【作者】 欧阳艳琼；

【导师】 陈汉平；

【作者基本信息】 华中科技大学 ， 妇产科学， 2007， 博士

【摘要】 研究背景子痫前期是严重危害孕产妇健康及造成围产期新生儿死亡的重要原因。近年来,对子痫前期的的病因和病理生理有了更深的认识,但其病因和确切发病机制至今尚未阐明,给子痫前期的预防和治疗带来困难。国内外学者提出内皮细胞激活损伤学说、免疫学说、氧化应激学说、炎症学说、一元化学说等试图来阐明子痫前期的发病机制。大量研究表明,子痫前期发病的中心环节为广泛的血管内皮损伤,而氧化应激及氧自由基的产生是内皮细胞受损的重要因素。在缺氧状态下氧化应激及氧自由基生成增加,并且与胎盘低灌注和子痫前期发生相联系。氧化应激及氧自由基进一步加重内皮细胞的损伤,氧自由基和脂质过氧化物能抑制前列环素的产生,促进血栓素A2(TXA2)和一氧化氮(NO)的释放。氧化应激与神经内分泌系统过度激活、内皮功能障碍等病理生理机制相互作用,共同促进疾病的发生、发展,通过多个途径诱发子痫前期。近年来,子痫前期发病的炎症机制再次受到重视,越来越多的学者认识到,子痫前期是母体对妊娠的一种过度性炎症反应,血管内皮细胞损伤是子痫前期患者白细胞、凝血系统和补体系统等多种因素共同参与的血管内过度性炎症反应的一部分。脂联素(adiponectin)作为一种炎性细胞因子与子痫前期的关系引起关注。脂联素作为一种新型脂源性激素,不仅参与调节内皮细胞炎症反应,而且参与脂质代谢和内皮细胞功能的病理生理过程,与炎症反应、胰岛素抵抗、动脉粥样硬化等均有密切关系。许多研究证实子痫前期存在明显的脂质代谢紊乱、胰岛素抵抗、动脉粥样硬化和内皮细胞功能障碍。瘦素(leptin)是脂肪细胞分泌的蛋白质类激素,它在人体内多种组织脏器中均有表达,通过与其受体结合发挥多种生物学效应。瘦素与子痫前期之间的关系已有较多报道,但结论分歧较大。越来越多的证据显示环氧合酶-2(cyclooxyygenase-2,COX-2)与炎症及动脉粥样硬化有关。研究还证实,子痫前期存在炎症和动脉粥样硬化。研究表明COX-1和COX-2在胎盘组织中均有表达,而且,在正常妊娠孕妇胎盘组织中COX-1和/或COX-2水平升高。然而,国外有关子痫前期孕妇胎盘组织中COX-1和COX-2水平或活性的研究,结论分歧较大,既有子痫前期孕妇胎盘组织中COX水平增加的报道,也有研究认为COX在子痫前期胎盘组织中的表达不变,还有学者的结论是降低。国内尚无子痫前期孕妇胎盘组织中COX-1和COX-2水平或活性的研究。我们推测,COX-2作为前列腺素生物合成的限速酶,可能在子痫前期发生和发展中起到重要作用,因而,COX-2在胎盘组织中的水平或活性是否升高值得研究。本课题采用分子生物学和免疫学实验技术检测正常妊娠和子痫前期患者血浆高敏C-反应蛋白(hs-CRP)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)等炎症标志物和8-异前列腺素(8-isoprostane)、丙二醛(MDA)、氧化修饰低密度脂蛋白(ox-LDL)等氧化应激产物的浓度,以求进一步探讨氧化应激和炎症反应在子痫前期中的作用及其相互关系。同时测定血清脂联素和瘦素等脂源性细胞因子在子痫前期中的变化,探索脂源性细胞因子在子痫前期中的作用,为临床预防和治疗子痫前期提供理论依据。本课题还应用逆转录聚合酶链式反应(reverse transcriptional-polymerase chain reaction, RT-PCR)法和免疫组织化学法分别检测子痫前期患者和正常孕妇胎盘组织中COX-2 mRNA和蛋白的表达,以期进一步探讨COX-2在子痫前期发病机制中的作用。本研究分为以下三部分,现分述如下:第一部分氧化应激和炎症反应在子痫前期中的作用及其相互关系目的通过测定子痫前期患者氧化应激产物和炎症标志物的水平,进一步研究子痫前期的发病机制。方法以53例子痫前期孕妇为研究组(其中轻度子痫前期32例、重度子痫前期21例),20例同期分娩的正常孕妇为对照组。检测血浆8-异前列腺素(8-isoprostane)、丙二醛(MDA)、氧化修饰低密度脂蛋白(ox-LDL)和高敏C-反应蛋白(hs-CRP)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的浓度。结果①子痫前期患者血浆8-isoprostane、MDA、ox-LDL分别为(153.07±44.84)pg/ml、(5.25±0.11)μmol/L、(772.04±151.00)μg/L均高于对照组[(82.86±20.91)pg/ml、(4.67±0.38)μmol/L、(431.45±200.69)μg/L,P<0.01、P>0.05和P<0.05];②子痫前期患者血浆hs-CRP、IL-6、TNF-α水平分别为(2.17±1.29)mg/L、(26.49±12.73)pg/ml、(18.47±4.17)pg/ml均明显高于对照组[(1.46±1.00)mg/L、(13.35±5.01)pg/ml、(8.86±1.39)pg/ml,P值均<0.01];③子痫前期患者8-isoprostane与hs-CRP、IL-6、TNF-α呈显著正相关,而MDA及ox-LDL均与hs-CRP、IL-6、TNF-α没有相关性。结论氧化应激及炎症反应可能在子痫前期中发挥重要的作用,干预氧化应激及炎症反应可能有利于控制子痫前期的发生和发展。第二部分脂源性细胞因子与子痫前期关系的研究目的探讨子痫前期患者血清脂源性细胞因子(脂联素、瘦素)水平的变化及其意义。方法以53例子痫前期孕妇为研究组(其中轻度子痫前期32例、重度子痫前期21例),20例同期分娩的正常孕妇为对照组。采用ELISA法检测血清脂联素和瘦素水平。同时检测血清甘油三脂(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平。结果①轻度、重度子痫前期患者血清脂联素水平分别为(8.88±4.67)ng/ml及(5.14±2.79)ng/ml,明显低于对照组(11.61±2.90ng/ml),差异有统计学意义(P均<0.01)。而轻度、重度子痫前期患者血清瘦素水平为(21.79±15.19)ng/ml及(27.27±18.38)ng/ml,明显高于对照组(12.35±6.51ng/ml),差异有统计学意义(P<0.05, P<0.01)。②子痫前期患者血清脂联素与TG、TC、LDL-C、HDL-C均呈显著相关(r值分别为-0.658、-0.624、-0.419、0.461),瘦素水平也与上述指标呈显著相关(r值分别为0.534、0.707、0.418、-0.513)。③子痫前期患者血清脂联素及瘦素水平呈高度负相关(r=-0.760, P<0.01)。结论脂联素、瘦素等脂源性细胞因子在子痫前期的发病中可能发挥一定的作用。第三部分环氧合酶-2在子痫前期患者胎盘组织中的表达目的研究环氧合酶-2(cyclooxyygenase-2,COX-2)在子痫前期患者胎盘组织中的表达,探讨其在子痫前期发病机制中的作用。方法采用免疫组化SP法检测32例子痫前期患者(轻度18例,重度14例)及20例正常孕妇胎盘组织中COX-2的表达。应用逆转录多聚酶链反应(RT-PCR)检测30例子痫前期患者及12例正常孕妇的胎盘组织中COX-2 mRNA的表达。结果(1) COX-2主要表达于胎盘血管内皮细胞。子痫前期孕妇及正常孕妇胎盘组织中COX-2蛋白的阳性表达率分别为65.5%和50.0%,差异无显著性(P>0.05)。(2)子痫前期组COX-2 mRNA表达水平为0.50±0.22,对照组为0.44±0.17,两组比较,差异无显著性(P>0.05)。结论COX-2在子痫前期的作用尚难确定。更多还原

