雄性大鼠Inhibinα亚基的表达及其启动子表达载体的构建
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摘要
目的:确定雄性大鼠Inhibinα亚基的mRNA及蛋白在各种组织中有无表达及相对表达量。
方法:选用成年SD雄性大鼠,10%水合氯醛麻醉后处死,立即取睾丸、附睾、肾、肾上腺及肝组织,采用免疫组化法和荧光定量PCR方法分别检测Inhibinα蛋白和mRNA在大鼠各组织中的表达情况。
结果:Inhibinα蛋白在睾丸、附睾、肾、肾上腺组织中均有表达,在肝脏组织中无表达。InhibinαmRNA在各组织中的表达相对量依次为:肾>肾上腺>睾丸>附睾。
结论:本实验从定性和定量两方面检测了Inhibinα亚基的表达情况,结果提示Inhibinα亚基不仅仅在生殖系统有表达,在其它系统也有表达。
目的:将大鼠Inhibinα基因的启动子区分段克隆后分别构建真核表达载体,为进一步筛选出其核心启动子做好准备。
方法:利用已有文献及网络资源进行Inhibinα启动子区的预测及生物信息学分析,用基因重组技术将预测的启动子区分段克隆到真核表达载体pEGFP-N1中,并对其测序验证。
结果:成功克隆了大鼠Inhibinα基因的启动子区的395bp、606bp、775bp大小的片段,并分别构建了这三种片段的真核表达载体。
结论:对预测的大鼠Inhibinα基因的启动子区分段克隆,并成功构建了含有这些克隆片段的真核表达载体。
Objective: To examine the expression of Inhibinαprotein and the relative expression levels of its mRNA in various tissues.
Methods: Adult male SD rats were killed immediately after anesthesia with 10% chloral hydrate.The testis, epididymis, kidney, adrenal and liver were isolated, The immunohistochemistry and fluorescence quantitative PCR technology was performed to detect the Inhibinαprotein and mRNA in the rat tissues.
Results: Inhibinαprotein was expressed in testis,epididymis, kidney adrenal gland and pituitary but not in liver. The relative expression levels of inhibinαmRNA were as follows: kidney> adrenal> testis> epididymis.
Conclusion: Both qualitative and quantitative detection of Inhibinαsubunit in this experiment suggest that the Inhibinαsubunit is not only expressed in tissues of reproductive system but also in other tissues. Objective: To construct eukaryotic expression vector of rat Inhibinαgene promoter region respectively after fragmental cloning, in order to prepare for selecting its core promoter.
Methods: Papers and network resources had been used for the prediction and the bioinformatic analysis of Inhibinαpromoter region.Gene recombinant technology was used to clone the fragmental promoter region into the eukaryotic expression vector ------pEGFP-N1, then the sequences were verified.
Results: 395bp, 606bp and 775bp fragments of the rat Inhibinαgene promoter region were cloned successfully, moreover, the eukaryotic expression vectors containing this three fragments were constructed respectively. Conclusion: The predicted rat Inhibinαgene promoter region is successfully cloned and the eukaryotic expression vectors containing the fragmental promoter are successfully constructed.
方法:选用成年SD雄性大鼠,10%水合氯醛麻醉后处死,立即取睾丸、附睾、肾、肾上腺及肝组织,采用免疫组化法和荧光定量PCR方法分别检测Inhibinα蛋白和mRNA在大鼠各组织中的表达情况。
结果:Inhibinα蛋白在睾丸、附睾、肾、肾上腺组织中均有表达,在肝脏组织中无表达。InhibinαmRNA在各组织中的表达相对量依次为:肾>肾上腺>睾丸>附睾。
结论:本实验从定性和定量两方面检测了Inhibinα亚基的表达情况,结果提示Inhibinα亚基不仅仅在生殖系统有表达,在其它系统也有表达。
目的:将大鼠Inhibinα基因的启动子区分段克隆后分别构建真核表达载体,为进一步筛选出其核心启动子做好准备。
方法:利用已有文献及网络资源进行Inhibinα启动子区的预测及生物信息学分析,用基因重组技术将预测的启动子区分段克隆到真核表达载体pEGFP-N1中,并对其测序验证。
结果:成功克隆了大鼠Inhibinα基因的启动子区的395bp、606bp、775bp大小的片段,并分别构建了这三种片段的真核表达载体。
结论:对预测的大鼠Inhibinα基因的启动子区分段克隆,并成功构建了含有这些克隆片段的真核表达载体。
Objective: To examine the expression of Inhibinαprotein and the relative expression levels of its mRNA in various tissues.
