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目的:本实验探讨人羊膜上皮细胞(human amniotic epithelial cells,h AECs)预防RAW264.7细胞在脂多糖(LPS)刺激后向M1极化的作用以及可能机制。方法:采用流式细胞仪检测细胞凋亡,划痕实验检测细胞迁移,ELISA检测细胞释放NO浓度,Real-time PCR检测白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、一氧化氮合成酶(inducible nitric oxide synthase,i NOS)、精氨酸酶1(arginase-1,Agr-1)及甘露糖受体(mannose receptor,MR又称CD206)等基因表达情况,Western blotting检测RAW264.7胞质蛋白p-IκBα以及胞核蛋白NF-κB的表达。结果:RAW264.7培养基组与h AECs条件培养基干预组的凋亡率分别为5.68%±2.3%、6.68%±2.1%(p>0.05)。LPS刺激组与h AECs条件培养基干预组两组的迁移率分别为42.03±0.07%、14.71±0.04%(p<0.05);LPS刺激组与h AECs干预组两组NO释放量分别为27.73±10μM、13.33±6.43μM(p<0.05);Real Time-PCR结果显示,h AECs干预组M1型巨噬细胞相关基因如IL-1β、TNF-α、iNOS以及INF-β的表达显著下调,M2型巨噬细胞相关基因如Arg-1、CD206、CD36等表达上调(p<0.01)。Western blotting结果显示,hAECs干预组RAW264.7中胞质蛋白p-IκBа以及胞核蛋白NF-κB的蛋白含量降低。结论:h AECs与RAW264.7预培养能有效预防LPS刺激下RAW264.7向M1极化,其机制可能是通过抑制IκBα蛋白磷酸化来降低核内NF-κB的含量,从而抑制了M1型相关基因的表达。
OBJECTIVE: To investigate the role of human amniotic epithelial cells (h AECs) in preventing the polarization of RAW264.7 cells after M1 stimulation by lipopolysaccharide (LPS) and its possible mechanism. Methods: Cell apoptosis was detected by flow cytometry. Scratch assay was used to detect the migration of cells. The concentration of NO was detected by ELISA. The expression of interleukin-1β (IL-1β) and tumor necrosis factor- α, TNF-α, inducible nitric oxide synthase (iNOS), arginase-1 (Agr-1) and mannose receptor , MR also known as CD206) and other gene expression, Western blotting detection of RAW264.7 cytosolic protein p-IκBα and nuclear protein NF-κB expression. Results: The apoptotic rates of RAW264.7 group and h AECs conditioned medium group were 5.68% ± 2.3% and 6.68% ± 2.1%, respectively (p> 0.05). The migration rates of LPS stimulation group and h AECs conditioned medium intervention group were 42.03 ± 0.07% and 14.71 ± 0.04% (p <0.05), respectively. The NO release of LPS stimulation group and h AECs intervention group were 27.73 ± Real-time PCR results showed that the expressions of M1-related macrophages such as IL-1β, TNF-α, iNOS and INF-β in h AECs intervention group were significantly down-regulated while that in M2 Type macrophages related genes such as Arg-1, CD206, CD36 expression was increased (p <0.01). The results of Western blotting showed that the protein content of p-IκBa and NF-κB in RAW264.7 cells was reduced by hAECs intervention. CONCLUSION: h AECs and RAW264.7 pre-culture can effectively prevent the polarization of RAW264.7 to M1 induced by LPS, which may be due to the inhibition of IκBα phosphorylation to reduce the content of NF-κB in the nucleus, Gene expression.