睾丸支持细胞的功能、分离纯化及鉴定研究进展

doi:10.16431/j.cnki.1671-7236.2021.08.027

摘要/Abstract

摘要： 支持细胞对维持精子形成过程中的微环境起决定作用，它可以通过分泌功能、细胞间连接形成的血睾屏障功能以及吞噬功能等来促进精子的形成过程，其发育异常会导致不同程度的雄性生殖缺陷。基于支持细胞在雄性动物生殖过程中的作用，体外培养高纯度支持细胞可成为研究睾丸两大核心功能-精子发生和性激素分泌功能相关调节机制重要的细胞模型。此外，体外培养睾丸支持细胞也可作为生殖毒理学等新兴热点领域的细胞模型，为评估和研究环境因素对雄性生殖的影响提供便利。因此，作者系统地归纳、总结了目前关于动物支持细胞生物功能的研究及常用的体外分离纯化、培养及鉴定方法，以期为利用动物支持细胞开展雄性生殖领域的研究提供参考。

关键词: 支持细胞; 功能; 分离纯化; 体外培养; 鉴定

Abstract: Sertoli cells play a decisive role in maintaining the microenvironment in the process of spermatogenesis.They can promote the process of spermatogenesis through secretion function, blood testis barrier function formed by intercellular connection and phagocytosis, abnormal development of them will lead to different degrees of male reproductive defects.Based on the role of Sertoli cells in the reproductive process of male animals, highly purified Sertoli cells cultured in vitro can be an important cell model to study the two core functions of testis spermatogenesis and sex hormone secretion.In addition, purified Sertoli cells cultured in vitro can also be used as a cell model in toxicology and other newly developing hot fields to provide convenience for assessing and researching on the effects of environmental factors on male reproduction.In this article, the current research progress on the function of Sertoli cell and its commonly methods of in vitro isolation, purification, culture and characterization were systematically summarized to provide references for the use of animal Sertoli cells in the studies of male reproduction.

Key words: Sertoli cells; function; isolation and purification; in vitro culture; identification

中图分类号:

S827

伊敏娜, 才文道力玛, 陶力, 王希生, 神英超, 杜明, 任宏, 芒来, 格日乐其木格. 睾丸支持细胞的功能、分离纯化及鉴定研究进展[J]. 中国畜牧兽医, 2021, 48(8): 2947-2956.

YI Minna, Ulaangerel Tseweendolmaa, Toli Davshilt, WANG Xisheng, SHEN Yingchao, DU Ming, REN Hong, MANG Lai, BOU Gerelchimeg. Research Progress on Function,Isolation,Purification and Identification of Sertoli Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2947-2956.

