17-β-雌二醇和孕酮对体外培养奶牛输卵管上皮细胞中环氧合酶表达影响
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摘要
环氧合酶(cyclooxygenase ,COX)是动物组织合成前列腺素(prostaglandin, PG)的限速酶,目前已知它存在两个同功酶(COX-1和COX-2)。许多对前列腺素的研究已经证实,前列腺素是哺乳动物体内作用极为广泛的局部激素,影响生殖过程的多个环节,在哺乳动物的排卵、受精、分娩、着床等生殖过程中发挥重要的作用,其中也涉及到对输卵管功能的调节。但是对于前列腺素(prostaglandin, PG)合成的研究主要还是集中在环氧合酶(cyclooxygenase ,COX)上,目前尚未有关于雌性动物输卵管上皮细胞的环氧合酶表达和雌激素、孕激素关系的研究报道。为阐明雌激素对输卵管上皮细胞中环氧合酶的表达调控机理,在本文应用实时荧光定量RT-PCR技术(real-time RT-PCR)和蛋白印迹技术(Western blot),采用体外培养奶牛输卵管上皮细胞(bovine oviduct epithelial cells,BOECs)并且建立传代细胞系的方法,运用体外方法研究了雌激素和孕激素对奶牛输卵管上皮细胞环氧合酶表达的影响。在体外培养的BOECs中,分不同浓度、不同时间添加17β-雌二醇、孕酮,研究BOECs中环氧合酶的表达量和雌激素、孕激素的关系。实验结果表明,添加不同浓度的17-β-雌二醇培养24 h,COX-1mRNA在100pg/ml(10-10g/mL)时表达量最高, COX-2 mRNA在100 pg/mL(10-10g/ml)时表达量达到最高值。添加不同浓度的孕酮诱导对体外培养的BOECs中COX-1、COX-2 mRNA转录,COX-1在1ng/ml(10-9 g/ml)时高表达, COX2 mRNA在100pg/ml(10-10g/ml)时高表达。结果与在奶牛的体内正常的雌、孕激素生理浓度相关。而添加10pg/mL的17-β-雌二醇、10ng/mL孕酮诱导诱导培养不同时间,对BOECs中COX-1、COX-2mRNA转录有影响,2h时表达量均较高。上述结果提示, COX-1和COX-2的表达受到雌、孕激素浓度的调节,而且17-β-雌二醇影响比孕酮明显, COX-2受的影响要比COX-1大,与COX-2是诱导型酶受诱导因子的影响较大的原因有关。
Cyclooxygenase (COX) is a rate-limiting enzyme for animal tissue produced prostaglandin. It has two currently known isoenzymes COX-1 and COX-2. Many research results show that prostaglandin is a local hormone performing a wide range of functions in mammals. It plays an important role in many stages of the birth process such as ovulation, fecundation, labor and rooting, etc., including the regulation of fallopian tube functions. Studies into the adjusting function of PG have mainly focused on COXs, and there has been no report about research on the interrelations between the COX expression of female oviduct epithelial cells, and estrogen and progesterone. In order to clarify how estrogen regulates COX expression of female animal oviduct epithelial cells, this research adopts the real-time RT-PCR and Western blot methods. BOECs are cultivated extra-corporally and method to establish the regenerating cells is found. Extra-corporal method is used to discover the impact of estrogen and progesterone on the COX expression of female animal oviduct epithelial cells. BOECs cultured in vitro are of different concentrations.
17β-estradiol and progesterone are treated at different times. The purpose is to find out the interrelation between the expressed amount of COX and estrogen as well as progesterone. Study results show adding 17β-estradiol of different concentrations, and after 24 hours of culturing, COX-1mRNA reaches the highest point in 100pg/ml(10-10g/ml). COX-2 mRNA reaches the highest point in 1000 pg/ml(10-9g/ml). Copying COX-1, COX-2 and mRNA in BOECs is affected by the time when 17β-estradiol is added. However, there is no obvious regularity regarding concentration of liquid. Adding progesterone of different concentrations can help copy COX-1, COX-2 and mRNA in BOECs. COX-1runs high in 1ng/ml(10-9 g/ml)while COX-2 mRNA in 10ng/ml. Statistics acquired from adding 10ng/mL progesterone at different times demonstrate less regularity than concentration. Study results show the expression of COX-1 and COX-2 are regulated by the concentration of estrogen and progesterone. And COX-1 is more seriously affected than COX-2. 17β-estradiol makes a greater change than progesterone. This is because COX-2 is more sensitive to inducing agents. As the amount of progesterone in the subject animal is regulated by estradiol, much of its power depends on estradiol.
