摘要
目的:观察体外培养的牙髓细胞向成牙本质细胞分化过程中碱性磷酸酶的表达和矿化结节形成能力,建立成牙本质细胞样细胞体外培养体系。方法:实验于2004-07/2005-07在中山大学光华口腔医学院完成。利用组织块酶解法培养人牙髓细胞并进行鉴定,取第3代牙髓细胞,胰酶消化后进行细胞计数,连续观察7d,绘制生长曲线。第3代牙髓细胞,使用矿化诱导液诱导其向成牙本质细胞样细胞分化,观察细胞形态改变和碱性磷酸酶活性变化,用Von kossa染色法观察诱导后的细胞形成矿化结节的能力。结果:①获得的牙髓细胞均为波形丝蛋白阳性,角蛋白阴性,证明为中胚层来源的细胞。②诱导分化的牙髓细胞在2周左右进入复层生长期;3周左右开始有细胞结节形成,周围细胞呈放射状排列,部分细胞出现细长突起并呈极性排列。4周左右细胞团中央Von Kossa染色为阳性。③与未诱导的细胞相比,矿化诱导开始3周碱性磷酸酶活性逐渐升高,但到第4周后活性有所降低。结论:实验成功建立了成牙本质细胞样细胞体外培养体系,所获得的成牙本质细胞样细胞具有成牙本质细胞的部分形态和生物学功能,是研究成牙本质细胞的较好模型。
AIM: To observe the expression of alkaline phosphatase and nodal formation in the differentiation of dental pulp cells cultured In vitro into odontoblast and to establish In vitro culture system of odontoblast-like cells. METHODS: The experiment was performed at the Guanghua Stomatologic College, Sun Yat-sen University from July 2004 to July 2005. Human dental pulp cells were cultured by tissue enzymatic digestion method and identified. The third passage cells were obtained. Cell counting was conducted after trypsinization, successively for 7 days. Growth curve was drawn. The third passage dental pulp cells were induced to differentiate into odontoblast-like cells with mineralization induction liquor. At the same time, changes of cell morphology and alkaline phosphatase activity (APA) were observed. Yon kossa staining was applied to observe the nodal formation of odontoblast-like cells after induction. RESULTS: ①Dental pulp cells obtained were all vimentin positive and keratin negative staining. It verified that the cells were from mesoblast. ②Around the second week, dental pulp cells were stratified, then they had node, arranged radiated shape, and partial cells had slender evection and were polarized at about the third week. Yon kossa staining became positive at about the Fourth week. ③APA increased gradually at week 3, but decreased from the fourth week. CONCLUSION: In vitro cultured system of odontoblast-like cells is successfully established. The obtained odontoblast-like cells that have the partial appearance and biological function of odontoblast are better models for odontoblast study.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第11期2005-2008,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research