体外培养人牙周膜细胞方法的探讨.docVIP

体外培养人牙周膜细胞方法的探讨 nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 作者：张丽，王永，张军梅，侯豫，吴承龙，王文秀 【摘要】nbsp; 　　目的： 研究如何提高体外培养人牙周膜细胞（hPDL）的成功率，为简便、快速地获得大量成活率高的hPDL膜细胞，建立体外细胞模型提供一定的实验方法。方法： 利用3种不同方法（组织块法、酶消化组织块法、全牙消化法）进行牙周膜细胞的原代培养，用形态学、免疫细胞化学染色等方法鉴定细胞来源；通过生长曲线的测定研究体外细胞生长特性。结果： 3种方法获得的细胞形态和生物学行为大致相同；波丝蛋白抗体染色阳性，角蛋白抗体染色阴性。组织块法培养的细胞同源性好，但初代培养时间长；全牙消化法的原代培养成功率高于其他2种方法。结论： 通过对牙周膜细胞培养方法的改良，可以显著提高hPDL原代培养的成功率。 【关键词】nbsp; 牙周膜； 细胞培养； 免疫组织化学 　　［Abstract］ Objective: To establish a simple, fast, and efficient technique for human periodontal ligament （hPDL） cell in vitro cultivation with higher success rate. Methord: Primary hPDL cells were cultured using three different methods （tissue explant, explant with enzymatic digestion, and tooth enzymatic digestion）. Morphological analysis and immunocytochemical staining were used to characterize the cell lineage, and growth curve assay was adopted to evaluate the biological features of hPDL cells. Results: Morphological and biological characteristics of cells cultured with the 3 kinds of method were similar. They were spindle-shaped, and showed positive reaction to antibodies against vimentin, and negative reaction to antibodies against keratin. Cells obtained with tissue explant showed better homology, but need longer culture time; The success rate of primary hPDL cell culture in tooth enzymatic digestion group was significantly higher than those in the other 2 groups. Conclusion: By making improvement in specimen collecting and culturing methods, the success rate of primary culture of hPDL cells can be noticeably increased. 　　［Key words］ periodontal ligament; cell culture; immunohistochemistry nbsp;nbsp;nbsp; 细胞体外分离培养是研究复杂的生物系统的重要手段，细胞培养条件下，将细胞从复杂的体内环境转移到较为简单的体外环境，可对细胞学研究中的变量进行分离和控制。上世纪70年代以来，国外学者对牙周膜细胞（Periodontal LigamentCells PDL）分离纯化进行了大量的探索和研究，且成功地从猴、猪、人等牙周膜分离培养出牙周膜细胞［1～3］。人牙周膜细胞（hPDL）分离培养主要有酶消化法和组织贴块法［4］。国内外学者对这2种方法评价不一，并且在这2种方法的基础上进行改进提出了酶消化组织块法和全牙消化法［5］。对组织块法、酶消化组织块法