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）。打开灌注泵和设置灌注率0.5毫升/分钟。 <ol><li>冲洗各用50毫升70％乙醇后，再用100毫升高压灭菌超纯水清洗两者水夹套的加热线圈的，附着油管和25号插管。 </li><li>清除加热线圈通过推动空气用注射器，用10ml Hank氏平衡盐溶液（HBSS）洗无酚红。在加热线圈和管道离开的HBSS。 </li><li>附加填充有室温下的HBSS无酚红到管道10ml无菌鲁尔锁注射器。确保附接HBSS灌封注射器时，因为这可以在消化过程中导致空气栓子到血管平滑肌细胞分离产生不利影响没有气泡被截留在管和加热线圈的内部。 </li></ol></li><li>在另一10ml无菌注射器填充有冰冷的HBSS无酚红移除的加热线圈和地点25号插管。保持这个冰上待用。重复其他套管。 </li><li>准备酶消化解决方案。 <ol><li>根据特定的批号权衡型胶原酶2 75毫升消化溶液达到300单位/毫升的最终浓度。 </li><li>在75ml的HBSS用酚红溶解胶原酶。接下来，添加加入1.5ml 0.1毫克/毫升大豆胰蛋白酶抑制剂和75微升的1M氯化钙2到混合物中。 </li></ol></li><li>如果立即用消化解决方案，它加热至37℃。如果不是，将其放置在4℃下长达6小时。该解决方案必须由每日新鲜。 </li><li>准备一站式解决方案/平板培养基。 <ol><li>加入100 ml热灭活的FBS，加入5ml L-谷氨酰胺，5毫升的非必需氨基酸，将5ml的HEPES，和1ml primocin的至500ml高葡萄糖（4.5克/升）的DMEM。建议除菌过滤。 </li><li>等分试样6毫升在4℃终止液成每十的15毫升锥形管，编号为A1-A6和B1-B6，和地点。等分试样50毫升的停止溶液到50毫升锥形并发生在37℃培养箱中。 注意:此步骤也可在必要时当天制备隔离之前。此终止溶液将用作以下细胞分离电镀介质。 </li></ol></li><li>准备两个1毫升注射器配备了为30G针头用100微升肝素（1000单位/ ml）。 </li><li>切5厘米长5-0手术丝绸。只要两次扳平一松上手结在外科丝一端为其他。放置到25号插管，但不要拧紧。重复其他地下25插管。 </li><li>用2ml无菌，冰冷，HBSS不含酚红的装满四35毫米无菌培养皿中（＃1-4）。置于冰上。 </li></ol> 2.安乐死，心脏分离和插管<ol><li>心脏隔离之前，确保终止液消解液和50毫升的锥形是温暖的。删除与来自滴定管夹（或环形支架钳）冰和地点上环支架连接地下25插管HBSS充式注射器。 </li><li>麻醉，用3％异氟烷的小鼠。确认小鼠是通过在每个后肢趾触摸麻醉。如果腿部抽搐，鼠标还没有麻醉所以等待1分钟，重复触脚尖。 </li><li>通过去除皮草露出颈静脉第二皮肤上用剪刀颈部的前部。接下来，使用镊子小心地移除颈静脉任何脂肪。去除脂肪后，缓缓注入肝素100微升（1000单位/ ml）。放置海绵手术在静脉，避免流血过多。 </li><li> 1分钟后，开胸暴露心脏，心脏电梯用弯钳和用剪刀切除它，确保保持升主动脉完好无损通过第一头臂分支。立即将心脏到35毫米的菜用冰冷的HBSS（＃1）无酚红和冲洗。 注意:请用解剖范围以下4个步骤。 </li><li>位置被安装在环上的地下25插管站在一个45°角，使尖端刚好位​​于冰冷的HBSS的表面下在培养皿＃2和显然通过解剖显微镜可见。的心脏转移到菜＃2。 </li><li>使用杜蒙钳，由经钝性分离至P轻轻去除多余的脂肪暴露主动脉r事件刺入或主动脉撕裂。接下来，使用镊子在地下25插管主动脉滑动。 </li><li>通过主动脉，接近，但不超过主动脉瓣，这将损害阀的完整性并因此，逆行灌注向下引导插管。一旦插管就位，在主动脉滑动预先打结丝，并拧紧。领带的第二结以形成围绕主动脉平结，以确保心脏。 </li><li>一旦主动脉被牢固地捆绑，轻轻地从在附着在地下25插管注射器与冰冷的HBSS冠脉循环冲洗剩余的血液。小心不要推太多，以避免过多的压力冠心病。只有非常缓慢轻柔冲洗才能完成冲洗。这个温柔的冲洗过程中也可以观察到无泄漏的主动脉插管。 </li><li>打开灌注泵开始HBSS的流动。取出地下25插管（附带的心脏）从HBSS充式注射器，同时注意保持充满HBSS套管座，并将其连接到温暖的HBSS填充加热线圈。以0.5毫升/分钟为8分钟的速度开始与灌注泵的心脏灌注。 </li><li>第一个心脏插管和系统上正在灌注后，重复步骤2.1-2.8的第二心脏。这些隔离会错开每一个心脏，因此需要仔细的规划和时间安排。 </li></ol> 3.消化<ol><li>用HBSS冲洗后，在泵目前改变至10毫升HBSS注射器一个填充用10ml预热的消化溶液。以0.5毫升/分钟的速率灌注用12分钟的消化溶液每个心脏。不收集分数从这个第一所产生的消化，因为这将包含任何残血细胞和内皮细胞。 </li><li> 12分钟后，改变它的灌注率0.4毫升/分钟，将装有冷停止SOLUT 15毫升的锥形（管A1或B1，对应于第一或第二心脏对于每个消化）离子进入，以便开始VSMC富集灌流收集冰桶的心脏下方。 </li><li> 15分钟后，这两种级分的采集后立即在89 XG改变集合锥形到管A2或B2和自旋锥形管A1和B1 10分钟。当填充有泵上的消化溶液的注射器具有小于1毫升溶液，替换的消化溶液新鲜充10毫升注射器。 </li><li>离心后，从每个采集管吸出上清液，留下大约为0.5毫升从A1重悬沉淀用2ml的暖终止溶液（或电镀介质）。从A1管加入悬浮到B1管与管的地方在37℃孵化器与帽松动。 </li><li>重复步骤3.3和3.4，直到所有的管（至A6和B6）被用于90分钟的总采集时间。在80分钟的消化，切开心脏顶点冲洗掉可能被困心室内的任何单元格，并收集这些细胞在试管A6和B6，respe沉着应对。 注意:有时心脏就会脱落之前的90分钟时间点套管。如果发生这种情况，检索心脏和地方进入无菌35毫米培养皿填充用2ml消化溶液。切开心脏的顶点，并轻轻漂洗消化解决方案的心脏。接着，通过无菌100微米的细胞滤网过滤的2ml消化溶液到50毫升锥形，并加入到管A6或B6。 </li><li>随后管（A2和B2）的每个自旋后，加入再悬浮的血管平滑肌细胞到先前收集管（组合A1和B1）。另外，注意不要让泵消解液用完了，整个切换消化必要的10毫升注射器。 </li><li>所有管进行组合后，离心富VSMC-悬浮液在89×g离心10分钟。 </li><li>从收集管尽可能吸出尽可能多的上清液。悬浮颗粒残留在2毫升电镀媒体和板在一次35 100mm培养碟。地方培养皿我N 37℃培养箱中24小时。 </li></ol> 4.细胞培养注:完成生物安全柜中的剩余步骤。 <ol><li> 24小时后，轻轻地吸出镀介质。培养通常会有大量的碎片。洗涤细胞，加入2毫升温，无菌PBS和而在顺时针运动旋转轻轻拍打菜针对两个手指。 </li><li>旋转360°后，吸出PBS，重复第二次。吸出PBS，并添加另外2毫升无菌，温暖的PBS。检查在显微镜下的培养皿，以确保有一点要上盘无杂物。细胞融合，也可以在此时确定的。 </li><li>如果碎片留在培养皿中，重复步骤4.2。如果培养皿是明确的碎片，吸出PBS并加入2毫升温镀媒体。 </li><li> 24小时后，用温，无菌PBS再次洗涤细胞以除去从培养的任何最终碎片或死细胞。吸出PBS和p花边对细胞另外2毫升温镀媒体。 </li><li>每天检查细胞和饲料每48小时用新鲜的温热镀介质。在这点上，细胞可以用于标准细胞培养测定。血管平滑肌细胞应该是大约80％汇合4-5天电镀后，直到该过程被完善尽管可能需要长达7天。如果拆分到后续段落，最多使用1:435毫米菜（或到培养皿相当的区域）。 </li></ol>

