AIM:To study the expression of Runx2 in the dental follicle cells(DFCs) of mouse tooth germ at bell stage.METHODS:Mandibles including the first molar germs were removed from 5~7-day-old BALB/c mice and 5 μm sections were prepared for the following experiments.In situ hybridization and immunohistochemical (ICH) techniques were used to determine the tissue distribution and cellular localization of Runx2 mRNA and protein in dental follicle tissue.Then, loose dental follicle tissue around mandibular first molar germs at bell stage were isolated under stereomicroscope from the mice.Primary DFCs were obtained by trypsin digestion and cultured in vitro.Total RNA and protein were respectively extracted from cultured DFCs.RT-PCR and Western blot were adopted to observe the expression of Runx2 mRNA and protein in DFCs.RESULTS:Mandibular first molar germs of 5~7-day-old BALB/c mice were at later bell stage, the epithelial root sheath had already innerly folded, however, the root still could not be seen.Dental follicle tissue surrounding the developing dental germ was loose fibrous connective tissue.In situ hybridization showed Runx2 mRNA expression in DFCs,but IHC showed no Runx2 protein expression in the cells.RT-PCR showed Runx2 mRNA experssion in DFCs, while Western blot showed no Runx2 protein expression in the cultured DFCs.CONCLUSION:Runx2 may have a role in the development and differentiation of DFCs, but not at dental follicle of pre-root-formation.%目的:研究Runt相关基因2(Runx2)在小鼠牙胚钟状期牙囊细胞中的表达.方法:取出生后5~7 d 的BALB/c小鼠含下颌第一磨牙牙胚的下颌骨,并采用原位杂交和免疫组化的方法,观察Runx2 mRNA及其蛋白在牙囊组织中的表达;随后再取小鼠下颌第一磨牙牙胚的牙囊组织,原代培养牙囊细胞后,分别采用RT-PCR和Western blot法检测Runx2 mRNA及其蛋白在体外培养牙囊细胞中的表达.结果:出生后5~7 d 的BALB/c小鼠下颌第一磨牙牙胚处于钟状晚期,经原位杂交和免疫组化染色观察发现,Runx2 mRNA在牙囊细胞的胞浆中有表达,而Runx2蛋白未见表达.体外培养的牙囊细胞经RT-PCR和Western blot检测发现,Runx2 mRNA呈阳性表达,但未检测到Runx2蛋白的表达,与体内牙囊细胞的表达相一致.结论:Runx2对牙根形成前期牙囊的生长发育可能具有一定的潜在作用,但可能并未直接参与.
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