Cyclooxygenase (COX) is a rate-limiting enzyme for animal tissue produced prostaglandin. It has two currently known isoenzymes COX-1 and COX-2. Many research results show that prostaglandin is a local hormone performing a wide range of functions in mammals. It plays an important role in many stages of the birth process such as ovulation, fecundation, labor and rooting, etc., including the regulation of fallopian tube functions. Studies into the adjusting function of PG have mainly focused on COXs, and there has been no report about research on the interrelations between the COX expression of female oviduct epithelial cells, and estrogen and progesterone. In order to clarify how estrogen regulates COX expression of female animal oviduct epithelial cells, this research adopts the real-time RT-PCR and Western blot methods. BOECs are cultivated extra-corporally and method to establish the regenerating cells is found. Extra-corporal method is used to discover the impact of estrogen and progesterone on the COX expression of female animal oviduct epithelial cells. BOECs cultured in vitro are of different concentrations.
17β-estradiol and progesterone are treated at different times. The purpose is to find out the interrelation between the expressed amount of COX and estrogen as well as progesterone. Study results show adding 17β-estradiol of different concentrations, and after 24 hours of culturing, COX-1mRNA reaches the highest point in 100pg/ml(10-10g/ml). COX-2 mRNA reaches the highest point in 1000 pg/ml(10-9g/ml). Copying COX-1, COX-2 and mRNA in BOECs is affected by the time when 17β-estradiol is added. However, there is no obvious regularity regarding concentration of liquid. Adding progesterone of different concentrations can help copy COX-1, COX-2 and mRNA in BOECs. COX-1runs high in 1ng/ml(10-9 g/ml)while COX-2 mRNA in 10ng/ml. Statistics acquired from adding 10ng/mL progesterone at different times demonstrate less regularity than concentration. Study results show the expression of COX-1 and COX-2 are regulated by the concentration of estrogen and progesterone. And COX-1 is more seriously affected than COX-2. 17β-estradiol makes a greater change than progesterone. This is because COX-2 is more sensitive to inducing agents. As the amount of progesterone in the subject animal is regulated by estradiol, much of its power depends on estradiol.
引文
1.杨增明孙青原夏国良主编生殖生物学[M]北京2005
2.朱辉杨增明环氧合酶的研究进展[J]生理科学进展2004,29(2):36-39
3.徐志斌环氧合酶-2抑制剂的设计与合成[D]中国协和医科大学,[博士研究生学位论文],2003年6月·北京
4.汪凤颖新型COX-2选择性抑制剂的分子设计研究[D] [硕士研究生学位论文],北京2005年5月
5. Vane J Rbert,Inhibition of prostaglandin synthesis a same chanism of action for a spirin-like drugs,Nature New Biology,1971,231(19),232-236
6.吕苗苗COX-2抑制剂塞来昔布合成方法的研究硕士学位论文北京2007年5月
7.倪华前列腺素E合成酶与小鼠胚胎着床和蜕膜化的关系东北农业大学博士学位论文哈尔滨2002年5月
8.刁红录COX-2在大鼠排卵着床和蜕膜化过程中的作用东北农业大学博士学位论文哈尔滨2006年5月
