摘要
目的:
1.对比观察米非司酮对孕7-9周人胎盘绒毛中前列腺素合成及表达、氧化/抗氧化状态和间质微血管形态及数量的影响,从前列腺素合成调控、氧化应激和胎盘绒毛间质微血管变化三方面探讨米非司酮终止早孕的可能机制。
2.进一步完善米非司酮在药物流产中作用机理的认识,为临床上合理扩大米非司酮终孕应用范围提供理论基础和实验参考依据,同时为其在其它领域的应用提供理论依据。
方法:
1.应用免疫组织化学(SP)法检测COX-2、COX-1、NF-κB在米非司酮药流组与人流对照组(孕7-9周各12例)胎盘绒毛滋养细胞中表达量的差异;应用酶联免疫吸附定量(ELISA)法检测PGF_(2α)在上述两组胎盘绒毛中含量的差异,了解米非司酮对人早孕7-9周胎盘绒毛前列腺素合成及表达的影响。
2.应用化学比色法检测MDA、T-AOC、CuZn-SOD和MDA/T-AOC比值在上述两组胎盘绒毛中含量的差异,了解米非司酮对人早孕7-9周胎盘绒毛氧化/抗氧化平衡状态的影响。
3.应用免疫组化法定位HO-1在上述两组胎盘绒毛中的表达部位及检测其在滋养细胞中表达量的差异;蛋白质印迹(Western blot)法检测HO-1在上述两组胎盘绒毛中蛋白表达量的差异;逆转录酶—聚合酶链式反应(RT—PCR)法检测HO-1mRNA在上述两组胎盘绒毛中RNA转录量的差异,通过HO-1将前列腺素合成调控、氧化应激和胎盘绒毛间质微血管变化三方面相互作用联系起来探讨米非司酮终止早孕的可能机制。
4.应用免疫组化法检测VEGF在上述两组胎盘绒毛滋养细胞中表达量的差异;间接免疫荧光组织化学方法及激光共聚焦成像技术,观察分析上述两组胎盘绒毛间质中CD34荧光标记的微血管形态及数量的差异,准确直观地了解米非司酮对孕7—9周人胎盘绒毛间质微血管形成的影响。
结果:
1.COX-2主要强表达于绒毛表面的合体滋养细胞胞浆中,细胞滋养细胞胞浆弱表达,米非司酮药流组阳性表达较人流对照组强(P＜0.01)。NF-κB主要强表达于绒毛表面的合体滋养细胞胞浆中,细胞滋养细胞胞浆表达稍弱,米非司酮药流组阳性表达较人流对照组强(P＜0.01)。COX-1强表达于米非司酮药流组与人流对照组胎盘绒毛合体及细胞滋养细胞胞浆中,两组表达无明显差别(P=0.064,P＞0.05)。米非司酮药流组胎盘绒毛中PGF_(2α)的表达较人流对照组明显增多(P＜0.01)。
2.米非司酮药流组胎盘绒毛中MDA含量较人流对照组明显增多(P＜0.01)。米非司酮药流组胎盘绒毛中T-AOC含量较人流对照组明显减少(P＜0.01)。米非司酮药流组胎盘绒毛中CuZn-SOD含量较人流对照组明显减少(P＜0.01)。米非司酮药流组胎盘绒毛中MDA/T—AOC比值较人流对照组明显增大(P＜0.01)。
3.HO-1主要强表达于绒毛的滋养细胞胞核及胞浆中,部分血管内皮细胞和间质细胞胞核、胞浆也有表达。米非司酮药流组滋养细胞胞核、胞浆阳性表达较对照组强(P＜0.01)。Western blot法检测HO-1在上述两组胎盘绒毛中蛋白表达量,米非司酮药流组较对照组增多(P＜0.01)。RT-PCR法检测HO-1mRNA在上述两组胎盘绒毛中RNA转录量,米非司酮药流组较对照组增多(P＜0.01)。
4.米非司酮药流组与人流对照组均可见血管内皮细胞在FITC-CD34标记下呈清晰明亮的绿色荧光,人流对照组毛细血管的形态规则,管腔清晰,管径大小正常。米非司酮药流组毛细血管的数目增多,形态不规则,管腔不同程度扭曲扩张。米非司酮药流组微血管长度密度(Lv)较人流组增高(P=0 043,P＜0.05)。米非司酮药流组微血管体积密度(Vv)较人流组增高(P=0.031,P＜0.05)。VEGF表达于上述两组胎盘绒毛滋养细胞、血管内皮细胞胞浆中,米非司酮药流组滋养细胞阳性表达较人流对照组减弱(P＜0.01)。
结论:
1.米非司酮激活NF-κB,使COX-2表达增多,继而使PGs(尤其PGF_(2α))合成增多,PGF_(2α)引起血管收缩,蜕膜组织变性坏死继而直接或间接影响绒毛组织的血液供应,刺激产生更多ROS,进一步加重氧化损伤,同时激活子宫平滑肌收缩及扩张宫颈,导致正常妊娠无法维持而终孕。
2.人早孕7-9周胎盘绒毛中存在氧化应激现象。米非司酮可使MDA含量增加,T-AOC和CuZn-SOD含量减少,MDA/T-AOC比值增大,加重氧化/抗氧化失衡,促进ROS产生和NF-κB激活,使PGs(尤其PGF_(2α))合成及表达增多,血管收缩使血管内皮细胞和线粒体功能受损,又进一步促进ROS的产生,加重氧化/抗氧化失衡。
3.米非司酮促进ROS产生,ROS通过激活HO-1基因启动区的NF-κB上调HO-1mRNA转录和蛋白合成。HO-1发挥其抗氧化应激、扩血管改善胎盘循环和胎儿生长发育、细胞保护、抗炎、抑制血小板凝集、抑制凋亡等作用来对抗米非司酮的作用。PGF_(2α)与HO-1相互竞争,究竟是使氧化/抗氧化在新的水平上保持平衡,妊娠得以维持,还是PGs最终引起血管收缩,血管内皮细胞损伤,进一步促进ROS的产生,导致氧化/抗氧化完全失衡,这种竞争的结果就决定了妊娠的结局。
