Name: electrophysiological recordings of single-cell ion currents under well-defined shear stress
Uploaded: 2019-08-02T13:00:00
Description: Watch this Scientific Journal Video about Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress at JoVE.com

这项工作由国家心肺血液研究所(R01 HL073965,IL)和(T32 HL007829-24,ISF)资助。作者还感谢芝加哥伊利诺伊大学科学机器商店生成了我们最新的MPP流动室。

伊利诺伊大学芝加哥动物护理委员会(#16-183)批准了动物在我们的研究中的使用。
1. 改进平行板流室的装配
注:有关 MPP 流室件件的 D,请参阅表 1和图1。请参阅图1,详细说明装配室件的方向。
<ol><li>要将矩形盖玻璃 D 粘附到 C 件的底部,首先通过将 500 μL 硅胶弹性体固化剂彻底混合成 5 mL ...

<ol>
	<li>Green, D. J., Hopman, M. T., Padilla, J., Laughlin, M. H., Thijssen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Adaptation+to+Exercise+in+Humans:+Role+of+Hemodynamic+Stimuli.">Vascular Adaptation to Exercise in Humans: Role of Hemodynamic Stimuli.</a> Physiological Reviews. 97 (2), 495-528 (2017).</li><li>Gimbrone, M. A., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction,+hemodynamic+forces,+and+atherogenesis.">Endothelial dysfunction, hemodynamic forces, and atherogenesis.</a> Annals of the New York Academy of Sciences. 902, 230-239 (2000).</li><li>Olesen, S. P., Clapham, D. E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemodynamic+shear+stress+activates+a+K++current+in+vascular+endothelial+cells.">Haemodynamic shear stress activates a K+ current in vascular endothelial cells.</a> Nature. 331 (6152), 168-170 (1988).</li><li>Barakat, A. I., Lieu, D. K., Gojova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secrets+of+the+code:+do+vascular+endothelial+cells+use+ion+channels+to+decipher+complex+flow+signals?.">Secrets of the code: do vascular endothelial cells use ion channels to decipher complex flow signals?.</a> Biomaterials. 27 (5), 671-678 (2006).</li><li>Beech, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Piezo1+channels+as+sensors+of+exercise.">Endothelial Piezo1 channels as sensors of exercise.</a> Journal of Physiology. 596 (6), 979-984 (2018).</li><li>Ahn, S. 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=Inwardly+rectifying+K(+)+channels+are+major+contributors+to+flow-induced+vasodilatation+in+resistance+arteries.">Inwardly rectifying K(+) channels are major contributors to flow-induced vasodilatation in resistance arteries.</a> Journal of Physiology. 595 (7), 2339-2364 (2017).</li><li>Fancher, I. S., 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=Hypercholesterolemia-Induced+Loss+of+Flow-Induced+Vasodilation+and+Lesion+Formation+in+Apolipoprotein+E-Deficient+Mice+Critically+Depend+on+Inwardly+Rectifying+K(+)+Channels.">Hypercholesterolemia-Induced Loss of Flow-Induced Vasodilation and Lesion Formation in Apolipoprotein E-Deficient Mice Critically Depend on Inwardly Rectifying K(+) Channels.</a> Journal of the American Heart Association. 7 (5), (2018).</li><li>Rode, B., 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=Piezo1+channels+sense+whole+body+physical+activity+to+reset+cardiovascular+homeostasis+and+enhance+performance.">Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance.</a> Nature Communications. 8 (1), 350 (2017).</li><li>Li, 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=Piezo1+integration+of+vascular+architecture+with+physiological+force.">Piezo1 integration of vascular architecture with physiological force.</a> Nature. 515 (7526), 279-282 (2014).</li><li>Levitan, I., Helmke, B. P., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chamber+to+permit+invasive+manipulation+of+adherent+cells+in+laminar+flow+with+minimal+disturbance+of+the+flow+field.">A chamber to permit invasive manipulation of adherent cells in laminar flow with minimal disturbance of the flow field.</a> Annals of Biomed Engineering. 28 (10), 1184-1193 (2000).</li><li>Fang, Y., 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=Hypercholesterolemia+suppresses+inwardly+rectifying+K++channels+in+aortic+endothelium+in+vitro+and+in+vivo.">Hypercholesterolemia suppresses inwardly rectifying K+ channels in aortic endothelium in vitro and in vivo.</a> Circulation Research. 98 (8), 1064-1071 (2006).</li><li>Shetty, S., Weston, C. J., Adams, D. H., Lalor, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+adhesion+assay+to+study+leucocyte+recruitment+to+human+hepatic+sinusoidal+endothelium+under+conditions+of+shear+stress.">A flow adhesion assay to study leucocyte recruitment to human hepatic sinusoidal endothelium under conditions of shear stress.</a> Journal of Visualized Experiments. (85), e51330 (2014).</li><li>Man, H. S. 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=Gene+Expression+Analysis+of+Endothelial+Cells+Exposed+to+Shear+Stress+Using+Multiple+Parallel-plate+Flow+Chambers.">Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers.</a> Journal of Visualized Experiments. (140), e58478 (2018).</li><li>White, L. 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=The+Assembly+and+Application+of+'Shear+Rings':+A+Novel+Endothelial+Model+for+Orbital,+Unidirectional+and+Periodic+Fluid+Flow+and+Shear+Stress.">The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress.</a> Journal of Visualized Experiments. (116), e54632 (2016).