两步酶法在小鼠睾丸支持细胞分离与培养过程中的研究
|
摘要
目的:探讨两步酶法分离小鼠睾丸支持细胞以及在体外培养、纯化、鉴定过程中的优越性。方法:选取7~8d雄性昆明小鼠,用胶原酶Ⅳ及胰蛋白酶两步酶法分离小鼠睾丸组织获得支持细胞,利用细胞差速贴壁特性纯化支持细胞,利用倒置显微镜、PI染色、特异性抗体标记的方法从多个维度鉴定小鼠睾丸支持细胞。结果:培养72 h后支持细胞呈片状平铺于培养皿底层,相互接触,连接成片。用波形蛋白特异性鉴定证实本实验分离所获得的小鼠睾丸支持细胞。经过PI染色提示这些支持细胞为具有活性的支持细胞。结论:本研究提示两部酶法是一种简便且高效的分离小鼠睾丸支持细胞的有效方法,值得推广。
Objective To investigate the advantages of the two step enzyme method to Isolated mouse Sertoli cells and culture in vitro,purify and identify. Methods Selection the Kun Ming male mice which born 7-8 days,using collagenase IV and trypsin to digestive testis tissue to get the sertoli cell and spermatogonium. Purification of Sertoli cells using differential adherent characteristics of the two cells. Sertoli cells were identified in several ways by using inverted microscopy,PI staining,and specific antibody labeling.Results After 72 hours of culture,The Sertoli cell spread at the bottom of the culture dish in? patches,and there are no obvious spermatogonial stem cells here whice looks like round. The PI staining indicated that these Sertoli cells are active. Vimentin specific identification proved that the isolated Testis Sertoli cells were obtained in this study. To verify what we got from the seperation experiment were the sustentacular cells of mouse testicles through vimentin specificity identification. Conclusion This research suggests the two enzymes is a simple and efficient method for separating Sertoli cells from mouse testis,the method is worth to be spread.
Objective To investigate the advantages of the two step enzyme method to Isolated mouse Sertoli cells and culture in vitro,purify and identify. Methods Selection the Kun Ming male mice which born 7-8 days,using collagenase IV and trypsin to digestive testis tissue to get the sertoli cell and spermatogonium. Purification of Sertoli cells using differential adherent characteristics of the two cells. Sertoli cells were identified in several ways by using inverted microscopy,PI staining,and specific antibody labeling.Results After 72 hours of culture,The Sertoli cell spread at the bottom of the culture dish in? patches,and there are no obvious spermatogonial stem cells here whice looks like round. The PI staining indicated that these Sertoli cells are active. Vimentin specific identification proved that the isolated Testis Sertoli cells were obtained in this study. To verify what we got from the seperation experiment were the sustentacular cells of mouse testicles through vimentin specificity identification. Conclusion This research suggests the two enzymes is a simple and efficient method for separating Sertoli cells from mouse testis,the method is worth to be spread.
引文
[1]郭杰,方南洙,李钟淑,等.睾丸支持细胞结构、功能及研究进展[J].畜牧兽医杂志,2008,27(1):40-42.
[2]Orth J M,Gunsalus G L,Lamperti A A.Evidence from Sertoli cells depleted rats indicate that spermatid number in adults depends on number of Sertoli cells produced during perinatal development[J].Endocrinology,1988,122(3):787-794.
[3]Knobil E,Neill JD.The Physiology of Reproduction[M].New York:Raven Press,1994.
[4]Wu Z,Templeman J L,Smith R A,et al.Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro[J].Journal of Anatomy,2005,206(2):175-184.
[5]Kubota H,Avarbock M R,Brinster R L.Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(47):16489-16494.
[7]胡建新,孙兆林,何坚,等.胶质细胞源性神经营养因子对体外培养小鼠精原干细胞增殖分化作用的影响[J].中华泌尿外科杂志,2010,31(4):227-230.
[8]Anthony C T,Danzo B J,Orgebincrist M C.Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the restricted rat[J].Endocrinology,1984,114(4):1419-25.
[9]Allan C M,Garcia A,Spaliviero J,et al.Complete Sertoli cell proliferation induced by follicle-stimulating hormone(FSH)independently of luteinizing hormone activity:evidence from genetic models of isolated FSH action[J].Endocrinology,2004,145(4):1587.
[10]张学明,赖良学,李德雪,等.小鼠精原细胞的分离和纯化[J].解剖学报,2000,31(3):235-238.
[11]Vergouwen R P,Jacobs S G,Huiskamp R,et al.Proliferative activity of gonocytes,Sertoli cells and interstitial cells during testicular development in mice[J].Journal of Reproduction&Fertility,1991,93(1):233-243.
[12]Raychoudhury S S,Kubinski D.Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response[J].Environmental Health Perspectives,2003,111(1):33-38.
[13]丁训诚,蒋学之.男性生殖毒理学[M].北京:中国人口出版社,1997:230.
[14]章静波.细胞生物学实用方法与技术[M].北京:北京医科大学,中国协和医科大学联合出版社,2005:8-10.
[2]Orth J M,Gunsalus G L,Lamperti A A.Evidence from Sertoli cells depleted rats indicate that spermatid number in adults depends on number of Sertoli cells produced during perinatal development[J].Endocrinology,1988,122(3):787-794.
[3]Knobil E,Neill JD.The Physiology of Reproduction[M].New York:Raven Press,1994.
[4]Wu Z,Templeman J L,Smith R A,et al.Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro[J].Journal of Anatomy,2005,206(2):175-184.
[5]Kubota H,Avarbock M R,Brinster R L.Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(47):16489-16494.
[7]胡建新,孙兆林,何坚,等.胶质细胞源性神经营养因子对体外培养小鼠精原干细胞增殖分化作用的影响[J].中华泌尿外科杂志,2010,31(4):227-230.
[8]Anthony C T,Danzo B J,Orgebincrist M C.Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the restricted rat[J].Endocrinology,1984,114(4):1419-25.
[9]Allan C M,Garcia A,Spaliviero J,et al.Complete Sertoli cell proliferation induced by follicle-stimulating hormone(FSH)independently of luteinizing hormone activity:evidence from genetic models of isolated FSH action[J].Endocrinology,2004,145(4):1587.
[10]张学明,赖良学,李德雪,等.小鼠精原细胞的分离和纯化[J].解剖学报,2000,31(3):235-238.
[11]Vergouwen R P,Jacobs S G,Huiskamp R,et al.Proliferative activity of gonocytes,Sertoli cells and interstitial cells during testicular development in mice[J].Journal of Reproduction&Fertility,1991,93(1):233-243.
[12]Raychoudhury S S,Kubinski D.Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response[J].Environmental Health Perspectives,2003,111(1):33-38.
[13]丁训诚,蒋学之.男性生殖毒理学[M].北京:中国人口出版社,1997:230.
[14]章静波.细胞生物学实用方法与技术[M].北京:北京医科大学,中国协和医科大学联合出版社,2005:8-10.