Name: human saphenous vein endothelial cell isolation and exposure to controlled levels of shear stress and stretch
Uploaded: 2023-04-21T14:35:00
Description: Watch this Scientific Journal Video about Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch at JoVE.com

JEK得到了圣保罗国家宪法权利保护基金会[FAPESP-INCT-20214/50889-7和2013/17368-0]和全国发展委员会（INCT-465586/2014-7和309179/2013-0）的资助。AAM得到了圣保罗国家宪法权利保护基金会（FAPESP 2015/11139-5）和全国公民和技术发展委员会（环球-407911/2021-9）的资助。

未使用的隐静脉段是从圣保罗大学医学院心脏研究所（InCor）接受主动脉冠状动脉搭桥手术的患者那里获得的。所有个体都知情同意参与该研究，该研究由当地伦理委员会审查和批准。
1. 原代人隐静脉内皮细胞 （hSVEC） 的分离、培养和表征
<ol><li>制备<ol><li>高压灭菌一对直镊子或弯曲的镊子和组织剪刀（7-8厘米）。</li>
		<li>准备无菌明胶。将0.1g（0.1%w / v）或3g?...

<ol>
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J., Zakkar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+inflammation+of+the+venous+endothelium+in+vein+graft+disease.">Activation and inflammation of the venous endothelium in vein graft disease.</a> Atherosclerosis. 265, 266-274 (2017).</li><li>Ward, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-&#954;B+inhibition+prevents+acute+shear+stress-induced+inflammation+in+the+saphenous+vein+graft+endothelium.">NF-&#954;B inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium.</a> Scientific Reports. 10 (1), 15133 (2020).</li><li>Golledge, J., Turner, R. J., Harley, S. L., Springall, D. R., Powell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumferential+deformation+and+shear+stress+induce+differential+responses+in+saphenous+vein+endothelium+exposed+to+arterial+flow.">Circumferential deformation and shear stress induce differential responses in saphenous vein endothelium exposed to arterial flow.</a> The Journal of Clinical Investigation. 99 (11), 2719-2726 (1997).</li><li>Gir&#227;o-Silva, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+stretch+induces+endothelial+dysfunction+accompanied+by+oxidative+stress+and+actin+remodeling+in+human+saphenous+vein+endothelial+cells.">High stretch induces endothelial dysfunction accompanied by oxidative stress and actin remodeling in human saphenous vein endothelial cells.</a> Scientific Reports. 11 (1), 13493 (2021).</li><li>Ataollahi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+the+isolation+of+endothelial+cells+from+large+vessels.">New method for the isolation of endothelial cells from large vessels.</a> Cytotherapy. 16 (8), 1145-1152 (2014).</li><li>Torres, C., Machado, R., Lima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+the+saphenous+veins+endothelial+cells+in+patients+with+chronic+venous+disease+and+in+patients+undergoing+bypass+surgery:+an+exploratory+study.">Flow cytometric characterization of the saphenous veins endothelial cells in patients with chronic venous disease and in patients undergoing bypass surgery: an exploratory study.</a> Heart and Vessels. 35 (1), 1-13 (2020).</li><li>Aird, W. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-arrestin+is+critical+for+early+shear+stress-induced+Akt/eNOS+activation+in+human+vascular+endothelial+cells.">&#946;-arrestin is critical for early shear stress-induced Akt/eNOS activation in human vascular endothelial cells.</a> Biochemical and Biophysical Research Communications. 483 (1), 75-81 (2017).</li><li>Davis, M. E., Cai, H., Drummond, G. R., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+regulates+endothelial+nitric+oxide+synthase+expression+through+c-Src+by+divergent+signaling+pathways.">Stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways.</a> Circulation Research. 89 (11), 1073-1080 (2001).</li><li>Dekker, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+fluid+shear+stress+induces+a+distinct+set+of+endothelial+cell+genes,+most+specifically+lung+Kruppel-like+factor+(KLF2).">Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2).</a> Blood. 100 (5), 1689-1698 (2002).</li><li>Hamik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kruppel-like+factor+4+regulates+endothelial+inflammation.">Kruppel-like factor 4 regulates endothelial inflammation.</a> The Journal of Biological Chemistry. 282 (18), 13769-13779 (2007).</li><li>Beamish, J. A., He, P., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+contractile+smooth+muscle+cell+phenotype:+implications+for+vascular+tissue+engineering.">Molecular regulation of contractile smooth muscle cell phenotype: implications for vascular tissue engineering.</a> Tissue Engineering. Part B, Reviews. 16 (5), 467-491 (2010).</li></ol>

