子宫内膜中NO含量与功能失调性子宫出血的关系
摘要
目的
本研究通过测定功血患者和正常人子宫内膜组织和外周血中一氧化氮(NO)的含量来探讨NO在子宫出血中可能起到的作用，以及NO与功能失调性子宫出血的关系。
材料与方法
1．材料
子宫内膜标本实验组采集2002年10月至2003年12月就诊于中国医科大学附属第一临床学院的功能失调性子宫出血(功血)患者的子宫内膜标本共60例，其中增生期子宫内膜34例，子宫内膜单纯型增生16例，子宫内膜复杂型增生10例；对照组为同期采集的正常的增生期子宫内膜组织标本19例。血清实验组标本是与内膜组同期采集的功血患者的空腹静脉血25例，对照组采集的是2003年8月到中国医科大学附属第一临床学院体检中心体检的健康、未绝经妇女的空腹静脉血32例。要求两组实验对象为未绝经成年妇女，3个月内未接受过激素替代疗法、未服用过避孕药，2周内未食用腌菜等富含NO_x~-丰富的食物，并且要求取内膜组织的子宫无宫内节育器。
2．实验方法
2．1 标本的采集和处理：
采患者(或正常人)的子宫内膜组织100mg加入1ml生理盐水，用匀浆机搅碎内膜组织制成混浊液，用超声粉碎仪破坏细胞膜，以10000转／分的速度离心10分钟取上清液保存于-70℃的冰箱中，待测NO_2~-／NO_3~-的含量。采患者(或正常人)的肘静脉血5ml在试管中静置3小时后离心取上清，保存于-70℃的冰箱中，待测NO_2~-／NO_3~-的含量。
2．2 检测方法
患者或正常人的子宫内膜和外周血中NO的测定用NO_2~-／NO_3~-的含量来反应，采用改良的镀铜镉粒还原法测定，内膜组同时测定标本中蛋白的
浓度，以蛋白的浓度作为标准来反应NO的含量，内膜组NO的单位为
nmoFmsPr;血清组NO的单位为卜moFL。
2.3统计分析
应用SSPS 12 .0软件进行两个独立样本的T一检验分析。
结果
1.功血患者子宫内膜组织中NO含量明显高于正常人子宫内膜组织
中NO含量，二者之间的差异有显著性意义(p<0.05)。
2.功血患者外周血中NO的含量与正常人外周血中NO的含量没有明
显差异(P>0.05)。
3.功血患者子宫内膜各种病理类型间两两比较都没有明显的差异(P
>0·05)。
结论
功血患者子宫内膜组织中NO含量明显高于正常人，二者之间的差异
具有统计学意义(p<0.05)，而功血患者体内NO含量在外周血中的增高并
不明显(p>0.05)，提示No是通过改变子宫局部微环境来影响子宫出血
的，子宫内膜组织中NO含量的增高可导致子宫出血时间过长或流血过多。
功血患者子宫内膜各种病理类型间两两比较都没有明显的差异(p>
0.05)，提示子宫内膜的病理类型与NO含量无密切关系。
Objective
To explore the relation between nitric oxide (NO) and disfunctional uterine bleeding ( DUB) , we determined the NO in endometrium and blood.
Material and Method
Material
All tissue samples and blood samples were collected from women undergoing a gynaecological operation at the first clinical hospital China Medical University dating from Oct. 2002 to Dec. 2003. Two groups of women participated in the study of endometrium: 1) study group: 60 women, of all these tissue samples 34 of them are proliferative phase endometrium, 16 of them are simple hy-perplasia, 10 of them are complex hyperplasia; 2) control group: 19 women. Two groups of women participated in the study of blood, too: 1) study group: 25 women; 2) control group: 32 women who were testified healthy in this hospital. All the women of control groups participated in the study were adults with normal mestrual cycles. All the women recruited to the study had not been exposed to steroid for three months prior to operation, did not have either an intra-uterine contraceptive device (IUD) in situ or take too much food full of NOX-.
Experiment method
Endometrium samples were taken 100mg from every sample mixed with 1ml 0. 9% natricholoridi by machine and the cell membrane were destroyed by ultrasonic , the mixtures were centrifuged at 10000 rpm for 10min. The upper liquid layers were stored at -70 C until use. Blood samples were taken from women' s vein who were empty. The blood samples were left untouched for 3 hours, the
upper layers were stored at -70C until use.
Experiment Method
We determined the concentration of N02-/N03- in the endometrium and blood samples that have been worked to reflect the concentration of NO by improved cuprum electroplated cadmium method. At the same time we determined the concentration of protein in the endometrium. The concentration of NO in the endometrium is expressed by nmol/mgPr as unit; the concentration of NO in blood is expressed by mol/L as unit.
