Name: scratch migration assay and dorsal skinfold chamber for in vitro and in vivo analysis of wound healing
Uploaded: 2019-09-26T13:00:00
Description: Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

我们感谢佩特拉·韦斯伯格博士和卫生院SPF动物设施的转基因单位（SFB 894项目P2）和洪堡萨尔兰大学医学院临床和实验外科研究所的动物设施。我们感谢安德里亚斯·贝克博士对手稿的批判性阅读。这项研究由德国金融管理局（DFG）Sonderforssssbereich（SFB）894资助，A3项目对A.B.和V.F.）。

所有实验程序均根据道德条例和萨尔兰大学和萨尔兰大学的动物福利委员会获得批准和实施。
1 原细胞培养和siRNA转染
注：在所述方法中，使用初级成纤维细胞。这些细胞在伤口愈合和组织重塑中起着至关重要的作用。在这个实验中，编码高压门控钙通道12的Cav_3亚单元的Cacnb3基因被降压，从而显示出它在体外细胞...

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在这份手稿中，我们描述了体外和体内伤口愈合测定，并关联了获得的结果。对于体外测定，我们使用原位小鼠成纤维细胞4，14，15，在伤口愈合和组织重塑11中发挥重要作用。其他作为单层生长的粘附细胞类型（例如，上皮细胞、内皮细胞、角蛋白细胞）也可用于。电镀相同数量的活细胞和健康细胞，并在?...

皮肤伤口愈合在皮肤损伤后立即开始，以恢复皮肤的完整性，并保护生物体免受感染。伤口愈合过程经历四个重叠阶段;凝固，炎症，新的组织形成，和组织重塑1。在这些阶段，细胞迁移至关重要。炎症细胞、免疫细胞、角蛋白细胞、内皮细胞和成纤维细胞在不同的时间点被激活并侵入伤口区域2。研究体外和体内伤口愈合的方法不仅对了解其基本机制非常感兴趣，而且对测试新药和开发旨在改善和加速皮肤伤口愈合的新策略都具有重大意义。
要监视和分析单元格迁移，可以使用临时迁移分析。它通常被称为体外伤口愈合测定。此方法需要细胞培养工具3。这是一个简单的程序，不需要高端设备，大多数细胞生物学实验室都可以进行检测。在此测定中，通过汇体细胞单层的机械破坏（优选上皮或内皮状细胞或成纤维细胞）的机械破坏而创建无细胞区域。划痕边缘的单元格将迁移以重新填充创建的间隙。随时间减少的无细胞区域的量化类似于迁移速率，并指示细胞需要缩小间隙的时间。为此，研究人员可以使用WT小鼠的急性分离细胞或缺乏4基因的小鼠，或从可靠的细胞储存库获得的永生细胞。划痕测定允许研究药理活性化合物的影响或转染的cDNA或siRNA对细胞迁移的影响。
在体内，伤口愈合是一个复杂的生理过程，需要不同的细胞类型，包括角蛋白细胞、炎症细胞、成纤维细胞、免疫细胞和内皮细胞，以便尽快恢复皮肤的物理完整性1.在过去的5、6、7、8，研究体内伤口愈合的不同方法已经开发并使用了。本文中描述的背皮折叠室以前用于伤口愈合测定9。它用作小鼠的改性背皮折叠室制剂。改性皮肤折叠室模型具有几个优点。1） 它最大限度地减少皮肤收缩，这阻止观察伤口愈合过程，并可能影响小鼠的伤口修复。2） 本室使用玻璃盖玻片覆盖伤口，减少组织感染和干燥，这可能延迟愈合过程。3） 血流量和血管化可以直接监测。4）它允许重复局部应用药理活性化合物和试剂，以治疗伤口和加速愈合9，10。
我们使用两种独立的方案，即体外划痕迁移测定和体内背皮折叠室模型，将高压门控钙通道（Cav_3）的β3亚单位确定为影响皮肤伤口愈合的潜在靶分子。对于体外测定，我们使用原发性成纤维细胞，这些细胞确实表达了编码Cav_3蛋白的Cacnb3基因，但缺乏去极化引起的Ca2+流入或电压依赖的Ca2+电流。我们描述了Cav_3在这些成纤维细胞中的新功能：Cav_3与肌醇1，4，5-三磷酸盐受体（IP3R）结合，并约束内质神经质的钙释放。在小鼠中删除Cacnb3基因可提高IP3R对IP3的敏感性，增强细胞迁移和增加皮肤伤口修复4。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9 % NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD Plastipak</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Kai Industries</td>
			<td>48201</td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Cacnb3 Mouse siRNA Oligo Duplex (Locus ID 12297)</td>
			<td>Origene</td>
			<td>SR415626</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilation cream</td>
			<td></td>
			<td></td>
			<td>any depilation cream</td>
		</tr>
		<tr>
			<td>Dexpanthenol 5% (BEPANTHEN)</td>
			<td>Bayer</td>
			<td>3400935940179.00</td>
			<td>(BEPANTHEN)</td>
		</tr>
		<tr>
			<td>Dihydroxylidinothiazine hydrochloride (Xylazine)</td>
			<td>Bayer Health Care</td>
			<td></td>
			<td>Rompun 2%</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco by life technologies</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco by life technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexagon full nut</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Zoetis</td>
			<td></td>
			<td>KETASET</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Keyence, Osaka, Japan</td>
			<td>BZ-8000</td>
			<td>Similar microscopes might be used</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778075</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Home-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>plan apo 10x/0.45</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco by life technologies</td>
			<td>51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene sutures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screwdriver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skin disinfectant (octeniderm)</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td>118212</td>
			<td></td>
		</tr>
		<tr>
			<td>Slotted cheese head screw</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap ring</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap ring plier</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope with camera</td>
			<td>Leica</td>
			<td>Leica M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium frames for the skinfold chamber</td>
			<td>IROLA</td>
			<td>160001</td>
			<td>Halteblech M</td>
		</tr>
		<tr>
			<td>Wire piler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

