诱导骨髓SSEA-1^+干细胞向卵母细胞分化的初步研究  

Derivation of oocyte-like cells from SSEA-1^+ stem cells of mouse bone marrow

作　　者：范静[1] 姜宏[1] 殷慧群[1] 汪存利[1] 何瑞冰[1] 

机构地区：[1]解放军第105医院生殖医学中心,合肥230031

出　　处：《生殖与避孕》2015年第8期538-543,560,共7页Reproduction and Contraception

基　　金：南京军区医学科技创新重点项目;项目编号:11Z010

摘　　要：目的:探讨骨髓SSEA-1+干细胞向卵母细胞分化的潜能。方法:采用免疫磁珠分选系统(MACS)分选骨髓中SSEA-1+干细胞,接种于小鼠成纤维细胞(mE Fs)饲养层上,在含1 000 U/ml白血病抑制因子(LIF)的DMEM/F12培养液中将SSEA-1+干细胞传代培养至第2代(P2),撤去饲养层,在含20 ng/ml骨形成蛋白4(BMP4)的培养液中进行定向诱导,并分阶段加入体积分数10%人卵泡液、0.005 IU/ml卵泡刺激素(FSH)和0.003 IU/ml黄体生成素(LH)。光学显微镜下观察细胞形态学变化,检测不同发育阶段生殖细胞相关特异性基因Dazl、Fragilis、Mvh、Nobox、Oct4、Stella、STRA8、Gdf9的表达。结果:骨髓SSEA-1+干细胞生殖细胞相关特异性基因表达水平均显著高于骨髓单个核细胞和SSEA-1-细胞(P<0.05)。小鼠骨髓SSEA-1+干细胞与m EFs共培养后呈圆形,碱性磷酸酶染色阳性,显示细胞处于未分化状态。BMP4诱导7 d SSEA-1+干细胞形成细胞集落,14 d可见类胚体(EB)样结构形成,21 d可观察到卵母细胞样结构,且随诱导时间的延长生殖细胞相关特异性基因尤其卵母细胞减数分裂基因Stra8和Gdf9表达水平显著升高。结论:骨髓SSEA-1+干细胞具备向类卵母细胞样细胞分化的潜能。Objective： To explore the derivative potential of stage specific embryonic antigen（SSEA）-1＋stem cells of mouse bone marrow toward oocyte-like cells in vitro. Methods： SSEA-1＋ stem cells were separated from mouse bone marrow by magnetic activated cell sorting（MACS）, and cultured on mouse embryonic fibroblasts feeder layer（mE Fs） in DMEM/F12 medium contained leukemia inhibitory factor（LIF）. The Passage2（P2） SSEA-1＋ cells were removed from m EFs and induced to differentiate toward germ line under 20 ng/ml bone morphogenetic protein（BMP）4, then 10% human follicular fluid, 0.005 IU/ml follicle stimulating hormone（FSH） and 0.003 IU/ml luteinizing hormone（LH） were added to the medium at different differentiation stages.Meanwhile, the morphological observation and detection of early germ cell-specific genes related to germ cell differentiation such as Dazl, Fragilis, Mvh, Nobox, Oct4, Stella, STRA8 and Gdf9 were performed at different differentiation stages. Results： The expression levels of all selected early germ cell-specific genes were significantly higher in SSEA-1＋ stem cells than those in whole bone marrow cells and SSEA-1- cells（P〈0.05）.Mouse bone marrow SSEA-1＋ stem cells grew well and maintained undifferentiated state on m EFs feeder, with roughly circle shapes and positive alkaline phosphatase staining. The cell colonies, embryoid body（EB） structures,oocyte-like cells from P2 SSEA-1＋ stem cells could be seen on day 7, 14 and 21 respectively after induction of BMP4, and the expression levels of germ cell specific genes were increased gradually, especially for STRA8 and Gdf9. Conclusion： Under the induction of BMP4, SSEA-1＋ bone marrow mesenchymal stem cells could differentiate into oocyte-like cells in vitro.

关 键 词：骨髓间充质干细胞(BMSCs) 卵母细胞样细胞 骨形成蛋白4(BMP4) 细胞分化 QRT-PCR 

分 类 号：R392.6[医药卫生—免疫学]