阿司匹林对人胃黏膜上皮细胞生长和occludin蛋白表达的影响及其机制研究

背景：研究显示ERK信号通路激活可促进细胞生长,上调紧密连接蛋白oceludin表达,而阿司匹林可抑制ERK的磷酸化激活。目的：探讨阿司匹林对人胃黏膜上皮细胞生长和occludin蛋白表达的影响及其可能机制。方法：建立人胃黏膜上皮细胞株GES-1单层细胞模型．M1Tr法检测阿司匹林（5-20mmol／L）和MEK抑制剂U0126（10～40μmol／L）对GES-1细胞的生长抑制作用。将GES-1细胞分为阿司匹林（8．78mmol／L）组、U0126（14．91μmol／L）组、联合组（U0126＋阿司匹林）和阴性对照组,倒置相差显微镜下观察细胞形态学变化,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测P-ERK1／2、occludin蛋白表达。结果：阿司匹林和U0126均能剂量依赖性地抑制GES．1细胞生长,联合组细胞凋亡较两药单用更为显著,贴壁细胞数量减少更为明显．细胞变圆、悬浮。阿司匹林组、U0126组和联合组P-ERK1／2、occludin蛋白表达量均显著低于阴性对照组,U0126组和联合组降低更为明显。结论：阿司匹林可抑制人胃黏膜上皮细胞生长．下调occludin蛋白表达,其机制至少部分与抑制ERK信号通路激活有关。     

麝香黄芪复方滴丸对体外缺血缺氧血脑屏障通透性及相关调节蛋白的影响

目的探究麝香黄芪复方滴丸调控体外缺血缺氧性血脑屏障(blood-brain barrier,BBB)模型的作用机制。方法利用大鼠脑微血管内皮细胞(brain microvessel endothelial cells,BMEC)与星形胶质细胞(astrocytes,AS)共培养建立体外BBB模型及缺血缺氧BBB模型。随机分为正常对照组、模型组、麝香黄芪复方滴丸高剂量组(500μg/mL)、中剂量组(100μg/mL)、低剂量组(10μg/mL)及依达拉奉对照组。通过跨内膜电阻(TEER)、辣根过氧化物酶法(HRP)检测BBB通透性变化情况。Western Blot法检测紧密连接(tight junction,TJ)相关蛋白闭合蛋白-5(Claudin-5)、连接黏附分子-A(junctional adhesion molecule,JAM-A)、跨膜蛋白的咬合蛋白(Occludin)和闭锁小带蛋白-1(zonula Occludens,ZO-1)表达水平。结果TEER及HRP实验显示,与正常对照组相比,模型组血脑屏障通透性增加(P<0.05);麝香黄芪复方滴丸高、中、低剂量组和依达拉奉组1 d、2 d、3 d各组通透性明显降低(P<0.05);WB结果显示,模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达较正常组显著降低(P<0.05);麝香黄芪复方滴丸高、中剂量组较模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达升高(P<0.05)。结论麝香黄芪复方滴丸能降低缺血缺氧性BBB模型的通透性,其作用机制与调控紧密连接蛋白而达到修复BBB的作用有关。     

回药扎里奴思方联合BMSCs移植对脑缺血再灌注大鼠BBB紧密连接蛋白的影响

目的观察扎里奴思方联合BMSCs移植对MCAO模型大鼠BBB上Occludin和Claudin mRNA表达的影响。方法 250只SD大鼠随机分为假手术组、模型组、扎方组、移植组和联合组,除假手术组10只外,其余各组15只;线栓法制备MCAO模型,体外全骨髓贴壁法培养及扩增BMSCs;大鼠灌胃给药[14.6 g/(kg·d)],BMSCs悬浮液经颈内动脉移植入脑(2×106/200μl);移植后1 d、3 d、7 d、14 d取材,干湿重法检测脑含水量,Real timePCR技术检测Occludin和Claudin mRNA表达。结果模型大鼠脑含水量较假手术组增加(P0.01),Occludin和Claudin mRNA表达降低(P0.01);与模型组比较,扎方、移植及联合各3 d、7 d、14 d组脑含水量降低(P0.01),各组各时间点Occludin和Claudin mRNA表达增高(P0.01);与移植组比较,扎方1 d组脑含水量降低(P0.05),3 d组Occludin mRNA表达增高(P0.01),7 d组表达降低(P0.01),扎方1 d组Claudin mRNA表达增高(P0.05),7 d、14 d组表达降低(P0.01),联合各组脑含水量均降低,以1 d、14 d明显(P0.01),各组Occludin和Claudin mRNA表达均增高(P0.01);扎方与联合组比较,联合各组脑含水量降低,以7 d、14 d组明显(P0.01,P0.05),Occludin和Claudin mRNA表达增高(P0.01);同组间比较,均以7 d组变化显著,脑含水量呈先增后减趋势,Occludin和Claudin mRNA表达呈先减后增趋势,14 d有明显改善(P0.01)。结论脑缺血再灌注损伤后BBB受损,通透性改变,脑水肿形成,以7 d最为明显;扎里奴思方和BMSCs移植均可不同程度改善脑缺血后脑水肿程度,以二者联合应用作用显著,其机制可能与干预Occludin和Claudin mRNA动态表达有关。     

