不同培养液添加物对尼罗罗非鱼精原干细胞生长增殖的影响
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摘要
【目的】探讨不同血清和细胞因子等培养液添加物对尼罗罗非鱼(Oreochromisniloticus)精原干细胞(Spermatogonial stem cells,SSCs)生长增殖的影响。【方法】无菌取发育第Ⅲ期的尼罗罗非鱼精巢组织,用酶消化法结合差速贴壁法获得尼罗罗非鱼SSCs,在有支持细胞饲养层条件下,L-15培养液中分别添加体积分数2%、5%、10%新生牛血清(New bovine serum,NBS),体积分数1%、2%、3%罗非鱼血清以及5、10、15 ng/mL白血病抑制因子(Leukemia inhibitor factor,LIF),比较不同添加物对尼罗罗非鱼SSCs生长增殖的影响。【结果】在支持细胞饲养层上,SSCs分裂指数明显高于无饲养层的SSCs,支持细胞饲养层明显促进精原干细胞分裂增殖(P <0.01);与2%、10%NBS组相比,添加体积分数5%NBS可显著促进精原干细胞增殖(P <0.01);添加体积分数1%、2%、3%尼罗罗非鱼血清对SSCs无显著的促增殖作用(P> 0.05);添加5、10 ng/mL LIF可促进SSCs增殖,添加10 ng/mL LIF效果更佳,添加15 ng/mL LIF则抑制SSCs增殖。【结论】使用尼罗罗非鱼精巢支持细胞作饲养层,用添加体积分数5%NBS及10 ng/mL LIF的L-15培养液培养,有助于促进尼罗罗非鱼精原干细胞生长增殖。
【Objective】To investigate the effects of different medium additives on growth and proliferation of the spermatogonial stem cells(SSCs) of Nile tilapia(Oreochromis niloticus).【Methods】The SSCs were obtained from fresh testis of Nile tilapia in the development stageⅢ by pancreatic enzyme digestion and followed differential adhesion.With the Sertoli cells as feeder layer,the growth and proliferation of SSCs were investigated by culturing in L-15 culture medium supplemented with 2%,5%,10% NBS(new bovine serum),or 1%,2% and 3% Nile tilapia serum,or 5,10 and 15 ng/mL LIF(leukemia inhibitor factor),in different combinations.【Results】The splitting index of SSCs with Sertoli cell feeder was significantly higher than those without the feeder(P<0.01).Sertoli cell feeder layer significantly could speed up division and proliforation of spermatogonial stem cells significantly.Compared to L-15 with 2% and 10% new bovine serum,proliferation of spermatogonial stem cells was respectively increased(P<0.01) in 5% new bovine serum medium.However,supplementation of 1%,2% and 3% Nile tilapia serum in culture medium had no effect on proliferation of SSCs.Proliferation of spermatogonial stem cells was increased in culture medium supplemented with 5 ng/mL and 10 ng/mL leukemia inhibitor factor and 10 ng/mL had better result,but decreased with 15 ng/mL LIF.【Conclusions】The proliferation of Nile tilapia SSCs was improved by using Sertoli cells as feeder layer and cultured in L-15 cell culture medium with 5% new bovine serum and 10 ng/mL leukemia inhibitor factor.
【Objective】To investigate the effects of different medium additives on growth and proliferation of the spermatogonial stem cells(SSCs) of Nile tilapia(Oreochromis niloticus).【Methods】The SSCs were obtained from fresh testis of Nile tilapia in the development stageⅢ by pancreatic enzyme digestion and followed differential adhesion.With the Sertoli cells as feeder layer,the growth and proliferation of SSCs were investigated by culturing in L-15 culture medium supplemented with 2%,5%,10% NBS(new bovine serum),or 1%,2% and 3% Nile tilapia serum,or 5,10 and 15 ng/mL LIF(leukemia inhibitor factor),in different combinations.【Results】The splitting index of SSCs with Sertoli cell feeder was significantly higher than those without the feeder(P<0.01).Sertoli cell feeder layer significantly could speed up division and proliforation of spermatogonial stem cells significantly.Compared to L-15 with 2% and 10% new bovine serum,proliferation of spermatogonial stem cells was respectively increased(P<0.01) in 5% new bovine serum medium.However,supplementation of 1%,2% and 3% Nile tilapia serum in culture medium had no effect on proliferation of SSCs.Proliferation of spermatogonial stem cells was increased in culture medium supplemented with 5 ng/mL and 10 ng/mL leukemia inhibitor factor and 10 ng/mL had better result,but decreased with 15 ng/mL LIF.【Conclusions】The proliferation of Nile tilapia SSCs was improved by using Sertoli cells as feeder layer and cultured in L-15 cell culture medium with 5% new bovine serum and 10 ng/mL leukemia inhibitor factor.
引文
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[14]柴玮杰,王玉燕,高珉之,等.以支持细胞为饲养层小鼠精原干细胞体外培养的研究[J].畜牧兽医科技信息,2008(12):33-34.
[15]IZADYAR F,DEN OUDEN K,CREEMERS L B,et al.Proliferation and differentiation of bovine type a spermatogonia during long-term culture[J].Biol Reprod,2003,68(1):272-281.