参考文献

[1] YANG H,MA J Y,WAN Z,et al.Characterization of sheep spermatogenesis through single-cell RNA sequencing[J].FASEB Journal,2021,35(2):e21187.
[2] 余晓晖,胡云飞.睾丸支持细胞的生物学特性及研究进展[J].中国组织工程研究与临床康复,2010,14(44):8311-8314. YU X H,HU Y F.Biological characteristics and research progress of Sertoli cells[J].Chinese Journal of Tissue Engineering Research,2010,14(44):8311-8314.(in Chinese)
[3] SAPSFORD C S.The development of the Sertoli cell of the rat and mouse:Its existence as a mononucleate unit[J].Journal of Anatomy,1963,97(Pt 2):225-238.
[4] CAMERON D F,MUFFLY K E.Hormonal regulation of spermatid binding[J].Journal of Cell Science,1991,100(Pt3):623-633.
[5] WU S,LYU L,LI L,et al.KIF15 supports spermatogenesis via its effects on Sertoli cell microtubule,actin,vimentin,and septin cytoskeletons[J].Endocrinology,2021,162(4):bqab010.
[6] BERGER T,TANG S,TU L,et al.Changes in testicular gene expression following reduced estradiol synthesis:A complex pathway to increased porcine Sertoli cell proliferation[J].Molecular and Cellular Endocrinology,2021,523:111099.
[7] WEN Q,TANG E I,GAO Y,et al.Signaling pathways regulating blood-tissue barriers-lesson from the testis[J].Biochimica et Biophysica Acta,2018,1860(1):141-153.
[8] DANCE A,KASTELIC J,THUNDATHIL J.A combination of insulin-like growth factor I (IGF-I) and FSH promotes proliferation of prepubertal bovine Sertoli cells isolated and cultured in vitro[J].Reproduction Fertility and Development,2017,29(8):1635-1641.
[9] 国晓瞳,张倩,于枫,等.黄芩苷对热应激犊牛睾丸支持细胞胶质细胞源性神经生长因子(GDNF)和干细胞因子(SCF)表达的影响[J].农业生物技术学报,2015,23(4):441-449. GUO X T,ZHANG Q,YU F,et al.Baicalin Affects Glial Cell Line-derived Neurotrophic Factor(GDNF) and Stem Cell Factor (SCF) Expression in Heat-stressed Calf (Bos taurus) Sertoli Cells[J].Journal of Agricultural Biotechnology,2015,23(4):441-449.(in Chinese)
[10] 王婷婷,张继东.TGF-β1调控哺乳动物睾丸发生的研究进展[J].遵义医科大学学报,2020,43(2):264-270. WANG T T,ZHANG J D.Research progress of TGF-β1 in the regulation of mammalian testis[J].Journal of Zunyi Medical University,2020,43(2):264-270.(in Chinese)
[11] REHMAN Z U,WORKU T,DAVIS J S,et al.Role and mechanism of AMH in the regulation of Sertoli cells in mice[J].Journal of Steroid Biochemistry and Molecular Biology,2017,174:133-140.
[12] SIEMANN D N,STRANGE D P,MAHARAJ P N,et al.Zika virus infects human Sertoli cells and modulates the integrity of the in vitro blood-testis barrier model[J].Journal of Virology,2017,91(22):e00623-17.
[13] 秦俊昌.支持细胞紧密连接复合体在氧化应激损伤小鼠睾丸精子发生中的作用[D].郑州:郑州大学,2012. QIN J C.Role of Sertoli cell tight junctions in the pathogenesis of male reproductive dysfunction induced by oxidative stress in mice[D].Zhengzhou:Zhengzhou University,2012.(in Chinese)
[14] 张倩,周可柔,胡倩倩,等.大鼠睾丸支持细胞的分离培养及鉴定[J].安徽科技学院学报,2018,32(2):1-6. ZHANG Q,ZHOU K R,HU Q Q,et al.Isolation and identification of Sertoli cells in rats[J].Journal of Anhui Science and Technology University,2018,32(2):1-6.(in Chinese)
[15] COUTURE R,MARTIN L J.The transcription factors SF-1 and SOX8 cooperate to upregulate Cx43 expression in mouse TM4 sertoli cells[J].Biochemistry and Biophysics Reports,2020,24:100828.
