芥子氣經(jīng)腹腔和氣管致大鼠急性肺損傷炎性反應(yīng)的比較研究*

貝媛媛，朱雙雙，張長(zhǎng)堯，趙　建，鐘玉緒，韓　瑋，劉　菲，趙玉玲，祝筱姬△

(1.濰坊醫(yī)學(xué)院研究生部，山東濰坊 261042；2.解放軍第八十九醫(yī)院呼吸科，山東濰坊 261021；3.軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所，北京 100850)

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·論著·doi:10.3969/j.issn.1671-8348.2016.26.003

芥子氣經(jīng)腹腔和氣管致大鼠急性肺損傷炎性反應(yīng)的比較研究*

貝媛媛1，朱雙雙1，張長(zhǎng)堯2，趙建3，鐘玉緒3，韓瑋2，劉菲2，趙玉玲2，祝筱姬2△

(1.濰坊醫(yī)學(xué)院研究生部，山東濰坊 261042；2.解放軍第八十九醫(yī)院呼吸科，山東濰坊 261021；3.軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所，北京 100850)

目的經(jīng)腹腔和氣管建立大鼠芥子氣(SM)肺損傷的動(dòng)物模型，比較兩種大鼠急性肺損傷模型炎性反應(yīng)的差異。方法選取Sprague Dawley大鼠136只，分為5組，正常對(duì)照組8只,其他4個(gè)組(腹腔SM組、腹腔丙二醇對(duì)照組、氣管SM組、氣管丙二醇對(duì)照組)每組32只。腹腔SM組腹腔內(nèi)注入稀釋的SM 0.1 mL(0.96 LD50= 8 mg/kg)，氣管SM組氣管內(nèi)注入稀釋的SM 0.1 mL(0.98 LD50=2 mg/kg)，正常對(duì)照組不做任何處理。ELISA法檢測(cè)支氣管肺泡灌洗液和血液標(biāo)本，HE染色和免疫組織化學(xué)判斷炎性反應(yīng)情況。結(jié)果腹腔SM組各時(shí)間段支氣管肺泡灌洗液蛋白含量和細(xì)胞計(jì)數(shù)與氣管SM組相比顯著升高(P<0.05)；腹腔SM組各時(shí)間段血清TNF-α、IL-1β、IL-6與氣管SM組相比顯著升高(P<0.05)；腹腔SM組各時(shí)間段肺泡間隔T、B淋巴細(xì)胞和巨噬細(xì)胞陽性表達(dá)率與氣管SM組相比顯著增加(P<0.05)。結(jié)論大鼠在SM LD50相似的情況下，腹腔SM組支氣管肺泡灌洗液、肺泡間隔及血清炎性反應(yīng)指標(biāo)明顯高于氣管SM組。

芥子氣；肺/損傷；炎性反應(yīng)；大鼠

芥子氣(Sulfur mustard,SM)是一種親脂性烷化劑，可迅速穿透上皮組織導(dǎo)致皮膚或呼吸道損傷[1-2]。皮膚、眼睛和呼吸道是SM攻擊的主要靶器官，其損傷程度與劑量和持續(xù)時(shí)間密切相關(guān)[3]。SM肺損傷早期死亡原因?yàn)榉尾扛腥竞秃粑ソ遊4]。SM可觸發(fā)促炎反應(yīng)通路，炎性因子介導(dǎo)炎性細(xì)胞肺浸潤(rùn)，并貫穿于肺損傷的全過程[5-6]。SM經(jīng)皮膚、皮下、口服使小鼠染毒，以經(jīng)皮膚致肺損傷的組織學(xué)改變最明顯[7]。有關(guān)SM肺損傷炎性反應(yīng)的實(shí)驗(yàn)指標(biāo)，國(guó)內(nèi)文獻(xiàn)報(bào)道甚少。本文通過建立經(jīng)腹腔和氣管SM肺損傷大鼠模型，比較支氣管肺泡灌洗液和血清及肺泡間隔的炎性反應(yīng)指標(biāo)，旨在評(píng)估2種途徑SM肺損傷的差異性。

