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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: To explore the isolation and culture of Sertoli cells (SCs) and female bone marrow stem cells (FBMSCs) in vitro. To observe the effect of SCs on the growth and proliferation of FBMSCs. To observe the effect of SCs and all-trans retinoic acid all-trans retinoic acid (ATRA) on the differentiation of FBMSCs into FSHR + and stra8 + cells. Methods Whole-bone marrow adherent method was used to separate FBMSCs of young SD rats. The third generation of FBMSCs were subcultured and divided into four groups: 1.SCs co-culture group, 2.SCs co-culture + ATRA group, 3.ATRA group, 4 .FMSCSs group. The ATRA group was added to the control group at a final concentration of 10-5 mol / l. ATRA was used to detect the effect of co-culture of supportive cells and retinoic acid on the survival and proliferation of bone marrow stem cells by MTT assay. To detect the differentiation of female bone marrow stem cells, RT-PCR was used to detect the change of germline stem cells from mitosis to pre-meiotic expression stra8 mRNA and fshr m RNA expression of ovarian granulosa cells. RESULTS: Female bone marrow stem cells cultured in the group of cell-coculture and retinoic acid partially differentiated into large, round oocyte-like cells surrounded by many small cells surrounding the differentiated cells. Control group cells are more homogeneous. RT-PCR showed stra8 m RNA and FSHR m RNA were expressed in the bone marrow stem cells of the coculture group and the retinoic acid group, respectively, but not in the control group. Conclusion Both ATRA and SCs can induce the differentiation of bone marrow stem cells into stra8 + And FSHR ~ + cells.