尼古丁调控人牙周膜细胞自噬水平的实验研究

目的 探讨尼古丁对人牙周膜细胞（hPDLCs）自噬水平的影响。方法 选取因正畸治疗而拔除的前磨牙,采用组织块法分离培养hPDLCs。通过Western blot法筛选尼古丁影响hPDLCs自噬的最佳作用时间及浓度,使用透射电子显微镜（TEM）和免疫荧光染色法检测该作用时间及浓度下hPDLCs自噬体形成情况和自噬标志蛋白LC3的表达情况。结果 LC3Ⅱ蛋白表达在尼古丁作用的12 h内持续升高,从而确定12 h为最佳作用时间;LC3Ⅱ蛋白表达上调具有尼古丁浓度依赖性,1×10^-5 mol·L^-1为尼古丁最佳作用浓度。TEM和免疫荧光染色证实尼古丁在此浓度及作用时间下hPDLCs细胞质内自噬体的数量增加,自噬标志蛋白LC3表达升高。结论 在一定条件下,尼古丁能够上调hPDLCs自噬水平,为进一步研究细胞自噬与吸烟相关牙周炎的关系奠定了基础。

A preliminarystudyon the autophagy level of human periodontal ligament cells regulated by nicotine

Objective To explore the effect of nicotine on the autophagy level of human periodontal ligament cells(hPDLCs). Methods Periodontal tissues collected from premolars for orthodontic treatment reasons were used to culture hPDLCs. Western blot analysis was performed to test the most optimal time and concentration of nicotine on the autophagy level of the hPDLCs. Transmission electron microscope and immunofluorescence observation were carried out to detect the form of autophagosomes and expression of autophagy related protein LC3 in hPDLCs under this optimal condition. Results Protein expression of LC3Ⅱwas up regulated with the 12 h nicotine stimulating. Besides that, the up regulation of the protein expression of LC3Ⅱwas concentration dependent and nicotine with a concentration of 1×10-5 mol·L-1 was the most optimal condition. Transmis-sion electron microscope and immunofluorescence observations indicated that nicotine would activate the autophagy level of hPDLCs by increasing the number of autophagosomes and up regulating the expression of autophagy related protein LC3. Conclusion Nicotine could increase autophagy level of hPDLCs, thus affecting the occurrence and development of smoking related periodontitis.