非甾体类抗炎药对人牙髓细胞的抗炎修复作用

doi:10.19723/j.issn.1671-167X.2020.01.004

摘要/Abstract

摘要：

目的:研究不同非甾体类抗炎药(non-steroidal anti-inflammatory drugs,NSAIDs)对人牙髓细胞(human dental pulp cells,hDPCs)的抗炎和修复作用,探索可适宜于牙髓炎活髓保存治疗的药物。方法:取新鲜拔除的人类第三磨牙的牙髓进行hDPCs原代培养,消化传代至4~6代,向培养基中加入1 mg/L脂多糖(lipopolysaccharide,LPS)培养24 h,获得LPS刺激后的hDPCs(LPS-hDPCs),实验组使用含有不同浓度(1、10、100 μmol/L)NSAIDs(阿司匹林或美洛昔康)的培养基培养LPS-hDPCs,普通培养基作为阴性对照组。采用MTT法在第1、3、5、7天对细胞增殖活力进行检测,采用real-time PCR法于6 h检测炎症基因白细胞介素6(interleukin-6,IL-6)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的表达;培养7 d后检测成牙分化基因牙本质基质蛋白(dentin matrix protein-1,DMP-1)、牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)基因表达;采用茜素红染色法于第14天检测矿化结节形成,氯化十六烷基吡啶进行矿化结节钙半定量检测。结果:MTT结果显示,1~100 μmol/L阿司匹林或美洛昔康对hDPCs增殖具有显著促进作用(P<0.05),该促进作用呈浓度依赖性。1~100 μmol/L美洛昔康或100 μmol/L阿司匹林均可显著下调LPS-hDPCs炎症基因TNF-α和 IL-6表达(P<0.05),且100 μmol/L美洛昔康的该作用效果显著强于100 μmol/L阿司匹林(P<0.05)。100 μmol/L美洛昔康可以显著促进LPS-hDPCs成牙向分化基因DSPP、DMP-1的表达和矿化(P均<0.05)。结论:美洛昔康可促进hDPCs增殖,在有效抑制炎症因子升高的同时,可促进炎症状态下hDPCs的成牙本质向分化和矿化,美洛昔康可能在牙髓炎症中发挥抗炎和促进修复的作用。

关键词: 消炎药, 非甾类, 脂多糖类, 牙髓, 细胞, 培养的, 美洛昔康

Abstract:

Objective: To study the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on anti-inflammation and repair of human dental pulp cells (hDPCs). Methods: Primary hDPCs from the freshly extracted human third molars were cultured and passaged in vitro, and the following experiments were performed using the 4th-6th generations of hDPCs. HDPCs were cultured in Dulbecco’s modified eagle medium (DMEM) containing 1 mg/L lipopolysaccharide (LPS) to obtain LPS irritated hDPCs (LPS-hDPCs), which served as the inflammatory positive group. LPS-hDPCs in the experimental group were cultured in DMEM containing different concentrations (1, 10, and 100 μmol/L) of NSAIDs (aspirin or meloxicam). HDPCs cultured in DMEM were used as the negative control group. The effects of NSAIDs on the proliferation of hDPCs were assessed on the 1st, 3rd, 5th, and 7th day by MTT assay. The effects of NSAIDs on the expression of inflammation related genes interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of LPS-hDPCs were detected at the 6th hour by real-time PCR. The expression of diffe-rentiation related markers dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) were detected on the 7th day by real-time PCR. The effects of NSAIDs on the mineralization of LPS-hDPCs were assesd on the 14th day by alizarin red staining. Calcium mineralized nodules were semi-quantitatively determined by cetyl pyridine chloride. Results: MTT assay showed that 1-100 μmol/L aspirin or meloxicam significantly promoted the proliferation of hDPC in a concentration dependent manner (P<0.05). Real-time PCR showed that 1-100 μmol/L meloxicam or 100 μmol/L aspirin down-regulated significantly the mRNA expression of TNF-α and IL-6 of LPS-hDPCs (P<0.05), and 100 μmol/L meloxicam down-regulated IL-6 and TNF-α more significantly than 100 μmol/L aspirin of LPS-hDPCs (P<0.05). Real-time PCR showed that 100 μmol/L meloxicam up-regulated the mRNA expression of DMP-1 and DSPP of LPS-hDPCs significantly (P<0.05). Alizarin red staining showed the meloxicam at the concentration of 100 μmol/L significantly promoted the mineralization of LPS-hDPCs (P<0.05). Conclusion: In this study, meloxicam promoted the proliferation of hDPCs, inhibited the inflammatory reaction and promoted differentiation and mineralization of hDPCs under LPS irritation. The present results suggest that meloxicam may play a role in anti-inflammation and repair of pulp inflammation.

Key words: Anti-inflammatory agents, non-steroidal, Lipopolysaccharides, Dental pulp, Cells, cultured, Meloxicam

中图分类号:

R781.31

李婧宜,王赛楠,董艳梅. 非甾体类抗炎药对人牙髓细胞的抗炎修复作用[J]. 北京大学学报（医学版）, 2020, 52(1): 24-29.

Jing-yi LI,Sai-nan WANG,Yan-mei DONG. Anti-inflammatory and repaired effects of non-steroidal anti-inflammatory drugs on human dental pulp cells[J]. Journal of Peking University(Health Sciences), 2020, 52(1): 24-29.

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Gene	Sequence (5'-3')
DMP-1	Forward: AGGAAGTCTCGCATCTCAGAG Reverse: TGGAGTTGCTGTTTTCTGTAGAG
DSPP	Forward: ATATTGAGGGCTGGAATGGGGA Reverse: TTTGTGGCTCCAGCATTGTCA
IL-6	Forward: CCACTCACCTCTTCAGAACG Reverse: CATCTTTGGAAGGTTCAGGTTG
TNF-α	Forward: CCTCTCTCTAATCAGCCCTCTG Reverse: GAGGACCTGGGAGTAGATGAG
GAPDH	Forward: GAAGGTGAAGGTCGGAGTC Reverse: GAGATGGTGATGGGATTTC