Effects of Nitric Oxide on Stem Cell Differentiation

描述(由申请人提供)： 在一项初步研究中，我们发现在干细胞体外分化为内皮细胞的过程中，内皮型一氧化氮合酶(ENOS)呈动态表达。在干细胞分化初期，eNOS的表达下调。研究还发现，干细胞在一氧化氮合酶抑制剂N-硝基-L-精氨酸甲酯(L-NAME)孵育24小时内失去其特异性表面标记SSEA-1。本研究旨在验证干细胞分化为内皮细胞需要下调eNOS表达的假说。本研究要解决的具体问题如下：1)NO-cGMP信号转导通路在维持干细胞特性中起什么作用？2)一氧化氮合酶抑制剂能否促进干细胞向内皮细胞分化？3)干细胞表面标志SSEA-1的丢失在一氧化氮合酶抑制剂处理后可逆吗？细胞能否恢复其多能干细胞特性？4)由NO供体提供的外源性NO能否阻止干细胞的分化？5)转eNOS基因的干细胞是否会失去分化能力？6)NOS抑制剂能否增加循环内皮祖细胞的数量并促进体内受损动脉壁的再内皮化？如果eNOS蛋白表达下调对其他细胞系的干细胞分化至关重要，那么预计像L-NAME这样的一氧化氮合酶抑制剂将加速干细胞的分化过程。相反，我们预计NO供体将阻止或推迟干细胞分化的开始。同样，只要eNOS基因在细胞中活跃表达，转eNOS基因的干细胞就有望失去分化能力。体内研究将确定一氧化氮合酶抑制剂是否增加循环内皮祖细胞的数量，并促进血管损伤部位的再内皮化。这些研究的数据将提供关于干细胞分化调控机制的新信息，并可能有助于开发新的方法来调节体内和体外干细胞分化的方向和速度。

DESCRIPTION (provided by applicant): In a pilot study we showed that endothelial nitric oxide synthase (eNOS) was dynamically expressed during the course of stem cell differentiation into endothelial cells in vitro. The expression of eNOS was down-regulated at the beginning of stem cell differentiation. It was also found that stem cells lost their specific surface marker SSEA-1 within 24 hours of incubation with the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). The present study is proposed to test the hypothesis that down-regulation of eNOS expression is required for stem cells to differentiate into endothelial cells. The specific questions to be addressed in this study are as follows: 1) What is the role of the NO-cGMP signal transduction pathway in the maintenance of characteristics of stem cells? 2) Can NOS inhibitors promote the differentiation of stem cells into endothelial cells? 3) Is the loss of the stem cell surface marker SSEA-1 reversible after treatment with NOS inhibitors (i.e., can the cells recover their multipotent stem cell characteristics? 4) Can exogenous NO supplied by NO donors prevent the differentiation of stem cells? 5) Will stem cells transfected with the eNOS gene lose their capability to differentiate? 6) Can NOS inhibitors increase the number of circulating endothelial progenitor cells and facilitate re-endothelialization of the injured arterial wall in vivo? If down-regulation of eNOS protein expression is critical for the differentiation of stem cells in other cell lines, it is anticipated that NOS inhibitors such as L-NAME will accelerate the stem cell differentiation process. Conversely, we expect that a NO donor will prevent or delay the onset of stem cell differentiation. Similarly, stem cells transfected with the eNOS gene are expected to lose their capability to differentiate as long as the eNOS gene is expressed actively in the cells. In vivo studies will determine whether NOS inhibitors increase the number of circulating endothelial progenitor cells and facilitate the re-endothelialization at sites of vascular injury. The data from these studies will provide new information concerning the regulatory mechanisms for stem cell differentiation, and may help to develop new approaches to modulate the direction and rate of stem cell differentiation both in vivo and in vitro.