雄激素和氢化可的松对山羊附睾头上皮细胞增殖的影响
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摘要
为研究雄激素和氢化可的松对山羊附睾上皮细胞生长的作用模式,本研究利用酶标仪检测附睾头部上皮细胞增殖情况,实时荧光定量PCR及ELISA检测雄激素受体(AR)的表达,检测睾酮与氢化可的松对山羊附睾上皮细胞体外增殖的作用以及对AR表达的影响。结果表明:100nmol/L睾酮对附睾头上皮细胞增殖的促进效应最高且与对照组差异极显著(P<0.01),200nmol/L氢化可的松促进附睾头上皮细胞增殖效应最佳并与对照组差异显著(P<0.05),前者的效应明显且可以被AR阻断剂阻断;睾酮和氢化可的松对附睾头上皮细胞增殖表现为协同作用;100nmol/L睾酮与200nmol/L氢化可的松均使附睾头上皮细胞的AR mRNA和蛋白表达量上调,与对照组差异显著(P<0.05),且二者共存时附睾头上皮细胞的AR mRNA和蛋白表达量极显著高于对照组(P<0.01)。本研究表明睾酮和氢化可的松对附睾头上皮细胞体外增殖均有促进作用,呈明显的浓度依赖性,且二者之间存在协同作用,这为进一步研究附睾上皮细胞增殖及功能的调节机理提供了基础。
To study the effects of androgen and hydrocortisone on the growth of epididymis epithelial cells in goats,the proliferation of epididymal epithelial cells was detected by microplate reader and androgen receptor(AR)and its expression was detected by Real-time fluorescence quantitative PCR and ELISA in this study.The effects of testosterone and hydrocortisone on the proliferation of caput epididymal epithelial cells and the expression of AR in goat were studied.The results showed that:The concentration of 100 nmol/L testosterone had the highest promoted effect on caput epididymal epithelial cells proliferation,which was significantly different from that of the control group(P<0.01).The 200 nmol/L hydrocortisone promotes the proliferation of caput epididymal epithelial cells was the best and which was significantly different from that of the control group(P< 0.05),the effect of former is obvious and could be blocked by AR blocker;The effect of both testosterone and hydrocortisone on the proliferation of epididymal epithelial cells was synergistic;The 100 nmol/L testosterone and 200 nmol/L hydrocortisone increased the expression of AR mRNA and protein in the caput epididymal epithelial cells,and was significantly different from the control group(P<0.05);The expression of AR mRNA and protein in the caput epithelial cells was significantly higher than that in the control group when the two coexisted(P< 0.01).This study indicated that testosterone and hydrocortisone could promote theproliferation of caput epididymal epithelial cells in vitro,showing a concentration dependent manner,and the synergistic effect was shown in the coexistence of androgen and hydrocortisone,which provided a basis for further study on the regulation mechanism of epididymal epithelial cell proliferation and function.
To study the effects of androgen and hydrocortisone on the growth of epididymis epithelial cells in goats,the proliferation of epididymal epithelial cells was detected by microplate reader and androgen receptor(AR)and its expression was detected by Real-time fluorescence quantitative PCR and ELISA in this study.The effects of testosterone and hydrocortisone on the proliferation of caput epididymal epithelial cells and the expression of AR in goat were studied.The results showed that:The concentration of 100 nmol/L testosterone had the highest promoted effect on caput epididymal epithelial cells proliferation,which was significantly different from that of the control group(P<0.01).The 200 nmol/L hydrocortisone promotes the proliferation of caput epididymal epithelial cells was the best and which was significantly different from that of the control group(P< 0.05),the effect of former is obvious and could be blocked by AR blocker;The effect of both testosterone and hydrocortisone on the proliferation of epididymal epithelial cells was synergistic;The 100 nmol/L testosterone and 200 nmol/L hydrocortisone increased the expression of AR mRNA and protein in the caput epididymal epithelial cells,and was significantly different from the control group(P<0.05);The expression of AR mRNA and protein in the caput epithelial cells was significantly higher than that in the control group when the two coexisted(P< 0.01).This study indicated that testosterone and hydrocortisone could promote theproliferation of caput epididymal epithelial cells in vitro,showing a concentration dependent manner,and the synergistic effect was shown in the coexistence of androgen and hydrocortisone,which provided a basis for further study on the regulation mechanism of epididymal epithelial cell proliferation and function.
