Study on The Dentinal Native Non-collagenous Protease

本课题的研究目标是：1)从牙本质中提取牙本质非胶原蛋白水解蛋白酶；2)研究蛋白酶对牙本质蛋白和蛋白聚糖的水解能力；3)建立矿化细胞培养；4)研究不同牙本质粘附机制对剪切和疲劳结合强度的影响。实验结果如下：1。从人和牛牙本质中提取了几种蛋白酶。59KDa （59K p酶）的近似分子量显示其主要酶活性，其最适pH为9.59K， p酶被EDTA、TIMP和裂解骨桥蛋白抑制。提示59K p酶是一种光蛋白蛋白酶，在牙本质基质发育和降解过程中均起重要作用。EDTA和氯化胍提取的牙本质蛋白聚糖均被59K p酶降解成小片段。推测59K p酶是一种金属蛋白酶而非胶原酶，在牙本质矿化和成熟过程中起重要作用。得到了几种牙本质蛋白聚糖，SDS/PAGE分子量分别为150 ~ 180kd、300KD、130 ~ 150kd和180 ~ 210kd。59K p酶降解了所有这些蛋白聚糖。E-ext蛋白多糖有几个核心蛋白，用Stains-all染色，这些核心蛋白也被59K p酶切割。这些结果表明59K - p酶具有类似基质溶解素的活性。来源于牛门牙的成牙细胞在基膜组分重组凝胶上培养，产生牙本质特异性蛋白磷酸holyn并生长矿化结节。基质中相关酶的最高浓度出现在第22天至第36天，与矿化开始一致。所描述的矿化牙本质细胞培养中蛋白质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。比较了第三代牙本质粘结剂KB-110与传统牙本质粘结剂的剪切和疲劳剪切强度。KB-110的剪切强度和疲劳结合强度显著高于常规材料，但随着贮存时间的延长，其疲劳强度迅速下降。这些结果表明，疲劳强度比剪切结合强度更准确地反映了牙本质基质的保持性能。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