</li><li>Franzoni, M., 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=Design+of+a+cone-and-plate+device+for+controlled+realistic+shear+stress+stimulation+on+endothelial+cell+monolayers.">Design of a cone-and-plate device for controlled realistic shear stress stimulation on endothelial cell monolayers.</a> Cytotechnology. 68 (5), 1885-1896 (2016).</li><li>Dewey, C. F., Bussolari, S. R., Gimbrone, M. A., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+response+of+vascular+endothelial+cells+to+fluid+shear+stress.">The dynamic response of vascular endothelial cells to fluid shear stress.</a> Journal of Biomechanical Engineering. 103 (3), 177-185 (1981).</li><li>Hoger, J. H., Ilyin, V. I., Forsyth, S., Hoger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+regulates+the+endothelial+Kir2.1+ion+channel.">Shear stress regulates the endothelial Kir2.1 ion channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (11), 7780-7785 (2002).</li><li>Moccia, F., Villa, A., Tanzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-activated+Na(+)and+K(+)Current+in+cardiac+microvascular+endothelial+cells.">Flow-activated Na(+)and K(+)Current in cardiac microvascular endothelial cells.</a> Journal of Molecular and Cellular Cardiology. 32 (8), 1589-1593 (2000).</li><li>Crane, G. J., Walker, S. D., Dora, K. A., Garland, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+differential+cellular+distribution+of+inward+rectifier+K+channels+in+the+rat+isolated+mesenteric+artery.">Evidence for a differential cellular distribution of inward rectifier K channels in the rat isolated mesenteric artery.</a> Journal of Vascular Research. 40 (2), 159-168 (2003).</li><li>Hannah, R. M., Dunn, K. M., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+SK(Ca)+and+IK(Ca)+channels+regulate+brain+parenchymal+arteriolar+diameter+and+cortical+cerebral+blood+flow.">Endothelial SK(Ca) and IK(Ca) channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood flow.</a> Journal of Cereberal Blood Flow and Metabolism. 31 (5), 1175-1186 (2011).</li><li>Lane, W. 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=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of Visualized Experiments. (59), e3349 (2012).</li><li>Lieu, D. K., Pappone, P. A., Barakat, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+membrane+potential+and+ion+current+responses+to+different+types+of+shear+stress+in+vascular+endothelial+cells.">Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells.</a> American Journal of Physiology-Cell Physiology. 286 (6), C1367-C1375 (2004).</li><li>Le Master, E., 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=Proatherogenic+Flow+Increases+Endothelial+Stiffness+via+Enhanced+CD36-Mediated+Uptake+of+Oxidized+Low-Density+Lipoproteins.">Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 38 (1), 64-75 (2018).</li><li>Kim, J. G., 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=Measurement+of+Ion+Concentration+in+the+Unstirred+Boundary+Layer+with+Open+Patch-Clamp+Pipette:+Implications+in+Control+of+Ion+Channels+by+Fluid+Flow.">Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow.</a> Journal of Visualized Experiments. (143), e58228 (2019).</li><li>Kim, J. G., 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=Fluid+flow+facilitates+inward+rectifier+K(+)+current+by+convectively+restoring+[K(+)]+at+the+cell+membrane+surface.">Fluid flow facilitates inward rectifier K(+) current by convectively restoring [K(+)] at the cell membrane surface.</a> Scientific Reports. 6, 39585 (2016).</li><li>Malek, A. M., Alper, S. L., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+shear+stress+and+its+role+in+atherosclerosis.">Hemodynamic shear stress and its role in atherosclerosis.</a> Journal of the American Medical Association. 282 (21), 2035-2042 (1999).</li><li>Jacobs, E. R., 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=Shear+activated+channels+in+cell-attached+patches+of+cultured+bovine+aortic+endothelial+cells.+Pflugers+Archiv.">Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells. Pflugers Archiv.</a> European Journal of Physiology. 431 (1), 129-131 (1995).</li><li>Barakat, A. I., Leaver, E. V., Pappone, P. A., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow-activated+chloride-selective+membrane+current+in+vascular+endothelial+cells.">A flow-activated chloride-selective membrane current in vascular endothelial cells.</a> Circulation Research. 85 (9), 820-828 (1999).</li><li>Fitzgerald, T. N., 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=Laminar+shear+stress+stimulates+vascular+smooth+muscle+cell+apoptosis+via+the+Akt+pathway.">Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the Akt pathway.</a> Journal of Cellular Physiology. 216 (2), 389-395 (2008).</li><li>Ueba, H., Kawakami, M., Yaginuma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+as+an+inhibitor+of+vascular+smooth+muscle+cell+proliferation.+Role+of+transforming+growth+factor-beta+1+and+tissue-type+plasminogen+activator.">Shear stress as an inhibitor of vascular smooth muscle cell proliferation. Role of transforming growth factor-beta 1 and tissue-type plasminogen activator.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17 (8), 1512-1516 (1997).</li></ol>

