缝隙连接阻断剂对体外培养的大鼠血管平滑肌细胞表型转化的影响

目的探讨缝隙连接阻断剂对大鼠血管平滑肌细胞表型转化的影响。方法采用贴块法培养大鼠主动脉平滑肌细胞,细胞免疫荧光染色法检测大鼠主动脉平滑肌细胞缝隙连接蛋白43的表达,荧光漂白后恢复技术检测缝隙连接介导的细胞间通讯。四唑盐比色法测定细胞增殖能力,逆转录聚合酶链反应法检测平滑肌α-肌动蛋白的表达,并观察缝隙连接特异性阻断剂18α-甘草次酸对上述指标的影响。结果大鼠血管平滑肌细胞体外培养3天后可表达缝隙连接蛋白43。荧光物质能够通过缝隙连接在相邻细胞间进行传递,孤立细胞荧光恢复率显著低于相邻细胞(7.30%±0.58%比80.61%±6.57%,P<0.01);而18α-甘草次酸能够抑制缝隙连接介导的细胞间通讯,18α-甘草次酸组荧光恢复率显著低于对照组(61.43%±7.62%比80.61%±6.57%,P<0.05)。18α-甘草次酸可抑制血管平滑肌细胞增殖,并且可促进平滑肌α-肌动蛋白信使核糖核酸的表达。结论缝隙连接阻断剂可促进大鼠血管平滑肌细胞由合成型向收缩型转化,提示缝隙连接在调节血管平滑肌细胞表型转化过程起一定作用。     

缝隙连接细胞间通讯在炎症、损伤修复和疾病中的作用

缝隙连接是相邻细胞间的通道结构,其主要功能是细胞通讯,又称缝隙连接细胞间通讯。缝隙连接细胞间通讯是细胞间最重要的信息交流形式,可调节组织细胞的生长、分化,在组织保持内稳态中处于核心地位。本文就缝隙连接细胞间通讯在炎症、损伤修复和疾病中的作用等方面作一综述。     

缝隙连接在维持血管张力及损伤血管修复中的作用

目的探讨缝隙连接在维持血管张力及损伤血管修复中的作用。方法取大鼠主动脉制成血管环分别测量在18α-甘草次酸(18α-GA)作用前后血管环对去甲肾上腺素(NE)和乙酰胆碱(Ach)反应性变化;建立大鼠颈动脉损伤模型,给予生胃酮3 mg/(kg·d)腹腔注射,对照组给予生理盐水腹腔注射(2 m L/d),14天后处死动物,用HE和DAPI-伊文思蓝染色观察新生内膜厚度,细胞免疫荧光染色法及Western blot检测靶血管缝隙连接蛋白43(Cx43)的表达。结果单纯给予18α-GA处理血管环并未出现明显的收缩或舒张反应。对照组给予去甲肾上腺素或乙酰胆碱后血管环发生明显的收缩或舒张反应,而经18α-GA预处理后,去甲肾上腺素或乙酰胆碱引起的血管环收缩或舒张反应显著降低(去甲肾上腺素:0.60±0.03比0.21±0.04;乙酰胆碱:0.15±0.01比0.62±0.03; P0.05)。损伤2周生胃酮干预组血管新生内膜增生明显减少,血管腔狭窄减轻。生胃酮干预组新生内膜细胞核数量显著低于对照组(89±28比236±15,n=5,P0.01)。免疫荧光染色显示,在形成的新生内膜中Cx43表达丰富。Western blot结果显示,生胃酮干预组Cx43的表达显著低于对照组(0.38±0.11比0.93±0.06,n=3,P0.01)。结论缝隙连接在生理条件下参与维持及调节血管张力,在病理条件下能够促进血管损伤后新生内膜的过度增生,在血管损伤性疾病的发生、发展过程中具有重要作用。     