1　材料與方法

1.1材料所有動(dòng)物經(jīng)濰坊醫(yī)學(xué)院動(dòng)物倫理委員會(huì)批準(zhǔn)。選取健康雄性Sprague Dawley大鼠(SPF級(jí),中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心，合格證號(hào)：0015902)136只,體質(zhì)量280～300 g,年齡15周。將大鼠分為腹腔SM組(32只)、腹腔丙二醇組(32只)、氣管SM 組(32只)、氣管丙二醇組(32只)、正常對(duì)照組(8只)。

1.2方法

1.2.1動(dòng)物模式的建立SM液(純度>90%)臨用前用丙二醇稀釋至所需濃度。(1)氣管途徑染毒動(dòng)物模型建立:實(shí)驗(yàn)前氣管SM組和氣管丙二醇組皮下注射阿托品(0.05 mg/kg)，30 min后腹腔內(nèi)注射鹽酸氯胺酮(100 mg/kg)實(shí)施麻醉，氣管內(nèi)注入稀釋的SM 0.1 mL(0.98 LD50=2 mg/kg)，氣管丙二醇組注入丙二醇0.1 mL。(2)腹腔途徑染毒動(dòng)物模型建立:同上方法實(shí)施麻醉。腹腔SM組大鼠腹腔內(nèi)注入稀釋的SM 0.1 mL(0.96 LD50= 8 mg/kg)，腹腔丙二醇組注入丙二醇0.1 mL，正常對(duì)照組不做任何處理。1，2-丙二醇溶液由天津致遠(yuǎn)化學(xué)有限公司提供。

1.2.2支氣管肺泡灌洗液測(cè)定腹腔和氣管SM組大鼠，在染毒6、24、48、72 h后，腹腔注射3%戊巴比妥(30 mg/kg)，麻醉后打開胸腔，心臟抽血2 mL，放血處死，然后結(jié)扎右側(cè)肺門。氣管做“T”形切口，靜脈導(dǎo)管(外徑1.8 mm)插入左主支氣管。抽取預(yù)熱(37.3～37.5 ℃)生理鹽水2.5 mL，緩慢注入，然后回抽灌洗液。反復(fù)抽注10次，每只大鼠灌洗5次，抽液注入離心管內(nèi)(冰浴)。標(biāo)本4 ℃ 離心(223.6×g離心10 min)，上清液肝素抗凝，-80 ℃ 保存?zhèn)溆?。采用全自?dòng)生化免疫一體機(jī)(COBAS 8000型，德國(guó)羅氏公司)進(jìn)行蛋白含量測(cè)定。1 mL磷酸鹽緩沖液(PBS)再懸浮細(xì)胞沉淀，用臺(tái)盼藍(lán)染色，取10 μL加入細(xì)胞計(jì)數(shù)板，光鏡(BX51型，日本奧林巴斯公司)下細(xì)胞計(jì)數(shù)。

1.2.3血清炎性因子測(cè)定將腹腔SM組和氣管SM組不同時(shí)間段獲取的大鼠血2 mL，37 ℃ 水浴1 h，4 ℃ 過夜，然后223.6×g離心10 min，取上清液，分裝在無菌小瓶中，-80 ℃ 保存?zhèn)溆?。采用酶?biāo)儀(Versa Max型，美國(guó)Molecular Devices公司)，檢測(cè)血清腫瘤壞死因子α(TNF-α)、白細(xì)胞介素(IL)-1β、IL-6濃度。ELISA試劑盒由深圳科潤(rùn)達(dá)生物工程有限公司提供，所有流程嚴(yán)格按說明書進(jìn)行操作。

1.2.4免疫組織化學(xué)每一個(gè)標(biāo)本切取15份，每5份一組進(jìn)行免疫組化染色。pH 8.5,乙二胺四乙酸(EDTA)抗原修復(fù)，0.3% H2O2和山羊血清封閉，免疫組織化學(xué)采用SP法，一抗4 ℃孵育過夜(兔抗大鼠CD4單克隆抗體標(biāo)記T淋巴細(xì)胞，兔抗大鼠CD20單克隆抗體標(biāo)記B淋巴細(xì)胞，兔抗大鼠CD68單克隆抗體標(biāo)記巨噬細(xì)胞)，DAB顯色，蘇木素復(fù)染，封片。陰性對(duì)照以PBS代替一抗。CD4、CD20、CD68試劑盒由北京中杉金橋生物技術(shù)有限公司提供。

