人精原干细胞分离、培养与鉴定

发布时间：2018-07-15 08:42

【摘要】：目的 :分离、鉴定和培养人精原干细胞(SSC),以获得纯化、富集的人SSC并为其研究与应用奠定基础。方法:通过免疫荧光方法检测睾丸组织中CD90表达情况;应用两步酶消化和差速贴壁法分离人睾丸生精细胞,以CD90为人SSC标志,用免疫磁珠分选技术(MACS)对人睾丸生精细胞进行分选,并利用RT-PCR和免疫细胞化学对分选所得CD90+细胞进行鉴定;同时用SG培养液将CD90+细胞与支持细胞进行体外共培养。结果:MACS分选所得CD90+细胞大小、形态特征较为均一,RT-PCR检测其表达人SSC标记性基因,免疫荧光检测其高表达人SSC特异标记GFRA-1、GPR125和UCHL-1,阳性率达90.5%,其在SG培养液中与支持细胞共培养14 d后仍可保持良好活性。结论:CD90可作为人SSC特异性标志之一,结合差速贴壁和MACS可高效分选人SSC,分选所得SSC可在SG培养液中进行体外培养。
[Abstract]:Objective: to isolate, identify and culture human spermatogonial stem cells (SSC) in order to obtain purified and enriched human SSC and lay a foundation for its research and application. Methods: the expression of CD90 in testicular tissue was detected by immunofluorescence method, and human testicular spermatogenic cells were isolated by two-step enzyme digestion and differential adherence method. CD90 was used as a SSC marker. Human testicular spermatogenic cells were separated by immunomagnetic bead sorting (Macs). CD90 cells were identified by RT-PCR and immunocytochemistry, and CD90 cells and Sertoli cells were co-cultured in SG medium in vitro. Results the size and morphological characteristics of CD90 cells were homogenous. RT-PCR was used to detect the expression of human SSC marker gene. The positive rate of GPR125 and UCHL-1 was 90.5, and the positive rate of GPR125 and UCHL-1 was 90.5. The positive rate of GPR125 and UCHL-1 was 90.5%. The positive rate of GPR125 and UCHL-1 was 90.5%. The positive rate of GPR125 and UCHL-1 was 90.5%. Conclusion: the specific marker of human SSC can be used as one of the specific markers of human SSC. Human SSC can be efficiently separated by differential adhesion and Macs. The isolated SSC can be cultured in the medium of SG in vitro.
【作者单位】：上海交通大学医学院附属仁济医院泌尿外科;上海交通大学医学院附属仁济医院临床干细胞中心;
【基金】：国家自然科学基金(31230048) 国家973重大科学研究计划(2013CB947901)~~
【分类号】：R321.1

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