妊娠肝内胆汁淤积症NO/ET、PGI_2/TXA_2测定与表达的初步研究
|
摘要
妊娠肝内胆汁淤积症（intrahepatic cholestasis of pregnancy，ICP）是一种妊娠特发性肝脏疾病，主要发生于妊娠中晚期，以皮肤瘙痒、黄疸、血清胆汁酸升高、肝功轻至中度异常为主要特点，发病具有明显的地域性和季节性差异。ICP对孕妇的影响较轻，皮肤瘙痒和生化异常多于产后2-4周自行恢复，未见孕妇死亡的报道。其主要危害在胎儿，可造成羊水粪染、早产、胎儿窘迫和围生儿死亡等多种不良结局，由于发病机制不清，难以采取有效的监护手段预测胎儿的宫内状况。
胎盘作为母胎连接的桥梁和胎儿营养供应的唯一中介，其功能状态与胎儿发育密切相关，胎盘血流量的调节更是其中的中心环节，在胎儿的成长过程中具有不可替代的重要作用。由于胎盘是一个无神经支配的器官，局部分泌的血管舒缩激素水平可通过内分泌和旁分泌途径直接影响胎盘血管的舒缩和血流量的调节，与胎儿的营养供应和生长发育密切相关。在胎盘分泌的血管舒缩激素中，一氧化氮（nitric oxide，NO）/内皮素（endothlin，ET）、以及前列环素（prostacyclin，PGI2）/血栓烷A2（thromboxane A2，TXA2）是具有代表性的成员，其功能紊乱可导致胎盘血流量异常，是妊高症、早产、IUGR等多种母婴并发症的发病原因之一。
为研究母体血清、脐血清、胎盘NO/ET、PGI2/ TXA2含量变化和ICP围生儿不良结局之间的关系，本实验通过建立ICP大鼠动物模型，采用放射免疫、免疫组化、半定量反转录PCR等方法，测定ICP大鼠和部分ICP患者血清、脐血清以及胎盘NO/ET、PGI2/ TXA2的表达变化，结果如下：
1. 皮下注射EE可成功建立大鼠ICP模型，动物出现血清生化改变和胆酸水平升高，电镜下病变肝脏可见胆小管扩张、线粒体变性等形态学改变，同时孕鼠的胆汁淤积可累及胎鼠肝脏，即ICP大鼠的子代也存在肝内胆汁淤积。
2. ICP孕妇血清舒缩因子与对照组相比无明显变化，但脐血清NO水平下降、ET水平显著升高，与对照组相比，有极显著性差异，表明ICP患者存在胎盘、脐带局部的血管舒缩激素失衡，并可能为ICP胎儿并发症的原因之一。ICP孕妇血清ALP和TBiL与胎儿体重呈显著负相关，可部分反映胎儿的宫内状况。
3. ICP患者胎盘存在eNOS、iNOS和ET表达异常，其中eNOS、iNOS表达显著下降，而ET表达水平则明显上升，与脐血NO、ET水平的变化一致，可能是脐血清
血管舒缩激素异常的重要原因之一。
本实验结果表明：EE可建立稳定的大鼠ICP动物模型；ICP患者胎盘存在一定程度的内分泌功能紊乱，并导致了胎儿-胎盘局部血管阻力的异常升高，为探讨ICP母儿并发症的发生机制增添了一个新的研究途径。ICP导致血管舒缩因子分泌失调的具体机制尚不明确，是否与母体内胆汁酸浓度异常升高有关、以及NO、ET的分泌异常与母儿结局的关系仍需进一步研究。
Intrahepatic cholestasis of pregnancy(ICP) ,or obstetric cholestasis,is a liver disease characterized by generalized skin pruritus with an increase in serum bile acids that appears during the second half of pregnancy ,lasts until the end of gestation,and disappears a few days after delivery.A mild abnormality in alanine aminotransferase may occur. There is considerable geographical and seasonal variation in the incidence of ICP. In the viewpoint of maternity, it is benign except that pruritus is severe. In the fetus, however, the course is not the case. The disease is closely related to a high incidence of perinatal complications, including an increased fetal distress and preterm delivery, a higher incidence of meconium passage and an increased perinatal mortality rate. Unfortunately,conventional fetal surveillance ,such as weekly nonstress tests and amniotic fluid volume assessment lack the ability to predict impending fetal demise in this condition.
