低切应力对体外培养血管中膜平滑肌细胞原癌基因c-Fos和c-Myc表达的影响

为了探讨切应力影响血管重建的机制,观察了低切应力对完整血管中膜平滑肌细胞原癌基因蛋白c-Fos和c-Myc表达的影响。应用血管体外应力培养系统,将一段完整的猪颈总动脉在体外进行培养,控制血管内培养液为定常流, 压力为1.33×105 Pa,切应力分别为2 Pa(S20组)、0.5 Pa(S5组)和0 Pa(S0组),培养时间分别为1、6和24 h,采用免疫组化和计算机图像分析方法观察血管中膜平滑肌细胞c-Fos和c-Myc的表达。结果显示,S20组仅有少量的表达,而S5组和S0组,血管壁的平滑肌细胞短时间(1 h)内即出现c-Fos和c-Myc表达增高,与S20组相比有显著差异(P<0.01),随后,c-Fos又很快降低(6 h),24 h时已降至基线水平,而c-Myc的表达则持续相对较长的一段时间,且与c-Fos相反,1 h的表达低于6 h。结果表明,在低切应力作用下,血管平滑肌细胞的c-fos和c-myc基因被激活,调节转录,从而调节切应力-血管平滑肌细胞的信号转导通路,进而影响血管平滑肌细胞的增殖和凋亡。     

缺氧条件下血管活性因子对血管内皮细胞中VEGF表达的影响

 探讨在缺氧条件下人脐静脉血管内皮细胞对血管内皮生长因子 (vascular endothelialgrowth factor,VEGF)表达及缩血管活性物质内皮素 (ET)、舒血管活性物质一氧化氮 (NO)和 NO抑制剂 LNNA对 VEGF基因表达的影响 .体外培养人脐静脉血管内皮细胞 ,经缺氧及血管活性物质处理 .Northern杂交、酶联免疫检测和计算机图象分析等观察 VEGF m RNA和蛋白表达水平 .发现缺氧 6h内皮细胞可见 VEGF表达 .ET可促进 VEGF m RNA的表达 ,NO可明显抑制 VEGFm RNA的表达 ,NO抑制剂 LNNA也影响 VEGF m RNA的表达 .ELISA检测 VEGF蛋白水平分别为 6h组 8.2± 1 .1 ng/ L,ET+6h组 9.37± 1 .0 2 ng/ L,NO+6h组 2 .86± 0 .91 ng/ L,L - NNA+6h组 1 4.75± 1 .87ng/ L.缺氧可诱导人脐静脉血管内皮细胞分泌 VEGF并受血管活性物质ET和 NO的调控 ,ET促进其表达 ,NO抑制其表达 .     

周期性张应力刺激下成肌细胞钙网蛋白的表达及变化

目的：检测成肌细胞钙网蛋白（CRT）在循环拉伸应力刺激下的表达变化。方法：体外构建面颌部成肌细胞力学刺激模型。加力组以0．5赫兹的加载频率和10％细胞拉伸变形幅度对细胞进行加力培养1h,6h,12h,24h,运用实时荧光定量PCR技术跟Westem Blot技术分别检测在周期性张应力作用下成肌细胞CRT在基因水平及蛋白水平的表达变化。结果：当对细胞加力6h后,CRTmRNA及蛋白表达量开始增多,加力12h后CRTmRNA及蛋白表达量到达最多（P〈0．01）,加力0h组与加力12h组之间差异有显著的统计学意义（P〈0．01）。结论：持续的周期性张应力刺激下CRTmRNA及蛋白表达增加。     

缺氧对世居高原藏族人脐静脉内皮细胞ET和NO水平的影响

目的: 观察世居高原藏族人静脉血ET和NO含量和缺氧对培养脐静脉内皮细胞ET和NO水平的影响,以期从胎儿生长发育的角度探讨人类高原适应的机制.方法: 分别以放射免疫分析法和Greiss法测定世居高原藏族、移居高原汉族和平原汉族人静脉血ET和NO含量.体外培养世居高原藏族和移居高原汉族新生儿UVECs,分为4组:①世居高原藏族人UVECs常氧组(TC);②世居高原藏族人UVECs缺氧组(TH);③移居高原汉族人UVECs常氧组(HC);④移居高原汉族人UVECs缺氧组(HH),收集培养上清液测定ET和NO含量.结果: 世居高原藏族人静脉血NO水平显著高于移居高原汉族人,而ET水平显著低于移居高原汉族人.体外低氧(0.5% O2)条件培养12 h和24 h时,TH组ET浓度显著低于HH组,而HH组与TH组和HC组与TC组相应时间点之间的NO浓度差异无显著性意义.结论: 缺氧时世居高原藏族人UVECs ET分泌增高的程度较低,可能有助于保持相对低的血管张力,有利于胎儿血液供应.     