隐静脉段应至少为 2 cm，以成功分离 hSVEC。小段难以处理并绑住血管末端以维持胶原酶溶液以分离细胞。减小的管腔表面积不能产生足够的细胞来扩增培养物。为了尽量减少非EC污染的风险，在整个过程中，大隐静脉段的操作需要非常轻柔。将移液器吸头引入管腔表面以去除血液和引入胶原酶溶液时要小心。应很好地控制酶溶液的暴露（不超过1小时），以减少非EC对培养物的污染。使用补充生长因...

内皮细胞 （EC） 功能障碍是隐静脉移植失败的关键因素1，2，3，4。剪切应力和周期性拉伸的持续增加诱导人隐静脉内皮细胞（hSVEC）的促炎表型3，4，5，6。潜在的分子途径仍未完全了解，体外研究的标准化方案可能会利用该领域的新见解。在这里，我们描述了一个简单的方案来分离，表征和扩增hSVEC，以及如何将它们暴露于不同水平的剪切应力和循环拉伸，模拟静脉和动脉血流动力学状况。
hSVEC通过胶原酶孵育分离，可使用至第8代。与其他可用的方案7相比，该协议需要较少的血管操作，从而减少了平滑肌细胞和成纤维细胞的污染。另一方面，它需要至少 2 厘米的较大容器段才能进行有效的 EC 提取。在文献中，已经报道了来自大型容器的EC也可以通过机械去除获得7，8。虽然有效，但物理方法具有EC产量低和成纤维细胞污染较高的缺点。为了提高纯度，需要使用磁珠或细胞分选的额外步骤，由于获取磁珠和抗体7，8，增加了方案的成本。酶法在EC纯度和活力方面具有更快更好的结果7，8。
研究内皮功能障碍最常用的EC是人脐静脉内皮细胞（HUVECs）。众所周知，EC表型在不同的血管床中发生变化，因此必须开发代表所研究血管的方法9，10。在这方面，建立分离hSVEC并在机械应力下培养的方案是了解hSVEC功能障碍在静脉移植疾病中的贡献的宝贵工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-0.02% EDTA solution</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>15 &#181; slide I 0.4 Luer&#160;</td>
			<td>Ibidi</td>
			<td>80176</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dilactate (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>6-wells equibiaxial loading station of 25 mm&#160;</td>
			<td>Flexcell International Corporation</td>
			<td>LS-3000B25.VJW</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide with removable well</td>
			<td>Thermo Fisher Scientific</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Millipore</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic sheet 1 cm thick</td>
			<td>Plexiglass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>Abcam</td>
			<td>ab24590</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31, FITC antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MHCD3101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-VE-cadherin antibody</td>
			<td>Cell Signaling</td>
			<td>2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioflex plates collagen I</td>
			<td>Flexcell International Corporation</td>
			<td>BF3001C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin solution</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton suture EP 3.5 15 x 45 cm</td>
			<td>Brasuture</td>
			<td>AP524</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclophilin forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclophilin reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 basal medium</td>
			<td>Lonza</td>
			<td>CC3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 SingleQuots supplements</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexcell FX-5000 tension system</td>
			<td>Flexcell International Corporation</td>
			<td>FX-5000T</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount aqueous mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium from porcine intestinal mucosa 5000 IU/mL</td>
			<td>Blau Farmac&#234;utica</td>
			<td>SKU 68027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi pump system (Pump + Fluidic Unit)</td>
			<td>Ibidi</td>
			<td>10902</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF2 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF2 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF4 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF4 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>NOA 280 nitric oxide analyzer</td>
			<td>Sievers Instruments</td>
			<td>NOA-280i-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NOS3 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>NOS3 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion set 15 cm, ID 1.6 mm, red, 10 mL reservoirs</td>
			<td>Ibidi</td>
			<td>10962</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin - Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin - Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>221945</td>
			<td></td>
		</tr>
		<tr>
			<td>QuanTitec SYBR green PCR kit</td>
			<td>Qiagen</td>
			<td>204143</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 12K flex platform&#160;</td>
			<td>Applied Biosystems</td>
			<td>4471087</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy micro kit&#160;</td>
			<td>Quiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide glass (24 mm x 60 mm)</td>
			<td>Knittel Glass</td>
			<td>VD12460Y1D.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>31443</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV first-strand synthesis system</td>
			<td>Thermo Fisher Scientific</td>
			<td>18091200</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Type II collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
	</tbody>
</table>

human saphenous vein endothelial cell isolation and exposure to controlled levels of shear stress and stretch

