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

1.(1)对体外正常人胚胎（卡耐基期13 ~ 23、妊娠4 ~ 8周、冠臀长5.1 ~ 28.0 mm）进行连续石蜡切片光镜观察和电镜观察，建立人胚胎发育过程中系统凋亡模式图。在人类胚胎组织和器官中，包括唾液腺原基、视网膜、下颌骨中线细胞和前胸壁细胞，均观察到细胞凋亡，但未见文献记载。(2)将观察到的细胞凋亡的系统性模式与流行病学资料中人类胚胎畸形合并多种异常的组合模式进行了比较，并提出了这些模式之间可能存在的关系。(3)光镜和电镜观察到小鼠胚胎的系统凋亡模式与人基本一致。(4)利用免疫组织化学和原位杂交技术，初步检测了小鼠胚胎中bcl-2、bcl-X等细胞死亡相关基因的表达规律。引入并改进了小鼠体外发育系统，实验分析了上述细胞凋亡的系统模式。妊娠第13天操作后，妊娠第18天的活胚率增加到>90%，第11天操作后，>70%。此外，已尝试在培养基中培养包括心脏在内的躯干的小鼠躯干培养，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。

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.