冷冻保存新生牛睾丸组织中支持细胞的分离纯化
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摘要
睾丸支持细胞(sertoli cells,SCs)是生精上皮中的多功能体细胞,其分离培养对体外探讨精子发生机理至关重要。因牛新鲜睾丸组织取材不便,本试验在前期基础上探讨了从冷冻保存的新生牛睾丸组织中分离纯化SCs的可行性。将冻存的1日龄牛睾丸组织复苏,进行形态学检查和SCs分离培养。石蜡切片苏木精-伊红(HE)染色显示,曲细精索及睾丸间质保存完好。二步酶消化结果显示,生精上皮解离良好。利用差异贴壁法将原代细胞纯化后扩大培养,绝大多数细胞呈不规则多边形,3d即可生长至汇合。RT-PCR分析显示,贴壁细胞表达SCs阳性分子标记胶质细胞源神经营养因子(GDNF)和雄性激素结合蛋白(ABP)而不表达精原细胞分子标记PLZF。流式细胞术检测显示,所获细胞中ABP和GDNF阳性细胞数分别为99.7%和99.8%。以上结果表明,从冻存的新生牛睾丸组织中可获高纯度的SCs。本试验结果为后续相关试验奠定了基础。
Sertoli cells(SCs)are multi-functional somatic cells in the seminiferous epithelium.The isolation and in vitro culture of SCs are of great importance to probe the spermatogenesis mechanism.Since it is inconvenient to get bovine testicular tissues,here we investigated the possibility of SCs isolation and enrichment from cryopreserved neonatal bovine testis based on our previous work.The cryopreserved testicular tissues of postnatal 1-day-old bulls were thawed for morphological examination and SCs separation.Hematoxylin-eosin(HE)staining of the paraffin sections showed that the seminiferous cords and interstitium were well preserved.The two-step enzymatic digestion showed that the seminiferous epithelia were dissociated completely.The attached primary cells were enriched by differential plating,and expanded subsequently.Most of them were in irregular polygon shape and grew to confluence within 3days.RT-PCR analysis revealed that they expressed SCs marker GDNF and ABP but not spermatogonial marker PLZF.Flow cytometry analysis showed that the enriched cells contained high purified ABP+cells(99.7%)and GDNF+cells(99.8%).The above data indicate that high purified SCs can be obtained from cryopreserved testicular tissues of neonatal bulls,which lays a solid foundation for our next step.
Sertoli cells(SCs)are multi-functional somatic cells in the seminiferous epithelium.The isolation and in vitro culture of SCs are of great importance to probe the spermatogenesis mechanism.Since it is inconvenient to get bovine testicular tissues,here we investigated the possibility of SCs isolation and enrichment from cryopreserved neonatal bovine testis based on our previous work.The cryopreserved testicular tissues of postnatal 1-day-old bulls were thawed for morphological examination and SCs separation.Hematoxylin-eosin(HE)staining of the paraffin sections showed that the seminiferous cords and interstitium were well preserved.The two-step enzymatic digestion showed that the seminiferous epithelia were dissociated completely.The attached primary cells were enriched by differential plating,and expanded subsequently.Most of them were in irregular polygon shape and grew to confluence within 3days.RT-PCR analysis revealed that they expressed SCs marker GDNF and ABP but not spermatogonial marker PLZF.Flow cytometry analysis showed that the enriched cells contained high purified ABP+cells(99.7%)and GDNF+cells(99.8%).The above data indicate that high purified SCs can be obtained from cryopreserved testicular tissues of neonatal bulls,which lays a solid foundation for our next step.
引文
[1]TANG K,WANG L,JIN Y,et al.GDF9affects the development and tight junction functions of immature bovine Sertoli cells[J].Reprod Domest Anim,2017,52(4):640-648.
[2]TANG K,JIN Y,CHEN F,et al.Overexpression of C/EBPβaffects the cell cycle regulators and spermatogenesis related genes expression and function of bovine Sertoli cells[J].Reprod Domest Anim,2016,51(4):591-596.
[3]高一,秦立红,房希碧,等.PDGFRA基因对牛未成熟支持细胞体外增殖的影响[J].吉林农业大学学报,2018,40(3):352-357.
[4]胡晓鹏,杨凡,吕忠显.小鼠睾丸支持细胞标志蛋白表达的研究[J].生殖与避孕,2015,35(8):509-516+524.
[5]张学明,李德雪,文兴豪,等.小鼠支持细胞体外培养的一般特性[J].黑龙江畜牧兽医,2000(2):34-35.
[6]LAKPOUR M R,AGHAJANPOUR S,KORUJI M,et al.Isolation,culture and characterization of human sertoli cells by flow cytometry:development of procedure[J].J Reprod Infertil,2017,18(2):213-217.
[7]CHEN L Y,WILLIS W D,EDDY E M,et al.Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development[J].Proc Natl Acad Sci USA,2016,113(7):1829-1834.
[8]赵佳,金洁,梁元晶,等.睾丸支持细胞对体外培养鼠胎脑皮质细胞的影响[J].中国组织工程研究与临床康复,2008(43):8490-8494.
[9]刘闯,赵晓娥,马保华,等.新生犊牛睾丸支持细胞的高效分离培养与鉴定[J].西北农林科技大学学报(自然科学版),2010,38(1):6-10+16.
