Elucidation of the caries-inducing mechanism by S.mutans and development of a diagnostic method to estimate caries-risk.

最近，我们构建了分别表达编码GTF-I、-SI和-S酶的变形链球菌gtfB、gtfC和gtfD的米勒链球菌转化子KSB 8、KSC 43和NH 5，并建立了产生对这三种GTF单特异性的单克隆抗体的杂交瘤。在这项研究中，在体外和体内致龋性的三个转化子进行了比较，阐明致龋的变异链球菌的机制。此外，使用单特异性单克隆抗体，一个高灵敏度的Gross-dot方法测定的三个GTF。主要研究结果如下：1.从感染KSC 43.2的大鼠的口腔中重新分离更稳定的表达gtfC的pETKSC 43-RT。通过定殖试验检查转化体的体外致龋性。结果表明，只有KSC 43-RT细胞能在玻璃表面形成定植.通过大鼠模型系统测试转化体的体内致龋性。结果表明，KSC 43-RT细胞能诱导颊面龋和沟龋，KSB 8细胞能诱导邻面龋，而NH 5细胞不能诱导邻面龋.建立并应用高灵敏度的Gross-dot法评价变形链球菌的致龋性.通过该方法分析了来自各种变形链球菌临床分离株的细胞相关GTF。结果表明，不同菌株的GTFs含量及其比值存在明显差异，提示gtfC产物在变形链球菌致龋过程中起重要作用，高灵敏度的Gross-dot法可用于准确评价变形链球菌的致龋性。

Recentry, we have constructed S.milleri transformants KSB8, KSC43 and NH5 which individually express S.mutans gtfB,gtfC and gtfD coding GTF-I,-SI and -S enzymes, and established hybridoma which produce monoclonal antibodies mono-specific to the three GTFs. In this study, in vitro and in vivo cariogenicities of the three transformants were compared to elucicate the caries-inducing mechanism by S.mutans. Furthermore, using the mono-specific MAbs, a high-sensitive Gross-dot method for determination of the three GTFs was developed. The results obtained were as follows :1. More stable gtfC-expressing transformant KSC43-RT was reisolated from an oral cavity of rat infected with KSC43.2. In vitro cariogenicity of the transformants was examined by a colonization test. The results showed that only KSC43-RT cells could makedly colonized on glass surfaces.3. In vivo cariogenicity of the transformants was tested by a rat model system. The results showed that the KSC43-RT cells could induced the buccal and sulcal caries, and the KSB8 cells could induced the proximal caries, but the NH5 cells did not.4. High-sensitive Gross-dot assay method was established and applied in order to evaluate the cariogenicity of S.mutans strains.5. Cell-associated GTFs from various clinical isolates of S.mutans were analyzed by the method. The results showed that the levels of three GTFs and those ratio were markedly different among the isolates.These results suggest that the gtfC product was especially important for caries development caused by S.mutans, and that the high-sensitive Gross-dot method may be useful to estimate the exact cariogenicity of individual strain of S.mutans.