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目的比较人羊膜上皮细胞((human amniotic epithelial cell,H-AEC))、羊膜间充质细胞(human amniotic mesenchymal cell,HA-MSC)和脐带间充质细胞(Umbilical cord mesenchymal stem cells,UC-MSC)分泌的细胞因子对脂多糖刺激巨噬细胞系RAW264.7炎症状态的影响。方法将LPS刺激的RAW264.7细胞炎症模型作为对照组,比较H-AEC、HA-MSC、UC-MSC和RAW264.7共培养或条件培养基培养RAW264.7对RAW264.7炎症状态的影响。比较各组RAW264.7细胞的迁移能力;检测各组细胞分泌一氧化氮(NO)的水平;用实时定量多聚酶链反应(RT-PCR)检测各组细胞经典激活的巨噬细胞(classically activated macrophage,M1 macrophage)相关的促炎基因如白介素-1β(IL-1β)、肿瘤坏死基因а(TNFа)、一氧化氮合成酶-2(NOS-2)以及M2 macrophage相关的抑炎基因如精氨酸酶(Arg-1)、甘露糖受体基因CD206、B类清道夫受体CD36)表达情况。结果 (1)H-AEC、HA-MSC、UC-MSC处理后RAW264.7的迁移率分别为14.7%±4.5%、9.6%±0.7%、13.0%±0.9%,与对照组(31.1%±11.0%)相比,3种细胞的条件培养基处理后RAW264.7的迁移率均降低,差异具有显著性(P<0.05);(2)H-AEC、HA-MSC、UCMSC共培养后RAW264.7细胞分泌NO的水平分别为24.26±0.72、44.52±2.51、42.25±0.76μmol/L,与对照组(45.65±1.78μmol/L)相比,H-AEC组细胞分泌的NO有显著性下降(P<0.05);(3)促炎基因与抑炎基因的表达改变:(A)H-AEC处理组促炎基因IL-1β、TNFа、NOS-2、INFβ的表达下调,与对照组相比有显著差别;HA-MSC、UC-MSC处理组促炎基因INFβ表达下调显著,其余基因均上调表达;抑炎相关基因如Arg-1、CD206、CD36均上调;(B)3组细胞干预后抑炎症相关基因Arg-1、CD206、CD36表达均上调,与对照组有显著差异。结论人羊膜上皮细胞、羊膜间充质细胞和脐带间充质细胞可以促进巨噬细胞向M2型分化,但其效果和机制存在不同。
Objective To compare the effect of human amniotic epithelial cell (H-AEC), human amniotic mesenchymal cell (HA-MSC) and umbilical cord mesenchymal stem cells (UC-MSCs) ) Secreted cytokines on the inflammatory state of macrophage cell line RAW264.7 stimulated by lipopolysaccharide. Methods RAW264.7 cells induced by LPS were used as control group. The effects of RAW264.7 cultured in H-AEC, HA-MSC, UC-MSC and RAW264.7 co-culture or conditioned media on the inflammatory status of RAW264.7 cells were compared. The migration ability of RAW264.7 cells in each group was compared. The levels of nitric oxide (NO) secreted by each group of cells were measured. The expressions of classically activated macrophage , M1 macrophage) such as interleukin-1β (IL-1β), tumor necrosis gene а (TNFа), nitric oxide synthase-2 (NOS-2) and M2 macrophage- (Arg-1), mannose receptor gene CD206, class B scavenger receptor CD36) expression. Results (1) The migration rates of RAW264.7 cells treated with H-AEC, HA-MSC and UC-MSC were 14.7% ± 4.5%, 9.6% ± 0.7% and 13.0% ± 0.9% (P <0.05). (2) Compared with RAW264 cells cultured with H-AEC, HA-MSC and UCMSC, the migration rate of RAW264.7 cells in conditioned medium of three kinds of cells decreased, .7 The level of NO secreted by H-AEC cells was 24.26 ± 0.72,44.52 ± 2.51,42.25 ± 0.76μmol / L, respectively. Compared with the control group (45.65 ± 1.78μmol / L), NO secretion in H-AEC group was significantly decreased (P <0.05). (3) The expression of proinflammatory gene and proinflammatory gene changed: (A) The expression of proinflammatory cytokines IL-1β, TNFα, NOS-2 and INFβwere downregulated in H- (P <0.05). The expression of INFβ was down-regulated in HA-MSC and UC-MSC groups, and the other genes were up-regulated. The levels of anti-inflammation related genes such as Arg-1, CD206 and CD36 were upregulated. After suppression of inflammation-related genes Arg-1, CD206, CD36 expression were up-regulated, and the control group were significantly different. Conclusion Human amniotic membrane epithelial cells, amniotic mesenchymal cells and umbilical cord mesenchymal cells can promote the differentiation of macrophages to M2, but their effects and mechanisms are different.