【作者】 李静；

【导师】 王谢桐；

【作者基本信息】 山东大学 ， 妇产科学（产科学）（专业学位）， 2017， 博士

【摘要】 背景:子痫前期是一种妊娠特发性疾病,目前约有4%的孕妇患有子痫前期,严重威胁孕产妇及围产儿的健康,导致多种发病率包括早产率的升高。子痫前期的高危因素包括肥胖、高龄产妇、孕妇糖尿病、种族、抗磷脂抗体综合征/易栓症、家族子痫前期病史等,其病因学一直是围产医学研究的热点,但其明确病因目前尚不清楚。近年来研究发现子痫前期的发病与氧化应激、免疫反应、炎性反应等多种病理生理反应有关,而氧化应激是众多影响因素中最重要的一项。血红素氧化酶-1(hemeoxygenase-1,HO-1)属于血红素氧化酶的一个亚型,是一种诱导酶,它的常见功能是在游离的亚铁血红素转变成胆红素时对限速步骤起催化作用,将血红素转变为胆绿素,胆绿素是在转换成胆红素过程最后一步中胆绿素还原酶作用的底物。血红素氧化酶有不同的生物学功能,HO-1能够调节并参与机体抗炎、抗凋亡及抗氧化应激作用,并且有助于维持孕期子宫血管的稳定性并参与孕期胎盘的形成,与子痫前期的发病关系密切。近期学者们关于HO-1在子痫前期病人血清或胎盘组织中的表达水平及作用机制等方面的研究较多,但是结果及观点并不统一。核因子 E2 相关因子 2(Nuclear factor erythroid 2-related factor-2,Nrf2)属于CNC-bZIP(cap’n’collar subfamily of basic leucine-zipper),也就是 CNC 亮氨酸拉链转录激活因子家族,可以被机体的氧化应激反应激活。早前有研究证实Nrf2在氧化应激过程中参与调节机体细胞的保护性基因,保护机体免受氧化应激的侵害,并且Nrf2在保护机体抵御致癌物质、亲电子试剂及活性氧等毒性侵害的过程中扮演重要角色。在机体多种氧化应激反应中,Nrf2可以激活机体保护基因包括HO-1基因,而在子痫前期病人的氧化应激反应中,二者是否共同参与疾病的病理生理过程并且是否存在信号通路或者关联作用,并未得到证实。我们推断Nrf2和HO-1在子痫前期的氧化应激反应中起到重要的作用。或许Nrf2作为HO-1重要的上游调节因子,在子痫前期发病的病理生理过程中也起到对HO-1的调节作用,二者作为一个信号通路参与了子痫前期的发病。子痫前期的另外一个临床特点是在治疗方面:目前没有预防和治疗子痫前期的有效措施,只有终止妊娠,胎盘娩出后,子痫前期的症状才可以得到抑制和改善,所以胎盘因素一直被当作参与子痫前期发病的重要原因。现在盛行的假设是子痫前期是由异常的滋养细胞迁移和侵入母体子宫螺旋动脉不足即胎盘浅着床导致的子宫胎盘单位血循环降低和胎盘缺血导致的。实际上,子痫前期病人与正常妊娠孕妇相比,的确子宫动脉管径更细,阻力更大。但目前胎盘滋养细胞迁移和侵袭的作用机制仍不清楚,也没有针对此方面的靶向治疗。因此,从胎盘滋养细胞侵袭能力方面入手,如果探寻到可以影响胎盘滋养细胞侵入子宫螺旋动脉的因素,除了对子痫前期病因学方面有重要意义外,对于子痫前期的靶向治疗也具有较大意义。基于上述基础,我们如果证实Nrf2作为上游因子调控HO-1参与子痫前期发病的病理生理过程,对于后续HO-1在子痫前期病人的发病及治疗中的研究将具有重要意义。若能进一步证实滋养细胞中HO-1蛋白表达水平的改变能够对滋养细胞的侵袭性造成影响,对于子痫前期病因学和靶向治疗等方面将具有重要价值。第一部分子痫前期病人胎盘组织中HO-1和Nrf2升高的协同作用目的:检测重度子痫前期病人胎盘组织中HO-1和Nrf2的表达水平差异,并且验证HO-1和Nrf2在胎盘组织中的表达是否有相关性,探讨HO-1和Nrf2在子痫前期发病中的作用,并且通过HTR-8/SVneo细胞系培养,进一步探讨Nrf2对HO-1的调控效应,验证在子痫前期发病中Nrf2是否为HO-1的上游调控因子。方法:1.共选取60例住院分娩孕妇作为研究对象,其中30例重度子痫前期患者作为研究组,30例健康足月妊娠孕妇作为对照组。2.用ELISA方法检测两组孕妇血清中HO-1蛋白的表达水平。3.采用Western blot方法检测两组孕妇胎盘组织中HO-1蛋白、Nrf2蛋白表达水平。4.用real-time PCR方法检测两组孕妇胎盘组织中HO-1 mRNA的表达水平。5.培养HTR-8/SVneo细胞系,免疫荧光定位细胞中HO-1、Nrf2蛋白的表达。6.采用 Nrf2 诱导因子 tert-butylhydroquinone(tBHQ)诱导 HTR-8/SVneo 细胞8小时后,分别检测细胞中Nrf2蛋白和HO-1 mRNA、HO-1蛋白的表达水平变化情况。结果:1.在重度子痫前期组,FGR的发生率为16.7%,其中20%的病例发生了 HELLP综合征,1例患者(3.3%)孕期发生胎儿宫内死亡。其中25例病人因重度高血压(收缩压160≥mmHg和/或舒张压>11OmmHg)接受降压治疗。另外,30例病人均接受硫酸镁解痉治疗,23例病人因为终止妊娠孕周<34进行地塞米松促胎肺成熟治疗。2.与正常妊娠组相比,重度子痫前期组血清HO-1蛋白水平明显降低(n = 30,P<0.01)。3.重度子痫前期组胎盘组织HO-1蛋白、HO-1 mRNA水平较正常妊娠组升高(n = 30,P<0.01;n = 30,P<0.05)。4.重度子痫前期组胎盘组织Nrf2蛋白的表达水平较正常妊娠孕妇增高(n =30,P<0.01)。5.采用Pearson’s相关性检验检测两组孕妇胎盘组织中HO-1蛋白和Nrf2蛋白水平的相关性,结果显示HO-1蛋白和Nrf2蛋白水平成正相关(r = 0.503,P<0.01)。采用Spearman等级相关性分析检测两组孕妇胎盘组织中Nrf2蛋白与HO-1 mRNA的相关性,同样发现Nrf2蛋白与HO-1 mRNA成正相关性(r = 0.488,P<0.01)。6.免疫荧光定位显示HTR-8/SVneo细胞核与细胞浆中均可见Nrf2蛋白表达,HO-1蛋白主要表达于HTR-8/SVneo细胞的胞浆。7.在HTR-8/SVneo细胞培养液中加入Nrf2诱导物tBHQ刺激细胞8小时后,发现细胞核中Nrf2蛋白表达量较对照细胞升高(n = 6,P<0.05)。并且接受tBHQ孵育的HTR-8/SVneo细胞中HO-1 mRNA的表达水平也较对照细胞升高(n = 6,P<0.01)。结论:1.与正常妊娠妇女相比较,重度子痫前期患者血清中HO-1水平降低。而重度子痫前期患者胎盘组织中HO-1蛋白和mRNA的表达均增高。胎盘和血清中H0-1表达水平不一致的原因可能是HO-1可以由机体多个器官合成,而妊娠期胎盘合成的HO-1并不是血清中HO-1的主要来源。