Some reports showed perinatal mortality ranges from 11%-20% in ICP women when untreated. In a review of published cases before 1988,the reported still birth rate in women of cholestasis was 10 of 1000 births, meconium staining in 27%, fetal distress occurred in 18% cases.The pathogenesis of fetal complicatioons has not been fully determined.
As the singulare source of fetal oxygen and nutrition ,the role of placenta is very important in fetal growth and development. Many studies of the mechanism of fetal asphyxia in ICP have been performed in the aspects of placenta. Bile acid and meconium could result in umbilical vessels and placental chorionic veins constriction which may aggrevate fetal hypoxia. It has been reported that different kinds of vasoactive substances can be secreted by human placenta trophoblast, which took part in controlling blood flow by autocrine, paracrine and endocrine pathway.
In order to explore the pathogenesis of fetal distress in , we measured the levels of nitric oxide /endothelin, prostacylin /thromboxane A2 in serum which drawn from ICP rat and ICP patients as well as umbilical vein. The expression of endothlin nitric oxide synthase, inducible nitric oxide synthase and endothelin in human placenta were investigated used immunohistochemical method. The semi-quantative reverse polymerase chain reaction were
used to investigated the expression of induced nitric oxide synthase in human placenta.
The following conclusion may be achieved in this study:
1.ICP rats model may be successfully established by treated with 17-а-ethinylestradiol(EE), inject subcutaneously for five days, 2.5mg/kg/day. Compared with control group,the serum levels of ALP and ALT in EE group increased significantly. There was no marked change in hepatocytes of EE group under hematoxylin-eosin staining and the same as bile canaliculi under silver nitrate staining. In hepatocyte of EE group, mitochondria swelling and bile canaliculi dilated under electron microscope. The same ultrastructure changes have been seen in hepatocyte of their fetal rats.Correspondingly, body weight and live birth of the fetal rats in EE group were lighter and shorter than those of control group significantly.
2.Compared to control group,the values of vasoactive substances in serum of ICP rats and ICP patients were not significantly different, except that the levels of NO in ICP rats was significantly higher than those of control rats,which is probably resulted from the application of estrogens.But in serum of umbilical blood of ICP patients, the values of NO decreased and ET increased significantly than those of normal pregnancy, which may effect the regulation of placental blood and may be one of the causes for human placenta dysfunction and high impedance of fetoplacental circulation. In addition, the relationship between levels of ALP and TBiL in serum of ICP patients and fetal weight were significantly negative, which indicated ALP and TBiL may be useful in the prediction of fetal conditions.
3. The expression of inducible and endothlin nitric oxide synthase as well as t
胎盘作为母胎连接的桥梁和胎儿营养供应的唯一中介，其功能状态与胎儿发育密切相关，胎盘血流量的调节更是其中的中心环节，在胎儿的成长过程中具有不可替代的重要作用。由于胎盘是一个无神经支配的器官，局部分泌的血管舒缩激素水平可通过内分泌和旁分泌途径直接影响胎盘血管的舒缩和血流量的调节，与胎儿的营养供应和生长发育密切相关。在胎盘分泌的血管舒缩激素中，一氧化氮（nitric oxide，NO）/内皮素（endothlin，ET）、以及前列环素（prostacyclin，PGI2）/血栓烷A2（thromboxane A2，TXA2）是具有代表性的成员，其功能紊乱可导致胎盘血流量异常，是妊高症、早产、IUGR等多种母婴并发症的发病原因之一。
为研究母体血清、脐血清、胎盘NO/ET、PGI2/ TXA2含量变化和ICP围生儿不良结局之间的关系，本实验通过建立ICP大鼠动物模型，采用放射免疫、免疫组化、半定量反转录PCR等方法，测定ICP大鼠和部分ICP患者血清、脐血清以及胎盘NO/ET、PGI2/ TXA2的表达变化，结果如下：
1. 皮下注射EE可成功建立大鼠ICP模型，动物出现血清生化改变和胆酸水平升高，电镜下病变肝脏可见胆小管扩张、线粒体变性等形态学改变，同时孕鼠的胆汁淤积可累及胎鼠肝脏，即ICP大鼠的子代也存在肝内胆汁淤积。