4.在顿服米非司酮150mg后24-48h而未加用米索前列醇的情况下,人早孕7-9周胎盘绒毛出现了PGs合成及表达增多、氧化/抗氧化失衡状态,但同时也出现HO-1mRNA转录和蛋白合成增加来对抗米非司酮上述作用,胎盘绒毛微血管出现数目增多,管腔不同程度扭曲扩张,可能反映此时胎盘绒毛处于一种代偿状态而非耗竭现象。此时需加服前列腺素抑制HO-1的代偿作用或适当延长米非司酮作用时间和剂量,以加重氧化/抗氧化失衡状态,刺激产生更多ROS,进一步加重氧化损伤,激活子宫平滑肌收缩及扩张宫颈,以达到预期的药物流产效果。
Objectives:
1.To investigate the synthesis and regulation of prostaglandins, oxidative stress and microvascular changes in the mesenchyma of human placental villi in order to explain the possible mechanism of mifepristone used for medical abortion.To observe the effect of mifepristone on the synthesis and expression of PGF_(2α),the balance of oxidation/antioxidation,the configuration and number of the mesenchyma micrangium in human placental villi during 7-9 week's pregnancy.
2.To improve the knowledge of action mechanism of mifepristone and rationally expand the use of mifepristone in abortion,offer theoretical basis and lab reference for its application even in other fields.
Methods:
1.To study the effect of mifepristone on synthesis and expression of PGF_(2α) in human placental villi during early pregnancy(7-9 weeks). The patients were divided into mifepristone group(n=12) and induced abortion group(n=12) randomly.Immunohistochemistry (streptavidin-peroxidase,S-P) was used to detect the expression of COX-2,COX-1 and NF-κB in human placental villous trophoblast between mifepristone group and induced abortion group.The expression level of PGF_(2α) was determined by enzyme linked immunosorbent assay(ELISA).
2.To investigate the effect of mifepristone on oxidation/antioxidation balance of human placental villi during early pregnancy(7-9 weeks). Colorimetry was used to detect the level of MDA,T-AOC,CuZn-SOD and MDA/T-AOC in above mentioned groups.
3.Immunohistochemistry(streptavidin-peroxidase,S-P) was used to locate and detect the expression level of HO-1 in trophoblast of two groups.The protein expression level of HO-1 was determined by Western blot;Reverse transcription-polymerase chain reaction (RT-PCR) was applied to measure the mRNA level of HO-1 in two groups;Thus we can investigate the possible mechanism of mifepristone by analyzing interactions among PGF_(2α) synthesis and regulation,oxidation stress and mesenchyma microvacular changes of human placental villi through HO-1.
