中国实用口腔科杂志

中国实用口腔科杂志

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细菌脂多糖对人根尖牙乳头干细胞自噬作用研究

雷双，毕静，庄雪莹，刘尧，陈旭   

  1. 中国医科大学口腔医学院·附属口腔医院儿童口腔科，辽宁省口腔疾病重点实验室，辽宁省口腔疾病临床医学研究中心，辽宁 沈阳 110002
  • 出版日期:2020-02-15 发布日期:2020-03-06
  • 基金资助:
    国家自然科学基金面上项目（81771059）；辽宁省科学技术计划项目（2018225061）；辽宁省高等学校基本科研项目（LQNK201723）

  • Online:2020-02-15 Published:2020-03-06

摘要： 目的　研究细菌脂多糖（lipopolysaccharide，LPS）对人根尖牙乳头干细胞（stem cells from apical palilla，SCAP）自噬的作用及影响，以探讨年轻恒牙根尖周炎损伤与修复的分子机制。方法　原代分离培养人SCAP，采用不同质量浓度（0.05、0.5、5 μg/mL）的细菌LPS处理SCAP记为LPS处理组，未加入细菌LPS的记为对照组。Western blot检测各组自噬相关基因5（autophagy related gene 5，Atg5）及自噬调节蛋白P62的表达情况，透射电子显微镜观察细菌LPS对SCAP自噬泡形态及数量的影响，数据采用SPSS18.0软件进行统计学分析。结果　原代培养的人SCAP表达间充质干细胞表面标志物CD73、CD90、CD105，不表达造血干细胞表面标记物CD45，体外诱导培养具有成骨向分化潜能。Western blot结果显示，各组间Atg5和P62的表达总的比较差异具有统计学意义（F值分别为118.227、74.144，P < 0.05）。Atg5的表达随处理组细菌LPS质量浓度的增大而升高，其中0.05 μg/mL LPS处理组Atg5相对表达量低于对照组，5 μg/mL LPS处理组Atg5相对表达量高于对照组，差异均具有统计学意义（均P < 0.05）。与对照组相比，0.5、5 μg/mL LPS处理组P62的表达降低，其中5 μg/mL LPS组P62相对表达量最低，差异均有统计学意义（均P < 0.05）。透射电子显微镜观察发现，与对照组相比，LPS处理组自噬泡数量显著增加。结论　细菌LPS能够促进人SCAP自噬的发生，提示在炎性环境下自噬可能对人SCAP生物学性能的调控发挥重要作用。

关键词: 根尖牙乳头干细胞, 自噬, 脂多糖, 根尖周炎

Abstract: Objective　To study the effect of bacterial lipopolysaccharide（LPS）on autophagy of human stem cells from apical papilla（SCAP），in order to explore the molecular mechanism of the damage and repair of immature permanent teeth affected with periapical periodontitis. Methods　Human primary SCAP was isolated. Different doses of LPS（0.05，0.5 and 5 μg/mL）were added into the culture media （LPS group）. Normal culture media was used as control. The expression of autophagy related gene-5（Atg5）and P62 were detected by Western blot. Autophagosomes were detected by transmission electron microscopy（TEM）after treatment with 5 μg/mL LPS. The data were analyzed by SPSS18.0 software. Results　Primary SCAP positively expressed mesenchymal stem cells markers（CD73，CD90，CD105）and negatively expressed hematopoietic stem cell surface markers CD45. SCAP possessed osteogenic differentiation potential in vitro. Western blot analysis showed that the differences in the expression of Atg5 and P62 between groups were statistically significant（F = 118.227，74.144，respectively，P < 0.05）. Atg5 expression was increased with increasing doses of LPS while the relative expression at 0.05 μg/mL was lower than that of the control group. The relative expression of Atg5 at 5 μg/mL was higher than that of the control group（P < 0.05）. P62 expression at 0.5 and 5 μg/mL group was decreased compared to the control group，and the 5 μg/mL group was the lowest in the four groups（P < 0.05）. TEM indicated that autophagosome number was significantly elevated. Conclusion　LPS promotes autophagy of SCAP，which indicates that autophagy may be involved in the regulation of SCAP biological property in inflammatory environment.

Key words: stem cells from apical papilla, SCAP；autophagy；lipopolysaccharide, LPS；periapical periodontitis