<ol>
	<li>Katz, P. 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=Coronary+arterioles+in+type+2+diabetic+(db/db)+mice+undergo+a+distinct+pattern+of+remodeling+associated+with+decreased+vessel+stiffness.">Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct pattern of remodeling associated with decreased vessel stiffness.</a> Basic Res Cardiol. 106 (6), 1123-1134 (2011).</li><li>Husarek, K. 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=The+angiotensin+receptor+blocker+losartan+reduces+coronary+arteriole+remodeling+in+type+2+diabetic+mice.">The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice.</a> Vascul Pharmacol. , (2015).</li><li>Teng, B., Ansari, H. R., Oldenburg, P. J., Schnermann, J., Mustafa, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+coronary+endothelial+and+smooth+muscle+cells+from+A1+adenosine+receptor-knockout+mice.">Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.</a> Am J Physiol Heart Circ Physiol. 290 (4), H1713-H1720 (2006).</li><li>Oldenburg, P. J., Wyatt, T. A., Sisson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+attenuates+contraction+of+primary+cultured+rat+airway+smooth+muscle+cells.">Ethanol attenuates contraction of primary cultured rat airway smooth muscle cells.</a> Am J Respir Cell Mol Biol. 43 (5), 539-545 (2010).</li><li>Xu, 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=Intracellular+two-phase+Ca2++release+and+apoptosis+controlled+by+TRP-ML1+channel+activity+in+coronary+arterial+myocytes.">Intracellular two-phase Ca2+ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes.</a> Am J Physiol Cell Physiol. 304 (5), C458-C466 (2013).</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Owens, G. K., Kumar, M. S., Wamhoff, B. R. <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+vascular+smooth+muscle+cell+differentiation+in+development+and+disease.">Molecular regulation of vascular smooth muscle cell differentiation in development and disease.</a> Physiol Rev. 84 (3), 767-801 (2004).</li><li>Li, L., Miano, J. M., Cserjesi, P., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SM22+alpha,+a+marker+of+adult+smooth+muscle,+is+expressed+in+multiple+myogenic+lineages+during+embryogenesis.">SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis.</a> Circ Res. 78 (2), 188-195 (1996).</li><li>Santiago, J. 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=Cardiac+fibroblast+to+myofibroblast+differentiation+in+vivo+and+in+vitro:+expression+of+focal+adhesion+components+in+neonatal+and+adult+rat+ventricular+myofibroblasts.">Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts.</a> Dev Dyn. 239 (6), 1573-1584 (2010).</li><li>Fisher, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+phenotypic+diversity+and+function.">Vascular smooth muscle phenotypic diversity and function.</a> Physiol Genomics. 42A (3), 169-187 (2010).</li><li>Archer, S. 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. W., Hort, W., Lichti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diameter+of+coronary+arteries+in+36+species+of+mammalian+from+mouse+to+giraffe.">Diameter of coronary arteries in 36 species of mammalian from mouse to giraffe.</a> Basic Res Cardiol. 79 (2), 199-206 (1984).</li></ol>

作者宣称，他们没有竞争的经济利益。

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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
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</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 ...

isolation-of-murine-coronary-vascular-smooth-muscle-cells

Research

JoVE Journal

Biology

14.0K 次观看.  The Ohio State University College of Medicine. 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。 

视频: Isolation of Murine Coronary Vascular Smooth Muscle Cells

Focal Ca2+ Transient Detection in Smooth Muscle

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+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

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+ ...

科研

生物学

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

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 and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

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

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

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 ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

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 ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

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

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

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 ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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 of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

小鼠主动脉夹层损伤:高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

鼠冠状动脉血管平滑肌细胞的分离

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