Structure and Function of Macromolecules Construction Hemopoietic Microenvironment

一些报道表明，造血器官基质细胞产生的蛋白聚糖参与造血调节。然而，目前还没有阐明蛋白多糖是如何做到这一点的。利用小鼠前脂肪细胞克隆细胞系MC3T3-G2/PA6，我们研究了细胞产生的蛋白聚糖的性质及其在造血中的作用，MC3T3-G2/PA6已被证明具有体外支持造血干细胞自我更新和分化的能力。结果表明：细胞产生了四种分子量不同的蛋白聚糖。其中三个与细胞层相关联，一个被释放到介质中。前者的主要糖胺聚糖为硫酸肝素，后者的主要糖胺聚糖为硫酸软骨素。β - d -木糖苷是一种糖胺聚糖延伸的人工引发剂，在β - d -木糖苷的存在下培养的细胞刺激了体外造血的能力。为了阐明在该系统中哪一种蛋白聚糖参与了支持造血，我们试图从它们的生化性质上分离蛋白聚糖。将蛋白多糖涂于Octyo-Sepharose CL-4B柱上，用Triton X-100线性梯度洗脱柱（0 - 0.5%）。整个释放到培养基中的蛋白多糖通过柱，表明它们的核心蛋白中没有疏水结构域。相反，细胞层蛋白多糖被分离成三个部分，即55%的蛋白多糖通过柱，15%的蛋白多糖结合并用0.03% Triton X-100洗脱，25%的蛋白多糖用0.2% Triton X-100洗脱。这些结果强烈提示PA6细胞至少产生两种类型的蛋白多糖，即基底膜型蛋白多糖（0.03% Triton X-100组分）和细胞膜型蛋白多糖（0.2% Triton X-100组分）。我们现在正试图制造针对这些蛋白多糖的单克隆抗体。它们在造血中的作用将通过使用这些工具来证明。少

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