血管紧张素Ⅱ AT1受体自身抗体在子痫前期患者血清中的分布及其对血管活动的影响
|
摘要
研究背景
子痫前期(Preeclampsia，PE)是妊娠期特发的全身性疾病，多在妊娠20周以后发病。临床表现以高血压、蛋白尿为特征，并伴有全身多脏器的损害，其基本病理生理改变是全身小动脉的痉挛性收缩。流行病学调查结果显示，子痫前期的发病率为5-10％，是母婴发病率及死亡率较高的主要原因。子痫前期对孕产妇的影响主要表现为胎盘早剥、肺水肿、凝血功能障碍、产后出血及产后血循环衰竭等并发症，严重者可致死亡：对胎儿多是由于胎盘供血不足，胎盘功能减退导致胎儿窘破、胎儿发育迟缓、死胎、死产或新生儿死亡。目前，对子痫前期普遍认同的病因学说主要有免疫学说、子宫-胎盘缺血学说、血管调节物质异常、遗传学说等，但是相关治疗措施虽能改善病人急性和慢性期的症状，但不能从根本上逆转疾病的进展和最终防止子痫前期的发生，提示在子痫前期发生发展过程中可能还有其它未知因素的参与。
20世纪90年代早期，Hanssens和Haller等先后发现并证实，子痫前期患者血液中存在一种作用类似血管紧张素Ⅱ(AngiotensinⅠ，Ang-Ⅱ)的循环因子，使血管紧张素Ⅱ-1型受体(AngiotensinⅡType 1 Receptor，AT1R)功能增强。1999年，Wallukat等证实这种类Ang-Ⅱ因子是抗ATlR自身抗体(AngiotensinⅡtype 1 Receptor Autoantibodies，AT1-AA)。随后的研究也相继证实AT1-AA存在于子痫前期患者的血清中，但是，研究的标本数较少，此外，没有该抗体存在与子痫前期体征关系的分析，即缺乏AT1-AA是否与子痫前期有关的分析资料，而这一问题的解决是研究AT1-AA在子痫前期发病中作用的前提。
随后的研究表明，这种自身抗体通过专一识别AT1R的细胞外第二环功能表位肽段(thesecond extracellular loop of the angiotensinⅡtype 1 receptor，AT1R-EC_Ⅱ，人鼠同源性为99％)促进血管平滑肌细胞增殖、NADPH氧化酶活化、ROS生成、细胞浆内Ca~(2+)浓度增加和激活纤溶酶原激活物抑制因子1。这些研究结果均提示，AT1-AA的过度活动与许多重要的病理生理改变有关，可能在子痫前期的发病中起着重要作用。由于子痫前期危害性极大而病因又极其诡异，因而这一成果的重要性和亟待被深入研究的必要性被国内的学者迅速而敏锐地捕捉到，并对此展开了广泛的研究。其中尤以廖玉华实验室的研究最为深入，他们的研究证实，AT1-AA通过激活AT1受体诱导大鼠。肾脏和心肌组织的免疫损伤并促进大鼠血管平滑肌细胞增殖。我实验室的前期研究也发现，抗AT1R-EC_Ⅱ抗体通过作用AT1R增强离体灌流心脏的心功能、升高心肌细胞内游离钙含量、增加心肌细胞I_(Ca-L)电流、激动I_(Na／Ca)电流和减小I_(k1)电流等。这些结果进一步提示，AT1-AA具有AT1受体类激动剂样效应。
虽然AT1R广泛分布于人体各组织器官，介导了目前已知Ang-Ⅱ的所有作用(如收缩血管、促进醛固酮分泌、刺激血管平滑肌细胞增殖和水钠潴留等)，但AT1R对脉管系统的作用尤为突出。因此我们推测，具有AT1受体激动剂样效应的AT1-AA是否也具有同样的血管效应呢?如果是，那么AT1-AA是否象AT1受体激动剂那样，也具有致血管病变的病理意义呢?但是到目前为止，已有的研究报道(包括我们的前期工作)仅局限于通过急性实验来探讨AT1-AA作用的微观机制，但缺乏AT1-AA对各类血管(尤其是阻力血管)作用的直接证据，而对这一问题的阐明是认识AT1-AA在子痫前期发生发展中作用和研究AT1-AA作用机制的关键。