冠状动脉旁路移植术（CABG）是一种对缺血性心肌进行血运重建的手术。尽管与动脉导管相比，长期通畅性降低，但隐静脉仍用作冠状动脉旁路移植术导管。与移植物动脉化相关的血流动力学应激突然增加导致血管损伤，尤其是内皮损伤，这可能会影响隐静脉移植物 （SVG） 的低通畅性。在这里，我们描述了人隐静脉内皮细胞（hSVEC）的分离，表征和扩增。通过胶原酶消化分离的细胞显示出典型的鹅卵石形态，并表达内皮细胞标志物CD31和VE-钙粘蛋白。为了评估机械应力的影响，本研究使用协议来研究动脉化SVG上的两种主要物理刺激，剪切应力和拉伸。 hSVEC在平行板流动室中培养以产生剪切应力，显示流动方向的对齐和KLF2，KLF4和NOS3的表达增加。hSVEC也可以在硅膜中培养，该硅膜允许模拟静脉（低）和动脉（高）拉伸的受控细胞拉伸。内皮细胞的F-肌动蛋白模式和一氧化氮（NO）分泌通过动脉拉伸相应地调节。总之，我们提出了一种分离hSVEC的详细方法，以研究血流动力学机械应激对内皮表型的影响。

通常，粘附的EC可以在提取后3-4天观察到。hSVEC最初形成细胞簇并显示出典型的“鹅卵石”形态（图1B）。它们表达EC标志物CD31（图1C，D）和VE-钙粘蛋白（图1D）。hSVEC可以在非包被处理的细胞培养皿上轻松繁殖，并且在培养物中保留内皮表型多达八代。
hSVEC在剪切应力下培养时，沿流动方向对齐（...

Watch this Scientific Journal Video about Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch at JoVE.com

