STAT3通路参与白细胞介素-22促进人结肠癌细胞系HT29紧密连接蛋白的表达

目的探讨白细胞介素-22(IL-22)对肠上皮屏障紧密连接的影响及机制。方法将人结肠癌细胞系HT29分为对照组、IL-22组(100 ng/mL)、FLLL32组(IL-22干预前使用FLLL32预处理)、colivelin组(IL-22干预前使用colivelin预处理)。转染处理组:shNC组、shNC+IL-22组(IL-22干预前转染阴性对照)、shSTAT3组(转染LV-STAT3-RNAi)、shSTAT3+IL-22组(IL-22干预前转染LV-STAT3-RNAi)。用Western blot、real-time PCR检测HT29细胞STAT3、p-STAT3和紧密连接蛋白ZO-1、occludin、claudin-1和claudin-2及其mRNA的表达;用透射电镜观察HT29细胞紧密连接结构。结果相比于对照组,IL-22组的STAT3、p-STAT3、 ZO-1、occludin、claudin-1和claudin-2蛋白及mRNA表达水平明显升高(P0.05),且紧密连接更致密。相比于IL-22组,FLLL32组中上述指标的蛋白及mRNA表达量明显下调(P0.05),紧密连接较差,colivelin组则表达上调(P0.05),紧密连接完好。相比于shNC组,shNC+IL-22组的所有紧密连接的蛋白及mRNA表达量明显增加(P0.05),紧密连接结构更致密;而相比于shSTAT3组,shSTAT3+IL-22组的所有紧密连接的蛋白及mRNA表达量的差异无统计学意义。结论 STAT3通路参与IL-22促进HT29细胞紧密连接蛋白ZO-1、occludin、claudin-1和claudin-2的表达。     

肿瘤细胞内皮蛋白C受体表达的意义

目的:检测活化蛋白C(rhAPC)对体外培养内皮细胞增殖的影响,并观察内皮蛋白C受体(EPCR)在内皮细胞及多种肿瘤细胞系的表达,初步探讨其与肿瘤生物学特性的关系。方法:MTT比色法测定APC对内皮细胞EAhy926的增殖作用;ELISA测定APC刺激内皮细胞上清IL-6和IL-8;RT-PCR分析EPCR基因在24种肿瘤细胞系和3种内皮细胞的表达水平。流式细胞术和Western blot检测部分肿瘤细胞EPCR表达。结果:APC显著提高内皮细胞增殖率,同时刺激其产生及释放IL-6和IL-8。91.7%(11/12)的实瘤细胞系和66.7%(8/12)的血液肿瘤细胞系均有EPCR基因的表达。结论:活化蛋白C可刺激内皮细胞增殖,EPCR基因高表达是多种肿瘤细胞的重要特征。     

血必净注射液通过降低细胞紧密连接的通透性减轻LPS诱导的脓毒症大鼠肺微血管内皮细胞损伤

目的 探究血必净注射液(XBJ)对脂多糖诱导的脓毒症大鼠肺微血管内皮细胞损伤的作用及其对细胞紧密连接通透性的影响。方法 选择大鼠肺微血管内皮细胞，利用5μg/mL的脂多糖(LPS)处理大鼠肺微血管内皮细胞24 h,构建大鼠脓毒症肺损伤细胞模型，XBJ治疗组采用生药浓度2.5、5、10、25、50 mg/mL的XBJ共同进行干预24 h。CCK-8检测各组细胞活性，筛选最佳XBJ浓度；Hoechst染色检测各组细胞凋亡率；Western blot检测各组细胞凋亡相关蛋白Bax、Bcl-2和cleaved caspase3的表达；Transwell小室检测单层大鼠肺微血管内皮细胞通透性；Western blot检测紧密连接相关蛋白ZO-1、ZO-2和occludin的表达。结果 CCK8实验结果显示，XBJ的最佳浓度为10mg/mL。LPS处理后，大鼠细胞凋亡率明显升高，凋亡相关蛋白Bax/Bcl-2的比值和cleaved caspase3的表达水平增加；而XBJ治疗后，细胞凋亡率下降。LPS处理导致FITC-dextran含量显著增加，ZO-1、ZO-2和Occludin的蛋白表达水平降...     