[16] KATO T,MIZUNO K,NISHIO H,et al.Disorganization of claudin-11 and dysfunction of the blood-testis barrier during puberty in a cryptorchid rat model[J].Andrology-Us,2020,8(5):1398-1408.
[17] WU H,JIANG X,GAO Y,et al.Mumps virus infection disrupts blood-testis barrier through the induction of TNF-alpha in Sertoli cells[J].FASEB Journal,2019,33(11):12528-12540.
[18] GAO Y,MRUK DD,CHENG C Y.Sertoli cells are the target of environmental toxicants in the testis-A mechanistic and therapeutic insight[J].Expert Opinion on Therapeutic Targets, 2015,19(8):1073-1090.
[19] DE MICHELE F,POELS J,GIUDICE M G,et al.In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue:Comparison with a large cohort of pre/peripubertal boys[J].Molecular Human Reproduction,2018,24(5):271-282.
[20] GERBER J,HEINRICH J,BREHM R.Blood-testis barrier and Sertoli cell function:Lessons from SCCx43KO mice[J].Reproduction,2016,151(2):R15-R27.
[21] HOLLENBACH J,JUNG K,NOELKE J,et al.Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics[J].PLoS One,2018,13(6):e0198100.
[22] MA B,ZHANG J,ZHU Z,et al.Luteolin ameliorates testis injury and blood-testis barrier disruption through the Nrf2 signaling pathway and by upregulating Cx43[J].Molecular Nutrition & Fod Research,2019,63(10):e1800843.
[23] MILLER S R,CHERRINGTON N J.Transepithelial transport across the blood-testis barrier[J].Reproduction,2018,156(6):R187-R94.
[24] MRUK DD,CHENG C Y.The mammalian blood-testis barrier:Its biology and regulation[J].Endocrine Reviews,2015,36(5):564-591.
[25] BOYER A,GIRARD M,THIMMANAHALLI D S,et al.MTOR regulates gap junction alpha-1 protein trafficking in Sertoli cells and is required for the maintenance of spermatogenesis in mice[J].Biology of Reproduction,2016,95(1):13.
[26] GAO Y,CHEN H,LUI W Y,et al.Basement membrane laminin alpha2 regulation of BTB dynamics via its effects on F-Actin and microtubule cytoskeletons is mediated through mTORC1 signaling[J].Endocrinology,2017,158(4):963-978.
[27] LAPLANTE M,SABATINI D M.MTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[28] 董合玲.Rictor/mTORC2调节睾丸支持细胞骨架结构与精子发生[D].广州:南方医科大学,2015. DONG H L.Rictor/mTORC2 regulates cytoskeleton organization of testicular Sertoli cell and spermatogenesis[D].Guangzhou:Southern Medical University,2015.(in Chinese)
[29] MOK K W,MRUK D D,CHENG C Y.Regulation of blood-testis barrier (BTB) dynamics during spermatogenesis via the "Yin" and "Yang" effects of mammalian target of rapamycin complex 1(mTORC1) and mTORC2[J].International Review of Cell and Molecular Biology,2013,301:291-358.
[30] MOK K W,MRUK D D,CHENG C Y.RpS6 regulates blood-testis barrier dynamics through Akt-mediated effects on MMP-9[J].Journal of Cell Science,2014,127(Pt22):4870-4882.
[31] LI S Y T,YAN M,CHEN H Q,et al.MTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo[J]. American Journal of Physiology-Endocrinology and Metabolism,2018,314(2):E174-E190.