[10]叶小琳,潘阳阳,王亚营,等.牦牛CLDN3基因特征分析及FSH对其在睾丸支持细胞表达的影响[J].农业生物技术学报,2018,26(3):437-446.
[11]王亚营,潘阳阳,叶小琳,等.牦牛Cyclin D1基因的克隆及IGF-I对其在睾丸支持细胞表达的影响[J].畜牧兽医学报,2018,49(2):310-317.
[12] RAN M,LI Z,CAO R,et al.miR-26asuppresses autophagy in swine Sertoli cells by targeting ULK2[J].Reprod Domest Anim,2018,53(4):864-871.
[13] WU J Y,HU T J,GUO B,et al.Cryopreservation of adult bovine testicular tissue for spermatogonia enrichment[J].Cryo Lett,2011,32(5):402-409.
[14] WU J Y,SUN Y X,WANG A B,et al.Effect of newborn bovine serum on cryopreservation of adult bovine testicular tissue[J].Andrologia,2014,46(3):308-312.
[15] CAI H,WU J Y,AN X L,et al.Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue[J].Anim Reprod Sci,2016,166:109-115.
[16] CAI H,TANG B,WU J Y,et al.Enrichment and in vitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls[J].Andrology,2016,4(6):1150-1158.
[17]司蕾,张学明,岳占碰,等.睾丸支持细胞对精原干细胞发育的调节[J].细胞生物学杂志,2009,30(4):479-482.
[18]邓洁,张会琼,黄莎,等.TNF-α对仔猪睾丸支持细胞中Fas/FasL表达的调节作用[J].中国兽医学报,2018,38(6):1214-1221.
[19] BERNARDINO R L,ALVES M G,OLIVEIRA P F,et al.Evaluation of the purity of Sertoli cell primary cultures[J].Methods Mol Biol,2018,1748:9-15.
[2]TANG K,JIN Y,CHEN F,et al.Overexpression of C/EBPβaffects the cell cycle regulators and spermatogenesis related genes expression and function of bovine Sertoli cells[J].Reprod Domest Anim,2016,51(4):591-596.
[3]高一,秦立红,房希碧,等.PDGFRA基因对牛未成熟支持细胞体外增殖的影响[J].吉林农业大学学报,2018,40(3):352-357.
[4]胡晓鹏,杨凡,吕忠显.小鼠睾丸支持细胞标志蛋白表达的研究[J].生殖与避孕,2015,35(8):509-516+524.
[5]张学明,李德雪,文兴豪,等.小鼠支持细胞体外培养的一般特性[J].黑龙江畜牧兽医,2000(2):34-35.
[6]LAKPOUR M R,AGHAJANPOUR S,KORUJI M,et al.Isolation,culture and characterization of human sertoli cells by flow cytometry:development of procedure[J].J Reprod Infertil,2017,18(2):213-217.
[7]CHEN L Y,WILLIS W D,EDDY E M,et al.Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development[J].Proc Natl Acad Sci USA,2016,113(7):1829-1834.
[8]赵佳,金洁,梁元晶,等.睾丸支持细胞对体外培养鼠胎脑皮质细胞的影响[J].中国组织工程研究与临床康复,2008(43):8490-8494.
[9]刘闯,赵晓娥,马保华,等.新生犊牛睾丸支持细胞的高效分离培养与鉴定[J].西北农林科技大学学报(自然科学版),2010,38(1):6-10+16.
[10]叶小琳,潘阳阳,王亚营,等.牦牛CLDN3基因特征分析及FSH对其在睾丸支持细胞表达的影响[J].农业生物技术学报,2018,26(3):437-446.
[11]王亚营,潘阳阳,叶小琳,等.牦牛Cyclin D1基因的克隆及IGF-I对其在睾丸支持细胞表达的影响[J].畜牧兽医学报,2018,49(2):310-317.
[12] RAN M,LI Z,CAO R,et al.miR-26asuppresses autophagy in swine Sertoli cells by targeting ULK2[J].Reprod Domest Anim,2018,53(4):864-871.
[13] WU J Y,HU T J,GUO B,et al.Cryopreservation of adult bovine testicular tissue for spermatogonia enrichment[J].Cryo Lett,2011,32(5):402-409.
[14] WU J Y,SUN Y X,WANG A B,et al.Effect of newborn bovine serum on cryopreservation of adult bovine testicular tissue[J].Andrologia,2014,46(3):308-312.
[15] CAI H,WU J Y,AN X L,et al.Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue[J].Anim Reprod Sci,2016,166:109-115.
[16] CAI H,TANG B,WU J Y,et al.Enrichment and in vitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls[J].Andrology,2016,4(6):1150-1158.
[17]司蕾,张学明,岳占碰,等.睾丸支持细胞对精原干细胞发育的调节[J].细胞生物学杂志,2009,30(4):479-482.
[18]邓洁,张会琼,黄莎,等.TNF-α对仔猪睾丸支持细胞中Fas/FasL表达的调节作用[J].中国兽医学报,2018,38(6):1214-1221.
[19] BERNARDINO R L,ALVES M G,OLIVEIRA P F,et al.Evaluation of the purity of Sertoli cell primary cultures[J].Methods Mol Biol,2018,1748:9-15.
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