血管系统不断暴露于活跃的血液动力学力量,激活肌敏性电通道3,22,但这些通道在剪切应力引起的回激转导的生理作用只是开始出现4,6,8。负责剪切应力激活通道的机械敏感性的机制仍然未知。本文详述的协议描述了实时对层状剪切应力暴露的美感敏感子通道进行直接调查的方法。
...

众所周知,血流产生的血动力在内皮和血管功能1、2中起着主要作用。众所周知,几种类型的子通道对剪切应力3、4、5的变化有剧烈反应,导致理论认为,子通道可以是初级剪切应力传感器。最近,我们和其他人表明,对剪切应力的血管敏感通道在若干剪切应力敏感血管功能中起着关键作用,包括对剪切应力6、7、8的血管活性反应,发育性血管生成9。然而,离音通道的剪切应力灵敏度机制几乎完全未知。这种知识差距可能是由于在明确定义的剪切应力下进行电生理记录的技术困难。因此,在本文中,我们提供了一个逐步的详细协议,在我们的实验室中例行执行,以实现这个目标6,7,10,11。
该方法的总体目标是允许在生理范围内明确定义的剪切应力下对电导道机活化进行实时调查。这是通过修改标准平行板流室来实现的,使电生理移液器能够降入腔室,并在实时接触流量时进入底部板上生长的细胞,从而提供了独特的方法来实现这一点进球6,7,11 。相比之下,在以前的出版物中描述的标准平行板流室可用于对暴露于剪切力12或其他非侵入性方法的细胞进行实时成像分析,但不适用于电 生理。同样,锥体和板装置,另一种强大的方法,使细胞暴露在剪切应力15,16也不适合电生理记录。因此,这些流动装置不允许研究光通道的剪切应力敏感性。在流下进行电生理记录的困难是缺乏关于这些关键影响的机制的信息的主要原因。
就实现同一目标的替代方法而言,没有一种方法准确或控制。一些早期的研究试图通过将细胞暴露在来自另一个移液器的液体流中来记录流中的离子电池通道活动,这些液体从17、18号进入细胞附近。这是高度非生理的,因为在这些条件下产生的机械力与血管中剪切应激的生理特征几乎没有共同之处。类似的关注也适用于通过灌注开放腔来模拟生理剪切应力的尝试。正如我们早期研究10中详细讨论的,一个开放的液体-空气界面会产生多重干扰和再循环,这是非生理的。为了解决所有这些问题,我们设计了一个经过改进的平行板(MPP)流动室,在早先的研究中,我们也被称为"微创流量装置",在6、7、10、11中,我们制造了从丙烯酸和广泛使用在我们的实验室。然而,尽管设计的原始描述已经发表近20年,并且是唯一允许在定义明确的剪切应力下进行电生理记录的流装置,但这种方法并没有其他实验室采用,只有极少数研究试图记录流下的电流。因此,我们相信,提供使用MPP流动室的详细描述,将非常有助于研究那些对美能敏感性水道和血管生物学感兴趣的人。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m sterile syringe filters</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td>Used for filtering electrophysiolgoical pipette solution</td>
		</tr>
		<tr>
			<td>5 grade forceps</td>
			<td>Fine Scientific Tools</td>
			<td>1252-30</td>
			<td>Used for transferring digested arteries to fresh solution</td>
		</tr>
		<tr>
			<td>9&#34; Pasteur Pipet</td>
			<td>Fisher Scientifc</td>
			<td>13-678-20D</td>
			<td>Used for mechanically disrupting digested arteries and transferring freshly isolated endohtelial cells&#160;</td>
		</tr>
		<tr>
			<td>12 mm diameter Cover glass circles</td>
			<td>Fisher Scientifc</td>
			<td>12-545-80</td>
			<td>For use with studies involving cultured cells and multiple treatments. Cells adhered to the cover glass are used for patch clamp analyses</td>
		</tr>
		<tr>
			<td>24 x 40 mm Rectangluar Cover glass</td>
			<td>Sigma-Aldrich</td>
			<td>CLS2975224</td>
			<td>Cover glass to be added to MPP flow chamber pieces C (Figure 1)</td>
		</tr>
		<tr>
			<td>24 x 50 mm Rectangluar Cover glass</td>
