Poly Ⅰ:C对急性期溃疡性结肠炎小鼠肠黏膜屏障的影响*

赵红伟，岳月红，郎晓猛，和丽丽，朱叶珊

(1.河北省人民医院消化内科,石家庄 050051；2.河北省人民医院神经内科,石家庄 050051；3.河北省中医院脾胃科，石家庄 050051；4.河北省人民医院老年病科，石家庄 050051；5.河北省唐山市中医医院脾胃科 063000)

论著·基础研究

Poly Ⅰ:C对急性期溃疡性结肠炎小鼠肠黏膜屏障的影响*

赵红伟1，岳月红2，郎晓猛3，和丽丽4，朱叶珊5△

(1.河北省人民医院消化内科,石家庄 050051；2.河北省人民医院神经内科,石家庄 050051；3.河北省中医院脾胃科，石家庄 050051；4.河北省人民医院老年病科，石家庄 050051；5.河北省唐山市中医医院脾胃科 063000)

目的 研究Poly Ⅰ:C对急性期溃疡性结肠炎模型小鼠肠道黏膜屏障调节作用。方法 30只小鼠随机分为正常对照组、模型组、Poly Ⅰ:C组，建立急性溃疡性结肠炎动物模型；观察小鼠一般活动情况及结肠病理学变化、应用免疫组织化学荧光染色法测定结肠组织水闸蛋白(claudin-1)、闭锁小带(zo-1)蛋白表达的变化。结果 实验期间，Poly Ⅰ:C干预治疗后模型小鼠的一般情况有明显改善；模型组小鼠结肠黏膜缺损，腺体破坏或消失，可见黏膜、黏膜下层甚至肌层大量炎性细胞浸润；Poly Ⅰ:C组可见炎性细胞浸润少，炎症程度较模型组明显减轻。与模型组比较，经过Poly Ⅰ:C治疗后结肠组织黏膜中claudin-1、zo-1蛋白的荧光表达明显增强。结论 Poly Ⅰ:C 对急性溃疡性结肠炎模型小鼠肠道黏膜屏障有调节作用。

结肠炎，溃疡性；结肠疾病；疾病模型，动物；黏膜;Poly Ⅰ:C;肠道黏膜屏障

溃疡性结肠炎(ulcerative colitis，UC)是一种高复发的肠道炎性疾病。近年来，研究学者认为本病发病基础是：肠黏膜屏障削弱，肠道致病菌群及其有害成分穿过黏膜屏障，引发局部免疫反应[1-2]。Poly Ⅰ:C(polyinosinic polycytidylic acid)可诱导分泌的Ⅰ型干扰素，可增强机体的初期抗体应答[3]，对急性UC模型小鼠的肠道黏膜屏障是否有调节作用仍需进一步研究。

1 材料与方法

1.1 材料 右旋葡聚糖硫酸钠(DSS，Sigma公司，美国)；Poly Ⅰ:C(Sigma公司，美国)；兔多抗zo-1抗体(invitrogen)；鼠抗claudin-1抗体(Santa Cruz公司，美国)；Cy3标记的山羊抗小鼠免疫球蛋白G(IgG)抗体、异硫氰酸荧光素(FITC)标记的山羊抗兔IgG抗体(碧云天分进口产品)。

1.2 方法

1.2.1 急性期造模及Poly Ⅰ:C干预 30只雄性C57BL/6小鼠(体质量18～22 g，7～12周)，购于北京维通利华实验动物技术有限公司。按随机数字表法分入正常对照组、模型组和Poly Ⅰ:C组，每组10只。模型组自由饮用2%DSS 7 d，正常对照组和Poly Ⅰ:C组小鼠自由饮用蒸馏水7 d；Poly Ⅰ:C组只在造模前给予0.3 mg/kg Poly Ⅰ:C，一次性肌内注射给药；空白对照组和模型组同时给予生理盐水同等剂量肌内注射作为对照，时间均为1周，第8天处死所有小鼠。

1.2.2 小鼠一般情况及结肠病理组织染色 每天观察小鼠的精神状态、体质量、活动情况、毛发光泽度、食欲、大便性状等。在实验结束时处死大鼠，取部分肠段置于多聚甲醛内固定，包埋、切片，苏木素-伊红(HE)染色，行组织病理学观察。

