【作者】 邓洋； 闫萍；

【Author】 Deng Yang;Yan Ping;School and Hospital of Stomatology,Wuhan University;

【机构】 武汉大学口腔医院；

【摘要】 目的:探讨根管充填材料三氧化矿物凝聚体(mineral trioxide aggregate,MTA)对三丁基过氧化氢(t-BHP)诱导的衰老小鼠骨髓巨噬细胞(bone marrow derived macrophages,BMMs)分泌因子的影响。方法:用不同浓度t-BHP(20、40、60、80、100μmol/L)作用不同时间(2、4、6h)诱导年幼C57BL/6小鼠骨髓来源巨噬细胞衰老,通过β-半乳糖苷酶染色法(SA-β-Gal)鉴定细胞衰老,MTT法测定细胞活力,流式细胞术(FCM)分析细胞周期,蛋白质印迹法检测衰老相关信号蛋白P16、P21、P53水平,根据各项检测结果,选取最佳作用浓度和作用时间。将小鼠原代骨髓巨噬细胞(对照组)及tBHP体外诱导衰老巨噬细胞(实验组)分别接种于六孔板中培养,待细胞贴壁并接近融合时,加入新鲜调制并固化的MTA,细胞培养24h后,收集细胞培养液,使用酶联免疫吸附测定法(ELISA)检测细胞分泌因子情况。结果:通过4h,80μmol/L的t-BHP诱导刺激,BMMs增值能力显著下降,SA-β-Gal染色阳性率显著上升,处于G1期的细胞比例增高,P16、P21、P53水平明显上调,细胞活力无明显变化。与MTA接触培养后,衰老细胞组TNF-α、IL-1β、IL-6的表达较对照组明显增多,而IL-4、IL-10的表达与对照组无明显差异。结论:本实验结果说明在衰老的BMMs细胞与MTA接触后较年幼细胞分泌更多的促炎因子(TNF-α、IL-1β、IL-6)。更多还原

【Abstract】 Objective: The aim of this study was to investigate the effects of root–end filling materials(Mineral trioxide aggregate, MTA) on the cytokine production of the aged mouse bone marrow derived macrophages(BMMs) induced by tert-butyl hydroperoxide(t-BHP). Methods: Different concentrations of t-BHP(20、40、60、80、100μ mol/L) were added into the culture medium for different processing time(2、4、6 h)to induce C57 BL/6 BMMs senescence. Cell scenescence was identified by senescence-associatedβ-galactosidase( SA-β-Gal). Cell survival was assessed by MTT staining.The distribution of cell cycle was detected by flow cytometry(FCM). The expression level of senescence-related signaling proteins P16, P21, P53 were detected by Western Blotting. And the optimum concentration and processing time were determined according to the results of all the tests to establish the aging model of BMMs. Then the primary BMMs(control group) and aging BMMs(experiment group) were seeding in sixwell plates and allowed to attach overnight. Freshly mixed or set specimens of MTA were placed into the cell culture plates with already attached BMMs. After 24 h incubation, cell culture media were collected for cytokine assay by using enzyme-linked immunosorbent assay(ELISA).Results: After 4 h co-cultured with 80μ mol/L, the BMMs in the aging model group displayed a significant decrease in proliferation; and the percentage of positive cells expressed SA-β-Gal and the number of cells entered G1 phase of the cell cycle increased significantly. The expression of P16、P21、P53 protein was obviously enhanced, and no significant change of the viability was observed. After incubation with MTA, the production of TNF-α、IL-1β、IL-6 in experience group were significant higher than that in control group, and the production of IL-4、IL-10 were no significant different between these two groups.Conclusion: This in vitro study demonstrated that after contacted with MTA,the aging BMMs secreted more proinflammatory cytokine(TNF-α、IL-1β、IL-6)than the primary BMMs.更多还原