趙紅偉，岳月紅，郎曉猛，和麗麗，朱葉珊

(1.河北省人民醫(yī)院消化內(nèi)科,石家莊 050051；2.河北省人民醫(yī)院神經(jīng)內(nèi)科,石家莊 050051；3.河北省中醫(yī)院脾胃科，石家莊 050051；4.河北省人民醫(yī)院老年病科，石家莊 050051；5.河北省唐山市中醫(yī)醫(yī)院脾胃科 063000)

論著·基礎(chǔ)研究

Poly Ⅰ:C對急性期潰瘍性結(jié)腸炎小鼠腸黏膜屏障的影響*

趙紅偉1，岳月紅2，郎曉猛3，和麗麗4，朱葉珊5△

(1.河北省人民醫(yī)院消化內(nèi)科,石家莊 050051；2.河北省人民醫(yī)院神經(jīng)內(nèi)科,石家莊 050051；3.河北省中醫(yī)院脾胃科，石家莊 050051；4.河北省人民醫(yī)院老年病科，石家莊 050051；5.河北省唐山市中醫(yī)醫(yī)院脾胃科 063000)

目的 研究Poly Ⅰ:C對急性期潰瘍性結(jié)腸炎模型小鼠腸道黏膜屏障調(diào)節(jié)作用。方法 30只小鼠隨機分為正常對照組、模型組、Poly Ⅰ:C組，建立急性潰瘍性結(jié)腸炎動物模型；觀察小鼠一般活動情況及結(jié)腸病理學(xué)變化、應(yīng)用免疫組織化學(xué)熒光染色法測定結(jié)腸組織水閘蛋白(claudin-1)、閉鎖小帶(zo-1)蛋白表達的變化。結(jié)果 實驗期間，Poly Ⅰ:C干預(yù)治療后模型小鼠的一般情況有明顯改善；模型組小鼠結(jié)腸黏膜缺損，腺體破壞或消失，可見黏膜、黏膜下層甚至肌層大量炎性細胞浸潤；Poly Ⅰ:C組可見炎性細胞浸潤少，炎癥程度較模型組明顯減輕。與模型組比較，經(jīng)過Poly Ⅰ:C治療后結(jié)腸組織黏膜中claudin-1、zo-1蛋白的熒光表達明顯增強。結(jié)論 Poly Ⅰ:C 對急性潰瘍性結(jié)腸炎模型小鼠腸道黏膜屏障有調(diào)節(jié)作用。

結(jié)腸炎，潰瘍性；結(jié)腸疾?。患膊∧Ｐ?，動物；黏膜;Poly Ⅰ:C;腸道黏膜屏障

潰瘍性結(jié)腸炎(ulcerative colitis，UC)是一種高復(fù)發(fā)的腸道炎性疾病。近年來，研究學(xué)者認為本病發(fā)病基礎(chǔ)是：腸黏膜屏障削弱，腸道致病菌群及其有害成分穿過黏膜屏障，引發(fā)局部免疫反應(yīng)[1-2]。Poly Ⅰ:C(polyinosinic polycytidylic acid)可誘導(dǎo)分泌的Ⅰ型干擾素，可增強機體的初期抗體應(yīng)答[3]，對急性UC模型小鼠的腸道黏膜屏障是否有調(diào)節(jié)作用仍需進一步研究。

1 材料與方法

1.1 材料 右旋葡聚糖硫酸鈉(DSS，Sigma公司，美國)；Poly Ⅰ:C(Sigma公司，美國)；兔多抗zo-1抗體(invitrogen)；鼠抗claudin-1抗體(Santa Cruz公司，美國)；Cy3標記的山羊抗小鼠免疫球蛋白G(IgG)抗體、異硫氰酸熒光素(FITC)標記的山羊抗兔IgG抗體(碧云天分進口產(chǎn)品)。