1.2.3 肠组织免疫荧光染色 新鲜肠组织以5 μm的厚度进行连续冰冻切片，按以下步骤进行闭锁小带(zo-1)、水闸蛋白(claudin-1)的免疫荧光染色：4%多聚甲醛固定10 min，免疫染色洗涤液洗5 min 3次，封闭液封闭，室温 1 h，加兔抗zo-1多克隆抗体/小鼠抗claudin-1单克隆抗体，4 ℃过夜，洗涤液洗5 min 3次，滴加标记绿色荧光的抗兔FITC/标记红色荧光的抗小鼠Cy3，室温 1 h。绿色荧光/红色荧光为阳性表达。阴性对照用磷酸盐缓冲液(PBS)替代一抗，其余步骤同上。

2 结 果

2.1 动物的一般状况 实验期间，正常对照组小鼠大小便正常，体质量持续增加，毛发有光泽，活动、精神状态均正常。急性期模型组动物饮用2%DSS水后第1天，体质量增长，饮水、进食较多；饮用2%DSS第3天，开始精神萎靡，懒于活动，被毛松散无泽，食量下降，拉黏液血便，体质量下降，粪便潜血试验呈强阳性；实验第8天模型组小鼠体质量明显减轻，肛周可见肉眼血便。Poly Ⅰ:C组第5～7天，便稀，但未见脓血便，状态接近正常，但体质量仍明显减轻。

2.2 Poly Ⅰ:C对小鼠结肠组织病理学变化的影响 正常对照组肠黏膜光滑，无水肿、充血等表现；模型组小鼠结肠黏膜缺损，可见黏膜、黏膜下层甚至肌层大量炎性细胞浸润。Poly Ⅰ:C组的小鼠结肠病理较模型组明显改善，见图1。

2.3 免疫荧光染色检测结肠黏膜紧密连接蛋白(TJ)的定位分布 与免疫组织化学相比，TJ的免疫荧光表达更具有特异性。激光共聚焦显微镜下见zo-1蛋白呈现绿光，claudin-1蛋白呈现红光。急性期正常对照组zo-1、claudin-1蛋白表达可见荧光沿胞膜分布，荧光强度强，边缘光滑；模型组荧光分布较正常对照组分散，荧光强度减弱，边缘粗糙呈锯齿状；Poly Ⅰ:C组荧光仍沿胞膜分布，强度较正常对照组稍减弱，但仍强于模型组，见图2、3。

A:正常对照组；B：模型组；C：Poly Ⅰ:C组。

图1 各组小鼠结肠组织病理学变化(×200)

A:正常对照组；B：模型组；C：Poly Ⅰ:C组。

图2 各组小鼠结肠组织zo-1蛋白表达(×200)

A:正常对照组；B：模型组；C：Poly Ⅰ:C组。

图3 各组小鼠结肠组织claudin-1蛋白表达(×200)

3 讨 论

目前研究显示，UC的发病机制可能与肠道黏膜屏障功能减弱，诱导局部免疫反应有关[4]。Poly Ⅰ:C是一种人工合成的dsRNA，可形成长期的免疫记忆，对多种免疫系统疾病均有治疗作用[5-7]。

黏膜屏障功能缺陷和TJ蛋白的减少均有利于微生物抗原进入肠黏膜固有层，诱发异常的黏膜免疫应答。故此，肠道黏膜屏障的功能受损可以被认为是UC发病的始动因素之一[8-10]。肠上皮TJ对于屏障功能的维持和TJ的完整性具有重要作用，肠黏膜上皮TJ是由一系列的细胞质蛋白、细胞骨架元素和几种跨膜蛋白组成[11]。UC发生时，与肠黏膜通透性密切相关的TJ的zo-1和claudin-1首先从位置分布上发生了改变，正常情况下分布于细胞边缘，沿细胞膜分布；而UC发生时zo-1和claudin-1分布不均，染色变淡，线条模糊，边缘粗糙有刺状突起，分布散乱，稀疏，结肠黏膜紧密连接蛋白的表达明显下降，经过Poly Ⅰ:C治疗后，结肠黏膜的上皮层紧密连接蛋白zo-1和claudin-1分布均匀，线条清晰，边缘整齐。可见Poly Ⅰ:C在急性UC模型小鼠保护肠黏膜屏障过程中起重要作用，为临床上UC治疗提供理论依据。

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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

河北省卫生厅科研基金项目(20160483)。 作者简介：赵红伟(1979-)，主治医师，博士，主要从事消化病方面研究。△

R574.1

A

1671-8348(2017)03-0299-03

2016-07-24

2016-09-10)