2. ICP孕妇血清舒缩因子与对照组相比无明显变化，但脐血清NO水平下降、ET水平显著升高，与对照组相比，有极显著性差异，表明ICP患者存在胎盘、脐带局部的血管舒缩激素失衡，并可能为ICP胎儿并发症的原因之一。ICP孕妇血清ALP和TBiL与胎儿体重呈显著负相关，可部分反映胎儿的宫内状况。
3. ICP患者胎盘存在eNOS、iNOS和ET表达异常，其中eNOS、iNOS表达显著下降，而ET表达水平则明显上升，与脐血NO、ET水平的变化一致，可能是脐血清
血管舒缩激素异常的重要原因之一。
本实验结果表明：EE可建立稳定的大鼠ICP动物模型；ICP患者胎盘存在一定程度的内分泌功能紊乱，并导致了胎儿-胎盘局部血管阻力的异常升高，为探讨ICP母儿并发症的发生机制增添了一个新的研究途径。ICP导致血管舒缩因子分泌失调的具体机制尚不明确，是否与母体内胆汁酸浓度异常升高有关、以及NO、ET的分泌异常与母儿结局的关系仍需进一步研究。
Intrahepatic cholestasis of pregnancy(ICP) ,or obstetric cholestasis,is a liver disease characterized by generalized skin pruritus with an increase in serum bile acids that appears during the second half of pregnancy ,lasts until the end of gestation,and disappears a few days after delivery.A mild abnormality in alanine aminotransferase may occur. There is considerable geographical and seasonal variation in the incidence of ICP. In the viewpoint of maternity, it is benign except that pruritus is severe. In the fetus, however, the course is not the case. The disease is closely related to a high incidence of perinatal complications, including an increased fetal distress and preterm delivery, a higher incidence of meconium passage and an increased perinatal mortality rate. Unfortunately,conventional fetal surveillance ,such as weekly nonstress tests and amniotic fluid volume assessment lack the ability to predict impending fetal demise in this condition.
Some reports showed perinatal mortality ranges from 11%-20% in ICP women when untreated. In a review of published cases before 1988,the reported still birth rate in women of cholestasis was 10 of 1000 births, meconium staining in 27%, fetal distress occurred in 18% cases.The pathogenesis of fetal complicatioons has not been fully determined.
As the singulare source of fetal oxygen and nutrition ,the role of placenta is very important in fetal growth and development. Many studies of the mechanism of fetal asphyxia in ICP have been performed in the aspects of placenta. Bile acid and meconium could result in umbilical vessels and placental chorionic veins constriction which may aggrevate fetal hypoxia. It has been reported that different kinds of vasoactive substances can be secreted by human placenta trophoblast, which took part in controlling blood flow by autocrine, paracrine and endocrine pathway.
In order to explore the pathogenesis of fetal distress in , we measured the levels of nitric oxide /endothelin, prostacylin /thromboxane A2 in serum which drawn from ICP rat and ICP patients as well as umbilical vein. The expression of endothlin nitric oxide synthase, inducible nitric oxide synthase and endothelin in human placenta were investigated used immunohistochemical method. The semi-quantative reverse polymerase chain reaction were
used to investigated the expression of induced nitric oxide synthase in human placenta.
The following conclusion may be achieved in this study:
1.ICP rats model may be successfully established by treated with 17-а-ethinylestradiol(EE), inject subcutaneously for five days, 2.5mg/kg/day. Compared with control group,the serum levels of ALP and ALT in EE group increased significantly. There was no marked change in hepatocytes of EE group under hematoxylin-eosin staining and the same as bile canaliculi under silver nitrate staining. In hepatocyte of EE group, mitochondria swelling and bile canaliculi dilated under electron microscope. The same ultrastructure changes have been seen in hepatocyte of their fetal rats.Correspondingly, body weight and live birth of the fetal rats in EE group were lighter and shorter than those of control group significantly.