慢病毒载体介导人缝隙连接蛋白43基因在大鼠骨髓间充质干细胞中的表达

目的构建带有缝隙连接蛋白43基因的慢病毒载体,并实现该基因在大鼠骨髓间质干细胞中的表达。方法通过逆转录聚合酶链反应获得人缝隙连接蛋白43基因,利用infusion技术重组构建慢病毒载体质粒FUGW-缝隙连接蛋白43,在脂质体介导下与包装质粒和包膜质粒VSVG共转染293T细胞包装生产慢病毒。所获慢病毒感染大鼠骨髓间充质干细胞后,在荧光显微镜下观察缝隙连接蛋白43蛋白表达情况。结果所获缝隙连接蛋白43基因经测序后与GeneBank报道序列完全一致;重组慢病毒载体质粒FUGW-缝隙连接蛋白43经鉴定正确;三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为1×1011TU/L;感染大鼠骨髓间充质干细胞后荧光显微镜观察到胞膜位置点线状荧光分布。结论成功构建带有缝隙连接蛋白43基因的慢病毒载体并实现在大鼠骨髓间充质干细胞中的表达,为间充质干细胞移植治疗心肌梗死的应用奠定了基础。     

血管内皮生长因子在肺动脉平滑肌细胞重构中的作用

目的 :探讨血管内皮生长因子 （VEGF）在野百合碱 （MCT）性肺动脉高压 （PH）中的作用。方法 :用 MCT复制大鼠慢性 PH病理模型 ,用免疫组化法和图像分析技术测定肺组织中 VEGF的表达。结果 :发现 VEGF可在正常大鼠肺血管平滑肌、支气管平滑肌和软骨组织中表达 ,并且 MCT组 VEGF表达的相对含量在肺血管平滑肌 （177±7）和支气管平滑肌 （172± 6）均较正常组 （血管平滑肌 13 6± 8,支气管平滑肌 13 8± 12 ）呈显著增强 （P<0 .0 5）。结论 :MCT性 PH中 VEGF在肺血管平滑肌的过度表达参与 MCT性 PH的发病过程     

11β-HSD2活性抑制在大鼠血管重构中的作用研究

目的：观察11β-羟类固醇脱氢酶（HSD2）活性抑制对大鼠血管重构的影响以及其在血压升高中的作用。方法：将24只SD大鼠分为对照组（C组）和甘草次酸组（M组）,分别给予普通饲料、甘草次酸饲喂六周。然后经颈动脉测量血压、采血测量血中11β-HSD2活性;以及测定血浆和主动脉中内皮素及一氧化氮的含量,并石蜡包埋切取的主动脉,切片染色后观察形态学改变。结果：甘草次酸组大鼠血压为（172.9±6.1）mmHg,较对照组（143.4±9.5）mmHg明显升高（P〈0.01）;而血浆11β-HSD2活性较对照组明显抑制[（0.5±0.1）∶（0.3±0.1）]（P〈0.05）;M组主动脉和血浆中内皮素含量增加（P〈0.05）,而一氧化氮含量减少（P〈0.05）。光镜下C组主动脉平滑肌排列整齐规则,管腔内皮完整,无增生;M组中膜明显增厚[（126.7±10.8）∶（94.8±8.9）]（P〈0.01）,平滑肌排列紊乱,但管腔增大不明显[（1006.7±86.5）∶（1108.4±108.9）]（P〉0.05）。结论：11β-HSD2活性抑制导致血管重构,可能是血压升高的机制之一。     

腺相关病毒介导血管抑素对血管内皮生长因子和细胞间黏附分子1表达的影响

目的探讨腺相关病毒介导血管抑素(rAAV-AS)基因对人脐静脉内皮细胞(HUVECs)血管内皮生长因子(VEGF)和细胞间黏附分子1(ICAM-1)表达的影响,以探讨rAAV-AS抗糖尿病动脉粥样硬化作用。方法选择体外培养的HUVECs传至第5代,随机分为6组:正常组、高糖组、高糖+rAAV-010~6 v.g./cell组、高糖+rAAV-AS10~4 v.g./cell组、高糖+rAAV-AS10~5 v.g./cell组、高糖+rAAV-AS10~6 v.g./cell组。分别检测rAAV-AS干预后24、48、72 h HUVECs凋亡及VEGF、ICAM-1的表达。结果与正常组比较,高糖组和高糖+rAAV-010~6 v.g./cell组不同时间HUVECs增殖明显,VEGF和ICAM-1表达明显升高(P<0.05);与高糖组比较,高糖+rAAV-AS10~4 v.g./cell组、高糖+rAAV-AS10~ 5v.g./cell组、高糖+rAAV-AS10~6 V.g./cell组增殖明显降低,VEGF和ICAM-1表达明显降低,并呈剂量依赖性(P<0.05)。结论 rAAV-AS对糖尿病大血管病变具有保护作用,其可能机制为下调内皮细胞中VEGF和ICAM-1的表达,从而减少内皮细胞增殖,抑制炎性反应。     

