Molecular Biology of Differentiation of Tooth Forming Cells

1.骨连接蛋白的免疫组织化学和免疫组织化学定位：应用免疫组织化学和免疫组织化学方法对不同组织中骨连接蛋白的表达进行研究。在活跃的成骨细胞和成骨前体细胞以及年轻的骨细胞中均可见骨连接蛋白的表达。然而，骨连接蛋白的表达并不局限于矿化组织，而是分布在许多软组织中。从牛成牙本质细胞表达文库中克隆出骨连接蛋白基因。为了验证其与SPARC的同源性，从牛成牙本质细胞(未培养细胞)的表达文库中克隆了骨连接蛋白基因。从LIBRAFY中分离到一个编码骨连蛋白的2.1kbpcDNA，并对其进行了测序。该核苷酸序列预测骨连蛋白含有304个氨基酸，其中包括17个残基的信号肽。骨连接蛋白序列与另一种蛋白SPARC.3有近90%的同源性。SPARC/Osteonectin转录本的表达：利用Osteonectin cDNA分析相应转录本在不同牛胎儿组织中的表达。几种非生物组织，包括皮肤、肌肉、肌腱，表达该转录本的程度低于骨组织。研究发现，经转化生长因子处理的MG-63和HOS人骨肉瘤细胞中，α2(1)胶原和骨结素的mRNA表达显著增加。在1，25(OH)_2D_3处理的细胞中，这些蛋白质的信息量也增加。人釉原蛋白基因。牛釉原蛋白基因被用来从人类基因组DNA片段文库中分离出釉原蛋白基因，该文库包含跨越约13kb的部分釉原蛋白编码区。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、猪和小鼠釉原蛋白有很高的同源性，并具有亲水性的羧基末端端肽。

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.