉平滑肌细胞(PASMCs)中Toll样受体(TLR)4表达情况,以期为探讨TLR4信号通路在COPD炎性反应及免疫应答中的作用提供理论基础。方法 2015年12月—2016年3月,选取SPF级Wistar大鼠24只,雌雄对半,6~8周龄。适应性饲养大鼠1周后,将其随机分为模型组和正常组,各12只。模型组大鼠于实验第1、14天经气道注入1μg/ml的LPS 200μl,置于自制有机玻璃舱,烟熏1 h/d,共8周;正常组大鼠于实验第1、14天经气道注入等量0.9%氯化钠溶液,与模型组大鼠在同等条件下饲养8周。8周后,两组分别随机选取2只大鼠,开胸取肺组织,分别进行HE染色观察肺组织病理学改变和免疫组织化学染色观察肺动脉平滑肌层TLR4表达情况。从模型组剩余大鼠中随机选取6只,分离、培养远端PASMCs,选用第3~6代(对数生长期)细胞,光镜下(×100)观察PASMCs形态,采用免疫荧光法(荧光显微镜下,×200)观察其α-肌动蛋白表达情况,并随机分为对照组(不进行干预)、LPS 12 h组(加入1μg/ml的LPS 10μl作用12 h)、LPS 24 h组(加入1μg/ml的LPS 10μl作用24 h)、LPS 48 h组(加入1μg/ml的LPS 10μl作用48 h)、LPS 72 h组(加入1μg/ml的LPS 10μl作用72 h),采用Western blotting法检测各组PASMCs中TLR4表达水平。结果肺组织病理学改变:模型组肺泡壁断裂,肺泡融合,肺大泡形成,肺动脉平滑肌层明显增厚,大量炎性细胞浸润,病理表现符合典型的COPD病理改变。肺动脉平滑肌层TLR4表达情况:模型组倒置相差显微镜下可见TLR4表达阳性,且肺动脉平滑肌层染成黄色较正常组颜色明显加深。PASMCs形态及其α-肌动蛋白表达情况:光镜下PASMCs呈梭形、不规则生长,随着培养时间的延长,层数增多,堆积形成"峰-谷"状;荧光显微镜下可见PASMCs胞质α-肌动蛋白表达阳性,胞质中有向细胞两极呈放射状分布的条丝状物,与细胞长轴平行,形态清晰。LPS 12 h组、LPS 24 h组、LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于对照组(P0.05);LPS 24 h组、LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于LPS 12 h组(P0.05);LPS 48 h组、LPS 72 h组PASMCs中TLR4表达水平均高于LPS 24 h组(P0.05)。结论 LPS诱导下COPD大鼠模型远端PASMCs中TLR4表达水平升高,猜测LPS可能通过TLR4信号通路诱导PASMCs的合成分泌功能,从而加重炎性反应及肺血管重塑。
[Abstract]:Background chronic obstructive pulmonary disease (COPD) is a chronic bronchitis and emphysema characterized by incomplete reversible airflow limitation, which is also one of the main causes of pulmonary hypertension. Objective to investigate the expression of Toll like receptor (TLR4) in the distal pulmonary artery smooth muscle cells of COPD rats induced by lipopolysaccharide (LPS), so as to provide a theoretical basis for the study of the role of TLR4 signaling pathway in the inflammatory response and immune response of COPD. Methods from December 2015 to March 2016, 24 Wistar rats of SPF grade were selected. After one week of adaptive feeding, the rats were randomly divided into model group and normal group, each with 12 rats. Rats in the model group were injected with LPS 200 渭 l of 1 渭 g/ml through the airway on the 1st day of the experiment and placed in a self-made plexiglass chamber for 1 h / d for 8 weeks, while the normal rats were injected the same amount of 0.9% sodium chloride solution through the airway on the 1st and 14th day of the experiment, and the rats in the control group were injected with 0.9% sodium chloride solution through the airway on the 1st and 14th day of the experiment. Two rats were randomly selected from the two groups after 8 weeks of feeding under the same conditions as the model group. The pathological changes of lung tissue and the expression of TLR4 in pulmonary artery smooth muscle layer were observed by HE staining and immunohistochemical staining respectively. From the remaining rats in the model group, 6 rats were randomly selected to isolate and culture the distal PASMCs, the 3rd passage (logarithmic growth phase) cells were selected, the morphology of PASMCs was observed under light microscope (脳 100), and the expression of 伪 -actin was observed by immunofluorescence method (fluorescence microscope, 脳 200). They were randomly divided into two groups: control group (LPS 10 渭 l added 1 渭 g/ml for 12 h) (LPS 10 渭 l added 1 渭 g/ml for 24 h) and LPS-48 h group (LPS 10 渭 l with 1 渭 g/ml for 48 h) (LPS 10 渭 l with 1 渭 g/ml for 72 h) The expression of TLR4 in PASMCs was detected by Western blotting method. Results in the model group, the alveolar wall rupture, alveolar fusion, alveolar formation, pulmonary smooth muscle layer thickening and inflammatory cell infiltration were observed. The pathological findings were consistent with the typical pathological changes of COPD. The expression of TLR4 in pulmonary artery smooth muscle layer: the positive expression of TLR4 was observed under inverted phase contrast microscope in model group, and the color of pulmonary artery smooth muscle layer stained yellow was significantly deeper than that of normal group. The morphology of PASMCs and the expression of 伪 -actin in pulmonary artery smooth muscle layer: PASMCs was fusiform under light microscope. Irregular growth, with the extension of culture time, the number of layers increased, forming a "peak-valley" shape, PASMCs cytoplasm 伪 -actin expression was positive under fluorescence microscope, and there were stripes of filaments in the cytoplasm with radial distribution toward the two poles of the cells. Parallel to the long axis of the cell, The expression of TLR4 in PASMCs of LP12h group was higher than that of control group P0.05 LP24h group LP72h group PASMCs expression level was higher than that of LPS 12h group LP0.05LP48-h PASMCs group. The expression level of P0.05 was higher than that of LPS 24 h group. Conclusion the expression of TLR4 in distal PASMCs of COPD rat model induced by LPS is increased. It is speculated that LPS may induce the synthesis and secretion of PASMCs through TLR4 signaling pathway, thus exacerbating inflammatory reaction and pulmonary vascular remodeling.
【作者单位】：桂林医学院附属医院呼吸科;桂林市人民医院;桂林医学院附属医院病理科;
【基金】：国家自然科学基金资助项目(81360010) 广西壮族自治区卫生厅重点科研课题(重2012004)
【分类号】：R-332;R563.9

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