間質(zhì)性肺疾病患者血清和支氣管肺泡灌洗液中內(nèi)皮素1活性的變化

1、間質(zhì)性肺疾病患者血清和支氣管肺泡灌洗液中內(nèi)皮素1活性的變化【摘要】目的評(píng)價(jià)內(nèi)皮素對(duì)肺纖維化發(fā)生、發(fā)展作用的影響。方法利用同位素放射免疫直接測(cè)定法, 檢測(cè)10例肺結(jié)節(jié)病和8例特發(fā)性肺纖維化(IPF)患者外周血和支氣管肺泡灌洗液(BALF)中內(nèi)皮素1(ET-1)的活性,并與8名健康非吸煙者進(jìn)行對(duì)照。結(jié)果肺結(jié)節(jié)病和IPF患者血清和BALF中的ET-1活性分別為(6229) ng/L,(17.02.4) ng/L和(7771) ng/L、(103) ng/L,與正常對(duì)照組(208) ng/L、（4.00.6） ng/L比較，差異有顯著性（P0.01);血清中ET-1活性與動(dòng)脈血氧分壓(PaO2)呈明顯

2、負(fù)相關(guān)(r=-0.538，P0.01);結(jié)節(jié)病組和IPF組BALF中的ET-1水平與BALF中細(xì)胞總數(shù)呈正相關(guān)(r=0.649, P0.01),肺結(jié)節(jié)病患者、IPF患者BALF中ET-1與淋巴細(xì)胞、中性粒細(xì)胞呈正相關(guān)(r=0.712，0.813, P均0.01)。結(jié)論ET-1在肺結(jié)節(jié)病和IPF發(fā)病機(jī)制中起著重要作用,并可作為疾病活動(dòng)性判定的一項(xiàng)重要參考指標(biāo)?！娟P(guān)鍵詞】肺疾病，間質(zhì)性內(nèi)皮素支氣管肺泡灌洗液Changes of the activities of ET-1 in the serum and BALF of ILD patients and their clinical signif

3、icanceLI Zhenhua, XIA Shuyue, HOU Xianming, et al. Institute of the Respiratory Disease, China Medical University, Shenyang 110001【Abstract】ObjectiveTo evaluate the roles of the Endothelin-1 (ET-1) in pathogenesis of the patients with interstitial lung disease (ILD). MethodsThe ET-1 activities in th

4、e serum and BALF from 10 patients with sarcoidosis, 7 with idiopathic pulmonary fibrosis (IPF) and 8 normal subjects non-smokers were determined using the radio-immunoassay. ResultsIn the activities of ET-1 in the serum and BALF in the patients with sarcoiosis (6229 and 17.02.4) ng/L and in the IPF

5、(7771 and 103) ng/L. was higher than those in the normal control subjects (208 and 4.00.6) ng/L. The activities of ET-1 in serum was negative correlated with PaO2(r=-0.538,P0.01). The level of ET-1 in the BALF of the patient was positive correlated with total cells in BALF (r=0.649, P0.01). The leve

6、ls of ET-1 in the BALF in the sarcoidosis was positive correlated with the percentage of lymphocyte (r=0.712, P0.01) but in patients with IPF was positive correlated with the percentage of neutrophil (r=0.813, P0.01). ConclusionsET-1 might play an important role in pathogenic of the patients with sa

7、rcoidosis and IPF. The levels of ET-1 can act as the markers of activity of disease.【Key words】Lung disease, interstitialEndothelin-1Bronchoalveolar lavage fluid彌漫性間質(zhì)性肺疾病(ILD)是一組以肺泡壁病變?yōu)橹鞯募膊∪?近年來(lái)發(fā)病率有明顯上升趨勢(shì),而發(fā)病機(jī)制至今尚不完全清楚,復(fù)雜的細(xì)胞因子網(wǎng)絡(luò)的相互作用在ILD肺纖維化的形成中可能起著重要作用。為進(jìn)一步探討內(nèi)皮素1(ET-1)在間質(zhì)性肺疾病發(fā)生發(fā)展過(guò)程中的作用,我們對(duì)10例肺結(jié)節(jié)病和8

8、例特發(fā)性肺纖維化(IPF)患者血清和支氣管肺泡灌洗液(BALF)中IL-1水平做了檢測(cè)。 對(duì)象與方法一、對(duì)象1.結(jié)節(jié)病組10例，男6例, 女4例, 平均年齡437歲。據(jù)中華醫(yī)學(xué)會(huì)呼吸病學(xué)分會(huì)1993年第三次修訂結(jié)節(jié)病診斷標(biāo)準(zhǔn), 其中期5例, 期4例和期1例。結(jié)核菌素試驗(yàn)陰性,血清血管緊張素轉(zhuǎn)換酶升高, 有6例經(jīng)支氣管肺活組織檢查; 3例經(jīng)淋巴結(jié)和1例經(jīng)皮膚活檢證實(shí)符合結(jié)節(jié)病的病理改變。IPF 8例，男5例，女3例，平均年齡4611歲，診斷標(biāo)準(zhǔn): 臨床有干咳和進(jìn)行性呼吸困難,杵狀指（趾）, 肺部聞及帛裂音, 胸部X線和CT呈彌漫間質(zhì)性改變,肺功能呈限制性通氣功能障礙, 彌散功能降低, 肺泡動(dòng)脈氧

