本文選題：肺疾病 + 慢性阻塞性��； 參考：《中國全科醫(yī)學(xué)》2017年21期

【摘要】：背景慢性阻塞性肺疾病(COPD)是以不完全可逆的氣流受限為特征的慢性支氣管炎和肺氣腫,也是導(dǎo)致肺動脈高壓的主要原因之一,但其發(fā)生發(fā)展機(jī)制尚未完全清楚。目的探討脂多糖(LPS)誘導(dǎo)下COPD大鼠模型遠(yuǎn)端肺動脈平滑肌細(xì)胞(PASMCs)中Toll樣受體(TLR)4表達(dá)情況,以期為探討TLR4信號通路在COPD炎性反應(yīng)及免疫應(yīng)答中的作用提供理論基礎(chǔ)。方法 2015年12月—2016年3月,選取SPF級Wistar大鼠24只,雌雄對半,6~8周齡。適應(yīng)性飼養(yǎng)大鼠1周后,將其隨機(jī)分為模型組和正常組,各12只。模型組大鼠于實(shí)驗(yàn)第1、14天經(jīng)氣道注入1μg/ml的LPS 200μl,置于自制有機(jī)玻璃艙,煙熏1 h/d,共8周;正常組大鼠于實(shí)驗(yàn)第1、14天經(jīng)氣道注入等量0.9%氯化鈉溶液,與模型組大鼠在同等條件下飼養(yǎng)8周。8周后,兩組分別隨機(jī)選取2只大鼠,開胸取肺組織,分別進(jìn)行HE染色觀察肺組織病理學(xué)改變和免疫組織化學(xué)染色觀察肺動脈平滑肌層TLR4表達(dá)情況。從模型組剩余大鼠中隨機(jī)選取6只,分離、培養(yǎng)遠(yuǎn)端PASMCs,選用第3~6代(對數(shù)生長期)細(xì)胞,光鏡下(×100)觀察PASMCs形態(tài),采用免疫熒光法(熒光顯微鏡下,×200)觀察其α-肌動蛋白表達(dá)情況,并隨機(jī)分為對照組(不進(jìn)行干預(yù))、LPS 12 h組(加入1μg/ml的LPS 10μl作用12 h)、LPS 24 h組(加入1μg/ml的LPS 10μl作用24 h)、LPS 48 h組(加入1μg/ml的LPS 10μl作用48 h)、LPS 72 h組(加入1μg/ml的LPS 10μl作用72 h),采用Western blotting法檢測各組PASMCs中TLR4表達(dá)水平。結(jié)果肺組織病理學(xué)改變:模型組肺泡壁斷裂,肺泡融合,肺大泡形成,肺動脈平滑肌層明顯增厚,大量炎性細(xì)胞浸潤,病理表現(xiàn)符合典型的COPD病理改變。肺動脈平滑肌層TLR4表達(dá)情況:模型組倒置相差顯微鏡下可見TLR4表達(dá)陽性,且肺動脈平滑肌層染成黃色較正常組顏色明顯加深。PASMCs形態(tài)及其α-肌動蛋白表達(dá)情況:光鏡下PASMCs呈梭形、不規(guī)則生長,隨著培養(yǎng)時間的延長,層數(shù)增多,堆積形成"峰-谷"狀;熒光顯微鏡下可見PASMCs胞質(zhì)α-肌動蛋白表達(dá)陽性,胞質(zhì)中有向細(xì)胞兩極呈放射狀分布的條絲狀物,與細(xì)胞長軸平行,形態(tài)清晰。LPS 12 h組、LPS 24 h組、LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于對照組(P0.05);LPS 24 h組、LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于LPS 12 h組(P0.05);LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于LPS 24 h組(P0.05)。結(jié)論 LPS誘導(dǎo)下COPD大鼠模型遠(yuǎn)端PASMCs中TLR4表達(dá)水平升高,猜測LPS可能通過TLR4信號通路誘導(dǎo)PASMCs的合成分泌功能,從而加重炎性反應(yīng)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 偽 -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 偽 -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 偽 -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.
【作者單位】： 桂林醫(yī)學(xué)院附屬醫(yī)院呼吸科;桂林市人民醫(yī)院;桂林醫(yī)學(xué)院附屬醫(yī)院病理科;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(81360010) 廣西壯族自治區(qū)衛(wèi)生廳重點(diǎn)科研課題(重2012004)
【分類號】：R-332;R563.9

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