锌指蛋白185在小鼠睾丸间质细胞的表达研究
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摘要
目的优化睾丸间质细胞分离纯化方法,研究锌指蛋白(zinc finger protein,ZNF)185在间质细胞中的表达。方法选用4-5周雄性小鼠,取睾丸,Ⅳ型胶原酶消化分离间质细胞,差速贴壁法纯化后,3β-羟基类固醇脱氢酶染色法鉴定细胞纯度,PCR、Western blot和免疫荧光法研究ZNF185在间质细胞中的表达。结果成功分离纯化小鼠睾丸间质细胞,有纯化组细胞的纯度(x珋=98%)显著高于无纯化组(x珋=22%)(P<0.001)。PCR和Western blot显示,ZNF185在间质细胞中高表达。免疫荧光显示,ZNF185定位于间质细胞的细胞质。结论通过该方法可获得高纯度间质细胞;ZNF185主要表达于间质细胞的细胞质,为进一步研究该蛋白的功能提供了实验基础。
Objective To optimize the method of isolating and purifying Leydig cells of male mouse,and investigate the expression of ZNF185 in Leydig cells. Methods The 4-5-week-old male mice were used,and testes were collected and digested with type Ⅳ collagenase,purified by differential attachment of cells,and identified by 3β-hydroxy-steroid-dehydrogenase staining method. The expression of ZNF185 was detected by PCR,Western blot and immunocytochemical analysis. Results The Leydig cells of mouse testis were successfully isolated and purified,and the purity in purification group( x珋= 98%) was significently higher than that in non-purification group( x珋= 22%)( P < 0. 001). PCR and Western blot results showed that ZNF185 was highly expressed in Leydig cells. ZNF185 was localized in the cytoplasm of Leydig cells by immunocytochemical analysis. Conclusion This method can successfully produce highly purified Leydig cells. ZNF185 is mainly localized in cytoplasm of Leydig cells,which provides evidences for the further investigation of its function.
Objective To optimize the method of isolating and purifying Leydig cells of male mouse,and investigate the expression of ZNF185 in Leydig cells. Methods The 4-5-week-old male mice were used,and testes were collected and digested with type Ⅳ collagenase,purified by differential attachment of cells,and identified by 3β-hydroxy-steroid-dehydrogenase staining method. The expression of ZNF185 was detected by PCR,Western blot and immunocytochemical analysis. Results The Leydig cells of mouse testis were successfully isolated and purified,and the purity in purification group( x珋= 98%) was significently higher than that in non-purification group( x珋= 22%)( P < 0. 001). PCR and Western blot results showed that ZNF185 was highly expressed in Leydig cells. ZNF185 was localized in the cytoplasm of Leydig cells by immunocytochemical analysis. Conclusion This method can successfully produce highly purified Leydig cells. ZNF185 is mainly localized in cytoplasm of Leydig cells,which provides evidences for the further investigation of its function.
引文
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[9]卿利娟,魏泓,李婧,等.仔猪睾丸间质细培养方法的比较[J].中国兽医杂志,2013,49(11):29-35.
[10]张柳平,邵根宝,潘耀谦.成年小鼠睾丸间质细胞的分离、鉴定及功能检测[J].动物医学进展,2013,34(9):43-46.
[11]赵为民,杨建英,代涛,等.小鼠睾丸间质细胞体外原代培养方法的建立[J].中国组织工程研究与临床康复,2011,15(46):8665-8667.
[12]张斌,宋国华.小鼠睾丸间质细胞培养及冻存与复苏的初步研究[J].山西医科大学学报,2011,42(5):387-390.
[13]关聪会,张志刚,孙应刚,等.硫酸镍对体外培养大鼠睾丸间质细胞毒作用的实验研究[J].毒理学杂志,2011,24(4):284-287.
[14]郑菁,张志刚,孙应彪,等.硫酸镍对体外培养大鼠干完间质细胞超微结构的影响[J].工业卫生与职业病,2012,38(1):29-32.
[15]孙新明,杨亚军.原代培养小鼠睾丸间质细胞的甘草毒性研究[J].中成药,2012,34(9):1801-1804.
[16]Li D,Sekhon P,Barr KJ,et al.Connexins and steroidogenesis in mouse Leydig cells[J].Can J Physiol Pharmacol,2013,91(2):157-164.