2.Compared to control group,the values of vasoactive substances in serum of ICP rats and ICP patients were not significantly different, except that the levels of NO in ICP rats was significantly higher than those of control rats,which is probably resulted from the application of estrogens.But in serum of umbilical blood of ICP patients, the values of NO decreased and ET increased significantly than those of normal pregnancy, which may effect the regulation of placental blood and may be one of the causes for human placenta dysfunction and high impedance of fetoplacental circulation. In addition, the relationship between levels of ALP and TBiL in serum of ICP patients and fetal weight were significantly negative, which indicated ALP and TBiL may be useful in the prediction of fetal conditions.
3. The expression of inducible and endothlin nitric oxide synthase as well as t
引文
1. Kroumpouzos G. Intrahepatic cholestasis of pregnancy, what's new? J Eur Acad Dermatol Venereol, 2002,16(4):316-318
2. Fisk NM, Storey GN. Fetal outcome in obstetric cholestasis. British J Obstet Gynaecol, 1988,95:1137-1143
3. 胡建铭，黄沁，杨伟雯等.妊娠肝内胆汁淤积症86例临床分析.实用妇产科杂志，1997，13（5）：266-267
4. 吴味辛，熊昌碧，毕婵琴等. 妊娠期肝内胆汁淤积症.中华妇产科杂志，1984，19（增刊）:17-19
5. Heinonen S, Kirkinen P. Pregnancy outcome with intrahepatic cholestasis. Obstet Gynecol, 1999, 94(2): 189-193
6. 谭世桥，曾蔚越，熊炬等. 妊娠期肝内胆汁淤积症对母儿的危害分析. 华西医科大学学报，1990，30（2）：210-213
7. 刘伯宁，沈宁，戴钟英等. 妊娠期肝内胆汁淤积症胎盘的组织计量测定. 中华妇产科杂志，1998，23（1）:361-368
8. 王晓东，刘淑芸，衡正昌.妊娠期肝内胆汁淤积症胎儿宫内缺氧机理的初步探讨.中华妇产科杂志，1998，33（2）：68-70
9. 蔡文琴，王伯沄主编.实用免疫细胞化学与核算分子杂交技术.1994，成都，四川科学技术出版社
10. 徐先明，庄依亮，杨幼明等. 乙炔雌二醇诱发孕鼠肝内胆汁淤积. 中华围产医学杂志，2000，3(2): 112-115
11. 徐先明，庄依亮，杨幼明等. 熊去氧胆酸对抗乙炔雌二醇诱发孕大鼠肝内胆汁瘀积.上海医科大学学报，1999，26(5): 349-352
12. 徐先明, 庄依亮, 杨幼明等. 乙炔雌二醇诱发的孕鼠肝内胆汁淤积. 上海实验动物科学，1997，17(2): 71-73
13. Koopen NR, Post SM, Wolters H, et al. Differential effects of 17alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat. J Lipid Res,1999 , 40(1): 100-108
14. Koopen NR, Wolters H, Havinga R, et al. Impaired activity of the bile canalicular organic anion transporter (Mrp2/cmoat) is not the main cause of ethinylestradiol-induced cholestasis in the rat. Hepatology, 1998 , 27(2): 537-545
Laatikainen T, Tulenheimo A. Maternal serum bile acid levels and fetal distress in
15. cholestasis of pregnancy. Int J Gynaecol Obstet, 1984, 22(2): 91-94
16. 何晓红，郑伟，董 岳.妊娠期肝内胆汁淤积症患者胆酸测定及其临床意义. 中级医刊，1996，31（5）：278-279
17. Fisk NM, Bye WB, Storey GN. Maternal features of obstetric cholestasis: 20 years experience at King George V Hospital. Aust N Z J Obstet Gynaecol, 1988 ,28(3): 172-176
18. Sepulveda WH, Gonzalez C, Cruz A, et al. Vasoconstrictive effect of bile acids on isolated placental chorionic veins. Eur J Obstet Gynaecol Reprod Biol,1991,42:211-215
19. Campos GA, Guerra FA, Israel EJ. Effects of cholic acid infusion in fetal lambs. Acta Obstet Gynecol Scand, 1986,65: 23-26
20. Furchgott RF, Zawadswi JV. The obligatory role of endothelial cell in the relaxation of arterial smooth muscle by acetylcholine. Nature , 1980,288:373-376
21. Weiner CP, Lizasoain I, Baylis SA, et al. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA. 1994, 91(11): 5212-5216
22. Hishikawa K, NakakiT, Marumo T . Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett, 1995，360(3): 291-293