【Abstract】 Background Preeclampsia (PE ) is a complication unique to human pregnancy which cause largely remains to be understood. Although the mechanisms underlying the pathogenesis are not fully understood, oxidative stress and a generalized inflammatory state are features of the maternal syndrome.Up to date, many theories regarding its etiology and pathogenesis have been proposed. Of these theories, endothelial dysfunction, existed before the onset of PE, has been widely considered as playing an important role in the progression of PE. Several lines of evidence support oxidative stress and systemic inflammation are involved in the pathogenesis of PE. Some authorities believe that a systemic maternal inflammatory response to pregnancy is responsible for the endothelial dysfunction. Previous studies have provided evidence that PE results from exaggeration of a maternal systemic inflammatory response common to all pregnancies. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone.The theory is consistent with many of the clinical observations and associations of PE. However, little is known about the association between oxidative stress and levels of inflammatory markers for PE. On the assumption that the effects of inflammatory reaction and oxidative stress and their interactions on vascular endothelial cells in PE patients and consequently contribute to the development of PE, changes in the production of oxidative stress (MDA,ox-LDL and 8-isoprostane)and inflammatory reaction markers (hs-CRP,IL-6,TNF-α) were measured in the plasma to investigate whether these changes would contribute to the occurrence or the development of PE.In addition, PE has many of the same pathophysiologic features as atherosclerosis. Endothelial dysfunction, insulin resistance and inflammation have been recognized features in PE. Many of adipocytes hormones such as tumor necrosis factor (TNF)- , leptin, adiponectin , and interleukin (IL)-6, collectively called adipokines, play important roles in the inflammatory and atherosclerotic processes. Adiponectin, one of the most abundant adipose tissue-specific proteins, is exclusively expressed and secreted from adipose tissue, which has been considered to improve insulin sensitivity and inhibit vascular inflammation and have anti-atherogenic effects. Hypoadiponectinemia has been found and considered as an independent risk factor in hypertension, especially in PE.Like adiponectin, leptin is yet another adipocytokine that is thought to have some role in regulating insulin sensitivity. Previous studies have suggested that leptin is increased in PE. The present study therefore hypothesized that there would be an inverse correlation between adiponectin and