Takagi M, Parmley R T, Toda Y, Denys F R
Lab Invest. 1982 Mar;46(3):288-97.
The role of osteoclasts or chondroclasts in degradation and synthesis of complex carbohydrates was investigated using the high iron diamine-thiocarbohydrazide-silver proteinate method (HID-TCH-SP) for sulfated glycoconjugates and the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained the calcified cartilage matrix, the osteoclast ruffled border, vacuoles and heterophagosomes but not the Golgi apparatus and primary lysosomes. The size and number of HID-TCH-SP stain deposits progressively decreased from the calcified cartilage matrix to the ruffled border (p less than 0.001). Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits averaging 13 nm. in diameter in the extracellular matrix, cytoplasmic vacuoles, and heterophagosomes of osteoclasts. Only sparse stain deposits averaging 8 nm. in diameter and presumed to be keratan sulfate remained in these sites after enzyme digestion. Osteoclast heterophagosomes contained the highest concentration of hyaluronidase-resistant material suggesting delayed degradation of keratan sulfate at this site. PA-TCH-SP strongly stained collagen fibrils in the calcified cartilage matrix. Reactive collagen fibrils were also observed in extracellular channels but only rarely were identifiable collagen fibrils observed in cytoplasmic vacuoles. A progressive decrease in the diameter of PA-TCH-SP reactive collagen fibrils was observed between the calcified cartilage matrix and the ruffled border region (p less than 0.001). PA-TCH-SP stained cisternae of rough endoplasmic reticulum, Golgi saccules, and primary lysosomes consistent with the synthesis and packaging of glycoprotein enzymes at these sites. These results indicate that the dissolution of sulfated glycoconjugates requires osteoclastic engulfment of degraded material and subsequent intracellular digestion, whereas the dissolution of collagen fibrils appears to be completed extracellularly.
采用高铁二胺-硫代碳酰肼-蛋白银法（HID-TCH-SP）检测硫酸化糖缀合物，以及高碘酸盐-硫代碳酰肼-蛋白银法（PA-TCH-SP）检测含邻位二醇的糖缀合物，研究破骨细胞或破软骨细胞在复合碳水化合物降解和合成中的作用。HID-TCH-SP可对钙化软骨基质、破骨细胞的皱襞缘、液泡和异噬体进行染色，但不能对高尔基体和初级溶酶体进行染色。从钙化软骨基质到皱襞缘，HID-TCH-SP染色沉积物的大小和数量逐渐减少（p小于0.001）。用睾丸透明质酸酶进行酶消化可去除大部分HID-TCH-SP染色沉积物，这些沉积物在细胞外基质、破骨细胞的细胞质液泡和异噬体中的平均直径为13nm。酶消化后，这些部位仅残留平均直径为8nm的稀疏染色沉积物，推测为硫酸角质素。破骨细胞异噬体中含有最高浓度的抗透明质酸酶物质，表明该部位硫酸角质素的降解延迟。PA-TCH-SP可强烈染色钙化软骨基质中的胶原纤维。在细胞外通道中也观察到反应性胶原纤维，但在细胞质液泡中仅偶尔能识别出胶原纤维。在钙化软骨基质和皱襞缘区域之间，观察到PA-TCH-SP反应性胶原纤维的直径逐渐减小（p小于0.001）。PA-TCH-SP可对粗面内质网的池、高尔基体囊泡和初级溶酶体进行染色，这与这些部位糖蛋白酶的合成和包装一致。这些结果表明，硫酸化糖缀合物的溶解需要破骨细胞吞噬降解物质并随后进行细胞内消化，而胶原纤维的溶解似乎在细胞外完成。