卡托普利对血管平滑肌细胞内皮素基因表达的影响

用反向PCR结合计算机图象处理技术检测卡托普利(CaP)对培养家兔主动脉平滑肌细细内皮素-1(ET-1)mRNA表达的影响。扩增的ET-1cDNA为425bp,经HindⅢ酶切后为279bg和146bg。血管紧张素Ⅱ(Ang-Ⅱ)引起ET-1mRNA表达,诱导表达有时间依赖性(作用3h,表达明显增加,作用5h,表达到高峰,作用7h,表达开始下降)。CaP(1和10μmolL~(-1))明显抑制Ang-Ⅱ引起的ET-1mRNA表达,抑制分别达73％和84％。这些结果表明CaP能直接抑制血管平滑肌细胞的ET—1基因表达。     

高压力对体外培养动脉中膜平滑肌细胞增殖及增殖相关蛋白和生长因子的影响

为了探讨力学因素在血管重建中的作用和机制,观察在单纯高压力条件下血管平滑肌细胞增殖及其相关蛋白和生长因子的变化。应用血管体外应力培养系统,在施加单纯压力的条件下培养猪颈总动脉。按压力大小,将培养的血管分为高压力(21．3kPa)组和正常压力(13．3kPa)组。两组血管均分别培养1、4和7d。免疫组织化学检测血管中膜平滑肌细胞的α-肌动蛋白,增殖细胞核抗原、血小板源性生长因子A、转化生长因子13l及P53蛋白的变化。结果显示：在高压力的作用下,随着培养时间的延长,α-肌动蛋白呈减少的趋势;增殖细胞核抗原,转化生长因子131、P53持续增多;血小板源性生长因子A先增加而后有所减少。说明高压力可明显促进血管平滑肌细胞表型的转变,发生增殖现象。提示高压力可能通过调节血小板源性生长因子A、转化生长因子131及P53蛋白的表达来调控血管平滑肌细胞的增殖。     

切应力作用下与血管平滑肌细胞联合培养的内皮细胞PDGF-B含量的变化

目的：探讨切应力作用下联合培养的血管平滑肌细胞(VSMC)对内皮细胞(EC)PDGF-B含量变化的影响,为组织工程血管和预防血管移植物再缩窄研究提供一些实验资料。方法应用免疫细胞化学方法和图像分析技术,以静态条件下单独培养的EC和联合培养的EC为两对照组,研究了切应力作用下单独培养的EC和与VSMC联合培养EC的PDGFB含量的变化。结果：静态条件下联合培养EC的PDGF-B的含量比单独培养的EC减少;切应力作用下,联合培养EC的PDGF-B含量在切应力作用1h左右有瞬时上升,随后下降至低于联合培养条件下的静态水平,且瞬时上升的时间点比单独培养的EC提前。结论：切应力作用下,与VSMC联合培养EC的PDGF-B含量下降,这可能有利于抑制VSMC的增殖,提示在组织工程血管构建时VSMC与EC联合种植有利于预防血管移植后的再狭窄     

Dexmedtomidine对SAP大鼠胃肠黏膜屏障保护作用研究

为探讨右美托咪定(dexmedtomidine,DEX)对SAP(重症急性胰腺炎)大鼠胃肠功能障碍的影响,本研究选取SD大鼠50只,随机分为五组,每组10只:即假手术组(S组),SAP组(C组)及小剂量药物DEX(D1)治疗组模型、中剂量药物DEX(D2)、大剂量药物DEX(D3)治疗组。每个实验组各取5只在造模并推药后12 h、24 h两个时间处死,并检测血清中D-乳酸、内毒素(ET)变化,并在显微镜下观察HE染色后的大鼠胰腺和回肠组织,对比其组织形态变化。实验中发现,在12 h和24 h时间段,各实验组的D-乳酸和ET水平均显著升高,与S组比较有统计学意义;3个治疗组与C组比较,血清D-乳酸和ET有降低,具有统计学意义(p0.05);胰腺组织学评分和回肠组织学评分显示,在12 h和24 h时间段,S组评分低,各时间点无明显变化,C组、D1、D2、D3组评分与S组比较有统计学意义;D1、D2、D3组与C组比较,评分有降低,具有统计学意义(p0.05)。本研究说明,右美托咪定可减轻SAP大鼠胃肠粘膜的损伤,同时小、中、大剂量右美托咪定对胃肠粘膜屏障具有保护作用,但保护作用与剂量变化并无显著关联,提示右美托咪对胃肠粘膜屏障的保护作用可能与抑制炎性反应有关。     