引文
[1] Ribeiro C M,Silva Erick J R,Hinton B T.β-defensins and the epididymis:Contrasting influences of prenatal,postnatal,and adult scenarios[J].Asian Journal of Andrology,2016,18(2):323-328
[2] Fang F,Ni K,Cai Y T,Zhao Q,Shang J,Zhang X K,Shen S L,Xiong C L.Busulfan administration produces toxic effects on epididymal morphology and inhibits the expression of ZO-1and vimentin in the mouse epididymis[J].Bioscience Reports,2017,37(6):BSR20171059
[3] Cornwall G A.New insights into epididymal biology and function[J].Human Reproduction Update,2009,15(2):213-227
[4] Arrotéia K F,Garcia P V,Barbieri M F,Justino M L,Pereira,L A V.The Epididymis:Embryology,Structure,Function and Its Role in Fertilization and Infertility[M].In:Pereira L A V,eds.Embryology-Updates and Highlights on Classic Topics,Croatia:InTech,2012:41-66
[5] Dacheux J L,Dacheux F. New insights into epididymal function in relation to sperm maturation[J].Reproduction,2014,147(2):R27
[6] Leir S H,Browne J A,Eggener S E,Harris A.Characterization of primary cultures of adult human epididymis epithelial cells[J].Fertility and Sterility,2015,103(3):647-654
[7] Thimon V,Koukoui O,Calvo E,Sullivan R.Region-specific gene expression profiling along the human epididymis[J].Molecular Human Reproduction,2007,13(10):691-704
[8] Jalkanen J,Kotimki M,Huhtaniemi I,Poutanen M.Novel epididymal protease inhibitors with Kazal or WAP family domain[J]. Biochemical and Biophysical Research Communications,2006,349(1):245-254
[9] Turner T T.On the epididymis and its role in the development of the fertile ejaculate[J].Journal of Andrology,1995,16(4):292
[10] Roy S K. Regulation of transforming growth factor-β-receptor type I and type II messenger ribonucleic acid expression in the hamster ovary by gonadotropins and steroid hormones[J].Biology of Reproduction,2000,62(6):1858
[11] Sorrell J M,Brinon L,Baber M A,Caplan A I.Cytokines and glucocorticoids differentially regulate APN/CD13 and DPPIV/CD26enzyme activities in cultured human dermal fibroblasts[J].Archives of Dermatological Research,2003,295(4):160-168
[12] Zhong C C,Tang Y,Shen Y,Chen H H,Liang W G,Li S M.Effect of insulin,hydrocortisone and their combined application on the proliferation of chondrocytes[J].Chinese Journal of Clinical Rehabilitation,2005,9(22):242-244
[13]卫俊霞,冯光珍,孙红,于大山,汪培铭,李冬梅.氢化可的松对体外培养的皮肤成纤维细胞增殖及胶原合成的影响[J].第四军医大学学报,2004,25(19):1785-1787Wei J X,Feng G Z,Sun H,Yu D S,Wang P M,Li D M.Effects of hydrocortisone on proliferation and collagen synthesis of cultured skin fibroblasts in vitro[J].Journal of the Fourth Military Medical University,2004,25(19):1785-1787(in Chinese)
[14]田青,王洪荣.胰岛素、催乳素和氢化可的松对奶牛乳腺上皮细胞增殖和凋亡的影响[J].中国饲料,2013(2):8-12Tian Q, Wang H R.Effects of insulin,prolactin and hydrocortisone on the proliferation and apoptosis of mammary gland epithelial cells in dairy cows[J].China Feed,2013(2):8-12(in Chinese)
[15] Rheinwald J G,Green H.Serial cultivation of strains of humanepidermalkeratinocytes:Theformationof keratinizing colonies from single cells[J].Cell,1975,6(3):331-343
[16] Yu M,Bojic S,Figueiredo G S,Rooney P,de Havilland J,Dickinson A,Figueiredo F C,Lako M.An important role for adenine,cholera toxin,hydrocortisone and triiodothyronine in the proliferation,self-renewal and differentiation of limbal stem cells in vitro[J].Experimental Eye Research,2016,152:113-122
[17] Huber B,Czaja A M,Kluger P J.Influence of epidermal growth factor(EGF)and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering[J].Cell Biology International,2016,40(5):569
[18] Qu B,Gu Y,Shen J,Qin J,Bao J,Hu Y,Zeng W,Dong W. Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer[J].Plos One,2014,9(3):e92483.
