目的: 探究小胶质细胞在急性低氧后DNA甲基转移酶的变化情况。方法: 以BV2细胞为研究对象,将其分为对照组(control)、H-0组(低氧12 h不进行复氧)、H-2组(低氧12 h复氧2 h)、H-4组(低氧12 h复氧4 h)、H-8组(低氧12 h复氧8 h)、H-12组(低氧12 h复氧12 h)、H-24组(低氧12 h复氧24 h)。将BV2细胞低氧12 h,在不同的时间点进行复氧构建模型,通过荧光倒置显微镜明场观察细胞形态;采用Real-time PCR检测DNMT1、DNMT3A、DNMT3B在mRNA水平上的表达变化;采用Western blot检测DNMT1、DNMT3A、DNMT3B在蛋白水平上的表达变化。结果: 与control组相比,H-0组的细胞数量减少,细胞形态皱缩,大部分细胞触角伸长,细胞暗淡无光,折光度降低。随着复氧时间的增加(H-2、H-4、H-8、H-12、H-24),贴壁细胞逐渐减少。与control组相比,DNMT1在H-0组中蛋白的表达水平显著上升(P＜0.05),H-8、H-12、H-24组DNMT1的mRNA和蛋白水平表达显著降低(P＜0.05)。DNMT3A在H-8、H-12组中的mRNA和蛋白水平表达显著降低(P＜0.05)。DNMT3B在H-0、H-4、H-8、H-12、H-24组细胞中的mRNA和蛋白水平无显著变化。结论: 在低氧后小胶质细胞中DNMT1、DNMT3A发挥了重要作用。
Objective: To investigate the changes of DNA methyltransferase in microglia after hypoxia. Methods: BV2 cells were used as the research object, and they were divided into the control group, H-0 group (hypoxia for 12 h without reoxygenation), H-2 group (hypoxia for 12 h with reoxygenation for 2 h), H-4 group (hypoxia for 12 h with reoxygenation for 4 h), H-8 group (hypoxia for 12 h with reoxygenation for 8 h), H-12 group (hypoxia for 12 h with reoxygenation for 12 h), and H-24 group (hypoxia for 12 h with reoxygenation for 24 h). The BV2 cells were cultured under hypoxia condition for 12 h and reoxygenated at different time points to construct the model, and the cell morphology was observed in the bright field by fluorescence inverted microscope. The expression changes of DNMT1, DNMT3A, and DNMT3B at the mRNA level were detected by Real-time PCR, and the expression changes of DNMT1, DNMT3A, and DNMT3B at the protein level were detected by Western blot. Results: Compared to the Control group, the H-0 group had a decreased number of cells, wrinkled cell morphology, and elongated tentacles in most cells. The cells were dull and the refractive index was reduced. With the reoxygenation time increasing (H-2, H-4, H-8, H-12, H-24), the adherent cells gradually decreased. Compared with the control group, the protein expression level of DNMT1 was significantly increased in the H-0 group (P＜0.05), and the mRNA and protein expression level of DNMT1 was significantly decreased in the H-8, H-12, and H-24 groups (P＜0.05). mRNA and protein expression level of DNMT3A was significantly decreased in the H-8, H-12 groups (P＜ 0.05). mRNA and protein expression of DNMT3B in cells of H-0, H-4, H-8, H-12, and H-24 groups did not change significantly. Conclusion: DNMT1 and DNMT3A play important roles in microglia after hypoxia.
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