Name: isolation of murine coronary vascular smooth muscle cells
Uploaded: 2016-05-30T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Coronary Vascular Smooth Muscle Cells at JoVE.com

这项工作是由美国国立卫生研究院（R01HL056046 PAL并K99HL116769到AJT）的研究所在全国儿童医院（PAL并AJT）的支持，。

伦理学声明：这项研究是根据健康指南全国学院进行，它是在全国儿童医院批准机构的动物护理和使用委员会。 1.准备/设置注意：此隔离技术需要定位侧由端上的环待机2的Langendorff加热线圈，和并联连接到一个循环水浴中。 <ol><li>开启循环水浴和调节温度的的Langendorff插管的尖端达到33-34℃的最终温度（在此所用的水浴设定为40℃，由于一个6-7℃的损失在整个管道温度）。打开灌注泵?...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+phenotype+and+function+of+vascular+smooth+muscle+cells.">Diversity of phenotype and function of vascular smooth muscle cells.</a> J Lab Clin Med. 127 (6), 524-529 (1996).</li><li>Souza-Smith, F. 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=Mesenteric+resistance+arteries+in+type+2+diabetic+db/db+mice+undergo+outward+remodeling.">Mesenteric resistance arteries in type 2 diabetic db/db mice undergo outward remodeling.</a> PLoS One. 6 (8), e23337 (2011).</li><li>Thuroff, J. 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这项研究的目的是适应现有的细胞分离协议，以增加冠状血管的产量从小鼠心脏流畅。大部分血管平滑肌生物学的开拓性工作与培养的大鼠主动脉平滑肌细胞进行。这些研究提供了控制血管平滑肌细胞生长，迁移和肥厚7分子机制的基础知识。然而，如该领域的进展，它变得明显VSMC表型和功能是由许多血管床的具体因素来控制。例如，相比于周围血管，冠状动脉显示不同的功能反应，损伤...

该方法的目标是分离来自鼠冠状循环的血管平滑肌细胞（VSMC），用于在细胞培养物和标准细胞培养测定中使用。我们开发这种技术来评估在糖尿病血管重塑的分子机制。之前我们已经内向肥厚性重塑报道在1型糖尿病的的db / db小鼠模型中间隔冠状小动脉。由于在小鼠中隔冠状动脉发现组织的有限数量，标准的实验技术调查的db / db小鼠和对照小鼠的蛋白质变化（ 如免疫印迹）难以在最好的。此外，我们以前曾表明，血管紧张素受体 ​​阻断剂（ARB）氯沙坦降低db / db小鼠2中观察到的重塑。因此，从冠脉循环主血管平滑肌细胞的隔离允许我们进一步调查在血管平滑肌细胞的表型或活化信号转导途径在糖尿病小鼠的变化，这可能是contribu婷不良冠脉动脉​​重塑。 许多研究已阐明了利用啮齿类动物主动脉，血管平滑肌细胞中分离规范的信号转导通路，而不是在每一个具体的血管床。然而，我们已经证明血管床在冠状动脉，主动脉具体重塑，和db / db小鼠1的肠系膜环流，这表明在各血管床的血管平滑肌细胞可以是不同的。因此，有必要分离来自每个血管床血管平滑肌，以便更好地理解在每一组的VSMCs发生的病理变化。有不同的方法进行分离和培养平滑肌细胞主动脉过多。然而，目前，仅存在一个已发布对来自小鼠冠脉循环3平滑肌细胞的分离研究。腾等 。是第一个报道的方法分离从鼠标冠脉循环血管平滑肌细胞;但是，我们有显著修订了协议，他们还分离内皮CELLS。其他实验室还使用从腾等的协议。分离冠状动脉细胞和气道平滑肌细胞4,5。我们已掺入的变动将产生高度富集用于从所述冠脉循环血管平滑肌细胞的细胞群。 在分离的哺乳动物心脏或者Langendorff灌注技术的逆行灌注，由奥斯卡的Langendorff成立于1897年6，目前仍在广泛使用的今天心血管细胞的分离。这里介绍的技术，加上现代的小鼠遗传修饰的进步，提供了从冠脉循环血管平滑肌细胞的分子行为的调查，接近一个有价值的工具。

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fetal bovine serum</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">HEPES 1M solution</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">MT-25-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primocin - 20mL</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (High Glucose, Sodium Pyruvate, L-Glutamine)&#160;</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM NEAA 10 mM 100X</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glut 200 mM - Gibco</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Cell Strainer 100um nylon mesh</td>
			<td>Fisher</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunclon Polystrene dish with lid, sterile, 35 mm</td>
			<td>Fisher</td>
			<td>12-565-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Apparatus black silk suture 5-0</td>
			<td>Fisher</td>
			<td>14-516-124</td>
			<td></td>
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			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type-2&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor 25mg</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid (clear)</td>
			<td style="width:143px;">Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid (phenol red)</td>
			<td style="width:143px;">Life Technologies</td>
			<td>14170-161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
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			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5.0 ml heating coil with degassing bubble trap</td>
			<td>Radnoti</td>
			<td>158830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">11 plus pump</td>
			<td style="width:143px;">Harvard Apparatus</td>
			<td style="width:119px;">70-2208</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Circulating heated water pump</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>any brand will work</td>
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	</tbody>
</table>

isolation of murine coronary vascular smooth muscle cells

虽然分离及大血管的血管平滑肌细胞（VSMC）的文化是公认的，我们试图从冠脉循环分离培养血管平滑肌细胞。用完整主动脉弓心中取出并经由逆行的Langendorff用含有300单位/ ml胶原酶II型，0.1毫克/毫升大豆胰蛋白酶抑制剂和1M 的 CaCl 2消化溶液灌注。所述灌流液分别以15分钟的间隔为90分钟，通过离心沉淀收集，再悬浮于电镀媒体，并接种在组织培养皿。通过SM22α的存在下，α-SMA，波形蛋白和血管平滑肌细胞进行表征。之一的使用这种技术的主要优点是，从小鼠的冠脉循环隔离血管平滑肌细胞的能力。虽然获得可以限制一些可用于其中细胞的应用程序单元的数量少，分离的冠状血管平滑肌细胞可以在多种成熟的细胞培养技术和阿萨用于YS。研究调查转基因小鼠的血管平滑肌细胞可以提供有关的结构功能和血管病变相关的信号传导过程的详细信息。

由于我们的冠状动脉平滑肌细胞分离技术的新颖方面，我们试图确定该细胞分离的纯度。根据它们的形态和免疫荧光染色到通道2.基于细胞培养的第一次洗涤后的形态学小鼠冠状动脉平滑肌进行了鉴定，分离过程中有效地除去心肌细胞和内皮细胞。的血管平滑肌细胞保留其形态高达通道2（ 图1）。然而，有污染的由外膜和间质成纤维细胞的可能性。为了排除这...

Watch this Scientific Journal Video about Isolation of Murine Coronary Vascular Smooth Muscle Cells at JoVE.com

Isolation of Murine Coronary Vascular Smooth Muscle Cells

此协议的目的是演示从冠脉循环鼠主要血管平滑肌细胞的分离和培养技术（血管平滑肌细胞）。一旦血管平滑肌细胞已被分离，它们可用于许多标准培养技术。

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

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Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

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The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

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分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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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 ...

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

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

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Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Mouse Coronary Endothelial Cells

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...