9、分壓差增大和動(dòng)脈血氧分壓(PaO2)降低。病理組織學(xué)證實(shí)符合肺間質(zhì)纖維化改變。2.對(duì)照組8名，男5名，女3名，年齡2552歲，均因有胸痛或咽部異物感等自覺(jué)癥狀而要求作纖維支氣管鏡(纖支鏡)檢查，經(jīng)肺功能、胸部X線和纖支鏡檢查無(wú)異常所見(jiàn)的非吸煙者。二、方法1.支氣管肺泡灌洗(BAL)和灌洗液細(xì)胞總分?jǐn)?shù)檢測(cè):應(yīng)用日本Olympus P30型纖支鏡常規(guī)氣道檢查后, 于活檢和刷檢前做BAL, 所有病例均選用右肺中葉(B4或B5)或左肺舌葉做灌洗,將纖支鏡頂端嚴(yán)密楔入段或亞段支氣管開(kāi)口處,從活檢孔分2次注入37滅菌生理鹽水100 ml,立即在80100 mm Hg(1 mm Hg=0.133 kPa)負(fù)

10、壓下吸引回收液,一般回收液可達(dá)40%60%,回收液經(jīng)雙層紗布過(guò)濾，4，1 200 r/min，離心10分鐘, 分離細(xì)胞,上清液置于-70保存?zhèn)溆?。離心沉淀細(xì)胞用Hanks液沖洗離心12次，并調(diào)整細(xì)胞數(shù)為5109/L, 在Neubauer數(shù)臺(tái)上計(jì)細(xì)胞總數(shù); 然后用特制的細(xì)胞離心支架,在4，1 200 r/min, 離心10分鐘，將一定量的支氣管肺泡灌洗液(BALF)細(xì)胞直接均勻地平鋪于載玻片上。經(jīng)冷風(fēng)吹干,用HE染色后, 在高倍鏡下計(jì)數(shù)200個(gè)細(xì)胞進(jìn)行分類計(jì)數(shù)。2.外周血和BALF中ET-1含量測(cè)定: 取靜脈血23 ml,注入含10%依地酸二納(EDTA) 30 l和抑肽酶40 l的試管中混勻,

11、在4，3 000 r/min，離心10分鐘,分離血清，-70保存, 將上述-70保存的BALF上清取1 ml進(jìn)行冷凍干燥濃縮,用ET-1放射免疫試劑盒(美國(guó)Penisular公司)直接測(cè)定法同步測(cè)定ET-1活性，以ng/L表示。3.質(zhì)量控制和統(tǒng)計(jì)學(xué)處理: 為消除批間誤差, 采取統(tǒng)一藥盒, 一批檢測(cè)。所有數(shù)據(jù)以s表示, 分別進(jìn)行兩組均值t檢驗(yàn)和相關(guān)系數(shù)檢驗(yàn)。結(jié)果一、BALF中細(xì)胞總數(shù)和分類計(jì)數(shù)肺結(jié)節(jié)病組和IPF組BALF中細(xì)胞總數(shù)均明顯高于對(duì)照組(P0.01)。在細(xì)胞分類計(jì)數(shù)中,肺結(jié)節(jié)病組淋巴細(xì)胞和IPF組中性粒細(xì)胞，顯著高于對(duì)照組(P0.01),肺結(jié)節(jié)病組和IPF組BALF中巨噬細(xì)胞雖低于對(duì)照

12、組0.900.05, 但其絕對(duì)值(肺結(jié)節(jié)病組為19.07107/L,IPF組為23.0107/L)高于對(duì)照組(18.0107/L，表1)。表1三組患者BALF中細(xì)胞總分?jǐn)?shù)分析(s)組別例數(shù)細(xì)胞總數(shù)(107/L)細(xì)胞分類巨噬細(xì)胞中性粒細(xì)胞淋巴細(xì)胞肺結(jié)節(jié)病組102610*0.720.13*0.0340.012*0.260.07*肺纖維化組83313*0.710.11*0.1600.120*0.090.06對(duì)照組82080.900.050.0130.070.100.07注：與對(duì)照組比較差異有顯著性?P0.05，?P0.01二、血清和BALF中ET-1活性檢測(cè)肺結(jié)節(jié)病組和IPF組血清和BALF中ET-

