摘要
目的探索睾丸支持细胞(Sertoli cells,SCs)和雌性骨髓干细胞(female bone marrow stem cells,FBMSCs)的分离和体外培养技术;观察SCs对FBMSCs生长、增殖的影响;观察SCs和全反式维甲酸(all-trans retinoic acid,ATRA)对FBMSCs向FSHR+和stra8+细胞分化的影响。方法利用全骨髓贴壁法分离青年SD大鼠FBMSCs,传代培养第三代FBMSCs,将其分为四组:1.SCs共培养组,2.SCs共培养+ATRA组,3.ATRA组,4.FMSCSs组。ATRA组在对照组的基础上加入的了终浓度为10-5 mol/l。ATRA用MTT比色法检测支持细胞共培养及维甲酸对骨髓干细胞的存活及增殖的影响;为检测雌性骨髓干细胞分化情况,用RT-PCR检测生殖干细胞由有丝分裂转变为减数分裂前特异表达基因stra8 m RNA及卵巢颗粒细胞特异表达基因fshr m RNA的表达。结果支持细胞共培养组和维甲酸组培养的雌性骨髓干细胞部分分化成为大而圆的卵母细胞样细胞,分化的细胞周围有许多小细胞环绕。对照组细胞比较均一。RT-PCR显示支持细胞共培养组和维甲酸组的骨髓干细胞均有stra8 m RNA、FSHR m RNA表达,对照组没有表达;结论 ATRA和SCs均能体外诱导雌性大鼠骨髓干细胞分化为stra8^+及FSHR^+细胞。
Objective： The aim is to establish the culture systems of sertoli cells （SCs） and female bone marrow stem cells （FBMSCs） in vitro, investigate whether adult female BMSCs are able to differentiate into Follicle-stimulating hormone receptor （FSHR, a GCs specific marker in the ovary cells） positive GCs and stra8 （stimulated by retinoic acid gene 8, a specific expression gene in mammalian germ cell＇ s transition from mitosis to meiosis） expression oogonia in vitro by two different treatments： all-trans retinoic acid （RA） and indirect co-cultured with Sertoli ceils （SCs） .Methods： FBMSCs were separated from adult female SD rat bone marrow.FBMSCs the 3nd passage divided four groups in random. 1. co-culture with SCs, 2. coculture with SCs and 10^-5mol/L ATRA, 3. co-culture with 10^-5mol/L ATRA, 4. control group： FBMSCs were cultured in DMEM supplemented with 15% fetal bovine serum MTT colorimetric assay was used to observe the survival and proliferation of SCs and FBMSCs in vitro respectively and of FBMSCs in indirect co-culture with SCs.RT-PCR was performed to detect the expression of stra8 mRNA, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into female germ cells. Results： In all of the two treatments, some of the FBMSCs were large and round and had a oocyte-like cell appearance. Some were FSHR positive by RT-PCR and the cells were small or middle and round. In addition, the FBMSCs also expressed stra8 by RT-PCR. Conclusions： These results suggest that RA and the soluble factors secreted by SCs could all induce FBMSC to differentiate into stra8^＋ Cells and FSHR＋ Cells in vitro.
出处
《中国优生与遗传杂志》
2016年第1期43-46,共4页
Chinese Journal of Birth Health & Heredity
