低氧处理的hAMSCs外泌体对巨噬细胞M1/M2分型影响及其分子机制

EFFECT OF HYPOXIA-TREATED HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELL-DERIVED EXOSOMES ON M1/M2 TYPING CHANGES OF MACROPHAGES AND ITS MOLECULAR MECHANISM

目的探究低氧处理的人脂肪间充质干细胞(hAMSCs)外泌体对巨噬细胞M1/M2分型的影响及其分子机制。方法提取低氧处理的hAMSCs外泌体与M1巨噬细胞共培养,采用流式细胞术检测共培养后巨噬细胞表型M1/M2比例,实时荧光定量PCR(qPCR)检测炎症相关因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、一氧化氮合酶2(NOS-2)和白细胞介素12(IL-12)的表达,Western blot检测核因子κB(NF-κB)通路中P65和p-P65蛋白的表达。结果加入低氧处理的hAMSCs外泌体后,巨噬细胞M1型比例下降,M2型比例增加(t=3.293、10.242,P<0.05),炎症相关因子TNF-α、IL-1β、NOS-2以及IL-12的表达下调(F=10.820~222.942,P<0.05),p-P65/P65蛋白表达比值下降(F=375.634,P<0.05)。结论低氧处理的hAMSCs外泌体能够通过抑制NF-κB通路使巨噬细胞表型由M1型向M2型转化。

Objective To explore the effect of exosomes derived from hypoxia-treated human adipose-derived mesenchymal stem cells(hAMSCs)on the M1/M2 typing changes of macrophages and its molecular mechanism.Methods Hypoxia-treated hAMSC-derived exosomes were extracted and co-cultured with M1 macrophages.Flow cytometry was used to determine the M1/M2 ratio of the co-cultured macrophages.Quantitative real-time polymerase chain reaction was used to measure the expression of inflammation-related factors,i.e.,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),nitric oxide synthase-2(NOS-2),and interleukin-12(IL-12).Western blot was used to measure the protein expression of P65 and p-P65 in the nuclear factor-kappa B(NF-κB)pathway.Results After addition of hypoxia-treated hAMSC-derived exosomes,the proportion of M1 macrophages was significantly reduced,while the proportion of M2 macrophages was significantly increased(t=3.293,10.242;P<0.05);inflammation-related factors TNF-α,IL-1β,NOS-2,and IL-12 had significantly down-regulated expression(F=10.820-222.942,P<0.05),and p-P65/P65 protein expression ratio was significantly reduced(F=375.634,P<0.05).Conclusion Exosomes derived from hypoxia-treated hAMSCs can transform the phenotype of macrophages from M1 to M2 by inhibiting the NF-κB pathway.