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目的探究骨形成蛋白4(BMP4)对小鼠胚胎干细胞向生殖细胞分化的调控作用。方法体外培养小鼠胚胎干细胞(m ESC),细胞被分为对照组、BMP4低剂量组(低BMP4组)和BMP4高剂量组(高BMP4组);低BMP4组和高BMP4组m ESC使用BMP4处理,剂量水平分别为5 ng/ml和10 ng/ml。干预72 h后,采用碱性磷酸酶(AP)染色法检测各组细胞内AP水平;采用Real-time PCR和Western印迹检测各组细胞多能性相关基因Oct4、c-Myc及生殖相关基因Vasa、Scp3、Stra8的表达水平。结果 AP染色结果显示低BMP4组和高BMP4组细胞AP水平显著低于对照组(P<0.05),且高BMP4组下降更为显著(P<0.05);Real-time PCR和Western印迹结果显示低BMP4组和高BMP4组细胞Oct4、c-Myc表达水平显著低于对照组(P<0.05),Vasa、Scp3、Stra8表达水平显著高于对照组(P<0.05),且高BMP4组这些变化更为显著(P<0.05)。结论 BMP4具有促进小鼠胚胎干细胞向生殖细胞分化的功能,可为应用BMP4诱导ESCs向生殖细胞定向分化提供理论基础。
Objective To investigate the regulation of bone morphogenetic protein 4 (BMP4) on the differentiation of mouse embryonic stem cells into germ cells. Methods Mouse embryonic stem cells (m ESC) were cultured in vitro. The cells were divided into control group, BMP4 low dose group (low BMP4 group) and BMP4 high dose group (high BMP4 group). BMP4 group and high BMP4 group Treatment, dose levels were 5 ng / ml and 10 ng / ml. The levels of AP in each group were detected by alkaline phosphatase (AP) staining 72 h after intervention. Real-time PCR and Western blotting were used to detect the expression of Oct4, c-Myc and reproductive-related genes Vasa , Scp3, Stra8 expression levels. Results The results of AP staining showed that the levels of AP in low BMP4 group and high BMP4 group were significantly lower than those in control group (P <0.05), and decreased significantly in high BMP4 group (P <0.05). Real-time PCR and Western blotting showed low The expression levels of Oct4 and c-Myc in BMP4 group and high BMP4 group were significantly lower than those in control group (P <0.05), and those in Vasa, Scp3 and Stra8 were significantly higher than those in control group (P <0.05) As significant (P <0.05). Conclusion BMP4 can promote the differentiation of mouse embryonic stem cells into germ cells and provide a theoretical basis for BMP4-induced directional differentiation of ESCs into germ cells.