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Structure and Function of Macromolecules Construction Hemopoietic Microenvironment

大分子构建造血微环境的结构与功能

基本信息

批准号：
63570582
负责人：
OKAYAMA Minoru
金额：
$ 1.28万
依托单位：
Research Institute, International Medical Center of Japan
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

Several reports have suggested that proteoglycans produced by stromal cells in a hemopoietic organ are involved in a regulation of hemopoiesis. It has not been, however, elucidated what and how proteoglycans do that. Using a clonal cell line of murine preadipocyte, MC3T3-G2/PA6, which has been demonstrated to have an ability to support self-renewal and differentiation of hemopoietic stem cells in vitro, we have studied the nature of the proteoglycans produced by the cells and their roles on hemopoiesis. The results obtained revealed the followings : The cells produced four molecular species of proteoglycans with different molecular weights. The three of them were associated with the cell layer and the one was released into the medium. The major glycosaminoglycans of the formers were heparan sulfate, whereas that of the latter was chondroitin sulfate. The cells cultured in the presence of beta-D-xyloside, an artificial initiator of glycosaminoglycan elongation, stimulated the ability to … More support hemopoiesis in vitro.To elucidate which type of the proteoglycans were involved in supporting hemopoiesis in this system, we have tried to separate the proteoglycans on their biochemical properties. The proteoglycans were applied to a column of Octyo-Sepharose CL-4B and the column was eluted with a linear gradient-concentration of Triton X-100 (0 - 0.5%). The whole proteoglycans released into the medium passed through the column, indicating that they have no hydrophobic domain in their core proteins. On the contrary, the cell layer-proteoglycans were separated into three fraction, i. e., 55% of them passed through the column, 15% of them bound and eluted with 0.03% Triton X-100, and 25% eluted with 0.2% Triton X-100. These results strongly suggested that PA6 cells produced at least two types of proteohlycans, nanely, basement membrare-type proteohlycans (0.03% Triton X-100 fraction) and cell membrane-type proteoglycans (0.2% Triton X-100).We are now trying to produce monoclonal antibodies against these proteoglycans. Their roles on hemopoiesis will be demonstrated by using these tools. Less

一些报道表明，造血器官中基质细胞产生的蛋白多糖参与造血的调节。然而，还没有阐明蛋白聚糖是什么以及如何做到这一点的。利用小鼠前脂肪细胞的克隆细胞系MC 3 T3-G2/PA 6，它已被证明有能力支持造血干细胞的自我更新和分化，我们研究了细胞产生的蛋白多糖的性质及其在造血中的作用。结果表明：该细胞产生四种分子量不同的蛋白多糖。三个都与细胞层相连，一个被释放到培养基中。前者的主要糖胺聚糖是硫酸乙酰肝素，而后者是硫酸软骨素。在β-D-木糖苷（一种糖胺聚糖延伸的人工引发剂）存在下培养的细胞刺激了细胞的能力， ...更多信息为了阐明哪种类型的蛋白聚糖参与了该系统中支持造血的作用，我们试图根据蛋白聚糖的生物化学性质对其进行分离。将蛋白聚糖上样到Octyo-Sepharose CL-4 B柱上，用线性梯度浓度的Triton X-100（0 - 0.5%）洗脱该柱。释放到培养基中的整个蛋白聚糖通过柱，表明它们的核心蛋白中没有疏水结构域。相反，细胞层蛋白聚糖被分成三个部分，即：例如，其中55%通过柱，15%结合并用0.03%Triton X-100洗脱，25%用0.2%Triton X-100洗脱。这些结果表明，PA 6细胞至少产生两种类型的蛋白聚糖，即基膜型蛋白聚糖（0.03%Triton X-100组分）和细胞膜型蛋白聚糖（0.2%Triton X-100组分）。通过使用这些工具，将证明它们在造血中的作用。少