9.王亮发情周期中青年母牛卵巢、雌二醇和孕酮的变化规律硕士学位论文扬州2009年5月
10.感感保,周虚.动物繁殖学「M].吉林,吉林科学技术出版社,1999
11.感忠诚.家畜繁殖学(第四版) [M].北京,中国农业出版社,2004
12.赵兴绪.兽医产科学「M].北京,中国农业出版社,2002
13.刘莹前列腺素E合成酶-2在小鼠输卵管中的表达与调节[D] [硕士学位论文],东北农业大学:2005年5月
14.唐博蒙古绵羊雌性生殖道β-防御素(sBD-1)表达的研究[D] [内蒙古农业大学博士论文] ,呼和浩特:2007年5月
15.徐直蒋振国徐一树等黑白花奶牛发情周期血液中各种生殖激素含量及其变化天津农业科学[J]1998 ,10(4)
16.朱玉哲,夏成,感感友,徐闯荷斯坦泌乳牛情期体内生殖激素与生化指标关系的研究[J]中国牛业科学2008 Vol.35(4):35-39
17.唐博,李子义,锡林高娃,付本懂,曹贵方17-β-雌二醇对培养的绵羊输卵管上皮细胞β-防御素( sBD21) mRNA表达的影响[J]中国兽医学报2009,29(8)
18.陈洋赵杰感红琴周欢敏成年绵羊皮肤成纤维细胞系的建立[J]内蒙古农业大学学报2006,27,(2)
19.李淑凤曹贵方雌二醇对蒙古绵羊输卵管上皮细胞内β-防御素( sBD21)表达的影响[J]动物学杂志2008 ,43 (4) :41-47
20.唐博,白萨日娜,李淑凤,曹贵方蒙古绵羊输卵管上皮细胞培养体系的建立[J]中国兽医杂志2009 ,45 (8):
21.唐博孕酮和米非司酮对蒙古绵羊输卵管上皮细胞β-防御素mRNA表达的影响[J]动物学杂志2009 ,44 (6) :151-155
22.唐博孕酮对蒙古绵羊输卵管上皮细胞β-防御素mRNA表达的影响[J]动物学研究2009,Oct. 30(5): 497-502
23.赵晓娥小鼠输卵管上皮细胞的原代培养及纯化方法研究[J]动物医学进展,2009 ,30 (2) :30-34
24. Simone Odau, Christoph Gabler, Christoph Holder and Ralf Einspanier Differential expression of cyclooxygenase 1 and cyclooxygenase 2 in the bovine oviduct. Journal of Endocrinology [J],2006, 191, 263感274
25. S. Perez Martneza, M. Hermoso b, M. Farina 17β-Estradiol upregulates COX-2 in the rat oviduct Prostaglandins & other Lipid Mediators, [J],006,80: 155-164
26. Perez Martinez S, Viggiano JM, Franchi A, et al. Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. J Reprod Fertil [J], 2000;118:11-17.
27. Morita I Distinct functions of COX-1 and COX-2. Prostaglandins and Other Lipid Mediators [J], 2002 ,(68感69) :165感175.
28. Wollenhaupt K, Tomek W, Brussow KP, et al. Effects of ovarian steroids and epidermal growth factor (EGF) on expression and bioactivation of specic regulators of transcription and translation in oviductal tissue in pigs. Reproduction [J] ,2002,123(1):87-96.
29. S. P感erez Mart感?nez 17β-Estradiol upregulates COX-2 in the rat oviduct Prostaglandins & other Lipid Mediators Chile [J] ,2006, 80:155感164
30. Perez Martinez S, Franchi A, Viggiano JM, Herrero MB, Gimeno M. Effect of prostaglandin F2 on oviductal nitric oxide synthase activity:possible role of endogenous NO on PG F2-induced contractions in rat oviduct. Prostaglandins Other Lipid Mediat[J] 1998;56: 155-66.
31. Akarasereenont P, Techatraisak K, Thaworn A, Chotewuttakorn S. The induction of cyclooxygenase-2 by 17beta-estradiol in endothelial cells is mediated through protein kinase C. Inamm Res [J]2000;49(9):460-5.
32. Perez Martinez S, Viggiano JM, Franchi A, et al. Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. J Reprod Fertil [J]2000;118:11-17.
33. Hermoso M, Barrera N, Morales B, Perez S, Villal感on M. Platelet activatingfactor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2. Pugers Arch[J] 2001;442:336-45.
34. Wijayagunawardane MP, Miyamoto A. Tumor necrosis factor alpha system in the bovine oviduct: a possible mechanism for embryo transport.J Reprod Dev[J] 2004;50(1):57-62.
