王鵬雁，蔣 明，王昌明*，韓旭惠，李 璐

·論著·

脂多糖誘導(dǎo)下慢性阻塞性肺疾病大鼠模型遠(yuǎn)端肺動脈平滑肌細(xì)胞中Toll樣受體4表達(dá)情況研究

王鵬雁1，蔣 明1，王昌明1*，韓旭惠1，李 璐2

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1 μg/ml的LPS 200 μl，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1 μg/ml的LPS 10 μl作用12 h)、LPS 24 h組(加入1 μg/ml的LPS 10 μl作用24 h)、LPS 48 h組(加入1 μg/ml的LPS 10 μl作用48 h)、LPS 72 h組(加入1 μg/ml的LPS 10 μl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(shí)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(P<0.05)；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(P<0.05)；LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于LPS 24 h組(P<0.05)。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。

肺疾病,慢性阻塞性；脂多糖類；肌細(xì)胞,平滑肌；肺動脈；Toll樣受體4

王鵬雁,蔣明,王昌明,等.脂多糖誘導(dǎo)下慢性阻塞性肺疾病大鼠模型遠(yuǎn)端肺動脈平滑肌細(xì)胞中Toll樣受體4表達(dá)情況研究[J].中國全科醫(yī)學(xué)，2017，20(21)：2603-2608.[www.chinagp.net]

WANG P Y,JIANG M,WANG C M,et al.Expression of the Toll-like receptor-4 in distal pulmonary artery smooth muscle cells of lipopolysaccharide-induced chronic obstructive pulmonary disease model rats[J].Chinese General Practice，2017，20(21)：2603-2608.

慢性阻塞性肺疾病(COPD)是一種常見的慢性疾病，其主要病理特征為血管炎性反應(yīng)和血管重塑。肺動脈平滑肌細(xì)胞(PASMCs)是肺血管重塑的主要效應(yīng)細(xì)胞，在炎癥、創(chuàng)傷等刺激下，其合成、分泌功能會受到影響。Toll樣受體(TLR)是一類天然免疫受體，其突出的生物學(xué)功能是促進(jìn)細(xì)胞因子的合成與釋放，引發(fā)炎性反應(yīng)。本研究組前期研究顯示，在哮喘的氣道重塑中TLR4 /核因子κB(NF-κB)信號通路可以調(diào)控支氣管平滑肌細(xì)胞合成、分泌炎性因子[1]。但目前TLR4在PASMCs中的功能尚未見報(bào)道。本研究通過建立COPD大鼠模型，分離培養(yǎng)大鼠模型遠(yuǎn)端PASMCs，探究COPD大鼠肺動脈平滑肌層TLR4表達(dá)情況以及脂多糖(LPS)誘導(dǎo)下COPD大鼠模型遠(yuǎn)端PASMCs中TLR4表達(dá)水平，旨在為探討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)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

1.1.1 實(shí)驗(yàn)動物 2015年12月—2016年3月，選取SPF級Wistar大鼠24只，雌雄對半，體質(zhì)量(200±20)g，6～8周齡，購自桂林醫(yī)學(xué)院科學(xué)實(shí)驗(yàn)中心，動物合格證號：SCXKG 2014-0001。

1.1.2 主要儀器及試劑 LPS(美國Sigma 公司)，翻蓋紅色金牌黃果樹香煙(貴陽卷煙廠，焦油量15 mg，煙氣煙堿量1.2 mg)，DMEM培養(yǎng)基、胎牛血清(美國Gibco公司)，TLR4鼠單克隆抗體(美國Abcam公司)，辣根過氧化物酶標(biāo)記的羊抗鼠二抗、β-actin小鼠單克隆抗體(北京中杉金橋生物技術(shù)有限公司)，總蛋白提取試劑盒(上海碧云天生物科技有限公司)，DAB顯色試劑盒(武漢博士德生物工程有限公司)，自制有機(jī)玻璃艙(91 cm×42 cm×62 cm)，倒置相差顯微鏡(日本OLYMPUS公司)，蛋白電泳儀、蛋白轉(zhuǎn)膜儀、全自動酶標(biāo)儀(美國Bio-Rad公司)。

