维甲酸定向诱导小鼠核移植胚胎干细胞向雄性生殖细胞分化

Effect of retinoic acid and induction of nuclear transfer embryonic stem cells to differentiate into male germ cells

目的初步探讨体外条件下定向诱导小鼠核移植胚胎干细胞分化为雄性生殖细胞的条件,建立有效诱导分化技术平台。方法分离BALB/c小鼠NT-ESCs样集落并鉴定。NT-ESCs消化后分4组:1)采用维甲酸诱导NT-EBs向雄性生殖细胞方向分化;2)未分化的NT-ESCs;3)分化培养前的NT-EBs;4)自主分化的NT-EBs;5)小鼠胚胎成纤维细胞。RT-PCR法检测各组Oct-4、VASA、Scp3、Sry、Tex14和β-actin的mRNA表达;免疫荧光染色法检测VASA、Scp3蛋白表达。结果 NT-ESCs集落性生长,符合小鼠胚胎干细胞的一系列特性。NT-EBs维甲酸诱导组Oct-4、VASA、Scp3、Sry、Tex14和β-actin均有表达,对照组为阴性;NT-EBs细胞诱导分化后Scp3荧光强度为(+);实验组VASA荧光强度(+)也优于对照组的(±)。结论采用维甲酸诱导可促进核移植胚胎来源NT-ESCs分化为雄性生殖细胞。

Objective To set up a feasible method of differentiation-oriented induction of male germ cells from mouse nuclear transfer embryonic stem cell.Methods The NT-ESCs were identified.The NT-ESCs were digested and divided into 4 groups：group 1 was induced to form embryoid bodies and cultured in 2 μmol/L retinoic acid（RA） inducing them to proliferate and differentiate into male germ cells; Then passage 1 NT-ESCs as group 2 ; group 3 underwent EB culture ; group 4 underwent EB culture and were cultured.The mouse embryonic fibroblast cell as group 5.mRNA expression （Oct-4,VASA,Scp3,Sry,Tex14 and β-actin） were detected by RTPCR.The proteins of VASA and Scp3 were analyzed by immunofluorescence assay.Results Most of NT-ESCs had typical characteristics of mouse embryonic stem cells.The genes related to male germ cells development were expressed in the NT-EBs cells,and the mRNA expression of Oct-4,VASA,Scp3,Sry,Tex14 and β-actin in the induced NT-EBs were positive.Those in control groups were negative.The fluorescence intensity of Scp3 in the induced NT-EBs was high（＋） ;The fluorescence intensity of VASA in the induced NT-EBs was higher（＋） than that in the control group（±）.Conclusions NT-ESCs may differentiate into male germ cells in vitro induced by retinoic acid.