23. Devin EE, Guadalupe B, Frenette Luc, et al. 17-Beta-estradiol protects the liver against warm ischemia/reperfusion injury and is associated with increased serum nitric oxide and decreased tumor necrosis factor-alpha. Surgery, 2002, 132(2): 302-309
24. M. Faxén，H. Nisell，KR. Kublickiene. Altered mRNA expression of ecNOS and iNOS in myometrium and placenta from women with preeclampsia. Arch Gynecol Obstet ,2001, 265:45-50
25. 杨居祥,王一理，司履生等. 逆转录-聚合酶链反应测定多药耐药基因.中华医学检验杂志,1996,(19)2:99-102
26. Maeda T, Yoshimura T, Okamura H. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in maternal and fetal circulation. J Soc Gynecol Investig, 2003,10(1):2-4
27. Myatt L,Brockman DE, Eis ALW,et al. Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta,1993,14:487-495
28. Ramsay B, Sooranna SR, Johnson MR. Nitric oxide synthase in human myometrium and villous trophoblast throughout pregnancy. Obstet Gynecol, 1996,87:249-253
Dotsch J, Hogen N, Nyul Z, et al. Increase of endothelial nitric oxide synthase and endothelin-1 mRNA expression in human placenta during gestation. Eur J Obstet
29. Gynecol Reprod Biol, 2001, 97(2): 163-167
30. Takizawa T, Yoshikawa H, Yamada M, et al. Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta. Am J Physiol Cell Physiol, 2002, 282(4): C762-767
31. Matsubara S, Takizawa T, Takayama T, et al. Immuno-electron microscopic localization of endothelial nitric oxide synthase in human placental terminal villous trophoblasts-normal and pre-eclamptic pregnancy. Placenta., 2001, 22(8-9):782-786
32. Bnnes WJB, Slater DM, Bennett PR. Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition. Bio Chem Biophysic Res Commun, 1997, 233: 276-278
33. Galan HL, Regnault TR, Le Cras TD, et al. Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth restriction. J Appl Physiol, 2001, 90(6):2420-2426
34. Hefler LA, Reyes CA, O'Brien WE, et al. Perinatal development of endothelial nitric oxide synthase-deficient mice. Biol Reprod, 2001, 64(2):666-673
35. Tefler JF, Lyall F, Norman JE, et al. Identification of nitric oxide synthase in uterus. Human Reprod, 1995, 10:19-23
36. Sladek SM，Regenstein AC，Lykins D，et al. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol,1993, 169(5): 1285-1291
37. Norman JE, Thompson AJ, Telfer JF, et al. Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy. Mol Hum Reprod,1999, 5(2): 175-181
38. Ayuk PTY, Theophanous D, D'Souza SW, et al. L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction. J Clin Endocrinol Metab, 2002, 87(2): 747-751
39. Escobales N, Rivera-Correa M, Altieri PI, et al. Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells. Amino Acids, 2000, 19:451-468
Closs EI, Scheld JS, Sharafi M, et al. Substrate supply for nitric oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters. Mol
40. Pharmacol, 2000, 57:68-74
41. Ayuk PT, Sibley CP, Donnai P, et al. Development and polarization of cationic amino acid transporters and regulators in the human placenta. Am J Physiol, 2000, 278:C1162-C1171
42. Maul H, Longo M, Saade GR, et al. Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Des, 2003, 9(5):359-80
43. Shi L, Shi SQ, Saade GR, et al. Studies of cervical ripening in pregnant rats: effects of various treatments. Mol Hum Reprod, 2000, 6:382-389
44. Tschugguel W, Schneeberger C, Lass H, et al. Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression. Biol Reprod, 1999, 60:1367-1372
