Molecular Cloning to Dentin Morphogenetic Protein (DMP)

牙本质形态发生蛋白 (DMP) 的分子克隆

• 批准号: 07044279
• 负责人: NAKASHIMA Misako
• 金额: $ 2.43万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044279/
• 关键词: reparative dentin; pulpal wound healing; bone morphogenetic protein; dentin morphogenetic protein (DMP); molecular cloning; polymerase chain reaction (PCR)

Dental pulp cells have the potential to differentiate into odontoblasts during pulpal wound healing process. Demineralized dentin matrix induces reparative dentin formation in the amputated pulp and new bone in ectopic sites indicating the presence of bone morphogenetic protein (BMP) activity. We hypothesize that there is "dentin morphogenetic protein (DMP)" in dentin matrix belonging to BMPs and/or TGF-beta superfamily and they play a critical role in pulpal wound healing and reparative dentin formation. Therefore, we tried to identify and purify DMP.Demineralized dentin matix was extracted in 4M quanidine HCl, loaded onto hydroxyapatite column and the 100 mM PO_4 eluate was loaded onto heparin-Sepharose CL-6B.The 0.5M NaCl eluate was then loaded in TSK-C18-4PW column by reversed phase high pressure liquid chromatography (HPLC). The collected samples were assayd by BMP activity using subcutaneous implantation. The active fraction was finally purified by Smart System reversed phase HPLC.The sample with a high alkaline phospatase stimulating activity was subjected to amino acied sequence analysis. The any sequences, however were not related to those of known TGF-beta superfamily members.An alternative approach to the purification of novel DMP was the cloning of BMPs by polymerase chain reaction (PCR). The rat incisor pulp was extracted and poly A^+ RNA was prepared to synthesize first strand cDNA as templete. PCR was performed with SJL 151-159 and 187-201 as 3'primer, SJL 160 as 5'primer. We identified BMP-2, -4, -6, -7, -8, Inhibin-beta, Growth and Differentiation Factor (GDF) -1, -5, -6, -8 and -11 from 350 plasmid-clones using an automatic sequencer. We are now trying to localize these factors in embryonic tooth and reparative dentin by in situ hybridization to determine whether these factors were concerned in differentiation of odontoblasts.

牙髓细胞在牙髓创伤愈合过程中具有向成牙本质细胞分化的潜能。脱矿的牙本质基质诱导离断牙髓中的修复性牙本质形成和异位部位的新骨，表明骨形态发生蛋白（BMP）活性的存在。我们推测牙本质基质中存在属于BMPs和/或TGF-β超家族的“牙本质形态发生蛋白（dentinmorphogeneticprotein，DMPs）”，它们在牙髓创伤愈合和修复性牙本质形成中起重要作用。本实验采用4 M盐酸胍提取脱矿牙本质基质，上样于羟基磷灰石柱，100mMPO_4上样于肝素-SepharoseCL-6 B柱，0.5MNaCl上样于TSK-C18-4PW柱，采用反相高效液相色谱法（HPLC）进行分离纯化。采用皮下埋植法测定骨形态发生蛋白（BMP）活性。活性组分经Smart System反相高效液相色谱纯化后，对具有较高碱性磷酸酶活性的样品进行氨基酸序列分析。然而，这些序列与已知的TGF-β超家族成员的序列不相关。提取大鼠切牙牙髓，制备poly A^+ RNA，合成第一链cDNA作为模板。以SJL 151-159和187-201为3 ′引物，SJL 160为5 ′引物进行PCR。我们使用自动测序仪从350个质粒克隆中鉴定了BMP-2、-4、-6、-7、-8、抑制素-β、生长和分化因子（GDF）-1、-5、-6、-8和-11。我们正试图通过原位杂交的方法在胚胎牙和修复性牙本质中定位这些因子，以确定这些因子是否参与成牙本质细胞的分化。