【作者】 郑静洁；

【导师】 丁依玲；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 子痫前期是严重威胁母儿健康的妊娠期特有疾病,围产儿病死率高。子痫前期根据病情严重程度分为轻度与重度,重度子痫前期患者血压、尿蛋白升高更明显,可伴多器官功能障碍。子痫前期比较明确的病理生理机制包括胎盘床血管重塑不足、胎盘缺血缺氧和系统性血管内皮损伤和(或)激活,但其起始发病原因目前仍不十分清楚。系统性血管内皮功能损伤是子痫前期病理生理机制的中心环节。一氧化氮(NO)作为内皮功能的重要标志物,其表达的减少和(或)活性的降低参与了子痫前期的病理过程。不对称二甲基精氨酸(asymmetric dimethylarginine, ADMA)是一种内源性NOS抑制剂,可减少NO的产生,与多种心血管疾病的发生相关,比如高血压病、糖尿病、高脂血症等。已有研究显示ADMA在子痫前期患者外周血中水平升高,可能通过影响L-精氨酸-eNOS-NO通路参与子痫前期的发病,但其升高的具体机制尚不清楚。最新的研究显示在子痫前期中升高的ADMA可能来源于胎盘组织。二甲基精氨酸二甲基氨基水解酶(dimethylarginine dimethylaminohydrolase, DDAH)作为ADMA的主要降解酶,在子痫前期胎盘组织中表达减少且活性减弱,可能是ADMA含量增多的原因。近年来的研究结果提示子痫前期中雌激素水平下降而孕激素水平升高,但其具体作用尚不清楚。不过有文献报道雌激素作为一种血管内皮保护因子,其水平的减退与多种心血管疾病的发生相关,雌激素可增加NO的产生,故其水平的降低可能通过NO产生减少而参与子痫前期的病理生理学改变过程,但是作用机制尚不明确。至于孕激素对心血管功能的影响的相关报道目前仍存在争议,研究发现高浓度的孕激素对雌激素有拮抗作用,可通过结合糖皮质激素受体而减少NO的合成,从而对抗雌激素的内皮保护功能。最新的研究证实雌二醇有调节ADMA表达的功能,可通过上调ADMA降解酶的活性而抑制ADMA水平升高,但雌二醇在子痫前期中与ADMA的关系尚未见文献报道。重度子痫前期是子痫前期的严重的一种类型,具有病情重、并发症多、围生期结局差、发生子痫风险高等特点,其临床表现如血压明显升高达到160/110mmHg以上,尿蛋白高达5g/24hr以上,以及眼底视网膜小动脉出现痉挛,均为重度子痫前期的重要诊断及监测指标。由于重度子痫前期患者容易并发脑血管意外、心力衰竭、胎盘早剥、产后出血、DIC等并发症,一旦出现,救治难度大大增加,存在较高的致死率和致残率,因此应对其进行及时诊断和积极治疗。最新的子痫前期临床指南提倡通过筛查方法做好早期诊断工作,为此,临床医生需要更便捷、快速、可靠、敏感度和特异度均较高的指标对疾病进行诊断、监测以及评价治疗效果。胎盘的病理生理改变与子痫前期的发病密切相关,缺血缺氧的胎盘组织中ADMA的表达水平如何?与其在血清的表达是否存在不同或相关性?胎盘缺血缺氧导致的合体滋养层细胞功能异常,是否合成和分泌水平异常的雌孕激素到母体外周循环中?其含量的变化与ADMA之间是否存在关系?而外周血中ADMA、雌孕激素与重度子痫前期的重要临床症状是否相关?可否用于疾病的诊断?目前尚无文献报道。故本课题拟通过一系列的研究对上述问题进行探讨,并试图为诊断重度子痫前期及监测病情进展和治疗效果提供新指标。第一章重度子痫前期患者外周血及胎盘组织中ADMA的表达目的：研究重度子痫前期患者外周血及胎盘组织中ADMA的表达水平及二者的相关性,探讨ADMA在重度子痫前期中的作用及可能来源。方法：1.对象分组：选择2012年7月至2013年3月在湖南省中南大学湘雅二医院门诊就诊或住院治疗并诊断为重度子痫前期的单胎孕妇共62例(P组),选择同期在我院进行产前检查或住院分娩的血压正常的单胎正常妊娠妇女(C组),共75例。2.资料与标本的收集与处理：(1)记录入选对象的年龄、籍贯、孕周等信息；(2)收集入选孕妇外周静脉血标本,离心后取血清待测；(3)选择上述入组对象中在我院行子宫下段横切口剖宫产者30例,其中重度子痫前期15例,正常对照15例,胎盘娩出后,立即在无菌条件下采集新鲜胎盘脐带根部周围大小约1cm×1cm×2cm的组织,匀浆后取上清液待测。3.血清及胎盘匀浆液ADMA的检测：采用酶联免疫吸附法(ELISA)测定。4.统计学分析：采用Excel2007和PASW Statistics18软件进行统计学分析。两两比较采用独立样本和配对样本-t检验或Mann-Whitney u检验。相关性分析采用Spearman相关分析。所有检验结果以P<0.05为差异有统计学意义。结果：P组血清ADMA水平为1.90±0.21μmol/l,C组血清ADMA水平为1.30±0.16μmol/l,两者间差异有统计学意义(P=0.000)。P组和C组胎盘组织匀浆液(n=15,15)中ADMA含量分别为2.42±0.23pmol/l、1.98±0.19μmol/l,二者有显著性差异(P=0.000)。另外,P组胎盘匀浆液中ADMA的含量明显高于其血清中的浓度(P=-0.000),且两者呈显著正相关(r=0.79P=-0.000)。结论：重度子痫前期孕产妇胎盘组织匀浆及母体外周血ADMA浓度均明显增高,前者显著高于后者,二者呈显著正相关,提示重度子痫前期患者ADMA可能主要为胎盘分泌,并在其发病中起作用。第二章重度子痫前期患者外周血中雌二醇、孕酮的表达目的：研究重度子痫前期患者体内雌激素与孕激素表达水平随着孕周的变化趋势及二者之间是否存在异常比例关系,并结合本课题第一章ADMA的研究结果,研究雌孕激素与ADMA之间的相关性,初步探讨雌孕激素在重度子痫前期中对ADMA的作用及可能机制。方法：1.对象分组：选择2012年7月至2013年3月在湖南省中南大学湘雅二医院门诊就诊或住院治疗并诊断为重度子痫前期的单胎孕妇共62例(P组),按照孕周分为P1小组(孕28+1-32周,n=22)、P2小组(孕32+1-36周,n=20)、P3小组(孕36+1-40周,n=20)。