流体切应力对内皮细胞IL-8基因表达的影响及信号转导

目的流体切应力对于维持血管的稳定有着重要的作用.另外在一些病理情况下,例如动脉粥样硬化、高血压等心血管疾病的发病过程中,流体切应力也扮演着关键角色.我们以往的实验结果证实:以低流体切应力(4.2dyne/cm2)处理培养的人脐静脉内皮细胞1 h,其IL-8mRNA的表达上调;处理2 h其表达量上调更为明显.在本实验中我们将进一步阐明流体切应力对培养的人脐静脉内皮细胞IL-8基因表达、蛋白生成的影响及其信号转导途径.方法分别用实时定量RT-PCR及定量三明治ELISA检测IL-8基因表达及蛋白生成;通过基因转染及流式细胞数检测IL-8报告基因pEGFP1-IL8USCS经流体切应力作用后的激活情况;用免疫荧光化学染色观测对照和层流切应力处理以后NF-κB核转移情况;将对照和层流切应力处理后的内皮细胞裂解物进行I-κB和磷酸化的I-κB的免疫印迹实验,以检测切应力诱导的I-κB磷酸化和降解的情况;RT-PCR、Northern杂交和免疫荧光细胞化学染色观察TLR-4和TLR-2在人脐静脉内皮细胞上的表达;用RT-PCR技术从血管内皮细胞扩增胞内区段缺失突变TLR-4 cDNA克隆于真核表达质粒pcDNA3,构建重组TLR-4胞内缺失突变基因真核表达质粒pcDNA3-mTLR4,与pEGFP1-IL8USCS共转染内皮细胞,流式细胞术观察mTLR4对切应力诱导IL-8报告基因表达的影响.结果和讨论(1)未用切应力处理的内皮细胞没有IL-8基因的表达;切应力处理内皮细胞后,1 h IL-8 mRNA表达增加,2 h IL-8 mRNA表达量至最高值,3 h IL-8 mRNA表达量开始下降,4 h后IL-8 mRNA持续相对高表达;各实验组(2.23、4.20、6.08 dyne/cm2)均表现出相同的IL-8 mRNA随时间的变化规律,即IL-8的表达对切应力有时间依赖性.(2)未用切应力处理的内皮细胞没有IL-8基因的表达;切应力处理内皮细胞后,低切应力时IL-8 mRNA表达量明显增加,高切应力时IL-8 mRNA表达量较低.IL-8 mRNA的表达量与内皮细胞所施加的切应力强度呈反变关系;不同的切应力作用时间(1、2 h)均表现出相同的IL-8 mRNA随切应力强度的变化规律.提示流体切应力诱导内皮细胞表达IL-8,不仅与切应力的作用时间有关,而且IL-8的表达量与切应力作用强度有关,两者之间具有明显的直线负相关.直线回归方程:1 h时为y=7.57-0.11x,相关系数r=-0.97;2 h时为y=7.92-0.10 x,相关系数r=-0.96.(3)未用切应力处理的内皮细胞只有极少量的IL-8蛋白质生成;切应力处理内皮细胞1 h后,IL-8蛋白质生成量增加,处理5 h后IL-8蛋白质生成量增加至最高值,处理8 h后IL-8蛋白质生成量下降,10 h后IL-8蛋白质生成量维持在一个较高的水平.各实验组(2.23、4.20、6.08 dyne/cm2)均表现出相同的IL-8蛋白质生成量随切应力作用时间的变化规律.提示流体切应力确实可以诱导内皮细胞生成IL-8,并且IL-8的生成量与切应力的作用时间有关,呈双相性变化.(4)未用切应力处理的内皮细胞只有极少量的IL-8蛋白质生成;切应力处理内皮细胞后,低切应力(2.23 dyne/cm2)时IL-8蛋白质生成量明显增加,为高切应力(19.29 dyne/cm2)时IL-8蛋白质生成量的约6(作用5h)或7倍(作用6h).IL-8蛋白质生成量与内皮细胞所施加的切应力强度呈反变关系;直线回归方程:5 h时为y=760.12-36.06x,相关系数r=-0.978;6 h时为y=781.87-36.66x,相关系数r=-0.980.(5)pEGFP1-IL8USCS转染细胞,层流切应力刺激3 h后IL-8报告基因绿色荧光蛋白表达增强.(6)NF-κB p65免疫荧光细胞化学染色显示,切应力刺激0.5 h,胞核即出现阳性反应,刺激1.5 h后,胞核呈强阳性染色.(7)细胞裂解物免疫印迹显示,刺激10 min时磷酸化IκB即显著增强,1 h后磷酸化IκB印迹强度降到无,而IκB随刺激时间延长而逐渐降低,0.5和1 h后降至测不到IκB.(8)免疫荧光细胞化学染色显示脐静脉血管内皮细胞膜表达TLR-4.RT-PCR和Northern杂交显示,人脐静脉血管内皮细胞表达TLR-2和TLR-4 mRNAs;当切应力刺激1 h后,TLR-4 mRNA表达明显增强.pcDNA3-mTLR4和pEGFP 1-IL8USCS共转染细胞,层流切应力刺激3 h后荧光蛋白表达未明显增强.本实验中我们采用的切应力为2.23、4.20、6.08 dyne/cm2,这些切应力与动脉血管分叉处等低切应力区生理切应力大小相似,在这些低切应力区,血管内皮细胞可分泌IL-8,IL-8转而激活中性粒细胞和单核细胞,调控它们和内皮细胞的黏附,这将触发一系列的病理变化,例如:动脉粥样硬化等.结论流体切应力诱导内皮细胞IL-8 mRNA的表达及IL-8蛋白的生成,在感染及动脉粥样硬化等病理过程中扮演着重要角色.     

