山羊精原干细胞的鉴定及培养体系优化的研究
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摘要
该研究优化了山羊精原干细胞(goat spermatogonial stem cells,g SSCs)培养体系,使山羊精原干细胞能在体外长期培养,维持自我更新的能力并保持未分化状态。取3~5月龄山羊睾丸,采用两步酶消法结合差速贴壁方法得到山羊精原干细胞悬液,分别通过形态学观察、碱性磷酸酶(alkaline phosphatase,AKP)染色、特异基因表达及蛋白质水平的分析对培养的细胞进行鉴定;并以山羊睾丸支持细胞(goat sertoli cells,g SCs)、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)和层黏连蛋白(laminin,L)为饲养层,观察饲养层对山羊精原干细胞体外增殖的影响。结果表明,山羊精原干细胞体外增殖形成克隆簇,AKP染色呈阳性。经RT-PCR检测,Oct-4、C-myc、Cyclin D1、Ngn3和TERT等干细胞特异基因均有表达。细胞免疫组化结果显示,Oct-4、SSEA-1、α6-integrin、Vasa和Thy-1蛋白质呈阳性。克隆簇统计显示,在山羊睾丸支持细胞上形成的山羊精原干细胞(goat spermatogonial stem cells,g SSCs)克隆数与其他两组比较差异显著(P<0.05)。山羊睾丸支持细胞饲养层上的精原干细胞可在体外传3~4代,培养时间为2个月。结果证明,通过两步酶消法和差速贴壁法可以分离获得山羊精原干细胞,且山羊睾丸支持细胞能够促进g SSCs的增殖。
This study developed an optimized stem cell culture method. With this method goat spermatogonial stem cells(g SSCs) can be effi ciently isolated and cultured in vitro for a long time to maintain self-renewal and remain undifferentiated state. Single g SSC suspension was obtained from testis of 3-5 month old goat by twostep enzymatic digestion and differential adhesion. The g SSCs were identifi ed by morphology observation, alkaline phosphatase(AKP) staining and cell immunohistochemistry. The g SSCs were co-cultured with the feeder layer of goat sertoli cells(g SCs), mouse embryonic fi broblast(MEFs) and laminin. The results showed that g SSC colonies were formed in vitro, and were positive AKP staining. The gene expressions of Oct-4, C-myc, Cyclin D1, Ngn3 and TERT were identifi ed by RT-PCR in gSSCs. The protein levels of Oct-4, SSEA-1, α6-integrin were also identifi ed by immunohistochemistry staining. According to statistics of g SSCs colonies, the results showed that the number of colonies on sertoli cell feeder layer was more than on the other two feeder layer, and the difference was signifi cant(P<0.05). The g SSCs were cultured for 3-4 generations as well as 2 months in vitro on sertoli cell feeder layer. All the above showed that the g SSCs can be obtained by the methods of two-step enzyme digestion and differential attachment, and the cells can be well cultured in vitro for proliferation on the feeder layer of sertoli cell.
This study developed an optimized stem cell culture method. With this method goat spermatogonial stem cells(g SSCs) can be effi ciently isolated and cultured in vitro for a long time to maintain self-renewal and remain undifferentiated state. Single g SSC suspension was obtained from testis of 3-5 month old goat by twostep enzymatic digestion and differential adhesion. The g SSCs were identifi ed by morphology observation, alkaline phosphatase(AKP) staining and cell immunohistochemistry. The g SSCs were co-cultured with the feeder layer of goat sertoli cells(g SCs), mouse embryonic fi broblast(MEFs) and laminin. The results showed that g SSC colonies were formed in vitro, and were positive AKP staining. The gene expressions of Oct-4, C-myc, Cyclin D1, Ngn3 and TERT were identifi ed by RT-PCR in gSSCs. The protein levels of Oct-4, SSEA-1, α6-integrin were also identifi ed by immunohistochemistry staining. According to statistics of g SSCs colonies, the results showed that the number of colonies on sertoli cell feeder layer was more than on the other two feeder layer, and the difference was signifi cant(P<0.05). The g SSCs were cultured for 3-4 generations as well as 2 months in vitro on sertoli cell feeder layer. All the above showed that the g SSCs can be obtained by the methods of two-step enzyme digestion and differential attachment, and the cells can be well cultured in vitro for proliferation on the feeder layer of sertoli cell.
引文
1 Tegelenbosch RA,de Rooij DG.A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101F1 hybrid mouse.Mutat Res 1993;290(2):193-200.
2 Rooij DG,Russell LD.All you wanted to know about spermatogonia but were afraid to ask.J Andro 2000;21(6):776-98.
3 Meachem S,von Schonfeldt V,Schlatt S.Spermatogonia stem cells with a great perspective.Reproduction 2001;121(6):825-34.
4 de Rooij DG.Proliferation and differentiation of spermatogonial stem cells.Reproduction 2001;121(121):347-54.
5 Kokkinaki M,lee TL,He Z,Jiang J,Golestaneh N,Hofmann MC,et al.The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis.Biol Reprod 2009;80(4):707-17.
6 Meistrich ML.Separation of spermatogenic cells and nuclei from rodent testes.Methods Cell Biol 1977;15(15):15-54.
7 Koh KB,Komiyama M,Toyama Y,Adachi T,Mori C.Percoll fractionation of adult mouse spermatogonia improvesgerm cell transplantation.Asian J Androl 2004;6(2):93-8.