[19]钟济华,成玲剑,周文,张义炜,钟华,黄洪晖,陈芳源.CCK法检测紫草素及衍生物对血液肿瘤细胞株的增殖抑制作用[J].中国实验诊断学,2012,16(8):1366-1369Zhong J H,Cheng L J,Zhou W,Zhang Y W,Zhong H,Huang H H,Chen F Y.Inhibitory effect of vegetarian and its derivatives on proliferation of blood tumor cell lines by CCK[J].Chinese Journal of Laboratory Diagnosis,2012,16(8):1366-1369(in Chinese)
[20] Heinlein C A,Chang C.The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions[J].Molecular Endocrinology,2002,16(10):2181-2187
[21] Lutz L B,Jamnongjit M,Yang W H,Jahani D,Gill A,Hammes S R. Selective modulation of genomic and nongenomic androgen responses by androgen receptor ligands[J].Molecular Endocrinology,2003,17(6):1106-1116
[22] Hu S G,Zou M,Yao G X,Ma W B,Zhu Q L,Li X Q,Chen Z J,Sun Y.Androgenic regulation of beta-defensins in the mouse epididymis[J]. Reproductive Biology and Endocrinology,2014,12(1):76
[23] Ghyselinck N B,Jimenez C,Lefran9ois A M,Dufaure J P.Molecular cloning of a cDNA for androgen-regulated proteins secreted by the mouse epididymis[J].Journal of Molecular Endocrinology,1990,4(1):5
[24] Lareyre J J,Zheng W L,Zhao G Q,Kasper S,Newcomer M E,Matusik R J,Ong D E,Orgebin-Crist M C.Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid[J].Endocrinology,1998,139(6):2971-2981
[25] Eberspaecher U,Roosterman D,Krtzschmar J,Haendler B, Habenicht U F, Becker A, Quensel C, Petri T,Schleuning W D,Donner P. Mouse androgen-dependent epididymal glycoprotein CRISP-1(DE/AEG):Isolation,biochemical characterization,and expression in recombinant form[J].Molecular Reproduction and Development,1995,42(2):157-172
[26] Planz B,Wang Q,Kirley S D,Lin C W,Mcdougal W S.Androgen responsiveness of stromal cells of the human prostate:Regulation of cell proliferation and keratinocyte growth factor by androgen[J].The Journal of Urology,1998,160(5):1850-1855
[27]王越.雄激素、雌激素与IL-6和IL-8促卵巢癌细胞增殖分子机制的研究以及Raf激酶抑制蛋白与卵巢癌转移关系的初步探讨[D].天津:天津医科大学,2006Wang Y.Regulation of androgen,estrogen,IL-6and IL-8in the proliferation of ovarian cancer and the effect of Raf kinase inhibitor protein on metastasis of ovarian carcinoma[D].Tianjin:Tianjin Medical University,2006
[28] Bennett N C,Gardiner R A,Hooper J D,Johnson D W,Gobe G C. Molecular cell biology of androgen receptor signaling[J].International Journal of Biochemistry and Cell Biology,2010,42(6):813
[29] Shu Y,Ming J,Grabowska M M,Li J,Connelly Z M,Zhang J, Hayward S W,Cates J M, Han G,Yu X.Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo[J].Oncotarget,2016,7(43):70404
[30] Lin H Y,Sun M Z,Lin C,Tang H Y,London D,Shih A,Davis F B,Davis P J.Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and-negative breast cancer cells[J].The Journal of Steroid Biochemistry and Molecular Biology,2009,113(3-5):182-188
[31] Bjornson B H,Harvey J M,Rose L.Differential effect of hydrocortisone on eosinophil and neutrophil proliferation[J].Journal of Clinical Investigation,1985,76(3):924-929
[32] Volz A C,Huber B,Schwandt A M,Kluger P J.EGF and hydrocortisone as critical factors for the co-culture of adipogenic differentiated ASCs and endothelial cells[J].Differentiation,2017,95:21
[33] Khan D,Vasu S,Moffett R C,Gault V A,Flatt P R,Irwin N.Locally produced xenin and the neurotensinergic system in pancreatic islet function andβ-cell survival[J].Biological Chemistry,2017,399(1):79-92
[34] Pearl C A,At-Taras E,Berger T,Roser J F.Reduced endogenous estrogen delays epididymal development but has no effect on efferent duct morphology in boars[J].Reproduction,2007,134(4):593-604
[35] Yurtsever T,Schilling T M,K9lsch M,Turner J D,Meyer J,Schchinger H,Schote A B.The acute and temporary modulation of PERIOD genes by hydrocortisone in healthy subjects[J].Chronobiology International,2016,33(9):1-13
