Molecular Biology of Differentiation of Tooth Forming Cells

1.骨粘连蛋白的免疫化学和免疫组织化学定位：通过免疫化学和免疫组织化学方法研究各种组织中骨粘连蛋白的存在。骨粘连蛋白可在活跃的成骨细胞和骨祖细胞以及年轻的骨细胞中表达。骨连接素的表达并不局限于矿化组织，而是分布于许多软组织中.从牛成牙本质细胞的表达文库中克隆骨连接蛋白的cDNA为了检测骨连接蛋白与牛成牙本质细胞的同一性，从牛成牙本质细胞（未培养的细胞）的λ gtll表达文库中克隆骨连接蛋白的cDNA。从该文库中分离到一个2.1kbp的编码骨连接素的cDNA，并对其进行了序列测定。核苷酸序列预测骨粘连蛋白含有304个氨基酸，包括17个残基的信号肽。骨粘连蛋白序列与另一种蛋白SPARC显示出接近的同一性（>90%）。骨连接蛋白/骨连接蛋白转录本的表达骨连接蛋白cDNA用于分析相应转录本在各种牛胎儿组织中的表达。在皮肤、肌肉、肌腱等非骨组织中，该基因的表达量低于骨组织.影响骨连接素表达的因素：TGF-β、MG-63和HOS人骨肉瘤细胞中，α 2（1）胶原和骨连接素的mRNA表达均显著增加。在1，25（OH）_2D_3处理的细胞中，这些蛋白的信息量也增加.人釉原蛋白基因：牛釉原蛋白cDNA用于从人基因组DNA片段文库中分离釉原蛋白基因，所述人基因组DNA片段含有跨越约13个酶的釉原蛋白编码区的一部分。对该基因的分析表明至少存在四个外显子。结果表明，人釉原蛋白与牛、猪和鼠釉原蛋白具有高度同源性，并且具有亲水性羧基端肽。

1. Immunochemical and immunohistochemical localization of osteonectin.Various tissues were investigated for the presence of osteonectin by means of immunochemical and immunohistochemical methods. Osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes. Osteonectin expression, however, was not confined to mineralizing tissue, but was distributed in many soft tissues.2. cDNA cloning of osteonectin from expression libraries of bovine odontoblast.To examine the identity of osteonectin with SPARC, cloning of osteonectin cDNA from lambda gtll expression library of bovine odontoblasts (non-cultured cells) was carried out. A 2.lkbp cDNA coding for osteonectin, isolated from the librafy, was sequenced. The nucleotide sequence predicted that osteonectin contained 304 amino acids including 17-residue signal peptide. The osteonectin sequence showed near identity (>90%) with another protein, SPARC.3. Expression of SPARC/osteonectin transcript.Osteonectin cDNA was used to analyze the expression of the corresponding transcript in various bovine fetal tissues. Several non-botie tissues, including skin, muscle, tendon, expressed the transcript in lesser extent than bone tissue.4. Factors effect on the expression of osteonectin.It was found that mRNA for alpha2(1) collagen and osteonectin were markedly increased in TGF-,beta -treated MG-63 and HOS human osteosarcoma cells. The messages of these proteins also increased in 1,25(OH)_2D_3 treated cells.5. Human amelogenin gene.Bovine amelogenin cDNA was used to isolate the amelogenin gene from a library of human genomic DNA fragments which contained a part of amelogenin coding region spanning approximately 13 kilobases. Analysis of this gene indicated the presence of four exons at least. It was revealed that the human amelogenin had a high homology with bovine, porcine, and murine amelogenins and had also hydrophilic carboxyterminus telopeptide.