1.2 實(shí)驗(yàn)方法

1.2.1 COPD大鼠模型的建立 大鼠適應(yīng)性飼養(yǎng)(飼養(yǎng)溫度保持在20～25 ℃，相對濕度維持在50%～65%，自由進(jìn)食、飲水)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1 μl/ml的LPS 200 μl，置于自制有機(jī)玻璃艙，采用翻蓋紅色金牌黃果樹香煙煙熏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。

1.2.2 HE染色觀察模型組和正常組肺組織病理學(xué)改變、免疫組織化學(xué)染色觀察模型組和正常組肺動脈平滑肌層TLR4表達(dá)情況 8周后，兩組分別隨機(jī)選取2只大鼠，腹腔注射10%水合氯醛(3～4 ml/100 g)麻醉，75%乙醇浸泡15 min，開胸取心肺，4%多聚甲醛固定24 h，組織脫水、石蠟包埋、切片、脫蠟。兩組分別取一部分肺組織進(jìn)行HE染色，倒置相差顯微鏡(×100)下觀察大鼠肺組織病理學(xué)改變。分別取兩組剩余肺組織，采用10%胎牛血清封閉液封閉孵育30 min，TLR4一抗(TLR4鼠單克隆抗體，10%胎牛血清封閉液稀釋，滴度1∶800)孵育4 ℃過夜，辣根過氧化物酶標(biāo)記的羊抗鼠二抗稀釋液(滴度1∶200)室溫孵育30 min；予DAB顯色試劑盒進(jìn)行顯色，中性樹膠封固，倒置相差顯微鏡(×400)下觀察正常組、模型組大鼠肺動脈平滑肌層TLR4表達(dá)情況。胞質(zhì)呈黃色定義為TLR4表達(dá)陽性。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

1.2.3 遠(yuǎn)端PASMCs分離、培養(yǎng)及鑒定 8周后，從模型組剩余大鼠中隨機(jī)選取6只，腹腔注射10%水合氯醛(3～4 ml/100 g)麻醉，75%乙醇浸泡15 min，開胸取心肺，取肺動脈3級以下分支，分離血管中膜，將中膜組織剪成盡可能碎的組織塊，加入0.2%Ⅰ型膠原酶，37 ℃水浴消化15～20 min，1 000 r/min離心5 min(離心半徑8 cm)，吸出消化酶，加入含25%胎牛血清的DMEM培養(yǎng)基，置于37 ℃、5% CO2培養(yǎng)箱培養(yǎng)，4 d左右可見細(xì)胞從組織塊周圍萌出，10 d左右細(xì)胞生長融合達(dá)培養(yǎng)瓶的90%以上時(shí)即消化傳代，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)鑒定其α-肌動蛋白表達(dá)情況(胞質(zhì)染成紅色，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)胞長軸平行定義為α-肌動蛋白表達(dá)陽性)；并隨機(jī)分為對照組(不進(jìn)行干預(yù))、LPS 12 h組(加入1 μg/ml的LPS 10 μl作用12 h)、LPS 24 h組(加入1 μg/ml的LPS 10 μl作用24 h)、LPS 48 h組(加入1 μg/ml的LPS 10 μl作用48 h)、LPS 72 h組(加入1 μg/ml的LPS 10 μl作用72 h)，用于下述實(shí)驗(yàn)。

1.2.4 Western blotting法檢測對照組、LPS 12 h組、LPS 24 h組、LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平 取對照組、LPS 12 h組、LPS 24 h組、LPS 48 h組、LPS 72 h組PASMCs接種于10 cm的培養(yǎng)皿中，當(dāng)細(xì)胞數(shù)量達(dá)70%時(shí)，各組加入蛋白裂解液裂解細(xì)胞，采用總蛋白提取試劑盒提取總蛋白，采用全自動酶標(biāo)儀檢測蛋白濃度；采用蛋白電泳儀進(jìn)行聚丙烯酰胺凝膠電泳：取10 μg蛋白質(zhì)樣品上樣，先以80 V電壓電泳，溴酚藍(lán)進(jìn)入分離膠后，改電壓為120 V；采用蛋白轉(zhuǎn)膜儀進(jìn)行濕式轉(zhuǎn)膜，取出聚偏氟乙烯(PVDF)膜，加入封閉液，將其放入搖床上室溫孵育2 h；加入一抗〔TLR4鼠單克隆抗體(稀釋濃度為1∶500)和β-actin小鼠單克隆抗體(稀釋濃度為1∶1 000)〕，以β-actin為內(nèi)參照，4 ℃搖床過夜；TBST漂洗3次，10 min/次，加入二抗(羊抗鼠IgG，稀釋濃度為1：5 000)，搖床上室溫孵育1 h；TBST漂洗3次，10 min/次；加入A、B顯色液，采用ECL超敏發(fā)光儀檢測各組PASMCs中TLR4表達(dá)水平。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

