Name: isolation of mouse interstitial valve cells to study the calcification of the aortic valve in vitro
Uploaded: 2021-05-10T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro at JoVE.com

注：这里描述的所有动物程序都已获得西奈山伊坎医学院机构核心和使用委员会的批准。
1. 在从成年小鼠中分离出瓣膜细胞之前的准备
<ol><li>使用 70% v/v 乙醇清洁和消毒 图 1A 中显示的所有手术器械，然后用 70% 乙醇自动清洁手术工作空间 30 分钟。</li>
	<li>加入500微升青霉素-链霉素至50ml的10毫米赫佩斯。准备 50 mL 的 1 倍磷酸盐缓冲盐水 （PBS） 的碱度?...

<ol>
	<li>Rostagno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+disease+in+elderly.">Heart valve disease in elderly.</a> World Journal of Cardiology. 11 (2), 71-83 (2019).</li><li>Stewart, B. 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=Clinical+factors+associated+with+calcific+aortic+valve+disease.+Cardiovascular+Health+Study.">Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.</a> Journal of the American College of Cardiology. 29 (3), 630-634 (1997).</li><li>Marquis-Gravel, G., Redfors, B., Leon, M. B., G&#233;n&#233;reux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+treatment+of+aortic+stenosis.">Medical treatment of aortic stenosis.</a> Circulation. 134 (22), 1766-1784 (2016).</li><li>Spitzer, 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=Aortic+stenosis+and+heart+failure:+disease+ascertainment+and+statistical+considerations+for+clinical+trials.">Aortic stenosis and heart failure: disease ascertainment and statistical considerations for clinical trials.</a> Cardiac Failure Review. 5 (2), 99-105 (2019).</li><li>Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+structure+and+function+in+development+and+disease.">Heart valve structure and function in development and disease.</a> Annual Review of Physiology. 73, 29-46 (2011).</li><li>Simionescu, D. T., Chen, J., Jaeggli, M., Wang, B., Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+follows+function:+advances+in+trilayered+structure+replication+for+aortic+heart+valve+tissue+engineering.">Form follows function: advances in trilayered structure replication for aortic heart valve tissue engineering.</a> Journal of Healthcare Engineering. 3 (2), 179-202 (2012).</li><li>Bouchareb, 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=Activated+platelets+promote+an+osteogenic+programme+and+the+progression+of+calcific+aortic+valve+stenosis.">Activated platelets promote an osteogenic programme and the progression of calcific aortic valve stenosis.</a> European Heart Journal. 40 (17), 1362-1373 (2019).</li><li>Rutkovskiy, 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=Valve+interstitial+cells:+the+key+to+understanding+the+pathophysiology+of+heart+valve+calcification.">Valve interstitial cells: the key to understanding the pathophysiology of heart valve calcification.</a> Journal of the American Heart Association. 6 (9), (2017).</li><li>Bosse, Y., Mathieu, P., Pibarot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics:+the+next+step+to+elucidate+the+etiology+of+calcific+aortic+valve+stenosis.">Genomics: the next step to elucidate the etiology of calcific aortic valve stenosis.</a> Journal of the American College of Cardiology. 51 (14), 1327-1336 (2008).</li><li>Drexler, H. G., Uphoff, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+contamination+of+cell+cultures:+Incidence,+sources,+effects,+detection,+elimination,+prevention.">Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.</a> Cytotechnology. 39 (2), 75-90 (2002).</li><li>Richards, 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=Side-specific+endothelial-dependent+regulation+of+aortic+valve+calcification:+interplay+of+hemodynamics+and+nitric+oxide+signaling.">Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling.</a> American Journal of Pathology. 182 (5), 1922-1931 (2013).</li><li>Bouchareb, 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=Mechanical+strain+induces+the+production+of+spheroid+mineralized+microparticles+in+the+aortic+valve+through+a+RhoA/ROCK-dependent+mechanism.">Mechanical strain induces the production of spheroid mineralized microparticles in the aortic valve through a RhoA/ROCK-dependent mechanism.</a> Journal of Molecular and Cellular Cardiology. 67, 49-59 (2014).</li><li>Lerman, D. A., Prasad, S., Alotti, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcific+aortic+valve+disease:+molecular+mechanisms+and+therapeutic+approaches.">Calcific aortic valve disease: molecular mechanisms and therapeutic approaches.</a> European Cardiology. 10 (2), 108-112 (2015).</li><li>Janssen, J. W., Helbing, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenazo+III:+an+improvement+of+the+routine+calcium+determination+in+serum.">Arsenazo III: an improvement of the routine calcium determination in serum.</a> European Journal of Clinical Chemistry and Clinical Biochemistry. 29 (3), 197-201 (1991).</li><li>Ortlepp, J. 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=Lower+serum+calcium+levels+are+associated+with+greater+calcium+hydroxyapatite+deposition+in+native+aortic+valves+of+male+patients+with+severe+calcific+aortic+stenosis.">Lower serum calcium levels are associated with greater calcium hydroxyapatite deposition in native aortic valves of male patients with severe calcific aortic stenosis.</a> Journal of Heart Valve Disease. 15 (4), 502-508 (2006).</li></ol>