低强度周期性静水压力对人膝关节软骨细胞的影响

目的:探讨低强度周期性静水压力对体外培养的人膝关节软骨细胞增殖、凋亡,以及细胞Ⅱ型胶原分泌表达的影响。方法:体外酶消化法分离培养成人膝关节正常软骨细胞,将培养的第3代软骨细胞分为两组:正常对照组、3.0MPa组压力实验组,应用多功能恒温体外细胞培养中高压静水压力加载装置加载低强度周期性压力,共5d,每天2h。Ⅱ型胶原免疫组织化学染色法和甲苯胺蓝染色法鉴定软骨细胞,流式细胞术检测细胞凋亡,四甲基偶氮唑蓝(MTT)法绘制细胞生长曲线,qRT-PCR、Western-Blot检测Ⅱ型胶原的分泌和表达。结果:软骨细胞Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色均显示为阳性。与正常对照组相比,3.0MPa组表现出促进软骨细胞增殖,抑制细胞凋亡,且Ⅱ型胶原的合成分泌明显升高(P0.05)。结论:通过体外模拟人生理情况下较低强度(3.0MPa)的周期性静水压力对人软骨细胞增殖、凋亡水平及周围基质分泌合成功能的影响,初步证实了较低强度压力有助于软骨自我修复和自身保护作用的发挥。     

周期性张应力对大鼠髁突软骨细胞Ⅱ型胶原表达的影响

目的:在成功构建髁突软骨细胞体外培养-力学刺激模型的基础上,探讨周期性张应力对髁突软骨细胞主要细胞外基质(Ⅱ型胶原)合成的影响.方法:本研究采用FX-5000T应力加载系统对体外培养的第3代大鼠髁突软骨细胞分别施加1h、6h、12h和24 h的周期性张应力,应力刺激强度为10％1 HZ.加力完成后即刻收集加力细胞,提取细胞总RNA反转录成cDNA,应用RT-PCR技术检测髁突软骨细胞主要细胞外基质Ⅱ型胶原(type-Ⅱ collagen,Col-Ⅱ)mRNA的表达变化情况.结果:与对照组(0h组)相比,加力1 h时Col-Ⅱ的表达增加,但无统计学意义;加力6h时Col-Ⅱ表达显著增加(P＜0.05);加力12h时Col-Ⅱ表达开始下降;当加力至24h时表达量显著降低(P＜0.05).结论:周期性张应力可以影响髁突软骨细胞主要细胞外基质的合成,在一定范围内随加力时间的延长基质合成逐渐增强;进一步延长加力时间,基质的合成受到明显抑制.     

血管内皮细胞内皮素代谢的数值分析

将培养的人胚肾小球血管单层内皮细胞置于剪应力分别为6．5dyn/cm^2和13．0dyn/cm^2的定常层流中剪切10小时,样品中的内皮素（ET）分泌量用放射免疫法测定。结果表明,剪切应力和剪切作用时间对内皮细胞内皮素的代谢均有显著的影响,其影响不是简单的线性增减,而呈较复杂的非线性特征,内皮素累积含量和分泌速率,随剪切时间的变化的关系可用Logistic方程来描述,获得了反映内皮素（ET）分泌规律的大量特征方程和特征数,为了解体内发生于血管壁的病理生理过程中内皮素分泌规律提供了实验数据。     

剪切应力下内皮细胞内皮素及其mRNA的表达

应用Ｎｏｒｔｈｅｒｎ印迹方法研究高血压（ＳＨＲ）和正常（ＷＫＹ）大鼠脑微血管培养内皮细胞,在剪切应力０、０．５、１、２Ｐａ作用下２４ｈ后检测内皮素及其基因ｍＲＮＡ表达上的区别。结果表明,在剪切应力０、０．５、１Ｐａ下,ＷＫＹ大鼠的内皮细胞随着剪切应力加大,其内皮素水平及其基因的ｍＲＮＡ的表达均比ＷＫＹ相应组的为高。在剪切应力２Ｐａ时,ＷＫＹ和ＳＨＲ大鼠的内皮细胞内皮素及其基因的ｍＲＮＡ表达水平不同     

Vitronectin enhances adhesion force and t-PA production of weakly adherent 293 cells exposed to a shear stress

