Molecular Biology of Differentiation of Hard Tissue Forming Cells

1. 原位杂交和免疫组织化学分析淀粉原蛋白基因表达。为了阐明大鼠牙胚中淀粉原蛋白基因表达的时空格局，我们进行了原位杂交和免疫组织化学研究。用1%戊二醛灌注Wistar大鼠，解剖上门牙及周围组织。将组织脱钙并进行石蜡切片处理。在pSPT18中亚克隆了860 bp的全长cDNA模板，利用T7聚合酶合成了Amelogenin反义RNA。[α ^<35>S]将UTP加入到反应混合物中，得到比活性为5 × 10^8 cpm/mug的探针。在成釉细胞分泌前期至成熟初期均检测到杂交信号。中层细胞和星状网细胞没有杂交信号。切片免疫组化染色，成釉细胞从分泌前期到成熟后期均有阳性信号。这些结果表明，成釉细胞在分泌前期到成熟初期表达淀粉原蛋白基因，但在成熟后期不表达，成熟阶段的成釉细胞吸收淀粉原蛋白分子。胶原酶基因在人骨肉瘤源性成骨细胞系中的表达调控。用Northern分析法研究了胶原酶基因在人成骨肉瘤源性成骨细胞MG-63、U2-OS和人成纤维细胞IMR-90中的表达调控。将静止的MG-63、U2-OS和IMR-90细胞暴露于TPA和10% FCS中可诱导编码胶原酶的mRNA。EGF在IMR-90细胞中诱导胶原酶mRNA表达，而在MG-63和U2-OS细胞中无诱导作用。在IMR-90和MG-63细胞中，EGF刺激编码与人胶原酶基因启动子区直接相互作用的转录因子的c-fos和c-jun基因的转录。甲状旁腺激素和1,25 -二羟基维生素D_3在两种骨肉瘤细胞中均未增加胶原酶mRNA水平。2 . il -1 β在MG-63细胞中诱导胶原酶和c-jun mRNA，但不诱导c-fos mRNA。原位杂交法检测牛根吸收组织胶原酶mRNA。据报道，活跃的根吸收组织具有很强的胶原溶解活性。为了阐明胶原酶产生细胞的类型和胶原酶在根吸收中的作用，用地高高素标记的非放射性RNA探针对牛根吸收组织切片进行原位杂交。胶原酶mRNA在破牙细胞、巨噬细胞、成纤维细胞、成牙髓细胞和成骨水泥细胞中均有明显表达。少

1. Analysis of amelogenin gene expression by in situ hybridization and immunohistochemistry.To elucidate temporal and spatial patterns of amelogenin gene expression in the rat tooth germ, in situ hybridiazation and immunohistochemicalstudies were carried out. Wistar rats were perfused with 1% glutaraldehyde and the upper incisors with surrounding tissues were dissected. The tissues were decalcified and processed for paraffin sections. Amelogenin antisense RNA was synthesized by T7 polymerase from a 860 bp full-length cDNA template subcloned in pSPT18. [alpha^<35>S]UTP was added to the reaction mixture to yield probes with specific activities of 5x10^8 cpm/mug. The hybridization signals were detected over ameloblasts from presecretory to the begining of maturation stage. The cells of stratum intermedium and stellate reticulum did not show hybridization signals. In immunohistochemical staining of the sections, positive signals were observed in ameloblasts from presecretory t o late matur … More ation stage. These results indicate that ameloblasts from presecretory to the beginning of maturation stage express amelogenin gene but not in late maturation stage and ameloblasts of maturation stage resorb amelogenin molecules.2. Regulation of collagenase gene expression in human osteosarcoma-derived osteoblastic cell lines.The regulation of collagenase gene expression in the human osteosarcoma-derived osteoblastic cell lines MG-63, U2-OS and human fibroblasts cell line IMR-90 was investigated by Northern analysis. Exposure of quiescent MG-63, U2-OS and IMR-90 cells to TPA and 10% FCS resulted in the induction of mRNA encoding collagenase. EGF induced collagenase mRNA in the IMR-90 cell but not in the MG-63 and U2-OS cells. In the IMR-90 and MG-63 cells, EGF stimulated the transcription of the c-fos and c-jun genes encoding the transcriptional factors which interact directly with the promoter region of the human collagenase gene. PTH and 1, 25-dihydroxyvitamin D_3 did not increase the collagenase mRNA level in both osteosarcoma cells. IL-1beta induced collagenase and c-jun but not c-fos mRNA in the MG-63 cell.3. Detection of collagenase mRNA in bovine root resorbing tissue by in situ hybridization.It has been reported that active root resorbing tissue has a strong collagenolytic activity. in order to elucidate the type of collagenase-producing cells and the role of collagenase in root resorption, in situ hybridization of bovine root resorbing tissue sections were conducted with digoxigenin-labeled non-radioactive RNA probes. Collagenase mRNA expression was clearly observed in odontoclasts in addition to macrophages, fibroblasts, odontoblasts and cemento-blasts. Less