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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20μL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。
As a kind of adult stem cells, spermatogonial stem cells (SSCs) have the potential of self-renewal and differentiation and can transmit genetic information to offspring. To clarify its proliferation and differentiation characteristics of SSCs further application is of great significance. Micro-droplet culture studies of mouse SSCs have shown that droplet culture techniques have unique advantages over conventional culture methods. However, whether other SSCs can be cultured in microtitration remains to be confirmed. The aim of this study was to establish a technique for in vitro culture of rat SSCs using droplet culture. 5, 8, 10, 20 and 40 rat SSCs were respectively cultured in 20 μL droplet and treated with mitomycin-treated STO cells as trophoblast. Inverted microscope was used to observe the proliferation status of rat SSCs. One month later, the SSCs cultured in droplet were identified by double immunofluorescence staining. The results showed that in one droplet, 5 SSCs could inoculate and proliferate in vitro. After one month of culture, SSCs still express their specific marker gene molecules such as CDH1, OCT4, PLZF, Thy1 and Gfra1. In vitro induction analysis showed that rat SSCs cultured in droplets had the ability to differentiate into spermatocytes. The establishment of rat SSCs droplet culture method provides reference for the culture of SSCs of other species, and also provides a technological platform for the research in regenerative medicine and life science related fields.