免疫荧光双重标记结合激光共聚焦扫描显微镜观察Ⅰ型胶原和硫酸软骨素在人牙本质中的分布  

Analysis of type-Ⅰ collagen fibrils and chondroitin sulfate distribution in human dentin by confocal laser scanning microscopy combined with dual immunofluorescent labeling technique

作　　者：芦帅[1] 赵三军[1] 孙勇[2] 高宇[1] 李晓静[1] 陈吉华[1] 

机构地区：[1]第四军医大学口腔医学院修复科军事口腔医学国家重点实验室,西安710032 [2]成都军区机关医院口腔科

出　　处：《中华口腔医学杂志》2015年第12期746-750,共5页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81130078、81170985）;教育部“长江学者和创新团队发展计划”（IRT13051）

摘　　要：目的 介绍牙本质在不完全脱矿下免疫荧光双标染色方法,研究Ⅰ型胶原和硫酸软骨素在人牙本质中的分布情况,以期进一步理解牙本质湿粘接的机制.方法 选取新鲜拔除的无龋第三磨牙8颗,制备30 μm厚的牙本质切片40张,用37％磷酸凝胶酸蚀15 s,应用免疫荧光双重标记方法和激光共聚焦扫描显微镜,观察硫酸软骨素经甲苯磺酰苯丙氨酰氯甲酮（tosyl-phenylalanyl chloromethyl ketone,TPCK）处理的胰蛋白酶消化去除前后相对胶原纤维的分布位置.结果 硫酸软骨素分布于牙本质小管管腔以及管周牙本质,Ⅰ型胶原纤维在管间牙本质和管周牙本质均存在;经特异性酶处理去除硫酸软骨素后,荧光强度明显减弱甚至消失.结论 免疫荧光双重标记结合激光共聚焦扫描显微镜可以在牙本质不完全脱矿的情况下研究其内部有机成分的分布;硫酸软骨素分布于牙本质小管管腔及管周牙本质,Ⅰ型胶原纤维分布于管间牙本质和管周牙本质.Objective To introduce the method of dual immunofluorescence labeling of human dentin matrix without demineralization of the whole dentin fragments, and to analyze the distribution of type-Ⅰ collagen fibrils and chondroitin sulfate in human dentin.Methods Forty 30 μm-thick middle coronal dentin sections were obtained from 8 freshly extracted human third molars and etched with 37% phosphoric acid（PA） gel for 15 s.After preconditioning with or without tosyl-phenylalanyl chloromethyl ketone（TPCK） treated trypsin digestion, sections were subjected to dual immunofluorescent labeling and scanned by confocal laser scanning microscopy to identify the type-Ⅰ collagen fibrils and chondroitin sulfate.Results Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-Ⅰ collagen fibrils were localized in intertubular dentin and peritubular dentin.After preconditioning with TPCK treated trypsin digestion, the red fluorescence was decreased or disappeared.Conclusions The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without demineralization of the whole dentin fragments.Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-Ⅰ collagen fibrils were localized in intertubular dentin and peritubular dentin.

关 键 词：软骨素硫酸盐类 胶原Ⅰ型 牙本质 显微镜检查 共焦 免疫荧光双重标记 COLLAGEN type Ⅰ 

分 类 号：R781[医药卫生—口腔医学]