Methods: Adult male SD rats were killed immediately after anesthesia with 10% chloral hydrate.The testis, epididymis, kidney, adrenal and liver were isolated, The immunohistochemistry and fluorescence quantitative PCR technology was performed to detect the Inhibinαprotein and mRNA in the rat tissues.
Results: Inhibinαprotein was expressed in testis,epididymis, kidney adrenal gland and pituitary but not in liver. The relative expression levels of inhibinαmRNA were as follows: kidney> adrenal> testis> epididymis.
Conclusion: Both qualitative and quantitative detection of Inhibinαsubunit in this experiment suggest that the Inhibinαsubunit is not only expressed in tissues of reproductive system but also in other tissues. Objective: To construct eukaryotic expression vector of rat Inhibinαgene promoter region respectively after fragmental cloning, in order to prepare for selecting its core promoter.
Methods: Papers and network resources had been used for the prediction and the bioinformatic analysis of Inhibinαpromoter region.Gene recombinant technology was used to clone the fragmental promoter region into the eukaryotic expression vector ------pEGFP-N1, then the sequences were verified.
Results: 395bp, 606bp and 775bp fragments of the rat Inhibinαgene promoter region were cloned successfully, moreover, the eukaryotic expression vectors containing this three fragments were constructed respectively. Conclusion: The predicted rat Inhibinαgene promoter region is successfully cloned and the eukaryotic expression vectors containing the fragmental promoter are successfully constructed.
引文
1.吴惠梅李慕军.抑制素与人类辅助生殖技术.生殖与避孕,2006,26(3):169-174.
2. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J.Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature,1986,321:776–779.
3. Vale W, Rivier C, Hsueh A .Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res ,1988 ,44:1–34.
4. Haisenleder, J. C. Marshall. Production and actions of inhibin and activin during folliculogenesis in the rat.Molecular and Cellular Endocrinology , 2001,180:139– 144.
5. Halder A, Fauzdar A, Kumar A. Serum inhibin B and follicle-stimulating hormone levels as markers in the evaluation of azoospermic men. Andrologia. 2005 ,37(5): 173-179.
6. Cuevas P, Ying SY, Ling N, et al. Immunohistochemical detection of inhibin in the gonad. Biochemical and BiophysicalResearch Communications, 1987, 142(1): 23-30.
7. Risbridger GP, Clements J, Robertson DM, et al. Immunoandbioactive inhibin and inhibin alpha-subunit expression in rat Leydig cell cultures. Mol Cell Endocrinol, 1989, 66(1):119-122.
8. Gregor M, Allen S. M, Richard M. S,et al. Testicular Expression of Inhibin and Activin Subunits and Follistatin in the Rat and Human Fetus and Neonate and During Postnatal Development in the Rat.Endocrinology , 1997,138: 2136–2147.
9. C. Marchetti, M. Hamdane, V. Mitchell, K. Mayo, L,et al. Immunolocalization of Inhibin and ActivinαandβB Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis. Biology of reproduction, 2003,68, 230–235.
10. Andersson AM, Muller J & Skakkebaek NE. Different roles of prepubertal and post pubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels. J Clin Endocrinol Metab, 1998, 83(12):4 451-458.
11. Carolina J. Jorgez, Francesco J. De Mayo and Martin M. Matzuk.Inhibin a-iCre Mice: Cre Deleter Lines for the Gonads, Pituitary, and Adrenal. Genesis,2006,44:183–188.
