Claudin蛋白与肿瘤关系的研究进展

正常机体上皮组织细胞间存在着紧密连接（tight junction,TJ）复合物,位于复合物最顶端的就是TJ。TJ具有细胞黏附、维持细胞极性和通透性及辅助传递调节细胞增殖、分化信号等作用,对维持上皮组织细胞正常的结构及功能极为重要。Claudin蛋白是TJ的主要成分,参与维持TJ的各种功能,其表达异常与多种肿瘤密切相关。本文就Claudin蛋白与肿瘤关系的研究进展作一综述。     

冀南地区胃癌组织中 Claudin-7、MT1-MMP的表达及临床意义

胃癌是我国最常见的消化系统恶性肿瘤，在河北南部更是高发，多数患者发现时已是进展期，预后较差。紧密连接（tight junction，TJ）是最表层细胞间的连接，具有调节上皮细胞极性和分化的重要作用。细胞间连接的结构异常以及伴随发生的相关蛋白的表达变化可能导致恶性肿瘤细胞的分离和脱落，从而形成浸润和转移。浸润和转移是胃癌高死亡率的主要原因。癌细胞在转移形成之前，要多次穿过基底膜才能完成浸润，因此基底膜的完整性和酶对基质的降解是目前研究的热点。Claudin 蛋白是构成紧密连接的重要的跨膜糖蛋白，能够维持细胞的黏附和极性。Claudin7是其家族的一员，其异常表达与肿瘤发生、发展相关［1］。MT1-MMP 是基质金属蛋白酶家族中（MMPs）的一员，可以降解多种细胞外基质成分，在肿瘤的演进过程中也发挥着重要的作用。本实验联合检测 Claudin-7、MT1-MMP 蛋白在冀南地区胃癌中的表达，探讨其表达与该地区胃癌的临床病理特点的关系及意义。     

DEHP体外对大鼠睾丸支持细胞紧密连接作用及相关基因表达影响

目的探讨邻苯二甲酸二乙基己酯(di-2-ethylhexyl phthalate,DEHP)体外对大鼠睾丸支持细胞紧密连接的作用以及相关基因表达的影响。方法体外分离和原代培养大鼠睾丸支持细胞,建立支持细胞原代双室培养模型,以只加入二甲基亚砜(DMSO)的细胞孔作为对照,以10、50、100 nmol/L浓度的DEHP(DEHP低、中、高剂量组)染毒24 h。应用跨上皮电阻测定法检测单层细胞两侧跨上皮细胞电阻值(TER),荧光定量聚合酶链反应(PCR)法检测支持细胞闭锁蛋白(Occludin)、闭锁小带蛋白1(Zonula occluden-1,ZO-1)、紧密连接蛋白11(Claudin 11)基因mRNA表达,应用Weastern blot检测支持细胞Occludin、ZO-1、Claudin 11蛋白的表达。结果与对照组比较,不同浓度的DEHP各组细胞TER值均降低。同时大鼠睾丸支持细胞Occludin、ZO-1、Claudin 11基因mRNA表达随DEHP浓度增加而降低(P0.05),Occludin、ZO-1、Claudin 11蛋白表达也随DEHP浓度增加而降低(P0.05)。结论 DEHP可通过下调Occludin、ZO-1、Claudin 11基因表达而影响大鼠睾丸支持细胞的紧密连接,进而影响支持细胞的功能。     

磷酸弗林蛋白酶酸性氨基酸簇分选蛋白-2在代谢综合征及糖尿病肾病中的研究进展

磷酸弗林蛋白酶酸性氨基酸簇分选蛋白-2(phosphofurin acidic cluster sorting protein-2,PACS-2)是一种具有膜转运及维持线粒体相关内质网膜(mitochondria-associated endoplasmic reticulum membrane,MAM)结构与功能的多功能蛋白。近年研究发现PACS-2不仅参与调节细胞凋亡等细胞生理与病理活动,同时参与MAM结构调节及胰岛素功能调节,与代谢综合征及糖尿病肾病等多种疾病发病机制相关,并有望成为相关疾病的药物新治疗靶点。本文就国内外有关PACS-2分子特征、表达定位、生物学功能及其在代谢综合征与糖尿病肾病研究中的最新进展进行阐述。     

