Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix
Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix
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DOI：
10.4081/ejh.2009.e15
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发表时间：
2009-09
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通讯作者：
Alessandra Ruggeri;Giovanna Orsini;A. Mazzoni;F. Nato;Veronica Papa;M. Piccirilli;A. Putignano;Giovanni Mazzotti;E. D. S. Dorigo;Lorenzo Breschi
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作者：
Alessandra Ruggeri;Giovanna Orsini;A. Mazzoni;F. Nato;Veronica Papa;M. Piccirilli;A. Putignano;Giovanni Mazzotti;E. D. S. Dorigo;Lorenzo Breschi
本研究应用免疫组织化学方法，结合场发射透镜扫描电镜（FEI-SEM）、透射电镜（TEM）、荧光显微镜（FM）和生物化学方法，研究多功能蛋白聚糖在人牙本质有机基质中的分布。含有牙本质和前牙本质的标本从非龋性人牙齿获得，并分为三组：1）FEI-SEM组：切片暴露于包埋前免疫组织化学程序; 2）TEM组：标本固定，脱矿，包埋并提交包埋后免疫组织化学程序; 3）FM组：将切片矿化并进行荧光标记的预包埋免疫组织化学程序。将样本暴露于两种不同的抗体以测定多功能蛋白聚糖片段和整个多功能蛋白聚糖分子的分布。牙本质和牙髓提取物的蛋白质印迹分析也进行了。相关FEI-SEM、TEM和FM分析显示versican片段在前牙本质和牙本质中均呈阳性反应，而仅在TEM下在前牙本质中发现很少识别整个versican分子的金颗粒。FEI-SEM和FM分析未检测到多功能蛋白聚糖全分子的标记。免疫印迹分析证实了形态学结果。这项研究表明，在完全发育的人类牙齿中，多功能蛋白聚糖片段是人类牙本质和前牙本质有机基质的重要组成部分，而多功能蛋白聚糖整个分子只能在前牙本质中以稀缺的量可视化。versican片段在人牙本质有机基质中的作用有待进一步阐明。
Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.