胱抑素C对脑缺血再灌注损伤大鼠大脑皮质紧密连接的影响

目的:观察胱抑素C(Cys C)干预对脑缺血再灌注损伤(IRI)后大鼠脑皮质毛细血管内皮细胞紧密连接超微结构的形态学变化并探讨其影响机制。方法:雄性SD大鼠随机分为假手术组(Sham)、缺血再灌注损伤组(IRI)和胱抑素C(Cys C)干预组(根据药物浓度不同分为低、中、高3个亚组分别为Cys C-L、Cys C-M和Cys C-H),采用改良线拴法建立大鼠大脑中动脉缺血2 h再灌注24 h模型。透射电镜下观察内皮细胞紧密连接超微结构的改变,Western Blot观察损伤侧脑皮质ZO-1蛋白表达变化,免疫荧光检测Hsp20的表达与分布。结果:与Sham组相比,IRI组电镜下可见毛细血管内皮细胞紧密连接开放,ZO-1蛋白表达明显减少(P 0. 01),Hsp20细胞数目增多。与IRI组比较,Cys C-L、Cys C-M组紧密连接开放程度明显减轻,ZO-1蛋白表达增加(P 0. 01),Hsp20阳性细胞数也显著增多。而Cys C-H组紧密连接结构消失,毛细血管内皮细胞间完全开放、松散;ZO-1蛋白表达明显降低,Hsp20阳性细胞数也明显减少。结论:Cys C在一定浓度下干预能减轻脑IRI对紧密连接超微结构的破坏,其机制可能与ZO-1蛋白表达增加及Hsp20阳性细胞个数增多有一定的关系。     

