组织块法重复培养牙周膜干细胞的生物学功能研究

Study on biological function of periodontal ligament stem cells in repeated culture by tissue block method

目的对同一块牙周膜组织重复培养获得的原代牙周膜干细胞(periodontal ligament stem cell,PDLSC)的生物学特性和功能进行比较,评价重复培养得到的PDLSC能否用于实验研究和临床治疗。方法收集正畸治疗拔除的前磨牙,组织块法进行PDLSC原代培养。首次传代后收集剩余的牙周膜组织重复进行组织块法细胞培养,多次培养获得原代PDLSC。每次培养的原代PDLSC进行扩增培养,选取第1、3、5次培养获得的PDLSC分为A、B、C组。分别绘制细胞增殖曲线,STRO-1表面标记物表达的检测鉴定,细胞周期分布检测和观察成骨诱导,测定各组PDLSC端粒长度和端粒酶表达水平。结果三组细胞的STRO-1表达均呈阳性;生长趋势和扩增数量相似;A、B两组细胞周期分布无显著差异(P>0.05),C组与A、B组比较细胞周期分布差异显著(P<0.05);地塞米松诱导三组PDLSC成骨,21d时均有茜素红染色阳性的钙化结节;A、B两组PDLSC端粒长度、端粒酶表达水平均无显著差异(P>0.05),C组PDLSC端粒长度显著长于A、B组(P<0.05),端粒酶表达水平显著高于A、B组(P<0.05)。结论通过同一块牙周膜组织培养得到的PDLSC生物学功能相同,但在细胞安全性上是否能够用于实验研究和临床治疗有待进一步的探讨。

Objective To compare the biological characteristics and functions of periodontal ligament stem cells(PDLSCs)obtained from repeated culture of the same periodontal ligament tissue,so as to evaluate whether the PDLSCs obtained from repeated culture can be used for experimental research and clinical treatment.Methods PDLSCs were isolated from orthodontic extracted premolars and cultured in vitro.After the first passage,the remaining periodontal ligament tissue was collected and repeated for cell culture by tissue block method to obtain primary PDLSCs.PDLSCs from each culture were amplified and cultured.PDLSCs obtained from the first,third,and fifth culture were divided into groups A,B,and C.The cell proliferation curve was drawn,the expression of STRO-1 surface markers was detected,the cell cycle distribution was detected,and the osteogenic induction was observed.The telomere length and telomerase expression level of PDLSCs in each group were measured.Results The expression of STRO-1 was positive in all three groups.The growth trend and number of amplifications were similar.There was no significant difference in cell cycle distribution between group A and group B(P>0.05),but there was significant difference in cell cycle distribution between group C and groups A and B(P<0.05).Dexamethasone induced osteogenesis of PDLSCs in three groups,and alizarin red staining showed positive calcified nodules at 21 days.The telomere length and telomerase expression level of PDLSCs in groups A and B had no significant difference(P>0.05),but the telomere length of PDLSCs in group C was significantly longer than that in groups A and B(P<0.05),telomerase expression level was higher than that in groups A and B(P<0.05).Conclusion The biological function of PDLSCs derived from the same periodontal ligament tissue culture is the same,but whether PDLSCs can be used for experimental research and clinical treatment in terms of cell safety needs to be further explored.