12. Tena-Sempere M, Kero J, Rannikko A, et al. The pattern of inhibin/activin alpha- and beta (B)-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate. Endocrinol, 1999, 140(12):5761-5770.
13. Bohring C, Schroeder-Printzen I, Weidner W, et al. Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are undergoing testicular sperm extraction. Fertil Steril, 2002, 78(6):1 195-1198.
14. S J Meachem, E Nieschlag, M Simoni.Inhibin B in male reproduction: pathophysiology and clinical relevance. European Journal of Endocrinology 2001, 145:561-571.
15. von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesisin men, but cannot predict the presence of sperm in testicular tissue samples. Journal of Clinical Endocrinology and Metabolism 1999 84 2496-2501.
16. Bohring C, Krause W. Serum levels of inhibin B in men with different causes of spermatogenic failure. Andrologia 1999, 31:137-141.
17. de Gouveia Brazao CA, Pierik FH, Erenpreiss Y, et al. The effect of cryptorchidism on inhibin B in a subfertile population. Clin Endocrinol, 2003, 59(1):136-141.
18. Brian M. Gummow, Jonathon N. Winnay, and Gary D. Hammer.Convergence of Wnt Signaling and Steroidogenic Factor-1 (SF-1) on Transcription of the Rat InhibinαGene .the journal of biological chemistry. 2003,278(29): 26572–26579.
19. Anna D. Burkart, Abir Mukherjee, Esta Sterneck, Peter F. Johnson, and Kelly E. Mayo.Repression of the Inhibinα-Subunit Gene by the Transcription Factor CCAAT/Enhancer-Binding Protein-β. Endocrinology, 2005,146(4):1909–1921.
20. Anna D. Burkart, Abir Mukherjee, and Kelly E. Mayo .Mechanism of Repression of the Inhibinα-Subunit Gene by Inducible 3’,5’-Cyclic Adenosine Monophosphate Early Repressor. Molecular Endocrinology ,2006,20(3):584–597.
1. Pierik FH, Vreeburg JTM, Stijnen T, De Jong FH, Weber RFA. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998; 83:3110–3114.
2. von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired sperma- togenesisin men, but cannot predict the presence of sperm in testicular tissue samples. Journal of Clinical Endocrinology and Metabolism ,1999 ,84:2496-2501.
3. Bohring C,Krause W. Serum levels of inhibin B in men with different causes of spermatogenic failure. Andrologia 1999, 31:137-141.
4. C. Marchetti, M. Hamdane, V. Mitchell, K. Mayo, L,et al. Immunolocalization of Inhibin and Activin a andβB Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis. Biology of reproduction, 2003,68, 230–235.
5. Gregor M, Allan S. M, Richard M. S,et al. Testicular Expression of Inhibin and Activin Subunits and Follistatin in the Rat and Human Fetus and Neonate and During Postnatal Development in the Rat.Endocrinology , 1997,138: 2136–2147.
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7. Carolina J. Jorgez, Francesco J. De Mayo and Martin M. Matzuk.Inhibin a-iCre Mice: Cre Deleter Lines for the Gonads, Pituitary, and Adrenal. Genesis, 2006,44:183–188.
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11. Haisenleder, J C. Marshall. Production and actions of inhibin and activin during folliculogenesis in the rat.Molecular and Cellular Endocrinology , 2001, 180: 139– 144.
12. Feng Z-M, Wu AZ, Chen C-LC. Testicular GATA-1 factor upregulates the promoter activity of rat inhibin a-subunit gene in MA-10 Leydig tumor cells. Mol Endocrinol , 1998,12:378–390.
13. Yomogida K, Ohtani H, Harigae H, Ito E, Nishimune Y, Engel JD, Yamamoto M. Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells. Development ,1994,120: 1759–1766.
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9. Anna D. Burkart, Abir Mukherjee, and Kelly E. Mayo.Mechanism of Repression of the Inhibinα-Subunit Gene by Inducible 3’5’-Cyclic Adenosine Monophosphate Early Repressor. Molecular Endocrinology 2006,20(3):584–597.