45. Vaisanen-Tommiska M, Nuutila M, Aittomaki K, et al. Nitric oxide metabolites in cervical fluid during pregnancy: further evidence for the role of cervical nitric oxide in cervical ripening. Am J Obstet Gynecol, 2003 ,188(3): 779-785
46. Tiboni GM，Giampietro F. Inhibition of nitric oxide synthesis causes preterm delivery in the mouse. Hum-Reprod, 2000, 15(8): 1838-1842
47. Bukowski R, Saade GR. New developments in the management of preterm labor. Semin Perinatol, 2001, 25(5): 272-294
48. Wetzka B, Schafer WR, Stehmans A, et al. Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women. Gynecol Endocrinol, 2001, 15(1): 34-42
49. Duckitt K, Thornton S. Nitric oxide donors for the treatment of preterm labour. Cochrane Database Syst Rev, 2002, (3): CD002860
50. Leszczynska Gorzelak B，Laskowska M，Marciniak B, et al. Nitric oxide for treatment of threatened preterm labor. Int J Gynaecol Obstet, 2001, 73(3): 201-206
51. Sagawa N，Hasegawa M，Itoh H，et al. Current topic: the role of amniotic endothelin in human pregnancy. Placenta, 1994, 15(6): 565-575
52. Handwerger S .Endothelins and the placenta. J Lab Clin Med, 1995, 125(6): 679-681
53. Poston L.The control of blood flow to the placenta. Exp Physiol, 1997, 82(2): 377-387
54. Ahmad Z, Reznil SE. Immunohistochemmical location of ECE-1 in the human placenta. Placenta, 2000, 21(2-3): 226-233
Barros JS, Bairos VA, Baptista MG, et al. Immunocytochemical localization of
55. endothelin-1 in human placenta from normal and preeclamptic pregnancies. Hypertens-Pregnancy, 2001, 20(1): 125-137
56. Myatt L, Langdon G, Brewer DE, et al. The comparative effects of big endothelins-a, endothelin 1, and endothelin 3 in the human fetal-placenta circulation. Am J Obstet Gynecol, 1992,167:1651-1657
57. Rowe J, Campbell S, Gallery ED. Effects of hypoxia on regulation of prostanoid production in decidual endothelial cells in normal and preeclamptic pregnancy. J Soc Gynecol Investig, 2000, 7(2): 118-124
58. 腊晓琳, 王冬梅, 朱启英.妊娠肝内胆汁淤积症胎盘雌激素受体及血管舒缩因子变化. 现代妇产科进展，2002，11(1)：48-50
59. 马志松，王晓铃. 妊娠肝内胆汁淤积症妇女血清一氧化氮水平的研究.中华妇产科杂志，1998，33（3）：148-149
60. 刘云，漆洪波，李小木，等. 妊娠肝内胆汁淤积症孕妇肝功能指标与围生儿预后的关系.中国实用妇科与产科杂志，2001，17(6): 350-352
2. Fisk NM, Storey GN. Fetal outcome in obstetric cholestasis. British J Obstet Gynaecol, 1988,95:1137-1143
3. 胡建铭，黄沁，杨伟雯等.妊娠肝内胆汁淤积症86例临床分析.实用妇产科杂志，1997，13（5）：266-267
4. 吴味辛，熊昌碧，毕婵琴等. 妊娠期肝内胆汁淤积症.中华妇产科杂志，1984，19（增刊）:17-19
5. Heinonen S, Kirkinen P. Pregnancy outcome with intrahepatic cholestasis. Obstet Gynecol, 1999, 94(2): 189-193
6. 谭世桥，曾蔚越，熊炬等. 妊娠期肝内胆汁淤积症对母儿的危害分析. 华西医科大学学报，1990，30（2）：210-213
7. 刘伯宁，沈宁，戴钟英等. 妊娠期肝内胆汁淤积症胎盘的组织计量测定. 中华妇产科杂志，1998，23（1）:361-368
8. 王晓东，刘淑芸，衡正昌.妊娠期肝内胆汁淤积症胎儿宫内缺氧机理的初步探讨.中华妇产科杂志，1998，33（2）：68-70
9. 蔡文琴，王伯沄主编.实用免疫细胞化学与核算分子杂交技术.1994，成都，四川科学技术出版社
10. 徐先明，庄依亮，杨幼明等. 乙炔雌二醇诱发孕鼠肝内胆汁淤积. 中华围产医学杂志，2000，3(2): 112-115
11. 徐先明，庄依亮，杨幼明等. 熊去氧胆酸对抗乙炔雌二醇诱发孕大鼠肝内胆汁瘀积.上海医科大学学报，1999，26(5): 349-352
12. 徐先明, 庄依亮, 杨幼明等. 乙炔雌二醇诱发的孕鼠肝内胆汁淤积. 上海实验动物科学，1997，17(2): 71-73
13. Koopen NR, Post SM, Wolters H, et al. Differential effects of 17alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat. J Lipid Res,1999 , 40(1): 100-108
14. Koopen NR, Wolters H, Havinga R, et al. Impaired activity of the bile canalicular organic anion transporter (Mrp2/cmoat) is not the main cause of ethinylestradiol-induced cholestasis in the rat. Hepatology, 1998 , 27(2): 537-545