内皮祖细胞在血管新生和抑制血管损伤后再狭窄中的作用

内皮祖细胞（EPCs）也叫成血管细胞，是一种成体干细胞。日益增多的证据显示，通过各种途径向体内移植或动员机体自身的EPCs可以提高缺血组织的血管新生能力，促进损伤血管的再内皮化、抑制新生内膜的增生，从而为治疗缺血性心脏病和血管介入治疗后再狭窄提供了新思路。     

基质细胞衍生因子-1及其受体CXCR4轴介导内皮祖细胞参与血管损伤修复的研究进展

<正>内皮祖细胞(endothelial progenitor cells,EPCs)能够参与血管损伤修复,给再生医学治疗带来了希望。Asahara等[1]从人类外周血中分离出内皮前体细胞,发现这种细胞参与缺血组织的血管新生。多项研究证实,EPCs通过分化成新生内皮细胞替代损伤的血管内皮,参与血管损伤修复[2-3]。EPCs参与血管损伤修复过程涉及EPCs动员、迁移、分化等一系列过程,期间多种因子通过多种途径参与调节此     

细胞连接通讯与动脉粥样硬化

动脉粥样硬化（AS）是临床心血管病的重要发展原因。它是一种多因素疾病,由多种因素作用于不同环节所致,其发病机理,至今尚未完全明确。各种因素和细胞成份之间的相互作用和相互影响构成了AS发病机制的中心环节。     

细胞连接通讯与动脉粥样硬化

动脉粥样硬化(atherosclerosis)是临床心血管病的重要发展原因,其发病机理,至今尚未完全明确。各种因素和细胞成份之间的相互作用和相互影响构成了动脉粥样硬化致病机制的中心环节。     

Omega-3 Polyunsaturated Fatty Acids Reduce Vascular Endothelial Growth Factor Production and Suppress Endothelial Wound Repair

Long-chain polyunsaturated omega-3 fatty acids (n-3 PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have diverse beneficial effects on cardiovascular diseases and have been used widely as supplements in reducing the risk of cardiovascular diseases. The beneficial effects are believed to be related to the anti-inflammatory and antioxidant action of n-3 PUFA. EPA and DHA can inhibit inflammatory cytokine-induced endothelial activation and reduce endothelial migration and proliferation. Revascularisation is the major therapeutic approach for end-stage cardiovascular diseases, and endothelial migration and proliferation are essential for the success of revascularisation. The aim of this study was to investigate the role of n-3 PUFAs on vascular endothelial wound repair. A scratch-wound repair assay was carried out in cultured human microvascular endothelial cells (HMEC-1) with and without different concentrations of DHA or EPA. The effect of DHA and EPA on HMEC-1 proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of DHA and EPA on vegf mRNA expression was detected by real-time RT-PCR and vascular endothelial growth factor (VEGF) protein secretion by enzyme-linked immunosorbent assay. DHA and EPA dose-dependently suppressed HMEC-1 cell proliferation and wound repair. DHA and EPA treatment did not induce significant HMEC-1 cell death. The treatment, however, significantly suppressed vegf mRNA expression and protein secretion in both normoxia and hypoxia culture conditions. The addition of exogenous VEGF prevented DHA- and EPA-mediated suppression of HMEC-1 cell proliferation. DHA and EPA have anti-angiogenic effect partially through vegf suppression. The use of DHA and EPA may benefit angiogenic diseases, but may have potential side effects to patients undergoing revascularisation therapy. Further studies will be required to confirm the effect of n-3 PUFAs on vascular repair.     

The Role of Endothelial Progenitor Cells in Vascular Repair after Arterial Injury and Atherosclerotic Plaque Development

Endothelial progenitor cells (EPCs) are under investigation due to their association with vascular injury. In response to chemotactic stimuli they are mobilized from bone‐marrow and nonbone marrow sites, they migrate, adhere and home to the injured vessel. Numerous molecular and cellular pathways participate and converge to the EPCs mediated vascular repair. However, the exact phenotypic properties, modes of functions and effects in vascular diseases and particularly in atherosclerosis are under investigation. EPCs represent a heterogeneous group of cells in different stages of differentiation, from hematopoietic bone marrow progenitors to mature endothelial cells that participate in adult vascular repair under ischemic or apoptotic stimuli. This review aims to provide an integrative view of EPC‐mediated vascular repair.     