1.2 方法

1.2.1 急性期造模及Poly Ⅰ:C干預(yù) 30只雄性C57BL/6小鼠(體質(zhì)量18～22 g，7～12周)，購于北京維通利華實驗動物技術(shù)有限公司。按隨機數(shù)字表法分入正常對照組、模型組和Poly Ⅰ:C組，每組10只。模型組自由飲用2%DSS 7 d，正常對照組和Poly Ⅰ:C組小鼠自由飲用蒸餾水7 d；Poly Ⅰ:C組只在造模前給予0.3 mg/kg Poly Ⅰ:C，一次性肌內(nèi)注射給藥；空白對照組和模型組同時給予生理鹽水同等劑量肌內(nèi)注射作為對照，時間均為1周，第8天處死所有小鼠。

1.2.2 小鼠一般情況及結(jié)腸病理組織染色 每天觀察小鼠的精神狀態(tài)、體質(zhì)量、活動情況、毛發(fā)光澤度、食欲、大便性狀等。在實驗結(jié)束時處死大鼠，取部分腸段置于多聚甲醛內(nèi)固定，包埋、切片，蘇木素-伊紅(HE)染色，行組織病理學(xué)觀察。

1.2.3 腸組織免疫熒光染色 新鮮腸組織以5 μm的厚度進行連續(xù)冰凍切片，按以下步驟進行閉鎖小帶(zo-1)、水閘蛋白(claudin-1)的免疫熒光染色：4%多聚甲醛固定10 min，免疫染色洗滌液洗5 min 3次，封閉液封閉，室溫 1 h，加兔抗zo-1多克隆抗體/小鼠抗claudin-1單克隆抗體，4 ℃過夜，洗滌液洗5 min 3次，滴加標記綠色熒光的抗兔FITC/標記紅色熒光的抗小鼠Cy3，室溫 1 h。綠色熒光/紅色熒光為陽性表達。陰性對照用磷酸鹽緩沖液(PBS)替代一抗，其余步驟同上。

2 結(jié) 果

2.1 動物的一般狀況 實驗期間，正常對照組小鼠大小便正常，體質(zhì)量持續(xù)增加，毛發(fā)有光澤，活動、精神狀態(tài)均正常。急性期模型組動物飲用2%DSS水后第1天，體質(zhì)量增長，飲水、進食較多；飲用2%DSS第3天，開始精神萎靡，懶于活動，被毛松散無澤，食量下降，拉黏液血便，體質(zhì)量下降，糞便潛血試驗呈強陽性；實驗第8天模型組小鼠體質(zhì)量明顯減輕，肛周可見肉眼血便。Poly Ⅰ:C組第5～7天，便稀，但未見膿血便，狀態(tài)接近正常，但體質(zhì)量仍明顯減輕。

2.2 Poly Ⅰ:C對小鼠結(jié)腸組織病理學(xué)變化的影響 正常對照組腸黏膜光滑，無水腫、充血等表現(xiàn)；模型組小鼠結(jié)腸黏膜缺損，可見黏膜、黏膜下層甚至肌層大量炎性細胞浸潤。Poly Ⅰ:C組的小鼠結(jié)腸病理較模型組明顯改善，見圖1。

2.3 免疫熒光染色檢測結(jié)腸黏膜緊密連接蛋白(TJ)的定位分布 與免疫組織化學(xué)相比，TJ的免疫熒光表達更具有特異性。激光共聚焦顯微鏡下見zo-1蛋白呈現(xiàn)綠光，claudin-1蛋白呈現(xiàn)紅光。急性期正常對照組zo-1、claudin-1蛋白表達可見熒光沿胞膜分布，熒光強度強，邊緣光滑；模型組熒光分布較正常對照組分散，熒光強度減弱，邊緣粗糙呈鋸齒狀；Poly Ⅰ:C組熒光仍沿胞膜分布，強度較正常對照組稍減弱，但仍強于模型組，見圖2、3。

A:正常對照組；B：模型組；C：Poly Ⅰ:C組。

圖1 各組小鼠結(jié)腸組織病理學(xué)變化(×200)

A:正常對照組；B：模型組；C：Poly Ⅰ:C組。

圖2 各組小鼠結(jié)腸組織zo-1蛋白表達(×200)

A:正常對照組；B：模型組；C：Poly Ⅰ:C組。

圖3 各組小鼠結(jié)腸組織claudin-1蛋白表達(×200)

