Name: breast milk enhances growth of enteroids: an ex vivo model of cell proliferation
Uploaded: 2018-02-15T12:30:00
Description: Watch this Scientific Journal Video about Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation at JoVE.com

MG 由国家卫生研究院赠款 K08DK101608 和 R03DK111473 支助, 3月的硬币基金会赠款 5-FY17-79, 华盛顿大学儿童发现研究所和圣路易斯儿童医院和部圣路易斯华盛顿大学医学院儿科。CJL 是由 R01DK104946 (PI:), 华盛顿大学儿童发现研究所和圣路易斯儿童医院支持。

这项研究的所有动物程序都是由圣路易斯大学动物保育和使用委员会 (20160187 号议定书) 或匹兹堡大学机构动物护理和使用委员会 (14103918 号议定书) 批准的。人类胎儿小肠在不到24周的妊娠是根据匹兹堡大学解剖组织采购指南后, 机构审查委员会批准 (礼宾 PRO14100537) 从大学匹兹堡健康科学组织银行通过一个诚实的经纪系统。未经匹兹堡大学机构审查委员会的同意, 获得了不确定的人母乳或巴氏杀菌?...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+lactoferrin+to+prevent+nosocomial+sepsis+and+necrotizing+enterocolitis+of+premature+neonates+and+effect+on+T-regulatory+cells.">Oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on T-regulatory cells.</a> Am J Perinatol. 31 (12), 1111-1120 (2014).</li><li>Amin, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+supplementation+prevents+necrotizing+enterocolitis+in+the+premature+infant.">Arginine supplementation prevents necrotizing enterocolitis in the premature infant.</a> J Pediatr. 140 (4), 425-431 (2002).</li><li>Rodgers, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+milk+collection+in+the+rat.">Practical aspects of milk collection in the rat.</a> Lab Anim. 29 (4), 450-455 (1995).</li><li>DePeters, E. J., Hovey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+collecting+milk+from+mice.">Methods for collecting milk from mice.</a> J Mammary Gland Biol Neoplasia. 14 (4), 397-400 (2009).</li><li>Willingham, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+collection+methods+for+mice+and+Reeves'+muntjac+deer.">Milk collection methods for mice and Reeves' muntjac deer.</a> J Vis Exp. (89), (2014).</li><li>Saxena, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Intestinal+Enteroids:+a+New+Model+To+Study+Human+Rotavirus+Infection,+Host+Restriction,+and+Pathophysiology.">Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology.</a> J Virol. 90 (1), 43-56 (2015).</li><li>Zachos, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Enteroids/Colonoids+and+Intestinal+Organoids+Functionally+Recapitulate+Normal+Intestinal+Physiology+and+Pathophysiology.">Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.</a> J Biol Chem. 291 (8), 3759-3766 (2016).</li><li>Drummond, C. 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肠道上皮是由许多细胞亚型组成的, 负责为病原体提供宿主防御, 维持肠道屏障完整性, 并可在多种疾病的发病机制中被突破。虽然动物模型可以重述这一疾病的某些方面, 但从小鼠和人类小肠中提取的 enteroids 的前体模型为测试各种治疗效果提供了一个平台。enteroid 模型的意义使研究人员能够将肠道干细胞培养并区分为所有上皮细胞亚型, 从幼稚或患病的动物和没有现有方法的人类。这种模?...