35. Arosh JA, Parent J, Chapdelaine P, Sirois J Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biology of Reproduction [J]2002,67 :161感169.
36. possible involvement in the regulation of the oviductal motility and embryo transport. Mol Reprod Dev [J] 2005,72(5)11-20.
37. Gimeno MF, Franchi AM, Chaud M, Gonz感alez ET, Viggiano M, Gimeno AL. Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts. Prostaglandins [J]1984;27: 737-52.
38. Gimeno MF, Franchi AM, Sterin-Speziale NB, Gonz感alez ET, Speziale EH, Gimeno AL. Sow (Sus scrofa) follicular uid: prostaglandin content and effect on the motility of isolated oviducts Prostaglandins [J]1985,29:831-46.
39. Wijayagunawardane MP, Miyamoto A, Cerbito WA, Acosta TJ, Takagi M &Sato K Local distributions of oviductal estradiol, progesterone,prostaglandins, oxytocin and endothelin-1 in the cyclic cow. Theriogenology [J],1998, 49:607-618.
40. Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques[J] 2000,29:332-337.
41. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol[J],2002,29:23-39.
42. Bas A, Hammarstrom SG, Hammarstrom ML. Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression. J Immunol[J],2003,171:3359感3371.
43. Fahlgren A, Hammarstrom S, Danielsson A,.Increased expression of antimicrobial peptides and lysozyme incolonic epithelial cells of patients with ulcerative colitis. Clin Exp Immunol[J],2003,131:90感101.
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2.朱辉杨增明环氧合酶的研究进展[J]生理科学进展2004,29(2):36-39
3.徐志斌环氧合酶-2抑制剂的设计与合成[D]中国协和医科大学,[博士研究生学位论文],2003年6月·北京
4.汪凤颖新型COX-2选择性抑制剂的分子设计研究[D] [硕士研究生学位论文],北京2005年5月
5. Vane J Rbert,Inhibition of prostaglandin synthesis a same chanism of action for a spirin-like drugs,Nature New Biology,1971,231(19),232-236
6.吕苗苗COX-2抑制剂塞来昔布合成方法的研究硕士学位论文北京2007年5月
7.倪华前列腺素E合成酶与小鼠胚胎着床和蜕膜化的关系东北农业大学博士学位论文哈尔滨2002年5月
8.刁红录COX-2在大鼠排卵着床和蜕膜化过程中的作用东北农业大学博士学位论文哈尔滨2006年5月
9.王亮发情周期中青年母牛卵巢、雌二醇和孕酮的变化规律硕士学位论文扬州2009年5月
10.感感保,周虚.动物繁殖学「M].吉林,吉林科学技术出版社,1999
11.感忠诚.家畜繁殖学(第四版) [M].北京,中国农业出版社,2004
12.赵兴绪.兽医产科学「M].北京,中国农业出版社,2002
13.刘莹前列腺素E合成酶-2在小鼠输卵管中的表达与调节[D] [硕士学位论文],东北农业大学:2005年5月
14.唐博蒙古绵羊雌性生殖道β-防御素(sBD-1)表达的研究[D] [内蒙古农业大学博士论文] ,呼和浩特:2007年5月
15.徐直蒋振国徐一树等黑白花奶牛发情周期血液中各种生殖激素含量及其变化天津农业科学[J]1998 ,10(4)
16.朱玉哲,夏成,感感友,徐闯荷斯坦泌乳牛情期体内生殖激素与生化指标关系的研究[J]中国牛业科学2008 Vol.35(4):35-39
17.唐博,李子义,锡林高娃,付本懂,曹贵方17-β-雌二醇对培养的绵羊输卵管上皮细胞β-防御素( sBD21) mRNA表达的影响[J]中国兽医学报2009,29(8)
18.陈洋赵杰感红琴周欢敏成年绵羊皮肤成纤维细胞系的建立[J]内蒙古农业大学学报2006,27,(2)
19.李淑凤曹贵方雌二醇对蒙古绵羊输卵管上皮细胞内β-防御素( sBD21)表达的影响[J]动物学杂志2008 ,43 (4) :41-47
20.唐博,白萨日娜,李淑凤,曹贵方蒙古绵羊输卵管上皮细胞培养体系的建立[J]中国兽医杂志2009 ,45 (8):
21.唐博孕酮和米非司酮对蒙古绵羊输卵管上皮细胞β-防御素mRNA表达的影响[J]动物学杂志2009 ,44 (6) :151-155
22.唐博孕酮对蒙古绵羊输卵管上皮细胞β-防御素mRNA表达的影响[J]动物学研究2009,Oct. 30(5): 497-502
23.赵晓娥小鼠输卵管上皮细胞的原代培养及纯化方法研究[J]动物医学进展,2009 ,30 (2) :30-34
24. Simone Odau, Christoph Gabler, Christoph Holder and Ralf Einspanier Differential expression of cyclooxygenase 1 and cyclooxygenase 2 in the bovine oviduct. Journal of Endocrinology [J],2006, 191, 263感274
25. S. Perez Martneza, M. Hermoso b, M. Farina 17β-Estradiol upregulates COX-2 in the rat oviduct Prostaglandins & other Lipid Mediators, [J],006,80: 155-164
26. Perez Martinez S, Viggiano JM, Franchi A, et al. Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. J Reprod Fertil [J], 2000;118:11-17.
