应力培养对血管内皮细胞形态结构的影响

为在体外形成一个抗流动切应力作用能力强的单层内皮细胞，在１５ｄｙｎｅｓ／ｃｍ＾２切应力的条件下，培养内皮细胞２４ｈ。细胞内皮细胞中央出现应力纤维，Ｇ０／Ｇ１期细胞增加，这一现象提示，在切应力条件下培养内皮细胞，可增强细胞抗血流冲击的能力。     

切应力对内皮细胞膜流动性的影响

目的：探讨切应力对内皮细胞膜流动性的影响。方法：选择１５ｄｙｎｅ／ｃｍ＾２、５０ｄｙｎｅ／ｃｍ＾２、３００ｄｙｎｅ／ｃｍ＾２三种切应力,分别模拟大中动脉处,大中动脉分叉处及病理状态下的切应力,以体外培养的人脐静脉内皮细胞作为实验对象,分别测定三组切应力作用０ｈ、１ｈ、２ｈ、４ｈ、６ｈ、１０ｈ的内皮细胞膜流动性。结果：切应力可降低内皮细胞膜液动性,且切应力越大,作用越强。切应力作用４ｈ,内皮细胞膜流     

切应力对培养人脐静脉内皮细胞摄取脂质的影响

了解切应力对血管内皮细胞脂质摄取的影响。方法可模拟生理及超生理范围脉动切应力的循环装置，分别模拟１９．２９、５６．２５、３８５．７１ｄｙｎｅｓ／ｃｍ＾２脉动应力作用培养人脐内皮细胞，免疫组化定量分析内细胞对低密度脂蛋白吞饮的影响。     

血流切应力作用于内皮细胞对脂质过氧化物产生的影响

目的：研究血流脉动切应力对循环液中脂质过氧化物产生的影响。材料与方法：本室已已建立怕体外模拟脉动循环装置及硫代巴比妥酸测定法，进行１９，２９与５６．２５ｄｙｎｅｓ／ｃｍ＾２两组切应力作用于培养人脐静脉内皮细胞０，１，２，４，６，１０，２４小时后循环液中脂质过氧化物含量变化的实验。结果：在两组模拟脉动切应力的作用下，循环液中ＬＰＯ含量均实验１小时开始增加，４小时达最高值，之后下降，于６小时后下降趋于     

用于内皮细胞与平滑肌细胞联合培养的流动腔系统

内皮细胞与平滑肌细胞的联合培养是体外研究这两种细胞生物学特性的重要手段。静态条件下联合培养的内皮细胞的形态及功能特性与在体条件下均有差异,这可能是没有血流切应力对内皮细胞直接作用的结果。为了了解切应力对与平滑肌细胞联合培养的内皮细胞的生物学作用,本文建立了用于多孔ＰＥＴ膜联合培养模型的力学系统,通过本系统可以产生０～４０ｄｙｎ／ｃｍ２的稳定层流切应力,并应用本系统观察了切应力对联合培养的内皮细胞的形态学影响,结果表明,在４０ｄｙｎ／ｃｍ２切应力作用下,与平滑肌细胞联合培养的内皮细胞沿流体方向发生了重排。这一系统的建立为研究切应力作用下与平滑肌细胞联合培养的内皮细胞的形态和功能提供了手段。     

毫米波辐照对小鼠EMT6乳腺癌细胞增殖活性的影响

作者用频率３６．１１ＧＨｚ，功率密度分别为１，３，５，７ｍＷ／ｃｍ＾２的毫米波辐照培养的小鼠ＥＭＴ６乳腺癌细胞，观察对其增生活性的影响。结果发现，５或７ｍＷ／ｃｍ＾２功率密度照射１５ｍｉｎ后，细胞增殖数目少于对照组，照射３０ｍｉｎ后，细胞的克隆形成数目少于对照组及１，３ｍＷ／ｃｍ＾２照射组。７ｍＷ／ｃｍ＾２照射后２４ｈ时，细胞的ＡｇＮＯＲ颗粒数虽多于对照组和其他照射组，但颗粒体积缩小。照射组细胞超     