如前所述，AT1R广泛分布于人体各组织器官。在血管系统，AT1R除分布在血管平滑肌外，还广泛分布在血管内皮细胞，后者在调节血管收缩舒张功能中具有重要的作用。如果存在于血液中的AT1-AA随血液循环在血管内流动时，由于其具有类AT1受体类激动剂样效应，那么AT1-AA应该可以和血管内皮细胞上的AT1R结合。但是，这一推测需要实验来证实。众所周知，在体情况下血管内皮损伤后，会导致流经损伤部位的血液中诸多血管活性物质的聚集(如血小板、凝血因子等)并引发随后的释放反应，这些病理过程都有利于动脉粥样硬化斑块的形成。在子痫前期患者发病过程中，一个典型的病理改变是子宫螺旋小动脉存在“类粥样硬化样”改变，因此导致胎盘血流量下降，直接危害到胎儿的健康；此外，另一个典型病理改变是肾小球血管内皮下大量纤维样物质沉积，进而造成血管管腔狭窄、肾脏血液灌注减少和肾小球滤过率下降等后果。但是，多年以来，导致这种血管内皮细胞病理改变的机制一直不清。因此，如果能够通过直接的实验证据说明AT1-AA是导致“类粥样硬化样”病变的原因将有助于认识子痫前期血管病变的机制。
综上所述，我们设计了本研究课题：(1)通过临床流行病学调查，追踪子痫前期患者血清AT1-AA的变化，观察该抗体水平和病情相关性，明确该抗体的临床意义。(2)在此基础上开展实验室研究，首先以人工合成的AT1R-EC_Ⅱ主动免疫大鼠制备抗AT1R-EC_Ⅱ抗体，然后通过离体大血管和微血管环技术明确该抗体对大鼠胸主动脉、主要脏器的小动脉(包括冠状动脉、大脑中动脉和肾动脉)、主要阻力血管(肠系膜动脉)和反应子宫-胎盘血流量的血管(子宫动脉)等不同类型小动脉的直接作用。(3)以血管内皮的结构和功能为切入点，通过离体细胞培养技术研究抗AT1R-EC_Ⅱ抗体对内皮细胞的损伤效应和形式；并将抗AT1R-EC_Ⅱ抗体转移进入正常大鼠体内建立大鼠被动免疫模型，然后通过检测血清中内皮素的含量来观察抗AT1R-EC_Ⅱ抗体对在体血管内皮的损伤作用；通过大血管和微血管环技术明确该抗体在在体情况对血管活动的影响。以期阐明AT1-AA致血管类动脉粥样硬化改变的可能性及其防治的意义。
本课题拟验证的假说：子痫前期患者血清中存在的AT1-AA，有可能通过影响血管结构和功能而与子痫前期发生发展相关。(1)在子痫前期患者血清中的AT1-AA与该病的某些特殊指征呈一定的相关性；(2)AT1-AA对大血管具有直接的收缩效应，但对不同小动脉可能表现各不相同；(3)AT1-AA通过作用于AT1R细胞外第二环，引起血管内皮细胞损伤，或许可以进而诱发血管“类动脉粥样硬化”改变。所有这些结构和功能变化可能与子痫前期患者所表现的血压升高、组织器官血流量下降以及和其它病理学改变密切相关。
一、子痫前期患者血清中血管紧张素ⅡAT1受体自身抗体的检测及其免疫学特征分析
目的
本工作首次报道了AT1-AA在子痫前期患者血清中的分布规律并分析了该抗体与子痫前期患者典型体征的关系。
方法
1．检测对象：子痫前期患者82例，年龄26-33岁，正常孕妇56例，年龄23-34岁，未孕健康妇女48例，年龄24-23岁。子痫前期的诊断符合ISSHP2001年标准，即妊娠20周以后出现，血压≥140／90mmHg，且尿蛋白≥0．3g／24h或(+)，可伴有上腹部不适、头痛、视力模糊等症状(见表1)。以上三组均排除内分泌及自身免疫性疾病。
2．AT1受体抗原多肽的合成：按照人AT1受体细胞外第二环功能表位肽段序列(165-191位，见表2)，由西安联美生物科技有限公司合成(肽段合成的质量报告单见附录)。用于血清抗体的定性与定量测定、血清抗体的功能测定。
