Establishment of the culture method to mimic the development of neural nucleus of alimentary center in mammals

模拟哺乳动物消化中枢神经核发育培养方法的建立

基本信息

批准号：
22659334
负责人：
IMAI Hajime
金额：
$ 2.1万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

In this study, we initially established the 12 day-culture system by the whole rat embryo culture (WEC) for 3 days followed by the organ culture of craniofacial rudiments for 9days. The embryos in WEC for 3 days could mimic the appearance of crucial molecules, i.e., Lhx1, Foxa2, Isl-1, pitx2, Nkx2.1, NeuroD1, Tpit, Lhx3, Shh, Bmp2, Bmp4, Fgf8, which were necessary for a development of Hypothalamus and adenohypophysis. The craniofacial rudiments following 9 days could mimic the appearance of adenohypophysis hormones, i.e., α subunit, POMC, LHβ and TSHβ. Moreover, the application of removal experiment of anterior axial mesendoderm (AME) to the culture system showed that AME was essential for the appearance of Lhx1, Foxa2, isl-1, Shh, Fgf8, Bmp4, Bmp2 and Nkx2.1. In addition, the loss-of-function using an anti-Shh antibody inthe culture system resulted in the reduction of the cells expressing the Nkx2.1, suggesting that the Shh-secreting AME were essential for the development of arcuate nucleus, the presumptive alimentary center. However, the arcuate nucleus cultured in the 12 day-culture system failed to differentiate into the NPY-neuron and αMSH-neuron due to the oxygen deficiency in the 12day culture system. Therefore, we started to establish the whole rat embryo culture for 6 days in order to mimic the development of neuron in the presumptive arcuate nucleus. The heartbeat were not maintained in 19 of the 25 embryos cultured for 6 days under the previous culture condition, whereas their heartbeat were maintained for 6 days in 8 of the 12 embryos cultured in novel improved condition. At present, we are establishing the organ culture in which the arcuate nucleus in explants differentiates into NPY-neuron and αMSH-neuron.

在本研究中，我们首先建立了12天培养系统，先进行3天的全大鼠胚胎培养，然后进行9天的颅面雏形器官培养。胚胎在WEC中培养3天可以模拟关键分子的出现。，即 Lhx1、Foxa2、Isl-1、pitx2、Nkx2.1、NeuroD1、Tpit、Lhx3、Shh、Bmp2、 Bmp4、Fgf8是下丘脑和腺垂体发育所必需的，9天后的颅面雏形可以模拟腺垂体激素的出现，即α亚基、POMC、LHβ和TSHβ。此外，前轴去除实验的应用。中内胚层 (AME) 对培养系统的影响表明 AME 对于 Lhx1 的出现至关重要， Foxa2、isl-1、Shh、Fgf8、Bmp4、Bmp2 和 Nkx2.1 此外，在培养系统中使用抗 Shh 抗体导致功能丧失，导致表达 Nkx2.1 的细胞减少。这表明分泌Shh的AME对于弓状核（假定的消化中心）的发育至关重要，然而，弓状核是在12天的培养系统中培养的。由于12天的培养系统中缺氧，未能分化为NPY神经元和αMSH神经元因此，我们开始建立为期6天的全大鼠胚胎培养物，以模拟假定弓状核中神经元的发育。在之前的培养条件下培养了6天的25个胚胎中，有19个胚胎没有维持心跳，而在12个胚胎中培养的12个胚胎中，有8个胚胎的心跳维持了6天。目前，我们正在建立外植体中的弓状核分化为NPY神经元和αMSH神经元的器官培养物。