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

从鸡胚成纤维细胞（CCFN）制备的细胞纤维连接已被证明具有促进鸡胚肢芽间充质细胞体外聚集和随后聚集细胞软骨增生的活性物质。通过Sepharose CL 2b柱层析，我们发现CCFN在sds -聚丙烯酰胺凝胶电泳时，在低分子量部分有活性，在Mr= 15000左右出现了几个糖蛋白带。Kasai等人（Teikyo University）已经证明在对照胚胎中存在两种Mr=16,000和Mr=14,000的β -半乳糖苷结合凝集素。用这些凝集素和它们的特异性抗体观察CCFN中的活性物质是否与这些凝集素相同。利用这些抗体进行sds -聚丙烯酰胺凝胶电泳和随后的免疫印迹分析表明，CCFN中不仅存在Mr=16,000和Mr=14,000的凝集素，而且在22-23期的检查胚胎肢芽洗涤液中也存在Mr=16,000的凝集素。将纯化的卵磷脂直接加入到肢体芽间充质细胞的培养基中，促进了细胞的聚集。同时加入含有CCFN的抗体可以中和诱导的细胞聚集。此外，将抗体添加到没有CCFN的培养物中，导致缓慢的细胞聚集完全消失，这可能是由于内源性合成的选择。因此，CCFN中的活性物质很可能是由污染物Mr= 16000和14000 β -半乳糖苷结合凝集素引起的，前者凝集素存在于肢芽中，在肢芽间质凝聚过程中对细胞聚集起重要作用。

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.