内皮细胞间连接的研究进展

内皮细胞形成了大分子物质和循环细胞从知液到细胞的最主要屏障,内皮细胞的通航性主要是通过内皮细胞间连接进行调控的,本文从内容细胞间连接的几种方式,信号的传导,连接变化的调控,篾这中液体的流动及中性粒细胞渗出对内皮细胞间连接的影响进行阐述,讨论了目前内皮细胞间连接的研究进展,提出内皮细胞间连接和骨架结构对血管通透性的调控,中性粒细胞的渗出和血管内皮细胞间连接的重建都具有非常重要的作用,其中内皮细胞的渗     

体外研究血管内皮细胞单层通透性的新方法

血管壁通透性增高足创伤、烧伤、感染、休克和炎症等病理过程的重要变化和特征,其机理尚不十分清楚。内皮细胞是血管壁的主要通透屏障,研究内皮单层的通透性特征及其在病理情况下的变化机制对人们了解血管壁的通透性增高机制和寻找临床防治方法有着极为重要的意义。我们建立了用体外培养的内皮细胞单层研究通透性的装置,可以较为准确地测定内皮单层对液体的滤过系数Kf,便于体外研究各种体液因子和炎症介质对血管通透性的影响及其机制,也可用于研究白细胞-内皮细胞的相互作用。     

肺微血管内皮细胞通透性调控的信号转导机制

肺微血管内皮细胞通透性增加是急性呼吸窘迫综合征等疾病的病理基础,多种信号转导系统参与其通透性调控,如细胞内Ca2 、蛋白激酶C、环磷酸腺苷、丝裂原激活蛋白激酶、小G蛋白.这些信号转导系统的激活和调控机制各异,并且相互关联组成复杂的信号网络.肺微血管内皮细胞中信号转导的相互作用将是研究方向之一.     

低氧时肺动脉内皮细胞单层通透性变化

目的和方法：研究肺动态内皮细胞（ＰＡＥＣ）在低氧性肺水肿（ＨＰＥ）发生中的作用机制。采用ＯＰＡＥＣ体外培养的方法，观察了ＰＡＥＣ生长状态和特征性蛋白因子ⅧＲ：Ａｇ的变化，并利用ＰＡＥＣ融合单层通透性模型研究了低氧对ＰＡＣＥ融合单层的通透性的影响。结果：ＰＡＥＣ生长数量无明显变化，但细胞的生长质量，ⅧＲ：Ａｇ阳性细胞数明显下降，ＰＡＣＥ通透性明显增加，ＣａＭ阻断剂ＴＦＰ只能部分抑制０这种通透性增加。     

粘附分子在组织内淋巴管内皮和体外培养淋巴管内皮细胞的表达

目的研究粘附分子PECAM-1、ICAM-3、VCAM-1和CD44在组织淋巴管和培养淋巴管内皮细胞(LECs)的表达.方法取狗的空肠和腹前壁皮肤,作冰冻切片,用免疫组织化学染色法标记粘附分子在组织淋巴管内皮的表达.分离和培养狗胸导管的LECs,用免疫荧光染色法标记粘附分子在培养LECs的表达,在共聚焦激光扫描显微镜下观察.用图像分析系统分析粘附分子表达的光密度值(OD值),并与肿瘤坏死因子α(TNF-α)或脂多糖(LPS)刺激细胞的表达进行比较.结果组织内淋巴管内皮的PECAM-1和ICAM-3呈免疫反应阳性.培养LECs表达PECAM-1、ICAM-3和CD44,而VCAM-1的表达不明显.用TNF-α刺激细胞后,PECAM-1和ICAM-3表达的OD值与正常对照组无明显差别,CD44的OD值低于正常对照组.用LPS刺激细胞后,PECAM-1、ICAM-3和CD44表达的OD值均无显著性变化.用TNF-α或LPS刺激LECs后,VCAM-1呈弱表达.结论粘附分子PECAM-1、ICAM-3、VCAM-1和CD44的表达在培养LECs和组织内淋巴管内皮细胞存在着差异.用TNF-α或LPS刺激后,LECs的VCAM-1表达增强.这可能与淋巴管内皮细胞的功能状态有关.     

