Name: effect of hyaluronic acid 35 kda on an in vitro model of preterm small intestinal injury and healing using enteroid-derived monolayers
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此内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。HC得到了美国国立卫生研究院P20GM134973的资助。KB由儿童医院基金会（CHF）和长老会健康基金会（PHF）资助。癌症功能基因组学核心提供的活细胞成像服务部分得到了美国国立卫生研究院国家普通医学科学研究所拨款P20GM103639和国家癌症研究所拨款P30CA225520的支持，授予俄克拉荷马大学健康科学中心斯蒂芬森癌症中心。

本研究中的所有动物程序均已获得俄克拉荷马大学健康科学中心机构动物护理和使用委员会的批准。经机构批准，在安乐死后获得来自早产非人灵长类动物（NHP，90%妊娠，橄榄狒狒， Papio anubis）的胎儿小肠便利样本，以进行另一项研究（协议#101523-16-039-I）20。
1.早产非人灵长类动物三维肠样的建立
<ol><li>培养基制备 注意：可以按照标...

<ol>
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早产儿的胃肠道因反复暴露于与生态失调、炎症性细菌代谢物和毒素以及间歇性缺氧相关的环境损害而承受持续的再生压力23，24。不幸的是，早产儿的肠上皮无法迅速建立功能完整性23，导致屏障功能障碍，肠道通透性增加，严重时，肠道炎症和NEC发展猖獗。
糖胺聚糖（GAGs）是一类在母乳中普遍存在的多糖，是?...

坏死性小肠结肠炎（NEC）是早产儿最常见的胃肠道急症之一1。该疾病的特征是严重的肠道炎症，可迅速恶化为肠道坏死、败血症和潜在的死亡。虽然病因尚不清楚，但有证据表明 NEC 是多因素的，是喂养、异常细菌定植和未成熟肠上皮2，3 复杂相互作用的结果。早产儿肠道通透性增加，细菌定植异常，肠细胞再生能力低4，5，增加肠屏障功能障碍、细菌易位和NEC发展的风险。因此，确定加速肠上皮成熟和促进肠上皮再生或愈合的策略或干预措施对于预防这种致命疾病至关重要。
研究表明，母乳（HM）对早产儿6，7，8，9，10，11具有NEC的保护作用。人类和动物研究都表明，牛配方可增加肠道通透性，对肠上皮细胞有直接毒性2，12。虽然尚未完全阐明，但有证据表明HM的保护作用是通过乳铁蛋白，免疫球蛋白A（IgA）和HM低聚糖等生物活性成分介导的13。HM还富含透明质酸（HA），这是一种独特的非硫酸化糖胺聚糖，具有重复的D-葡萄糖醛酸和N-乙酰基-D-葡萄糖胺二糖14，15。重要的是，我们已经证明口服35 kDa HA（HA35），一种HM HA模拟物，可以减轻肠道损伤的严重程度，防止细菌易位，并降低小鼠NEC样肠损伤模型的死亡率16，17。
在这里，进一步研究了HA35对 体外 肠道愈合和再生的影响。目前，用于肠道损伤和修复的最广泛使用的 体外 测定是在结直肠癌（CRC）细胞单层中进行的划痕伤口测定。这种模型与早产儿肠道的生理相关性有限，因为CRC细胞的伤口修复在很大程度上依赖于癌细胞的高度增殖性质，而不是干细胞驱动的修复过程18。为了克服这一限制，这里描述了建立2D肠样划痕伤口模型，包括从早产非人灵长类动物（NHP）中分离和维持原代干细胞来源的小肠类肠道的过程。鉴于早产NEC最常在远端小肠中报告，与使用传统结直肠单层的现有模型相比，在肠道损伤和修复模型中使用原代上皮细胞类器官提供了更具生理可转化的 体外 模型18，19。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipet</td>
			<td>Fisher Scientific</td>
			<td>13-675-49</td>
			<td></td>
		</tr>
		<tr>
			<td>100x21mm Dish, Nunclon Delta</td>
			<td>ThermoFisher Scientific</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well, TC-Treated, Flat Bottom Plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;M Reversible Cell Strainer</td>
			<td>STEMCELL Technologies</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Sterile Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>FB22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>100-0485</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte S3 Live-Cell Analysis Instrument</td>
			<td>Sartorius</td>
			<td>4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Scratch Wound Analysis Software Module</td>
			<td>Sartorius</td>
			<td>9600-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>STEMCELL Technologies</td>
			<td>06010</td>
			<td>This is HOGMY, but without the Y-27632 or antibiotics. Also used as base for HOGM, but then only missing the antibiotics.</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography</td>
			<td>Millipore Sigma</td>
			<td>L3012-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>ThermoFisher Scientific</td>
			<td>136101</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-Buffered Saline), 1X [-] Calcium, Magnesium, pH 7.4</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>This is broad-spectrum antibiotics</td>
		</tr>
		<tr>
			<td>Sodium Hyaluronate, Research Grade, HA20K</td>
			<td>Lifecore Biomedical</td>
			<td>HA20K-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Company: Bio-Rad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1X, 0.25% Trypsin</td>
			<td>Fisher Scientific</td>
			<td>MT25053CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of hyaluronic acid 35 kda on an in vitro model of preterm small intestinal injury and healing using enteroid-derived monolayers

