Molecular Biology of Differentiation of Hard Tissue Forming Cells

1.釉原蛋白基因表达的原位杂交和免疫组织化学分析为了阐明釉原蛋白基因在大鼠牙胚中表达的时间和空间模式，我们进行了原位杂交和免疫组织化学研究。Wistar大鼠用1%戊二醛灌注，解剖上切牙及其周围组织。将组织脱钙并处理用于石蜡切片。通过T7聚合酶从亚克隆在pSPT 18中的860 bp全长cDNA模板合成釉原蛋白反义RNA。将[α ^<35>S]UTP加入到反应混合物中，得到比活度为5 × 10^8 cpm/mug的探针。从分泌前期到成熟初期的成釉细胞均检测到杂交信号。中间层和星网状层细胞未显示杂交信号。免疫组化染色显示，从分泌前期到成熟晚期，成釉细胞中均可见阳性信号， ...更多信息 化阶段。这些结果表明，成釉细胞从分泌前到成熟初期表达釉原蛋白基因，而成熟晚期不表达，成熟期的成釉细胞吸收釉原蛋白分子.人骨肉瘤成骨细胞系中胶原酶基因表达的调控通过北方分析研究人骨肉瘤成骨细胞系MG-63、U2-OS和人成纤维细胞系IMR-90中胶原酶基因表达的调控。将静止的MG-63、U2-OS和IMR-90细胞暴露于TPA和10%FCS导致编码胶原酶的mRNA的诱导。EGF可诱导IMR-90细胞胶原酶mRNA的表达，但对MG-63和U2-OS细胞胶原酶mRNA的表达无明显影响。在IMR-90和MG-63细胞中，EGF刺激编码转录因子的c-fos和c-jun基因的转录，所述转录因子直接与人胶原酶基因的启动子区域相互作用。PTH和1，25-二羟维生素D_3均不增加两种骨肉瘤细胞胶原酶mRNA的表达。IL-1 β可诱导MG-63细胞胶原酶和c-jun mRNA的表达，但对c-fos mRNA的表达无影响.牛牙根吸收组织中胶原酶mRNA的原位杂交检测研究发现，活跃的牙根吸收组织具有较强的胶原酶活性。为了阐明胶原酶产生细胞的类型和胶原酶在牙根吸收中的作用，用地高辛标记的非放射性RNA探针对牛牙根吸收组织切片进行原位杂交。胶原酶mRNA表达在破牙本质细胞、巨噬细胞、成纤维细胞、成牙本质细胞和成牙骨质细胞中均清晰可见。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