选择同期在我院进行产前检查或住院分娩的血压正常的单胎正常妊娠妇女(C组),共75例,亦按照孕周分为C1小组(孕28+1-32周,n=25)、C2小组(孕32+1-36周,n=25)、C3小组(孕36+1-40周,n=25)。2.资料和标本的收集与处理：(1)记录入选对象的年龄、籍贯、孕周等信息；(2)收集入选孕妇外周静脉血标本,离心后取血清待测。3.血清雌二醇、孕酮的检测：采用化学发光免疫法(CLIA)测定。4.血清及胎盘匀浆ADMA的检测：同第一章。5.统计学分析：采用Excel2007和PASW Statistics18软件进行统计学分析。两样本比较采用独立样本和配对样本t检验或Mann-Whitney u检验,多样本比较采用方差分析或Kruskal-Wallis检验。相关性分析采用Spearman相关分析。所有检验结果以P<0.05为差异有统计学意义。结果：1.两组血清样本中雌二醇、孕酮含量的比较在正常妊娠组中,C1小组(孕28+1-32周,n=25)、C2小组(孕32+1-36组,n=25)、C3小组(孕36+1-40周,n=25)的血清雌二醇含量分别为：13.31±2.20ng/ml、19.13±1.81ng/ml、22.76±3.17ng/ml,孕酮的含量分别为：105.76±14.56ng/ml、138.76±13.72ng/ml、182.68±17.81ng/ml,二者均呈递增趋势。而在重度子痫前期分组中,P1小组(孕28+1-32周,n=22)、P2小组(孕32+1-36组,n=20)、P3小组(孕36+1-40周,n=20)的血清雌二醇含量分别为：11.71±1.97ng/ml、13.33±2.60ng/ml、17.28±2.88ng/ml,孕酮的含量分别为：117.03±17.47ng/ml、149.39±27.70ng/ml、192.16±30.55ng/ml,二者的递增趋势仍然存在,然而,P1与C1小组之间、P2与C2小组之间、P3与C3小组之间,雌二醇含量分布均存在显著差异(P<0.05)；对于孕酮,仅在P1与C1小组之间存在显著差异(P<0.05),而在P2与C2小组之间、P3与C3小组之间,虽有升高,但差异不显著(P>0.05)。2.两组血清样本中孕酮/雌二醇比值的差异比较将每个血清样本中测得的孕酮和雌二醇做比值运算,结果得出重度子痫前期组(P组,n=62)孕酮/雌二醇(Pg/E2)比值为10.70(10.14-11.52),正常妊娠对照组(C组,n=75)Pg/E2比值为7.62(7.30-8.14),两者比较差异非常显著(P=0.000)。3.两组血清样本中雌二醇、孕酮、Pg/E2比值与ADMA含量之间的相关性分析重度子痫前期组(P组,n=62)和正常妊娠对照组(C组,n=75)中,单独E2或Pg与血清ADMA之间均无显著性相关(r=0.04P=0.785, r=0.015P=0.897; r=0.24P=0.058, r=0.20P=0.094),而Pg/E2比值与ADMA水平之间均存在显著正相关性(r=0.77、0.57P=-0.000)。结论：1.重度子痫前期患者外周血中雌二醇水平显著降低,孕酮水平仅在孕28+1-32周间有显著升高,32周以后无显著性变化,而孕酮/雌二醇比值显著增加,故雌二醇水平降低、雌孕激素比例失调可能参与重度子痫前期的发病过程。2.重度子痫前期患者外周血中孕酮/雌二醇比值与外周血中ADMA浓度呈显著性高度正相关,提示雌孕激素比例失调可能通过加重ADMA水平的升高而参与重度子痫前期的发生发展过程。第三章外周血ADMA及孕酮/雌二醇比值与重度子痫前期临床表现的相关性及其在疾病评估中的应用目的：研究ADMA的表达水平及孕酮/雌二醇比值的变化与重度子痫前期患者临床症状的相关性,为评估疾病进展及治疗效果提供新指标,并为寻找诊断重度子痫前期的理想指标提供初步依据。方法：1.研究对象及分组：实验分组同第一章。2.研究方法：2.1资料与标本的收集与处理：(1)记录入选对象的年龄、籍贯、孕周等信息；(2)测量入选所有孕妇的右肱动脉血压(休息状态下反复测量3次,取平均值),同时检测重度子痫前期组患者24小时尿蛋白水平。另外,对重度子痫前期组对象进行眼底检查,观察视网膜小动脉的痉挛程度,视网膜动静脉比值1：2以上、视乳头水肿、絮状渗出或出血以及视网膜剥离等病理改变视为眼底血管病变阳性,记为1,无上述病理改变视为眼底血管病变阴性,记为0。2.2血清及胎盘匀浆液ADMA的测定：同第一章。2.3血清雌二醇、孕酮的测定：同第二章。3.统计学分析：采用Exce120、PASW Statistics18和MedCalc V9.6.4.0软件进行统计学分析。两样本比较采用独立样本和配对样本t检验或Mann-Whitney u检验。相关变量之间的相关性分析采用Spearman相关分析。利用MedCalc统计学软件进行诊断重度子痫前期的ROC曲线分析。所有检验结果以P<0.05为差异有统计学意义。结果：1.一般资料的比较及临床表现分析P组收缩压、舒张压水平分布明显高于C组,而且,P组24小时尿蛋白含量为3891.60(1127.24-7940.65)mg。有61例重度子痫前期患者进行了眼底检查,其中16例患者眼底血管病变阳性,发生率为25.8%。2.重度子痫前期组患者血清ADMA水平及Pg/E2比值与其临床表现之间的关系重度子痫前期组患者血清ADMA含量与患者收缩压、舒张压的相关系数分别为0.422(P=0.001)、0.292(P=0.021),血清Pg/E2比值与患者收缩压的相关系数为0.260(P=0.041),而ADMA含量与Pg/E2比值与患者24hr尿蛋白与眼底血管病变之间并无显著性相关(P>0.05)。3.血清ADMA水平及Pg/E2比值对重度子痫前期的诊断价值ROC曲线分析结果显示：血清ADMA的ROC曲线下面积(AUC)为0.997(P=0.000,CI：0.968-1.0),最佳筛选界值为1.56μmol/l,敏感度为98.4%,其特异度为86.0%。而血清Pg/E2比值的ROC曲线下面积(AUC)为0.993(P=0.000,CI：0.961-0.999),最佳筛选界值为9.05,敏感度为98.4%,特异度为95%。所以我们认为外周血ADMA和Pg/E2比值可能是诊断重度子痫前期的较理想指标,当外周血清中ADNA水平超过1.56μmol/l或者Pg/E2比值超过9.05时,要警惕重度子痫前期的发生。结论：1.重度子痫前期外周血ADMA表达水平与收缩压、舒张压呈正相关,Pg/E2比值与收缩压呈正相关,提示二者在一定程度上可反映疾病血压变化情况。2.外周血ADMA表达水平及Pg/E2比值可能对重度子痫前期具有潜在诊断价值。更多还原

