Study on The Dentinal Native Non-collagenous Protease

本研究的目的是：1）从牙本质中提取牙本质非胶原蛋白水解蛋白酶，2）研究该蛋白酶对牙本质蛋白和蛋白多糖的水解能力，3）建立矿化细胞培养体系，4）研究牙本质不同粘附机制对剪切和疲劳粘结强度的影响，得到以下结果：1.从人牙本质和牛牙本质中提取了几种蛋白酶。59 KDa左右的P-ase为主要酶活性，其最适pH为9. 59 KP-ase可被EDTA和TIMP抑制，并裂解骨桥蛋白。结果表明：59 K P-ase是一种磷酸蛋白酶，在牙本质基质的形成和降解过程中起重要作用。推测59 K P酶可能是金属蛋白酶B的一种 ...更多信息 3.从牙本质中分离得到几种蛋白多糖，SDS/PAGE分析分子量分别为150- 180 KD、300 KD、130- 150 KD和180- 210 KD。E-ext蛋白聚糖具有几个核心蛋白，这些核心蛋白被59 K P-ase酶切，并被蓝染色。这些结果表明，59 K P-ase具有基质溶解素样活性。4.牛切牙成牙本质细胞在基底膜成分的重组凝胶上培养，产生牙本质特异性蛋白磷酸化蛋白，并生长矿化结节。在第22天和第36天之间观察到基质中相关酶的最高浓度，与矿化开始一致。所描述的蛋白质在矿化牙本质细胞培养中的发育表达序列与骨中的非常相似，这表明骨和牙本质中基质矿化的共同机制。KB-110的剪切强度和疲劳强度均显著高于常规粘结剂，但其疲劳强度随贮存时间的延长而迅速下降。这些结果表明，疲劳强度比剪切粘结强度更准确地反映了牙本质基质的固位性能。少

The objectives of this research project were 1) to extract the non-collagenous dentin protein hydrolyzable protease from dentin, 2) to study the hydrolytic ability of the protease against the dentin proteins and proteoglycans, 3) to establish the mineralizing cell culture and 4) to investigate the influence of different dentin adhering mechanisms on shear and fatigue bond strength.The results obtained were as follows :1.Several proteases were extracted from human and bovine dentin. The approximate molecular weight of 59KDa (59K P-ase) showed the major enzymatic activity and its optimum pH was 9.59K P-ase was inhibited by EDTA and TIMP, and cleaved osteopontin. It was suggested that 59K P-ase is a phophoprotein protease and play an important role in both dentin matrix development and degradation process.2.Both the EDTA and guanidium chloride extracted dentin proteoglycans were degraded into small fragments by 59K P-ase. It was conceivable that the 59K P-ase was one of metalloproteases b … More ut not a collagenase, and play an important role in the process of mineralization and maturation of dentin.3.Several dentin proteoglycans were obtained, ther molecular weights were 150-180KD, 300KD, 130-150KD and 180-210KD on SDS/PAGE.59K P-ase degraded all of these proteoglycans. E-ext proteoglycan has several core proteins which were stained blue with Stains-all and these core proteins were also cleaved by 59K P-ase. These results demonstrate that the 59K P-ase has a stromelysin-like activities.4.Odontoblast derived from bovine incisors was cultured on a reconstituted gel of basement membrane components, produced a dentin-specific protein phosphopholyn and grew mineralized nodules. The highest concentration of the enzyme related in the matrix was observed between day 22 and day 36 coinciding with the initiation of mineralization. The described sequence of developmental expression of proteins in this mineralizing dentin cell culture is very similar to that in bone which suggests common mechanisms of matrix mineralization in bone and dentin.The shear and fatigue shear bond strength of a third generational dentin bonding agent (KB-110) was compared with a conventional one. KB-110 showed significantly higher shear and fatigue bond strength than conventional, though its fatigue strength decreased rapidly as the storage period prolonged. These results suggest that the fatigue strength reflects more accurately the retentive properties of the dentin matrix than the shear bond strength. Less