The Molecular Basis of Anisotropy: Role of Gap Junctions

Role of Gap Junctions in Anisotropic Conduction. Electrical activation of the heart requires transfer of current from one discrete cardiac myocyte to another, a process that occurs at gap junctions. Recent advances in knowledge have established that, like most differentiated cells, individual cardiac myocytes express multiple gap junction channel proteins that are members of a multigene family of channel proteins called connexins. These proteins form channels with unique biophysical properties. Furthermore, functionally distinct cardiac tissues such as the nodes and bundles of the conduction system and atrial and ventricular muscle express different combinations of connexins. Myocytes in these tissues are interconnected by gap junctions that differ in a tissue-specific manner in terms of their number, size, and three-dimensional distribution. These observations suggest that both molecular and structural aspects of gap junctions are critical determinants of the anisotropic conduction properties of different cardiac tissues. Expression of multiple connexins also creates the possibility that "hybrid" channels composed of more than one connexin protein type can form, thus greatly increasing the potential for fine control of intercellular ion flow and communication within the heart.     

Vascular Endothelial Cadherin (VE-Cadherin): Cloning and Role in Endothelial Cell-Cell Adhesion

Objective: To identify proteins responsible for intercellular junction integrity in human umbilical vein endothelial cells (HUVEC), we produced a monoclonal antibody that recognized an endothelial cell-specific, junctionally restricted protein. We characterized and cloned the antigen to study its functional properties. Methods: The size and cellular distribution of the antigen were determined by immunofluorescence and immunoprecipitation. The molecule was cloned and transfected into cell lines, and its role in cell-cell adhesion and growth rate was determined. Results: Monoclonal antibody heel recognizes VE-cadherin, an endothelial cell-restricted cell adhesion molecule. VE-cadherin is localized to the borders between apposing endothelial cells but is diffusely distributed on subconfluent or migrating cells. Transfection of fibroblasts with VE-cadherin imparts to them the ability to adhere to each other in a calcium-dependent homophilic manner. Expression of VE-cadherin over a several-log range does not change the growth rate of these cells. Conclusions: Despite the fact that VE-cadherin is a “nonclassical” cadherin by structure, it functions as a classic cadherin by imparting to cells the ability to adhere in a calcium-dependent, homophilic manner. On HUVEC it appears to play a role in maintaining monolayer integrity.     

Pathophysiology of Gap Junctions in Heart Disease

Cardiac Gap Junctions. Electrical coupling between cardiac muscle cells is mediated by specialized sites of plasma membrane interaction termed gap junctions. These junctions consist of clusters of membrane channels that directly link the cytoplasmic compartments of neighboring cells. Each gap-junctional channel consists of two connexons, one from each of the interacting plasma membranes, extending across the narrow extracellular gap. Connexons are constructed from connexins. a multigene family of conserved proteins. Different connexins confer specific electrophysiologic characteristics on the assembled channel protein. The major connexin of the mammalian heart is connexin43, although other types of connexins are also expressed, notably connexin40 in myocytes of the atrioventricular conduction system. Confocal laser scanning microscopy of anti–connexin43 immunolabeled samples reveals two major abnormalities in myocardial gap junctions in ischemic heart disease: loss of the usual ordered distribution of gap junctions at border zones adjacent to infarct scars, and reduction in the quantity of connexin43 gap junctions in myocardium distant from the infarct. These and other changes reported in myocardial gap-junctional communication pathways following infarction may result in heterogeneous anisotropic conduction and reduced conduction velocity, (hereby forming a proarrhythmic substrate. Current evidence suggests that reduction in conuexin43 content is a general pathogenetic feature of cardiac disease, and that changes in the expression levels of other connexin types may contribute to altered electrophysiologic function in the diseased heart.     

再生内皮细胞表型改变在血管损伤性疾病中的作用

血管内皮细胞(endothelialcell;EC)再生是血管内膜损伤或剥脱后的主要修复方式。然而,Koo等[1]于1989年应用血管体外培养发现,球囊拉伤后再生EC存在功能紊乱,并促进了再狭窄病变的发生发展。近来研究表明,EC再生的过程不仅参与血管内膜的修复,而且也参与动脉粥样硬化和再?..