13、1水平均顯著高于對(duì)照組(P0.01，表2)。表2三組患者血清和BALF中ET-1活性分析(s)組別例數(shù)血清(ng/L)BALF(ng/L)肺結(jié)節(jié)病組106229*17.02.4*肺纖維化組87771*10.03.1*對(duì)照組82084.00.6注：?與對(duì)照組比較，P0.01三、血清ET-1與PaO2、BALF中ET-1與細(xì)胞數(shù)相關(guān)分析肺結(jié)節(jié)病組和IPF組中血清ET-1與PaO2呈負(fù)相關(guān)(r=-0.538，P0.01),BALF中ET-1與細(xì)胞總數(shù)呈正相關(guān)(r=0.649，P0.01),結(jié)節(jié)病組ET-1與淋巴細(xì)胞和IPF與中性粒細(xì)胞呈明顯正相關(guān)(r分別=0.712，0.813，P均0.01)。討論

14、自Yandgisme等1于1988年從牛內(nèi)皮細(xì)胞分離出ET-1以來(lái),ET被認(rèn)為是一種極強(qiáng)的血管收縮因子，在哮喘、肺動(dòng)脈高壓和急性肺損傷等許多疾病的發(fā)病機(jī)制上起著重要作用。ET也是促進(jìn)細(xì)胞有絲分裂的細(xì)胞因子,能促使血管平滑肌細(xì)胞和肺成纖維細(xì)胞增殖,特別是肺成纖維細(xì)胞增殖活化,使膠原分泌合成增加,這是肺纖維化重要環(huán)節(jié)2。本組結(jié)果表明:肺結(jié)節(jié)病組和IPF組外周血和BALF中有較高的ET-1活性,進(jìn)一步提示ET促使肺血管平滑肌細(xì)胞和肺成纖維細(xì)胞增殖,說(shuō)明ET與肺纖維化密切相關(guān)。ET是由多種細(xì)胞合成和分泌的，如血管平滑肌細(xì)胞、內(nèi)皮細(xì)胞、氣道上皮細(xì)胞、肉芽腫細(xì)胞和肺泡巨噬細(xì)胞。肺免疫組化染色也發(fā)現(xiàn)在上皮細(xì)

15、胞、血管內(nèi)皮細(xì)胞和浸潤(rùn)的巨噬細(xì)胞中有較高的ET表達(dá)3。在肺結(jié)節(jié)病和IPF患者肺內(nèi)有較多的炎性效應(yīng)細(xì)胞,使ET分泌合成增加,因此在BALF中有較高的ET活性,其增高水平與細(xì)胞數(shù)有明顯正相關(guān),說(shuō)明肺內(nèi)ET-1活性與疾病活動(dòng)狀態(tài)密切相關(guān)。然而間質(zhì)性肺疾病肺內(nèi)炎性效應(yīng)細(xì)胞所釋放的各種細(xì)胞因子是直接作用于成纖維細(xì)胞,還是通過(guò)內(nèi)皮素作用于成纖維細(xì)胞。這種復(fù)雜相互作用及鏈鎖反應(yīng)的機(jī)制是不清楚的,有人將肺泡巨噬細(xì)胞和血管內(nèi)皮細(xì)胞一起培養(yǎng),結(jié)果ET分泌含量明顯增多4。推測(cè)肺泡巨噬細(xì)胞釋放細(xì)胞因子(IL-1,轉(zhuǎn)化生長(zhǎng)因子和腫瘤壞死因子)首先作用于內(nèi)皮細(xì)胞,使ET分泌合成增加,ET進(jìn)一步作用于肺內(nèi)血管平滑肌細(xì)胞和成

16、纖維細(xì)胞,使之增殖活化5。因此進(jìn)一步說(shuō)明ET參與了間質(zhì)性肺疾病肺纖維化的發(fā)生發(fā)展過(guò)程。此外,缺氧、酸中毒及內(nèi)毒素可損傷血管內(nèi)皮細(xì)胞,導(dǎo)致ET-1合成釋放增加。在彌漫性間質(zhì)性肺疾病中、晚期均有不同程度的低氧血癥,是間質(zhì)性肺疾病血清和BALF中ET-1活性增高的另一個(gè)原因。本組結(jié)果顯示，血清中ET-1增加與PaO2呈負(fù)相關(guān)。綜上所述,本組結(jié)果提示:ET-1在肺結(jié)節(jié)病和IPF發(fā)病機(jī)制中起著重要作用,并可做為判定疾病活動(dòng)性的一項(xiàng)重要指標(biāo)。本課題受省科委基金資助(基金編號(hào)：972258)作者單位：110001 沈陽(yáng)，中國(guó)醫(yī)科大學(xué)呼吸疾病研究所參考文獻(xiàn)1Yanagisawa MH, Karihara S,

17、 Kimura Y, et al. A novel potent vasoconstrictor peptides produced by vascular endothelial cells. Nature Land, 1988, 332:411-415.2Cambrey AD, Harrison NK, Dawes KE, et al. Increased levels of endothelin-1 in brochoalveolar lavage fluid from patients with systemic sclerosis contributes to fibroblast mitogenic activity in vitro. Am J Respir Cell Mol Biol, 1994, 11:439-445.3Giaid ARP, Michel DJ, Steward M, et al. Expression of endothelin-1 in lungs of patients with crytogenic fibro