流体低剪切力上调内皮细胞IL-8基因表达的细胞内信号转导途径分析

目的　利用流室系统，体外研究低剪切力上调内皮细胞IL-8基因表达的胞内信号转导途径。方法　以培养的人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)为研究对象，采用定量RT-PCR检测HUVECs经低剪切力(0.42 Pa)刺激后IL-8基因的表达情况及多种阻断剂对其的干预作用。结果　(1)KT5720(蛋白激酶A抑制剂)、Neomycin（磷脂酶C抑制剂）、Calphostin C（蛋白激酶C抑制剂）、Tyrphostin-25（酪氨酸蛋白激酶抑制剂）均不同程度地抑制低剪切力对内皮细胞IL-8基因的上调；(2)Ca(上标 2+)可促进低剪切力上调内皮细胞IL-8 mRNA表达；(3)非水解的GDP类似物－GDPβS亦可抑制低剪切力诱导的内皮细胞IL-8基因的上调；(4)蛋白激酶G抑制剂－KT5823对低剪切力诱导的IL-8基因表达无明显影响。结论　低剪切力上调内皮细胞IL-8基因表达的胞内信号转导途径非常复杂，涉及到多种信号分子。     

RNA干扰沉默神经轴突导向因子受体4基因增加血肿瘤屏障通透性

目的研究神经轴突导向因子受体(Robo4)对血肿瘤屏障(BTB)通透性的影响。方法建立了体外BTB模型,应用Real-time PCR和Western blot检测Robo4在正常人脑微血管内皮细胞和胶质瘤微血管内皮细胞中的表达变化。设计合成针对Robo4基因的小干扰RNA,转染至人脑微血管内皮h CMEC/D3细胞,下调体外血肿瘤屏障模型内皮细胞中Robo4的表达,跨内皮电阻测量系统和辣根过氧化物酶渗透试验分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白occludin和ZO-1的表达和分布变化。结果和正常人脑微血管内皮细胞相比,Robo4在胶质瘤微血管内皮细胞中的表达显著上调。下调体外血肿瘤屏障模型内皮细胞Robo4的表达后,TEER值显著降低,辣根过氧化物酶透过率显著增高;同时胶质瘤微血管内皮细胞中紧密连接相关蛋白occludin和ZO-1的表达显著降低,在细胞膜上呈不连续分布。结论 RNA干扰沉默Robo4表达能够显著降低紧密连接相关蛋白occludin和ZO-1的表达,增加BTB通透性。     

IL-18BP重组蛋白对TNF-α诱导的人脐静脉内皮细胞的作用研究

目的探讨人重组白细胞介素-18结合蛋白(rhIL-18BP)对肿瘤坏死因子(TNF-α)诱导的内皮细胞炎症、凋亡的影响。方法体外培养人脐静脉内皮细胞(HUVECs),用20 ng/ml的TNF-α和不同质量浓度的rhIL-18BP孵育相应时间,提取mRNA并用PCR及实时定量PCR检测细胞因子(IL-6、IL-8)、黏附分子(ICAM-1、VCAM-1)的mRNA表达;ELISA法检测细胞上清液中细胞因子(IL-6、IL-8)、可溶性黏附分子(sICAM-1、sVCAM-1)的蛋白含量;流式细胞术检测细胞凋亡的变化。结果 1)IL-18BP可以抑制TNF-α诱导的人脐静脉内皮细胞中炎症因子的表达;2)IL-18BP能抑制TNF-α诱导的HUVECs的凋亡。结论 rhIL-18BP对TNF-α诱导的内皮炎症反应有抑制作用,并可以抑制TNF-α诱导的内皮细胞凋亡。     

miR-579-3p/DEFA3调控脓毒血症的炎症反应及靶向关系验证北大核心CSCD

目的:研究miR-579-3p对脓毒血症小鼠炎症因子分泌和内皮细胞焦亡的影响机制。方法:基于过表达miR-579-3p转基因小鼠和敲减α-防御素3(DEFA3)基因的转基因小鼠构建脓毒血症小鼠模型;qRT-PCR检测小鼠血浆中miR-579-3p表达;ELISA检测小鼠血清中TNF-α、IL-8和IL-6的表达;流式细胞术检测小鼠内皮细胞焦亡;Western blot检测小鼠血浆中DEFA3蛋白表达;双荧光素酶报告基因实验检测miR-579-3p与DEFA3的结合力。结果:miR-579-3p、DEFA3在脓毒血症小鼠血浆中的表达水平均显著升高(P<0.05)。过表达miR-579-3p转基因脓毒血症小鼠和敲减DEFA3基因的转基因脓毒血症小鼠血清中炎症因子TNF-α、IL-8和IL-6的表达均明显降低,内皮细胞焦亡率明显降低(P<0.05)。miR-579-3p与DEFA3存在靶向作用。结论:miR-579-3p可抑制脓毒血症小鼠的炎症因子分泌和内皮细胞焦亡,其机制与靶向DEFA3相关,可为脓毒血症的治疗提供靶标。     

