全反式维甲酸对M2型巨噬细胞极化的影响

Effect of All-trans Retinoic Acid on Polarization of M2 Macrophages

目的建立M2型巨噬细胞极化的体外模型,研究全反式维甲酸(all-trans-retinoic acid,ATRA)对白细胞介素4(interleukin-4,IL-4)诱导的M2型巨噬细胞极化的影响。方法选取8周龄C57BL/6雌性小鼠。经腹腔灌洗贴壁纯化后,将小鼠腹腔巨噬细胞在体外进行原代培养。小鼠随机分为空白对照组、M2型极化模型组、DMSO对照组和ATRA处理组。白细胞介素-4(IL-4,20 ng/mL)诱导小鼠腹腔巨噬细胞构建M2型巨噬细胞极化模型,45μg/mL ATRA孵育24 h。在倒置显微镜下观察细胞形态学表现,采用Annexin V-PE/7-AAD双染法测验ATRA对巨噬细胞凋亡的影响,流式细胞仪检测F4/80+CD206+M2型巨噬细胞的表达,实时荧光定量PCR(RT-qPCR)法检测白细胞介素-10(IL-10)、精氨酸酶1(Arg1)和转化生长因子-β1(TGFβ1)的mRNA表达。结果倒置显微镜下观察,对照组可见细胞主要呈圆形或椭圆型,M2极化模型组可见细胞主要呈圆盘状;与M2极化模型组比较,ATRA处理组可见圆盘状细胞增多变大;各组细胞间巨噬细胞凋亡率比较无明显差异(P>0.05);与空白对照组相比较,M2极化模型组中F4/80+CD206+M2表型巨噬细胞表达水平提高(P<0.01),M2极化模型组中Arg1,IL-10,TGF-β1 mRNA的表达增高(P<0.01);与M2极化模型组相比较,ATRA处理组中F4/80+CD206+M2表型巨噬细胞表达水平显著升高(P<0.01),ATRA处理组中IL-10、Arg1和TGFβ1 mRNA的表达水平显著升高(P<0.01)。结论ATRA(45μg/mL)对巨噬细胞凋亡没有明显影响,并且促进巨噬细胞向M2表型极化。

Objective To establish an in vitro model of M2-type macrophage polarization,and investigate the effect of all-trans retinoic acid(ATRA)on the M2-type macrophage polarization induced by interleukin-4(IL-4).Methods The eight-week-old C57BL/6 female mice were selected.After peritoneal lavage and adherent purification,mouse peritoneal macrophages were subjected to primary culture in vitro.The mice were randomly divided into blank control group,M2 polarization group,DMSO control group and ATRA treatment group.Interleukin 4(IL-4,20 ng/mL)was used to induce mouse peritoneal macrophages to establish the macrophage M2 polarization model.45μg/mL ATRA was incubated for 24 h.The morphology of cells was observer by inverted microscope.The expression of Annexin V-PE/7-AAD and F4/80+CD206+M2 macrophages was detected by flow cytometry.The mRNA expression levels of interleukin-10(IL-10),arginase 1(Arg1)and transforming growth factorβ1(TGFβ1)were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.Results Under the inverted microscope,the cells in the control group were mainly round or elliptic,while the cells in the M2 polarization group were mainly disc-shaped.Compared with M2 polarization group,ATRA treated group showed increased and larger disk cells.There was no significant difference in the apoptosis rate of macrophages among all groups(P>0.05);compared with blank control group,the proportion of F4/80+CD206+M2 macrophages in M2 polarization group was significantly increased(P<0.01),and the mRNA expressions of Arg1,IL-10 and TGF-β1 in M2 polarization group were increased significantly(P<0.01);compared with M2 polarization group,the proportion of F4/80+CD206+M2 macrophages in ATRA treatment group was significantly increased(P<0.01),and the mRNA expressions of Arg1,IL-10 and TGF-β1 in ATRA treatment group were increased significantly(P<0.01).Conclusion ATRA(45μg/mL)has no cytotoxic effect on macrophages and promotes the polarization of macrophages to the M2 type.