| 陆文玲 何妲 彭琳 黄生建 侯伟榕 王建.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[J].,2016,16(30):5819-5823 |
| 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 |
| The Preventive Effect of Human Amniotic Epithelial Cells on LPS-inducedRAW264.7 to M1 Polarization and its PreliminaryMolecular Mechanism |
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| DOI： |
| 中文关键词: 羊膜上皮细胞 巨噬细胞极化 M1 巨噬细胞 NF-κB |
| 英文关键词: Human amniotic epithelial cells Macrophage polarization M1 macrophages NF-κB |
| 基金项目:中南大学硕士生自主探索创新项目（2014zzts289）；长沙科技计划项目（K1203005-31）；中南大学教师研究基金项目（905010092） |
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| 中文摘要: |
| 目的：本实验探讨人羊膜上皮细胞（human amniotic epithelial cells，hAECs）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-1beta（interleukin-1beta，IL-1beta）、肿瘤坏死因子-alpha（tumor necrosis factor-alpha，TNF-alpha）、一氧化氮合成酶
（inducible nitric oxide synthase，iNOS）、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-资B 的表达。结果：RAW264.7培养基组与hAECs 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hAECs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hAECs 干预组两组NO 释放量分别为27.73± 10 滋M、13.33±
6.43 滋M（p<0.05）；Real Time-PCR结果显示，hAECs干预组M1 型巨噬细胞相关基因如IL-1茁、TNF-琢、iNOS 以及INF-茁的表达显
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：hAECs 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。 |
| 英文摘要: |
| Objective:To investigate the preventive effect of human amniotic epithelial cells (hAECs) on the RAW264.7 to M1
polarization after stimulated by lipopolysaccharide (LPS) and the possible mechanism of this process.Methods:Flow cytometry was
taken to detect the rate of cell apoptosis. The scratch assay was used to detect the cell migration. The release of nitric oxide (NO) was
measured by nitrate reductase assay. And the real-time PCR was performed to analyze the expressions of the pro-inflammatory and
anti-inflammatory genes in RAW264.7 cells after co-culturing with hAECs, such as IL-1beta, TNF-α, iNOS, Agr-1 and CD206. The
expressions of cytoplasmic protein p-IκBαand nuclear protein NF-κB in RAW264.7 were analyzed by western blotting analysis.Results:The apoptosis rates of RAW264.7 group and hAECs conditioned media group were 5.68 % ± 2.3 %and 6.68 % ± 2.1 %(p>0.05). The
mobilities of LPS-stimulated group and hAECs conditioned media group were 42.03 ± 0.07 % and 14.71 ± 0.04% (p<0.05). The
release of NO in LPS-stimulated group and hAECs co-culture group were 27.73 ± 10 uM, 13.33 ± 6.43 uM (p<0.05). After
co-culturing with hAECs, the expression of M1-related genes in LPS-induced RAW264.7 (IL-1β, TNF-α, iNOS and INF-β) was
dramatically decreased, while the expression of M2-related genes (CD206, Arg-1, CD36, etc.) was up regulated (p<0.01). The protein
content of NF-κB cytoplasmic protein p-IκBа and nuclear protein NF-κB (both were classic inflammatory pathway-related proteins) in
LPS-induced RAW264.7 was decreased after hAECs intervening.Conclusion:The results showed that hAECs could effectively prevent
LPS-stimulated RAW264.7 polarization to M1 and up-regulate M2 related gene expressions. The possible mechanism may bepre-incubated with hAECs inhibited the protein phosphorylation of IκBα, and then prevent NF-κB protein from entering into the
RAW264.7 cell nucleus to start M1-related gene expressions |
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