缓激肽对脑胶质瘤大鼠紧密连接影响的形态学观察

目的研究缓激肽(BK)对脑胶质瘤大鼠血肿瘤屏障紧密连接的影响。方法采用伊文氏兰(EB)法检测缓激肽作用后血肿瘤屏障(BTB)通透性的变化;应用透射电镜(TEM)观察BK作用后内皮细胞间紧密连接的变化,同时应用硝酸镧[La(NO3)3]和辣根过氧化物酶(HRP)作示踪剂,检测缓激肽作用后,小分子和大分子示踪剂通过紧密连接的情况。结果缓激肽可使血肿瘤屏障对伊文氏兰的通透性增加,在15min时达到高峰,以后逐渐下降。透射电镜显示缓激肽作用15min时,肿瘤组织毛细血管内皮细胞间紧密连接的完整性明显破坏,缝隙指数显著增加,同时可见硝酸镧和辣根过氧化物酶在紧密连接处沉积。结论缓激肽能够通过开放紧密连接选择性增加血肿瘤屏障的通透性。     

Aberrant expression of tight junction-related proteins ZO-1, claudin-1 and occludin in synovial sarcoma: an immunohistochemical study with ultrastructural correlation.

Synovial sarcoma demonstrates epithelial differentiation, either by light microscopy (biphasic synovial sarcoma) or by immunohistochemical/ultrastructural methods only (monophasic) and poorly differentiated synovial sarcoma. Although the glands of synovial sarcoma are known to have tight junction-like structures, far less is known about junction formation in the spindled component of synovial sarcomas. Additionally, it is unknown whether the tight junctions of synovial sarcoma are normally constituted. The tight junction is a multiprotein complex consisting of numerous proteins that include ZO-1, claudin-1 and occludin. A total of 35 cases of synovial sarcoma (13 biphasic, 14 monophasic and eight poorly differentiated) were immunostained for ZO-1, claudin-1 and occludin using commercially available antibodies, heat-induced epitope retrieval and standard avidin-biotin technique. When available, corresponding electron micrographs were reviewed. For five cases, the presence of either an SYT-SSX1 (three cases) or SYT-SSX2 (two cases) gene fusion was known. Positive cases showed particulate membrane staining. The glands of biphasic synovial sarcomas expressed ZO-1 (13/13), claudin-1 (12/13) and occludin (11/13) in a manner identical to normal glandular epithelia, at the apical portion of the lateral membrane. The spindle cells of biphasic synovial sarcomas showed abnormal circumferential membranous expression of ZO-1 (12/13), claudin-1 (6/13) and occludin (3/13). Monophasic synovial sarcomas expressed ZO-1 in a circumferential pattern (13/14) but less often claudin-1 (4/14) or occludin (3/14). Poorly differentiated synovial sarcomas expressed ZO-1 (8/8) and claudin-1 (6/8) but only rarely occludin (2/8). By electron microscopy, recognizable tight junctions were seen only in glands. No correlation was seen between histologic subtype or fusion type and expression of tight junction proteins. We conclude that the glands of biphasic synovial sarcomas show well-organized, true epithelial tight junctions. In contrast, the spindled cells of all synovial sarcomas show significant abnormalities in the expression and localization of tight junction proteins, suggesting partial and/or aberrant epithelial differentiation.     

Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study.