2 結(jié)果

2.1 肺組織病理學(xué)改變 正常組：肺泡結(jié)構(gòu)完整，無炎性細(xì)胞浸潤(見圖1A，本文圖1～4彩圖見本刊官網(wǎng)www.chinagp.net電子期刊相應(yīng)文章)；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(見圖1B)。

2.2 肺動脈平滑肌層TLR4表達(dá)情況 正常組：倒置相差顯微鏡下可見少量TLR4表達(dá)陽性(見圖2A)。模型組：倒置相差顯微鏡下可見TLR4呈陽性表達(dá)，且其肺動脈平滑肌層染成黃色部分較正常組顏色明顯加深(見圖2B)。

注：1A為正常組，1B為模型組

圖1 正常組和模型組大鼠肺組織病理學(xué)改變(HE染色，×100)

Figure 1 Pathological changes in lung tissues of normal group and model group

注：2A為正常組，2B為模型組，圖中箭頭指向肺動脈平滑肌層

圖2 正常組和模型組大鼠肺動脈平滑肌層TLR4表達(dá)情況(免疫組織化學(xué)染色，×400)

Figure 2 TLR4 expression in pulmonary artery smooth muscle layer of normal group and model group

2.3 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(shí)間的延長，層數(shù)增多，堆積形成“峰-谷”狀(見圖3)。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(見圖4)。

2.4 PASMCs中TLR4表達(dá)水平 對照組、LPS 12 h組、LPS 24 h組、LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中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，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；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，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于LPS 24 h組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表1、圖5)。

3 討論

COPD是一種以氣流受限為特征的肺部疾病，主要表現(xiàn)為肺功能加速下降，嚴(yán)重影響患者生活質(zhì)量，并給患者帶來沉重經(jīng)濟(jì)負(fù)擔(dān)[2]，血管炎性反應(yīng)和肺血管重塑是其主要病理特征。肺血管重塑主要為內(nèi)皮損傷、平滑肌細(xì)胞增殖、細(xì)胞外基質(zhì)重塑；而小血管重塑主要表現(xiàn)為平滑肌細(xì)胞增殖，研究表明，當(dāng)受到致病因素刺激時(shí)，PASMCs發(fā)生表型轉(zhuǎn)化，細(xì)胞的增殖活性、合成分泌基質(zhì)蛋白及炎性因子的活性增加，并促進(jìn)PASMCs的增殖和遷移，成纖維細(xì)胞分化，進(jìn)而促進(jìn)肺血管重塑[3-5]。

