尼古丁调控人牙周膜细胞自噬水平的实验研究

doi:10.7518/hxkq.2017.02.017

华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (2): 198-202.doi: 10.7518/hxkq.2017.02.017

尼古丁调控人牙周膜细胞自噬水平的实验研究

杜样¹(), 袁帅¹, 周志斐¹, 邬礼政², 汪璐璐¹, 吴兴安³, 王小竞¹()

1.军事口腔医学国家重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病临床医学研究中心,第四军医大学口腔医院儿童口腔科,西安 710032
2.武警后勤学院附属医院口腔科,天津 300300
3.第四军医大学微生物学教研室,西安 710032

收稿日期:2016-11-05 修回日期:2017-01-02 出版日期:2017-04-01 发布日期:2017-04-01
作者简介:
杜样,硕士,E-mail：duyangzizi@126.com
基金资助:
国家自然科学基金面上项目（81470743,81670988）

A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine

Yang Du¹(), Shuai Yuan¹, Zhifei Zhou¹, Lizheng Wu², Lulu Wang¹, Xing’an Wu³, Xiaojing Wang¹()

1. State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shanxi Clinical Research Center for Oral Diseases, Dept. of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, China
2. Dept. of Stomatology, Affiliated Hospital of Logistics University of People’s Armed Police Force, Tianjin 300300, China
3. Dept. of Microbiology, The Fourth Military Medical University, Xi’an 710032, China

Received:2016-11-05 Revised:2017-01-02 Online:2017-04-01 Published:2017-04-01
Supported by:
The General Program of National Nature Science Foundation of China (81470743)

摘要/Abstract

摘要：

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^-5mol·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。

关键词: 人牙周膜细胞, 尼古丁, 自噬

Abstract:

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 LC3Ⅱ was up regulated with the 12 h nicotine stimulating. Besides that, the up regulation of the protein expression of LC3Ⅱ was concentration dependent and nicotine with a concentration of 1×10^-5mol·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.

Key words: human periodontal ligament cells, nicotine, autophagy

中图分类号:

R780.2

杜样, 袁帅, 周志斐, 邬礼政, 汪璐璐, 吴兴安, 王小竞. 尼古丁调控人牙周膜细胞自噬水平的实验研究[J]. 华西口腔医学杂志, 2017, 35(2): 198-202.

Yang Du, Shuai Yuan, Zhifei Zhou, Lizheng Wu, Lulu Wang, Xing’an Wu, Xiaojing Wang. A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine[J]. West China Journal of Stomatology, 2017, 35(2): 198-202.

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尼古丁调控人牙周膜细胞自噬水平的实验研究

A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine

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