Laatikainen T, Tulenheimo A. Maternal serum bile acid levels and fetal distress in
15. cholestasis of pregnancy. Int J Gynaecol Obstet, 1984, 22(2): 91-94
16. 何晓红，郑伟，董 岳.妊娠期肝内胆汁淤积症患者胆酸测定及其临床意义. 中级医刊，1996，31（5）：278-279
17. Fisk NM, Bye WB, Storey GN. Maternal features of obstetric cholestasis: 20 years experience at King George V Hospital. Aust N Z J Obstet Gynaecol, 1988 ,28(3): 172-176
18. Sepulveda WH, Gonzalez C, Cruz A, et al. Vasoconstrictive effect of bile acids on isolated placental chorionic veins. Eur J Obstet Gynaecol Reprod Biol,1991,42:211-215
19. Campos GA, Guerra FA, Israel EJ. Effects of cholic acid infusion in fetal lambs. Acta Obstet Gynecol Scand, 1986,65: 23-26
20. Furchgott RF, Zawadswi JV. The obligatory role of endothelial cell in the relaxation of arterial smooth muscle by acetylcholine. Nature , 1980,288:373-376
21. Weiner CP, Lizasoain I, Baylis SA, et al. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA. 1994, 91(11): 5212-5216
22. Hishikawa K, NakakiT, Marumo T . Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett, 1995，360(3): 291-293
23. Devin EE, Guadalupe B, Frenette Luc, et al. 17-Beta-estradiol protects the liver against warm ischemia/reperfusion injury and is associated with increased serum nitric oxide and decreased tumor necrosis factor-alpha. Surgery, 2002, 132(2): 302-309
24. M. Faxén，H. Nisell，KR. Kublickiene. Altered mRNA expression of ecNOS and iNOS in myometrium and placenta from women with preeclampsia. Arch Gynecol Obstet ,2001, 265:45-50
25. 杨居祥,王一理，司履生等. 逆转录-聚合酶链反应测定多药耐药基因.中华医学检验杂志,1996,(19)2:99-102
26. Maeda T, Yoshimura T, Okamura H. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in maternal and fetal circulation. J Soc Gynecol Investig, 2003,10(1):2-4
27. Myatt L,Brockman DE, Eis ALW,et al. Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta,1993,14:487-495
28. Ramsay B, Sooranna SR, Johnson MR. Nitric oxide synthase in human myometrium and villous trophoblast throughout pregnancy. Obstet Gynecol, 1996,87:249-253
Dotsch J, Hogen N, Nyul Z, et al. Increase of endothelial nitric oxide synthase and endothelin-1 mRNA expression in human placenta during gestation. Eur J Obstet
29. Gynecol Reprod Biol, 2001, 97(2): 163-167
30. Takizawa T, Yoshikawa H, Yamada M, et al. Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta. Am J Physiol Cell Physiol, 2002, 282(4): C762-767
31. Matsubara S, Takizawa T, Takayama T, et al. Immuno-electron microscopic localization of endothelial nitric oxide synthase in human placental terminal villous trophoblasts-normal and pre-eclamptic pregnancy. Placenta., 2001, 22(8-9):782-786
32. Bnnes WJB, Slater DM, Bennett PR. Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition. Bio Chem Biophysic Res Commun, 1997, 233: 276-278
33. Galan HL, Regnault TR, Le Cras TD, et al. Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth restriction. J Appl Physiol, 2001, 90(6):2420-2426
34. Hefler LA, Reyes CA, O'Brien WE, et al. Perinatal development of endothelial nitric oxide synthase-deficient mice. Biol Reprod, 2001, 64(2):666-673
35. Tefler JF, Lyall F, Norman JE, et al. Identification of nitric oxide synthase in uterus. Human Reprod, 1995, 10:19-23
36. Sladek SM，Regenstein AC，Lykins D，et al. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol,1993, 169(5): 1285-1291