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2. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J.Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature,1986,321:776–779.
3. Vale W, Rivier C, Hsueh A .Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res ,1988 ,44:1–34.
4. Haisenleder, J. C. Marshall. Production and actions of inhibin and activin during folliculogenesis in the rat.Molecular and Cellular Endocrinology , 2001,180:139– 144.
5. Halder A, Fauzdar A, Kumar A. Serum inhibin B and follicle-stimulating hormone levels as markers in the evaluation of azoospermic men. Andrologia. 2005 ,37(5): 173-179.
6. Cuevas P, Ying SY, Ling N, et al. Immunohistochemical detection of inhibin in the gonad. Biochemical and BiophysicalResearch Communications, 1987, 142(1): 23-30.
7. Risbridger GP, Clements J, Robertson DM, et al. Immunoandbioactive inhibin and inhibin alpha-subunit expression in rat Leydig cell cultures. Mol Cell Endocrinol, 1989, 66(1):119-122.
8. Gregor M, Allen S. M, Richard M. S,et al. Testicular Expression of Inhibin and Activin Subunits and Follistatin in the Rat and Human Fetus and Neonate and During Postnatal Development in the Rat.Endocrinology , 1997,138: 2136–2147.
9. C. Marchetti, M. Hamdane, V. Mitchell, K. Mayo, L,et al. Immunolocalization of Inhibin and ActivinαandβB Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis. Biology of reproduction, 2003,68, 230–235.
10. Andersson AM, Muller J & Skakkebaek NE. Different roles of prepubertal and post pubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels. J Clin Endocrinol Metab, 1998, 83(12):4 451-458.
11. Carolina J. Jorgez, Francesco J. De Mayo and Martin M. Matzuk.Inhibin a-iCre Mice: Cre Deleter Lines for the Gonads, Pituitary, and Adrenal. Genesis,2006,44:183–188.
12. Tena-Sempere M, Kero J, Rannikko A, et al. The pattern of inhibin/activin alpha- and beta (B)-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate. Endocrinol, 1999, 140(12):5761-5770.
13. Bohring C, Schroeder-Printzen I, Weidner W, et al. Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are undergoing testicular sperm extraction. Fertil Steril, 2002, 78(6):1 195-1198.
14. S J Meachem, E Nieschlag, M Simoni.Inhibin B in male reproduction: pathophysiology and clinical relevance. European Journal of Endocrinology 2001, 145:561-571.
15. von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesisin men, but cannot predict the presence of sperm in testicular tissue samples. Journal of Clinical Endocrinology and Metabolism 1999 84 2496-2501.
16. Bohring C, Krause W. Serum levels of inhibin B in men with different causes of spermatogenic failure. Andrologia 1999, 31:137-141.
17. de Gouveia Brazao CA, Pierik FH, Erenpreiss Y, et al. The effect of cryptorchidism on inhibin B in a subfertile population. Clin Endocrinol, 2003, 59(1):136-141.
18. Brian M. Gummow, Jonathon N. Winnay, and Gary D. Hammer.Convergence of Wnt Signaling and Steroidogenic Factor-1 (SF-1) on Transcription of the Rat InhibinαGene .the journal of biological chemistry. 2003,278(29): 26572–26579.
19. Anna D. Burkart, Abir Mukherjee, Esta Sterneck, Peter F. Johnson, and Kelly E. Mayo.Repression of the Inhibinα-Subunit Gene by the Transcription Factor CCAAT/Enhancer-Binding Protein-β. Endocrinology, 2005,146(4):1909–1921.
20. Anna D. Burkart, Abir Mukherjee, and Kelly E. Mayo .Mechanism of Repression of the Inhibinα-Subunit Gene by Inducible 3’,5’-Cyclic Adenosine Monophosphate Early Repressor. Molecular Endocrinology ,2006,20(3):584–597.
1. Pierik FH, Vreeburg JTM, Stijnen T, De Jong FH, Weber RFA. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998; 83:3110–3114.
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