比较矿物三氧化物凝聚体及山东蜂胶乙醇提取物对牙髓成纤维细胞生物学性能的影响

doi:10.19723/j.issn.1671-167X.2019.06.023

摘要/Abstract

摘要：

目的比较矿物三氧化物凝聚体(mineral trioxide aggregate,MTA)与山东蜂胶乙醇提取物对牙髓成纤维细胞活性、矿化和趋化能力以及抗炎效应的影响。方法利用CCK-8法检测山东蜂胶提取物和MTA浸出液作用牙髓成纤维细胞1、5、7、9 d时的细胞毒性,各组同时作用15 h,Transwell法检测不同组的迁移细胞数目。矿化诱导21 d后,各组进行茜素红染色比较诱导硬组织沉积能力。按照1 mg/L浓度将脂多糖(lipopolysaccharide,LPS)加入两种材料后刺激细胞3 h,采用实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法检测不同组细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)三种炎症因子的表达量。采用单因素方差分析及非参数检验对组间差异进行分析(P<0.05)。结果蜂胶的细胞毒性大于MTA,蜂胶组迁移细胞数目(26.67±2.52)明显少于对照组(61.33±4.93)及MTA组(80.00±2.65),茜素红染色显示蜂胶组相较MTA组的钙沉积更多。LPS刺激细胞3 h后,蜂胶相比MTA可以显著降低IL-1β和IL-6的表达量。结论山东蜂胶相比MTA对牙髓细胞有一定的细胞毒性,对细胞迁移的促进作用不明显,但蜂胶具有良好的抗炎效果及诱导牙髓细胞成牙本质分化的能力,经处理后有望应用于感染牙髓的活髓保存治疗。

关键词: 蜂胶, 牙髓, 成纤维细胞, 抗炎作用, 矿物三氧化物凝聚体

Abstract:

Objective: To evaluate the effect of mineral trioxide aggregate (MTA) and propolis from Shangdong province on the cell viability, mineralization and migration and anti-inflammatory ability of dental pulp fibroblasts.Methods: The human dental pulp fibroblasts were cultured and subjected to 10 mg/L of propolis and 1 ∶8 dilution of MTA extraction. The cell viability was evaluated with cell counting kit-8 (CCK-8) after 1, 5, 7 and 9 days. The cells in the upper inserts and the test culture media on the bottoms of 24-well plates interacted for 15 hours. Then the numbers of cells migrated through the per-meable membranes were compared. The cells seeded in the 24-well plates were incubated in osteogenic medium with different materials for 21 days and stained with alizarin red S, then photographed. To evaluate the deposition of calcified matrix, the wells were destained with 100 mmol/L cetylpyridinium chloride. Finally, the cells were exposed to 1 mg/L lipopolysaccharide (LPS) to induce an inflammatory response, in the presence of propolis, MTA extraction. The cells were collected after 3 h, and the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined using real-time polymerase chain reaction (real-time PCR). Statistical analysis was performed by one-way ANOVA and nonparametric tests (P<0.05).Results: The cell viability of propolis group was significantly lower than those of MTA and control groups on days 5, 7 and 9, while MTA significantly increased the numbers of the viable cells on days 7 and 9. The migration cells of propolis group (26.67±2.52) were fewer than control group (61.33±4.93), and the cells of MTA group (80.00±2.65) were statistically more than those of the other two groups. The propolis group significantly induced more calcified matrix deposition than MTA group after 21 days of culture. Propolis significantly suppressed the expressions of IL-1β and IL-6 after LPS exposure compared with MTA and control groups.Conclusion: The propolis from Shandong compared with MTA showed a certain degree of cytotoxicity, and had no significant effect on cell migration. On the other hand, propolis exhibited significant anti-inflammatory and mineralization promotion effect, suggesting that the active ingredients of propolis could be introduced as a supplement of pulp capping materials, or used as an irrigant or intracanal medicament due to its excellent anti-inflammatory effect. Propolis may have potential in vital pulp treatment of young permanent tooth suffering pulp inflammation.

Key words: Propolis, Dental pulp, Fibroblasts, Anti-inflammatory effect, Mineral trioxide aggregate

中图分类号:

R788.2

石冰清,袁晓静,赵玉鸣. 比较矿物三氧化物凝聚体及山东蜂胶乙醇提取物对牙髓成纤维细胞生物学性能的影响[J]. 北京大学学报（医学版）, 2019, 51(6): 1108-1114.

Bing-qing SHI,Xiao-jing YUAN,Yu-ming ZHAO. Effects of mineral trioxide aggregate and ethanolic extracts of Shandong propolis on the biological properties of human dental pulp fibroblasts[J]. Journal of Peking University(Health Sciences), 2019, 51(6): 1108-1114.

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Gene	Primer sequences
TNF-α	F: CGAGTGACAAGCCTGTAGCC	R: TGAAGAGGACCTGGGAGTAGAT
IL-1β	F: AGCTCGCCAGTGAAATGATG	R: GCCCTTGCTGTAGTGGTGGT
IL-6	F: GAAAGCAGCAAAGAGGCACT	R: TTTCACCAGGCAAGTCTCCT