切应力作用下联合培养的血管平滑肌细胞对内皮细胞F-肌动蛋白构筑的影响

目的　探讨切应力作用下联合培养的血管平滑肌细胞对内皮细胞抗应力和粘附能力的影响 ,为改进血管内皮细胞种植的组织工程学技术提供生物力学基础。　方法　应用荧光标记和激光共聚焦扫描显微镜技术 ,以静态条件下单独培养的内皮细胞、联合培养的内皮细胞以及切应力作用下单独培养的内皮细胞为对照组 ,研究了切应力作用下与血管平滑肌细胞联合培养的内皮细胞的细胞骨架F 肌动蛋白构筑的变化。　结果　静态条件下单独培养的内皮细胞的F 肌动蛋白排列松散 ,不规则 ,微丝较细 ;联合培养的内皮细胞的F 肌动蛋白微丝明显增多增粗。切应力作用下 ,与血管平滑肌细胞联合培养的内皮细胞的F 肌动蛋白发生重排 ,并形成大量沿切应力方向排列的应力纤维 ,且发生重排的时间明显早于单独培养的内皮细胞。　结论　在切应力作用和血管平滑肌细胞的影响下 ,内皮细胞F 肌动蛋白构筑的变化有利于增强内皮细胞的抗应力和粘附能力     

内皮细胞种植的组织工程

组织工程（ｔｉｓｓｕｅｅｎｇｉｎｅｅｒｉｎｇ）又称细胞工程（ｃｅｌｕｌａｒｅｎｇｉｎｅｅｒｉｎｇ），其实质是应用活的功能细胞改进人工器官的功能。本文介绍了在心血管移植物表面种植内皮细胞的研究概况，从内皮细胞的获取，移植物表面的预处理，内皮细胞的生物力学及内皮细胞的基因修饰等几个方面阐述了这一研究进展，并提出，进一步探讨在体外给培养的单层内皮细胞一个应力环境和／或与平滑肌联合培养可能有利于形成耐血流冲击的单层内皮细胞     

切应力对联合培养的血管内皮细胞F-肌动蛋白排列的影响

与平滑肌细胞联合培养的血管内皮细胞在静态时形态就开始发生变化,即由单独培养条件下在的多边形到联合培养条件下的长梭形。切应力作用下,联合培养的内皮细胞的形态在较短时间内发生更进一步的变化。F－actintlEylli持细胞形态及使内皮细胞与细胞外基质的粘附上起着重要的作用。为了探讨切应力对联合培养的血管内皮细胞卜肌动蛋白的排列的影响,本文应用平滑肌细胞与内皮细胞联合培养模型,将牛主动脉平滑肌细胞和内皮细胞进行联合培养,待内皮细胞形成单层后,用手行平板流动胜,将内皮细胞置于40dyn／cm’稳定层流切应力之下12、24／…     

原代培养人脐动脉内皮细胞的生长与增殖

在未来贴附基质和生长因子情况下，成功地培养了人脐动脉内皮细胞（ＨＵＡＥＣ）。接种密度为１×１０＾５／ｃｍ＾２。采用ＨＥ染色、显微计量法及透射电镜术研究了内皮细胞的形态、生长行为与增殖。（１）接种后２４ｈ已出现岛状细胞团；４８ｈ细胞团增大，可区分出细胞密集的中央区及细胞密度较小的周边区；７２ｈ大部分细胞团已汇合；２１６ｈ内皮细胞衰退、脱落。（２）ＨＵＡＥＣ呈较长的多边形，相邻细胞间以短突相连，内胞质     

Monocyte adhesion and changes in endothelial cell number, morphology, and F-actin distribution elicited by low shear stress in vivo.

