肠上皮细胞紧密连接的研究进展
2015-03-13张金卫林汉杰韩凌
张金卫 林汉杰 韩凌
1.广州中医药大学第二临床医学院,广东广州510006; 2.广州中医药大学第二附属医院,广东广州510006
肠上皮细胞紧密连接的研究进展
张金卫1林汉杰1韩凌2
1.广州中医药大学第二临床医学院,广东广州510006; 2.广州中医药大学第二附属医院,广东广州510006
肠上皮细胞紧密连接(TJ)在肠道黏膜屏障中起着重要作用,其受损会导致细胞间的通透性增加,细菌、内毒素和大分子物质通过细胞旁路途径进入其他组织、器官或体循环,从而引发疾病。本文从蛋白角度和信号通路角度介绍肠上皮细胞TJ的研究进展,并进一步指出肠黏膜受到刺激后分泌大量的Zonulin蛋白,其与Zonulin受体结合,传导信号,调控TJ上Claudin、Occludin、JAM、ZOs、Cingulin等多种蛋白的表达,从而开放TJ。肿瘤坏死因子-α通过激活核转录因子κB p65/p50异源二聚体与启动子下游κB结合区域结合激活肌球蛋白轻链激酶转录启动子是TJ信号通路中较为成熟的通路。
紧密连接;肠上皮细胞;信号通路
肠屏障是指肠道能够防止肠内有害物质穿过肠黏膜进入其他组织、器官和血液循环的结构和功能的总和。肠屏障由机械屏障、化学屏障、免疫屏障和生物屏障共同构成,其中机械屏障最为重要。机械屏障由肠上皮细胞及其连接构成,调控着水和溶质的跨上皮转运。肠上皮细胞间的连接包括紧密连接(tightjunction,TJ)、缝隙连接(gapjunction,GJ)、黏附连接(adhesion junction,AJ)及桥粒(desmosome)等,其中,TJ在
肠屏障中发挥着重要作用。在透射电镜下观察,TJ位于上皮细胞顶端,呈箍状围绕在细胞的周围,线条清晰连续,边缘光滑流畅,可与下段复合连接勾勒出纤毛柱状上皮细胞的柱状形态。TJ由50多种蛋白组成,分为结构蛋白和功能蛋白。结构蛋白构成TJ的结构骨架;功能蛋白连接细胞骨架及膜蛋白,并传递信号[1]。TJ一方面调控着细胞的通透性,另一方面作为信号中心在细胞外环境和细胞内之间进行着双向信息传递,调节着细胞的生长以及细胞的极性、表型和信号转导等[2]。在生理情况下,离子及小分子物质能够通过TJ,毒性大分子和微生物则不能通过。如果TJ受损,会导致细胞通透性增加,细菌、内毒素和大分子物质通过旁路途径进入其他组织、器官或体循环,从而引发多种疾病,例如炎症性肠病、腹泻、乳糜泻、食物过敏等。当前国内外主要从蛋白角度和信息通路角度研究肠上皮细胞TJ,本文主从这两个角度介绍其研究进展。
1 从蛋白角度研究肠上皮细胞TJ现状
肠上皮细胞TJ由50多种蛋白组成,分为结构蛋白和功能蛋白,结构蛋白主要有Occludin、Claudin和JAM等,构成TJ的结构骨架;功能蛋白主要有ZO-1、ZO-2、ZO-3、Cingulin和Zonulin等,连接细胞骨架及膜蛋白,并传递信号[1]。目前,国内外研究的焦点是Occludin、Claudin、ZOs和Zonulin。
1.1 Occludin蛋白
Occludin蛋白是在TJ中第一个被发现的蛋白,其为4分子交联体蛋白,2个环分布在细胞外,N端和C端分布在细胞内,相对分子量为65 000[3]。Occludin蛋白是TJ的主要组成部分,其4次跨膜结构能够和Claudin-l、Claudin-2及其他外周膜蛋白结合[4],对维持TJ的结构和功能具有重要意义[5]。研究发现,在炎症性肠病中,肠黏膜Occludin蛋白表达水平下降[6]。Occludin蛋白可增强成纤维细胞间的黏附性,增加跨膜电阻[7],调节细胞间的通透性[8]。乳酸杆菌等益生菌干预大肠埃希菌(E.coli)感染的小肠上皮细胞,使Occludin蛋白和细胞骨架蛋白表达升高,从而修复了破坏的TJ[9]。研究表明,肠致病菌及其毒素通过下调Occludin蛋白表达的途径破坏TJ结构[10]。
1.2 Claudin蛋白
Claudin蛋白家族对TJ结构起到支柱作用[11]。Claudin蛋白参与了TJ上只允许离子和小分子物质通过的小“孔”的形成[8]。缺少Claudin-1的小鼠在出生数小时内因脱水而死亡,研究表明,表皮屏障因缺少Claudin-1而导致功能失常,水分由皮肤大量流失,进而引发小鼠死亡[12]。细胞转染试验也进一步证实了这一结论[13]。实验表明,缺少Claudin-1表达会使肠道屏障受损[14]。杨丽等[15]用应激因子肾上腺皮质激素释放因子(corticotrophin-releasingfactor,CRF)诱导了肠上皮细胞TJ相关蛋白Claudin-2表达升高,并且发现这种诱导作用与核转录因子κB(nuclear factor kappa B,NF-κB)激活有关。
1.3 ZOs蛋白