敲低脑内皮细胞粘附分子抑制HNSC细胞的侵袭能力

转移是包含头颈部鳞状细胞癌（head-and-neck squamous cell carcinoma, HNSC）在内的多种肿瘤复发和患者死亡的主要原因。目前,关于HNSC转移的网络调控机制仍有待进一步完善,以期降低HNSC死亡率。首先,通过在线数据库GEPIA统计后发现,脑内皮细胞粘附分子（cerebral endothelial cell adhesion molecule, CERCAM）在519例头颈部肿瘤中的相对表达量为4.98,是其在44例正常组织中（相对表达量为2.47）表达量的2.02倍。并且发现CERCAM表达量与头颈部肿瘤患者较差的预后正相关（P=0.015）。其次,在舌癌细胞SCC-9、喉癌细胞HEP2和鼻咽癌细胞CNE2敲低CERCAM的表达,发现上述3种细胞的侵袭能力均较对照组分别下降93.60%、37.18%和40.63%。最后,生物信息学预测结合Western印迹的结果证实,敲低CERCAM后,上调E-钙黏蛋白（E-cadherin）,同时下调锌指E盒结合蛋白(zinc-finger E-box-binding homeobox1, ZEB1)、波形蛋白(vimentin)和Twist相关蛋白1（Twist-related protein 1, TWIST1）。结果证实,CERCAM可能通过调控头颈部肿瘤细胞的上皮间充质转化（epithelial-mesenchymal transition, EMT）标志物来促进该类细胞发生侵袭。     

粘附分子与细胞凋亡

当贴壁生长的上皮细胞和内皮细胞丧失粘附特性时,细胞可发生凋亡,这一现象促进全们去认识细胞粘膜与凋亡的关系,粘附分子是细胞粘附（细胞－细胞,细胞－细胞外基质）的重要分子基础,并可能通过信号传递途径影响细胞内凋亡相关基因,调控细胞凋亡过程,尽管由粘附分子参与介导的细胞凋亡机理尚不完全清楚,然而其与细胞凋亡的生物学意义正经起广泛关注,本文简述粘附分子与细胞凋亡的研究现状。     

细胞粘附分子研究现状

细胞粘附分子是指由细胞合成、存在于细胞膜或胞外、可促进细胞粘附的一大类分子的总称。研究表明,细胞粘附分子在胚胎发育、伤口愈合、学习与记忆的神经机制以及病毒感染、肿瘤转移等多种生理和病理过程中均发挥重要作用。研究细胞粘附分子既可以帮助我们了解机体的重要生理过程和病理机制,并为肿瘤、ＡＩＤＳ的治疗提供新的手段。细胞粘附分子（ｃｅｌｌａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅｓ,ＣＡＭ）是多细胞生物的重要功能分子,在形成组织器官的正常结构与功能、细胞的游走与运动、机体的发育与成长、伤口的愈合、神经的再生、病毒感染、肿瘤转移等方面均有重要作用。     

蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

内皮祖细胞在血管组织工程研究中的应用现状

组织工程血管是修复紫绀型先心病右心室流出道的潜在替代材料,实验研究表明外周血内皮祖细胞(endothelial progenitor cell,EPC)可作为构建组织工程血管的良好种子细胞。现阶段国内外对组织工程血管的研究方兴未艾,EPC在血管组织工程研究中的应用还处在体外培养或动物实验阶段,尚无临床应用报道。近来某些基础研究的成果对于新生血管的形成和EPC的自动募集机制有了合理的解释,其中缺氧被认为是重要的始动因素,这些研究成果也为EPC作为种子细胞应用于血管组织工程提供了理论基础。所以,阐明EPC的低氧诱导机制及其在血管组织工程的应用必将有助于复杂紫绀型先心病的外科治疗。本文主要介绍了目前该领域研究现状及相关研究基础的进展,并总结提出了在EPC研究和未来临床应用中需解决的问题。     

Endothelial Cell Senescence and Age-Related Vascular Diseases

Advanced age is an independent risk factor for ageing-related complex diseases,such as coronary artery disease,stroke,and hypertension,which are common but life threatening and related to the ageing-associated vascular dysfunction.On the other hand,patients with progeria syndromes suffer from serious atherosclerosis,suggesting that the impaired vascular functions may be critical to organismal ageing,or vice versa.However,it remains largely unknown how vascular cells,particularly endothelial cell,become senescent and how the senescence impairs the vascular functions and contributes to the age-related vascular diseases over time.Here,we review the recent progress on the characteristics of vascular ageing and endothelial cell senescence in vitro and in vivo,evaluate how genetic and environmental factors as well as autophagy and stem cell influence endothelial cell senescence and how the senescence contributes to the agerelated vascular phenotypes.such as atherosclerosis and increased vascular stiffness,and explore the possibility whether we can delay the age-related vascular diseases through the control of vascular ageing.     