TLR是一類天然的免疫受體，可以通過調(diào)節(jié)抗原遞呈細(xì)胞誘導(dǎo)抗原特異性免疫反應(yīng)，成為連接天然免疫和獲得性免疫的橋梁。TLR4是TLR家族中的重要成員之一，也是第一個(gè)被發(fā)現(xiàn)的TLR，屬于細(xì)胞膜Ⅰ型跨膜糖蛋白受體，可被病原微生物的LPS及肽聚糖(PGN)等激活，產(chǎn)生細(xì)胞因子、趨化因子、黏附分子及急性期蛋白來調(diào)節(jié)炎性反應(yīng)。TLR4主要分布于氣道上皮細(xì)胞、肺巨噬細(xì)胞、中性粒細(xì)胞、血管內(nèi)皮細(xì)胞及氣道平滑肌細(xì)胞[6-8]，當(dāng)肺組織受到致病因素刺激時(shí)，炎性細(xì)胞及組織細(xì)胞上TLR4可識別相應(yīng)的配體，主要通過髓樣分化因子88(MyD88)依賴性通路和MyD88非依賴性通路活化NF-κB，從而介導(dǎo)生物局部炎癥激活炎性細(xì)胞因子。尤其是腫瘤壞死因子α(TNF-α)和白介素1(IL-1)等原發(fā)性遞質(zhì)的增加，誘導(dǎo)肺泡巨噬細(xì)胞產(chǎn)生大量繼發(fā)性炎性細(xì)胞因子和遞質(zhì)，如白介素6(IL-6)、白介素8(IL-8)等，進(jìn)一步誘導(dǎo)中性粒細(xì)胞和CD8+T淋巴細(xì)胞參與COPD的炎性反應(yīng)，與COPD的發(fā)病密切相關(guān)。有研究表明，TLR4信號通路可以調(diào)控氧化型低密度脂蛋白、血小板衍生生長因子(PDGF)等誘導(dǎo)大鼠血管平滑肌細(xì)胞合成分泌因子的釋放，從而參與冠狀動脈粥樣硬化的發(fā)生發(fā)展[9-10]，說明TLR4信號通路與血管重塑有密切關(guān)系。本實(shí)驗(yàn)首先構(gòu)建COPD大鼠模型，通過HE染色發(fā)現(xiàn)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，提示COPD模型組造模成功；在此基礎(chǔ)上再通過免疫組織化學(xué)染色觀察模型組和正常組肺動脈平滑肌層TLR4表達(dá)情況，結(jié)果顯示：模型組倒置相差顯微鏡下可見TLR4呈陽性表達(dá)，且其肺動脈平滑肌層染成黃色部分較正常組顏色明顯加深，說明肺動脈平滑肌中TLR4表達(dá)與COPD早期肺血管重塑也存在一定的關(guān)系。

圖3 光鏡下觀察PASMCs形態(tài)(×100)

Figure 3 Morphology of PASMCs observed under an optical microscope

圖4 熒光顯微鏡觀察PASMCs胞質(zhì)α-肌動蛋白表達(dá)情況(×200)

Figure 4 α-SM-actin expression in PASMCs observed under an immunofluorescence microscope

注：LPS=脂多糖，TLR=Toll樣受體

圖5 Western blotting法檢測PASMCs中TLR4表達(dá)水平的聚丙烯酰胺凝膠電泳圖

Figure 5 Polyacrylamide gel electrophoresis of TLR4 expression in PASMCs detected by Western blotting

Table 1 Comparison of TLR4 expression in PASMCs among control group,LPS 12 h group,LPS 24 h group,LPS 48 h group and LPS 72 h group

組別TLR4對照組0.13±0.03LPS12h組0.27±0.04aLPS24h組0.54±0.04abLPS48h組0.86±0.03abcLPS72h組0.80±0.03abcF值177.71P值<0.001