本文为原培养提供了鼠标瓣膜细胞隔离的详细方案。从8周大的小鼠的三个主动脉瓣被集中，以获得足够的细胞数量。此外，本协议描述了VIC表型的特征和体外矿化测定。该方法改编自马蒂厄等人先前描述的协议。
在主动脉瓣隔离期间，必须小心避免所有可能的传染源，以保护细胞免受细菌或支原体污染。事实上，在开始实验之前，对所有的手?...

钙化主动脉瓣病（CAVD）是西方人群中最常见的瓣膜心脏病，影响近2.5%的65岁以上的老年人。CAVD影响超过600万美国人，并与传单的机械性质的变化，损害正常的血液流过1，2。目前，没有药理治疗来阻止疾病的进展或激活矿物质回归。治疗CAVD的唯一有效疗法是主动脉瓣置换手术或导管主动脉瓣置换3。因此，必须研究导致阀门矿化的分子机制，以确定新的药理学目标。事实上，未经治疗的主动脉狭窄有若干不良后果，如左心室功能障碍和心力衰竭4。
主动脉瓣由三层组成，称为纤维瘤、海绵状和心室，其中含有以VC为主要细胞型的5。纤维瘤和心室被一层血管内皮细胞（VECs）5覆盖。VEC调节炎症细胞和寄生虫信号的渗透性。机械应力增加可能会影响VEC的完整性，扰乱主动脉瓣的平衡，导致炎症细胞入侵6。扫描电子显微镜分析显示，人类钙化主动脉瓣7的内皮被破坏。 
钙化组织的组织学分析揭示了骨细胞和骨囊的存在。此外，在体外和人体瓣膜组织8中观察到VIC的骨分化。这个过程主要是由Runt相关的转录因子2（Runx2）和骨形态遗传蛋白（BMPs）8，9精心策划的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 mm cutting edge scissors</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle Actin antibody</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse, Alexa Fluor 488 conjugate</td>
			<td>Cell Signaling</td>
			<td>4412</td>
			<td></td>
		</tr>
		<tr>
			<td>Arsenazo-III reagent set</td>
			<td>POINT SCIENTIFIC</td>
			<td>C7529-500</td>
			<td>a Kit to measure the concentration of calcium</td>
		</tr>
		<tr>
			<td>Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydroxide</td>
			<td>SIGMA -Aldrich 31219</td>
			<td>31219</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Novusbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I&#160; (125 units/mg)</td>
			<td>Thermofisher Scientific</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Tthermofisher</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine graefe forceps</td>
			<td>F.S.T</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco 16000044</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>SIGMA-ALDRICH</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M solution</td>
			<td>STEMCELLS TECHNOLOGIES</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100x</td>
			<td>Thermofisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycozap</td>
			<td>Lanza</td>
			<td>VZA-2011</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penecillin streptomycin 100x</td>
			<td>Thermofisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100 mM</td>
			<td>Thermofisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>&#160;F.S.T</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse interstitial valve cells to study the calcification of the aortic valve in vitro

主动脉瓣细胞的钙化是主动脉狭窄的标志，与瓣膜尖纤维化有关。瓣膜间质细胞（VICs）通过激活其去分化程序，在主动脉狭窄的钙化过程中发挥重要作用。小鼠 VIC 是用于阐明驱动主动脉瓣细胞矿化的信号通路的一个很好的 体外 工具。此处描述的方法，这些作者成功使用，解释了如何获得新分离的细胞。以1毫克/mL和4.5毫克/mL进行两步拼贴手术。第一步对于去除内皮细胞层和避免任何污染至关重要。第二个拼贴剂孵化是促进VIC从组织转移到板。此外，还讨论了分离小鼠瓣膜细胞表型特征的免疫荧光染色程序。此外，钙化检测是使用钙试剂测量程序和阿里扎林红色染色 在体外 进行的。使用小鼠瓣膜细胞原培养物对于测试新的药理靶点以抑制体外细胞矿化至关重要 。

由于木质主动脉瓣的直径通常为1毫米，因此必须集中至少三个阀门来收集一百万个可行的细胞，用于不同的实验程序。VIC 隔离过程的不同步骤显示在图1和图 2中。由于很难手动刮擦瓣膜组织，最好使用涡流产生的剪切应力来去除 VEC。事实上，CD31免疫荧光染色结果显示没有内皮细胞污染（图3D）。此外，小鼠VIC表达维他汀和α-SMA?...