体外 划痕伤口测定通常用于研究各种组织类型中上皮愈合的机制和特征。在这里，我们描述了一种协议，以从源自末端回肠肠隐窝的三维（3D）非人灵长类动物肠生成二维（2D）单层。然后将这些肠来源的单层用于 体外 划痕伤口测定，以测试透明质酸35 kDa（HA35）（一种人乳HA模拟物）促进细胞沿上皮伤口边缘迁移和增殖的能力。单层生长至汇合后，手动划伤并用HA35（50μg/ mL，100μg/ mL，200μg/ mL）或对照（PBS）处理。使用配备活细胞成像的透射光显微镜对细胞迁移和增殖到间隙中成像。伤口闭合被量化为使用ImageJ中的伤口愈合大小插件的伤口愈合百分比。在24小时内测量划痕面积和细胞迁移速率以及伤口闭合百分比。 HA35体 外 加速小肠样单层的伤口愈合，可能是通过伤口边缘的细胞增殖和向伤口区域的迁移的组合。这些方法有可能用作探索早产儿小肠肠道再生的模型。

HA对各种组织和器官的组织修复和伤口愈合的影响是有据可查的;然而，分子量为35 kDa的HA对胎儿或新生儿小肠愈合和再生的具体影响目前尚不清楚。为了测试HA35在胎儿或新生儿小肠模型中促进伤口愈合的能力，我们从NHP回肠组织中生成了3D肠样肠，并进一步将该组织解离成单细胞以创建2D肠衍生单层（图1A，B）。将单层生长至汇合并使用P200移液器吸头手动刮擦（...

Watch this Scientific Journal Video about Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers at JoVE.com