白血病和卵巢癌亲代及耐药细胞系细胞外调节蛋白激酶与端粒酶活性的变化

本研究观察白血病和卵巢癌亲代及耐药细胞系中端粒酶活性及细胞外调节蛋白激酶(extracelluar regulated protein kinases ERK)磷酸化蛋白表达水平的变化，探讨端粒酶与ERK在白血病及卵巢癌耐药中的作用。采用MTT法评价白血病和卵巢癌亲代和耐药细胞系对HRT或DDP的敏感性，用流式细胞术分析这两种细胞系间细胞周期分布的差别，用端粒酶重复扩增技术(TRAP)、生物发光分析法定量和定性检测端粒酶活性及用Western blot检测法检测磷酸化ERK1和ERK2蛋白的表达水平。结果表明：白血病和卵巢癌耐药细胞系处于G0／G1期的细胞比例增加，端粒酶活性和磷酸化ERK1/2蛋白表达水平耐药细胞系较亲代细胞系高。结论：耐药细胞系G0／G1期细胞比例增加可能是细胞产生耐药的一种标志。白血病和卵巢癌细胞系耐药的产生可能与端粒酶活性和磷酸化ERK1/2蛋白表达水平的增高有关。     

构建脑血管内皮细胞紧密连接模型对脑血管疾病发生发展机制研究的意义

目的：电镜下观察人脐静脉内皮细胞株ECV304细胞间紧密连接的形成，并建立脑血管内皮细胞紧密连接的模型，以期发现该模型的建立对发现脑血管病变早期发生及发展的意义。方法：培养ECV304细胞，在细胞生长接触到融合状态后，在透射电镜下观察细胞间紧密连接的形成情况。结果：透射电镜下可见到ECN304细胞间能够形成典型的紧密连接结构，以及内皮细胞所特有的W-P小体，吞饮小泡等结构。结论：ECV304细胞之间能够形成典型的紧密连接；可以利用ECV304细胞系建立研究脑血管内皮细胞间紧密连接的模型。     

紧密连接蛋白在非小细胞肺癌中的研究进展

紧密连接是细胞间连接的一种常见形式, 主要位于上皮细胞、内皮细胞间的连接复合体中,起到栅栏和屏障的作用。紧密连接蛋白(Claudins)是紧密连接的主要组成部分,可调节细胞渗透性,参与维持紧密连接的各种功能。非小细胞肺癌患者Claudins异常表达,表达水平可能上调或下调,甚至离域。Claudins的异常表达对非小细胞肺癌的发生、发展具有促进或抑制作用,并与患者预后显著相关。本研究通过对Claudin-1、Clauidn-3、Clauidn-6、Clauidn-7在非小细胞肺癌中的研究进展进行综述,旨在进一步明确Claudins在非小细胞肺癌中的作用,为非小细胞肺癌的基础研究及临床治疗提供依据。     

间隙连接与膀胱肿瘤关系的研究进展

间隙连接（gap junction，GJ）是相邻细胞间进行物质和信息交换的跨膜蛋白通道结构，细胞间隙连接通讯（GJIC）是相邻细胞间信息传递的一种通讯方式。研究发现，间隙连接蛋白（connexin，CX）的表达与膀胱肿瘤的发生发展关系密切。本文综述了GJIC的物质基础及其生物学功能，其与细胞增殖及膀胱肿瘤的关系，以及恢复GJIC在膀胱肿瘤治疗中的前景。     

mTOR信号通路在造血及其它系统衰老中作用的研究进展

哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）是一种丝/苏氨酸蛋白激酶,在调节细胞的生长、增殖和存活中起着重要的作用.mTOR复合体调控mRNA的转录、核糖体的合成及代谢相关基因的表达,通过磷酸化其下游的靶蛋白如S6蛋白激酶和4E-BP1等来调节细胞的活动.近年来,mTOR分子及其相关信号通路在衰老调控中所起的作用渐渐被人们所认识.对mTOR信号通路在衰老中的生物学功能和调节机制的研究,不仅可以深入了解细胞自我更新和分化的机制,而且对衰老及其相关疾病的治疗、预防及寻找潜在的治疗靶点与药物具有重要意义.本文主要介绍mTOR信号通路在造血系统及其他系统衰老中研究的最新进展.     

小窝蛋白1与肾脏疾病

小窝（caveolae）是近年新认识的一种膜特异性微区结构，小窝蛋白（caveotin，Cav）是形成小窝的重要结构蛋白，参与跨膜信号转导和调节、细胞外基质重塑及纤连蛋白基质的更新。小窝蛋白1（Cavl）是一个整合膜蛋白，作为与细胞黏附生长和存活有关的信号分子活动平台（包括激素、生长因子受体及细胞因子，在肾脏疾病的发生发展中起重要作用，我们对Cavl在肾脏疾病中的作用综述如下。     

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.     

Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer

Disruption of the cell-cell junction with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis. The role of adherent junction proteins has been studied extensively in cancer, but the roles of tight junction (TJ) proteins are less well understood. Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs. Recent studies show changes in expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established. Here, we report an increased expression of claudin-1 in human primary colon carcinoma and metastasis and in cell lines derived from primary and metastatic tumors. We also report frequent nuclear localization of claudin-1 in these samples. Genetic manipulations of claudin-1 expression in colon cancer cell lines induced changes in cellular phenotype, with structural and functional changes in markers of epithelial-mesenchymal transition. Furthermore, we demonstrate that changes in claudin-1 expression have significant effects on growth of xenografted tumors and metastasis in athymic mice. We further provide data suggesting that the regulation of E-cadherin expression and β-catenin/Tcf signaling is a possible mechanism underlying claudin-1–dependent changes.     

结肠直肠癌紧密连接相关蛋白的分布和表达

目的 探讨结肠直肠癌中紧密连接相关蛋白在癌组织和邻近正常组织中分布和表达的变化.方法 采用逆转录荧光定量实时PCR技术、Western blot技术以及免疫组织化学方法,分别对35份结肠直肠癌癌组织标本和邻近正常组织标本7种紧密连接相关蛋白occludin(OC)、ZO1、ZO2和claudin14(CL1～CL4)的分布和表达进行检测.结果 癌组织中claudin 1和claudin 2的mRNA表达水平分别为0.97和0.54,较正常组织显著增高,平均增高倍数分别为26.3倍和30.4倍.Western blot分析也证实了这两种蛋白的高表达.occludin、ZO2、claudin 1、claudin 2和claudin 4的免疫组织化学分析也揭示了它们在癌组织细胞和正常组织细胞中的不同分布.claudin 1在肠正常组织上皮细胞主要见于胞质,在癌组织主要沿着上皮细胞质膜的侧面分布.claudin 2在正常组织中主要位于黏膜下层,呈颗粒状不均匀分布,在癌组织上皮细胞、胞质及胞核均有着色,且在膜上的分布较为明显.结论 紧密连接相关蛋白分布和表达的异常与结肠直肠癌密切相关,有望作为结肠直肠癌的肿瘤标志物以及治疗的靶分子.     

尿酸钠结晶影响肾小管上皮细胞的紧密连接

背景：目前研究来看,尿酸性结石与紧密连接蛋白及肾间质纤维化的关系仍未明确。目的：观察尿酸钠结晶对肾小管上皮细胞紧密连接的影响。方法：配制单钠尿酸钠晶体。将正常大鼠肾小管上皮细胞随机分为对照组和尿酸钠结晶组,分别用无血清培养基和尿酸钠结晶进行培养。免疫荧光和RT-PCR方法检测24,48,72h紧密连接蛋白的表达。结果与结论：与对照组比较,尿酸钠结晶于不同时相刺激NRK-52E细胞后,紧密连接蛋白和mRNA表达下降（P〈0.05）,72h时下降显著（P〈0.01）,蛋白表达出现重新分布现象。说明尿酸钠结晶可破坏肾小管上皮细胞紧密连接蛋白的结构、功能及导致分布异常。     

IL-6基因结构和功能生物信息学预测

目的以白细胞介素(IL)-6为研究对象,对其基因结构和功能进行生物信息学分析。方法采用生物信息学软件对基因编码的蛋白质结构、理化性质、信号肽、跨膜结构、亚细胞定位、二级结构及高级结构进行生物信息学分析。结果 IL-6为稳定亲水性跨膜蛋白,定位于细胞外,二级结构以α-螺旋和无规则卷曲为主,有2个Ser和2个Thr可成为蛋白激酶磷酸化位点。结论分析预测IL-6,在研究其参与高血压、炎性反应等方面有重要意义。     

beta 2-Microglobulin: methods and clinical applications.

beta 2-Microglobulin is a low molecular weight protein that is found in most biological fluids. It was originally isolated from urine of cadmium-poisoned patients. Its amino acid sequence was established and shown to be structurally related to immunoglobulin constant domains. With the aid of antibodies specific against beta 2-microglobulin, the protein was detected on the membranes of all nucleated cells, normal and neoplastic. Measuring the quantity of beta 2-microglobulin showed that high levels are present in patients with renal tubular deficiencies and several other pathological conditions including neoplastic diseases. Extremely high levels were detected in seminal fluid and colostrum. Despite the structural relationship to immunoglobulins, no immunological relationship was demonstrated with these proteins using antibodies specific for beta 2-microglobulin. However, such antibodies are cytotoxic to all cells carrying beta 2-microglobulin on their surfaces. The discovery that beta 2-microglobulin is an integral part of the histocompatibility antigens of human and murine origin stimulated further research and interest in this molecule. Several groups of investigators have shown that beta 2-microglobulin is the low molecular weight chain and is noncovalently bound to a high molecular weight chain which carries the histocompatibility antigens. The structure of the histocompatibility antigens of lymphocytes (HLA) was shown by immunochemical as well as biological methods, and it is now well accepted. The antibodies against beta 2-microglobulin are extremely useful in the isolation of the histocompatibility antigens for sequence studies. Furthermore, the antibody to beta 2-microglobulin revealed that other structures may be bound to beta 2-microglobulin such as phytohemoagglutimin (PHA) receptors, mixed lymphocyte culture (MLC) antigens, etc. Murine thymus leukemia (TL) antigen also contains beta 2-microglobulin as an integral part of its structure; other tumor antigens may have a similar structure. Through all these studies, beta 2-microglobulin emerged as the best known membrane protein that can serve as a model for study of the arrangement and the function of the cell membrane.     