2　結(jié)　果

2.1支氣管肺泡灌洗液蛋白和細(xì)胞分析腹腔和氣管SM組支氣管肺泡灌洗液中蛋白含量和細(xì)胞計(jì)數(shù)均48 h達(dá)高峰。腹腔SM組各時(shí)間段蛋白含量和細(xì)胞計(jì)數(shù)與氣管SM組相比明顯升高(圖1A、B)。

A：支氣管肺泡灌洗液蛋白含量；B：支氣管肺泡灌洗液細(xì)胞計(jì)數(shù);C：血清TNF-α水平；D：血清IL-1β水平； E：血清IL-6水平；F：肺泡間隔CD4陽性表達(dá)率；G：肺泡間隔CD20陽性表達(dá)率；H：肺泡間隔CD68陽性表達(dá)率。a：P<0.05，與氣管SM組比較;b：P<0.05，與正常對(duì)照組比較。

圖1大鼠支氣管肺泡灌洗液和血清及肺泡間隔炎性反應(yīng)變化趨勢(shì)

a:CD4表達(dá);b:CD20表達(dá);c:CD68表達(dá)。A～D:6、24、48、72 h腹腔SM組陽性表達(dá)；E:正常對(duì)照組(箭頭示陽性表達(dá)，標(biāo)尺為20 μm)。F～I(xiàn):6、24、48、72 h氣管SM組陽性表達(dá)；J:正常對(duì)照組(箭頭示陽性表達(dá)，標(biāo)尺為20 μm)。K～N:6、24、48、72 h氣管丙二醇對(duì)照組；O:正常對(duì)照組(標(biāo)尺為20 μm)。

圖2大鼠肺泡間隔T淋巴細(xì)胞、B淋巴細(xì)胞、巨噬細(xì)胞表達(dá)(×400)

2.2血清炎性因子分析腹腔和氣管SM組血清TNF-α、IL-1β、IL-6水平24 h達(dá)高峰，腹腔SM組各時(shí)間段血清炎性因子水平與氣管SM組相比明顯升高(圖1C～E)。

2.3大鼠肺泡間隔炎細(xì)胞浸潤(rùn)

2.3.1腹腔SM組(CD4)6、24、48 h 肺泡間隔T淋巴細(xì)胞聚集成簇，72 h呈團(tuán)簇狀。氣管SM組(CD4)6、24、48、72 h肺泡間隔T淋巴細(xì)胞聚集成簇。丙二醇和正常對(duì)照組(CD4)呈零星分布(圖2a A～O)。腹腔SM組各時(shí)間段肺泡間隔T淋巴細(xì)胞陽性表達(dá)率與氣管SM組相比明顯增多(圖1F)。

2.3.2腹腔和氣管SM組(CD20)6 h肺泡間隔B淋巴細(xì)胞呈帶狀分布，24、48、72 h聚集成簇。丙二醇和正常對(duì)照組呈零星分布(圖2b A～O)。腹腔SM組各時(shí)間段肺泡間隔B淋巴細(xì)胞陽性表達(dá)率與氣管SM組相比明顯增多(圖1G)。

2.3.3腹腔和氣管SM組(CD68)6 h 肺泡間隔巨噬細(xì)胞呈散在分布，24 h增多，48、72 h明顯增多。丙二醇和正常對(duì)照組呈零星分布(圖2c A～O)。腹腔SM組各時(shí)間段肺泡間隔巨噬細(xì)胞陽性表達(dá)率與氣管SM組相比明顯增多(圖1H)。