[32] 刘民,罗兰.睾丸支持细胞功能的研究进展[J].实验动物科学,2012,29(1):52-56. LIU M,LUO L.Progress of Sertoli cells functions[J].Laboratory Animal Science,2012,29(1):52-56.(in Chinese)
[33] YAMADA A,ARAKAKI R,SAITO M,et al.Dual role of fas/fasL-mediated signal in peripheral immune tolerance[J].Front Immunol,2017,8:403.
[34] WANG M,SU P.The role of the fas/fasL signaling pathway in environmental toxicant-induced testicular cell apoptosis:An update[J].Systems Biology in Reproductive Medicine,2018,64(2):93-102.
[35] BERNARDINO R L,ALVES M G,OLIVEIRA P F.Establishment of primary culture of Sertoli cells[J].Methods in Molecular Biology,2018,1748:1-8.
[36] 丁天云,张明,杨洋,等.大鼠睾丸支持细胞的分离培养及鉴定[J].南昌大学学报(理科版),2016,40(5):493-496. DING T Y,ZHANG M,YANG Y,et al.Isolation culture and identification of testis Sertoli cells from rat[J].Journal of Nanchang University (Natural Science),2016,40(5):493-496.(in Chinese)
[37] 梁洺源.小鼠精原干细胞、支持细胞体外培养及白消安毒性影响研究[D].长春:吉林农业大学,2016. LIANG M Y.Study on the vitro culture of murine SSCs and SCs and the toxic effect of busulfan on them[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[38] HABAS K,BRINKWORTH M H,ANDERSON D.Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro[J].Food and Chemical Toxicology,2018,116(Pt B):182-188.
[39] 胡旭燕,伍琼芳,张学宏.成年小鼠睾丸支持细胞体外快速分离与培养[J].江西医药,2018,53(3):218-220. HU X Y,WU Q F,ZHANG X H.Separation and culture of adult mice Sertoli cells in vitro[J].Jiangxi Medical Journal,2018,53(3):218-220.(in Chinese).
[40] 南涛,张奎原,张虎,等.两步酶法在小鼠睾丸支持细胞分离与培养过程中的研究[J].中国民族民间医药,2018,27(6):41-44. NAN T,ZHANG K Y,ZHANG H,et al.The study on the two-step enzymic method used in the process of the isolation and culture of Sertoli cells in mice[J].Chinese Journal of Ethnomedicine and Ethnopharmacy,2018,27(6):41-44.(in Chinese).
[41] GERBER J,RODE K,HAMBRUCH N,et al.Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43[J].Cell and Tissue Research,2020,381(2):309-326.
[42] 宋连杰,崔迎迎,赵一萍,等.蒙古马睾丸支持细胞的体外分离培养与鉴定[J].中国畜牧兽医,2020,47(9):2751-2758. SONG L J,CUI Y Y,ZHAO Y P,et al.Isolation,culture and identification of testis Sertoli cells in Mongolian hprses in vitro[J].China Animal Husbandry & Veterinary Medicine,2020,47(9):2751-2758.(in Chinese).
[43] 王帅,贾琦珍,张玲,等.和田羊睾丸支持细胞的体外培养与分离鉴定[J].西南农业学报,2017,30(8):1918-1922. WANG S,JIA Q Z,ZHANG Q,et al.Culture and identification of testis Sertoli cells in hetian sheep in vitro[J].Southwest China Journal of Agricultural Sciences,2017,30(8):1918-1922.(in Chinese)
[44] 王菊花,刘丹丹,刘琦,等.山羊睾丸支持细胞分离培养及其氧化应激效果研究[J].中国农业大学学报,2019,24(2):99-106. WANG J H,LIU DD,LIU Q,et al.Isolation,culture and oxidative stress of goat Sertoli cells[J].Journal of China Agricultural University,2019,24(2):99-106.(in Chinese)
[45] 杜国玉,吴锦艳,尚振华,等.绵羊睾丸支持细胞的分离鉴定及其在山羊痘病毒疫苗株培养中的应用[J].中国兽医科学,2020,50(3):360-365. DU G Y,WU J Y,SHANG Z H,et al.Isolation and identification of sheep testis Sertoli cell and it's application in the culture of Goatpox virus vaccine strain[J].Chinese Veterinary Science,2020,50(3):360-365.(in Chinese)
