中国实用口腔科杂志

中国实用口腔科杂志 ›› 2022, Vol. 15 ›› Issue (2): 197-202.DOI: 10.19538/j.kq.2022.02.014

• 论著 • 上一篇    下一篇

热刺激对人牙周膜细胞NLRP3/Caspase-1信号通路调控影响的初步探索

王    飞1，陶    荣2，倪龙兴3   

  1. 1. 中国人民解放军联勤保障部队第904医院口腔科，江苏  无锡  214000；2. 南京大学医学院附属口腔医院，南京市口腔医院牙体牙髓病科，江苏  南京  210008；3. 军事口腔医学国家重点实验室，口腔疾病国家临床医学研究中心，陕西省口腔医学重点实验室，空军军医大学口腔医院牙体牙髓病科，陕西  西安 710032
  • 出版日期:2022-03-30 发布日期:2022-04-07
  • 基金资助:
    国家自然科学基金（面上）项目（81371139）；江苏省自然科学基金（面上）项目（BK20201119）

  • Online:2022-03-30 Published:2022-04-07

摘要： 目的    初步探索热刺激状态下，正常及炎症人牙周膜细胞（PDLCs）内NOD样受体蛋白3（NLRP3）/半胱天冬酶1（Caspase-1）信号通路的调控表达变化。方法    使用三磷酸腺苷（ATP）和脂多糖（LPS）制备炎症PDLCs模型，分别以45℃、5%CO2条件和37℃、5%CO2条件培养正常和炎症PDLCs，CCK8法检测细胞光密度（OD450）值。根据细胞刺激条件，分为3个实验组（ATP+LPS组、热刺激组、ATP+LPS+热刺激组）和1个对照组，采用荧光实时定量PCR（qRT-PCR）检测各组热休克RNA-1（HSR1）、热休克转录因子1（HSF-1）、NLRP3、Caspase-1、白介素（IL）-1β、IL-18的表达水平。结果    ①正常PDLCs活性检测结果显示，随着培养时间的延长，37℃培养条件下细胞OD450值逐渐增加，45℃培养条件下细胞OD450值逐渐降低（F = 36.309，P < 0.05）；炎症PDLCs活性检测结果显示，随着培养时间的延长，2种温度培养条件下细胞OD450值均逐渐降低（F = 23.353，P < 0.05）；且不同培养时间的2种温度培养条件下细胞OD450值比较，差异均有统计学意义（均P < 0.05）；随着培养时间的延长此差异越明显（均P < 0.05）。②各组测量指标mRNA相对表达量总的比较，差异均有统计学意义（均P < 0.05）。3个实验组各测量指标mRNA相对表达量分别与对照组比较，差异均有统计学意义（均P < 0.05）。且与ATP+LPS组比较，热刺激组和ATP+LPS+热刺激组的HSR1和HSF-1 mRNA相对表达量增高，NLRP3、Caspase-1、IL-1β、IL-18 mRNA相对表达量降低，差异均有统计学意义（均P < 0.05）。结论    HSR1可能负反馈调节着NLRP3/Caspase-1炎症小体通路，正常和炎症PDLCs抵抗热刺激时的免疫防御反应能力均减弱。

关键词: 牙周膜细胞；NOD样受体蛋白3, NLRP3；半胱天冬酶1, Caspase-1；热刺激

Abstract: Objective　To invastigate the effect of thermal stimulation on the regulation of NOD-like receptor protein 3（NLRP3）/ cysteinyl aspartate-specific protease-1（Caspase-1）signaling pathway in normal and inflammatory periodontal ligament cells （PDLCs）. Methods　Inflammatory PDLCs models were prepared using adenosine triphosphate（ATP）and lipopolysaccharide（LPS）. Normal and inflammatory PDLCs were both cultured at 45℃，5% CO2 conditions and 37℃，5% CO2 conditions，respectively. The optical density（OD450）value of cells was detected by CCK8 method. According to the cell stimulation conditions，they were divided into 3 experimental groups（ATP+LPS group，thermal stimulation group，ATP+LPS+thermal stimulation group）and 1 control group. Quantitative real-time PCR （qRT-PCR）was used to detect heat shock RNA-1（HSR1），heat shock transcription factor （HSF-1），NLRP3，Caspase-1，interleukin（IL）-1β and IL-18 mRNA expression in each group. Results　①The detection results of normal PDLCs activity showed that with the prolongation of culture time，the OD450 value of cells under 37°C culture condition gradually increased，and the cell OD450 value gradually decreased under 45°C culture condition（F = 36.309，P < 0.05）. The results of inflammatory PDLCs activity detection showed that with the prolongation of culture time，the OD450 values of cells under the two culture conditions gradually decreased （F = 23.353，P < 0.05）；there was statistical difference in OD450 between different culture time （all P < 0.05）；the difference became more obvious with the prolongation of culture time（all P < 0.05）. ②Comparison of the total relative mRNA expression of the measurement indicators in each group showed there were statistically significant differences（all P < 0.05）. The relative mRNA expression levels of each measurement index in the three experimental groups were compared with those in the control group，and there were statistically significant differences（all P < 0.05）. And compared with the ATP+LPS group，the relative expressions of HSR1 and HSF-1 mRNA in the thermal-stimulation group and the ATP+LPS+thermal stimulation group increased，while the relative expressions of NLRP3，Caspase-1，IL-1β and IL-18 mRNA decreased. All were statistically significant（all P < 0.05）. Conclusion　HSR1 may negatively regulate the NLRP3/Caspase-1 inflammasome pathway，and the immune defense response of normal and inflammatory PDLCs against thermal stimulation is weakened.

Key words: periodontal ligament cells；NOD-like receptor protein 3, NLRP3；cysteinyl aspartate-specific protease-1, Caspase-1；thermal stimulation