37. Norman JE, Thompson AJ, Telfer JF, et al. Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy. Mol Hum Reprod,1999, 5(2): 175-181
38. Ayuk PTY, Theophanous D, D'Souza SW, et al. L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction. J Clin Endocrinol Metab, 2002, 87(2): 747-751
39. Escobales N, Rivera-Correa M, Altieri PI, et al. Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells. Amino Acids, 2000, 19:451-468
Closs EI, Scheld JS, Sharafi M, et al. Substrate supply for nitric oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters. Mol
40. Pharmacol, 2000, 57:68-74
41. Ayuk PT, Sibley CP, Donnai P, et al. Development and polarization of cationic amino acid transporters and regulators in the human placenta. Am J Physiol, 2000, 278:C1162-C1171
42. Maul H, Longo M, Saade GR, et al. Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Des, 2003, 9(5):359-80
43. Shi L, Shi SQ, Saade GR, et al. Studies of cervical ripening in pregnant rats: effects of various treatments. Mol Hum Reprod, 2000, 6:382-389
44. Tschugguel W, Schneeberger C, Lass H, et al. Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression. Biol Reprod, 1999, 60:1367-1372
45. Vaisanen-Tommiska M, Nuutila M, Aittomaki K, et al. Nitric oxide metabolites in cervical fluid during pregnancy: further evidence for the role of cervical nitric oxide in cervical ripening. Am J Obstet Gynecol, 2003 ,188(3): 779-785
46. Tiboni GM，Giampietro F. Inhibition of nitric oxide synthesis causes preterm delivery in the mouse. Hum-Reprod, 2000, 15(8): 1838-1842
47. Bukowski R, Saade GR. New developments in the management of preterm labor. Semin Perinatol, 2001, 25(5): 272-294
48. Wetzka B, Schafer WR, Stehmans A, et al. Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women. Gynecol Endocrinol, 2001, 15(1): 34-42
49. Duckitt K, Thornton S. Nitric oxide donors for the treatment of preterm labour. Cochrane Database Syst Rev, 2002, (3): CD002860
50. Leszczynska Gorzelak B，Laskowska M，Marciniak B, et al. Nitric oxide for treatment of threatened preterm labor. Int J Gynaecol Obstet, 2001, 73(3): 201-206
51. Sagawa N，Hasegawa M，Itoh H，et al. Current topic: the role of amniotic endothelin in human pregnancy. Placenta, 1994, 15(6): 565-575
52. Handwerger S .Endothelins and the placenta. J Lab Clin Med, 1995, 125(6): 679-681
53. Poston L.The control of blood flow to the placenta. Exp Physiol, 1997, 82(2): 377-387
54. Ahmad Z, Reznil SE. Immunohistochemmical location of ECE-1 in the human placenta. Placenta, 2000, 21(2-3): 226-233
Barros JS, Bairos VA, Baptista MG, et al. Immunocytochemical localization of
55. endothelin-1 in human placenta from normal and preeclamptic pregnancies. Hypertens-Pregnancy, 2001, 20(1): 125-137
56. Myatt L, Langdon G, Brewer DE, et al. The comparative effects of big endothelins-a, endothelin 1, and endothelin 3 in the human fetal-placenta circulation. Am J Obstet Gynecol, 1992,167:1651-1657
57. Rowe J, Campbell S, Gallery ED. Effects of hypoxia on regulation of prostanoid production in decidual endothelial cells in normal and preeclamptic pregnancy. J Soc Gynecol Investig, 2000, 7(2): 118-124
58. 腊晓琳, 王冬梅, 朱启英.妊娠肝内胆汁淤积症胎盘雌激素受体及血管舒缩因子变化. 现代妇产科进展，2002，11(1)：48-50
59. 马志松，王晓铃. 妊娠肝内胆汁淤积症妇女血清一氧化氮水平的研究.中华妇产科杂志，1998，33（3）：148-149
60. 刘云，漆洪波，李小木，等. 妊娠肝内胆汁淤积症孕妇肝功能指标与围生儿预后的关系.中国实用妇科与产科杂志，2001，17(6): 350-352