JCOG trials of systemic chemotherapy for unresectable or recurrent gastric cancer

Background  Claudin, occludin, and zonula occludens (ZO)-1 are known as tight-junction-associated proteins. The aim of this study was to examine the expression of these proteins in gastric carcinoma. Methods  Gastric cancer tissues (n = 124) were obtained from 124 patients who underwent gastrectomy at our hospital between January 2000 and December 2004. The expression of the above tight-junction-associated proteins in carcinoma, normal mucosa, and metaplastic epithelium was examined using immunohistochemistry. In addition, the expression of claudin-4 mRNA was examined in fresh frozen tissue obtained from 34 patients. Results  Significant correlations were seen between the expression of claudin-4, occludin, and ZO-1. In regard to claudin-4, significant correlations were seen between the expression of claudin-4 evaluated by immunohistochemistry and the expression of claudin-4 mRNA. Claudin-4 expression was significantly decreased in tumors with undifferentiated-type adenocarcinoma, advanced T stage, lymph node metastasis, and peritoneal metastasis. Occludin and ZO-1 expression was significantly decreased in tumors with undifferentiated-type adenocarcinoma. Overall survival was significantly shorter in patients with low claudin-4 expression. Cox multivariate analysis revealed that low claudin-4 expression was independently associated with significantly decreased overall survival. Conclusion  Tight-junction-associated proteins, particularly claudin-4, may play important roles in determining invasiveness, metastatic potential, and survival in gastric cancer.     

The loss of αSNAP downregulates the expression of occludin in the intestinal epithelial cell of acute pancreatitis model

Background and objectiveIntestinal barrier damage is an important event during the occurrence and progression of severe acute pancreatitis. The expression of occludin, one of the main components of the intestinal barrier proteins, is regulated by various factors related to intestinal barrier formation and the remodeling process. The αSNAP, as a novel membrane protein, is ubiquitously expressed in intestinal epithelial cells. This study aimed to investigate the role of αSNAP in acute pancreatitis and the relationship between occludin and αSNAP.MethodsMild and severe acute pancreatitis models were established by retrograde injections of 0.5% and 3.8% sodium taurocholate solutions, respectively, into rat pancreaticobiliary ducts. The animals were killed at 1, 2, and 3 days after the injection, and the pathological changes of the pancreas and intestinal mucosa, the changes in intestinal permeability, and the protein expression of occludin and αSNAP were assessed. Cultured epithelial IEC-6 cells were further infected with lentiviral αSNAP shRNA, cell apoptosis was determined with flow cytometry (FCM), and any changes in occludin expression were detected by Western blotting and immunofluorescent staining.ResultsThis pathologic study of a rat acute pancreatitis model indicated pancreatic tissue necrosis and inflammatory cell infiltration; the intestinal villi in the severe acute pancreatitis (SAP) group demonstrated edema, lodging, atrophy, and intestinal epithelial cell necrosis, and shedding. The intestinal permeability in rats with pancreatitis increased significantly. The SAP group showed significantly increased levels of serum TNF-α and endotoxins. The results of immunofluorescent staining and Western blotting revealed that compared with the SO (sham operation) and MAP (mild acute pancreatitis) groups, the SAP group displayed significantly downregulated protein expressions of αSNAP and occludin in the intestinal epithelial cells. After the lentiviral transduction of αSNAP shRNA, apoptosis in IEC-6 cells was drastically increased, whereas the expression of occludin was decreased significantly.ConclusionThe downregulated expression of αSNAP in intestinal epithelial cells leads to reduced occludin expression and enhanced apoptosis of intestinal epithelial cells. Hence, the permeability of the intestinal barrier may be increased in a severe acute pancreatitis model.     

Alterations of the blood-brain barrier in cerebral white matter lesions in the ageing brain

White matter lesions (WML) are associated with dementia and are common in brain ageing. In order to determine whether alteration of the blood-brain barrier (BBB) may contribute to the pathogenesis of WML we assessed albumin leakage and expression of the tight junction (TJ) proteins claudin-5 (Cln-5), zona occludin-1 (ZO-1) and occludin in cases derived from the Medical Research Council Cognitive Function and Ageing Study. Albumin extravasation was widespread in the ageing brain and enhanced in WML, suggesting dysfunction of the BBB may contribute to the pathogenesis of WML. This was not accompanied by significant changes in the endothelial expression of TJ proteins. However, ZO-1 and occludin were expressed by glial cells throughout the parenchyma of both control white matter and WML, suggesting these TJ proteins may have other functions in the brain.     