【Abstract】 Preeclampsia is a pregnancy-specific disorder and an important cause of maternal and fetal morbidity and mortality worldwide. It can be further differentiated into mild and severe forms. Patients with severe preeclampsia have severe hypertension, proteinuria, or evidence of other end-organ dysfunction. It remains unclear about the detail pathogenesis of preeclampsia, which make this complex disorder has no effectively treatment approaches. Investigations into the pathophysiology of preeclampsia have provided exciting new insights into potential mechanisms and continue to move the field forward. The initiating event in preeclampsia is generally regarded to be placental-bed vascular insufficient remodeling and then placental ischemia/hypoxia, resulting in the transferring of a variety of factors from the placenta to maternal circulation and systemic vascular endothelium dysfunction and (or) activation.Asymmetric dimethylarginine (ADMA) is a critical endogenous inhibitor of nitric oxide synthetase (NOS). Elevations of ADMA have previously been associated with endothelial dysfunction in preeclampsia. ADMA is degraded mainly by dimethylarginine dimethylaminohydrolase (DDAH) which is reported as reduced expression and activity in preeclampsia.Recent studies show that estrogen and progesterone levels are abnormal in preeclampsia, but there lacks of strong evidence and the specific mechanism is unclear. Over the years generous literatures reported that decrescence of estrogen is related with a variety of cardiovascular diseases like hypertension. So far, the effects of progesterone on cardiovascular function remain controversial. Studies suggest that progesterone may adopt with estrogen synergistically or antagonistically to participate in the regulation of cardiovascular function, however, the mechanism of interaction of progesterone and estrogen in cardiovascular disease is not clear. Research has shown that high levels of progesterone may inhibit eNOS gene transcription and activity through combining with glucocorticoid receptor, and thus NO synthesis decreased, affecting the regulation of blood pressure. The latest research confirms estradiol alleviates asymmetric dimethylarginine (ADMA) increase responded on the reduced expression and activity of dimethylarginine dimethylaminohydrolase(DDAH) through estrogen receptora.During preeclampsia, pathophysiological