Name: isolation of primary myofibroblasts from mouse and human colon tissue
Uploaded: 2013-10-12T19:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

这项工作是由卫生部资助5KO8DK085136-02 JY国家机构的支持。

1。获得结肠组织鼠标的协议进行动物实验，详细介绍了基本原理和研究的目的，已提交并经我们的机构动物研究监督处。 <ol><li>安乐死鼠标与吸入异氟醚后颈椎脱位。 </li><li>使下腹正中切口，收回小肠路程，确定结肠。收回膀胱和子宫（如果存在的话）横向和识别耻骨，切细剪刀，露出直肠。 </li><li>解剖直肠背侧肠系膜断结肠，动员结肠从肛门至盲肠?...

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无论是使用鼠标或结肠癌时，通用的技术是相似的。这种技术也可以用于从小肠分离成肌纤维细胞。具体细节，肌纤维母细胞从1隔离。小鼠和2。人结肠癌在下面讨论。 1。小鼠结肠癌小鼠结肠灌溉是通过附加一个4，为16G波普尔钝头针头至结肠的一端提供便利。这也有助于纵向切割结肠时。您可以手风琴结肠组织到针，然后将剪一个尖与尖底起来，滑过剪?...

胃肠道（GI）道功能的调节涉及粘膜层与下伏间质之间复杂的，动态的相互作用。这些相互作用通常用来维持内环境稳定，但也可能导致疾病状态的发展病理条件下，1。肌成纤维细胞是位于下方的所在，以旁分泌方式进行通信及邻近细胞亚群调节若干关键胃肠道工序，包括粘膜再生，修复和纤维化2的上皮细胞层基质细胞亚群。 的18 CO细胞株是已被广泛用于研究肌成纤维细胞功能的人结肠肌成纤维细胞系的一个例子，因为它有许多共同的初级原位成肌纤维细胞的特性。这些包括的α-平滑肌肌动蛋白的蛋白表达（α-SMA）和波形蛋白，以及一个数的表达式细胞表面受体如表皮生长因子受体，或受体的溶血磷脂酸3,4。因为这些共享超微结构特征，以及在生物功能5的相似性，则18 CO细胞系已被广泛用于研究肌成纤维细胞的功能在许多疾病状态如炎性肠疾病或结肠直肠癌3,6的范围内。但是，也有使用的细胞系时固有的局限性和关注。这些包括基因型不稳定随着时间的推移区别于原代细胞的细胞系，从而改变细胞的表型和生物学功能，包括生长速率，并与其他细胞群的相互作用。细胞系也缺乏GI微环境（上皮，基质和血管成分），这是一个主要的限制其使用的正常组成部分。因此，从实验细胞株的研究得出的结论需要进一步的验证，以降低RISK误解。 初级肌成纤维细胞可以从人类和小鼠的结肠组织得到证实在细胞系7识别的实验结果。该方法最初是由马希达， 等。8描述的，和我们的协议是可以被用来从小鼠和人结肠9隔离初级肌成纤维细胞的许多良好描述的技术之一。对于结肠疾病的任何小鼠模型，这种技术可以用来隔离初级肌成纤维细胞，研究它们与邻近的细胞是如何相互作用或促使正常或病理性的GI处理10,11。这种技术可以用来研究肌纤维母细胞如何促进黏膜愈合，纤维化和结直肠腺瘤和癌的发展。初级肌成纤维细胞还可以从已经在切除操作的时间为良性（炎性肠病，狭窄，憩室炎）或恶性Ç人结肠组织获得onditions。来自人类和小鼠的结肠组织分离的原代细胞可以生长在细胞培养物和利用多通道的数量有限。

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

的肌成纤维细胞是胃肠（GI）道，已获得相当大的关注在许多胃肠功能的关键作用的基质细胞。虽然一些肌成纤维细胞的细胞系是可商购的，研究这些细胞在体外 ，离暴露实验的细胞培养条件的细胞系的研究成果具有因工作过于简单化的性质固有的局限性。使用初级肌纤维母细胞提供了在确认细胞系鉴定实验结果方面有很大的优势。从动物模型隔离初级肌纤维母细胞可为肌成纤维细胞的研究更紧密地模仿所研究的疾病状态条件下。从人结肠组织主要肌纤维母细胞的分离提供了可以说是最相关的实验数据，由于细胞直接来自患者的基础疾病。我们描述了一个行之有效的技术，可以为utilized从小鼠和人的结肠组织隔离初级肌成纤维细胞。这些分离的细胞已被鉴定为α-平滑肌肌动蛋白和波形蛋白阳性的，和结蛋白阴性的，与皮下肠肌成纤维细胞是一致的。初级肌成纤维细胞的细胞可以生长在细胞培养物和用作实验过通道的数量有限。

一旦分离，原代人成肌纤维细胞可以生长在细胞培养物和使用过的通道的一个有限数量（最多通道4）。这些细胞的特征在α- ​​平滑肌肌动蛋白和波形蛋白阳性，结蛋白阴性的（ 图1A），与肠上皮下成肌纤维细胞5,7一致。他们也有一个特点星状形态（ 图1B）。初级成肌纤维细胞随后可以用来确认在细胞系中发现的实验结果。这种方法被用来证明促炎细胞因子TNF-?...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

肌成纤维细胞是胃肠道，调节重要生理过程的正常和疾病状态的一个有影响力的基质细胞。我们描述了一种技术，它允许主肌纤维母细胞从小鼠和人结肠组织，它可以用于在体外实验的隔离。

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Research

JoVE Journal

Biology

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

科研

生物学

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

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 Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...