27. Morita I Distinct functions of COX-1 and COX-2. Prostaglandins and Other Lipid Mediators [J], 2002 ,(68感69) :165感175.
28. Wollenhaupt K, Tomek W, Brussow KP, et al. Effects of ovarian steroids and epidermal growth factor (EGF) on expression and bioactivation of specic regulators of transcription and translation in oviductal tissue in pigs. Reproduction [J] ,2002,123(1):87-96.
29. S. P感erez Mart感?nez 17β-Estradiol upregulates COX-2 in the rat oviduct Prostaglandins & other Lipid Mediators Chile [J] ,2006, 80:155感164
30. Perez Martinez S, Franchi A, Viggiano JM, Herrero MB, Gimeno M. Effect of prostaglandin F2 on oviductal nitric oxide synthase activity:possible role of endogenous NO on PG F2-induced contractions in rat oviduct. Prostaglandins Other Lipid Mediat[J] 1998;56: 155-66.
31. Akarasereenont P, Techatraisak K, Thaworn A, Chotewuttakorn S. The induction of cyclooxygenase-2 by 17beta-estradiol in endothelial cells is mediated through protein kinase C. Inamm Res [J]2000;49(9):460-5.
32. Perez Martinez S, Viggiano JM, Franchi A, et al. Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. J Reprod Fertil [J]2000;118:11-17.
33. Hermoso M, Barrera N, Morales B, Perez S, Villal感on M. Platelet activatingfactor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2. Pugers Arch[J] 2001;442:336-45.
34. Wijayagunawardane MP, Miyamoto A. Tumor necrosis factor alpha system in the bovine oviduct: a possible mechanism for embryo transport.J Reprod Dev[J] 2004;50(1):57-62.
35. Arosh JA, Parent J, Chapdelaine P, Sirois J Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biology of Reproduction [J]2002,67 :161感169.
36. possible involvement in the regulation of the oviductal motility and embryo transport. Mol Reprod Dev [J] 2005,72(5)11-20.
37. Gimeno MF, Franchi AM, Chaud M, Gonz感alez ET, Viggiano M, Gimeno AL. Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts. Prostaglandins [J]1984;27: 737-52.
38. Gimeno MF, Franchi AM, Sterin-Speziale NB, Gonz感alez ET, Speziale EH, Gimeno AL. Sow (Sus scrofa) follicular uid: prostaglandin content and effect on the motility of isolated oviducts Prostaglandins [J]1985,29:831-46.
39. Wijayagunawardane MP, Miyamoto A, Cerbito WA, Acosta TJ, Takagi M &Sato K Local distributions of oviductal estradiol, progesterone,prostaglandins, oxytocin and endothelin-1 in the cyclic cow. Theriogenology [J],1998, 49:607-618.
40. Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques[J] 2000,29:332-337.
41. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol[J],2002,29:23-39.
42. Bas A, Hammarstrom SG, Hammarstrom ML. Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression. J Immunol[J],2003,171:3359感3371.
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