			<td>Sigma-Aldrich</td>
			<td>CLS2975245</td>
			<td>Cover glass to be added to MPP flow chamber E (Figure 1)</td>
		</tr>
		<tr>
			<td>20 gauge syringe needles</td>
			<td>Becton Dickinson and Co</td>
			<td>305175</td>
			<td>For use in mechanical disruption of digested mesenteric arteries</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Genesee Scientific</td>
			<td>32-103</td>
			<td>For use in mechanical disruption of digested mesenteric arteries</td>
		</tr>
		<tr>
			<td>Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td>A9528-50MG</td>
			<td>Used for generating the perforated whole-cell patch configuration.</td>
		</tr>
		<tr>
			<td>collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>100 mg - LS004194</td>
			<td>Enzyme used in our laboratory as a brief digestion following the initial cocktail of neutral protease and elastase</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientifc</td>
			<td>67-68-5</td>
			<td>Solvent for Amphotericin B used in perforated whole-cell patch clamp</td>
		</tr>
		<tr>
			<td>elastase, lyophilized</td>
			<td>Worthington Biochemical</td>
			<td>25 mg - LS002290&#160;</td>
			<td>Enzyme used in our laboratory in a cocktail with neutral protease/dispase to begin digestion of arteries for endothelial cell isolation.</td>
		</tr>
		<tr>
			<td>Falcon Tissue culture Plate, 6-well, Flat Bottom with Low Evaporation Lid&#160;</td>
			<td>Corning</td>
			<td>353046</td>
			<td>For use with studies involving cultured cells and multiple treatments</td>
		</tr>
		<tr>
			<td>neutral protease/dispase</td>
			<td>Worthington Biochemical</td>
			<td>10 mg- LS02100 50 mg - LS02104</td>
			<td>Enzyme used in our laboratory in a cocktail with elastase to begin digestion of arteries for endothelial cell isolation</td>
		</tr>
		<tr>
			<td>SylGard&#160;</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Silicone elastomer for adhering the rectangular cover slip to the MPP flow chamber pieces C and E (Figure 1)</td>
		</tr>
		<tr>
			<td>Tygon ND 10-80 tubing</td>
			<td>Microbore Tubing</td>
			<td>AAQ04133</td>
			<td>ID: 0.05 in, OD: 0.09 in, inlet perfusion tubing for adminsitering flow to the chamber</td>
		</tr>
	</tbody>
</table>

