人羊膜上皮干细胞对脓毒症患者血清诱导RAW264．7细胞炎症状态的影响

Effects of human amniotie epithelial cells on the inflammatory status of RAW264.7 cells induced by sepsis patient＇s serum

目的评估人羊膜上皮干细胞分泌的细胞因子对脓毒症患者血清刺激巨噬细胞系RAW264．7炎症状态的影响，为人羊膜上皮干细胞在治疗炎症相关疾病的临床应用提供理论基础。方法通过机械法联合胰酶消化法分离人羊膜上皮干细胞，观察其细胞形态及增殖活力，用流式细胞技术对其干细胞标记物进行鉴定，同时评估其多向分化潜能。最后收集人羊膜上皮干细胞条件培养基作用于经脓毒症患者血清刺激的RAW264．7细胞，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（M1macrophage）相关的促炎基因及旁路活化的巨噬细胞（M2macrophage）相关的抑炎基因表达情况；同时用免疫荧光检测各组RAW264．7细胞经典激活的巨噬细胞M1、M2相关蛋白表达情况。结果①人羊膜上皮干细胞呈典型的鹅卵石样，细胞倍增时间约为3d。②人羊膜上皮干细胞表达间充质干细胞标记物CD29、CD90、CDl05，胚胎干细胞标记物SSEA-3、SSEA-4；不表达内皮祖细胞标记CD31，造血干细胞标记CD34，免疫标记物HLA—DR。人羊膜上皮干细胞具有良好的成骨和成脂分化能力。③与对照组相比，脓毒症患者血清能激活巨噬细胞M1相关促炎基因TNF-α、CCR7、IL-6的表达，而人羊膜上皮干细胞条件培养基可以促进脓毒症患者血清诱导RAW264．7细胞抑炎基因Arg-1、CD206和IL-10的表达。④免疫荧光实验表明，脓毒症患者血清能激活巨噬细胞M1相关蛋白TNF-α、CCR7的表达，而人羊膜上皮干细胞条件培养基可以促进脓毒症患者血清诱导巨噬细胞M2相关蛋白的Arg-1和CD206的表达。结论人羊膜上皮干细胞可以促进脓毒症患者血清诱导RAW264．7细胞M1型向M2型分化。

Objective To evaluate the effects of human amniotic epithelial cells （AECs） on the inflammatory status which is induced by sepsis patient＇s serum （SPS） in RAW264.7 cells. Methods Human AECs were isolated using 0.05% trypsin and 0.2 g/mL collagenase I and identified using fluorescence-activated cell sorting （FACS）. The differentiation potential of AECs was also evaluated. RAW264.7 cells stimulated with SPS were cultured in conditioned media of AECs to assess the changes of the inflammatory status. The expression of the pro-inflammatory and anti-inflammatory genes, including tumor necrosis factor- α （TNF- α） , chemokine receptor 7 （CCR7） and interleukin- 6 （IL- 6） of M 1 macrophages, and arginase 1 （Arg-1 ） , macrophage mannose receptor （CD206） and interleukin-10 （IL-10） of M2 macrophages were detected in the treatment of conditioned media of AECs. The expression of the pro-inflammatory and anti-inflammatory protein, including CCRT, TNF-α of M1 macrophages, and Arg- 1, CD206 of M2 macrophages were detected using immunofluorescence. Results （1） AECs showed a cobblestone-like morphology and AECs （Passage 2 ） have a doubling time about 3 days. （2）AECs were positive for mesenchymal stem cell markers CD29 （93.1%±4.1%）, CD90 （99.0±0.8%）, CD105 （98.7%±1.3%） ; embryonic stem cell markers SSEA-3 （75.4%±6.7%） ,SSEA-4 （84.1%±4.2%） ; and were negative for endothelial marker CD31 （0.8%±0.2%） ; hematopoietic marker CD34 （0.0%±0%） ; and immunological marker HLA-DR （0.2%±0.1% ）. （3） Compared with the control macrophages, RAW264.7 cells induced by SPS showed a significant up-regulation of TNF-α, CCR-7, IL-6 gene expression, and the conditioned media of AECs caused an up-regulation of Arg-1, CD206, IL-10 mRNA expression. （4）Immunofluorescent of RAW264.7 cells showed high expression of TNF-α, CCR-7 after the stimulation of SPS, and the conditioned media of AECs caused a down-regulation of TNF-α, CCR-7 and an up-regulation of Arg-1, CD206 expression. Conclusion AECs can promote sepsis patients serum induced RAW264.7 cells differentiation from M 1 to M2.