[36]彭苗苗.小剂量氢化可的松通过上调糖皮质激素受体-α表达对脓毒症大鼠早期肺损伤产生保护作用[D].山东:山东大学,2011Peng M M.The protective effects of low-dose hydrocortisone on sepsis-induced lung injury in rats model is by up-regulation expression of the glucocorticoid receptor alpha isoform[D].Jinan:Shandong University,2011
[2] Fang F,Ni K,Cai Y T,Zhao Q,Shang J,Zhang X K,Shen S L,Xiong C L.Busulfan administration produces toxic effects on epididymal morphology and inhibits the expression of ZO-1and vimentin in the mouse epididymis[J].Bioscience Reports,2017,37(6):BSR20171059
[3] Cornwall G A.New insights into epididymal biology and function[J].Human Reproduction Update,2009,15(2):213-227
[4] Arrotéia K F,Garcia P V,Barbieri M F,Justino M L,Pereira,L A V.The Epididymis:Embryology,Structure,Function and Its Role in Fertilization and Infertility[M].In:Pereira L A V,eds.Embryology-Updates and Highlights on Classic Topics,Croatia:InTech,2012:41-66
[5] Dacheux J L,Dacheux F. New insights into epididymal function in relation to sperm maturation[J].Reproduction,2014,147(2):R27
[6] Leir S H,Browne J A,Eggener S E,Harris A.Characterization of primary cultures of adult human epididymis epithelial cells[J].Fertility and Sterility,2015,103(3):647-654
[7] Thimon V,Koukoui O,Calvo E,Sullivan R.Region-specific gene expression profiling along the human epididymis[J].Molecular Human Reproduction,2007,13(10):691-704
[8] Jalkanen J,Kotimki M,Huhtaniemi I,Poutanen M.Novel epididymal protease inhibitors with Kazal or WAP family domain[J]. Biochemical and Biophysical Research Communications,2006,349(1):245-254
[9] Turner T T.On the epididymis and its role in the development of the fertile ejaculate[J].Journal of Andrology,1995,16(4):292
[10] Roy S K. Regulation of transforming growth factor-β-receptor type I and type II messenger ribonucleic acid expression in the hamster ovary by gonadotropins and steroid hormones[J].Biology of Reproduction,2000,62(6):1858
[11] Sorrell J M,Brinon L,Baber M A,Caplan A I.Cytokines and glucocorticoids differentially regulate APN/CD13 and DPPIV/CD26enzyme activities in cultured human dermal fibroblasts[J].Archives of Dermatological Research,2003,295(4):160-168
[12] Zhong C C,Tang Y,Shen Y,Chen H H,Liang W G,Li S M.Effect of insulin,hydrocortisone and their combined application on the proliferation of chondrocytes[J].Chinese Journal of Clinical Rehabilitation,2005,9(22):242-244
[13]卫俊霞,冯光珍,孙红,于大山,汪培铭,李冬梅.氢化可的松对体外培养的皮肤成纤维细胞增殖及胶原合成的影响[J].第四军医大学学报,2004,25(19):1785-1787Wei J X,Feng G Z,Sun H,Yu D S,Wang P M,Li D M.Effects of hydrocortisone on proliferation and collagen synthesis of cultured skin fibroblasts in vitro[J].Journal of the Fourth Military Medical University,2004,25(19):1785-1787(in Chinese)
[14]田青,王洪荣.胰岛素、催乳素和氢化可的松对奶牛乳腺上皮细胞增殖和凋亡的影响[J].中国饲料,2013(2):8-12Tian Q, Wang H R.Effects of insulin,prolactin and hydrocortisone on the proliferation and apoptosis of mammary gland epithelial cells in dairy cows[J].China Feed,2013(2):8-12(in Chinese)
[15] Rheinwald J G,Green H.Serial cultivation of strains of humanepidermalkeratinocytes:Theformationof keratinizing colonies from single cells[J].Cell,1975,6(3):331-343
[16] Yu M,Bojic S,Figueiredo G S,Rooney P,de Havilland J,Dickinson A,Figueiredo F C,Lako M.An important role for adenine,cholera toxin,hydrocortisone and triiodothyronine in the proliferation,self-renewal and differentiation of limbal stem cells in vitro[J].Experimental Eye Research,2016,152:113-122
[17] Huber B,Czaja A M,Kluger P J.Influence of epidermal growth factor(EGF)and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering[J].Cell Biology International,2016,40(5):569
[18] Qu B,Gu Y,Shen J,Qin J,Bao J,Hu Y,Zeng W,Dong W. Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer[J].Plos One,2014,9(3):e92483.
