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Systemic pattern of apoptosis in human embryos and establishment of mouse culture system to analyze its control mechanisms

人胚胎细胞凋亡的系统模式及小鼠培养体系的建立分析其调控机制

基本信息

批准号：
06807003
负责人：
OTANI Hiroki
金额：
$ 1.34万
依托单位：
Shimane Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807003/
关键词：
Apoptosis Human Embryo Mouse Embryo Exo utero Development Combined Malformations 重複奇形器官培養

项目摘要

1.Establishment of diagram of systemic pattern of apoptosis in human and mouse embryos : (1) Externally normal human embryos (Carnegie stage 13-23,4 to 8 weeks of gestation, 5.1-28.0 mm in crown-rump length) were analyzed by light microscopic observation on serial paraffin sections and by electron microscopic observation, and diagram of systemic pattern of apoptosis during human embryonic development was established. Apoptosis was observed in human embryonic tissues and organs, which have not been documented to exhibit apoptosis, including primordia of the salivary glands, retina, and midline cells in the mandible and anterior chest wall. (2) The observed systemic pattern of apoptosis was compared with epidemiological data on the combination pattterns of malformations in human embryos with multiple anomalies, and possible relationship between these patterns was suggested. (3) Systemic pattern of apoptosis in mouse embryos almost corresponding to that in humans was observed by light and electron microscopy. (4) Expression pattern of cell death-related genes such as bcl-2 and bcl-X was preliminarily examined in mouse embryos by immunohistochemistry and in situ hybridization.2.Mouse exo utero development system was introduced and improved to experimentally analyzed the systemic pattern of apoptosis described above. Ratio of viable embryos on day 18 of gestation increased to >90% after manipulation on day 13 of gestation, and >70% after day 11 manipulation. Further, mouse trunk culture in which the trunk including the heart is cultured in the medium has been tried to enable more precise manipulation and more continuous observation. Utilizing these culture system, we will examine systemic regulation of apoptosis and its possible relationship with combined malformations.

1.人胚胎和小鼠胚胎细胞凋亡系统图谱的建立：(1)对体外正常的人胚胎(Carnegie期13~23周，孕4~8周，头尾长5.1~28.0 mm)进行连续石蜡切片光镜观察和电子显微镜观察，建立人胚胎发育过程中细胞凋亡的系统图谱。在人类胚胎组织和器官中观察到了细胞凋亡，这些组织和器官包括唾液腺原基、视网膜以及下颌和前胸壁的中线细胞。(2)将观察到的全身性细胞凋亡模式与人类胚胎多畸形畸形组合模式的流行病学资料进行了比较，并提出了两者之间的可能关系。(3)在光镜和电子显微镜下观察到小鼠胚胎的全身性细胞凋亡模式与人类胚胎基本一致。(4)采用免疫组织化学和原位杂交的方法，初步检测了细胞死亡相关基因bcl2和bclX在小鼠胚胎中的表达模式。2.引入并改进了小鼠体外发育系统，对上述系统的细胞凋亡模式进行了实验分析。妊娠第18天的可用胚胎率在妊娠第13天达到90%，第11天达到70%。此外，还尝试了将包括心脏在内的树干培养在培养基中，以实现更精确的操作和更连续的观察。利用这些培养系统，我们将研究细胞凋亡的系统调控及其与合并畸形的可能关系。