Viability of microvascular endothelial cells to direct exposure of formalin,lambda-carrageenan,and complete Freund's adjuvant

We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood–brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, λ-carrageenan, or complete Freund's adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (34%) and a significant increase in cell permeability to [¹⁴C]sucrose at 120 min (137%). Brain perfusion using female Sprague–Dawley rats showed no difference in paracellular permeability to [¹⁴C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood–brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood–brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.     

鞘内注射右美托咪定对大鼠脊髓缺血再灌注损伤后FGFR3表达及血-脊髓屏障的影响

目的:观察鞘内注射右美托咪定(dexmedetomidine,Dex)对大鼠脊髓缺血再灌注损伤(spinal cord ischemia reperfusion injury,SCIRI)后细胞生长因子受体3(FGFR3)表达、血-脊髓屏障(blood-spinal cord barrier,BSCB)结构及其相关结构蛋白occludin的影响。方法 :120只SD大鼠随机分为4组:假手术组(Sham组)、缺血再灌注损伤组(IR组)、Dex预处理组(Dex组)和Dex+阿替美唑(ATIP)预处理组(Dex+ATIP组),Sham组和IR组分别于造模前3d开始鞘内生理盐水30μl、Dex组鞘内注射10μg DEX(30μl);Dex+ATIP组鞘内注射右美托咪定10μg+阿替美唑10μg(共30μl),1次/d,连续3d。Sham组仅暴露主动脉弓而不结扎,其他3组开胸后用无创动脉夹夹闭主动脉弓14min后再开放,建立SCIRI模型。分别于造模后12h和48h取L4~L6脊髓,采用干湿法测定脊髓含水量;伊文思蓝(evans blue,EB)染色测定BSCB完整性;Weston blot和RT-PCR测定脊髓组织中FGFR3和occludin含量。结果:造模后12h和48h,与Sham组比较,IR组、Dex组和Dex+ATIP组相应时间点脊髓组织中含水量、EB含量、FGFR3蛋白和基因表达均显著性增加,occludin表达显著性下降(P0.05);Dex组与相应时间点与IR组和Dex+ATIP组相比脊髓组织中含水量、EB含量和FGFR3蛋白和基因表达均显著性降低,而occludin表达增加(P0.05);Dex+ATIP组与IR组相应时间点比较均无显著性差异。造模后12h和48h,EB染色荧光显微镜下观察Sham组脊髓实质内几乎未见红色荧光,Dex组红色荧光有所增加,而IR组和Dex+ATIP组红色荧光显著增加,48h变化较12h更为显著。结论 :鞘内注射Dex可下调大鼠脊髓缺血再灌注损伤后脊髓组织中FGFR3蛋白表达,维持occludin蛋白含量,对血-脊髓屏障起到保护作用。     

血管通透性增高的基本机制

The increased microwascular permeability appears msinly in venule during inflammation,shock,and bums.Endothelial cells play an important role in venule permeability enhancement.There are two kinds of pathway for macromolecule extravasation.One is paracellular pathway and another is transcellular pathway,which are related to the formation of endothelial gap or transcellular openings seperately.The alter-ation of intercellular reated portein,such as occludin,claudin,zona occludens(ZO),junctional adhesion molecule(JAM),VE-cadherin,catenin,integrin,etc,and the alteration of endothelial cytoskeleton,such as rearrangement of actin filament,formation of stress fiber and focal ahdesion,etc,involve in the pathogenesis of increased microvascular permeability.     

Occludin expression and tight junction strand formation during replicative DNA synthesis in primary cultures of rat hepatocytes

Occludin, an integral membrane protein, is a candidate for forming the functional intercellular seal of the tight junction. In the present study, we examined the changes of occludin expression and tight junction strand in the cultured hepatocytes. In L-15 medium containing EGF with 2% DMSO and 10^–7M glucagon, the great majority of the cells were quiescent. Under this condition, occludin immunoreactivity was observed at cell-to-cell contacts. Tight junction strands formed well-developed networks in freeze-fracture replicas. When the cells entered S phase of the cell cycle, occludin immunoreactivity and the number of tight junction strands decreased. By contrast, when DNA synthesis was then inhibited by readdition of DMSO and glucagon, occludin immunoreactivity and the number of tight junction strands were restored. Regardless of DNA synthesis, however, there was no significant decrease in the protein and mRNA levels of occludin. These changes of occludin immunoreactivity were correlated to the organization of actin filaments. On the other hand, no change of ZO-1 immunoreactivity was observed. These results suggested that tight junctions of cultured hepatocytes regulated in a cell cycle-dependent fashion and correlated with the organization of circumferential actin bundles.