贴壁组织块反复消化法培养新生大鼠下颌骨成骨细胞及鉴定.doc

贴壁组织块反复消化法培养新生大鼠下颌骨成骨细胞及鉴定 贴壁组织块反复消化法培养新生大鼠下颌骨成骨细胞及鉴定 作者：鄂玲玲 刘洪臣 王东胜 作者单位：中国人民解放军总医院 口腔医学研究所，北京 100853 【摘要】 目的 探讨经济、高效的原代新生大鼠下颌骨成骨细胞培养方法。方法 将新生24 h SD大鼠处死并取出下颌骨，剪碎，用改良的贴壁组织块反复消化法培养细胞，噻唑蓝(MTT)比色法检测其增殖能力，通过相差显微镜观察，用骨钙素免疫荧光染色、碱性磷酸酶染色和钙结节染色方法对所获得的细胞进行鉴定。结果 MTT实验显示接种后1～3 d内细胞增殖缓慢，为细胞生长适应期，第7天达到高峰，以后增长速度逐渐减慢。所培养的细胞能形成矿化结节，碱性磷酸酶染色、骨钙素染色和钙结节染色均呈阳性，具有典型的成骨细胞形态特征。结论 改良的贴壁组织块反复消化法可获得大量且纯的新生大鼠下颌骨成骨细胞，所培养的细胞具有典型的成骨细胞形态和功能。 【关键词】 下颌骨 成骨细胞 细胞培养 　　Primary culture and identification of neonatal rat′s mandibular osteoblasts with modified repeating enzymatic digestion-adherent explants method E Ling-ling, LIU Hong-chen, WANG Dong-sheng. (Stomatologic Institute, General Hospital of Chinese PLA, Beijing 100853, China) 　　[Abstract] Objective To investigate an efficient method of primary mandibular osteoblasts(MO) culture. Methods The mandible harvested from 24 h SD rats was stripped off all soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then the bone block was subcultured in culture flask after digested with modified repeating enzymatic digestion-adherent explants method. The proliferation of the acquired cells were examined with assay of methyl thiazolyl tetrazolium(MTT) and identified with invert microscope, immunohistochemistry stain of osteocalcin, alkaline phosphorase(ALP) stain and stain of calcified nodules. Results MTT assay showed that the cells grew slowly in 1-3 days of post-inoculation, it was cell growth adaptation period. At seventh day, the cells growth reached highest, then the proliferation of the cells was slow gradually. The cells were identified to be osteoblasts by invert microscope, immunohistochemistry stain of osteocalcin, ALP stain and alizarin bordeaux stain of calcified nodule. Conclusion The modified repeating enzymatic digestion-adherent explants method is an ideal methodto obtain and culture neonatal rat′s MO having typical characteristics. 　　[Key words] mandible; o