change of the placenta is the initial onset. Whether there is a relationship of ADMA expression in the placenta and maternal circulation? Whether the dysfunction of syncytiotrophoblast caused by placental ischemia/hypoxia will induce abnormal synthesis and secretion of estrogen and progesterone into the maternal circulation or not? Whether the change of the concentration of estrogen and progesterone are correlated with ADMA level or not? Whether the changes of the levels of estrogen, progesterone and ADMA in maternal peripheral blood is relevant to the clinical manifestation or not? There are currently quite limited studies in the literature.Chapter I Serum and Placental Expression of ADMA in Severe PreeclampsiaOBJECTIVE:To research the serum and placental expression of ADMA in severe preeclampsia and their correlation and to approach the possible role of placenta on ADMA regulation in severe preeclampsia.METHODS:There were62severe preeclampsia patients selected from Second Xiangya Hospital outpatient. At the same period a total of75normotensive pregnant women were selected.30cases were selected among the above women who had a cesarean section in our hospital and their placenta samples were collected. Enzyme-linked immunosorbent assay (ELISA) method was used for measurement of ADMA in maternal serum and placenta.RESULTS:Serum ADMA levels in severe preeclampsia and normal pregnancy control group were1.90±0.21μmol/L and1.30±0.16μmol/L (P=0.000). Moreover, the ADMA content in the homogenate of placental in P and C groups (n=15,15) were2.42±0.23μmol/L,1.98±0.19pmol/L (P=0.000). The placental-homogenate ADMA level in P group is significantly higher than the maternal serum concentration (P=0.000) and both were positively correlated (r=0.65, P=0.009).CONCLUSION:Concentration of ADMA was significantly higher both in the homogenate of placenta and maternal peripheral circulation of severe preeclampsia, and higher content of ADMA in the homogenate of placenta than in the maternal serum was observed as well. There was a positive correlation between the level of ADMA in maternal serum and the homogenate of placenta of severe preeclampsia.Chapter II Serum Estradiol and Progesterone Expression in Severe PreeclampsiaOBJECTIVE:To study serum estradiol and progesterone expression in severe preeclampsia and to investigate their roles in the development of severe preeclampsia. Then to analyze the correlation of serum estradiol, progesterone and ADMA for further understanding of their effect and mechanism in the pathogenesis of severe preeclampsia.METHORDS:There