中国实用口腔科杂志

中国实用口腔科杂志

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年龄因素对人牙周膜干细胞生物学行为影响的研究

张凯1，2，杜婷婷3，张毅4，吴迎涛1，5   

  1. 1. 青岛大学口腔医学院，山东  青岛  266003；2. 寿光市妇幼保健院口腔科，山东  寿光  262700；3. 山东省立医院口腔正畸科，山东  济南  250021；4. 山东省泰安市中心医院口腔科，山东 泰安 271000；5. 青岛市口腔医院牙周科，山东  青岛  266000
  • 出版日期:2020-08-15 发布日期:2020-09-09

  • Online:2020-08-15 Published:2020-09-09

摘要： 目的　研究年龄因素对人牙周膜干细胞（periodontal ligament stem cells，PDLSC）生物学行为的影响。方法  选取2017年10月至2018年10月于青岛市口腔医院口腔外科就诊的因阻生齿或正畸需要拔除牙齿的患者18例，根据不同年龄段分为3组，分别为A组（18 ~ 20 岁）、B组（30 ~ 35岁）、C组（50 ~ 55岁），每组6例。原代培养人PDLSC，采用衰老相关β-半乳糖苷酶染色、MTT法检测各组细胞衰老程度和增殖能力。成骨诱导人PDLSC，采用碱性磷酸酶（ALP）染色和茜素红染色检测各组细胞的成骨分化能力。采用实时荧光定量PCR（qRT-PCR）检测各组多能性相关转录因子Nanog和Sox2的表达水平。结果　各组均成功原代培养出人PDLSC，经衰老相关β-半乳糖苷酶染色发现，A组阳性染色细胞率最低、B组次之、C组最高，组间差异均有统计学意义（均P < 0.05）。通过MTT法检测发现，不同年龄组OD值比较，差异有统计学意义（F = 22.354，P = 0.009），且各组OD值均随着培养时间的延长而增大（F = 28.367，P = 0.018），年龄与时间之间有明显的交互作用（F = 26.431，P = 0.015）。ALP染色和茜素红染色均发现，A组染色面积百分比大于B组和C组，B组染色面积百分比大于C组，差异均有统计学意义（均P < 0.05）。qRT-PCR结果显示，A组Nanog和Sox2的相对表达量均高于B组和C组，B组Nanog和Sox2的相对表达量高于C组，差异均有统计学意义（均P < 0.05）。结论　随着年龄增加，衰老的人PDLSC增多，其增殖能力、成骨分化能力以及多能性相关转录因子的表达水平也显著下降。

关键词: 年龄, 牙周膜干细胞, 增殖能力, 成骨分化

Abstract: Objective　To evaluate the effect of age on biological behavior of periodontal ligament stem cells（PDLSC）. Methods　From October 2017 to October 2018，18 patients who needed to remove healthy teeth due to impacted teeth or orthodontics were treated in Qingdao Stomatology Hospital，and they were included in the study. According to the age of patients，they were divided into three groups：group A（18 - 20 years old），group B（30 - 35 years old）and group C（50 - 55 years old）. PDLSC was cultured in primary culture. The aging and proliferation of PDLSC in each group were detected by aging-related β-galactosidase staining and MTT method. The ability of osteogenic differentiation of PDLSC was detected by ALP and alizarin red staining. Real time fluorescent quantitative PCR（qRT-PCR）was used to detect the expression of the pluripotent-related transcription factors （Nanog and Sox2） in each group. Results　Human PDLSC was successfully cultured in each group. Aging-related β-galactosidase staining showed that the staining rate of positive cells in group A was the lowest，group B was the second，group C was the highest，the difference being statistically significant（P < 0.05）. The results of MTT showed that there was a significant difference in the OD value of different age groups（F = 22.354，P = 0.009），and the OD value of each group increased with the prolongation of culture time（F = 28.367，P = 0.018）. There was a significant interaction between age and time（F = 26.431，P = 0.015）. ALP staining and alizarin red staining showed that the percentage of staining area in group A was greater than that in group B and C，and the percentage of staining area in group B was greater than that in group C，the difference being statistically significant（P < 0.05）. The qRT-PCR results showed that the relative expression of Nanog and Sox2 in group A was higher than that in group B and C，and the relative expression of Nanog and Sox2 in group B was higher than that in group C，the difference being statistically significant（P < 0.05）. Conclusion　With the increase of age，the number of aging human PDLSC increases，and the ability of cell proliferation and osteogenic differentiation and the expression of pluripotent-related transcription factors decrease significantly.

Key words: age；periodontal ligament stem cells, PDLSC；proliferation ability；osteogenic differentiation