胚胎干细胞向内皮细胞的分化及PECAM-1在胚胎干细胞分化过程中的表达规律研究

发布时间：2018-08-02 13:43

【摘要】：胚胎干细胞(embryonic stem cells，ES cells)是一类具有自我更新和发育全能性的早期胚胎细胞，本研究通过诱导小鼠ES细胞体外分化，建立了ES细胞体外血管生成和血管新生的序惯性模型，为研究血管形成的体内过程与血管形成有关的基因功能，评价血管形成促进和抑制因子提供了一个有用的工具；同时研究了血小板内皮细胞黏附分子(PECAM-1／CD31)在ES细胞的表达规律，探讨了PECAM-1在ES细胞分化过程中的作用。第一部分 (1) 体外培养ES细胞诱导形成胚胎小体(embryoid body，EB)，将11d的EB种植到胶原中诱发出芽性血管新生；通过免疫组织化学染色方法检测EB及其出芽向内皮细胞和功能血管的分化；(2) 通过悬滴培养的方法，将培养6d的EB在全反式维甲酸(All-trans retinoic acid，ATRA)的作用下向神经细胞诱导分化；(3) 将培养11d的EB消化成单个细胞后，种植到甲基纤维素中，向造血细胞诱导分化。结果显示，ES细胞在体外能自发形成EB，其内部含有血管样结构，并伴有平滑肌的分化；种植到胶原中的胚胎小体能再现出芽性血管新生(sprouting angiogenesis)；ES细胞在体外同时也能分化成神经和造血细胞。第二部分体外培养ES细胞，保持其未分化状态，利用免疫组织化学、流式细胞术、RT-PCR等方法检测ES细胞未分化标志SSEA-1、碱性磷酸酶、Oct-4及PECAM-1的表达情况，同时，用流式细胞仪检测PECAM-1与其他内皮细胞标志表达的关系。应用克隆分析的方法检测不同剪接体的表达分布。体外培养ES细胞，保持其未分化状态，同时在细胞因子的作用下诱导形成胚胎小体(EB)；然后将EB种植到胶原中，在细胞因子的作用下诱导出芽性血管新生。分别利用免疫组织化学、流式细胞术、逆转录-聚合酶链反应(RT-PCR)等方法检测ES细胞及其分化过程中PECAM-1、Oct-4及SSEA-1的表达。应用克隆分析的方法检测不同的PECAM-1剪接体在EB形成和出芽性血管新生过程中的表达分布。结果显示未分化的ES细胞表达较高水平的PECAM-1，表达8种PECAM-1的剪接体，包括全长、△12、△14、△15、△1214、△1215、△1415、△121415，其中△15和△1415表达水平最高。
[Abstract]:Embryonic stem cells (embryonic stem cells es cells) are a kind of early embryonic cells with self-renewal and developmental totipotency. In this study, we established an order inertia model of es cell angiogenesis and angiogenesis by inducing mouse es cells to differentiate in vitro. It provides a useful tool for studying the gene function related to angiogenesis in vivo and evaluating angiogenesis promoting and inhibiting factors. The expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31) in es cells and the role of PECAM-1 in es cell differentiation were studied. The results were as follows: (1) es cells were cultured in vitro to form embryoid body (EB), and 11 d EB was implanted into collagen to induce budding angiogenesis, and the differentiation of EB and its budding into endothelial cells and functional vessels was detected by immunohistochemical staining. (2) EB cultured for 6 days was induced to differentiate into neural cells by suspension culture, and EB cultured for 11 days was digested into a single cell, then implanted into methylcellulose to induce differentiation into hematopoietic cells. The results showed that the EBs cells formed spontaneously in vitro, which contained vascular-like structure and differentiation of smooth muscle. Small embryo implanted into collagen could reproduce budding angiogenesis (sprouting angiogenesis);. Es cells can also differentiate into nerve and hematopoietic cells in vitro. In the second part, es cells were cultured in vitro to maintain their undifferentiated state. The expression of undifferentiated markers SSEA-1, alkaline phosphatase Oct-4 and PECAM-1 were detected by immunohistochemistry, flow cytometry and RT-PCR. The relationship between PECAM-1 and other endothelial cell markers was detected by flow cytometry. Clone analysis was used to detect the expression distribution of different splicing bodies. Es cells were cultured in vitro to maintain their undifferentiated state. At the same time, embryonic body (EB); was induced by cytokines, then EB was implanted into collagen to induce budding angiogenesis. Immunohistochemistry, flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of PECAM-1 Oct-4 and SSEA-1 in es cells and their differentiation. Cloning analysis was used to detect the expression distribution of different PECAM-1 splicing bodies during EB formation and budding angiogenesis. The results showed that the undifferentiated es cells expressed a high level of PECAM-1, and expressed eight splicing bodies of PECAM-1, including the full length, 1214, 15, 1214, 1215, 1415, 121415, among which 15 and 1415 were the highest.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329

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