are62severe preeclampsia patients selected from Second Xiangya Hospital outpatient and they are divided into P1sub-group (28+1-32weeksof pregnancy, n=22), P2sub-group (32+1-36weeks of pregnancy, n=20), P3sub-group (36+1-40weeks of pregnancy, n=20) depending on the gestational age. At the same period a total of75normotensive pregnant women were selected and also divided by gestational age into C1sub-group (28+1-32weeks of gestation, n=25), C2sub-group (32+1-36weeks of gestation, n=25), C3sub-group (36+1-40weeks of pregnancy, n=25). Use the chemiluminescence immunoassay (CLIA) method for the determination of serum concentration of estradiol (E2) and progesterone (Pg).RESULTS:1. During the normal pregnancy group, the serum levels of E2in C1, C2, C3sub-groups were:13.31±2.20ng/ml,19.13±1.81ng/ml and22.76±3.17ng/ml respectively and the Pg concentrations were as follows:105.76±14.56ng/ml,138.76±13.72ng/ml and182.68±17.81ng/ml, both of E2and Pg showed an increasing trend following the increasing of gestational age. During severe preeclampsia group, serum estradiol levels of P1, P2, P3sub-groups were:11.71±1.97ng/ml,13.33±2.60ng/ml,17.28±2.88ng/ml, and progesterone content:117.03±17.47ng/ml,149.39±27.70ng/ml,192.16±30.55ng/ml. Both of E2and Pg levels in severe preeclampsia persist the increasing tendency with increasing gestational age, however, between P1and C1sub-groups, P2and C2sub-groups, P3and C3sub-groups, there are significant differences of the distribution of estradiol levels (P<0.05); for progesterone, the only significant difference was observed between P1and C1sub-groups (P<0.05); between P2and C2sub-groups, P3and C3sub-groups, the difference was not significant (P>0.05).2. Progesterone/estradiol (Pg/E2) ratio in severe preeclampsia group (P group, n=62) was10.70(10.14-11.52) which is significantly higher than the Pg/E2ratio in normal pregnancy control group (C group, n=75)(P=0.000).3. In both severe preeclampsia group (P group, n=62) and normal pregnancy control group (C group, n=75), separate E2or Pg has no significant correlation with serum ADMA (r=0.04P=0.785, r=0.015P=0.897; r=0.24P=0.058, r=0.20P=0.094), while there is a significant high positive correlation between Pg/E2ratio of serum ADMA levels (r=0.77,0.57P=0.000).CONCLUSION:1. The estradiol concentration in the peripheral blood serum of severe preeclampsia women continued persistently and significantly reduced level during the third trimester of pregnancy while the progesterone/estradiol (Pg/E2) ratio was significant higher than normal pregnancy women. Therefore, reduced serum estradiol