3　討　論

SM誘導(dǎo)肺損傷涉及炎性介質(zhì)和炎性細(xì)胞反應(yīng)。Mcclintock等[8]研究發(fā)現(xiàn)，大鼠氣管內(nèi)滴注2-氯乙基乙基硫醚(CEES)6 mg/kg，24 h肺泡內(nèi)可發(fā)生出血、水腫、巨噬細(xì)胞和單核細(xì)胞聚集。另有學(xué)者發(fā)現(xiàn)，CEES可誘導(dǎo)促炎因子IL-6 和 IL-1β上調(diào)，同時(shí)轉(zhuǎn)錄因子血清加速因子-1(serum accelerator factor-1,SAF-1)/癌基因相關(guān)鋅指蛋白(myc-associated zinc finger protein,MAZ)活性增加[9]。豚鼠CEES染毒后檢測(cè)血清發(fā)現(xiàn)，24 h 血清TNF-α、IL-1β、IL-6、IL-8水平升高[10]?？梢?，在SM誘導(dǎo)機(jī)體應(yīng)激狀態(tài)下，炎性細(xì)胞能釋放促炎介質(zhì)和細(xì)胞因子，刺激中性粒細(xì)胞的溢出和集聚[11-13]。在損傷部位，中性粒細(xì)胞也可通過脫顆粒和髓過氧化物酶的釋放來改變組織的微環(huán)境[14]。

本研究發(fā)現(xiàn)，腹腔和氣管SM組炎性反應(yīng)指標(biāo)的變化具有如下特點(diǎn)：(1)支氣管肺泡灌洗液蛋白含量和細(xì)胞計(jì)數(shù)48 h 達(dá)高峰；(2)血清促炎因子TNF-α、IL-1β、IL-6水平24 h 達(dá)高峰；(3)免疫組織化學(xué)顯示肺泡間隔T、B淋巴細(xì)胞和巨噬細(xì)胞浸潤(rùn)隨時(shí)間延長(zhǎng)增多；(4)上述炎性反應(yīng)指標(biāo)腹腔SM組與氣管SM組相比明顯升高。本研究支氣管肺泡灌洗液中蛋白含量和細(xì)胞計(jì)數(shù)與Anderson 等[15]和Calvet 等[16]報(bào)道一致。促炎因子水平與Yego 等[10]和Emad 等[17]報(bào)道一致，與Yaraee 等[18]和Pourfarzam 等[19]報(bào)道相反。筆者認(rèn)為，支氣管肺泡灌洗液蛋白含量和細(xì)胞計(jì)數(shù)增多，可能與肺間質(zhì)毛細(xì)血管和肺上皮細(xì)胞通透性增加有關(guān)，屬一種肺實(shí)質(zhì)伴隨肺結(jié)構(gòu)改變的炎性反應(yīng)。本研究還發(fā)現(xiàn)，SM致急性肺損傷炎細(xì)胞浸潤(rùn)以淋巴細(xì)胞為主，這與文獻(xiàn)報(bào)道以中性粒細(xì)胞和巨噬細(xì)胞浸潤(rùn)為主不一致[20]。分析可能與SM誘導(dǎo)細(xì)胞死亡，促炎介質(zhì)(TNF-α,IL-6,IL-1β,IL-8等)釋放到細(xì)胞外基質(zhì)中，激活巨噬細(xì)胞和肥大細(xì)胞，啟動(dòng)免疫反應(yīng)有關(guān)。與此同時(shí)，炎性細(xì)胞能釋放促炎介質(zhì)和化學(xué)引物，在損傷部位刺激中性粒細(xì)胞溢出與集聚[13,21]。本研究還顯示，兩種途徑和濃度SM致急性肺損傷動(dòng)物模型，肺泡間隔有大量淋巴細(xì)胞浸潤(rùn)，中量巨噬細(xì)胞浸潤(rùn)，其肺損傷程度與時(shí)間和細(xì)胞密度相關(guān)。這表明SM誘導(dǎo)急性肺損傷免疫反應(yīng)和炎性反應(yīng)共存，以免疫反應(yīng)為主導(dǎo)。文獻(xiàn)[22]報(bào)道，SM腹腔注射引起的肺損傷比經(jīng)皮下注射或口服途徑更嚴(yán)重。當(dāng)大鼠經(jīng)腹腔注射SM劑量高于10 mg/kg時(shí),就會(huì)出現(xiàn)大鼠死亡[23]。有學(xué)者發(fā)現(xiàn)，大鼠SM氣管內(nèi)吸入劑量(1.4 mg/kg),可產(chǎn)生明顯的肺臟炎性反應(yīng)[24]。所以,在預(yù)期實(shí)驗(yàn)設(shè)計(jì)的基礎(chǔ)上選擇SM劑量(0.96 LD50=8 mg/kg)腹腔造模和(0.98 LD50=2 mg/kg)氣管造模。本研究提示，大鼠在SM LD50相似的情況下，SM經(jīng)腹腔染毒肺炎性反應(yīng)指標(biāo)比經(jīng)氣管明顯升高。分析大鼠腹膜腔的腹膜對(duì)SM的接觸和吸收遠(yuǎn)遠(yuǎn)大于氣管的黏膜，由此存在毒素吸收入血的濃度差異，且SM的劑量與組織和血的炎性反應(yīng)程度呈正相關(guān)。SM腹腔染毒致大鼠急性肺損傷炎性反應(yīng)重，推測(cè)可能與腹膜腔對(duì)SM的快速吸收，血中SM的濃度迅速升高有關(guān)。在未來的戰(zhàn)爭(zhēng)和恐怖事件中，很難預(yù)測(cè)SM的染毒方式和劑量。本研究闡述的SM相關(guān)機(jī)制與獲得的參數(shù)，可為SM的預(yù)防與治療提供借鑒。

