共培养法分离培养不同遗传背景的小鼠精原干细胞  

Isolation and culturing of mouse spermatogonial stem cells from different genetic background mice by co-culture method

作　　者：范翠花[1,2] 邱东飚 兰风华[2,3] 张朵 Fan Cuihua;Qiu Dongbiao;Lan Fenghua;Zhang Duo(Department of Blood Transfusion,The First Afiliated Hospital of Fujian Medical University,Fuzhou,Fujian,350005,China;Fujian Provincial Key Laboratory of Transplant Biology,Fuzong Clinical College,Fujian Medical Uniersity,Fuzhou,Fujian,350025,China;Laboratory of Basic Medicine,Fuzong Teaching Hospital,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian,350025,China)

机构地区：[1]福建医科大学附属第一医院输血科,福建福州350005 [2]福建医科大学福总临床医学院(第九OO医院)移植生物学重点实验室,福建福州350025 [3]福建中医药大学福总教学医院(第九OO医院)基础医学实验室,福建福州350025

出　　处：《中国男科学杂志》2023年第2期35-41,共7页Chinese Journal of Andrology

基　　金：福建省自然科学基金面上项目(编号:2021J011267);联勤保障部队第九〇〇医院临床应用研究专项(编号:2020L14)。

摘　　要：目的 建立一种简便、高效的小鼠精原干细胞(mouse spermatogonial stem cell, mSSC)分离、培养方法。方法 选取昆明小鼠和C57BL/6小鼠的雄性幼鼠睾丸,通过机械分离和酶消化相结合的方法获得细胞悬液,随后将含有mSSC和支持细胞以及其它细胞的混合悬液以合适的密度接种至培养皿中共同培养,待mSSC形成克隆,但支持细胞还未进入大量增值期时,将mSSC移至饲养层细胞中扩大培养,最后通过特异性分子标志物检测和诱导分化等实验对分离的mSSC进行鉴定。结果 通过将mSSC和支持细胞共同培养的方法建立了mSSC体外分离培养的共培养法,该方法从昆明小鼠和C57BL/6小鼠两种不同遗传背景的幼鼠睾丸中成功分离mSSC。RT-PCR实验结果表明,本研究分离培养的mSSC表达mSSC的特异性分子标志物Plzf、Gfrα-1和Ngn3,使用维甲酸(retinoic acid, RA)诱导后可观察到精子样细胞的出现,使用流式细胞术进行倍体分析表明,诱导后的细胞中有9.52%为单倍体,而未诱导的细胞中检测不到单倍体细胞存在。RT-PCR实验结果表明,诱导后mSSC开始表达精原干细胞分化相关基因Acrosin、Th2b和Sycp3。结论 本研究建立了一种适合不同遗传背景mSSC的体外分离、培养方法,该方法既简化了分离步骤,又节约了培养成本,为广泛开展mSSC的研究提供了条件。Objective To establish a simple and efficient method for isolation and culture of mouse spermatogonial stem cell(mSSC)in vitro.Method(s)The cell suspension from testis of male fetal Kunming mice and C57BL/6mice was obtained by mechanical separation and enzyme digestion.The cell suspension containing mSSC cells and Sertoli cells and other cells was co-cultured until the clone of msSC formed.When Sertoli cells were not in period of proliferation,the clones of mSSC were removed to the feeding layer cells.The isolated mSSC cells were further identified by molecular markers and induced differentiation.Result(s)The mSSC cells isolated by this co-culturing method from different genetic backgrounds of male fetal Kunming and C57BL/6 mice could be cultured for several generations in vitro.The result of RT-PCR showed that specific molecular markers,such as Plzf,Gfrα-1 and Ngn3 were expressed in mSSC cells.mSSC cells treated with retinoic acid presented the phenotype of sperm-like cells.The amount of haploid in induced mSSC cells accounted for 9.52%,while no haploid in the control mSSC cells.The induced mSSC expressed differentiation-associated genes such as Acrosin,Th2b and Sycp3.Conclusion(s)In this study,we developed the co-culturing method which could isolate and culture mSSC cells from different genetic background mice.This method not only simplifies the separation steps,but also saves the cost of culture,which will provide basis for the study of mSSC cells.

关 键 词：精原干细胞 细胞培养技术 分子标志物 诱导分化 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R349.5[医药卫生—基础医学]