The development of the 'newly high sensitive biological evaluation method for dental organic materials

采用高效液相色谱法，建立了一种快速、灵敏地同时测定唾液中口腔有机物质及其代谢物浸出物的方法。说明双酚A有可能从聚碳酸酯正畸托槽中进入口腔，邻苯二甲酸二丁酯也有可能从临时填充物中进入口腔。从一个孩子身上可以明显看出，这些物质的日摄入量是不可忽视的。逆转录病毒载体导入雌激素反应元件（estrogen responsive element， ERE）和eGFP编码基因的HeLa细胞在雌二醇- 17 β刺激下表达荧光。雌激素最低浓度为1 ng/mL，刺激12小时后细胞表达GFP蛋白。可以建立人雌激素受体（hERα）上调、78-kDa葡萄糖调节蛋白（GRP78）下调的rsa源性细胞。用瓦巴因抗性试验进行突变分析，结果表明，hERα过量产生细胞对BPA致突变作用的敏感性与载体对照细胞相似。然而，grp78下调的细胞对BPA致突变性的敏感性高于对照细胞：在grp78下调的细胞中，浓度超过1 × 10^<-9> ~ 10^<-8> M时检测到致突变性。因此，grp78下调的细胞可能有助于评估环境化学物质在极低浓度下的致突变性。将一个基因构建体Choriogenin L 1.5Kb/GFP与另一个基因构建体emgb/RFP连接，将双融合基因构建体注入1或2细胞期胚胎，并在红系细胞中选择表达RFP的胚胎。当暴露于雌二醇-17β时，胚胎培养的成鱼显示肝脏特异性表达GFP，这表明1.5Kb的5'上游区域促进了雌激素依赖性chg-L基因的肝脏特异性表达。该转基因medaka可作为检测环境雌激素的实验动物之一。双酚a单独不诱导CYP1A1基因的转录激活，但双酚a与3-甲基胆蒽（MC）一起超诱导人CYP1A1基因。此外，BPA通过AhR/Arnt复合物增强了CYP1A1基因的转录激活。这些结果表明，BPA与3-MC对人CYP1A1基因转录的超诱导参与了细胞内Ca^<2+>浓度升高对肌内质网Ca2+- atp酶的抑制作用。从这些结果来看，本研究开发的每一种评价方法都可以被认为是一种新的高灵敏度的生物学评价方法，可以应用于牙科用有机材料的基础生物学试验。少

The simply simultaneous method to analyze in the high sensitivity to identify the leached substances from the dental organic materials immersed in saliva and its metabolites was developed using HPLC. It could be clarified the possibility that bisphenol A was immigrated from the polycarbonate orthodontic bracket into the oral cavity and that dibutyl phthalate did also from the temporary filling materials. It became obvious with a child that the daily intake of these substances was the quantity which could not be ignored. The HeLa cell that was introduced genes encoding estrogen responsive element (ERE) and eGFP by retrovirus expression vector, expressed fluorescence when they were stimulated with estradiol-l7β. The lowest concentration of estrogen was 1 ng/mL by which the cells expressed GFP protein 12 hr after the stimulation. RSa-derived cells with up-regulation of human estrogen receptor (hERα) and down-regulation of 78-kDa glucose regulated protein (GRP78) could be established. The … More hERα overproducing cells showed the similar sensitivity to the mutagenic effects of BPA with vector control cells in mutation analysis using ouabain resistance test. However, GRP78-down-regulated cells showed higher sensitivity to BPA mutagenicity than the control cells: the mutagenicity was detected at concentrations over 1 x 10^<-9>〜10^<-8> M in GRP78-down-regulated cells. Thus, GRP78-down-regulated cells may be useful for evaluating mutagenicity of environmental chemicals at very low concentrations. A gene construct Choriogenin L 1.5Kb/GFP to another construct emgb/RFP was linked, injected the double fusion gene construct into 1-or 2-cell-stage embryos, and selected embryos expressing RFP in erythroid cells. When exposed to estradiol-17β, adult fish raised from the embryos showed the liver-specific expression of GFP, suggesting that the 1.5Kb 5'-upstream region promotes estrogen-dependent liver-specific expression of the chg-L gene. This transgenic medaka may be useful as one of test animals for detecting environmental estrogens. BPA alone didn't induce the transcriptional activation of the CYP1A1 gene, but BPA together with 3-methylcholanthrene (MC), superinduced the human CYP1A1 gene. In addition, BPA enhanced the transcriptional activation of the CYP1A1 gene through the AhR/Arnt complex. These results suggested that the superinduction of the human CYP1A1 gene transcription by BPA together with 3-MC is involved in the increasing concentration of intracellular Ca^<2+> to the inhibition of the sarco-endoplasmic reticulum Ca2+-ATPases. From these results, each evaluation developed by this present research can be thought to be the high new sensitive biological evaluation method which can be applied to the basic biological test of the organic material for the dentistry. Less