2.重度子痫前期患者胎盘组织中HO-1和Nrf2水平同时增高,子痫前期孕妇可能通过胎盘组织中HO-1和Nrf2的高表达来保护子痫前期发病过程中氧化应激对机体的损伤。3.在子痫前期发病的病理生理过程中,胎盘滋养细胞中Nrf2可能作为HO-1的上游调控因子对HO-1的表达进行正向调节,激活HO-1,增加胎盘滋养细胞HO-1的表达量。第二部分HO-1对氧化应激环境下滋养细胞侵袭能力的影响目的:我们第一部分的研究提示:Nrf2在子痫前期发病中对HO-1进行正向调节,共同参与子痫前期发病的病理生理过程。而HO-1表达量的改变在氧化应激状态下是否对胎盘滋养细胞的侵袭能力造成影响,尚不明确。为进一步研究HO-1表达量的改变对胎盘滋养细胞生物学行为的影响,拟建立HTR-8/SVneo细胞氧化应激模型,然后分别抑制和诱导细胞中HO-1蛋白的表达,来探讨HTR-8/SVneo细胞在氧化应激环境下HO-1表达量的改变是否对HTR-8/SVneo细胞的侵袭能力造成影响。方法:1.培养HTR-8/SVneo细胞系,用100umol/lH202孵育12小时建立细胞氧化应激模型。2.将细胞模型分为三组:正常对照组、Znpp诱导组和Hemin诱导组,分别在三组细胞中加入相同体积的生理盐水、HO-1抑制剂Znpp和HO-1诱导剂Hemin,然后放置于37℃、5%C02的细胞培养箱中继续培养8小时备用。3.通过免疫荧光定位观察HTR-8/SVneo细胞氧化应激模型中HO-1蛋白的表达及定位。4.采用Western blot方法分别检测对照组、Znpp诱导组和Hemin诱导组HTR-8/SVneo细胞氧化应激模型中HO-1蛋白表达水平的变化。5.通过Transwell细胞侵袭实验观察在HO-1抑制剂Znpp和诱导剂Hemin作用下HTR-8/SVneo细胞氧化应激模型侵袭能力的改变。结果:1.在HTR-8/SVneo细胞系氧化应激模型中HO-1蛋白主要表达于细胞浆。2.在HO-1抑制剂Znpp的作用下,HTR-8/SVneo细胞氧化应激模型中HO-1蛋白的表达量较对照组降低(n = 6,P<0.05);在HO-1诱导剂Hemin的孵育下,HTR-8/SVneo细胞氧化应激模型中HO-1蛋白的表达量较对照组升高(n = 6,P<0.05)。3.HTR-8/SVneo细胞氧化应激模型在HO-1表达水平降低时其侵袭能力降低(n = 6,P 0.05);反之,在HO-1表达水平增高的情况下其侵袭能力有所提高(n=6,P<0.05)。结论:1.HO-1抑制剂Znpp能够有效抑制HTR-8/SVneo细胞氧化应激模型中HO-1的表达水平;同样,HO-1诱导剂Hemin能够有效诱导HTR-8/SVneo细胞氧化应激模型中HO-1的表达水平。2.在氧化应激环境中提高滋养细胞HO-1蛋白表达水平能够增加其侵袭能力,而降低HO-1蛋白表达水平则降低滋养细胞的侵袭能力,因此可尝试通过调控胎盘滋养细胞中HO-1的表达水平实现对子痫前期的靶向治疗。更多还原

【Abstract】 Background:Preeclampsia was defined following the occurrence of one or more of the following criteria after 20 weeks of pregnancy:the systolic pressure of the patients is equal to or greater than 140 mmHg;the diastolic blood pressure of the patients is equal to or greater than 90 mmHg;and the proteinuria level is equal to or greater than 300mg after 24 hours.The morbidity of preeclampsia is approximately 4%.As a high risk pregnancy,preeclampsia leads to various increasing of morbidities including premature delivery and threatens the health of maternal and infant seriously.It is generally recognized that the risk factors of preeclampsia includes obesity,advanced aged of pregnant women,maternal diabetes,racial differences,antiphospholipid syndrome/thrombophilia,family history of preeclampsia et al,however,the exact causes of preeclampsia is unclear.Recently researches indicated that oxidative stress,inflammation,endothelial dysfunction,and placental blood flow appear to be significant contributing factors of the physiopathology of preeclampsia,and oxidative stress appears to be the most important cause.Heme oxygenase-1(HO-1),is one of a functional isoforms of heme oxygenase.HO-1 is an inducible enzyme that catalyzes heme degradation.This degradation reaction triggers the synthesis of iron,biliverdin,and carbon monoxide,resulting in anti-inflammatory and anti-apoptotic effects,and oxidation resistance.Recent reports have suggested that heme oxygenase-1 plays an important