枸杞多糖对精原干细胞体外增殖的影响

Influence of lycium barbarum polysaccharides on proliferation of spermatogonial stem cells in vitro

背景:精原干细胞移植对不育具有潜在的临床应用价值,体外建立精原干细胞的培养系统获得数量较多的精原干细胞,仍是目前研究中亟待解决的问题。目的:观察枸杞多糖对精原干细胞体外增殖的影响。方法:采用两步酶消化法获取出生4～6d雄性C57BL/6小鼠睾丸Sertoli细胞与精原干细胞,将精原干细胞接种在Sertoli细胞饲养层上,再加入枸杞多糖或联合细胞因子添加到细胞培养液中。1周后以流式细胞仪检测细胞周期及细胞活性率,并检测各组精原干细胞GFRa-1、Thy-1、c-kit的阳性率。结果与结论:单独加入枸杞多糖后精原干细胞数量明显增加,增殖明显,联合加入胶质细胞源性神经营养因子与白血病抑制因子精原干细胞增殖更加明显(P<0.05)。并发现体外培养1周后的精原干细胞仍保持其睾丸组织内的精原干细胞特征,大多仍维持在未分化状态。表明在枸杞多糖或枸杞多糖联合胶质细胞源性神经营养因子及白血病抑制因子作用下,可促进精原干细胞体外增殖。

BACKGROUND：It has potential clinical value to use spermatogonial stem cell transplantation to cure infertility. In order to obtain a large number of spermatogonial stem cells for transplantation,culture system in vitro need to be established,which is still an urgent problem at present. OBJECTIVE：To investigate the effect of lycium barbarum polysaccharides（LBP） on proliferation of spermatogonial stem cells in vitro. METHODS：Sertoli cells and spermatogonial stem cells were separated from testis of 4-6 days postpartum C57BL/6 male mice by two-step enzyme digestion method. The spermatogonial stem cells were seeded on the sertoli cells feeder layer and cocultured in vitro with LBP or cytokines in cell culture medium. After one-week coculture,the cell cycle and cell activity rate of spermatogonial stem cells were determined by flow cytometry. Positive rate of spermatogonial stem cells for GFRa-1,Thy-1,c-kit as cell markers were detected. RESULTS AND CONCLUSION：Adding LBP into the culture medium alone,the amount of spermatogonial stem cell was increased apparently,and spermatogonial stem cells proliferated significantly. Adding LBP,glial cell line-derived neurotrophic factor and leukemia inhibitory factor into the culture medium together,spermatogonial stem cells proliferated more significantly（P 0.05） . Also spermatogonial stem cells after 1-week in vitro culture maintained the feature of spermatogonial stem cells within the testicular tissue,most of them remained undifferentiated state. Results indicate that LBP or combination of some cytokines can promote the proliferation of spermatogonial stem cells in vitro.