[19]钟济华,成玲剑,周文,张义炜,钟华,黄洪晖,陈芳源.CCK法检测紫草素及衍生物对血液肿瘤细胞株的增殖抑制作用[J].中国实验诊断学,2012,16(8):1366-1369Zhong J H,Cheng L J,Zhou W,Zhang Y W,Zhong H,Huang H H,Chen F Y.Inhibitory effect of vegetarian and its derivatives on proliferation of blood tumor cell lines by CCK[J].Chinese Journal of Laboratory Diagnosis,2012,16(8):1366-1369(in Chinese)
[20] Heinlein C A,Chang C.The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions[J].Molecular Endocrinology,2002,16(10):2181-2187
[21] Lutz L B,Jamnongjit M,Yang W H,Jahani D,Gill A,Hammes S R. Selective modulation of genomic and nongenomic androgen responses by androgen receptor ligands[J].Molecular Endocrinology,2003,17(6):1106-1116
[22] Hu S G,Zou M,Yao G X,Ma W B,Zhu Q L,Li X Q,Chen Z J,Sun Y.Androgenic regulation of beta-defensins in the mouse epididymis[J]. Reproductive Biology and Endocrinology,2014,12(1):76
[23] Ghyselinck N B,Jimenez C,Lefran9ois A M,Dufaure J P.Molecular cloning of a cDNA for androgen-regulated proteins secreted by the mouse epididymis[J].Journal of Molecular Endocrinology,1990,4(1):5
[24] Lareyre J J,Zheng W L,Zhao G Q,Kasper S,Newcomer M E,Matusik R J,Ong D E,Orgebin-Crist M C.Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid[J].Endocrinology,1998,139(6):2971-2981
[25] Eberspaecher U,Roosterman D,Krtzschmar J,Haendler B, Habenicht U F, Becker A, Quensel C, Petri T,Schleuning W D,Donner P. Mouse androgen-dependent epididymal glycoprotein CRISP-1(DE/AEG):Isolation,biochemical characterization,and expression in recombinant form[J].Molecular Reproduction and Development,1995,42(2):157-172
[26] Planz B,Wang Q,Kirley S D,Lin C W,Mcdougal W S.Androgen responsiveness of stromal cells of the human prostate:Regulation of cell proliferation and keratinocyte growth factor by androgen[J].The Journal of Urology,1998,160(5):1850-1855
[27]王越.雄激素、雌激素与IL-6和IL-8促卵巢癌细胞增殖分子机制的研究以及Raf激酶抑制蛋白与卵巢癌转移关系的初步探讨[D].天津:天津医科大学,2006Wang Y.Regulation of androgen,estrogen,IL-6and IL-8in the proliferation of ovarian cancer and the effect of Raf kinase inhibitor protein on metastasis of ovarian carcinoma[D].Tianjin:Tianjin Medical University,2006
[28] Bennett N C,Gardiner R A,Hooper J D,Johnson D W,Gobe G C. Molecular cell biology of androgen receptor signaling[J].International Journal of Biochemistry and Cell Biology,2010,42(6):813
[29] Shu Y,Ming J,Grabowska M M,Li J,Connelly Z M,Zhang J, Hayward S W,Cates J M, Han G,Yu X.Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo[J].Oncotarget,2016,7(43):70404
[30] Lin H Y,Sun M Z,Lin C,Tang H Y,London D,Shih A,Davis F B,Davis P J.Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and-negative breast cancer cells[J].The Journal of Steroid Biochemistry and Molecular Biology,2009,113(3-5):182-188
[31] Bjornson B H,Harvey J M,Rose L.Differential effect of hydrocortisone on eosinophil and neutrophil proliferation[J].Journal of Clinical Investigation,1985,76(3):924-929
[32] Volz A C,Huber B,Schwandt A M,Kluger P J.EGF and hydrocortisone as critical factors for the co-culture of adipogenic differentiated ASCs and endothelial cells[J].Differentiation,2017,95:21
[33] Khan D,Vasu S,Moffett R C,Gault V A,Flatt P R,Irwin N.Locally produced xenin and the neurotensinergic system in pancreatic islet function andβ-cell survival[J].Biological Chemistry,2017,399(1):79-92
[34] Pearl C A,At-Taras E,Berger T,Roser J F.Reduced endogenous estrogen delays epididymal development but has no effect on efferent duct morphology in boars[J].Reproduction,2007,134(4):593-604
[35] Yurtsever T,Schilling T M,K9lsch M,Turner J D,Meyer J,Schchinger H,Schote A B.The acute and temporary modulation of PERIOD genes by hydrocortisone in healthy subjects[J].Chronobiology International,2016,33(9):1-13
[36]彭苗苗.小剂量氢化可的松通过上调糖皮质激素受体-α表达对脓毒症大鼠早期肺损伤产生保护作用[D].山东:山东大学,2011Peng M M.The protective effects of low-dose hydrocortisone on sepsis-induced lung injury in rats model is by up-regulation expression of the glucocorticoid receptor alpha isoform[D].Jinan:Shandong University,2011
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