Watch this Scientific Journal Video about Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro at JoVE.com

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

本文描述了通过两步拼贴程序分离小鼠主动脉瓣细胞。分离的小鼠瓣细胞对于执行不同的测定（如 体外 钙化检测）和研究导致主动脉瓣矿化的分子通路非常重要。

小鼠间质瓣细胞分离研究主动脉瓣体外钙化

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

使用分离的小鼠瓣膜细胞对于研究导致转基因小鼠使用转基因细胞时导致瓣膜钙化的信号通路至关重要。两步消化是一种快速高效的方法。该协议最具挑战性的部分是主动脉瓣与八周小鼠隔离。最好在老老鼠身上练习，以便更好地可视化主动脉瓣。在开始实验之前，用70%乙醇清洁和消毒所有的手术器械和工作空间，并高压手术器械30分钟。当初始设置准备好后，从使用乙醇清洁一只八周大的安乐死小鼠的胸部和腹部开始。使用剪刀，打开鼠标的腹部和胸部，然后用小手术剪刀在左中庭和左心室之间切割。通过香水10毫升的冷PBS，并切开心脏，保持3毫米的上升主动脉，从心脏中取出血液。在立体显微镜下，通过在心室中间水平切开心脏并切开左心室朝向主动脉，然后仔细解剖主动脉瓣来解剖主动脉瓣。将阀门集中到一个35毫米的小型组织培养皿中。用5毫升新鲜制备的10毫升冷EPES，辅以抗生素，在5毫升的拼贴剂1型中用5毫升的分离瓣膜清洗，在37摄氏度下持续摇晃30分钟。孵化后，离心管5分钟，在150倍G和洗颗粒一次用两毫升10毫摩尔HPES与涡流在高速30秒。将由此产生的悬浮物从管子中收集到 35 毫米培养皿中，并使用薄钳小心地将组织碎片转移到新鲜管中。然后在连续搅拌35分钟的连续搅拌下，在15毫升管中孵育颗粒，在37摄氏度下用5毫升的拼贴剂类型1。在摄氏4度下，用1毫升移液器和离心机以150倍G为5分钟，将细胞分离。在两毫升完整的DMEM中重新吸收分离的颗粒，并在4摄氏度下以150倍G为离心，在5分钟内清洁细胞。对于电镀，将颗粒重新用一毫升的完整介质重新使用，并将其添加到六井细胞培养皿的一口井中，以最小的介质量。保持板不受干扰在37摄氏度与5%的二氧化碳。经过三天的孵化，通过观察显微镜下的细胞来确认组织碎片附近的生长。一旦1000个细胞可见，小心地用自动切开的钳子去除组织碎片，并改变介质。在达到70%的汇流后尝试细胞，并将其转移到75毫升组织培养皿中。在开始检测之前，用 70% 乙醇清洁生物安全罩，并将 DMEM 中到 37 摄氏度加热。接下来，将每种情况下的10万个细胞种子放入6个井板中，在37摄氏度的完全DMEM和培养中。24小时后，用钙化介质替换超高介质，在37摄氏度下孵育细胞7天，第三天替换介质。七天后，使用阿尔塞纳佐三号试剂测量96井板中的钙浓度，并将吸附量记录在650纳米处。在代表性分析中，经过三到五天的培养，各种瓣膜细胞表型被鉴定为免疫荧光染色。小鼠VIC表达的维他汀和αSMA，瓣膜细胞的重要标记。此外，CD31免疫荧光染色验证了VIC培养中内皮细胞的污染。用富含磷酸盐的钙化介质培养VIC刺激细胞中的钙化，进一步确认钙节点的红色阳性染色。该协议基于来自不同小鼠的三到五个阀门池。使用垃圾伴侣和三种不同的生物复制物对于验证这些发现非常重要。使用小鼠瓣膜细胞对于了解通过将细胞与转基因小鼠隔离而导致主动脉狭窄的分子途径至关重要。本协议以前曾用于研究钙化主动脉瓣矿物质回归中受体通路的含义。

Research

JoVE Journal

Biology

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

原原小鼠肝细胞的分离和3D胶原蛋白三明治培养，研究细胞骨架在胆汁形成中的作用

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

分离人原代瓣膜细胞进行体外疾病建模

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...