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，内皮型一氧化氮合酶（eNOS）的动态表达过程中的干细胞分化成内皮细胞在体外。 eNOS在干细胞分化初期表达下调。 还发现干细胞在与NOS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志物SSEA-1。 本研究旨在验证eNOS表达下调是干细胞向内皮细胞分化所必需的假说。 本研究的具体问题如下：1）NO-cGMP信号转导通路在干细胞特性维持中的作用是什么？ 2)NOS抑制剂能否促进干细胞向内皮细胞分化？ 3)干细胞表面标志物SSEA-1的丧失在用NOS抑制剂处理后是否可逆（即，细胞能否恢复其多能干细胞的特性？ 4)NO供体提供的外源性NO能否阻止干细胞分化？ 5)转染了eNOS基因的干细胞会失去分化能力吗？ 6)一氧化氮合酶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蛋白表达的下调对于其他细胞系中干细胞的分化是关键的，则预计NOS抑制剂如L-NAME将加速干细胞分化过程。 相反，我们预计NO供体将阻止或延迟干细胞分化的发生。 类似地，预期用eNOS基因转染的干细胞会失去其分化能力，只要eNOS基因在细胞中活跃表达。 体内研究将确定NOS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。 从这些研究的数据将提供有关干细胞分化的调控机制的新信息，并可能有助于开发新的方法来调节干细胞分化的方向和速度在体内和体外。

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.