注：LPS=脂多糖，TLR=Toll樣受體；與對照組比較，aP<0.05；與LPS 12 h組比較，bP<0.05；與LPS 24 h組比較，cP<0.05

LPS是革蘭陰性菌細(xì)胞壁的主要成分，由類脂A、核心多糖和O-特異側(cè)鏈3部分組成。脂質(zhì)A(Lipid A)為構(gòu)成內(nèi)毒素活性的LPS。LPS可以激活與炎性反應(yīng)相關(guān)的細(xì)胞(如巨噬細(xì)胞、單核細(xì)胞、平滑肌細(xì)胞等)，引起這些細(xì)胞的細(xì)胞因子合成、釋放，從而導(dǎo)致炎性反應(yīng)的發(fā)生。大量研究顯示，生物的炎性反應(yīng)與LPS誘導(dǎo)下TLR4/NF-κB信號通路密切相關(guān)[11-13]。同時(shí)本研究組前期研究發(fā)現(xiàn)，LPS可促進(jìn)COPD大鼠模型PASMCs合成分泌炎性細(xì)胞因子，加重炎性反應(yīng)[1]。國外有研究顯示，在巨噬細(xì)胞表面TLR4與LPS相結(jié)合，啟動信號通路，產(chǎn)生大量的炎性細(xì)胞因子；其中某些細(xì)胞因子如IL-6、干擾素(IFN)β、IFN-γ等可以誘導(dǎo)負(fù)性調(diào)節(jié)分子〔如細(xì)胞因子信號抑制蛋白(SOCS-1)〕的產(chǎn)生；反過來，負(fù)性調(diào)節(jié)分子又反饋性通過阻斷TLR4/NF-κB信號通路活化，進(jìn)而抑制促炎因子的產(chǎn)生，防止巨噬細(xì)胞在細(xì)菌性感染時(shí)發(fā)生過度炎性反應(yīng)[14-15]。本研究構(gòu)建COPD大鼠模型后，分離培養(yǎng)并鑒定原代PASMCs，光鏡下可見PASMCs呈梭形、不規(guī)則生長，隨著培養(yǎng)時(shí)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；Western blotting法檢測PASMCs中TLR4表達(dá)水平，結(jié)果顯示，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，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，LPS 48 h組、LPS 72 h組PASMCs中TLR4表達(dá)水平均高于LPS 24 h組，LPS 48 h組PASMCs中TLR4表達(dá)水平與LPS 72 h組無差異，說明TLR4表達(dá)水平在48 h達(dá)高峰，可能與大量炎性遞質(zhì)釋放對PASMCs中TLR4的表達(dá)起負(fù)反饋調(diào)節(jié)作用有關(guān)。另一方面可能本研究使用的是原代PASMCs，其生長及繁殖有一定的局限性；LPS誘導(dǎo)期間未予以換液，亦不能排除藥物作用72 h后細(xì)胞狀態(tài)不佳的可能。故本研究結(jié)果說明LPS誘導(dǎo)可增加PASMCs中TLR4表達(dá)水平；高TLR4表達(dá)水平可能與PASMCs合成分泌功能有關(guān)，阻斷TLR4的表達(dá)有可能會控制過度炎性反應(yīng)的發(fā)生，從而保護(hù)機(jī)體免受傷害。

本研究僅是初步觀察COPD大鼠模型PASMCs中TLR4表達(dá)的變化，但TLR4在COPD發(fā)生發(fā)展中的具體作用機(jī)制尚有待進(jìn)一步研究。

綜上所述，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。故后續(xù)研究工作將深入了解TLR4信號通路在COPD大鼠模型PASMCs中的作用及機(jī)制，以LPS誘導(dǎo)下TLR4/NF-κB信號通路中的某些環(huán)節(jié)的干預(yù)為靶點(diǎn)研究新的藥物，為臨床COPD早期治療提供新思路。

作者貢獻(xiàn)：王昌明進(jìn)行文章的構(gòu)思與設(shè)計(jì)，負(fù)責(zé)文章的質(zhì)量控制及審校，對文章整體負(fù)責(zé)，監(jiān)督管理；蔣明、王昌明進(jìn)行研究的實(shí)施與可行性分析；王鵬雁進(jìn)行數(shù)據(jù)收集，統(tǒng)計(jì)學(xué)處理，結(jié)果的分析與解釋，撰寫論文，英文的修訂；王鵬雁、韓旭惠進(jìn)行數(shù)據(jù)整理；王昌明、蔣明進(jìn)行論文的修訂；李璐進(jìn)行病理圖片的審閱。

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(本文編輯：崔麗紅)

Expression of the Toll-like Receptor-4 in Distal Pulmonary Artery Smooth Muscle Cells of Lipopolysaccharide-induced Chronic Obstructive Pulmonary Disease Model Rats

WANGPeng-yan1,JIANGMing1,WANGChang-ming1*,HANXu-hui1,LILu2

1.DepartmentofRespiration,AffiliatedHospitalofGuilinMedicalUniversity,Guilin541001,China2.DepartmentofPathology,AffiliatedHospitalofGuilinMedicalUniversity,Guilin541001,China

*Correspondingauthor:WANGChang-ming,Chiefphysician;E-mail:wistarrat@163.com

Note:WANGChang-mingisnowworkingatGuilinPeople′sHospital

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.

Pulmonary disease,chronic obstructive;Lipopolysaccharides;Myocytes,smooth muscle;Pulmonary artery;Toll-like receptor 4

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)

R 563.9

A

10.3969/j.issn.1007-9572.2017.21.009

2017-01-11；

2017-03-23)

1.541001 廣西桂林市，桂林醫(yī)學(xué)院附屬醫(yī)院呼吸科

2.541001 廣西桂林市，桂林醫(yī)學(xué)院附屬醫(yī)院病理科

*通信作者：王昌明，主任醫(yī)師；E-mail：wistarrat@163.com

注：王昌明現(xiàn)工作單位為桂林市人民醫(yī)院