Left common carotid arteries of New Zealand white rabbits were ligated rostral to origin of the thyroid artery to reduce flow in the carotid upstream of this branch, and the vessels were examined 5 days later. Estimates of mean shear stress in the upstream carotid artery indicated a decrease of 73% (from 12.1 +/- 1.6 dynes/cm2 to 3.26 +/- 0.58 dynes/cm2). The contralateral common carotid artery carried collateral flow and experienced a 170% increase in shear stress (from 11.3 +/- 1.6 dynes/cm2 to 30.5 +/- 4.6 dynes/cm2). There was an adaptive reduction in the diameter in the left common carotid artery (low shear) from 2.07 +/- 0.06 mm to 1.75 +/- 0.12 mm, but the diameter of the right carotid was unchanged. Fluorescence microscopy and scanning electron microscopy of endothelium exposed to low shear revealed attachment of leukocytes (5.02 +/- 1.59 cells/mm2, mean +/- SE) that were identified as monocytes using the monoclonal antibody HAM 56. Laser confocal microscopy demonstrated that they were migrating across the endothelial cell monolayer. Fluorescence microscopy and scanning electron microscopy of left common carotid artery (low shear) also revealed cell morphology suggestive of endothelial cell desquamation. Endothelial cell loss was confirmed by morphometric determination of cell number (1.29 +/- 0.13 x 10(4) cells/mm length in experimental animals versus 1.71 +/- 0.08 x 10(4) cells/mm length in sham-operated animals). This endothelial cell loss may be an adaptation to a narrowing of carotid arteries exposed to low shear, which reduces luminal surface area of the vessel. Staining of F-actin with rhodamine phalloidin showed that endothelial cells exposed to low shear were less elongated and had fewer stress fibers than normal cells. By contrast, increasing shear stress by two- to threefold caused an increase in the number of stress fibers and a reduction in peripheral actin staining. Distal carotid ligation provided a consistent and well-defined in vivo technique for manipulating shear stresses imposed on a large population of endothelial cells.     

切应力对培养人脐静脉内皮细胞形态的影响

用可模拟生理及超生理切应力的平行板模拟循环流动装置进行实验，发现模拟生理脉动切应力及超生理脉动切应力均可使培养人脐静脉内皮细胞沿液体流动方向伸长。采用计算机半自动图像分析系统半定量处理，进一步发现内皮细胞伸长程度与切应力大小及作用时间呈正相关、并且，模拟超生理切应力对内皮细胞形态的影响较生理脉动切应力大。     

Effects of pulsatile shear stress on signaling mechanisms controlling nitric oxide production, endothelial nitric oxide synthase phosphorylation, and expression in ovine fetoplacental artery endothelial cells.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.     

In vitro study of endothelial cells lining vascular grafts grown within the recipient's peritoneal cavity

A living-tissue conduit with strong mechanical properties was used to produce small-diameter vessels. To improve blood compatibility, a shear-resistant confluent monolayer endothelium was formed on the luminal surface of the conduit. Under mechanical stimulation induced by pulsatile flow in a bioreactor, abrupt high-flow shear stress of 15.3 +/- 4.6 dynes/cm2 was applied to endothelial cells (ECs) seeded onto the lumen of a living-tissue conduit after 2 days of static culture. Scanning electron microscopy images revealed that most of the ECs were washed off after 3 days of dynamic culture. When shear stress was increased stepwise from 1.5 +/- 0.8 to 15.3 +/- 4.6 dynes/cm2 and applied to the ECs, scanning electron microscopy images of the luminal surface revealed that the confluent monolayer ECs were highly elongated and oriented to the flow direction, similar to findings in natural arteries in vivo. The results indicated that in vitro flow conditions played a key role in determining the durability of the EC layer. Careful design of the bioreactor and careful selection of the culture conditions will greatly improve the chances of producing a useful anti-thrombogenic surface for tissue-engineered small-diameter vessels.     

大鼠脑微血管内皮细胞应力敏感性钾通道的研究

为探讨脑微血管内皮细胞的电生理学特征,研究了脑微血管内皮细胞钾离子通道的应力反应性.1.建立了开放式切应力作用装置并进行了切应力值的计算; 2.培养大鼠脑微血管内皮细胞并种植到1 cm×1 cm玻片上,采用Axonpatch 200A型膜片钳放大器以及全细胞膜片钳技术记录脑微血管内皮细胞的应力敏感性钾通道.结果表明1.1 dynes/cm2剪切应力能激发脑微血管内皮细胞的应力敏感性钾通道,该通道电流与钳制电压有良好的相关性.脑血管内皮细胞膜上的各种应力反应与细胞膜上的应力敏感性钾通道相关.     