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A comparative study on inflammatory response due to sulfur mustard-induced acute lung injury in rat via the intraperitoneal and tracheal injection*

Bei Yuanyuan1,Zhu Shuangshuang1,Zhang Changyao2,Zhao Jian3,Zhong Yuxu3,HanWei2,LiuFei2,ZhaoYuling2,ZhuXiaoji2△

(1.DepartmentofGraduate,WeifangMedicalUniversity,Weifang,Shandong261042,China;2.DepartmentofRespiration,The89thHospitalofPLA,Weifang,Shandong261021,China;3.InstituteofPharmacologyandToxicology,AcademyofMilitaryMedicalSciences,Beijing100850,China)

ObjectiveThe purpose of this study was to establish animal model of sulfur mustard (SM)-induced acute lung injury in rats via the intraperitoneal and the tracheal injection,in order to compare the difference of inflammatory reaction.Methods136 male Sprague Dawley rats were selected,then were randomly divided into the five groups,the control group with 8 cases,other four groups (i.e.the intraperitoneal SM group,the intraperitoneal propylene glycol group,the tracheal SM group,the tracheal propylene glycol group) with 32 cases in each group.The intraperitoneal SM group were injected intraperitoneally with diluted SM 0.1 mL(0.96 LD50=8 mg/kg),the tracheal SM group were injected intratracheally with diluted SM 0.1 mL(0.98 LD50= 2 mg/kg),meanwhile the status quo was kept with the normal group.SM-induced inflammatory reaction was observed by bronchoalveolar lavage fluid (BALF),serum examination,Hematoxylin Eosin staining,and immunohistochemical staining.ResultsCompared with the tracheal SM group at different time,protein contents and cell counts of BALF in the intraperitoneal SM group were significantly inceased,respectively (P<0.05).Compared with the tracheal SM group at different time,the levels of serum TNF-α,IL-1β,IL-6 in the intraperitoneal SM group were significantly inceased,respectively (P<0.05).The positive expression ratio of T lymphocytes,B lymphocytes and macrophages in intraperitoneal SM group at different time were increased compared with the tracheal SM group,respectively (P<0.05).ConclusionUnder similar SM LD50in rat,in the intraperitoneal SM group,inflammatory reaction of BALF,alveolar septum,and serum were significantly higher than in the tracheal SM group.

mustard gas;lung/injuryies;inflammatory reaction;rat

國(guó)家“重大新藥創(chuàng)制”科技重大專項(xiàng)(2013ZX09J13013-01B)。作者簡(jiǎn)介：貝媛媛(1991-),在讀研究生，主要從事呼吸毒理學(xué)研究?！?/p>

，E-mail:xiaojizhu@163.com 。

R114

A

1671-8348(2016)26-3608-03

2016-03-18

2016-06-01)