ZOs是外周膜蛋白,能够与细胞质内Occludin蛋白的末端连接,与Occludin蛋白、肌动蛋白骨架构成稳定的连接系统[16]。ZO-1是连接跨膜蛋白Occludin、Claudin-5与细胞骨架蛋白F-actin的桥梁蛋白,其分布和表达影响着TJ的结构和功能[17]。孙兆瑞等[16]发现,ZO-1和Occludin主要定位在细胞膜上,呈边缘平滑的网状结构。Al-Sadi等[8]用肺炎克雷伯菌、乳酸杆菌、粪肠球菌和大肠埃希菌刺激Caco-2细胞,降低了TJ中ZO-1的表达。
1.4 Zonulin蛋白
Zonulin蛋白是最近发现的可调节肠道通透性的生理性蛋白,能够快速并且可逆的调控TJ[18]。Fasano[19]发现,肠黏膜受到细菌或麦胶蛋白刺激后分泌大量的Zonulin蛋白,其与Zonulin受体结合,传导信号,开放TJ,从而增加细胞的通透性。体外细胞实验表明,沙门菌和3种类型的大肠埃希菌都能够诱导Zonulin的释放、ZO-1蛋白重布和细胞通透性的增加[20]。另外,临床试验证明,口服益生菌能够降低血清中Zonulin的含量,进而有效地预防相关感染[21]。动物实验表明,糖尿病倾向大鼠肠腔内的Zonulin含量升高,阻断Zonulin受体能够降低糖尿病的发病率[22]。虽然肠道分泌过量的Zonulin蛋白会导致屏障受损,但是,生理情况下,Zonulin蛋白构成了机体的固有免疫[23]。肠上皮细胞受到肠内细菌的刺激后,释放Zonulin蛋白,开放TJ,增加肠腔内液体的分泌,从而有效地阻止小肠远端细菌向近端的定植[20],起到了免疫调节的作用。
2 从信号通路角度研究肠上皮细胞TJ现状
肠上皮细胞TJ一方面调控着细胞的通透性,另一方面作为信号中心在细胞外环境和细胞内之间进行着双向信息传递,调节着细胞的生长以及细胞的极性、表型和信号转导等[2]。在众多肠上皮细胞TJ信号通路中,肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)/ NF-κB/肌球蛋白轻链激酶(myosin lightchain kinase,MLCK)信号通路较为成熟。
2.1 信号通路概论
MLCK、磷脂酰肌醇三磷酸激酶/蛋白激酶B(phosphatidylinositol threekinase/proteinkinaseB,PI3K/AKT)、蛋白激酶C(protein kinase C,PKC)、丝裂原活化的蛋白激酶(mitogenactivated protein kinase,MAPK)、非受体蛋白酪氨酸激酶(non receptor protein tyrosine kinase,NRPTK)、Rho/Rho激酶(Rho/Rho associated kinase,ROCK)和蛋白激酶A(protein kinase A,PKA)等信号通路调控着TJ蛋白的表达[24]。这些信号通路调控着TJ蛋白的组装、分解和磷酸化,影响着其构象和生物化学特性,调节着TJ的开放,其中,PI3K/AKT、MAPK和PKC信号通路在调控TJ蛋白表达及其磷酸化过程中发挥着重要作用[24]。徐焰等[25]发现,细胞外信号调节激酶(extracellularsignal-regulated kinase,ERK1/2)和丝氨酸/苏氨酸蛋白激酶(protein kinase B,AKT)的抑制剂可部分回转铅诱导的紧密连接ZO-1蛋白、Pccludin蛋白和Claudin-5蛋白的下降。
2.2 TNF-α/NF-κB/MLCK信号通路
MLCK是Ca2+/钙调蛋白(calmodulin,CaM)依赖蛋白激酶家族的一员。Ca2+/CaM复合体是MLCK的重要元件[26],其与MLCK结合后,解离MLCK的天然抑制物[27],激活MLCK,形成MLCK前体。激活的MLCK使肌球蛋白轻链(myosin lightchain,MLC)的Thr18及Ser19磷酸化[28],改变MLC的空间构象[29]。同时,增强肌球蛋白与肌动蛋白丝之间的相互作用[30],使肌球蛋白和肌动蛋白丝收缩,增加TJ和细胞表面的张力,最终调控细胞骨架,开放TJ[31]。MLCK的活性决定着MLC磷酸化的程度[28],而在调控细胞骨架和细胞收缩等过程中MLC的磷酸化又起着举足轻重的作用[32]。研究进一步证明,MLC的磷酸化在TJ受损的过程中起着重要作用,肌动蛋白解聚剂能够开放TJ[33]。Zolotarevsk等[34]发现,MLC的磷酸化启动了TJ的开放。Shen等[35]证明了,在维护TJ结构及其功能的完整性过程中,MLCK发挥着重要作用。敲除MLCK基因或使用MLCK抑制剂都能够减轻TJ的损伤,这提示着MLCK在TJ调控中发挥着至关重要的作用[36]。
TNF-α具有激活MLCK活性、促进MLC磷酸酶转录、上调MLCK蛋白的作用,其影响着MLC的磷酸化及TJ蛋白的表达,动态调控着TJ的结构[37]。韩亮等[38]证实,抗TNF-α抗体通过抑制小肠黏膜上皮细胞的MLCK表达及其活性的途径修复受损的TJ。