physiological role in the body,effecting the maintenance of uterine vessel quiescence during pregnancy and placental development.Several recent studies have demonstrated that HO-1 is involved in the etiopathogenesis of preeclampsia.Nuclear factor erythroid 2-related factor-2(Nrf2)is belongs to a group of the cap’n’collar(CNC)subfamily of basic region leucine zipper(b Zip)transcription factors.It is widely activated following the induction of oxidative stress.This protein has been demonstrated to be involved in the regulation of cytoprotective genes that are mediated by oxidative stress.And Nrf2 is recognized as an essential upstream transcription factor that regulates HO-1.However,the specific role of Nrf2 in the regulation of HO-1 during preeclampsia has not yet been elucidated.If it is demonstrated that Nrf2 plays a synergistic role with HO-1 in the pathophysiology of preeclampsia,the research will be an interesting work.Part Ⅰ Increased HO-1 and Nrf2 in the Placenta Have a Cooperative Action on Preeclampsia.Objective:This study was conducted to explore the physiological roles of HO-1 and Nrf2 in preeclampsia,so we monitored HO-1 and Nrf2 levels in the placenta of patients suffering from severe preeclampsia,comparing with HO-1 and Nrf2 levels in tissue samples obtained from healthy pregnant females.And we tested the correlation between HO-1 and Nrf2 protein levels.Next,to investigate whether these factors are correlated in preeclampsia,we measured the changes of HO-1 mRNA and protein and Nrf2 protein in HTR-8/SVneo cell line following stimulation with tert-butylhydroquinone(tBHQ).Methods:Serum and placenta were collected from 30 patients presenting with severe preeclarnpsia.Samples were also collected from 30 healthy pregnant females.Serum HO-1 protein levels were determined using human enzyme-linked immunosorbent assay(ELISA)kits.HO-1 protein and HO-1 mRNA and Nrf2 protein expression levels in the placenta were measured.Following stimulation of the HTR-8/SVneo cell line with tert-butylhydroquinone(tBHQ),an Nrf2 activator,Nuclear Nrf2 protein and HO-1 mRNA expression levels were determined.Results:In the severe preeclampsia cases,fetal growth restriction(FGR)occurred in 16.7%of the cases,HELLP syndrome was observed in 20%of the cases,and stillbirth or neonatal birth occurred in 3.3%of the observed cases.Serum HO-1 protein levels were detected by ELISA.HO-1 protein levels were significantly lower in the serum of the severe preeclampsia group,compared to the normal pregnancy group(n = 30,P<0.01).HO-1 mRNA and HO-1 protein levels were increased in placental tissue from patients in the severe preeclampsia group compared with normal pregnant females(n = 30,P<0.05;n = 30,P<0.01).Nrf2 levels were increased in patients with severe preeclampsia compared with