【作者】 涂超；

【导师】 孙涛；

【作者基本信息】 华侨大学 ， 生物医学工程， 2023， 硕士

【摘要】 背景:脑干在调节和维持人体生命中发挥着至关重要的作用,涉及心脏、呼吸、消化、体温调节、睡眠等方面。然而,目前对于人类胚胎脑干细胞发育和多样性的研究仍十分有限。随着科学技术的不断进步,单细胞转录组测序技术被广泛应用于研究胚胎组织发育及相关领域。因此,我们借助单细胞转录组测序技术,对人类胚胎脑干的细胞分子多样性进行了深入研究。目的:本课题将采用单细胞转录组测序技术,对人类胚胎脑干细胞的多样性、发育模式以及分化过程进行研究,并详细探究胚胎期脑干组织的细胞分子特征。通过深入的细胞分子层面,本研究将为人类胚胎脑干的基础研究提供重要的理论依据,并为该领域的相关疾病的诊断和治疗提供有价值的参考。方法:本课题首先通过收集临床上孕期9周到12周的人类胚胎脑干样本,将收集的样本进行单细胞测序得到数据库,使用R语言对数据进行分析,主要包括:1.数据的质控和筛选去除低质量细胞数据;2.利用R包Harmony进行去批次效应;3.利用R包Seurat进行聚类和差异基因分析来研究胚胎脑干的细胞多样性和分子特征;4.使用R包SCORPIUS和monocle2数据进行拟时间分析来研究胚胎脑干细胞的发育规律;5.使用R包cluster Profiler和sc Metabolism研究本实验数据中的细胞生物学功能和代谢活性;6.最后使用免疫荧光的方法对部分标记基因进行验证。结论:通过对数据的分析,本课题鉴定了发育早期胚胎脑干的多种细胞类型。为了进一步了解早期人类胚胎脑干神经回路的细胞多样性,对神经祖细胞、谷氨酸能神经元和GABA能神经元进行了深入分析,本研究区分了中脑和脑桥-延髓这两个区域细胞类型,并详细阐述了中脑和脑桥-延髓区域的细胞多样性、发育模式和基因表达特征。此外,本研究还发现了胚胎脑干与阿尔茨海默症的关联性。本课题研究揭示了人类胚胎脑干的细胞分子多样性,为人类胚胎脑干的发育规律提供了基础证据,并为脑干相关疾病的研究提供理论依据。更多还原

【Abstract】 Background: The brainstem plays a crucial role in regulating and maintaining human life,involving aspects such as the heart,respiration,digestion,temperature regulation,sleep,etc.However,our current understanding of the development and diversity of human embryonic brainstem cells is still very limited.With the continuous development of scientific and technological research,single-cell transcriptome sequencing technology is increasingly being utilized in various studies of embryonic tissue development.Therefore,we employed single-cell transcriptome sequencing technology to investigate the cellular molecular diversity of human embryonic brainstem.Objective: This project will use single-cell transcriptome sequencing technology to study the diversity,development mode and differentiation process of human embryonic brainstem cells,and explore the cellular and molecular characteristics of embryonic brainstem tissue in detail.Through in-depth cell and molecular level,we will provide important theoretical basis for the basic research of human embryonic brainstem,and provide valuable reference for the diagnosis and treatment of related diseases in this field.Method: First of all,this project collected human embryonic brainstem samples from 9 to 12 weeks of gestation in clinical,sequenced the collected samples by single cell to obtain the database,and analyzed the data using R language,mainly including:1.data quality control and screening to remove low-quality cell data;2.Use R package Harmony to carry out de-batch effect;3.The cell diversity and molecular characteristics of embryonic brain stem were studied by clustering and differential gene analysis using R-packet Seurat;4.Use the R-packet SCORPIUS and monocle2 data to conduct pseudo-time analysis to study the development of embryonic brain stem cells;5.Use R package cluster Profiler and sc Metabolism to study the cell biological function and metabolic activity in the experimental data;6.Finally,some marker genes were verified by immunofluorescence.Conclusion: Through the analysis of the data,this project identified a variety of cell types of the early embryonic brain stem.In order to further understand the cell diversity of the neural circuit of the early human embryonic brain stem,this research conducted in-depth analysis of neural progenitor cells,glutamate neurons and GABAergic neurons.This research distinguished the cell types of the midbrain and pons-medulla oblongata,The cell diversity,developmental pattern and gene expression characteristics of the midbrain and pons-medulla region were described in detail.In addition,we also found the association between embryonic brain stem and Alzheimer’s disease.This research reveals the cell and molecular diversity of human embryonic brain stem,provides basic evidence for the understanding of human embryonic brain stem,and provides theoretical basis for the research of brainstem related diseases.更多还原