[46] 刘欢欢,余树民,夏娟,等.仔猪睾丸支持细胞的体外生物学特性研究[J].中国细胞生物学学报,2015,37(8):1122-1128. LIU H H,YU S M,XIA J,et al.Characterization of Sertoli cells from prepubertal porcine testis in vitro[J].Chinese Journal of Cell Biology,2015,37(8):1122-1128.(in Chinese)
[47] 李欣,赵中利,罗晓彤,等.松辽黑猪睾丸支持细胞的制备、体外培养及鉴定[J].黑龙江畜牧兽医,2019,5:55-57. LI X,ZHAO Z L,LUO X T,et al.Preparation,in vitro culture and identification of Sertoli cells of Songliao Black pig[J].Heilongjiang Animal Science and Veterinary Medicine,2019,5:55-57.(in Chinese)
[48] ZHANG H,LIU B,QIU Y,et al.Pure cultures and characterization of yak Sertoli cells[J].Tissue Cell,2013,45(6):414-420.
[49] 武海军.犊牛睾丸支持细胞的分离培养及其对淋巴细胞的影响[D].杨凌:西北农林科技大学,2007. WU H J.Isolation and cultivation of calf Sertoli cells and their effection on lymphocytes[D].Yangling:Northwest A&F University,2007.(in Chinese)
[50] 吴洪,李碧春,周冠月,等.鸡睾丸细胞分离方法与培养的研究[J].中国兽医杂志,2007,43(3):11-13. WU H,LI B C,ZHOU G Y,et al.Isolation and culture of chicken testicular cells[J].Chinese Journal of Veterinary Medicine,2007,43(3):11-13.(in Chinese)
[51] GUIBERT E,BRIERE S,PELLETIER R,et al.Characterization of chicken Sertoli cells in vitro[J].Journal of Poultry Science,2011,90(6):1276-1286.
[52] 张静,周智慧,邹海军,等.成年公兔睾丸支持细胞的分离纯化及鉴定[J].中国畜牧兽医,2007,34(5):63-66. ZHANG J,ZHOU Z H,ZOU H J,et al.Isolation,purification and identification of testis Sertoli cells in adult rabbit[J].China Animal Husbandry & Veterinary Medicine,2007,34(5):63-66.(in Chinese)
[53] SAEWU A,KONGMANAS K,RAGHUPATHY R,et al.Primary Sertoli cell cultures from adult mice have different properties compared with those derived from 20-day-old animals[J].Endocrinology,2020,161(1):bqz020.
[54] 付俊,罗艳云,缪时英,等.小鼠睾丸中细胞连接相关蛋白质的分析[J].中国医学科学院学报,2016,38(2):205-209. FU J,LUO Y Y,MIAO S Y,et al.Expression patterns of the proteins associated with cell junctions in mouse testes[J].Acta Academiae Medicinae Sinicae,2016,38(2):205-209.(in Chinese)
[55] SYED V,HECHT N B.Disruption of germ cell-Sertoli cell interactions leads to spermatogenic defects[J].Molecular and Cellelar Endocrinology,2002,186(2):155-157.
[56] 王雪,王子铭,李晓宇,等.犊牛睾丸支持细胞分离培养与纯度鉴定[J].黑龙江动物繁殖,2020,28(6):1-6. WANG X,WANG Z M,LI X Y,et al.Isolation,culture and purity identification of testis Sertoli cells in newborn calf[J].Heilongjiang Journal of Animal Reproduction,2020,28(6):1-6.(in Chinese)
[57] 唐丽娟,王庆忠,徐承水.高纯度分离睾丸支持细胞的简便方法[J].细胞生物学杂志,2009,31(1):123-124. TANG L J,WANG Q Z,XU C S.A simple method for isolating highly purified mouse Sertoli cells[J].Chinese Journal of Cell Biology,2009,31(1):123-124.(in Chinese)
[58] 周元陵,钟先玖,吴鑫,等.原代睾丸支持细胞分离纯化及双室培养模型的建立[J].环境与职业医学,2005,22(3):208-211. ZHOU Y L,ZHONG X J,WU X,et al.Establishment of dual-chamber model for rat Sertoli cells primary culture[J].Journal of Environmental and Occupational Medicine,2005,22(3):208-211.(in Chinese)
[59] 朱培元,黄宇烽.支持细胞体外培养研究进展[J].中华男科学,2000,(2):123-126. ZHU P Y,HUANG Y F.Advances in Sertoli cell cultures in vitro[J].National Journal of Andrology,2000,(2):123-126.(in Chinese)
[60] YANG Y F,HONARAMOOZ A.Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells[J].Reproduction Fertility and Development,2010,22(3):523-532.