Statistical Analysis
Independent - samples T test, SSPS 12. 0.
Result
The NO amounts in endometrial samples of study group (n = 60) were higher than those of the corresponding controls (n = 19) (p <0. 05) .
The NO amounts in blood samples of study group ( n = 25 ) were no higher than that of the corresponding controls ( n = 32) , but there is not significant difference between them ( p > 0. 05 ) .
There was not significant difference between any two groups of different pathological types of the three groups in the study group of endometrium ( p > 0.05).
Conclusion
The NO amounts in endometrial samples of disfunctional uterine bleeding group (n = 60) were higher than the corresponding controls (n = 19) (p< 0.05). Their difference have no significant means. The NO amounts in blood samples of disfunctional uterine bleeding group ( n = 25 ) were no higher than those of the corresponding controls ( n = 32) , but there is not significant difference between them (p >0. 05). This indicates that NO affect the uterine bleeding is by influence the local microcircumstance of uterine, the highter NO a-mounts lead to persistent bleeding of uterine or menorrhagia. There was not sig-
nificant difference between any two groups of different pathological types of the three groups in the study group of endometrium ( p > 0.05 ). This suggests that there is no closed relation between the NO amounts and pathological type of endometrium.
本研究通过测定功血患者和正常人子宫内膜组织和外周血中一氧化氮(NO)的含量来探讨NO在子宫出血中可能起到的作用，以及NO与功能失调性子宫出血的关系。
材料与方法
1．材料
子宫内膜标本实验组采集2002年10月至2003年12月就诊于中国医科大学附属第一临床学院的功能失调性子宫出血(功血)患者的子宫内膜标本共60例，其中增生期子宫内膜34例，子宫内膜单纯型增生16例，子宫内膜复杂型增生10例；对照组为同期采集的正常的增生期子宫内膜组织标本19例。血清实验组标本是与内膜组同期采集的功血患者的空腹静脉血25例，对照组采集的是2003年8月到中国医科大学附属第一临床学院体检中心体检的健康、未绝经妇女的空腹静脉血32例。要求两组实验对象为未绝经成年妇女，3个月内未接受过激素替代疗法、未服用过避孕药，2周内未食用腌菜等富含NO_x~-丰富的食物，并且要求取内膜组织的子宫无宫内节育器。
2．实验方法
2．1 标本的采集和处理：
采患者(或正常人)的子宫内膜组织100mg加入1ml生理盐水，用匀浆机搅碎内膜组织制成混浊液，用超声粉碎仪破坏细胞膜，以10000转／分的速度离心10分钟取上清液保存于-70℃的冰箱中，待测NO_2~-／NO_3~-的含量。采患者(或正常人)的肘静脉血5ml在试管中静置3小时后离心取上清，保存于-70℃的冰箱中，待测NO_2~-／NO_3~-的含量。
2．2 检测方法
患者或正常人的子宫内膜和外周血中NO的测定用NO_2~-／NO_3~-的含量来反应，采用改良的镀铜镉粒还原法测定，内膜组同时测定标本中蛋白的
浓度，以蛋白的浓度作为标准来反应NO的含量，内膜组NO的单位为
nmoFmsPr;血清组NO的单位为卜moFL。
2.3统计分析
应用SSPS 12 .0软件进行两个独立样本的T一检验分析。
结果
1.功血患者子宫内膜组织中NO含量明显高于正常人子宫内膜组织
中NO含量，二者之间的差异有显著性意义(p<0.05)。
2.功血患者外周血中NO的含量与正常人外周血中NO的含量没有明
显差异(P>0.05)。
3.功血患者子宫内膜各种病理类型间两两比较都没有明显的差异(P
>0·05)。
结论
功血患者子宫内膜组织中NO含量明显高于正常人，二者之间的差异
具有统计学意义(p<0.05)，而功血患者体内NO含量在外周血中的增高并
不明显(p>0.05)，提示No是通过改变子宫局部微环境来影响子宫出血
的，子宫内膜组织中NO含量的增高可导致子宫出血时间过长或流血过多。
功血患者子宫内膜各种病理类型间两两比较都没有明显的差异(p>
0.05)，提示子宫内膜的病理类型与NO含量无密切关系。
Objective
To explore the relation between nitric oxide (NO) and disfunctional uterine bleeding ( DUB) , we determined the NO in endometrium and blood.
