Molecular Biology of the Gene Expression of Hard Tissue Forming Cells

(1)克隆小鼠淀粉原蛋白染色体DNA并研究其结构。采用PCR技术分析了该基因的外显子-内含子结构，并对该基因第1外显子上游约3000个碱基对进行了测序。在基因表达控制区鉴定出TATA box、倒位CCAAT、GATA-1、AP- 1、GR结构。(2)以猪釉素PCR产物为探针，从牛成釉细胞cDNA文库中克隆牛釉素cDNA。将克隆的cDNA插入pET29c（+）表达载体，用大肠杆菌制备重组牛漆素肽。将该肽段免疫兔，获得了抗重组牛漆蛋白的抗体。该抗体用于牛牙胚免疫组织化学分析。釉质素的产生和分泌似乎与釉质基质的钙化有关。(3)通过比较牙根吸收组织的活性期和静息期来研究参与牙齿吸收的蛋白酶。从牛下颌骨根吸收组织中提取RNA。采用RT-PCR和Northern blot分析，研究了活性期和静息期组织中蛋白酶mrna的表达。结果显示两期均有mmp -1、MMP-2、MMP-9、MMP-13、MTl-MMP和组织蛋白酶K mrna。其中，MMP-1、MMP-9和组织蛋白酶K在活跃的根吸收组织中的表达量远高于静止组织。明胶酶谱分析表明，根吸收组织也具有溶明胶活性。由此可见，MMP-1、MMP-9和组织蛋白酶K在乳牙根吸收中起重要作用。

(1)Mouse amelogenin chromosomal DNA was cloned and investigated the structure. The exon-intron structure was analyzed by PCR technique, and the up-stream of 1st exon of the gene (about 3000 base pair) was sequenced. TATA box, inverted CCAAT, GATA-1, AP- 1, GR structure were identified in the region of gene expression controling regin.(2)Bovine enamelin cDNA was cloned from bovine ameloblast cDNA libraiy using PCR product of porcine enamelin as probe. The cloned cDNA was inserted into pET29c (+) expression vector, and the recombinant bovine enamelin peptide was produced by E.coli. The peptide was immunized to rabbit, antibody against recombinant bovine enamelin was obtained. The antibody was used in the immunohistochemical analysis of bovine tooth germ. The production and secretion of the enamelin was seemed to relate with calcification of the enamel matrix.(3)The proteinases involved in tooth resorption was investigated by comparing the active and resting phases of root-resorbing tissue. RNA was extracted from root-resorbing tissue of bovine mandible. Using RT-PCR and Northern blot analysis, the expression of proteinase mRNAs in both active- and resting-phase tissues was investigated. The results showed MMP-l, MMP-2, MMP-9, MMP-13, MTl-MMP, and cathepsin K mRNAs in both phases. Of these, MMP-1, MMP-9, and cathepsin K were expressed much more in the active root-resorbing tissue than in the resting tissue. Gelatin zymography showed that the root-resorbing tissue has gelatinolytic activity too. From these findings, it may be concluded that MMP-1, MMP-9, and cathepsin K play an important role in root resorption of deciduous teeth.