3 討 論

目前研究顯示，UC的發(fā)病機制可能與腸道黏膜屏障功能減弱，誘導(dǎo)局部免疫反應(yīng)有關(guān)[4]。Poly Ⅰ:C是一種人工合成的dsRNA，可形成長期的免疫記憶，對多種免疫系統(tǒng)疾病均有治療作用[5-7]。

黏膜屏障功能缺陷和TJ蛋白的減少均有利于微生物抗原進入腸黏膜固有層，誘發(fā)異常的黏膜免疫應(yīng)答。故此，腸道黏膜屏障的功能受損可以被認為是UC發(fā)病的始動因素之一[8-10]。腸上皮TJ對于屏障功能的維持和TJ的完整性具有重要作用，腸黏膜上皮TJ是由一系列的細胞質(zhì)蛋白、細胞骨架元素和幾種跨膜蛋白組成[11]。UC發(fā)生時，與腸黏膜通透性密切相關(guān)的TJ的zo-1和claudin-1首先從位置分布上發(fā)生了改變，正常情況下分布于細胞邊緣，沿細胞膜分布；而UC發(fā)生時zo-1和claudin-1分布不均，染色變淡，線條模糊，邊緣粗糙有刺狀突起，分布散亂，稀疏，結(jié)腸黏膜緊密連接蛋白的表達明顯下降，經(jīng)過Poly Ⅰ:C治療后，結(jié)腸黏膜的上皮層緊密連接蛋白zo-1和claudin-1分布均勻，線條清晰，邊緣整齊?？梢奝oly Ⅰ:C在急性UC模型小鼠保護腸黏膜屏障過程中起重要作用，為臨床上UC治療提供理論依據(jù)。

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Effects of Poly Ⅰ:C on intestinal mucosal barrier in mice acute ulcerative colitis*

ZhaoHongwei1,YueYuehong2,LangXiaomeng3,HeLili4,ZhuYeshan5△

(1.DepartmentofGastroenterology,HebeiProvincialPeople′sHospital,Shijiazhuang,Hebei050051,China;2.DepartmentofNeurology,HebeiProvincialPeople′sHospital,Shijiazhuang,Hebei050051,China;3.DepartmentofSpleenandStomach,HebeiProvincialHospitalofTraditionalChineseMedicine,Shijiazhuang,Hebei050051,China；4.DepartmentofGeriatrics,HebeiProvincialPeople′sHospital,Hebei050051,China;5.DepartmentofSpleenandStomach,TangshanMunicipalHospitalofTraditionalChineseMedicine,Tangshan,Hebei063000，China)

Objective To investigate the regulatory effects of polyinosinic polycytidylic acid (Poly Ⅰ:C) on the intestinal mucosal barrier in acute ulcerative colitis model mice.Methods Thirty mice were randomly grouped as the normal group,model group and Poly Ⅰ:C group.The acute ulcerative colitis animal model was established.The general condition and colon pathological changes in mice were observed.The expressions of claudin-1 proteins and zonula occluden-1 (zo-1) proteins were detected by immunohistochemical fluorescence staining.Results During the experiment,the general condition after Poly Ⅰ:C intervention in the model group was significantly changed,colonic mucous membrane were defected,glands were destroyed or disappeared,a large number of inflammatory cells infiltration could be seen in the mucous membrane,submucous layer and even muscle layer,but little inflammatory cells infiltration was seen in the Poly Ⅰ:C group,the inflammatory degree was significantly alleviated compared with the model group;compared with the model group,the fluorescence expression of claudin-1 and zo-1 protein after Poly Ⅰ:C treatment in colonic mucous membrane tissue was significantly enhanced.Conclusion PolyⅠ:C has the regulatory effect on the intestinal mucosal barrier in the acute ulcerative colitis model mice.

colitis,ulcerative;colonic disease;disease model,animal;mucous membrane;Poly Ⅰ:C;intestinal mucosal barrier

10.3969/j.issn.1671-8348.2017.03.004

河北省衛(wèi)生廳科研基金項目(20160483)。 作者簡介：趙紅偉(1979-)，主治醫(yī)師，博士，主要從事消化病方面研究。△

R574.1

A

1671-8348(2017)03-0299-03

2016-07-24

2016-09-10)