Ma等[39]研究发现,TNF-α诱导的TJ相关蛋白表达异常、细胞通透性升高与NF-κB的激活有关,并进一步证实了TNF-α引起MLCK转录启动子的激活,是由于NF-κBp65/p50异源二聚体与启动子下游κB结合区域结合引起的。
3 小结与展望
从蛋白角度研究肠上皮细胞TJ中Zonulin、Claudin、Occludin、JAM、ZOs和Cingulin等成为热点,然而,本文发现,Zonulin蛋白与其余蛋白的表达具有一致性,基于这一点,笔者提出:肠黏膜受到刺激后分泌大量的Zonulin蛋白,其与Zonulin受体结合,传导信号,调控TJ上Claudin、Occludin、JAM、ZOs和Cingulin等多种蛋白的表达,从而开放TJ。
从信号通路角度研究肠上皮细胞TJ中,多数是基于蛋白表达来证明的,鲜有从基因水平论证的,例如,Clayburgh等[36]敲除MLCK基因。从基因水平论证相关信号通路是最直接、最有力的证据,笔者认为从基因水平论证相关信号通路是今后的热点和难点,并指出TNF-α通过激活NF-κB p65/p50异源二聚体与启动子下游κB结合区域结合激活MLCK转录启动子是TJ信号通路中较为成熟的通路。
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Research progress of intestinal epithelial tight junctions
ZHANG Jinwei1LIN Hanjie1HAN Ling21.The Second Clinical Medical College of Guangzhou University of Chinese Medicine,Guangdong Province,Guangzhou 510006,China;2.The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangdong Province,Guangzhou510006,China
Intestinal epithelial tight junctions(TJ)plays an important role in the intestinal mucosal barrier,and the damage resultes in increased intercellular permeability,bacteria,endotoxin and macromolecules through the paracellular way into other tissues,organs or body circulation,causing disease.This article introduces the progress of studies on intestinal epithelial cell line TJ from the angle of view and protein signaling pathway,and further points out that the intestinal mucosa after stimulated to secrete large amounts of Zonulinprotein,combining with Zonulinreceptor,andzonulin receptor signal transduction,regulation of TJ on Claudin,Occludin,JAM,ZOs,the expression of Cingulin and other protein,so as to open TJ.Tumor necrosis factor-α through the activation of nuclear factor kappa B p65/p50 two dimer and heterologous promoter B binding domain binding activating myosin light chain kinase transcription promoter is the pathway of mature TJ signal pathway.
Tight junction;Intestinal epithelial cell;Signaling pathway
R574.1
A
1673-7210(2015)02(c)-0160-04
2014-11-23本文编辑:程铭)
国家自然科学基金资助项目(编号81202635)。
张金卫(1988-),男,广州中医药大学第二临床医学院2013级中西医结合基础专业在读硕士研究生;研究方向:补益药的免疫药理。
韩凌(1974-),女,博士,副研究员,硕士生导师;研究方向:补益药的免疫药理。