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.

在这项研究中，我们最初通过整个大鼠胚胎培养（WEC）建立了为期12天的培养系统，持续了3天，然后是颅面植物的器官培养9天。 WEC中的胚胎3天可以模仿至关重要的分子的出现，即LHX1，FOXA2，ISL-1，PITX2，NKX2.1，Neurod1，tpit，tpit，lhx3，shh，shh，shh，bmp2，bmp2，bmp4，bmp4，fgf8，这是针对Hypothalamus and adnyhohohohohospoph的开发所必需的。 9天后的颅面基础可能会模仿腺旋转恐怖的出现，即α亚基，POMC，LHβ和TSHβ。此外，在培养系统中的去除实验的应用在培养系统上的应用表明，AME对于LHX1，FOXA2，ISL-1，SHH，FGF8，BMP4，BMP2，BMP2和NKX2.1的外观至关重要。此外，在培养系统中使用抗SHH抗体的功能丧失导致表达NKX2.1的细胞的减少，这表明SHH分泌AME对于可推定的营养中心的Arcuate Nucius的发展至关重要。然而，由于为期12天的培养系统，在为期12天的培养系统中培养的弧形核us无法区分NPY-NEURON和αMSH-NEURON，这是由于12天培养系统中的氧缺乏。因此，我们开始建立整个大鼠胚胎培养6天，以模仿可假定的弧形核us中神经元的发展。在先前的培养条件下，在6天培养的25个胚胎中，没有保持心跳，而在新的改善状态中培养的12个胚胎中的8个中，他们的心跳保持了6天。目前，我们正在建立细胞培养物，在该器官培养物中，在该器官培养物中，外植物中的核us伸向了NPY-NEURON和αMSH-NEURON。