Name: isolation and cultivation of adult rat cardiomyocytes
Uploaded: 2017-10-19T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

作者感谢 Woitasky 和彼得沃尔克的技术援助。此外, 作者还感谢 Mrs. (医学作家 ACCEDIS) 在准备手稿方面的帮助。此手稿由 DFG (Schlu 324/7-1) 资助。

这项调查是根据美国国立卫生研究院 (NIH 出版物 No. 85-23, 修订 1996) 所出版的实验室动物的护理和使用指南进行的。通常, 雄性大鼠年龄在3到4月, 平均重量为 250-350 克, 用于本协议。一只老鼠的心脏是足够为20文化菜 (1 毫升每盘; 内径:35 毫米) 与近似细胞密度 1.5 x 10 4 cells/1000 mm 2 . 
 1. 培养基和试剂的制备 <ol> <li> 肌酸-肉碱-牛磺酸培养基 (cct 培养基) ...

<ol>
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成年心肌细胞的行为在体内受许多与其他细胞的相互作用 (如: 如, 神经元, 内皮细胞, 成纤维细胞, 炎症细胞) 和电合, 他们形成1。因此, 研究成年心肌细胞的应激适应需要 ARVC 的分离和培养。分离和培养 ARVC 的主要作用是: 1) 从细胞外基质和细胞间接触;2) 将它们与收缩刺激断开;3) 迫使他们从三维组织适应 two-dimensional 环境。在这些条件下, ARVC 开始 de-and re-differentiation...

成年心肌细胞在体内的工作, 作为一个电子合的基础上细胞细胞之间的联系。此外, 它们受邻近细胞的影响, 如心脏成纤维细胞、内皮细胞、神经元和炎症细胞1。为了研究心肌细胞适应其细胞内组织改变负荷状况的能力, 心肌肥厚是导致心力衰竭的最初步骤, 成年心室大鼠的分离和培养心肌细胞 (ARVC) 是必需的2,3,4。从历史上看, 心肌细胞首先从胚胎小鸡心脏中分离出来5,6。几年后, 首次分离的终末分化心肌细胞使用钙消耗7描述。然而, 这些成年心肌细胞不能耐受钙, 因此不能用于功能性化验。最后, 在 1976年, 一项新的协议使鲍威尔和扭转在生理情况下调查成年心室心肌细胞8。作为第一步, 他们在低钙浓度下分离成年心肌细胞, 然后在逐步的过程中增加钙对生理浓度。今天, 大多数用于分离和培养成年心肌细胞的协议与此钙协议一起使用, 胶原酶用于酶消化致密细胞-细胞接触1。
为成功的耕种, 胎儿小牛血清 (FCS) 或 oncostatin M (OSM) 是需要的。ARVC 执行 de-and re-differentiation 与广泛的结构变化, 包括肌拆卸和改革9,10,11,12。这一过程伴随着重新的胎儿型基因, 如β-肌球蛋白重链 (β-MHC), 如已知的肥大, 和形成的伪足样结构, 也称为传播4,11,13. 此外, swiprosin-1 (EFHD2), 一个新发现的蛋白质, 发挥了重要作用的过程中 re-differentiation 栽培 ARVC11。结果, ARVC 在文化中转化为广泛的多态细胞, 在培养的两个到三周后自发地出现收缩2,4,14。
最近的发现表明, 心脏 re-and de-differentiation, 因为它发生在文化条件下模仿功能在心脏重塑10,15期间看到的在体内。心脏重塑是心脏疾病的一个关键过程16。由于心脏疾病仍然是工业化社会的主要死亡原因, 对成人心肌细胞生物学的更好的了解是重要的 (卫生组织; 2015)。ARVC 的分离和培养有助于制定新的治疗心脏疾病的策略和药物。通过这份手稿, 为 ARVC 的分离和培养提供了一个协议。此外, 该方法的某些关键部分在讨论部分中进行了突出显示。

<table>
	<tbody>
		<tr>
			<td>Langendorff perfusion system</td>
			<td>inhouse construction</td>
			<td></td>
			<td>double-walled with a water 
			based heating system</td>
		</tr>
		<tr>
			<td>Tissue chopper Mc Ilwain</td>
			<td>Cavey Laboratory Engeneering Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic Cannula, OD 1,8 mm</td>
			<td>inhouse construction</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Abdominal shears</td>
			<td>Aeskulap</td>
			<td>BC772R</td>
			<td></td>
		</tr>
		<tr>
			<td>Capsule forceps</td>
			<td>Eickemeyer</td>
			<td>171307</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor large</td>
			<td>Aeskulap</td>
			<td>BC562R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor small</td>
			<td>Aeskulap</td>
			<td>BC163R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mash with Polyamid</td>
			<td>Neolab</td>
			<td>4-1413</td>
			<td>mash size 200 &#956;m</td>
		</tr>
		<tr>
			<td>plastic disc</td>
			<td>Cavey Laboratory Engeneering Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Typ II</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of adult rat cardiomyocytes

在一个完整的心脏, 相邻的细胞影响成年心肌细胞。利用成人心肌细胞的分离和培养方法, 对其在特定的治疗和环境下的行为进行精确的研究是可行的。本论文提出了一种成功的成年大鼠心室心肌细胞 (ARVC) 的分离和培养方案。
大鼠在深麻醉下被宫颈脱位所牺牲。然后, 心脏被提取, 主动脉被发现。随后, 对共生灌注系统进行钙耗和胶原酶处理。随后, 心室组织得到切碎, 循环, 并筛选, 随后三离心步骤, 逐步增加 CaCl2 , 直到达到生理钙浓度。ARVC 被镀在细胞培养皿上。在对细胞培养基进行清爽后, ARVC 可在不改变含血清培养基的情况下培养六天。ARVC 的分离是一个钙敏感的过程。细胞内钙离子浓度的微小变化会导致分离电池的质量和生存能力下降。
新鲜地被隔绝的 ARVC 是杆形状。在耕种的第一天他们丢失了杆形状的形态学并且形成伪足象结构 (传播)。在这种形态学形成过程中, ARVC 最初降低其收缩元素, 然后通过肌动蛋白应力纤维和de 从头sarcomerogenesis 进行改革。经过一个星期的耕种, 大多数 ARVC 显示一个广泛的外观与一个明确的检测交叉条纹。这个过程是敏感的细胞内钙浓度, 作为治疗与 ionomycin 减弱传播。de-and re-differentiation 的关键标志是β-肌球蛋白重链 (β-MHC)、oncostatin M (OSM) 和 swiprosin-1 (EFHD2)。最近的研究表明, 在培养条件下发生的心脏 re-and de-differentiation 模仿心脏重塑过程中出现的体内的特征。因此, ARVC 的分离和培养在了解心肌细胞生物学方面起着关键的作用。

文化中的成人心肌细胞:图 1显示了最新隔离的成人心肌细胞在最后一次洗涤后2小时的概况。大约75% 的所有心肌细胞都有一个杆状形态。其余的25% 显示了一个不寻常的外观与圆形的形态学和没有可检测完整的细胞膜 (图 1)。在耕种的末端 (天 6), 15% 所有心肌细胞显示传播, 大约10% 保持在一个圆的形态学, 不用伪足象?...

Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

Isolation and Cultivation of Adult Rat Cardiomyocytes

在这里, 我们提出了一个分离和培养成年大鼠心室心肌细胞 (ARVC) 的协议。隔离 ARVC 可用于短期和长期种植。ARVC 的分离和培养可在开发新的心脏疾病治疗方案中发挥关键作用。

成年大鼠心肌细胞的分离与培养

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A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...