Molecular Biology of Differentiation of Hard Tissue Forming Cells

1.用原位杂交和免疫组织化学方法分析釉原蛋白基因在大鼠牙胚中的表达。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，进行了原位杂交和免疫组化研究。用1%戊二醛灌流Wistar大鼠，解剖上切牙及其周围组织。组织脱钙后制成石蜡切片。用T7聚合酶从pSPT18中亚克隆的860bp全长cDNA模板中合成了釉原蛋白反义RNA。[Alpha^&lt；35&gt；S]在反应混合物中加入UTP，得到比活度为5×10~(-8)cpm/mug的探针。在成釉细胞上检测到从成熟前期到成釉细胞的杂交信号。中间层和星状网的细胞未见杂交信号。在切片的免疫组织化学染色中，从妊娠前期到晚期…的成釉细胞中可观察到阳性信号。更多的交流阶段。这些结果表明，成熟前期的成釉细胞表达釉原蛋白基因，而成熟后期的成釉细胞不表达釉原蛋白，成熟期的成釉细胞表达再吸收蛋白。人成骨肉瘤成骨细胞系胶原酶基因表达的调控。采用Northern杂交方法研究胶原酶基因在人成骨肉瘤成骨细胞系MG-63、U2-OS和人成纤维细胞系IMR-90中的表达。静止的MG-63、U2-OS和IMR-90细胞暴露于TPA和10%FCS后，可诱导编码胶原酶的mRNA表达。EGF可诱导IMR-90细胞胶原酶mRNA表达，但对MG-63和U2-OS细胞无明显影响。在IMR-90和MG-63细胞中，EGF刺激c-fos和c-jun基因的转录，这些转录因子直接与人胶原酶基因启动子区域相互作用。甲状旁腺激素和1，25-二羟基维生素D_3对骨肉瘤细胞胶原酶基因表达水平无明显影响。IL-1β可诱导MG-63细胞胶原酶和c-jun的表达，但不能诱导c-fos的表达。原位杂交法检测牛根吸收组织中胶原酶mRNA的表达据报道，活性根吸收组织具有很强的胶原酶分解活性。为了阐明胶原酶产生细胞的类型以及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。除巨噬细胞、成纤维细胞、成牙本质细胞和成牙本质细胞外，破牙本质细胞中也有胶原酶基因的表达。较少

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