8 Shinohara T,Avarbock MR,Brinster RL.Functional analysis of spermatogonial stem cells in steel and cryptorchid infertile mouse models.Develop Biol 2000;220(2):401-11.
9 Owen CS,Sykes NL.Magnetic labeling and cell sorting.J Immunol Methods 1984;73(1):41-8.
10 Shinohara T,Orwig KE,Avarbock MR,Brinster RL.Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells.Proc Natl Acad Sci USA 2000;97(15):8346-51.
11 Nagano M,Avarbock MR,Leonida EB.Culture of mouse spermatogonial stem cells.Tissue Cell 1998;30(4):389-97.
12 Kanatsu-Shinohara M,Miki H,Inoue K.Long-term culture of mouse male germline stem cells under serum-or feeder-freeconditions.Biol Reprod 2005;72(4):985-91.
13 Kanatsu-Shinohara M,Ogonuki N,Inoue K.Long-term proliferation in culture and germline transmission of mouse male germline stem cells.Biol Reprod 2003;69(2):612-6.
14 Honaramooz A,Behboodi E,Hausler CL,Blash S,Mengee SO,Dobrinski I.Germ cell transplantation in goats.Mol Reprod Dev2003;64(4):422-8.
15 姜颖,潘庆杰,张钦恺,李岩,徐君君.山羊睾丸支持细胞的分离培养及鉴定.中国畜牧杂志(Jiang Ying,Pan Qingjie,Zhang Qinkai,Li Yan,Xu Junjun.Separation,culture and identification of goat sertoli cells.Animal Science and Biotechology)2014;21:23-6.
16 Richards M,Fong C,Chan W,Wong P,Bongso A.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat Biotechnol 2002;20(9):933-6.
17 Wu J,Song W,Zhu H,Niu Z,Mu H,Lei A,et al.Enrichment and characterization of Thy1-positive male germline stem cells(m GSCs)from dairy goat(Capra hircus)testis using magnetic microbeads.Theriogenology 2013;80(9):1052-60.
18 Wu X,Schmidt JA,Avarbock MR,Tobias JW,Carlson CA,Kolon TF,et al.Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules.Proc Natl Acad Sci USA 2009;106(51):21672-7.
2 Rooij DG,Russell LD.All you wanted to know about spermatogonia but were afraid to ask.J Andro 2000;21(6):776-98.
3 Meachem S,von Schonfeldt V,Schlatt S.Spermatogonia stem cells with a great perspective.Reproduction 2001;121(6):825-34.
4 de Rooij DG.Proliferation and differentiation of spermatogonial stem cells.Reproduction 2001;121(121):347-54.
5 Kokkinaki M,lee TL,He Z,Jiang J,Golestaneh N,Hofmann MC,et al.The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis.Biol Reprod 2009;80(4):707-17.
6 Meistrich ML.Separation of spermatogenic cells and nuclei from rodent testes.Methods Cell Biol 1977;15(15):15-54.
7 Koh KB,Komiyama M,Toyama Y,Adachi T,Mori C.Percoll fractionation of adult mouse spermatogonia improvesgerm cell transplantation.Asian J Androl 2004;6(2):93-8.
8 Shinohara T,Avarbock MR,Brinster RL.Functional analysis of spermatogonial stem cells in steel and cryptorchid infertile mouse models.Develop Biol 2000;220(2):401-11.
9 Owen CS,Sykes NL.Magnetic labeling and cell sorting.J Immunol Methods 1984;73(1):41-8.
10 Shinohara T,Orwig KE,Avarbock MR,Brinster RL.Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells.Proc Natl Acad Sci USA 2000;97(15):8346-51.
11 Nagano M,Avarbock MR,Leonida EB.Culture of mouse spermatogonial stem cells.Tissue Cell 1998;30(4):389-97.
12 Kanatsu-Shinohara M,Miki H,Inoue K.Long-term culture of mouse male germline stem cells under serum-or feeder-freeconditions.Biol Reprod 2005;72(4):985-91.
13 Kanatsu-Shinohara M,Ogonuki N,Inoue K.Long-term proliferation in culture and germline transmission of mouse male germline stem cells.Biol Reprod 2003;69(2):612-6.
14 Honaramooz A,Behboodi E,Hausler CL,Blash S,Mengee SO,Dobrinski I.Germ cell transplantation in goats.Mol Reprod Dev2003;64(4):422-8.
15 姜颖,潘庆杰,张钦恺,李岩,徐君君.山羊睾丸支持细胞的分离培养及鉴定.中国畜牧杂志(Jiang Ying,Pan Qingjie,Zhang Qinkai,Li Yan,Xu Junjun.Separation,culture and identification of goat sertoli cells.Animal Science and Biotechology)2014;21:23-6.
16 Richards M,Fong C,Chan W,Wong P,Bongso A.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat Biotechnol 2002;20(9):933-6.
17 Wu J,Song W,Zhu H,Niu Z,Mu H,Lei A,et al.Enrichment and characterization of Thy1-positive male germline stem cells(m GSCs)from dairy goat(Capra hircus)testis using magnetic microbeads.Theriogenology 2013;80(9):1052-60.
18 Wu X,Schmidt JA,Avarbock MR,Tobias JW,Carlson CA,Kolon TF,et al.Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules.Proc Natl Acad Sci USA 2009;106(51):21672-7.
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