几种根管封闭剂对永生化人牙髓细胞的细胞毒性、遗传毒性和免疫炎症生物标志物活性的比较。
Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.
机构信息
Department of Restorative Dentistry, Endodontic Division, São José dos Campos Dental School, Paulista State University (UNESP), São José dos Campos, São Paulo, Brazil.
Department of Bioscience and Oral Diagnosis, Biochemistry Division, São José dos Campos Dental School, Paulista State University (UNESP), São José dos Campos, São Paulo, Brazil.
出版信息
Int Endod J. 2018 Jan;51(1):41-57. doi: 10.1111/iej.12785. Epub 2017 Jun 5.
AIM
To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers.
METHODOLOGY
Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level.
RESULTS
The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P < 0.05) and exhibited the highest inflammatory potential against the SV40 T-Ag-transfected cells (P < 0.05). All root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P < 0.05). Higher levels of IL-6, IL-8 and TNF-α were found in cell supernatant after stimulation with multimethacrylate (Real Seal) compared to all other sealers tested (P < 0.05). No differences were found comparing epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05).
CONCLUSIONS
A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit).
目的
建立 SV40 T-Ag 转染的人牙髓细胞系，以比较几种根管封闭剂的细胞毒性、遗传毒性，并研究其免疫生物标志物的活性。
方法
原代人牙髓细胞和转染细胞进行培养。通过 RT-PCR 和免疫组织化学分析细胞形态和增殖，并检测细胞特异性基因转录本和蛋白质的表达。转染人牙髓细胞后，得到了一株保留原代细胞表型特征的永生化细胞系。SV40 T-Ag 转染细胞经封闭剂（Apexit Plus、Real Seal、AH Plus 和 EndoREZ）培养和刺激，通过 MTT 和 MTN 测定分别评估细胞毒性和遗传毒性。通过 ELISA 测定免疫炎症生物标志物（IL6、IL8 和 TNF-α）。采用 Kruskal-Wallis 和 Dunn 检验，在 5%的显著性水平下对中位数进行差异统计学分析。
结果
细胞毒性测定表明，多甲基丙烯酸酯（Real Seal）是最具细胞毒性的封闭剂（P<0.05），对 SV40 T-Ag 转染细胞的炎症潜能最高（P<0.05）。所有测试的根管封闭剂均能刺激永生化牙髓细胞产生 IL-6、IL-8 和 TNF-α，与对照组相比差异有统计学意义（P<0.05）。多甲基丙烯酸酯（Real Seal）刺激后，细胞上清液中发现的 IL-6、IL-8 和 TNF-α水平高于所有其他测试的封闭剂（P<0.05）。在用环氧树酯基封闭剂（AHPlus）、单甲基丙烯酸酯封闭剂（EndoREZ）和氢氧化钙基封闭剂（Apexit Plus）进行比较时，无论研究的细胞因子如何，均未发现差异（均 P>0.05）。
结论
建立了 SV40 T-Ag 转染的人牙髓细胞系。与环氧树酯基封闭剂（AH Plus）、甲基丙烯酸酯基封闭剂（EndoRez）和氢氧化钙基封闭剂（Apexit Plus）相比，甲基丙烯酸酯树脂基封闭剂（Real Seal）对永生化牙髓细胞表现出最大的细胞毒性和炎症潜能。
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