leptin in PE.Cyclooxygenase is the key enzyme in the biosynthesis of prostaglandins and thromboxane from arachidonic acid. Two isoenzymes,COX-1 and COX-2, have been identified,the former as a regulator of physiologic functions and the latter as a mediator in pathophysiologic reactions such as inflammation. COX-1 and COX-2 are expressed in the placental tissue samples and several studies have reported that placental levels of COX-2 were increased in the placentas of healthy pregnant women in labor at term. But there are controversial data regarding COX-2 levels or activities in the placentas of women with PE, which have been found increased,unchanged or decreased.The present study is divided into three parts as follows. Part I The Study of Oxidative Stress and Inflammatory Reaction in PreeclampsiaObjective The purpose of the present study was to investigate the changes in plasma markers of oxidative stress and inflammatory reaction in preeclampsia(PE) and to evaluate their clinical significance and interactions in the pathogenesis of preeclampsia. Methods A prospective study was conducted involving 53 women with PE(study group:including 32 cases of mild PE and 21 severe PE) and 20 normotensive pregnant women(control group) in the third trimester. The plasma concentrations of high-sensitive C-reactive protein (hs-CRP),interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha),malondialdehyde(MDA),and 8-isoprostane were determined. Results①The plasma comcentrations of hs-CRP,IL-6,TNF-alpha, 8-isoprostane and ox-LDL were significantly higher in the study group [(2.17±1.29)mg/L, (26.49±12.73)pg/ml, (18.47±4.17)pg/ml,(153.07±44.84)pg/ml, (772.04±151.00)μg/L, respectively] than those of the control(P<0.01,or P<0.05),while no significant differences were noted between the plasma levels of MDA in these two groups [(5.25±0.11)μmol/L vs (4.67±0.38)μmol/L, P>0.05].②The plasma levels of hs-CRP,IL-6,TNF-alpha, and 8-isoprostane were markedly elevated in the severe PE group[(2.84±1.76)mg/L, (27.27±18.38)pg/ml, (22.13±12.35)pg/ml, (163.99±51.01) pg/ml, respectively] than those mild PE group (P<0.01,or P<0.05),while there were no significant differences between the plasma levels of MDA as well as ox-LDL between the MPE and SPE group[(5.49±0.15)μmol/L vs (5.08±0.19)umol/L,P>0.05; (809.29±211.83)μg/L vs (741.66±190.73)μg/L,P>0.05].③Plasma levels of 8-isoprostane were significantly correlated with the plasma levels of hs-CRP,IL-6,and TNF-alpha in PE women (r=0.618,P<0.01; r=0.689,P<0.01;r=0.712,P<0.01;respectively). But plasma levels of MDA were uncorrelated with the plasma levels of hs-CRP,IL-6,and TNF-alpha (r=0.168,P>0.05;r=0.113,P>0.05;and =0.110,P>0.05,respectively). In addition, no significant relationship was observed between plasma levels of ox-LDL and hs-CRP, IL-6 and TNF-alpha in PE women (r=0.140, P>0.05;r=0.134,P>0.05;and r=0.144, P>0.05, respectively). Conclusion Oxidative stress and inflammatory reaction are closely associated with PE, and the interactions