新生CD-1小鼠雌性生殖干细胞的建系及生物学特征

Establishment and characterization of CD-1 neonatal female germline stem cell lines

背景:传统观点认为雌性哺乳动物出生后不再产生新的卵母细胞,近年来多项研究表明出生后哺乳动物卵巢中存在能再生卵子的雌性生殖干细胞。目的:从新生CD-1小鼠卵巢中分离、纯化雌性生殖干细胞并建系及进行生物学特征研究。方法:通过两步酶消化法和免疫磁珠分选从新生CD-1小鼠卵巢中分离与纯化雌性生殖干细胞并体外长期培养,然后通过RT-PCR、细胞免疫荧光化学和核型分析鉴定其生物学特征。结果与结论:从3-5 d龄CD-1小鼠卵巢中分离出雌性生殖干细胞并建立细胞系,目前已在体外长期培养15个月,共传代70多次。该细胞系表达Mvh,Dazl,Oct-4,Stella,Blimp1,Fragilis等生殖细胞标志物,同时通过Fragilis和BrdU的双免疫荧光实验证实了其增殖活性和生殖细胞特性;核型分析发现该细胞系也具有正常的二倍体核型。因此CD-1小鼠卵巢中存在具有有丝分裂活性和正常的核型的雌性生殖干细胞。

BACKGROUND：In traditional view, the germ cels in mammal ovaries were considered to lose the capacity of self-renew after birth. However, recent studies have showed the existence of female germline stem cels （FGSCs） which take the responsibility for regenerating oocytes in the ovaries of postnatal mammals. OBJECTIVE：To establish the FGSC line and to explore the biological characterization of the FGSC line from neonatal CD-1 mouse ovaries. METHODS：Using two-step enzymatic digestion and immunomagnetic purification, Fragilis-positive cels were isolated from neonatal CD-1 mice ovaries and subjected to long-term culture. Then their biological characteristics were studies with RT-PCR, fluorescent immunocytochemistry and cytogenetic analysis. RESULTS AND CONCLUSION：FGSCs from neonatal CD-1 mice aged 3-5 days were isolated and long-term cultured for 70 passages to establish a cellline. This cellline expressed Mvh, Dazl, Oct-4, Stela, Blimp1, Fragilis, and was proven to be mitoticaly active germ cels by BrdU/Fragilis dual immunofluorescence analysis. Moreover, cytogenetic analysis by Gimsa staining confirmed their normal karyotype. In al, these results indicate the existence of FGSCs with mitotic activity in neonatal CD-1 mouse ovaries.