The analysis for role of enamel and enamel related protein in, during body formation

采用原位PCR方法对大鼠上磨牙组织中淀粉原蛋白的表达进行了研究，并对扩增基因进行了组织学检测，以探讨淀粉原蛋白在组织形成中的作用。本研究的结果证实了成釉细胞和初始未分化成牙细胞中有成釉原蛋白的表达。不仅在未分化的成牙细胞中，在一定程度上成熟的成牙细胞中也有微弱的淀粉原蛋白基因表达。然而，由于该方法快速地重复高温到低温的变化，导致组织切片出现了许多问题，如切片从切片草上移除和组织破坏，因此选择了一种新的基因扩增方法来代替PCR。近年来，LAMP方法采用了四种引物对基因进行扩增。本方法的特点是在75℃的恒温条件下，扩增效率比一般PCR方法高十倍到几百倍。本研究采用原位LAMP法检测组织切片中扩增的基因表达，与原位PCR法检测成牙髓细胞和牙髓细胞中淀粉原蛋白的基因表达。在此之前，一些研究者报道了原位LAMP方法对软组织的影响。然而，没有使用硬组织的报道。因此硬组织原位LAMP系统目前尚未建立，本研究被迫建立原位LAMP法硬组织原位LAMP系统。此外，本研究还检测了主要的牙釉质蛋白酶之一KLK4。从本研究结果来看，不仅在成釉细胞中，在口腔癌细胞中也观察到了KLK4基因的表达和蛋白的定位。这一结果表明，淀粉原蛋白可能对细胞的不同部位有不同的作用。

The expression of amelogenin in rats upper molar were studied by using in situ PCR method that detected amplified gene in histology to examine a role of amelogenin for tissue formation. Results of this study, the expression of amelogenin were confirmed in the ameloblasts and initial undifferentiational odontoblasts. Furthermore, the faintly amelogenin gene expression were observed not only in the undifferentiational but also in some degree of maturational odontoblasts. However, a lot of problems of tissue sections such as removing sections from a slide grasses and a destruction of the tissue were happened in this method because of repeating the temperatures change from high temperatures to a low temperatures quickly, Therefore a new gene amplified method was selected in stead of PCR. Recently, the LAMP method that was used four primers on gene amplification. The character of this methods are used a constant temperature of 75℃, and the amplification efficiency of this method is from ten to hundreds of times higher than a general PCR methods.In this study, in situ LAMP method that was detected amplified gene expression in the tissue section, same as in situ PCR method was tried to detect the gene expression of amelogenin in odontoblasts and pulp cells. Previously, some researchers were reported that using in situ LAMP method to soft tissue. However there were no reports for using hard tissues. Therefore the in situ LAMP system for hard tissues has not been established now, and this study was enforced to establish a system for hard tissues with in situ LAMP method.Moreover, one of main enamel proteases, KLK4 was also examined in this study. From the results of this study, KLK4 gene expression and protein localization were observed not only in ameloblasts, but also in oral cancer cells. This result indicated that amelogenin might has a several role for several areas of cells.