4.To study how mifepristone affect mesenchyma micrangium of human placental villi during early pregnancy(7-9weeks). Immunofluorescence histochemistry and Laser Scanning Confocal Fluorescence Microimaging System were used to detect configuration and number of vascular fluorescence labeled by CD34 in two groups.
Results:
1.COX-2 was strongly expressed mainly in cytoplasm of syncytrotrophoblast on the surface of villi,weakly expressed in cytoplasm of cytotrophoblast,and the expression in mifepristone group was significantly higher than that in artificial induced group (P＜0.01).NF-κB was strongly expressed mainly in cytoplasm of syncytrotrophoblast,weakly expressed in cytoplasm of cytotrophoblast, and the expression in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).COX-1 was strongly expressed in the cytoplasm of syncytrotrophoblast and cytrotrophoblast in both mifepristone group and induced abortion group.No statistically significant difference was found between two groups(P =0.064,P＞0.05).The expression of PGF_(2α) in villi in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).
2.The villus level of MDA in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).The villus level of T-AOC in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).The villus level of CuZn-SOD in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).The villus level of MDA/T-AOC in mifepristone group was significantly higher than that in induced abortion group(P＜0.01)
3.HO-1 was expressed mainly in cytoplasm and neucleus of trophoblast, partly in cytoplasm and neucleus of vascular endothelial cells and mesenchyma cells.The expression of HO-1 in trophoblast in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).Using Western blot,the protein level of HO-1 was significantly higher in mifepristone group than that in induced abortion group(P＜0.01).When detected by RT-PCR,the mRNA level of HO-1 was significantly higher in mifepristone group than that in induced abortion group(P＜0.01)
4.Clear and bright green fluorescence was found in the vascular endothelial cells labeled by FITC-CD34 in both groups.In induced abortion group,the capillary configuration was regular.The lumen was distinct and normal in size.While in mifepristone group,the number of mesenchyma micrangium increased.The configuration was irregular.The lumen was twisted and dilated in various degrees. The microvascular length density(Lv) was significantly higher in mifepristone group than that in induced abortion group(P=0.043,P＜0.05.The microvascular volume density(Vv) was significantly higher in mifepristone group than that in induced abortion group(P = 0.031,P＜0.05).VEGF was expressed in the cytoplasm of trophoblast and vascular endothelial cell in above mentioned groups, and the expression in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).
Conclution:
1.NF-κB is activated by mifepristone,the expression of COX-2,and more PGs(esp.PGF_(2α)) are synthesized,which consequently induces vessel contraction,degeneration and necrosis of decidua tissue and the villus blood supply is affected directly or indirectly.The inhibition of HO-1 produces more ROS by antioxidation stress which further aggrevates the oxidative damage and leads to uterine smooth muscle contraction,thus interrupts and hampers pregnancy.
2.There is oxidative stress in human villi during early pregnancy (7-9weeks).Mifepristone can further destroy the balance of oxidation/antioxidation,produce ROS and activate NF-κB, synthesize and express more PGs(esp.PGF_(2α)),then vessel contraction leads to the potential loss of vascular endothelial cells and mitochondrial,which produces more ROS,and oxidation/antioxidation balance is further destroyed.
3.Mifepristone promotes to produce ROS,which can increase HO-1mRNA transcription and protein synthesis through activational NF-κB in promoter region of HO-1 gene.To resist the effect of mifepristone,HO-1 decreases oxidative stress,dilates vessel to improve blood supply in placenta and infant growth and development,protects cell,inhibits inflammation,aggregation of platelet and apoptosis.The result of competition between PGF_(2α) and HO-1 decides whether prgnancy continues.If the balance of oxidation/antioxidation is maintained in a new degree,the pregnancy will continue.On the contrary,the vessel contracts,the endothelial cells are damaged,ROS is produced,the oxidation/antioxidation balance is completely destroyed.
4.24-48h after taking mifepristone 150mg(without adding misoprostol), the synthesis and expression level of PGs are increased and the balance of oxidation/antioxidaiton is destroyed in human placental villi during early pregnancy(7-9weeks).HO-1 mRNA and protein synthesis are also increased to resist the effects of mifepristone mentioned above.The number of capillary in villi is increased.The lumen is twisted and dilated in various degrees.Probably it is a compensatory rather than an exhausting phenomenon.Then PG is needed to inhibit the compensatory,effect of HO-1.Therefore,the usage time and dosage of mifepristone should be extended or increased in order to complete the medical abortion.
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