scratch migration assay and dorsal skinfold chamber for in vitro and in vivo analysis of wound healing

受损的皮伤愈合是糖尿病患者和老年人的主要关切，需要有效的治疗。适当的体外和体内方法对于确定新的靶分子以进行药物治疗以改善皮肤伤口愈合过程至关重要。我们确定电压门控钙通道（Cav_3）的β3亚单位是影响伤口愈合的潜在靶分子，在两个独立的测定中，即体外划痕迁移测定和体内背膜皮肤折叠室模型。从野生型 （WT） 和 Cav+3 缺陷小鼠 （Cav+3 KO） 急性分离的原源小鼠胚胎成纤维细胞 （MEF） 或从使用 siRNA 治疗的 WT 小鼠急性分离成纤维细胞，以降低Cacnb3基因的表达，使用编码 Cav=3。对汇体细胞单层进行了划痕，然后通过在规定的时间点拍摄微量图像，直到迁移细胞完全重新填充间隙。对这些图像进行了分析，并为每个情况确定了细胞迁移率。在体内测定中，我们在WT和Cav_3 KO小鼠上植入了背皮折叠室，应用了直径为2毫米的圆形伤口，用玻璃盖玻片覆盖伤口，以保护伤口免受感染和干燥，并监测宏观伤口闭合随着时间的推移。在Cacnb3-基因缺乏小鼠中，伤口闭合速度明显加快。由于体内和体外测定的结果关联性良好，因此，在验证体外伤口愈合模型对体外命中之前，体外测定法可能对高通量筛选有用。我们在这里展示的野生型和Cav=3缺陷小鼠或细胞可能也适用于Cav_3以外的特定分子。

划痕测定是在野生型和β3缺陷MEF的汇合细胞单层上进行的（图1c）。使用 200 μL 移液器尖端执行"划痕"后，两种基因型的细胞迁移到划痕区域并缩小间隙。图像是在 6、10 和 30 h 之后拍摄的 （图 1a）。细胞迁移按百分比（%）在执行划痕后 6 小时通过迁移单元格重新填充的划痕区域。迁移 Cav_3 缺陷 MEF 比野生型小鼠的 MEF 关闭划?...

Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

在这里，我们提出了使用原纤维细胞进行体外划痕测定和小鼠体内皮肤伤口愈合测定的协议。这两种检测都是评估体外和体内伤口愈合的直截了当的方法。

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

叛乱分子需要治疗皮肤伤口。为了识别药物治疗的目标分子，需要体外和体内测试系统。适当的系统是体外划痕测定，和体内后皮褶皱室。两者都是直接程序，监测细胞迁移和皮肤伤口愈合，在没有和存在药理活性化合物。虽然这两种技术都是直截了当的，但调查人员应该练习划痕应用。并进行后体皮肤折叠室植入，并损伤获得可靠且可重复的结果。手术的演示将由临床和实验外科研究所的外科医生和教授马蒂亚斯·拉施克博士完成。在开始该过程之前，使用超细永久标记来标记每个细胞类型一个六井板，每个井的底部有一条水平线来描绘每个培养物的划痕区域。下一盘原纤维爆炸从野生型和β3缺陷小鼠分离，在六井板中，每井浓度为5倍10至第五纤维爆炸。在2mL的DMEM辅以10%的胎儿牛血清。用细胞类型、基因型和日期标记板。将盘子放在细胞培养箱中24小时。第二天早上，将每根脂质的转染试剂加入9微升，加入每个管中含有150微升的微离心管，减少血清介质。将 1.5 微升 siRNA 添加到第二个微离心管中，每个转染试剂中每管含有 150 微升的减少血清介质。将每个 siRNA 溶液与单管转染试剂溶液混合。短暂涡旋后，将混合物在21摄氏度下放置5分钟。在孵育过程中，请小心地更换每一个井中的超生。井边有2.25mL的新鲜培养介质。在孵育结束时加入250微升的合适siRNA转染试剂混合物，将智慧滴入每个相应的细胞井中。将板返回细胞培养箱72小时。当细胞达到100%汇合时，丢弃每一个井的培养上能。并使用 200 微升移液器尖端在汇合单元单层中产生划痕。垂直于每一个井底部的标记水平线。然后小心冲洗每个受伤的井2次，每次洗涤2mL的PPS。从受损的细胞、松动的细胞或碎屑中去除任何释放的因素。第二次洗涤后，加入2mL的新鲜细胞培养培养，并辅以1%或10%的血清到每个井。并在光学显微镜的舞台上捕捉每个板的伤口图像。刮到 10 倍放大倍率后立即。然后将板返回细胞培养箱。继续在未来 30 小时的适当实验时间点捕获图像。在实验结束时，在6、10和30小时的培养后量化初始无细胞区域和剩余区域。确定迁移单元格相对于初始暂存区域重新填充的暂存区域的百分比。在确认对趾捏反应不足后，在野生型或贝塔三淘汰C57黑色六只鼠标后，小心剃光头。在动物的眼睛上涂抹软膏。涂抹脱毛霜，消除任何剩余的头发。10分钟后取出奶油。要准备钛合金腔室，请用螺母固定对称钛腔架的一部分。在腔室的两个对称部分之间保持 400 到 500 微米。避免皮肤中血囊的压缩。用70%乙醇对裸露的皮肤进行消毒。在光源前抓住一叠皮肤。将双层皮肤的中间线放置到钛室的植入位置。用聚丙烯缝合线将皮肤褶皱固定。拧紧金属架上缝合线的另一侧，以抬起折叠的皮肤。并调整机架的高度，让鼠标舒适地坐着。用聚丙烯缝合钛室的第一帧，其优越的边缘，以背皮褶皱的背面。重新确认后，使用细剪刀在皮肤上做小切口。并通过皮肤褶皱的底座将连接到钛室前半部分的两个连接螺钉。将鼠标从机架上取下，然后将动物放在横向位置。将钛合金室的后半部分放在 3 个连接螺钉的顶部。并手动施加轻微压力，通过这些螺钉穿过钛框架的后半部分。折叠的皮肤层现在夹在钛框架的两个对称的两半之间。然后用不锈钢螺母固定两个对称部件。使用标准化的 2mL 直径活检冲孔，在皮肤折叠室的观察窗口内标记皮肤中心的伤口区域。并使用精细钳子和剪刀去除标记内的整个表皮和真皮。创建圆形伤口。用 0.5mL 的消毒盐水清洁 3 个半到 4 米半的方形伤口。用玻璃盖玻片盖住伤口使用钛室上的扣环钳将盖玻片固定到位。并立即将鼠标转移到立体显微镜的成像阶段。然后以 40 倍的放大倍率对伤口进行成像。将鼠标放入带监控的保持架之前，直到完全恢复。如此证明，使用 200 微升移液器尖端创建划痕后，在此代表性实验中，来自两个基因型的细胞迁移到划痕区域并缩小了间隙。然后，在执行划痕后 6 小时通过迁移单元格重新填充的填充单元格迁移的百分比将单元格迁移量。例如，在这个实验中迁移 Cav beta 3 缺陷纤维爆炸关闭的划痕区域明显早于野生型小鼠的纤维爆炸。为了确认在Cana beta 3缺陷纤维爆炸中观察到的Cana beta 3依赖效应，野生型纤维爆炸与siRNA一起转染，以调节Canaβ3蛋白。使用 Cav beta 3 特定 siRNA 处理的光纤爆炸的行为类似于 beta 3 缺陷光纤爆炸。为了比较两种基因型之间的皮肤伤口愈合，皮肤折叠室的伤口区域在受伤后被直接拍摄。在接下来的两周里，伤口被成像了。伤口的大小在图像和伤口区域上测量，在给定的一天，表示为初始伤口面积的百分比。正如预期的那样，与野生类型对控相比，Cav beta 3缺陷小鼠的伤口闭合率有所提高。确保对移液器尖端施加相等的压力，对每个划痕，并尽可能将划痕与板底上的标记线相匹配。在愈合过程中，可以随时打开后体皮肤折叠室的观察窗口。允许不同药理活性化合物的局部应用。也可以在治疗过程的不同阶段切除受伤区域。用于分子蛋白生化或组织学分析。

Research

JoVE Journal

Medicine

Murine Model of Wound Healing

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

科研

医学

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

在背皮褶商会鼠标的缺血性组织损伤：一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...