切应力对与血管平滑肌细胞联合培养的内皮细胞微管骨架重构的影响

目的探讨切应力对与血管平滑肌细胞(VSMCs)联合培养的内皮细胞(ECs)中微管的聚集重构的影响,为阐明应力诱导血管重建的分子机制提供一些实验证据。方法应用ECs与VSMCs联合培养的平行平板流动腔系统,给ECs面施加15dyne/cm2的层流切应力,以静态条件下联合培养的ECs为对照组,用WesternBlot、免疫荧光细胞化学和图像分析等技术,研究切应力作用下与VSMCs联合培养的ECs的微管聚集的变化。结果静态联合培养组,ECs微管骨架的排列是稀疏、发散和无规律的。切应力诱导了ECs的微管的重构,,微管骨架变得有序,朝切应力的方向规律的排列。切应力能够促进ECs的微管聚集,与对照组相比,切应力作用下的ECs内多聚微管的数量增加,切应力作用3h,ECs内多聚微管的数量达到峰值,之后开始下降。结论切应力诱导和促进了EC的微管骨架发生重构(聚集)。结果提示:微管可能是机械应力刺激作用的靶标,应力可能通过它改变ECs的形态,影响细胞的黏附与迁移等功能。     

Electrical impedance of cultured endothelium under fluid flow

The morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro. Quantitative, real-time changes in the electrical impedance of endothelial monolayers were evaluated using a modified electric cell-substrate impedance sensing (ECIS) system. This ECIS/Flow system allows for a continuous evaluation of the cell monolayer impedance upon exposure to physiological fluid shear stress forces. Bovine aortic endothelial cells grown to confluence on thin film gold electrodes were exposed to fluid shear stress of 10 dynes/cm² for a single uninterrupted 5 h time period or for two consecutive 30 min time periods separated by a 2 h no-flow interval. At the onset of flow, the monolayer electrical resistance sharply increased reaching 1.2 to 1.3 times the baseline in about 15 min followed by a sustained decrease in resistance to 1.1 and 0.85 times the baseline value after 30 min and 5 h of flow, respectively. The capacitance decreased at the onset of flow, started to recover after 15 min and after slightly overshooting the baseline values, decreased again with a prolonged exposure to flow. Measured changes in capacitance were in the order of 5% of the baseline values. The observed changes in endothelial impedance were reversible upon flow removal with a recovery rate that varied with the duration of the preceding flow exposure. These results demonstrate that the impedance of endothelial monolayers changes dynamically with flow indicating morphological and/or functional changes in the cell layer. This in vitro model system (ECIS/Flow) may be a very useful tool in the quantitative evaluation of flow-induced dynamic changes in cultured cells when used in conjunction with biological or biochemical assays able to determine the nature and mechanisms of the observed changes. © 2001 Biomedical Engineering Society. PAC01: 8719Nn, 8719Uv, 8717-d     

流体切应力梯度对血管内皮细胞排列和形状的影响

目的研究不同梯度切应力作用下,血管内皮细胞(endothelial cells,ECs)排列和形状变化,旨在了解流体切应力梯度对ECs形态的影响,为进一步探讨其功能变化提供实验基础。方法建立可对体外培养ECs施加梯度切应力的流动腔装置,并应用该装置对人脐静脉ECs加载了大小在15dyn/cm2～6.6dyn/cm2(1dyn=10-5N)范围、梯度分别为1.5dyn/cm2和3dyn/cm2的切应力,加载时间均为6h。比较这两种不同切应力梯度对ECs的细胞方向角、细胞宽长比和细胞形态指数的影响。结果在不同切应力梯度作用下,ECs的细胞方向角分布散乱,细胞无排列规律。与3dyn/cm2相比,1.5dyn/cm2切应力梯度下ECs的宽长比和细胞形态指数明显减少,趋向于拉伸状态。结论在不同切应力梯度作用下,ECs均排列紊乱,无规律可循。然而,在相对较小的切应力梯度作用下,细胞容易被拉伸,细胞形状趋向于伸长,而较大切应力梯度作用下,细胞形状则趋向于圆形。     

Effect of Zinc and Nitric Oxide on Monocyte Adhesion to Endothelial Cells under Shear Stress

This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm² or 1 dyne/cm²) or reversing shear stress (time average 1 dyne/cm²) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike other shear stress regimes, reversing shear stress alone enhanced monocyte adhesion, which may be associated with increased H₂O₂ and superoxide together with relatively low levels of nitric oxide (NO) production. L-N^G-Nitroarginine methyl ester (L-NAME) treatment increased monocyte adhesion under 15 dynes/cm² and under reversing shear stress. After reversing shear stress, monocyte adhesion dramatically increased with heparinase III treatment followed by a zinc scavenger. Static culture experiments supported the reduction of monocyte adhesion by zinc following endothelial cell cytokine activation. These results suggest that endothelial cell zinc levels are important for the inhibition of monocyte adhesion to endothelial cells, and may be one of the key factors in the early stages of atherogenesis.