雷帕霉素对内皮细胞迁移和血管内皮生长因子表达的影响

目的：研究不同浓度的雷帕霉素对体外培养的人血管内皮细胞（rE）4移及血管内皮生长因子（VEGF）表达的影响。方法：用含10％胎牛血清的细胞培养基（DMEM）培养正常VE细胞,用10nM,50nM,100nM和200nM的雷帕霉素孵育vE细胞24h,Westernbloting测定雷帕霉素对VE中mTOR和VEGF表达的影响,Transwell迁移模型观察不同浓度的雷帕霉素对内皮细胞迁移影响。结果：①雷帕霉素可显著抑制VE的迁移,除了在100riM之外,基本呈浓度依赖性的。100nM雷帕霉素对VE迁移的抑制作用显著减弱（P〈0．01）。②雷帕霉素对mTOR和VEGF165的表达呈浓度依赖性的抑制;而VEGF121的表达则是先升高后降低,在100nM雷帕霉素时表达最高,远远高于该浓度雷帕霉素时VEGF165的表达,可以解释100nM雷帕霉素时VE迁移抑制显著减轻的现象。结论：雷帕霉素抑制了VEGF165的表达,并且其对VE迁移抑制的效应主要由VEGF165表达减少所介导。VEGF121的表达在一定雷帕霉素浓度范围内可显著上调,从而显著改善了雷帕霉素诱导的VEGF165表达减少所致的内皮细胞迁移抑制。     

雷帕霉素对内皮细胞迁移和血管内皮生长因子表达的影响

目的:研究不同浓度的雷帕霉素对体外培养的人血管内皮细胞(VE)迁移及血管内皮生长因子(VEGF)表达的影响。方法:用含10%胎牛血清的细胞培养基(DMEM)培养正常VE细胞,用10nM,50nM,100nM和200nM的雷帕霉素孵育VE细胞24 h,Western bloting测定雷帕霉素对VE中mTOR和VEGF表达的影响,Transwell迁移模型观察不同浓度的雷帕霉素对内皮细胞迁移影响。结果:①雷帕霉素可显著抑制VE的迁移,除了在100nM之外,基本呈浓度依赖性的。100nM雷帕霉素对VE迁移的抑制作用显著减弱(P<0.01)。②雷帕霉素对mTOR和VEGF165的表达呈浓度依赖性的抑制;而VEGF121的表达则是先升高后降低,在100nM雷帕霉素时表达最高,远远高于该浓度雷帕霉素时VEGF165的表达,可以解释100nM雷帕霉素时VE迁移抑制显著减轻的现象。结论:雷帕霉素抑制了VEGF165的表达,并且其对VE迁移抑制的效应主要由VEGF165表达减少所介导。VEGF121的表达在一定雷帕霉素浓度范围内可显著上调,从而显著改善了雷帕霉素诱导的VEGF165表达减少所致的内皮细胞迁移抑制。     

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Protein-tyrosine Phosphatase 4A3 (PTP4A3) Promotes Vascular Endothelial Growth Factor Signaling and Enables Endothelial Cell Motility

Protein-tyrosine phosphatase 4A3 (PTP4A3) is highly expressed in multiple human cancers and is hypothesized to have a critical, albeit poorly defined, role in the formation of experimental tumors in mice. PTP4A3 is broadly expressed in many tissues so the cellular basis of its etiological contributions to carcinogenesis may involve both tumor and stromal cells. In particular, PTP4A3 is expressed in the tumor vasculature and has been proposed to be a direct target of vascular endothelial growth factor (VEGF) signaling in endothelial cells. We now provide the first in vivo experimental evidence that PTP4A3 participates in VEGF signaling and contributes to the process of pathological angiogenesis. Colon tumor tissue isolated from Ptp4a3-null mice revealed reduced tumor microvessel density compared with wild type controls. Additionally, vascular cells derived from Ptp4a3-null tissues exhibited decreased invasiveness in an ex vivo wound healing assay. When primary endothelial cells were isolated and cultured in vitro, Ptp4a3-null cells displayed greatly reduced migration compared with wild type cells. Exposure to VEGF led to an increase in Src phosphorylation in wild type endothelial cells, a response that was completely ablated in Ptp4a3-null cells. In loss-of-function studies, reduced VEGF-mediated migration was also observed when human endothelial cells were treated with a small molecule inhibitor of PTP4A3. VEGF-mediated in vivo vascular permeability was significantly attenuated in PTP4A3-deficient mice. These findings strongly support a role for PTP4A3 as an important contributor to endothelial cell function and as a multimodal target for cancer therapy and mitigating VEGF-regulated angiogenesis.     

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.     

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.