小鼠雌性生殖干细胞体内分化鉴定方法

摘要

目的 建立一种小鼠卵巢雌性生殖干细胞(FGSCs)体内分化鉴定方法. 方法 从6周龄C57BL/6小鼠卵巢利用Fragilis抗体基于免疫磁珠分选(MACS)分离FGSCs细胞,并置于STO饲养层细胞上扩增培养,采用RT-PCR法检测生殖干性基因包括Fragilis、MVH、Prdm1和Tert,细胞免疫荧光法检测Fragilis和MVH蛋白表达,利用绿色荧光蛋白(GFP)慢病毒载体荧光标记FGSCs后将其植入受体鼠卵巢,在体内环境下促其分化发育为卵母细胞,至少12d后取卵巢置于载玻片上并压上盖玻片,直接置于荧光显微镜下观察. 结果 RT PCR结果显示FGSCs生殖干性基因包括Fragilis、MVH、Prdm1和Tert为阳性表达,而卵母细胞特异表达基因中Nobox为阴性,GDF9和ZP3为弱阳性;细胞免疫荧光结果显示FGSCs的MVH和Fragilis蛋白表达阳性,而且呈明显的膜表达.GFP-FGSCs植入6周龄雌性C57BL/6的卵巢中,至少12d以后将卵巢取出,压片后置于荧光显微镜下观测,结果显示有荧光阳性的卵母细胞. 结论 该鉴定方法简单直观,可作为FGSCs体内分化鉴定的可选方法.%Objective:To establish a method for identifying the differentiation of mouse female germline stem cells(FGSCs).Methods:Firstly the FGSCs were isolated with Fraggis antibody from the ovaries of 6-week-old C57BL/6 mouse based on immunomagnetic beads sorting(MACS).The isolated FGSCs were placed on STO feeder cells for expanded culture.Then RT-PCR was used to detect the female reproductionassociated stemness genes including Fragilis,MVH,Prdm1 and Tert.The expression of Fragilis and MVH proteins was detected by immunofluorescence assay.FGSCs were labeled with GFP lentiviral vector and implanted into the recipient mouse ovary for differentiating & developing to oocytes in vivo.After more than 12 days of development in vivo,the ovaries were dissected and put onto a slide and pressed against a cover slip to observe directly under fluorescence microscope.Results:The results of RT-PCR showed that Fragilis,MVH,Prdm1 and Tert were positive in FGSCs;while oocyte specific genes Nobox was negative,and GDF9 and ZP3 were weakly positive.The cellular immunofluorescence results showed that the protein expression of MVH,Fragilis and FGSCs was positive,and which presented membrane expression.GFP-FGSCs were implanted into the ovaries of the 6 week-old female C57BL/6.After at least 12 days,the ovaries were removed and compressed and observed under fluorescence microscopy.The results showed that there were oocytes with fluorescent positive.Conclusions:The method is simple and intuitive and can be used as an alternative method for FGSCs differentiation in vivo.