摘要
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 modulatory effects of human amniotic epithelial cells (H-AECs), human amniotic mesenchymal cell (HA-MSCs), umbilical mesenchymal ceils (UC-MSCs) on the inflammatory status of lipopolysaccharide (LPS)-induced RAW264.7 cells. Methods RAW264.7 cells stimulated with LPS were co-cultured with H-AECs, HA-MSCs, or UC-MSCs or cultured in conditioned media of the 3 stem cells to assess the changes of the inflammatory status of RAW264.7 cells. The migration ability, nitric oxide concentration, and expressions of the pro-inflammatory and anti-inflammatory genes, including intefleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and inducible nitric oxide synthase (NOS-2) of M1 macrophages, and Arg-1, CD206, and CD36 of M2 macrophages, were detected in the co-cultures. Results Compared with the control macrophages, RAW264.7 cells cultured in the conditioned media of H-AECs, HA-MSCs, and UC-MSCs all showed significantly lowered migration abilities (P〈0.05). Co-culture with H-AECs, but not the other two stem cells, resulted in a significant reduction of NO production (P〈0.05) and significant down-regulation of IL-1β, TNFα, NOS-2, and INFβ expressions in RAW264.7 cells; co-culture with HA-MSCs and UC-MSCs only caused a down-regulation of INFβ mRNA expression. In all the 3 RAW264.7 and stem cell co-cultures, the expressions of the inflammation related genes including Arg-1, CD206, and CD36 were up-regulated significantly. Conclusion H-AECs, HA-MSCs, and UC-MSCs can all prevent RAW264.7 cells from differentiating into M2 macrophages, but their effects and mechanisms are different from one another.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2014年第11期1627-1631,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30700272)
教育部新教师基金(20070533080)
湖南省科学基金(2009FJ3180)
长沙科技计划(K1203005-31)~~