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目的 :研究大鼠牙乳头细胞(rat dental papilla cells,RDPCs)对LPS活化的巨噬细胞分泌免疫因子的影响。方法:分离SD大鼠牙胚,酶消化法原代培养牙乳头细胞并进行矿化诱导和成脂诱导,茜素红染色观察矿化结节形成,油红O染色观察脂滴形成。CCK8法检测大鼠牙乳头细胞条件培养基(rat dental papilla cells’conditioned medium,RDPC-CM)对巨噬细胞增殖能力的影响,采用酶联免疫吸附实验(ELISA)测定RDPC-CM对巨噬细胞分泌炎症因子IL-1β、TNF-α和IL-6的影响,Griess reagent法检测RDPC-CM对巨噬细胞分泌炎症因子NO的影响。采用SPSS13.0软件包对数据进行统计学分析。结果:原代培养24 h后,大鼠牙乳头细胞从组织块中爬出,传代后可得到纯化大鼠牙乳头细胞;茜素红染色见矿化诱导组中出现散在的红色矿化结节;油红O染色后可见经成脂诱导的牙乳头细胞中脂滴形成;CCK8结果表明,RDPC-CM对巨噬细胞增殖能力无影响;ELISA结果显示,大鼠牙乳头细胞条件培养基可对LPS活化巨噬细胞分泌细胞因子产生作用,表现为条件培养基作用于巨噬细胞24h可减少细胞分泌TNF-α,而对IL-1β和IL-6分泌无影响;Griess reagent法结果显示,RDPC-CM对LPS活化的巨噬细胞分泌NO无影响。结论 :大鼠牙乳头细胞条件培养基作用于LPS活化的巨噬细胞,可使巨噬细胞分泌TNF-α下降,提示牙乳头细胞具有一定的免疫调控作用。
AIM: To investigate the effects of rat dental papilla cells (RDPCs) on the secretion of immune factors by LPS-activated macrophages. METHODS: SD rat tooth germ was isolated. The dental papilla cells were primarily cultured by enzymatic digestion and induced by mineralization and adipogenic induction. Alizarin red staining was used to observe the formation of mineralized nodules. The formation of lipid droplets was observed by oil red O staining. The effect of RDPC-CM on the proliferation of macrophages was detected by CCK8 assay. The effect of RDPC-CM on the proliferation of macrophages was determined by enzyme-linked immunosorbent assay (ELISA) Secretion of inflammatory cytokines IL-1β, TNF-α and IL-6, Griess reagent method to detect RDPC-CM macrophage inflammatory factor secretion of NO. SPSS13.0 software package for statistical analysis of the data. RESULTS: After primary culture for 24 h, rat dental papilla cells climbed out of the tissue block, and purified rat dental papilla cells were obtained after passage. Alizarin red staining showed sparse red mineralized nodules ; Lipid drop formation was observed in adipose-induced dental papilla cells after oil red O staining; CCK8 results showed that RDPC-CM had no effect on macrophage proliferation ability; ELISA results showed that rat papillary cells conditioned medium LPS-activated macrophages secrete cytokines, showing that conditional medium on macrophages 24h can reduce the secretion of TNF-α, but had no effect on the secretion of IL-1β and IL-6; Griess-reagent method showed that RDPC -CM had no effect on NO secretion by LPS-activated macrophages. CONCLUSION: Rat dental papilla cell conditioned medium acts on LPS-activated macrophages, which can decrease the secretion of TNF-α by macrophages, suggesting that dental papilla cells have certain immunomodulatory effects.