Extracellular Matrix Macromolecules of Developing Limbs

我们从各种先天性骨骼疾病，特别是肢体异常患者的病理软骨或骨组织中定量和定性地分析了蛋白多糖，发现在大多数情况下蛋白多糖的数量和/或结构异常。我们的观察结果支持了细胞外基质大分子（包括蛋白聚糖）参与骨骼组织细胞分化和形态发生的一般观点。为了阐明蛋白多糖在肢体形态发生中的作用，我们分离并鉴定了由发育中的大鼠肢体芽合成的蛋白多糖。从妊娠第13、14或15天的大鼠胎儿中分离出肢芽，在含有[<^(35)S>]硫酸盐的培养基中培养3小时。[<^(35)S>]标记的蛋白聚糖溶解在含有蛋白酶抑制剂和0.8% MEGA-9的洗涤剂的4 M胍HCl中。从第13天的断肢芽中分离出硫酸软骨素蛋白多糖和硫酸肝素蛋白多糖。此外，还分离到了硫酸乙酰肝素单链。我们的显微镜检查显示，在这个阶段可以观察到形成肢体骨骼原基的间充质细胞凝结。第14天首次检测到软骨型蛋白多糖，此后含量逐渐增加。这一发现表明，大鼠四肢的软骨形成始于妊娠第14天。通过蛋白水解获得的13天肢体芽多糖在醋酸纤维素膜上进行双向电泳，分离成4个阿利新蓝染色点；透明质酸，硫酸肝素，硫酸软骨素和一种未知成分。有趣的是，从相同发育阶段的大鼠胎儿的大脑或尾巴中获得的多糖制剂中均未发现该未知成分。虽然目前对该成分的表征并不令人满意，主要是由于可用量少，但该成分可能参与肢体形态发生。少

We have analyzed proteoglycans quantitatively as well as qualitatively in the pathological cartilaginous or osseous tissues excised from patients with various congenital skeletal disorders, especially limb anomalies, and found in most cases abnormalities in the amount and/or structure of proteoglycans. Our observations support the general idea that extracellular matrix macromolecules including proteoglycans are involved in cell differentiation and morphogenesis of skeletal tissues. To clarify the role of proteoglycans in limb morphogenesis, we isolated and characterized proteoglycans synthesized by developing limb buds of the rat.Limb buds, dissected from rat fetuses on day 13, 14 or 15 of gestation, were incubated in a culture medium containing [ <^(35)S> ]sulfate for 3 hr. [ <^(35)S> ]labeled proteoglycans were solubilized in 4 M guanidine HCl containing protease inhibitors and a detergent, 0.8% MEGA-9. A chondroitin sulfate proteoglycan and a heparan sulfate proteoglycan were isolat … More ed from day-13 limb buds. In addition, single chain heparan sulfate was also isolated. Our microscopic examination revealed that mesenchymal-cell condensation to form the primordia of the limb skeleton became observable at this stage. Cartilage-type proteoglycan could be detected for the first time on day 14, and the amount increased thereafter. This finding indicates that chondrogenesis in rat limbs begins on day 14 of gestation.Polysaccharide preparation obtained from day-13 limb buds by proteolysis was separated by two-dimensional electrophoresis on cellulose acetate film into four Alcian blue-stained spots; hyaluronate, heparan sulfate, chondroitin sulfate and an unknown component. Interestingly, the unknown component was not found in polysaccharide preparations obtained either from the brain or tail of rat fetuses at the same developmental stages. Although characterization of this component is not satisfactory at this moment mainly owing to the small amount available, the component could be involved in limb morphogenesis. Less