人羊膜上皮细胞对脂多糖刺激下的RAW264.7细胞向M1极化的预防作用及其初步作用机制

发布时间：2019-01-19 15:52

【摘要】：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%(p0.05)。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%(p0.05);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(p0.05);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(p0.01)。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。
[Abstract]:Aim: to investigate the effect of human amniotic epithelial cell (human amniotic epithelial cells,h AECs) on preventing RAW264.7 cells from polarization to M1 induced by lipopolysaccharide (LPS) and its possible mechanism. Methods: apoptosis was detected by flow cytometry, cell migration was detected by scratch test, NO concentration was detected by ELISA, interleukin-1 尾 (IL-1 尾) was detected by Real-time PCR, tumor necrosis factor- 伪 (tumor necrosis factor- 伪 was detected. TNF- 伪, (inducible nitric oxide synthase,i NOS), argininase 1 (arginase-1,Agr-1) and mannose receptor,MR receptor (CD206), etc. The expression of RAW264.7 cytoplasmic protein p-I 魏 B 伪 and nuclear protein NF- 魏 B was detected by Western blotting. Results: the apoptotic rates of RAW264.7 medium group and h AECs conditioned medium group were 5.68% 卤2.3, respectively. The migration rates of the two groups were 42.03 卤0.07 卤14.71 卤0.04% (p0.05) and 14.71 卤0.04% (p0.05), respectively. The release of NO in LPS stimulation group and h AECs intervention group was 27.73 卤10 渭 M, 13.33 卤6.43 渭 M (p0.05), respectively. Real Time-PCR results showed that the expression of M1 type macrophage associated genes such as IL-1 尾, TNF- 伪, iNOS and INF- 尾 were significantly down-regulated in, h AECs intervention group, while M 2 macrophage associated genes such as Arg-1,CD206, were significantly down-regulated. The expression of CD36 was up-regulated (p0.01). Western blotting results showed that the contents of p-I 魏 B protein and NF- 魏 B protein in RAW264.7 were decreased in hAECs intervention group. Conclusion: h AECs and RAW264.7 preculture can effectively prevent RAW264.7 polarization to M1 induced by LPS. The mechanism may be that the expression of M1 related genes can be inhibited by inhibiting the phosphorylation of I 魏 B 伪 protein and decreasing the content of NF- 魏 B in nucleus.
【作者单位】：中南大学基础医学院生殖与干细胞研究所;荆州市第一人民医院检验科;人类干细胞国家工程研究中心;
【基金】：中南大学硕士生自主探索创新项目(2014zzts289);中南大学教师研究基金项目(905010092) 长沙科技计划项目(K1203005-31)
【分类号】：R392

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