Material and Method
Material
All tissue samples and blood samples were collected from women undergoing a gynaecological operation at the first clinical hospital China Medical University dating from Oct. 2002 to Dec. 2003. Two groups of women participated in the study of endometrium: 1) study group: 60 women, of all these tissue samples 34 of them are proliferative phase endometrium, 16 of them are simple hy-perplasia, 10 of them are complex hyperplasia; 2) control group: 19 women. Two groups of women participated in the study of blood, too: 1) study group: 25 women; 2) control group: 32 women who were testified healthy in this hospital. All the women of control groups participated in the study were adults with normal mestrual cycles. All the women recruited to the study had not been exposed to steroid for three months prior to operation, did not have either an intra-uterine contraceptive device (IUD) in situ or take too much food full of NOX-.
Experiment method
Endometrium samples were taken 100mg from every sample mixed with 1ml 0. 9% natricholoridi by machine and the cell membrane were destroyed by ultrasonic , the mixtures were centrifuged at 10000 rpm for 10min. The upper liquid layers were stored at -70 C until use. Blood samples were taken from women' s vein who were empty. The blood samples were left untouched for 3 hours, the
upper layers were stored at -70C until use.
Experiment Method
We determined the concentration of N02-/N03- in the endometrium and blood samples that have been worked to reflect the concentration of NO by improved cuprum electroplated cadmium method. At the same time we determined the concentration of protein in the endometrium. The concentration of NO in the endometrium is expressed by nmol/mgPr as unit; the concentration of NO in blood is expressed by mol/L as unit.
Statistical Analysis
Independent - samples T test, SSPS 12. 0.
Result
The NO amounts in endometrial samples of study group (n = 60) were higher than those of the corresponding controls (n = 19) (p <0. 05) .
The NO amounts in blood samples of study group ( n = 25 ) were no higher than that of the corresponding controls ( n = 32) , but there is not significant difference between them ( p > 0. 05 ) .
There was not significant difference between any two groups of different pathological types of the three groups in the study group of endometrium ( p > 0.05).
Conclusion
The NO amounts in endometrial samples of disfunctional uterine bleeding group (n = 60) were higher than the corresponding controls (n = 19) (p< 0.05). Their difference have no significant means. The NO amounts in blood samples of disfunctional uterine bleeding group ( n = 25 ) were no higher than those of the corresponding controls ( n = 32) , but there is not significant difference between them (p >0. 05). This indicates that NO affect the uterine bleeding is by influence the local microcircumstance of uterine, the highter NO a-mounts lead to persistent bleeding of uterine or menorrhagia. There was not sig-
nificant difference between any two groups of different pathological types of the three groups in the study group of endometrium ( p > 0.05 ). This suggests that there is no closed relation between the NO amounts and pathological type of endometrium.
引文
1. Lgmiro LT; Buga Gm; Wood ks er al. Proc Natll Acad Sd 1987, 84; 9265.
2. Palmer RM; Ferrige AG; Moncadas. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987, 327: 524.
3. Moncada s; Pamer; Rm J; Higgs EA. Nitric oxide : Physiology pathophysiology and pharmacology. Pharmacological Review 1991, 43: 109-42.
4. Buhimschi I, Ali M, Jain V, et al. Differential regulation of nitric oxide in the rat uterus and cervix during pregnancy and labor. Hum Rep, 1996, 11: 1755-66.
5. Yallampalli C, Izumi H, et al. An L-argingine-nitric oxide-cyclic guanosine monophsophate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol, 1994, 170: 175-85.
6. Telfer JF, Lyall F, et al. Identification of nitric oxide synthase in human uterus. Hum reprod. 1995,10: 19-23.
7. Trudy L. Cornwell, Jie Li, et al. Regulation of cyclic guansine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle. Biology of Reproduction 64, 857-864(2001).
8. Lincoln TM, Comwell TL. Intracellular cyclic GMP receptor proteins. FASEB J 1993; 7: 328-338.
9. Francis S, Corbin JD. Cyclic nucleolide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 1999; 36: 275-328. ~
10. Cornwell TL, Lincoln TM. Regulation of intracellular Ca_(2+) levels by atriopeotin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem 1989; 264: 1146-1155.
11. Maul H, Longo M, et al. Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Des 2003, 9(5): 359-80.
12. Cameron IT, Campbell S. Nitric oxide in the endometrium. Hum Reprod Update 1998 Sep-Oct; (5): 565-9.
13. O. Khorram, M. Garthwaite, et al. Endometrial and myometrial expression of nitric oxide synthase isoforms in pre-and postmenopausal woman. J Clin Endo Meta, 1999, 84(6): 2226-32.