electrophysiological recordings of single-cell ion currents under well-defined shear stress

众所周知,流体剪切应力在内皮功能中起着重要作用。在大多数血管病床上,血流急剧增加引起的剪切应力升高会触发信号级联,导致血管扩张,从而减轻血管壁上的机械应力。众所周知,剪切应力模式也是动脉粥样硬化发展的关键因素,而层状剪切应力是具有英雄保护作用的,而受干扰的剪切应力是亲动脉粥样硬化的。虽然我们对各种中间细胞信号通路有详细的了解,但首先将机械刺激转化为化学中介的受体并不完全了解。干扰性电子通道对剪切的响应至关重要,并调节剪切诱导的细胞信号,从而控制血管活性介质的产生。这些通道是最早激活的剪切信号成分之一,通过促进一氧化氮生产(例如,向内矫正 K+ [Kir] 和瞬态受体电位 [TRP],与剪切引起的血管化相关通道)和内皮超极化因子(例如,基尔和钙激活K +[KCa]通道)和剪切引起的血管收缩,通过一个未确定的机制,涉及压电通道。了解这些通道被剪切力(即直接或通过初级机械受体)激活的生物物理机制,可以为解决与内皮功能障碍相关的病理生理学提供潜在的新靶点和英雄发生。利用电生理学实时记录流动引起的电通道活化仍然是一大挑战。将细胞暴露在定义良好的剪切应力下的标准方法,如锥体和板流变仪和闭合平行板流室,不允许实时研究电子通道激活。该协议的目的是描述一个经过修改的平行板流室,它允许在定义明确的剪切应力下实时电生理记录机械敏感电道。

图1显示了显微镜台上MPP流室的不同视图(上面板)和MPP流室(底部面板)的示意图表示。原理图详细说明了整个设备和流室的尺寸。图 2显示了我们实验室(上面板)MPP 流动室的重力灌注系统的照片。还显示了流系统(底部面板)的示意图,用于突出显示将流动室中的细胞与灌注系统输送溶液和溶液真空去除力分离的步骤。
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Watch this Scientific Journal Video about Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress at JoVE.com

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

在定义良好的剪切应力下单细胞电电流的电生理记录

该协议的目的是描述一个经过修改的平行板流室,用于研究通过剪切应力实时激活美能敏感子孔通道。

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...