Tight junction associated proteins are key molecular components governing cellular adhesion, polarity and glandular differentiation. Tight junction proteins also play critical roles in cellular proliferation and neoplastic pathways via their functions as couplers of the extracellular milieu to intracellular signaling pathways and the cytoskeleton. Neoplastic cells frequently exhibit structural and functional deficiencies in the tight junction. The purpose of this study was to determine the pattern of expression and prognostic value of four tight junction associated proteins, claudin-1, claudin-4, occludin and ZO-1 in a cohort of TNM stage II colon cancer using tissue microarray technology. In this study, we retrospectively analyzed, resected and otherwise untreated paraffin embedded specimens from 129 consecutive patients with TNM stage II colonic carcinomas for claudin-1, claudin-4, occludin and ZO-1 protein expression by immunohistochemistry. Seventy-five, 58, 56 and 44% of the tumors exhibited normal to elevated expression levels (+2 and +3 immunopositivity) of claudin-1, claudin-4, occludin and ZO-1 respectively. Low expression levels of claudin-1 and ZO-1 were directly associated with higher tumor grade (P=0.05 and 0.03 respectively). Multivariate analysis indicated that lymphovascular invasion (P=0.01) and low levels of claudin-1 (P=0.0001) expression were independent predictors of recurrence and that reduced claudin-1 expression (P=0.0001) was associated with poor survival. This study is the first to comprehensively examine the expression of several tight junction associated proteins in colonic neoplasms and to correlate their expression with disease progression. Loss of claudin-1 expression proved to be a strong predictor of disease recurrence and poor patient survival in stage II colon cancer.     

Amyloid beta-peptide1-42 alters tight junction protein distribution and expression in brain microvessel endothelial cells

Alzheimer's disease is characterised by neuronal loss, numerous intraneuronal deposits of neurofibrillary tangles, senile plaques, and cerebrovascular amyloid deposits. The major component of senile plaques and cerebrovascular deposits is the 39-43 amino acid beta-amyloid peptide (Abeta). The effects of Abeta on cerebral endothelium and thus the blood-brain barrier remain unclear. Utilising endothelial cells isolated from rat cerebral cortex microvessels, we have examined effects of Abeta peptides on tight junction protein behaviour. The transmembrane tight junction proteins occludin, claudin-1 and claudin-5, as well as the cytoplasmic accessory proteins ZO-1 and ZO-2 displayed a continuous distribution at cell boundaries. Endothelial cells exposed to Abeta1-42 (20 microM) for 3 days showed a disrupted plasma membrane pattern of claudin-5 and ZO-2 with relocation to the cytoplasm. These effects were not seen with Abeta25-35 or Abeta1-40[Gln22] (Dutch type). Abeta1-42 treatment altered also protein expression: occludin was lower at 1st day, claudin-1 increased at all times, and ZO-2 increased after 1 day and then decreased. These data suggest that Abeta1-42 effects on tight junction protein complexes may alter blood-brain barrier integrity and contribute to the neuropathological sequelae of Alzheimer's disease.     

青蒿素减轻LPS诱导的肠上皮细胞屏障功能损伤的实验研究

目的:探究青蒿素对脂多糖(lipopolysaccharide,LPS)诱导的大鼠肠上皮IEC-6细胞屏障功能损伤的影响。方法:体外培养IEC-6细胞,随机分为5组:对照组、LPS(100 mg/L)组和LPS+青蒿素(30、50和100μmol/L)组,MTT法检测各组细胞毒性变化,ELISA检测各组细胞分泌炎性因子TNF-α、IL-1β和IL-6水平的变化,电阻仪检测肠上皮细胞跨上皮电阻(TER),酶标仪检测单层细胞对辣根过氧化物酶(HRP)的通透性,RT-qPCR和Western blot检测各组细胞紧密连接蛋白(ZO-1、claudin-1和occludin)以及TLR4/My D88/NF-κB mRNA和蛋白表达的变化。结果:LPS与青蒿素在本实验浓度范围对IEC-6细胞均无毒性。与对照组相比,LPS处理下,细胞分泌TNF-α、IL-1β和IL-6水平以及TLR4/My D88/NF-κB的mRNA和蛋白表达明显增加,ZO-1、claudin-1和occludin的mRNA和蛋白表达降低。而青蒿素干预下,细胞分泌TNF-α、IL-1β和IL-6水平以及TLR4/My D88/NF-κB mRNA和蛋白表达明显降低,ZO-1、claudin-1和occludin的mRNA和蛋白表达升高(P0.05),均呈现浓度依赖性。结论:青蒿素可能通过抑制TLR4/My D88/NF-κB通路减轻LPS诱导的肠上皮细胞屏障功能损伤。     

Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell-cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as 'positive'. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell-cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.     

