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目的通过体外三维培养牙髓细胞,观察牙髓细胞形成细胞集落时炎症因子白细胞介素1β(IL-1β)的表达水平,探讨牙髓炎症发生的可能机制。方法用U形底铺有琼脂糖的96孔板形成非黏附表面,每孔接种104个细胞形成三维培养体系。在细胞接种的0、24、48、72、96 h收集细胞,实时荧光定量PCR(qRT-PCR)检测体系中牙髓细胞IL-1βmRNA的表达水平,ELISA检测体系中牙髓细胞IL-1β蛋白的水平。结果光镜下可见牙髓细胞接种20 h后即可形成紧密的细胞聚集球体,培养至40 h,细胞球体聚集更紧密。与培养0 h的牙髓细胞相比,实时定量PCR检测发现三维培养24、48、72、96 h的牙髓细胞IL-1βmRNA表达水平明显增加,培养72、96 h牙髓细胞IL-1βmRNA表达量高于培养24 h的牙髓细胞;ELISA检测发现三维培养24、48、72、96 h的牙髓细胞较培养0 h的细胞IL-1β蛋白水平明显增高,且培养72、96 h牙髓细胞IL-1β蛋白量高于培养24 h的牙髓细胞。结论三维培养牙髓细胞形成细胞集落能诱导牙髓细胞自发性表达和释放IL-1β。
Objective To investigate the expression of interleukin-1β (IL-1β) in dental pulp cells by three-dimensional culture in vitro and to explore the possible mechanism of dental pulp inflammation. Methods Non-adherent surfaces were formed on a 96-well U-shaped bottom plate with agarose, and 104 cells were seeded per well to form a three-dimensional culture system. The cells were harvested at 0,24,48,72,96 h after inoculation, and the expression of IL-1βmRNA in dental pulp cells was detected by real-time quantitative PCR (qRT-PCR). The levels of IL-1β s level. Results The dental pulp cells were seeded under light microscope for 20 hours to form compact cell aggregates. After cultured for 40 hours, the cell aggregates became more compact. Compared with 0 h cultured dental pulp cells, real-time quantitative PCR detected IL-1βmRNA expression level in dental pulp cells cultured for 24, 48, 72 and 96 h in three-dimensional culture, and expression of IL-1βmRNA in cultured 72 and 96 h dental pulp cells The amount of IL-1β protein in 24 h, 48 h, 72 h and 96 h three-dimensional cultures was significantly higher than that in 0 h cultured cells, and the dental pulp cells cultured for 72 and 96 h The amount of IL-1β protein in the cells was higher than that in the dental pulp cells cultured for 24 h. Conclusion Three-dimensional culture of dental pulp cells to form cell colonies can induce spontaneous expression of dental pulp cells and the release of IL-1β.