[16]张仕强,毕聪明,彭树英,等.不同培养条件对牛精原干细胞增殖的影响与特性鉴定[J].畜牧兽医学报,2007,38(6):542-547.
[17]李恩中,李德雪,张世庆,等.以支持细胞为饲养层培养小鼠精原干细胞[J].动物学报,2006(4):774-779.
[18]NAGANO M,BRINSTER R L.Spermatogonial transplantation and reconstitution of donor cell spermatogenesis in recipient mice[J].Apmis,1998,106(1):47-55;56-57.
[19]NIU Z W,MU H L,ZHU H J,et al.P38 MAPKpathway is essential for self-renewal of mouse male germline stem cells(mGSCs)[J].Cell Prolif,2017,50(1):1-9.DOI:10.111/cpr.12314.
[20]IZADYAR F,DEN OUDEN K,CREEMERS L B,et al.Proliferation and differentiation of bovine type a spermatogonia during long-term culture[J].Biol Reprod,2003,68(1):272-281.
[21]DE MIGUEL M P,DE BOER-BROUWER M,PANIAGUA R,et al.Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of sertoli cells and gonocytes in coculture system[J].Endocrinology,1996,137(5):1885-1893.
[22]李德雪,张学明,李子义,等.小鼠精原干细胞体外培养的一般特性[J].中国兽医学报,2001,32(2):160-163.
[2]WANG L,ZHU H J,WU J,et al.Characterization of embryonic stem-like cells derived from HEK293T Cells through miR302/367 expression and their potentiality to differentiate into germ-like cells[J].Cytotechnology,2014,66(5):729-740.
[3]NIU B W,LI B,WU C Y,et al.Melatonin promotes goat spermatogonia stem cells(SSCs)proliferation by stimulating glial cell line-derived neurotrophic factor(GDNF)production in Sertoli cells[J].Oncotarget,2016,7(47):77532-77542.
[4]SAKAI N.Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture[J].Development,2002,129(14):3359-3365.
[5]KURITA K,BURGESS S M,SAKAI N.Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm[J].Proc Natl Acad Sci USA,2004,101(5):1263-1267.
[6]WU J,SONG W C,ZHU H J,et al.Enrichment and characterization of thy1-positive male germline stem cells(m GSCs)from dairy goat(Capra hircus)testis using magnetic microbeads[J].Theriogenology,2013,80(9):1052-1060.
[7]ZHU H J,M A J,DU R,et al.Characterization of immortalized dairy goat male germline stem cells(mGSCs)[J].J Cell Biochem,2014,115(9):1549-1560.
[8]NIU Z W,ZHENG L M,WU S,et al.Ras/ERK1/2pathway regulates the self-renewal of dairy goat spermatogonia stem cells[J].Reproduction,2015,149(5):445-452.
[9]ZHENG L M,ZHU H J,MU H L,et al.CD49f promotes proliferation of male dairy goat germline stem cells[J].Cell Prolif,2016,49(1):27-35.
[10]HONG Y,LIU T,ZHAO H,et al.Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro[J].Proc Natl Acad Sci USA,2004,101(21):8011-8016.
[11]NASIRI Z,HOSSEINI S M,HAJIAN M,et al.Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro[J].Theriogenology,2012,77(8):1519-1528.
[12]刘茜,余荣娇,张凯,等.体外培养条件下Scf与Ra对昆明小鼠精原干细胞增殖的影响[J].四川解剖学杂志,2015,23(3):10-13.
[13]APONTE P M,SODA T,VAN DE KANT H,et al.Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor[J].Theriogenology,2006,65(9):1828-1847.
[14]柴玮杰,王玉燕,高珉之,等.以支持细胞为饲养层小鼠精原干细胞体外培养的研究[J].畜牧兽医科技信息,2008(12):33-34.
[15]IZADYAR F,DEN OUDEN K,CREEMERS L B,et al.Proliferation and differentiation of bovine type a spermatogonia during long-term culture[J].Biol Reprod,2003,68(1):272-281.
[16]张仕强,毕聪明,彭树英,等.不同培养条件对牛精原干细胞增殖的影响与特性鉴定[J].畜牧兽医学报,2007,38(6):542-547.
[17]李恩中,李德雪,张世庆,等.以支持细胞为饲养层培养小鼠精原干细胞[J].动物学报,2006(4):774-779.
[18]NAGANO M,BRINSTER R L.Spermatogonial transplantation and reconstitution of donor cell spermatogenesis in recipient mice[J].Apmis,1998,106(1):47-55;56-57.
[19]NIU Z W,MU H L,ZHU H J,et al.P38 MAPKpathway is essential for self-renewal of mouse male germline stem cells(mGSCs)[J].Cell Prolif,2017,50(1):1-9.DOI:10.111/cpr.12314.
[20]IZADYAR F,DEN OUDEN K,CREEMERS L B,et al.Proliferation and differentiation of bovine type a spermatogonia during long-term culture[J].Biol Reprod,2003,68(1):272-281.
[21]DE MIGUEL M P,DE BOER-BROUWER M,PANIAGUA R,et al.Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of sertoli cells and gonocytes in coculture system[J].Endocrinology,1996,137(5):1885-1893.
[22]李德雪,张学明,李子义,等.小鼠精原干细胞体外培养的一般特性[J].中国兽医学报,2001,32(2):160-163.
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