[17]Matzkin ME,Yamashita S,Ascoli M.The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells[J].Mol Cell Endocrinol,2013,370(1-2):130-137.
[18]Wang N,Zheng Q,Zhang JS,et al.Molecular cloning and characterization of a novel mouse actin-binding protein Zfp185[J].J Mol Histol,2008,39(3):295-302.
[19]Medina PP,Carretero J,Ballestar E,et al.Transcriptional targets of the chromatin-remodelling factor SMARCA4/BRG1 in lung cancer cells[J].Hum Mol Genet,2005,14:973-982.
[20]郑全辉,陆斌,葛淑芝,等.锌脂蛋白185通过与肌动蛋白纤维结合抑制肿瘤细胞增殖[J].中国老年学杂志,2012,32(21):4720-4722.
[2]He Z,Kokkinaki M,Dym M.Signaling molecules and pathways regulating the fate of spermatogonial stem cells[J].Microsc Res Tech,2009,72(8):586-595.
[3]谢天承.小鼠睾丸间质细胞分离培养方法的研究进展[J].临床与病理杂志,2015,35(4):569-572.
[4]Alexandrova ML,Bochev PG.Reduced extracellular phagocyte oxidative activity,antioxidant level changes and increased oxidative damage in healthy human blood as a function of age[J].Age(Dordr),2009,31(2):99-107.
[5]潘智芳,冯卫国,陈丽梅,等.ZNF185基因的克隆及在小鼠睾丸中的定位[J].细胞与分子免疫学杂志,2010,26(10):973-975.
[6]冯卫国,潘智芳,连波,等.锌脂蛋白(ZNF)185在小鼠不同发育阶段睾丸中的表达[J].生殖与避孕,2012,32(10):672-675.
[7]EI-Alfy M,Luu-The V,Huang XF,et al.Localization of type 517betahydroxysteroid dehydrogenase,3beta-hydroxysteroiddehydrogenase,and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry[J].Endocrinology,1999,140(3):1481-1491.
[8]邵冰玉,杨占清,吴建英,等.小鼠睾丸间质细胞的分离培养[J].畜牧兽医科学,2008,24(5):26-28.
[9]卿利娟,魏泓,李婧,等.仔猪睾丸间质细培养方法的比较[J].中国兽医杂志,2013,49(11):29-35.
[10]张柳平,邵根宝,潘耀谦.成年小鼠睾丸间质细胞的分离、鉴定及功能检测[J].动物医学进展,2013,34(9):43-46.
[11]赵为民,杨建英,代涛,等.小鼠睾丸间质细胞体外原代培养方法的建立[J].中国组织工程研究与临床康复,2011,15(46):8665-8667.
[12]张斌,宋国华.小鼠睾丸间质细胞培养及冻存与复苏的初步研究[J].山西医科大学学报,2011,42(5):387-390.
[13]关聪会,张志刚,孙应刚,等.硫酸镍对体外培养大鼠睾丸间质细胞毒作用的实验研究[J].毒理学杂志,2011,24(4):284-287.
[14]郑菁,张志刚,孙应彪,等.硫酸镍对体外培养大鼠干完间质细胞超微结构的影响[J].工业卫生与职业病,2012,38(1):29-32.
[15]孙新明,杨亚军.原代培养小鼠睾丸间质细胞的甘草毒性研究[J].中成药,2012,34(9):1801-1804.
[16]Li D,Sekhon P,Barr KJ,et al.Connexins and steroidogenesis in mouse Leydig cells[J].Can J Physiol Pharmacol,2013,91(2):157-164.
[17]Matzkin ME,Yamashita S,Ascoli M.The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells[J].Mol Cell Endocrinol,2013,370(1-2):130-137.
[18]Wang N,Zheng Q,Zhang JS,et al.Molecular cloning and characterization of a novel mouse actin-binding protein Zfp185[J].J Mol Histol,2008,39(3):295-302.
[19]Medina PP,Carretero J,Ballestar E,et al.Transcriptional targets of the chromatin-remodelling factor SMARCA4/BRG1 in lung cancer cells[J].Hum Mol Genet,2005,14:973-982.
[20]郑全辉,陆斌,葛淑芝,等.锌脂蛋白185通过与肌动蛋白纤维结合抑制肿瘤细胞增殖[J].中国老年学杂志,2012,32(21):4720-4722.
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