The effects of shear stress on the adhesion andproductivity of 293 cells were studied quantitativelyand compared with those of Vero and human liver cells.These cells were cultured in polystyrene dishes byusing shear stress exposing equipment. 50% of 293cells cultured in 2% FBS supplemented medium detachedfrom the dish after 29 h of exposure to a shear stressof 0.10 Pa. On the other hand, 90% of Vero and humanliver cells remained on the dish under the samecondition. Observations with scanning electronmicroscopy about cell adhesion plaques on the surfaceof the dish showed that the area covered withlamellipodia and the number of microspikes for 293cells were found to be less than those of the othercell lines. Several attachment factors, especiallyvitronectin, were found to enhance the number ofmicrospikes and the adhesion force of 293 cells.Almost 100% of 293 cells remained on thevitronectin-coated dish after 40 h under 0.10 Pa ofshear stress. A higher shear stress (greater than 0.10Pa) caused a decrease in tissue plasminogen activator(t-PA) productivity of 293 cells. But 0.03 Pastimulated the t-PA secretion on the non-coated dish.Vitronectin also enhanced the t-PA secretion evenunder 0.10 Pa. These results indicate that theadhesion force of 293 cells is obviously weaker thanthat of the other cell lines, and vitronectin enhancesthe adhesion force and the productivity of 293 cellsexposed to a shear stress.     

The biased lamellipodium development and microtubule organizing center position in vascular endothelial cells migrating under the influence of fluid flow*

Summary— To analytically study the morphological responses of vascular endothelial cells (ECs) to fluid flow, we designed a parallel plate flow culture chamber in which cells were cultured under fluid shear stress ranging from 0.01 to 2.0 Pa for several days. Via a viewing window of the chamber, EC responses to known levels of fluid shear stress were monitored either by direct observations or by a video-enhanced time-lapse microscopy. Among the responses of cultured ECs to flow, morphological responses take from hours to days to be fully expressed, except for the fluid shear stress-dependent motility pattern change we reported earlier which could be detected within 30 min of flow changes. We report here that ECs exposed to more than 1.0 Pa of fluid shear shear stress have developed lamellipodia in the direction of flow in 10 min. This is the fastest structurally identifiable EC response to fluid shear stress. This was a reversible response. When the flow was stopped or reduced to the level which exerted less than 0.1 Pa of fluid shear stress, the biased lamellipodium development was lost within several minutes. The microtubule organizing center was located posterior to the nucleus in ECs under the influence of flow. However, this position was established only in ECs responding to fluid shear stress for longer than 1 h, indicating that positioning of the microtubule organizing center was not the reason for, but rather the result of, the biased lamellipodium response. Colcemid-treated ECs responded normally to flow, indicating that microtubules were not involved in both flow sensing and the flow-induced, biased lamellipodium development.     

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.     

Sprint interval and traditional endurance training induce similar improvements in peripheral arterial stiffness and flow-mediated dilation in healthy humans

Low-volume sprint interval training (SIT), or repeated sessions of brief, intense intermittent exercise, elicits metabolic adaptations that resemble traditional high-volume endurance training (ET). The effects of these different forms of exercise training on vascular structure and function remain largely unexplored. To test the hypothesis that SIT and ET would similarly improve peripheral artery distensibility and endothelial function and central artery distensibility, we recruited 20 healthy untrained subjects (age: 23.3 +/- 2.8 yr) and had them perform 6 wk of SIT or ET (n = 5 men and 5 women per group). The SIT group completed four to six 30-s "all-out" Wingate tests separated by 4.5 min of recovery 3 days/wk. The ET group completed 40-60 min of cycling at 65% of their peak oxygen uptake (Vo2peak) 5 days/wk. Popliteal endothelial function, both relative and normalized to shear stimulus, was improved after training in both groups (main effect for time, P < 0.05). Carotid artery distensibility was not statistically altered by training (P = 0.29) in either group; however, popliteal artery distensibility was improved in both groups to the same degree (main effect, P < 0.05). We conclude that SIT is a time-efficient strategy to elicit improvements in peripheral vascular structure and function that are comparable to ET. However, alterations in central artery distensibility may require a longer training stimuli and/or greater initial vascular stiffness than observed in this group of healthy subjects.     

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When the liver is damaged, hepatic stellate cells (HSCs) can change into an activated, highly migratory state. The migration of HSCs may be affected by shear stress due not only to sinusoidal flow but also by the flow in the space of Disse because this space is filled with blood plasma. In this study, we evaluated the effects of shear stress on HSC migration in a scratch-wound assay with a parallel flow chamber. At regions upstream of the wound area, the migration was inhibited by 0.6 Pa and promoted by 2.0 Pa shear stress, compared to the static condition. The platelet-derived growth factor (PDGF)-BB receptor, PDGFR-β, was expressed in all conditions and the differences were not significant. PDGF increased HSC migration, except at 0.6 Pa shear stress, which was still inhibited. These results indicate that another molecular factor, such as PDGFR-α, may act to inhibit the migration under low shear stress. At regions downstream of the wound area, the migration was smaller under shear stress than under the static condition, although the expression of PDGFR-β was significantly higher. In particular, the migration direction was opposite to the wound area under high shear stress; therefore, migration might be influenced by the intercellular environment. Our results indicate that HSC migration was influenced by shear stress intensity and the intercellular environment.