play an important role in the pathogenesis of PE. Antioxidant treatment and anti-inflammatory treatment may prevent the onset and progression of PE.Part II The Study of Relationship between Adipocytokine and PreeclampsiaObjective To explore the role of adipocytokine, adiponectin and leptin,in the pathogenesis of preeclampsia(PE). Methods The serum levels of adiponectin and leptin were measured using the enzyme-linked immunosorbent assay in 53 PE patients and 20 normal pregnant women. Seventy-three women were divided into three groups: group A consisted of 20 normotensive pregnant women (NPW); group B consisted of 32 women with mild preeclampsia (MPE); and group C consisted of 21 women with severe preeclampsia (SPE). Results The serum levels of adiponectin in women with MPE or SPE were significantly lower (8.88±4.67 ng/ml,5.14±2.79 ng/ml,respectively)than those in NPW(11.61±2.90ng/ml; bothP<0.01) .While the serum levels of leptin in women with MPE or SPE were significantly higher than those in NPW(MPE: 21.79±15.19 ng/ml versus 12.35±6.51 ng/ml,P<0.05; SPE: 27.27±18.38 ng/ml versus 12.35±6.51 ng/ml,P<0.01). In the pre-eclamptic women serum levels of triglycerides(TG),total cholesterol(TC) and low-density lipoprotein -cholesterol(LDL-C) were significantly increased (P<0.01),while high-density lipoprotein -cholesterol(HDL-C) were significantly decreased compared to levels in NPW (P<0.01). Serum levels of adiponectin were correlated significantly with TG, TC, LDL-C, and HDL-C in pre-eclamptic women (r=-0.658,P<0.01;r=-0.624,P<0.01; r=-0.419,P<0.05; r= 0.461, P<0.05, respectively). In addition,serum levels of leptin were significantly correlated with the serum levels of TG, TC,LDL-C,and HDL-C in pre-eclamptic women (r=0.534,P<0.05;r=0.707,P<0.01;r=0.418, P<0.05;and r=-0.513, P<0.01, respectively). There was a negative correlation between the serum levels of adiponectin and leptin in preeclamptic women (r=-0.760,P<0.01). Conclusion These findings suggest that adiponectin and leptin may be involved in the pathogenesis of PE. Moreover,reduced serum concentrations of adiponectin and elevated leptin are associated with development of severe disease.Part III The Study of COX-2 in PreeclampsiaObjective The purpose of the present study was to investigate the expression of cyclooxyygenase-2(COX-2)in placentas with preeclapsia(PE) and to evaluate its clinical significance. Methods Placentas of 32 PE pregnancies(PE group) and 20 normotensive pregnancies(control group) were investigated for COX-2 protein and mRNA expression using immunohistochemistry and RT-PCR. Results There was no statistical difference in the expression positive rate of COX-2 protein between PE group and control group(65.5% vs 50%, P>0.05). Similarly,no significant difference was noted between the mRNA levels in the PE group and those in control group[(0.50±0.22) vs (0.44±0.17), P>0.05]. Conclusion Our findings suggest that the role of COX-2 in the pathogenesis of PE is uncertain and complicated.更多还原