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

我们描述了一种分离和培养人隐静脉内皮细胞（hSVEC）的方案。我们还提供了产生剪切应力和拉伸的详细方法，以研究hSVEC中的机械应力。

人隐静脉内皮细胞分离和暴露于受控水平的剪切应力和拉伸

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

冠状动脉搭桥手术后，隐静脉突然暴露于动脉疾病。了解其对机械应力的适应反应可能有助于设计治疗工具来预防静脉移植失败。这种内皮细胞分离方案非常简单，只需要与胶原酶一起孵育。通过最少的血管操作，这可以减少平滑肌细胞和成纤维细胞的污染。分离人类SVEC并在剪切应力和循环拉伸下培养它们的方案对于了解机械力在与隐静脉移植失败相关的内皮细胞功能障碍中的贡献至关重要。先前的内皮细胞培养对细胞培养基补充剂的要求非常高。必须添加生长因子才能成功分离出一种培养物，即人类SVEC。为了分离原代人隐静脉内皮细胞或人SVEC，收集至少两到三厘米长的隐静脉段。在无菌层流罩下工作，将静脉段转移到装有预热PBS的培养皿中。要从管腔内去除所有血液，请将连接到移液器并充满PBS的移液器吸头插入静脉的一端并轻轻冲洗。冲洗直至洗涤流完全清澈。然后用无菌棉缝合线关闭静脉的一端。小心地用每毫升1毫克的2型胶原酶溶液填充血管，并用棉缝线关闭容器的另一端。将带有胶原酶溶液的容器在5%二氧化碳的潮湿气氛中以37摄氏度孵育一小时。然后在15毫升管上切割血管的一端以收集胶原酶溶液，然后再切割另一端。用一到两毫升PBS冲洗管腔表面，用胶原酶收集管内所有分离的内皮细胞。将试管在室温下以400G离心五分钟。除去上清液后，将细胞沉淀重新悬浮在含有额外肝素溶液的三至四毫升完整内皮细胞培养基中。将细胞接种在预涂有每体积重量3%明胶的60毫米细胞培养皿中。在不更换培养基的情况下，在潮湿的气氛中孵育细胞四天。之后，每隔一天更换一次培养基，而不添加肝素补充剂。在显微镜下检查平板以确保红细胞被去除。提取后一到四天后，根据静脉段的大小和直径，通过相差显微镜观察人类SVEC，以评估汇合程度及其鹅卵石形态。继续每两天更换一次培养基，直到细胞达到70%至80%汇合。在流动室载玻片上涂上每体积重量为0.1%的明胶，并在37摄氏度下孵育至少30分钟。为了平衡流体单元和具有10毫米储液器的无菌灌注装置，请在潮湿的气氛中孵育过夜。对于剪切应力实验，将悬浮在100微升培养基中的200， 000个内皮细胞种子放入涂有明胶的流动室载玻片中。为了将细胞附着到培养表面上，将细胞孵育四个小时。将灌注装置放在流体单元上。用约12毫升预热的完整内皮细胞培养基填充储液器和灌注装置。从灌注装置中去除气泡，以平衡两个储液槽的液位为五毫升。将流体单元放在安装的灌注装置上，而无需培养箱中的腔室滑块，并将其电缆连接到计算机调节的泵。通过在泵控制软件中运行预定义的协议，去除储液罐和灌注装置中的所有气泡。将装有灌注装置的流体单元连接到层流罩上，并连接具有汇合细胞单层的载玻片。将带有载玻片的整个装置放入培养箱后，将其电缆连接到泵。在泵控制软件中，通过选择滑块类型并设置介质粘度来调整流体单元设置。在流动参数中，输入剪切应力值并设置循环持续时间和流动类型。然后点击播放按钮开始实验。请记住，使用用相同培养基处理的细胞来维护载玻片，不要像静电对照那样暴露于流动中。将有机硅润滑剂涂在 25 毫米 6 孔等双轴加载站的顶部和侧面。将悬浮在3毫升中的400，000个细胞接种到涂有胶原蛋白的6孔柔性底培养板的每个孔中。在潮湿的空气中孵育细胞，直到它们达到100%汇合。在开始拉伸方案之前，用预热的PBS洗涤细胞一次，每孔加入三毫升新鲜培养基。将所有润滑的加载站的底板插入培养箱中，并将管道与张力细胞拉伸生物反应器的控制器设备适当连接。将每个培养板连接到生物反应器系统提供的一个红色垫圈上。然后将它们放入培养箱的底板中。将所有四个培养板放在底板中，以实现适当的真空和拉伸负荷。打开控制器设备和计算机系统。打开 FlexCell 控制软件并配置所需的方案，包括伸长率百分比、波形形状、频率和时间。保存方案。打开真空系统，然后选择方案并单击计算机屏幕上的开始以运行拉伸。检查硅胶膜是否在移动和拉伸细胞。在提取后4至10天可以观察到粘附的内皮细胞。它们最初形成细胞簇，显示出典型的鹅卵石形态。他们表达EC标志物CD31和VE-钙粘蛋白。在剪切应力下培养的hSVEC沿流动方向排列。剪切应力为每平方厘米20达因，持续72 h诱导典型机械敏感基因KLF2、KLF4和NOS3的表达，表明剪切刺激的有效性。周期性拉伸结果取决于应用于人类SVEC的强度。低拉伸下的细胞显示出类似于静态细胞的皮质F-肌动蛋白模式。在长达 72 小时内一氧化氮释放没有变化的情况下，我们的动脉拉伸水平在 24 小时后重塑了肌动蛋白细胞骨架，并在 72 小时后减少了 NO 释放。除了机械应力研究外，研究人员还可以使用分离的细胞进行各种方法，以扩展对内皮细胞功能和功能障碍的了解。按照演示的方案，可以在剪切应力和拉伸下研究任何其他类型的内皮细胞。

Research

JoVE Journal

Medicine

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

科研

医学

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Controlled Cervical Laceration Injury in Mice

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

步骤人类隐静脉<em&gt;前体内</em&gt;灌注和外部强化

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...