mda-9/syntenin: recent insights into a novel cell signaling and metastasis-associated gene

PDZ (an acronym representing three proteins--postsynaptic density protein PSD95/SAP90, drosophila tumor suppressor DLGA, and tight junction protein ZO-1) domain containing proteins are adapter proteins that play indispensable roles in regulating cell growth, development, and differentiation, predominantly through their capacity to serve as central organizers of protein complexes at the plasma membrane. A recently identified member of this protein family is melanoma differentiation associated gene-9 (mda-9), also known as syntenin, which was first identified as a gene down-regulated during human melanoma differentiation as mda-9 and subsequently recognized as an interacting partner of the cell-surface heparan sulfate syndecans, syntenin. Interest in mda-9/syntenin is intensifying because of its involvement in organization of protein complexes in the plasma membranes, regulation of B cell development, intracellular trafficking and cell surface targeting, cancer metastasis, synaptic transmission, and axonal outgrowth. In this review, we discuss the identification, structure and function of mda-9/syntenin and delineate future studies to address its role in regulating key physiological and pathological processes.     

Intestinal cytoskeleton degradation precedes tight junction loss following hemorrhagic shock

Hemorrhagic shock (HS) leads to intestinal barrier loss, causing systemic inflammation, which in turn can ultimately lead to multiorgan dysfunction syndrome. Barrier function is based on tight junctions (TJs) between intact epithelial cells. These TJs are anchored in the cell via the filamentous actin (F-actin) cytoskeleton. We hypothesize that HS causes hypoperfusion, leading to loss of F-actin, via activation of actin-depolymerizing factor/cofilin (AC), and consequently TJ loss. This study is aimed at unraveling the changes in cytoskeleton and TJ integrity after HS in organs commonly affected in multiorgan dysfunction syndrome (liver, kidney, and intestine) and to elucidate the events preceding cytoskeleton loss. Adult rats were subjected to a nonlethal HS and sacrificed, along with unshocked controls, at 15, 30, 60, and 90 min after induction of shock. Cytoskeleton, TJ integrity loss, and its consequences were studied by assessment of globular actin, F-actin, AC, zonula occludens protein 1, claudin 3, and bacterial translocation. In the liver and kidney, TJ and the F-actin cytoskeleton remained intact at all time points studied. However, in the intestine, significant loss of F-actin and increase of globular actin was seen from 15 min after shock. This change preceded statistically significant loss of the TJ proteins claudin 3 and zonula occludens protein 1, which were observed starting at 60 min after induction of shock (P < 0.05 vs. controls). Early after induction of shock (15 and 30 min) the nonactive AC (phosphorylated AC) in the intestine was significantly decreased (by 21% and 27%, P < 0.05 vs. control), whereas total AC remained constant, reflecting an increase in activated AC in the intestine from 15 min after shock. Bacterial translocation to mesenteric lymph nodes, liver, and spleen was present from 30 min after shock. This study shows for the first time that HS results in AC activation, selective intestinal actin cytoskeleton disruption, and TJ loss very early after the onset of shock. Loss of this intestinal barrier results in translocation of toxins and bacteria, which enhances inflammation and leads to infections.     

Protein aggregation.

Protein aggregation occurs in vivo as a result of improper folding or misfolding. Diverse diseases arise from protein misfolding and are now grouped under the term "protein conformational diseases", including most of the neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, the prion encephalopathies and Huntington's disease, as well as cystic fibrosis, sickle cell anemia and other less common conditions. The hallmark event in these diseases is a change in the secondary and/or tertiary structure of a normal, functional protein, leading to the formation of protein aggregates with various supramolecular organizations. In most cases the aggregates are organized in structurally well-defined fibrils forming amyloid deposits. The crucial feature of the amyloidogenic proteins is their structural instability induced either by mutations, post-translational modifications, or local conditions, such as pH, temperature, and co-solutes. The conformational change may promote the disease either by gain of a toxic activity or by the lack of biological function of the natively folded protein. As different molecular mechanisms are involved in the formation of the various forms of protein aggregates, the laboratory diagnostic approach remains frequently elusive.