使用经过修改的平行板流室可以帮助研究人员回答与机械离子通道对剪切应力的功能反应有关的关键问题。该技术的主要优点是，它允许实时调查在易于组装的可重复使用的平行板流室中流动激活的离子通道。要开始该过程，在 C 片矩形空间的边缘周围涂抹一层薄薄的硅胶弹性体溶液，并轻轻地将矩形盖玻璃、片 D 直接放在弹性体溶液上，以完全覆盖片 C 的开放式矩形空间。然后，重复将矩形盖玻璃（F 片）粘附到片 E 底部的过程，并允许硅胶弹性体溶液在室温下通宵固化。现在，按顺序将 MPP 流室组装，将每一件放在上一个顶部，从底部腔室开始，然后从 C 件开始，然后在顶部放置件 A。接下来，将各件的螺杆孔与角上的螺杆孔对齐，将碎片紧密拧在一起，以防止泄漏发生，同时管理流向 MPP 流量室的流量。在六井板中，在每个井中放置四到五、12 毫米盖玻璃圈。保存10%至30%汇合之间的细胞，以便单细胞可用于电生理记录。随后，在标准培养条件下孵育细胞不少于两小时，使细胞粘附，且不超过24小时，因为内皮细胞在亚流体下播种超过24小时时将变得扁平且难以修补。接下来，从六井板的井中取出含有粘附细胞的盖玻璃，并在 PBS 中快速冲洗。然后，将带电池的盖玻璃转移到含有两毫升电生理 BAF 溶液的 35 毫米培养皿中。立即将带电池的盖玻璃转移到 MPP 流室。将盖玻璃圈转移到长方形盖玻璃 D 片上，该玻璃块粘附在 MPP 流室的 C 片上。添加 BAF 溶液，将盖玻璃圈和电池完全淹没。随后，将盖玻璃圆放在片件D上，使电池与片B的狭缝开口一样，确保盖玻璃圆通过溶液玻璃粘附粘附在片D上，避免通过流动应用破坏盖玻璃圆的位置。然后，按适当的顺序将部件拧在一起，组装 MPP 流室。将腔室转移到显微镜阶段，并立即使用 BAF 溶液对腔室进行透镜。接下来，为实验确定一个具有暗边框和明显细胞核的健康细胞。避免出现虚张声势的单元格或与其他细胞接触的单元格。为了控制剪切应力，通过将一个 30 毫升的分级注射器筒将一个 30 毫升的流式注射器筒连接到装有微孔管的三向 Luer 锁来建立重力灌注系统。接下来，使用双面胶带将分级圆柱体连接到电生理学钻机周围的法拉第笼上。在将管子插入 MPP 腔室之前，使用 BAF 溶液预填充注射器和管材。然后，将管子插入 MPP 流量室入口孔 A.用溶液预先填充 MPP 流量室，以便将其在真空储液罐中清除。停止流向腔室，将分级油缸重新填充到顶部标记。使用秒表手动计算流速，允许溶液以给定的注射器气缸高度流经腔室。升起或降低注射器以改变流量，并使用此方程计算平行腔室中的剪切应力。重复此过程，直到找到所需的剪切应力水平。随后，将包含粘附细胞的组装室转移到电生理学钻机的显微镜阶段。将预填充的 BAF 溶液的管子插入 A 片孔中，然后填充腔室，同时用 10 毫升的 BAF 溶液清洗细胞。一旦成功获得所需的贴片配置，让通道电流在室温下稳定在静电浴中。一旦电流稳定下来，请逐步应用剪切，使增加的电流在下一步剪切应力增加之前稳定下来。通过停止流向腔室，消除剪切接触到电池的剪切，使机械敏感通道电流回到静态浴中观察到的基线电流。此处显示的是来自原小鼠内肠内皮细胞的 Kir 电流的代表性原始记录，具有显著的线性向外泄漏电流。超过 400 毫秒，对贴片应用负 140 到正 40 毫伏的斜坡。泄漏电流可防止对实际 Kir 通道活动进行分析。要减去泄漏电流，首先计算泄漏电流的线性斜率传导。多个 G 斜率由整个原始跟踪的相应电压绘制原始数据上的泄漏电流。线应完全覆盖向外线性泄漏。然后，从整个轨迹中减去绘制的泄漏电流，使线性向外电流约为每皮科法拉德的零皮奥安佩雷斯，并可以分析真正的 Kir 电流。尝试此过程时，要记住的最重要的事情是将盖玻璃正确定位在腔室中，以便细胞可供实验使用。此外，请确保盖滑完全粘附在 D 片上，这样液体流动不会破坏包含电池的盖玻璃。

Research

JoVE Journal

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...