胚胎神经干细胞对巨噬细胞分泌炎性反应因子的影响

Effect of neural stem cells on IFN-γ-induced secretion of inflammatory cytokines from macrophages in vitro

目的探讨小鼠胚胎皮质来源的神经干细胞(neural stem cells,NSCs)对小鼠骨髓来源巨噬细胞炎性因子和一氧化氮(nitric oxide,NO)表达的影响。方法体外分离、培养NSCs与巨噬细胞。实验分组为NSCs组、小鼠骨髓来源巨噬细胞组(简称为巨噬细胞组)、小鼠骨髓来源巨噬细胞+NSCs非接触型共培养组(简称为非接触型共培养组)和小鼠骨髓来源巨噬细胞+NSCs接触型共培养组(简称为接触型共培养组)。培养24h后添加10ng/mL干扰素γ(interferon-γ,IFN-γ)再孵育24h后收集各组上清液,利用ELISA检测上清液炎性反应因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(IL-1β)和IL-10的表达,采用Griess法测量NO的表达水平。结果成功体外分离、培养原代NSCs和巨噬细胞。与巨噬细胞组相比,非接触型共培养组和接触型共培养组巨噬细胞分泌NO、TNF-α、IL-1β明显下降,而IL-10明显增加(均P<0.01)。与非接触型共培养组比较,接触型共培养组TNF-α降低〔(65.68±7.15)pg/mL比(90.99±5.57)pg/mL〕,IL-10升高〔(531.38±60.11)pg/mL比(324.32±45.41)pg/mL〕(均P<0.01),而IL-1β和NO表达无统计学差异(P>0.05)。结论 NSCs能够明显降低活化巨噬细胞NO和促炎性因子TNF-α、IL-1β的释放,增加抗炎性因子IL-10的分泌,减轻炎性反应程度,其机制可能主要通过NSCs分泌可溶性因子起作用。

Objective To investigate the effect of embryonic neural stem cells （NSCs） from C57BL/6 mouse on secretion of inflammatory cytokines and nitric oxide （NO ） from mouse bone marrow‐derived macrophages stimulated by interferon‐γ （IFN‐γ） . Methods NSCs were derived from the cerebral cortex of embryonic mice. They were collected on the 14th day of pregnancy and cultured in Dulbecco′s Modified Eagle′s Medium （DMEM ） /F12 medium. The medium was supplemented with 20 ng/mL epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） . Macrophages were isolated from bone marrow in vitro and cultured in DMEM medium with 15% L929 supernatant and 5% fetal bovine serum （FBS） . There were four groups in the experiment ：（1） NSCs were co‐cultured alone in the NSCs group. （2） Bone‐marrow derived macrophages were cultured alone without NSCs in the macrophage group. （3） Macrophages were co‐cocultured with NSCs by using the transwell system in the non‐contact type group. （4） Macrophages were directly co‐cocultured with NSCs in the contact type group. After 24 h of culture ,macrophages were treated with IFN‐γfor 24 h and the levels of tumor necrosis factor‐α （TNF‐α） ,interleukin‐1β （IL‐1β） ,interleukin‐10 （IL‐10） and NO in the supernatants were detected by using enzyme‐linked immunosorbent assay and Greiss assay. Results NSCs and macrophages were primarily isolated and cultured in vitro. Compared with the simple bone‐marrow derived macrophages group ,the level of TNF‐α,IL‐1β and NO significantly decreased （TNF‐α：F=55.98 , P〈0.01 ;IL‐1β：F=63.53 , P〈0.01 ;NO ：F= 31.32 , P〈 0.01） ,and IL‐10 significantly increased in the contact and non‐contact type groups （F=101.36 ,P〈0.01） . Compared with the non‐contact type group ,the level of TNF‐αwas lower in the contact type group [ （65.68 ± 7.15） pg/mL vs. （90.99 ± 5.57） pg/mL] and IL‐10 was higher [ （531.38 ± 60.11） pg/mL vs. （324.32 ± 45.41） pg/mL , P〈 0.01 ,respectively] and there was no statistically significant difference in expression of IL‐1βand NO （P〉0.05） . Conclusions NSCs can obviously reduce the levels of inflammatory cytokines （TNF‐α,IL‐1β） and increase anti‐inflammatory factor （IL‐10） level secreted by the activated macrophages to ameliorate inflammatory reactions , mainly through the secretion of soluble factors.