大鼠精原干细胞的微滴培养法

精原干细胞（spermatogonial stem cells，SSCs）作为成体干细胞的一类，既具有自我更新和分化的潜能，又可向子代传递遗传信息。阐明其增殖过程及分化特性对SSCs的进一步应用具有重要意义。小鼠SSCs的微滴培养研究显示，微滴培养技术与常规培养方法相比具有独特的优势。然而其他物种的SSCs能否实现微滴培养尚有待证实。该研究旨在利用微滴培养法建立大鼠SSCs体外培养技术。5、8、10、20、40个大鼠SSCs分别置于20此微滴中培养，用丝裂霉素处理的STO细胞作为滋养层。倒置显微镜观察记录大鼠SSCs的增殖状态。一个月后，对微滴培养的SSCs进行免疫荧光双标记染色鉴定。结果显示，一个微滴内接种5个SSCs就能实现扩增培养；培养一个月后，SSC仍然表达其特异的标记基因分子如CDH1、OCT4、PLZF、Thy1和Gfra1。体外诱导分析显示，微滴培养的大鼠SSCs具有分化为精母细胞的能力。大鼠SSCs微滴培养法的建立，为其他物种SSCs的培养提供了借鉴，也为再生医学和生命科学相关领域的研究提供了技术平台。

Microdrop Culture of Rat Spermatogonial Stem Cells

Spermatogonial stem cells （SSCs） not only possess the capacity for self-renewal and differentiation, but also can pass on genetic information to offspring. It has great significance to verify proliferation and differentiation of SSCs for further application. Research on microdrop culture of mouse SSCs showed that it had unique advantages compared to conventional culture methods. However, feasibility of the microdrop culture in other species remains to be confirmed. The purpose of this study was to establish rat SSCs culture system in vitro by the microdrop culture method. We transfered 5, 8, 10, 20, 40 rat SSCs into microdrops containing 20 μL medium, respectively, in which STO cells treated with mitomycin were used as the feeder layer. The proliferation status of the rat SSCs were observed and recorded using microscopy. After cultured for one month, immunofluorescence double- label staining and induction of differentiation in vitro were performed for analysis of the cultured SSCs. The results showed that inoculating at least five SSCs in a microdrop was able to achieve the SSC proliferation. After cultured in microdrop for one month, the rat SSCs still expressed the marker molecules such as CDHI, OCT4, PLZF, Thyl and Gfral. And they also possessed the ability to differentiate into spermatocytes in vitro. The microdrop culture technique for proliferation of rat SSCs had been established. It offers reference for cultivation of SSCs in other species, and provides a technology platform for related research field in regenerative medicine and life science.