睾丸Leydig干细胞分离、纯化及鉴定的体外研究

Research of Leydig Stem Cells:Separation,Purification and Identification In Vitro

目的改进Leydig干细胞的分离及纯化方法,观察其生物学特性并进行鉴定。方法通过胶原酶消化、差速贴壁法及双抗体免疫磁珠分选法,从大鼠睾丸内获得Leydig干细胞。以条件培养液进行培养,采用CCK法测定增殖能力。通过PDGFRα、LIFR及3β-HSD免疫组织化学染色鉴定Leydig干细胞。经定向诱导分化后,检测Leydig细胞的睾酮分泌能力。结果每个睾丸约可获得83 000个干细胞,经免疫组织化学染色分析,PDGFRα阳性率为(98.0±0.8)%。在条件培养液中培养的SLCs增殖明显,在1个月内能稳定维持其干性而未向Leydig细胞系分化。免疫组织化学染色结果表明,PDGFRα、LIFR阳性表达,3β-HSD为阴性表达。Leydig干细胞在含有T3和HCG的培养液中培养后,第5天即可测到睾酮分泌,分化后的细胞3β-HSD+。结论由差速贴壁法及双抗体免疫磁珠分选法能获得纯化的PDGFRα+/LHR-细胞,这些细胞符合SLCs所必须具有的特征。采用该方法获取SLCs,操作简便,细胞损伤小并且获取纯度高。

Objective To improve the methods for separation and purification of Leydig stem cells （SLCs） and observe its biological characteristics and to identify the cells. Methods Leydig stem cells were gained from the testes of Wistar rats by ways of collagenase digestion, differential adhesion and two-antibodies magnetic activated cell sorting. And then the cells were cultured in modified culture media and its reproductive activity was tested by cell counting kit （CCK） method. SLCs were identified by immunohistochemistry （IHC） staining with the detection of PDGFRα, LIFR and 3β-HSD. Testosterone level was detected by RIA method after SLCs were induced for differentiation. Results About 83 000 cells were available in each testis and the PDGFRα＋ SLCs could reach to 98% by IHC staining. SLCs proliferated actively in modified culture media and could maintain undifferentiated state within a month in vitro. IHC staining results showed that the expression of PDGFRα and LIFR were positive and the expression of 3β-HSD was negative. Testosterone secretion could be detected at day 5 while SLCs were cultured in media added with T3 and HCG. 3β-HSD of differentiated cells turned to be positive by IHC staining. Conclusion PDGFRα＋/LHR- cells could be obtained by differential adhesion and magnetic activated cell sorting method. The cells were in accordance with the characteristics necessary for SLCs. This method has the advantages of simple, small cell damage and high purity.