14. Tseng L, Zang J, et al. Cyclic expression of endothelial nitric oxide synthase mRNA in the epithelial glands of human endometrium. J Soc Gynecol Investig 1996 Jan-Feb; 3(1): 33-8.
15. Zervous S, Klentzeris LD, Old RW. Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia. Mol Hum Reprod, 1999 Nov; 5(11): 1048-54.
16. Tschugguel W, Schneeberger C, Unfried G, et al. Elevation of inducible nitric oxide synthase activity in human endometrium during menstruation. Biol Reprod, 1999, 60(2): 297-304.
17. Tchugguel W, Schneeberger C, et al. Induction of inducible nitric oxide synthase expression in human secretory endometrium. Hum Reprod, 1998;May; 13(5):1414.
18. Daniele Bani, Maria Caterina Baccari, et al. Relaxin up-regulates the nitric oxide biosynthetic pathway in the mouse uterus: involvement in the inhibirtion if myometrial contractility. Endocrinology Vol. 140, No. 10 4434-41.
19. Word RA, Cornwell TL. Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy. Am J Physiol 1998; 274: C748-C756.
20. Heidi L. Rupnow, Terrance M, et al. Endothelial vasodilator production by uterine and systemic arteries. Ⅶ. Estrogen and progesterone effects on eNOS. Am J Physiol Heart Circ Physiol 280: H1699-H1705, 2001; Vol.280, Issue 4, H1699-1750, April 2001.
2. Palmer RM; Ferrige AG; Moncadas. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987, 327: 524.
3. Moncada s; Pamer; Rm J; Higgs EA. Nitric oxide : Physiology pathophysiology and pharmacology. Pharmacological Review 1991, 43: 109-42.
4. Buhimschi I, Ali M, Jain V, et al. Differential regulation of nitric oxide in the rat uterus and cervix during pregnancy and labor. Hum Rep, 1996, 11: 1755-66.
5. Yallampalli C, Izumi H, et al. An L-argingine-nitric oxide-cyclic guanosine monophsophate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol, 1994, 170: 175-85.
6. Telfer JF, Lyall F, et al. Identification of nitric oxide synthase in human uterus. Hum reprod. 1995,10: 19-23.
7. Trudy L. Cornwell, Jie Li, et al. Regulation of cyclic guansine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle. Biology of Reproduction 64, 857-864(2001).
8. Lincoln TM, Comwell TL. Intracellular cyclic GMP receptor proteins. FASEB J 1993; 7: 328-338.
9. Francis S, Corbin JD. Cyclic nucleolide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 1999; 36: 275-328. ~
10. Cornwell TL, Lincoln TM. Regulation of intracellular Ca_(2+) levels by atriopeotin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem 1989; 264: 1146-1155.
11. Maul H, Longo M, et al. Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Des 2003, 9(5): 359-80.
12. Cameron IT, Campbell S. Nitric oxide in the endometrium. Hum Reprod Update 1998 Sep-Oct; (5): 565-9.
13. O. Khorram, M. Garthwaite, et al. Endometrial and myometrial expression of nitric oxide synthase isoforms in pre-and postmenopausal woman. J Clin Endo Meta, 1999, 84(6): 2226-32.
14. Tseng L, Zang J, et al. Cyclic expression of endothelial nitric oxide synthase mRNA in the epithelial glands of human endometrium. J Soc Gynecol Investig 1996 Jan-Feb; 3(1): 33-8.
15. Zervous S, Klentzeris LD, Old RW. Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia. Mol Hum Reprod, 1999 Nov; 5(11): 1048-54.
16. Tschugguel W, Schneeberger C, Unfried G, et al. Elevation of inducible nitric oxide synthase activity in human endometrium during menstruation. Biol Reprod, 1999, 60(2): 297-304.
17. Tchugguel W, Schneeberger C, et al. Induction of inducible nitric oxide synthase expression in human secretory endometrium. Hum Reprod, 1998;May; 13(5):1414.
18. Daniele Bani, Maria Caterina Baccari, et al. Relaxin up-regulates the nitric oxide biosynthetic pathway in the mouse uterus: involvement in the inhibirtion if myometrial contractility. Endocrinology Vol. 140, No. 10 4434-41.
19. Word RA, Cornwell TL. Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy. Am J Physiol 1998; 274: C748-C756.
20. Heidi L. Rupnow, Terrance M, et al. Endothelial vasodilator production by uterine and systemic arteries. Ⅶ. Estrogen and progesterone effects on eNOS. Am J Physiol Heart Circ Physiol 280: H1699-H1705, 2001; Vol.280, Issue 4, H1699-1750, April 2001.