標識抗体法による歯牙齲蝕症の病理学的研究

とくに齲蝕象牙質における唾液成分と微生物の局在性について

抄録

The hypothesis that immunologic mechanism may play an important role in modifying the carious process was suggested by Blechman and Mori (1965). Presently an attempt was made to find the localization of salivary components, immunoglobulins, serum albumin and oral microorganisms in the human sound and carious dentin with a fluorescent antibody technique (Coons and Kaplan, 1950), an enzyme (peroxidase-labeled) antibody technique (Nakane and Pierce, 1967) and other staining techniques, as well as by microradiography.
Seventy freshly extracted sound and carious human permanent teeth (Table 1) were immediately put in cold 95% ethanol for fixation. These were divided into 2 segments. A segment from each tooth was hand polished to a thickness of about 70μ, cooled with cold saline and used for microradiographic studies.
Then it was demineralized in 0.5M pH7.4 EDTA (2Na-salt) at 4°C for 2-3 days. Another segment was demineralized in the same solution at 4°C for 7-14 days and embedded with paraffin. Microtome sections were made from each segment to a thickness of 7μ(Table 2).
Rabbit antiserums to human saliva, human immunoglobulins*, human serum albumin* and oral microorganisms were prepared. Fluorescein isothiocyanate**, tetramethylrhodamine isothiocyanate** and horseradish peroxidase*** were conjugated with the globulin fraction of rabbit antiserum. Commercially available fluoresceinlabeled anti human immunoglobulins serum** was also used. Control studies of specificity were made with labeled antiserum from which specific antibody had been absorbed by the addition of antigen.
When stained with labeled anti human saliva serum, labeled anti human immunoglobulins serum and labeled anti human immunoglobulins serum and labeled anti human serum albumin serum, specific reaction was usually observed in dentinal tubules (Figs. 6 and 7), dentinal fibers, predentin and the odontoblast layer of dentin (Fig. 8). These labeled antiserums also resulted in the most intense reaction in dentinal tubules beneath a translucent zone in the ground section, a radiopaque zone in microradiogram, of carious dentin (Figs. 10, 11, 14, 16, 17 and 18). Azure A eosin B stain, PAS stain and bromphenol blue stain produced a staining similar to that given by these labeled antiserums (Figs. 14, 15 and 19). Moreover, labeled anti human saliva serum resulted in diffuse reaction in soft carious dentin (Figs. 9 and 19). Although microorganisms were localized in dentinal tubules and the matrix of carious dentin (Figs. 20 and 21), no specific relation was observed between the localization of salivary components and that of oral microorganisms.
*Nutritional Biochemicals Co., Cleveland, Ohio, U. S. A.
**Baltimore Biological Laboratory, Baltimore, Maryland, U. S. A.
***Sigma Chemical Co., St. Louis, Missouri, U. S. A.