[61] LIVERA G,ROUILLER-FABRE V,PAIRAULT C,et al.Regulation and perturbation of testicular functions by vitamin A[J].Reproduction,2002,124(2):173-180.
[62] YILMAZ B O,ERKAN M.Effects of Vitamin C on Sodium Fluoride-Induced Oxidative Damage in Sertoli Cells[J].Fluoride,2015,48(3):241-251.
[63] GONÇALVES R,ZAMONER A,ZANATTA L,et al.1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells:gamma-glutamyl transpeptidase and glucose metabolism[J].Journal of Cell Communication and Signaling,2017,11(3):233-243.
[64] GAO Y,LU W,JIAN L,et al.Vitamin E promotes ovine Sertoli cell proliferation by regulation of genes associated with cell division and the cell cycle[J].Animal Biotechnology,2020,2:1-9.
[65] GROOTEGOED J A,OONK R B,JANSEN R,et al.Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells[J].Journal of Reproduction and Fertility,1986,77(1):109-118.
[66] MOROHOSHI A,NAKAGAWA T,NAKANO S,et al.The ubiquitin ligase subunit beta-TrCP in Sertoli cells is essential for spermatogenesis in mice[J].Developmental Neurobiology,2019,445(2):178-188.
[67] SU H,LUO F,BAO J,et al.Long-term culture and analysis of cashmere goat Sertoli cells[J].In Vitro Cellular and Developmental Biology Animal,2014,50(10):918-925.
[68] LIU C,WANG H N,SHANG Y L,et al.Autophagy is required for ectoplasmic specialization assembly in Sertoli cells[J].Autophagy,2016,12(5):814-832.
[69] 曹安堂,赵升鹏,李希康.小鼠睾丸支持细胞的分离培养及其影响因素[J].黑龙江畜牧兽医,2012,7(13):155-156. CAO A T,ZHAO S P,LI X K.Isolation and culture of Sertoli cells from mouse testis and its influencing factors[J].Heilongjiang Animal Science and Veterinary Medicine,2012,7(13):155-156.(in Chinese)
[70] 张晓丽,高英茂,赵舒武,等.新生昆明小鼠睾丸支持细胞和精原细胞的共培养及支持细胞的作用[J].解剖学报,2009,40(2):240-245. ZHANG X L,GAO Y M,ZHAO S W,et al.Coculture of supporting cell and spermatogonia in testes of neonatal Kunming mice and the effect of Sertoli cell[J].Acta Anatomica Sinica,2009,40(2):240-245.(in Chinese)
[71] XIONG Z,WANG C X,WANG Z L,et al.Raptor directs Sertoli cell cytoskeletal organization and polarity in the mouse testis[J].Biology of Reproduction,2018,99(6):1289-1302.
[72] 黄东晖,赵虎,田永红,等.大鼠睾丸支持细胞的分离、纯化和鉴定[J].解剖学报,2007,38(2):246-249. HUANG D H,ZHAO H,TIAN Y Y,et al.Isolation,purification and identification of Sertoli cells from rat testis[J].Acta Anatomica Sinica,2007,38(2):246-249.(in Chinese)
[73] CHEN S R,TANG J X,CHENG J M,et al.Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling[J].Oncotarget,2015,6(35):37012-37027.
[74] ARATO I,LUCA G,MANCUSO F,et al.An in vitro prototype of a porcine biomimetic testis-like cell culture system:A novel tool for the study of reassembled Sertoli and leydig cells[J]. Asian Journal of Andrology,2018,20(2):160-165.
[75] LEE S,JANG H,MOON S,et al.Differential regulation of TLE3 in Sertoli cells of the testes during postnatal development[J].Cells-Basel,2019,8(10):1156.
[76] RODE K,SIEME H,RICHTERICH P,et al.Characterization of the equine blood-testis barrier during tubular development in normal and cryptorchid stallions[J]. Theriogenology,2015,84(5):763-772.