Name: an improved method for the preparation of type i collagen from skin
Uploaded: 2014-01-21T22:00:00
Description: Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

这项工作是由美国国立卫生研究院（HL068915到DBC），从长尾研究基金（以CAP）新研究员奖的研究经费，格兰特在急救来自美国心脏协会（12GRNT11910008到DBC）的支持从儿童心脏基金会（以DBC），并捐赠给波士顿儿童医院心脏传导基金，瑞恩家庭养老和由大卫普尔曼的研究经费。

在这里，我们将展示COL1从羔羊皮隔离。作为写入，该协议也可以用来成功地从兔和人的皮肤隔离COL1。 1。准备皮肤样本<ol><li>将所有溶液平衡至4℃后才能使用。 </li><li>冲洗皮肤样本（25-50克）在冰冷的DH 2 O和删除任何羊毛，毛皮，或头发脱毛膏。 </li><li>使用单刃刀片刮结缔组织和脂肪的样品净化。 </li><li>冲洗在冰冷的DH 2 O样品</li><li>切片皮肤样品切成1厘?...

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这种方法的一个关键组成部分是真皮样品的反复有效的压缩，去除多余的液体。关键是通过压缩样本以除去尽可能多的醋酸钠尽可能在步骤2.5。我们用一个抹刀压实对管侧的皮肤，但是，也可以采用粗棉布，虽然这将须除去样品从管中，并增加来执行这些步骤所需的时间。同样地，以压缩的样本，在步骤3.1，使柠檬酸钠的量是在随后的提取步骤准确是重要的。未能充分从在上述任何步骤除去样品?...

几十年来，研究人员和商业厂商已经分离出溶解的COL1从组织来源，包括皮肤和腱使用简单的酸提取协议接着进行中和，这导致组织COL1原纤维基质的重悬浮，可用于某些变异的分类为多种生物医学应用1-4。虽然有对COL1临床应用的例子很多，少数这些采用autologously衍生COL1因为准备这种蛋白质的需要冗长的提取需要几天或几周进行5-7。因此，研究调查员和医师通常使用COL1的正在使用非人组织如鼠，牛或猪的真皮或皮肤用从尸体取出或以下包皮环切制备昂贵，商业制剂。对于再生应用，这些产品很多变化表现出对于自己的能力，形成固体矩阵或能够支持细胞粘附和生长的组织结构。因此，存在一个明显的需要，它能够快速从方便和丰富的自体组织源中提取COL1一个新的标准化方法。 这种方法可以节省时间和金钱在研究环境，让COL1可以从感兴趣的动物模型中提取并用于组装的胶原蛋白为基础的脚手架工程化组织的临床前试验。与此同时，具有在临床使用迅速隔离，autologously衍生COL1的选项会增加，否则将接收同种异体或异种制剂的患者的整体安全性。用于接收自体制剂的患者，这个简化的方法可大大减少活检收集和胶原蛋白应用程序之间的时间间隔。基于这些原因，我们试图通过使用高速AG改善后一个历史悠久的COL1分离和提纯方法itation和尺寸排阻离心。这种方法简单执行使用标准缓冲液和设备在许多实验室中找到。通过采用高速搅拌，我们的协议减少所需的时间来分离可溶性的，真皮COL1从约10天到小于3小时8。重要的是，这种方法可以容易地执行在临床上以制备autologously衍生COL1用于在一组程序中的患者使用。

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

可溶性Ⅰ型胶原（COL1）被广泛地用作用于细胞培养的粘合剂基体和作为再生的应用程序的蜂窝式脚手架。临床上，这种蛋白是广泛用于美容手术，经皮注射，骨移植术，和重建手术。 COL1对这些程序的来源通常是非人，这增加了炎症和排斥反应的可能性，以及外源性疾病的传播。鉴于此，一种方法能够有效地和快速地从数量autologously衍生组织有限净化COL1会规避很多这样的问题，但是，标准隔离协议是漫长的，往往需要大量的胶原组织。这里，我们表明一个有效的COL1的提取方法，可降低从约10天分离并纯化该蛋白为小于3小时的时间。我们在许多外科手术的选择，因为它的可用性真皮作为我们的组织来源。 Ŧ他的方法是使用传统的提取缓冲液结合有力的搅拌和离心过滤从少量真皮的获得高纯度的可溶性COL1。简单地说，皮肤活检进行彻底的冰冷的DH 2 O去除脂肪，结缔组织，和头发后清洗。将皮肤样品剥离用0.5M的乙酸钠和一个高速台式匀浆非胶原蛋白质和多糖的。从残余的固体胶原随后与使用匀浆机以0.075M的柠檬酸钠缓冲液提取。这些提取物是使用100,000兆瓦截止离心过滤器能产生可比较的或优质的商业产品或那些使用传统方法得到的COL1制剂纯化。我们预计这种方法将有助于autologously衍生COL1的为众多的研究和临床应用的利用率。

这COL1隔离协议需要大约2小时，15分钟即可完成。图1示出的示意图，概述在此过程中的主要步骤。制备样品并进行高速鼓动的7个循环和漂洗用乙酸钠约需35分钟（图1A-C）。执行一个搅拌和冲洗周期与DH 2 O大约需要5分钟（图1D和1E）。添加柠檬酸钠和试样施加1搅拌和冲洗循环后搅拌6个周期需要大约45分钟（图1F和1G）。从提取液...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

对于I型胶原蛋白的制备皮肤方法的改进

对于可溶性Ⅰ型胶原（COL1）隔离的传统程序需要从开始10天左右，由于长时间的缓冲液孵育和纤丝费力再悬浮完成。在这里，我们描述了从真皮小活检净化COL1，在不到3小时的方法。

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Research

JoVE Journal

Bioengineering

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

在压缩的胶原组织糜：含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...