抑制DNMT3A促进E2F1诱导间充质干细胞向动脉内皮细胞分化及血管生成

Inhibition of DNMT3A promotes E2F1-induced differentiation of mesen⁃chymal stem cells into arterial endothelial cells and angiogenesis

目的:探讨敲减DNA甲基转移酶3A(DNMT3A)的表达是否促进细胞周期相关转录因子E2F转录因子1(E2F1)诱导人脐带间充质干细胞(hUCMSCs)向动脉内皮细胞分化及血管形成。方法:体外培养hUCMSCs,将细胞分为对照组、E2F1过表达组(E2F1组)、DNMT3A敲减组(siDNMT3A组)和联合诱导组(E2F1+siDNMT3A组);倒置显微镜下观察细胞形态变化;采用RT-qPCR和Western blot检测动脉特异性标志物ephrin-B2和HEY2的mRNA及蛋白水平;成管实验检测分化后hUCMSCs的体外管状结构形成能力;裸鼠基质胶塞实验结合CD31染色检测分化后hUCMSCs的体内生成血管情况。结果:RT-qPCR和Western blot结果显示,与对照组相比,siDNMT3A组ephrin-B2和HEY2的mRNA及蛋白水平均显著升高(P<0.01);与siDNMT3A组相比,E2F1+siDNMT3A组HEY2和ephrin-B2的mRNA及蛋白水平进一步升高(P<0.01)。体内和体外成管实验证明,除对照组外,其他3个诱导组的细胞均可形成管腔样结构,其中E2F1+siDNMT3A组的成管能力最强。结论:与单一过表达E2F1或敲减DNMT3A比较,敲减DNMT3A表达同时过表达E2F1的hUCMSCs向动脉内皮细胞分化和血管形成能力更强。

AIM:To investigate whether knock-down of DNA methyltransferase 3A(DNMT3A)promotes E2F transcription factor 1(E2F1)-induced differentiation of human umbilical cord mesenchymal stem cells(hUCMSCs)into arterial endothelial cells and angiogenesis.METHODS:The hUCMSCs were cultured and divided into control group,E2F1 over-expression group(E2F1 group),DNMT3A knock-down group(siDNMT3A group)and combination induction group(E2F1+siDNMT3A group).The morphological changes of hUCMSCs were observed under inverted microscope.The expression of arterial specific markers ephrin-B2 and HEY2 at mRNA and protein levels was determined by RT-qPCR and Western blot.The angiogenesis ability of differentiated hUCMSCs was detected by tube formation assay in vitro and by ma-trix plug test and CD31 staining in nude mice in vivo.RESULTS:The mRNA and protein levels of ephrin-B2 and HEY2 in siDNMT3A group were significantly higher than those in control group(P<0.01).Compared with siDNMT3A group,the mRNA and protein expression levels of HEY2 and ephrin-B2 in E2F1+siDNMT3A group were further increased(P<0.01).The results of in vivo and in vitro angiogenesis experiments showed that the cells in the 3 induction groups formed lumen-like structures,and the cells in E2F1+siDNMT3A group had the strongest angiogenesis ability.CONCLUSION:Compared with over-expression of E2F1 or knock-down of DNMT3A alone,inhibition of DNMT3A in hUCMSCs with E2F1 over-expression promotes their differentiation into arterial endothelial cells and angiogenesis.