Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

资金目前的协议的发展特别是“目标为一流大学计划”国立台湾大学，台湾（授权ID-104R350144），以及台湾科学技术部（授权ID的支持：科技部104 -2313-B-002-039-MY3）。我们特别感谢中心国立台湾大学生物技术用于提供动物房和实验室空间，目前的研究。

注：此步骤是Bauer 等人 2013年和中泽等人 ，2011年2,9制定了鸡模型文化协议的修改。 1.准备健康胚胎第5天的胚胎从日本鹌鹑<ol><li>将1男3女性成熟的日本鹌鹑在同一个笼子里。供应饲料自由采食和饮水 。调整的光在动物室的光的14小时和黑暗10小时。 </li><li>每天收集在塑料袋受精卵在下午和保存在16℃的冰箱延缓胚胎的生长速度。 </li...

<ol>
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因为以前的培养系统只有有限的成功，需要一个更好的培养系统。日本鹌鹑YSM内胚层需要与蛋白水解酶如胶原酶松开细胞 - 细胞连接以实现在离体的外植体更好的生长性能的治疗。我们的数据显示，从局部消化处理的细胞数接种2天（ 图1）后，比从未消化的组织培养更大。因此，部分水解消化是改善从YSM细胞产量的关键步骤。 在当前的文化协议的关键修改是部分...

禽流胚胎的主要营养资源是蛋黄，33％脂质，17％的蛋白质组成，和1％的灰分。1在胚胎发育期间，卵黄囊膜（YSM）从胚胎腹腔内生长并逐渐覆盖蛋黄表面。在2天胚胎开始，与脂质代谢和血管发生相关的基因的表达在YSM逐渐增加，并且YSM发展缓慢绒毛状突起。-8,9-这些突起增加蛋黄营养的吸收，以支持胚胎发育。该YSM是包含三个胚层，内胚层，中胚层和外胚层的胚外组织。14卵黄囊外胚层面临的蛋白和链接与卵黄膜轻轻地覆盖卵黄囊。内胚层上皮细胞直接面对朝蛋黄作为养分利用率的门户网站。6作为绝经年限的扩大，内胚层上皮细胞（EECS）可以通过十八分e和功能分为两组，区卵黄和面积vasculosa 7 区卵黄是由内胚层细胞是从胚胎遥远;区域vasculosa是由胚层细胞，并覆盖有血管和结缔组织有区别的EECS。通过5天的胚胎，蛋黄完全由YSM的外胚层和内胚层覆盖，血管面积增长迅速。在YSM吸收，重新组成并释放脂质（如蛋黄衍生的极低密度脂蛋白）和蛋白质进入胚胎循环系统9中，2。因此，我们建立了主日本鹌鹑胚胎内胚层上皮细胞培养系统，来研究脂质的机制利用率YSM禽流胚胎发育过程中。 脂质如甘油三酯，磷脂，磷脂和胆固醇酯（CE）是禽类胚胎的主要能源。在开发的早期阶段，蛋黄脂类是Composed只有1.3％，CE和它上升到10-15％的鸟类胚胎发育的3期中，11胆固醇酯是由胆固醇在鸡胚YSM固醇O-酰基转移酶1（SOAT1）合成。4 胆固醇的存储形式是CE，CE是脂蛋白携带和脂蛋白被循环输送到组织中。13一个星期前舱门有鸟类胚胎的快速增长。大约在蛋黄中剩余的脂肪含量68％，在这一阶段被吸收。10由蛋黄脂质利用可通过EEC研究模型加以澄清机制。阿鸡欧洲经济共同体培养协议的建立是为了实现这一研究的目标。2,9然而，由于组织外植体的成功率很低，需要一种改进的欧洲经济共同体的细胞培养过程来研究某些酶和蛋白质的功能在通过介导养分的利用在开发过程中禽流感的胚胎。

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

我们建立了一个内胚层上皮细胞培养模型（EEC）用于研究开发过程中由禽胚胎调解养分利用某些酶和蛋白质的功能。受精日本鹌鹑蛋在37℃下培养5天，然后卵黄囊膜（YSM）收集来建立欧洲经济共同体培养系统。我们从YSM分离胚胎内胚层层，和切片膜进入2 - 3毫米件和在24孔培养板播种前用胶原酶部分消化。该EECS繁殖出来的组织和准备​​好细胞培养研究。我们发现，EECS有体内 YSM的典型特征，例如，脂滴的累积，固醇O-酰基转移酶和脂蛋白脂肪酶的表达。部分消化处理显著上升EEC文化的成功率。利用EECS，我们证明了SOAT1的表达受cAMP的调节Ðependent蛋白激酶相关途径。这主要日本鹌鹑EEC培养体系是研究胚胎脂质运输和澄清参与禽流感在胚胎发育过程中的调解YSM养分的利用基因的作用的有用工具。

为了实现建立一个一致的和有用的细胞模型的目标，我们需要扩大和稳定增殖率和禽流EECS的性能。我们比较了没有酶消化与内胚层与蛋白水解酶，如胶原酶或胶原酶加分散酶的0.6单位部分消化的内胚层的直接温育。分散酶是一种氨基肽酶是水解的非极性氨基酸残基的N-末端肽键。而没有消化（ 图1）相比，蛋白酶消化处理增加的细胞增殖，细胞生长不五天后孵...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...