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Molecular chractrerization of cell aggregation factor(s) in celluar fibronectin prepatration - A possibility of a determinant chondrogene

细胞纤连蛋白制备中细胞聚集因子的分子表征 - 决定性软骨基因的可能性

基本信息

批准号：
61580136
负责人：
KIMATA Koji
金额：
$ 1.34万
依托单位：
Aichi Medical University (1987)Nagoya University (1986)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Cellular fibronection prepared from chick embryonic fibroblast cells (CCFN) has been shown to have the active suvstance(s) to promote in vitro aggregation of chick embryonic limb bud mesencymal cells and subsequent chondrogrmesis of the aggregated cells. When fractionated by Sepharose CL 2b column chromatography, we found the activity in the lower molecular weight fraction of CCFN which gave a few glycoprotein bands around Mr=15,000 on SDS-polyacrylamide gel electrohoresis. Kasai et al(Teikyo University) have demonstrated the existence of tw kinds of beta-galactoside-binding lectins having Mr=16,000 and Mr=14,000, respectively, in check embryo. These lectins and their specific antibodies were served to see whether the active substance(s)in CCFN is identical with these lectine ot not. SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting using these antibodies suggested the presence of not only both the Mr=16,000 and Mr=14,000 lectins in CCFN but also the Mr=16,000 lCtin in the detergent extract of check embryo limb buds at stages 22-23. The difect addition of the putified lectine to the culture medium of the limb bud mesenchymal cells prometed the cell aggregation. The simultaneous addition of either the antibodies with CCFN neutralized the induced cell aggregation. Furthermore, the addition of the antibodies to the culture without CCFN brought about the complete disappearance of the slowly going cell aggregation which was probably due to the endogenous synthesis of the lection. Therefore, it is likely that the active substrances in CCFN is caused by the contaminants, Mr=16,000 and 14,000 beta-galactoside-binding lectins and that the former lectin exists in the limb buds and plays an importanr role in the cell aggregation during limb bud mesenchymal condensation.

由鸡胚成纤维细胞制备的细胞纤维连接蛋白（Cellular Fibronectin，CCFN）具有促进鸡胚肢芽间质细胞体外聚集和随后的聚集细胞软骨形成的活性物质。当用Sepharose CL 2b柱层析分离时，我们发现CCFN的低分子量部分具有活性，其在SDS-聚丙烯酰胺凝胶电泳上在Mr= 15，000附近产生一些糖蛋白条带。加塞等人（帝京大学）已经证明在对照胚胎中存在两种分别具有Mr= 16，000和Mr= 14，000的β-半乳糖苷结合凝集素。用这些凝集素及其特异性抗体检测CCFN中的活性物质是否与这些凝集素相同。SDS-聚丙烯酰胺凝胶电泳和随后使用这些抗体的免疫印迹表明，CCFN中不仅存在Mr= 16，000和Mr= 14，000的凝集素，而且在22-23期的检查胚胎肢芽的洗涤剂提取物中也存在Mr= 16，000的凝集素。在肢芽间充质细胞培养液中添加不同浓度的凝集素可促进细胞聚集。同时添加任一抗体与CCFN中和诱导的细胞聚集。此外，向没有CCFN的培养物中加入抗体导致缓慢进行的细胞聚集完全消失，这可能是由于内源性合成的凝集素。因此，CCFN中的活性物质可能是由Mr= 16，000和14，000的β-半乳糖苷结合凝集素引起的，前者存在于肢芽中，并在肢芽间充质凝聚过程中的细胞聚集中起重要作用。