concentration and imbalanced Pg/E2radio may be involved in the pathogenesis of preeclampsia.2. The Pg/E2ratio was positively correlated with the serum ADMA concentration in the peripheral blood of women with severe preeclampsia. Chapter III Correlation between ADMA, Progesterone/Estradiol radio and Clinical Manifestations in Severe Preeclampsia and Their Possible Application in Disease AssessmentOBJECTIVE:To research the correlation between ADMA, Progesterone/Estradiol radio and clinical manifestations in severe preeclampsia for providing new indicators for the assessment of disease progression and treatment. Then to screen important factors in preeclampsia and to provide a preliminary basis for the establishment of sever preeclampsia ideal diadynamic criteria.METHODS:The patient group and normal control group were split as the same as Chapter II. Besides, blood pressure was measured for every woman. For patient group, proteinuria during24hr was also collected and fundus examination was undertaken. In addition, by fundus examination in severe preeclampsia group, observation of pathological changes such as retinal artery spasm, more than1:2of retinal arteriovenous ratio, papilledema, flocculent exudate or hemorrhage and retinal detachment was regarded as positive retinal blood vessels lesions, denoted by1, otherwise, it was treated as negative retinal blood vessels lesions, denoted by0.RESULTS:1. Serum ADMA level in patients with severe preeclampsia has a moderate positive correlation with systolic blood pressure(r=0.422P=0.001) and slight positive correlation with diastolic blood pressure (r=0.292P=0.021). The Pg/E2ratio in the maternal serum of severe preeclampsia has a low positive correlation with systolic blood pressure the patient, the correlation coefficient was0.260(P=0.041).2. ROC curve showed that the area under the curve (AUC) of serum ADMA was0.997(P=0.000,95%CI:0.968-1.0) with98.4%sensitivity and86.0%specificity; the AUC of serum Pg/E2radio was0.993(P=0.000, CI:0.961-0.999) with98.4%sensitivity and95%specificity.CONCLUSION:1. There was a significant positive correlation between serum ADMA level and systolic blood pressure and diastolic blood pressure of severe preeclampsia patients, and the Pg/E2radio was positively correlated with systolic blood pressure of severe preeclampsia patients, suggesting that serum Pg/E2ratio and ADMA levels may provide the basis for the assessment of disease severity.2. Maternal serum ADMA levels and Pg/E2ratio were closely related with severe preeclampsia and may have diagnostic value.7figures,6tables and54references更多还原