日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Dulbecco's Modified Eagle Medium | Gibco by Life technologies | 12800-017 10 X 1 L | For wash the EECs pellets |
| D-MEM/F-12 | Gibco by Life technologies | 12400-024 10 X 1 L | As the basal medium in culturing EECs |
| NBCS | Gibco by Life technologies | 16010-159 | As the supplyment serum in culturing EECs |
| Pen-Strep Ampho. Solution | BI (Biological Industries) | 03-033-1B 100ml | For attenuating the possible infection |
| Collagenase Type IV | Gibco by Life technologies | 17104-019 1g | 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. |
| 24 well plate | FALCON® | REF-353047 | For EECs to attach and extension |
| 50 ML PP centrifuge tubes | Corning® CentriStarTM | 430829 | For transportion of membranes and enzyme digestion |
| 50ML Conical bottomed Tube with Cap | PRO TECH | CT-50-PL-TW | For transportion of membranes and enzyme digestion |
| Reciprocal shaking bath | DEAGLE | SB302 | For better enzymatic digestion on membranes |
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