normal pregnant women(n = 30,P<0.01).The correlation between Nrf2 protein and HO-1 mRNA levels was tested using Spearman rank correlation analysis.HO-1 protein concentrations in placental tissue were positively correlated with Nrf2 protein levels(n = 60,r = 0.503,P<0.01).Similar results were observed between HO-1 mRNA levels and HO-1(n = 60,r = 0.352,P<0.01)and Nrf2 protein levels(n =60,r = 0.488,P<0.01).IF staining showed that HO-1 was located in the cytoplasm of the HTR-8/SVneo cells and Nrf2 was located in both the nucleus and cytoplasm of the HTR-8/SVneo cells.An increase in Nrf2 protein expression was observed in the HTR-8/SVneo cell line compared with the control cells following stimulation with tBHQ(n = 6,P<0.05).A similar result was observed for HO-1 mRNA levels when the two cell types were analyzed(n = 6,P<0.01).Conclusions:(1)Patients with severe preeclampsia may be protected from oxidative stress following the induction of increased HO-1 and Nrf2 expression.(2)As an upstream regulator of HO-1,Nrf2 is likely to play a synergistic role with HO-1 in the pathophysiology of preeclampsia.Part Ⅱ HO-1 Effects on the Trophoblast Invasion by Oxidative StressObjective:As we have confirmed in part I that Nrf2 is likely to play a synergistic role with HO-1 in the pathophysiology of preeclampsia.To continue to explore the following function of HO-1 in the pathogenesis of preeclampsia,so we established a trophoblastic oxidative stress model of a human first-trimester trophoblast/simian virus(HTR8/SVneo),and assessed the effect of HO-1 on the trophoblast invasion by oxidative stress.Methods:HTR-8/SVneo cell line was stimulated with 100umol/l H2O2 for 12 hours and established a trophoblastic oxidative stress model.The trophoblastic oxidative stress models were incubated with normal saline,Znpp,and Hemin respectively,and then the cells invasion ability of different group were detected by Matrigel invasion assay,and the HO-1 protein expression levels in the cells were measured with Western blot.Results:In the trophoblastic oxidative stress model,HO-1 protein levels were increased in the cells incubated with Hemin(n = 6,P<0.05),compared with the control group;while the HO-1 protein levels were decreased in the cells incubated with Znpp(n = 6,P<0.05).Comparing to the control group,the invasion ability of HTR-8/SVneo cells incubated with Hemin was increased(n = 6,P<0.05),and the the invasion ability of HTR-8/SVneo cells incubated with Znpp was decreased(n = 6,P<0.05).IF staining showed that HO-1 was located in the cytoplasm of the HTR-8/SVneo cells.Conclusions:(1)The inhibitor and inducer of HO-1 can increased and decreased the HO-1 expression in the trophoblastic oxidative stress model.(2)HO-1 can increase the trophoblastic invasion ability by oxidative stress.更多还原