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

该协议描述了一种在从非人灵长类动物回肠分离的三维（3D）肠衍生的二维（2D）单层上建立和执行划痕伤口测定的方法。

透明质酸35 kDa对肠源单层早产小肠损伤体 外 模型及愈合的影响

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

该协议扩展了用于再生研究的非常常见的划痕伤口测定，代表小肠所有主要细胞类型的肠来源单层。该技术能够在肠再生的早产2D肠样单层模型中实时可视化伤口闭合。我们的技术可以应用于早产哺乳动物肠道的研究，通常使用转化的结直肠癌细胞系进行不良建模。首先在冰上解冻96微升细胞外基质或基于ECM的水凝胶BME，在2至8摄氏度的冰上过夜。以1：50的比例稀释基于ECM的水凝胶和不含钙或镁的冰冷PBS。用200微升冰冷稀释的基于ECM的水凝胶涂覆24孔组织培养板的每个孔，并将包被板在37摄氏度和5%二氧化碳下孵育至少一小时。然后从含有用于单层的3D类肠的24孔培养板中吸出培养基，并用每孔500微升细胞解离试剂替换它。通过上下移液 8 到 12 次并将 12 个孔的内容物转移到 15 毫升锥形管中，手动破坏基于 ECM 的水凝胶圆顶。用250微升细胞解离试剂洗涤12个空孔，以确保已去除所有肠样，并将内容物转移到15毫升锥形管中。将锥形管在 300g 的温度下在 4 摄氏度下离心五分钟，然后弃去上清液。向锥形管中加入 10 毫升冰冷的 DMEM/F-12 洗涤缓冲液，并通过倒置管悬浮肠样颗粒。在4摄氏度下以300g离心5分钟，然后除去上清液。将细胞沉淀重悬于12毫升37摄氏度胰蛋白酶-EDTA中，并将试管置于37摄氏度的水浴中10分钟或直到细胞看起来可溶。向每个试管中加入三毫升 DMEM/F-12 洗涤缓冲液以稀释胰蛋白酶-EDTA。倒置试管混合并放在冰上。通过37微米网状细胞过滤器过滤解离的肠。将内容物收集到干净的 15 毫升锥形管中，并在 4 摄氏度下以 300g 离心管五分钟。从锥形管中取出上清液，并用六毫升HOMGY重悬细胞沉淀。接下来，从培养箱中取出基于ECM的水凝胶涂层板，并吸出任何多余的基于ECM的水凝胶溶液，而不会划伤涂层表面或完全干燥板。将500微升合并的12毫升HOMGY细胞悬液加入涂有ECM基水凝胶的24孔板的每个孔中。用盖子旋转板以确保孔内细胞均匀分布。将板在37摄氏度和5%二氧化碳下孵育，每两天更换一次HOMGY培养基，直到单层达到大于90%的汇合度。一旦单层达到大于90%汇合度，吸出培养基以去除任何无法附着的细胞。用溶解在HOMGY中的500微升HA35或PBS处理细胞24小时。之后，在不破坏单层表面的情况下取出介质。使用200微升移液器吸头，在每个孔中单层的整个表面直径上划伤，注意不要让单层干燥。用500微升1X PBS清洗划伤的单层一次，并加入500微升溶解在HOMGY中的HA35或PBS。将板转移到37摄氏度和5%二氧化碳培养箱内的活细胞分析仪器上。在活细胞分析软件中的计划图标下，单击启动添加新容器向导图标。选择按计划扫描，然后单击下一步。在创建容器下选择新建并单击下一步，然后在扫描类型下选择整井并单击下一步。为图像通道选择相位，为物镜选择4倍。从提供的列表中选择印版制造商和目录号，然后单击下一步。选择一个空容器位置，然后选择所需的扫描模式。在“名称”中输入板名称，然后选择加号以创建板布局。选择“小加号”以创建治疗名称。输入短名称，然后输入确定。突出显示与该处理相对应的板孔，并点击大加号以指定具有该处理的孔。输入化合物浓度，然后单击确定。板完成后，选择“确定”、“下一步”。选择“将分析推迟到以后”，然后选择“每隔 4 小时创建扫描的新计划”。在一天零小时停止扫描。确保摘要正确，然后添加到计划。在活细胞分析软件中，选择屏幕顶部的“查看”。从“容器名称”菜单中，导航到已完成的 24 小时扫描，然后双击容器名称。选择左侧的导出图像和动画图标，然后选择显示的选项。突出显示感兴趣的井，然后选择感兴趣的时间点和显示的图像系列。确保序列类型和扫描正确。选择所需的目标文件夹位置。从下拉菜单中选择 JPEG，然后选择导出。打开 ImageJ 软件并上传划痕伤口测定图像。选择“图像”，然后键入 8 位以将图像从 24 位 RGB 更改为图像。选择插件、宏、安装Wound_healing_size插件以安装伤口愈合大小插件。之后，将方差窗口半径设置为 20，阈值设置为 100，饱和像素百分比设置为 0.001。是 对于设置全局比例，然后单击确定。验证轮廓区域是否为伤口区域，并重复其他划痕。最后，使用屏幕上显示的方程式计算24小时内迁移或增殖细胞的划痕区域伤口愈合百分比。此处，T0 是零小时的区域，TC 是零小时、4 小时、12 小时或 24 小时的区域。这些图像显示了肠样生成和肠源性单层伤口愈合测定程序。三维非人灵长类肠的培养物用于解离成二维单层。将单层在24孔板中生长至大于90%汇合度，然后用HA35或PBS处理24小时。然后用P200移液器吸头刮擦单层。这里显示的是HA35在非人灵长类肠样单层对照治疗中的代表性图像。在使用P200移液器吸头进行划痕后的零小时，4小时，12小时和24小时获得图像。此处显示了HA35对伤口愈合和样单层膜的影响。肠样单层伤口愈合随时间的变化取决于HA35治疗浓度。与对照组相比，HA35在100微克/毫升和200微克/毫升下在4小时和12小时后显着增加了伤口愈合，但愈合率在24小时内在治疗之间趋同。在这个过程中要记住的最重要的事情之一是肠样单层是短暂的。因此，一旦它们达到90%的流体，研究人员应该寻求快速使用它们。这项技术已成为我们体外早产肠道再生的首选模型，特别是如果我们对更多的机械性而不是炎症性损伤感兴趣。

Research

JoVE Journal

Medicine

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

科研

医学

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. ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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 ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

一个算法的开发与高位脊髓损伤使用遥测装置进行自主神经异常反射的动物的综合研究

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Murine Model of Intestinal Ischemia-reperfusion Injury

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

透明质酸35 kDa对肠源单层早产小肠损伤体 外 模型及愈合的影响

透明质酸35 kDa对肠源单层早产小肠损伤体外模型及愈合的影响