坏死性小肠结肠炎 (NEC) 是早产儿胃肠道疾病死亡的主要原因, 影响近 1 10 婴儿出生前29周妊娠1,2,3。有一半的婴儿与 NEC 进展到最严重的形式, 其中生存只有 10-30%4,5。在美国, 估计有 2-30亿美元/年用于治疗婴儿与 NEC6,7, 但在过去30年中, 存活率和治疗都没有改变。NEC 的发病特点是肠道损伤和黏膜愈合受损8,9,10,11, 然而, 信号通路导致恶化炎症反应和机制逆转炎症仍然不完全地被了解。
人类母乳的管理已被发现是唯一的保护策略, 针对 NEC 早产儿。我们以前已经表明, 母乳通过表皮生长因子受体 (EGFR) 信号通路11抑制肠道上皮中先天免疫受体 4 (TLR4), 从而防止 NEC 的发展。将母乳补充到一个实验 nec 公式中, 通过抑制 enterocyte 细胞凋亡和恢复 enterocyte 增殖的方式, 减弱了 nec 中所看到的炎症反应, 这种方法依赖于表皮生长因子 (EGF) 和 EGFR11。另一项研究表明, 母乳中的另一成分硝酸盐通过调节肠道灌注来促进其保护性, 与缺乏硝酸盐的婴儿配方奶粉相比, 这可能有助于提高 NEC 的频率。公式喂养婴儿12,13。母乳中含有的其他化合物包括人乳寡糖、l-精氨酸、谷氨酰胺和乳铁蛋白14,15,16, 17,18,19。母乳中的这些有益成分揭示了它在预防 nec 方面的必要性, 同时也强调了研究母乳如何调解对 nec 的保护所涉及的机制、信号通路和细胞效应的重要性。.
为了进一步研究 NEC 的小鼠模型中母乳的保护特性, 我们开发了一种新颖的、易于使用的技术, 用电动人乳泵从麻醉大坝中提取小鼠母乳的方法11,12.这项获取小鼠母乳的策略是有利的, 这不仅是因为人类母乳泵在母乳的采购方面是现成的, 而且还因为这种方法允许对特定种类的母乳进行分析。因此, 我们可以比较小鼠母乳与表达的人母乳的影响, 以及巴氏杀菌的人供奶从牛奶银行在物种特定的模型。这项技术允许研究母乳成分与他们对 NEC 预防的贡献。其他调查人员已经开发出母乳提取方法, 但是, 这些技术是手动的, 通常需要多个实验室成员20,21,22。这里提供了一个简单的技术, 可用于修改人的电动母乳泵从鼠标收集牛奶。这种技术也可以应用于其他物种。
为了充分审问与 NEC 有关的信号通路, 需要建立模型系统, 以评估已知受疾病影响的所有不同细胞类型。在这里, 我们讨论一个这样的模型系统-enteroids 和他们的建立从老鼠和人类小肠。人类肠道 enteroids (HIEs) 特别是提供了重要的承诺, 因为它们提供了一个创新的, 基因多样的前体人类模型, 以帮助研究在胃肠道发生的病理生理过程23. Enteroids 已被发现长期养殖, 可冷冻供以后使用23, 与人类肠道 Organoids (HIOs) 不同, 其培养方法来自诱导多潜能干细胞, Enteroids 是由干细胞产生的。在孤立的肠道墓穴中24。Enteroids 需要较少的维护, 可以快速感染25, 并且可以很容易地建立, 因为肠道墓穴比 HIOs23更加区分。因此, HIEs 提供了许多优势超过现有的技术, 因为它们可以被开发展示区域特定的成分和功能性质的人胃肠上皮23。使用 enteroids 是一个非常有效的选择, 当需要一个人的肠道模型, 坚持区域特定的限制和易用性。在这里, 我们展示了从小鼠和早产儿中分离和维持原发性干细胞源性小肠 enteroids 的技术。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 4.5 g/L Glucose and L-Glutamine</td>
			<td>Lonza</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Humulin N (Insulin)</td>
			<td>Eli Lilly And Company</td>
			<td>0002-8315</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Advanced DMEM/F-12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3537</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>100x N-2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>50x B-27 Supplement Minus Vitamin A</td>
			<td>Gibco</td>
			<td>08-0085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>100x ROCK Inhibitor Y-27632, Dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Wnt3a Protein</td>
			<td>R&#38;D Systems</td>
			<td>1324-WN</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm Cell Culture Petri Dish</td>
			<td>Eppendorf</td>
			<td>0030700112</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf</td>
			<td>0030722116</td>
			<td></td>
		</tr>
		<tr>
			<td>48-Well Cell Culture Plate</td>
			<td>Eppendorf</td>
			<td>0030723112</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Well Nunc Lab-Tek II Chamber Slide System</td>
			<td>Thermo Scientific</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;M Sterile Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;M Sterile Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-Buffered Saline (PBS), pH 7.2</td>
			<td>Invitrogen</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum (NDS)</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope Leica TCS SP8 X</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CS6</td>
			<td>Adobe Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G 1.5 Inch Needle</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxytocin</td>
			<td>Sigma-Aldrich</td>
			<td>O3251</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Double Electric Breast Pump</td>
			<td>Lansinoh</td>
			<td>044677530163</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Test Tube</td>
			<td>Corning</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber Stoppers</td>
			<td>Frey Scientific</td>
			<td>560761</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67 Antibody</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Mki67 primer F: 5&#39;-GACCTCAAACTGGCTCCTAATC-3&#39; R: 5&#39;-GCTGCCAGATAGAGTCAGAAAG-3&#39;</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

breast milk enhances growth of enteroids: an ex vivo model of cell proliferation

人小肠 enteroids 来自于墓穴, 当生长在一个干细胞的利基包含所有的上皮细胞类型。建立人类 enteroid 体外培养系统的能力对于模拟肠道病理生理学和研究所涉及的特定细胞反应非常重要. 近年来, 来自老鼠和人类的 enteroids 正在被培养、传代, 并在世界各地的几个实验室中被储存起来供将来使用。这个 enteroid 平台可以用来测试各种治疗和药物的影响, 以及对不同的肠道细胞类型的影响。在此基础上, 建立了从新生小鼠和早肠中提取的原发性干细胞衍生小肠 enteroids 的方法。此外, 利用这种 enteroid 培养系统来检测特定品种母乳的效果。用改良的人乳泵可以有效地获得小鼠母乳, 并表达小鼠乳可以用于进一步的研究实验。我们现在展示了表达的小鼠, 人和供体母乳对新生小鼠或早产人小肠 enteroids 的生长和增殖的影响。

我们首先试图调查表达的人母乳或巴氏杀菌供乳是否对小肠 enteroids 有影响。事实上, 人类母乳和捐赠母乳增加了新生鼠的生长 (图 1A) 和过早的人类衍生 enteroids (图 1B)。
由于人类母乳增加小肠 enteroids 的生长, 我们接下来研究是否母乳对过早人类 enteroids 的增殖有影响。我们证明, 人...

Watch this Scientific Journal Video about Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation at JoVE.com

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

该协议描述了如何建立从新生鼠或早肠的 enteroid 培养系统以及从小鼠收集牛奶的有效方法。

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Research

JoVE Journal

Medicine

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

自发性乳腺癌转移的原位小鼠模型

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...