大鼠牙乳头细胞旁分泌效应对巨噬细胞分泌炎症因子的调控作用

The effect of rat dental papilla cells on macrophage secretion of cytokines though paracrine mechanism

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。

PURPOSE： To investigate the immunomodulatory effects of rat dental papilla cells （RDPCs） on lipopolysaccharide（LPS）-induced macrophages. METHODS： Dental papilla tissues from SD rats were isolated and cultured. Cells were passaged and purified using different digesting method. After osteogenic differentiation of rat dental papilla cells, mineralized nodules were assessed by Alizarin red S staining. Oil red-O staining was used to observe the lipid after adipogenic differentiation of rat dental papilla cells. The effect of rat dental papilla cells conditioned medium （RDPC-CM） on macrophages proliferation was assessed using a cell counting kit-8 （CCK-8）. LPS-stimulated macrophages were treated with RDPC-CM and the level of IL-1β, TNF-α, IL-6 and nitric oxide （NO） in the supernatants were evaluated by enzyme-linked immunosorbent assay （ELISA） and Greiss reagent. The results were analyzed by using SPSS 13.0 software package. RESULTS： RDPCs were found around dental papilla piece after 24h of cultures, the cells could be purified using different digesting methods. Aliarin red staining and Oil red-O staining certified the presence of mineralized nodules and lipid in rat dental papilla cells, respectively. CCK-8 assay results showed that the proliferation of macrophages was not affected by RDPC-CM. ELISA and Griess reagent assay revealed a significantly decreased level of TNF-α in the supernatants of LPS-stimulated macrophages upon RDPC-CM treatment compared with the control, but not the levels of IL-6, IL-1β and NO.