猪牙本质基质蛋白提取物的分离纯化及其活性检测

Extraction and bio-characteristic analysis of porcine dentin matrix protein components

目的:从猪牙胚中获得牙本质基质蛋白提取物(Dentin Matrix Protein Components,DMPCs)并检测其生物活性。方法:用4mol/L盐酸胍和0.5mol/L EDTA提取6月龄猪第二磨牙牙胚,获得DMPCs,用蛋白质液相色谱法分离纯化目的蛋白,SDS-PAGE检测分子量,MTT法测定DMPCs对人牙髓干细胞(humanDental Pulp Stem Cells,hDPSCs)生长的影响,同时测定DMPCs对hDPSCs碱性磷酸酶(ALP)活性的影响。结果:牙本质基质蛋白分子量为94×103、38×103、20×103以及一些小分子。体外实验证实,细胞培养1d,100μg/mL上调hDPSCs ALP活性(P<0.05);培养3d,100、150μg/mL组均可促进细胞增殖以及上调ALP活性(P<0.05)。结论:牙本质基质蛋白提取物可以促进hDPSCs的增殖,提示混合蛋白组分相互作用可以促进细胞增殖分化。

AIM： To extract dentin matrix protein components（DMPCs） from porcine dental germ and analyze their bio - characteristics. METHODS ： DMPCs were extracted from developing porcine molars using sequential 4M guanidine and 4M guanidine/0.5M EDTA extractions. They were purified according to liquid phase chromatography and analyzed by SDS - PAGE for molecular weight. The effect of DMPCs on the human dental pulp stem cells （ hDP- SCs） was evaluated by MTF method and the effect of DMPCs on the activity of ALP was also examined. RESUTLS- The major molecular weights of the DMPCs were 94Kda, 38Kda and 20Kda. When the hDPSCs were cultured for 1 d in vitro, 100p.g/mL DMPCs could up -regulate the activity of ALP （P 〈 0.05 ）. Three days later, the 100μg/mL and 150μg/mL DMPCs both could Up -regulate the activity of ALP and stimulate the proliferation of hDPSCs （P 〈 0.05）. CONCLUSION ： The DMPCs can both stimulate the growth and increase ALP activity of hDPSCs.