人牙髓细胞原代培养方法的研究

The Primary Culture of Human Tooth Pulp Cell

目的 确定酶解消化法牙髓细胞原代培养的合适条件与时间。方法 选取年轻患者因正畸拔除的完整和无龋坏牙齿 ,取出牙髓 ,分别采用胶原酶一步法消化牙髓组织 30分钟及 1、2、3小时 ,胶原酶分步消化法 ,组织块法和组织块酶解法进行原代培养。倒置显微镜台盼蓝染色观察细胞解离及存活状况 ,免疫组化法鉴定细胞来源。结果 胰酶消化 2小时后 ,组织块基本未解离。用 0 .1%胶原酶一步法消化 1、2、3小时后组织块均彻底解离为单细胞 ,基本无残余。 0 .1%胶原酶消化 30分钟后组织块解离不完全。 0 .1%胶原酶分步法消化 1小时后组织解离彻底。台盼蓝染色表明经胶原酶一步法消化获得的牙髓细胞被台盼蓝着色的比例随消化时间减短而下降。分步法消化细胞着色率低于一步法。组织块酶解法的组织块解离、贴附和细胞游出状况均优于组织块法。结论 型胶原酶在 37℃ ,持续振荡条件下消化 1小时是彻底酶解消化人牙髓的最短时间。分步酶解法能获得比一步酶解法更高的活细胞数量。而从效果、操作难易和耗时多少等多方面因素综合考虑 。

Objective In this study, we established the optimal condition and duration for the primary culture of the human tooth pulp cell with collagenase digestion method. Method Pulp tissue was removed from healthy young human teeth extracted for orthodontic purposes, and then the pulp was digested by collagenase separately in duration of 30mins, 1 hr, 2 hrs, 3 hrs. After the digestion , the viability and digestion efficiency were evaluated by microscopy and trypan-blue dying. The cytokeratin and vimentin were immunocytochemically detected to identify the cell phenotype. The tissue explant culture and trypsin digestion were set as controls. Results After digestion of 1hr, 2hrs, or 3hrs, little tissue residue was left, while there is still much tissue remained after digestion of 30mins. The viability decreased with the elongation of digestion duration. Conclusion 37℃, continuing stirring and 1hr of type Ⅰ collagenase digestion are the optimal conditions for the primary culture of human tooth pulp with digestion method.