Amino Acid Sequence of Pulp Cell Growth Factor (PGF)

对具有刺激牙髓细胞增殖和调节成牙本质分化能力的牙髓细胞生长因子和牙本质形态发生蛋白进行了纯化。用4 M胍盐酸解离提取牛脱矿牙本质基质，并将其装载到羟基磷灰石亲和团块上。在100mm磷酸钾溶液中浓缩洗脱液，将洗脱液装在herarin-Sepharose CL-6B柱上。经蒸馏水透析后冻干的0.5 M NaCl洗脱液，采用20% - 100%乙腈线性梯度反相高压液相色谱法进一步纯化。柱段13、14和15（乙腈48 ~ 69%）对原代牙髓细胞碱性磷酸酶活性有抑制作用，柱段14对DNA合成有促进作用。每个部分电泳切成薄片，在电泳浓缩器中洗脱约31 kda和25 kda区域的蛋白。部分13-2 （31 kDa）对碱性磷酸酶活性有抑制作用，部分14-4 （25 kDa）对DNA合成有刺激作用。25 kDa蛋白被分成两半，12.5 kDa和31 kDa未处于还原状态，说明这两种蛋白并不相同。我们现在正在测定这些蛋白质的氨基酸序列。

The purification of pulp cell growth factor and dentin morphogenetic protein were performed, shich have the ability to stimulate proliferation of dental pulp cells and regulate differentiation into odontoblasts. Bovine demineralized dentin matrix was dissociatively extracted in 4 M guanidine HCl, and loaded onto hydroxyapatite affinity clumn. The eluate from the column in 100 mM potassium phosphate was concentrated and loaded agein onto herarin-Sepharose CL-6B column. The 0.5 M NaCl eluate dialyzed against distillled water and lyophilized was further purified by reversed-phase high pressure liquid chromatography using a linear gradient of 20 %-100 % acetonitrile. The column fractions 13, 14 and 15 (acetonitrile 48-69 %) had an inhibitory affect on alkaline phosphatase activity and fraction 14 had a stimulatory effect on DNA synthesis in primary pulp cell culture. Each fraction was applied for electrophoresis to cut into slices, and around 31-kDa and 25-kDa region proteins were eluted in extraphor elecrophoretic concentrator. Fraction 13-2 (31 kDa) had an inhibitory effect on alkaline phosphatase activity and fraction 14-4 (25 kDa) had a stimulatory effect on DNA synthesis. The 25-kDa protein was divided into half, 12.5 kDa and 31-KDa was not under reduecd condition, suggesting that these two proteins were not identical each other. We are now determining of amino acid sequences of these proteins.