帕金森病模型大鼠十二指肠黏膜紧密连接蛋白表达下调

目的研究紧密连接蛋白在6-羟多巴(6-OHDA)制备的帕金森病(PD)大鼠模型十二指肠黏膜的表达变化。方法用6-OHDA损毁双侧中枢黑质多巴胺能神经元建立大鼠模型。用免疫荧光组织化学和蛋白免疫印迹检测紧密连接蛋白claudin-1、occludin和ZO-1肠黏膜的定位和表达。结果紧密连接蛋白claudin-1、occludin和ZO-1在PD大鼠模型十二指肠黏膜上均有表达,但仅ZO-1(P0.001)和occludin(P0.01)表达明显下调,而claudin-1无显著变化。结论 PD大鼠模型十二指肠黏膜紧密连接蛋白ZO-1、occludin的表达显著下调,可能与帕金森病十二指肠溃疡的发生发展相关。     

Alterations in tight junction molecules of uterine epithelial cells during early pregnancy in the rat

Distribution patterns of the tight junction associated proteins ZO-1, claudin-1 and occludin were investigated in rat uterine epithelial cells during early pregnancy. Light microscopy and immunohistochemical labelling were used to detect these proteins on days 1, 3, 6 and 7 of pregnancy. Intense staining of claudin-1 at the apical region of the lateral plasma membrane accompanied diffuse staining throughout the cytoplasm. ZO-1 was also localised in the apical region, but ZO-1 was not present in the lower two thirds of the lateral plasma membrane or in the cytoplasm. Occludin was present only on days 6 and 7 of pregnancy. Labelling was also localised in the apical region of the lateral plasma membrane where tight junctions are known to be present. Our results show that ZO-1, claudin-1 and occludin are present in the apical region of uterine epithelial cells, and appear to play a role in the very dynamic tight-junctional network of uterine epithelial cells during early pregnancy. In particular, occludin appears only during uterine receptivity for implantation.     

siRNA-Tie1下调紧密连接相关蛋白claudin-5、occludin和ZO-1增加血肿瘤屏障通透性

目的研究Tie1 AS和Tie1表达变化对血肿瘤屏障通透性的影响和相关机制。方法建立体外血脑屏障和血肿瘤屏障模型,Real-time PCR检测h CMEC/D3细胞中Tie1 AS和Tie1的表达水平。转染p IRES2-EGFPTie1 AS表达载体上调体外血肿瘤屏障模型h CMEC/D3细胞中Tie1 AS的表达,Western blot检测Tie1的表达变化。将si RNA-Tie1转染人h CMEC/D3细胞并建立体外血肿瘤屏障模型,跨内皮电阻测量系统分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白claudin-5、occludin和ZO-1的表达和分布变化。结果和正常脑微血管内皮细胞相比,体外血肿瘤屏障模型h CMEC/D3细胞中Tie1的表达显著增加,而Tie1 AS的表达显著降低。上调体外血肿瘤屏障模型内皮细胞中Tie1 AS的表达水平,能够显著降低Tie1的表达。下调体外血肿瘤屏障模型内皮细胞中Tie1的表达后屏障通透性显著下降,伴有claudin-5、occludin和ZO-1的表达下调以及在细胞膜上呈不连续分布。结论体外血肿瘤屏障模型内皮细胞中低表达的Tie1 AS能够负性调控Tie1的表达,进而通过调节紧密连接相关蛋白的表达和分布影响屏障的通透性。     

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.