Name: stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ
Uploaded: 2020-05-08T12:30:00
Description: Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In

这项工作得到了西班牙经济与竞争部（RTC-2014-2626-1至JE）和卡洛斯三世健康研究所（PI15/01458至JE）的资助。EC得到了Atracción de Talento Investigador拨款2017-T2 / BMD-5766（马德里市和UAM）的支持。

所有小鼠饲养和实验程序必须符合当地、国家、国际立法和动物实验指南。
1.诱导毛发生长，烧伤诱导和识别尾部皮肤上皮整体中的长期BrdU LRC
注意：使用10天或7周大的C57BL / 6小鼠，最好是同窝小鼠，用于下面描述的实验设计。在所有实验程序中，动物将通过3%异氟醚吸入麻醉或通过颈椎脱位安乐死，如所示。
<ol><li>在休止期（休息）阶段（出生后约50?...

<ol>
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在这里，我们提出了一种方法，该方法允许在小鼠皮肤中瞬时激活体内内源性ROS产生，并具有生理效应。该方法基于光动力程序，以诱导内源性光敏剂PpIX的受控和局部刺激（图1B）。这种实验方法是在体内实验系统中研究ROS生物学的有趣工具，与使用外部ROS源（通常是过氧化氢）的方法相比，该方法取得了重大进展，并允许在组织/样品中受控和局部产生ROS。
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活性氧（ROS）是分子氧化学还原形成水的结果，包括单线态氧、超氧阴离子、过氧化氢和羟基自由基1,2,3。由于其极强的化学反应性，ROS的寿命非常短。在需氧生物中，ROS是在线粒体中有氧呼吸（电子传递链）的主要泄漏副产物在细胞内偶然形成的。细胞中高水平的ROS的瞬时积累导致氧化应激条件，可能导致蛋白质，脂质和糖的不可逆失活，并在DNA分子中引入突变2,3,4,5。细胞，组织和整个生物体中氧化损伤的逐渐积累随着时间的推移稳步增加，并且与细胞死亡程序，几种病理和衰老过程的诱导有关2,3,4,6。
需氧生物已经稳步进化出有效的分子机制，以解决细胞和组织中过量的ROS积累。这些机制包括超氧化物歧化酶（SOD）蛋白家族的成员，其催化超氧化物自由基歧化为分子氧和过氧化氢，以及不同的过氧化氢酶和过氧化物酶，它们使用抗氧化剂库（谷胱甘肽，NADPH，过氧化物还原蛋白，硫氧还蛋白7,8）催化随后过氧化氢转化为水和分子氧。
然而，一些报告支持ROS作为调节关键细胞功能的分子回路的关键组成部分的作用，包括增殖，分化和迁移2,3,4。这一概念得到了需氧生物中专用ROS产生机制的初步鉴定和表征的进一步支持，包括脂氧合酶环氧合酶和NADPH氧化酶9,10。从这个意义上说，ROS在脊椎动物胚胎发育过程中表现出积极作用11,12,13，并且这些分子在调节特定体内生理功能中的关键作用已在不同的实验系统中报道，包括果蝇14中造血祖细胞的分化程序，斑马鱼的愈合诱导或非洲爪蟾蝌蚪的尾部再生15.在哺乳动物中，ROS参与了神经球模型中神经干细胞的自我更新/分化潜力16，以及结直肠癌起始期间肠道干细胞功能的失调17。在皮肤中，ROS信号传导与表皮分化以及皮肤干细胞生态位和毛囊生长周期的调节有关18,19。
从这个角度来看，确定ROS在正常或病理条件下在生物系统中的生理作用的一个主要实验限制是缺乏足够的实验工具来诱导细胞和组织中这些分子的受控产生，准确地类似于它们作为第二信号信使的生理产生。目前，大多数实验方法涉及外源性ROS的施用，主要以过氧化氢的形式。我们最近实施了一种实验方法，以在小鼠皮肤中开启内源性，非致命的内源性ROS的体内产生，基于内源性光敏剂原卟啉IX的前体（PpIX;例如氨基拉伏林酸或其甲基衍生物甲氨基乙酰丙酸）的施用，并用红光进一步照射样品以诱导细胞内分子氧原位形成ROS（图1).这种光动力程序可以有效地用于刺激常驻干细胞生态位，从而激活组织的再生程序19,20，并为皮肤再生医学中的新治疗方式开辟道路。在这里，我们提供了该方案的详细描述，显示了刺激干细胞生态位的代表性示例，测量为毛囊19,21的凸起区域中长期5-溴-2'-脱氧尿苷（BrdU）标记保留细胞（LRCs）数量的增加，以及随后由瞬态诱导的再生程序的激活（头发生长和烧伤愈合过程的加速）， C57Bl6小鼠品系皮肤中非致命ROS产生。

<table border="0" cellpadding="0" cellspacing="0" style="width:812px;" width="811">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">D6883-50MG</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5&#39;-bromo-2&#39;-deoxiuridine</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">B5002-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Anti-Bromodeoxyuridine-Fluorescein</td>
			<td style="width:104px;">Roche</td>
			<td>11202693001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream (e.g., Veet)</td>
			<td style="width:104px;">Veet</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Dihydroethidium</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">In Vivo imaging system, e.g., IVIS Lumina 2</td>
			<td style="width:104px;">Perkin Elmer</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td>
			<td style="width:104px;">Galderma</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Power energy meter (e.g., ThorLabs Model PM100D)</td>
			<td style="width:104px;">ThorLabs</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Red light source, e.g., 636 nm Aktilite LED lamp</td>
			<td style="width:104px;">Photocure ASA</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ

在这里，我们描述了一种在小鼠皮肤中诱导内源性活性氧（ROS）的可切换体内光生的方案。这种原位ROS的瞬时产生有效地激活了干细胞壁龛中的细胞增殖，并刺激组织再生，这通过加速烧伤愈合和毛囊生长过程得到了强烈体现。该方案基于可调节的光动力处理，该处理用内源性光敏剂原卟啉IX的前体处理组织，并在严格控制的物理化学参数下进一步用红光照射组织。总体而言，该协议构成了分析ROS生物学的有趣实验工具。

在小鼠背部和尾部皮肤中局部施用mALA前体导致PpIX在整个组织中显着积累，并且显着地在毛囊中，如该化合物在蓝光（407nm）激发下的红粉红色荧光所证明的那样（图2A，C）。随后以2.5−4J / cm 2的通量用红光（636nm）照射处理的组织，促进组织中ROS的瞬时产生，特别是在毛囊的凸起区域（图2B，D）。
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Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In 

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

该协议的目的是诱导小鼠皮肤中瞬时体内产生非致死水平的活性氧（ROS），进一步促进组织中的生理反应。

通过可切换原卟啉IX依赖性原位活性氧物种光生成刺激小鼠皮肤中的干细胞壁龛和组织再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

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Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

在蝾螈肢体再生过程中通过全皮瓣手术抑制伤口表皮的形成

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...