3．血清自身抗体的测定：用间接SA-ELISA(Streptavidin-ELISA)方法，抗原包被用量为AT1抗原10μg/ml。主要技术流程如下：包被合成的抗原多肽→加入人的血清→加入标记有生物素的抗人IgG抗体→加入标记有辣根过氧化物酶的亲和素→加入辣根过氧化物酶酶的底物→在酶标仪上(Molecular Devices Corp，Menio Park，CA，USA)测定反应物的光密度值(OD)，以OD值的大小代表人血清中抗多肽抗体的量。
4．结果判定：以P／N≥2．1为阳性，P／N≤1．5为阴性，P／N=(标本OD值-空白对照OD值)／(阴性对照OD值-空白对照OD值)；将血清标本从1：20起连续倍比稀释，以出现P／N≥2．1的最高稀释度作为该标本的效价。
5．统计处理：抗AT1R抗体在不同组间的分布用阳性率表示；每组抗体水平的平均值用几何平均数(Geometric mean)来表示；用有序2×C表资料直线回归的显著性检验进行抗体阳性率年龄趋势的检验；用U检验进行两样本阳性率的差别检验；两几何均数之间的差别检验用t检验：率的检验用经平方根反正弦变换后的数据进行；几何均数的检验用经对数转换后的数据进行。P值小于0．05认为有显著差异。
结果
1．82例子痫前期患者的血清中，抗AT1受体自身抗体的阳性率为69％±2．9％，显著高于56例正常孕妇和48例未孕健康妇女血清中AT1受体自身抗体的阳性率(分别为29％±2．1％和8.3％±0．9％，P<0．01)，正常孕妇和未孕健康妇女血清中AT1-AA的阳性率相比也有统计学差异(29％±2．1％vs．8．3％±0．9％，P<0．05)(见图1、图2)。
2．子痫前期患者抗体阳性血清的抗体效价为1：168．6±6．7，远高于正常孕妇和未孕健康妇女抗体阳性血清的抗体效价(分别为1：52．9±2．1和1：16．2±1．8，p<0．01)(图3)。
3．子痫前期患者血清中AT1受体自身抗体的滴度随孕妇收缩压的增高而呈上升趋势，(图4，r=0．856)。
4．子痫前期患者血清中AT1受体自身抗体的滴度和孕妇蛋白尿程度及血小板计数无相关(r=-0．013，r=0．205)。
结论
1．本研究结果证实，子痫前期患者血清中抗AT1-AA的分布特征是高阳性率、高抗体滴度，提示AT1-AA和子痫前期有着密不可分的关系；正常孕妇血清中AT1-AA的阳性率也明显高于同龄未孕健康妇女，提示怀孕过程中有免疫机制的参与；
2．子痫前期时AT1-AA滴度随孕妇收缩压的增高而呈上升趋势，提示AT1-AA可能与子痫前期患者血压增高有关；
3．AT1-AA滴度和子痫前期患者蛋白尿程度及血小板计数无相关性，这个结果提示：(1)可能样本例数较少，还不足以揭示AT1-AA滴度和蛋白尿程度及血小板计数之间真正的关系：(2)AT1-AA可能通过其他的途径导致了子痫前期患者肾脏的损害和凝血功能的障碍，而非直接引起蛋白尿的生成和血小板的异常。
二．血管紧张素ⅡAT1受体自身抗体对血管功能的影响
目的
本实验主要目的是：1)采用大血管环技术，观察AT1-AA对大鼠容量血管收缩功能的作用，分析该自身抗体对患者血压的影响；2)采用微血管环技术，观察AT1-AA对主要脏器的小血管(包括冠状动脉、大脑中动脉和肾动脉)的直接作用，明确该自身抗体对患者主要脏器的影响；3)采用微血管环技术，观察AT1-AA对子宫动脉的作用，阐明该抗体对胎盘血流量的影响；4)采用微血管环技术，观察AT1-AA对肠系膜动脉的作用，证实该抗体与外周阻力血管功能改变之间的关系。
方法
1．AT1-AA的制备：利用MAb Trap Kit试剂盒对子痫前期患者抗体阳性者血清中的抗体IgG进行提取和纯化。纯化后的抗体经PAGE胶凝胶电泳检测其纯度。具体方法如下：
(1)特异性亲和柱试剂盒提纯抗体：将合成的人ATlR-EC_Ⅱ抗原肽段连于亲和柱内，至少3ml结合缓冲液平衡亲和柱(流速0．5ml／min)，0．5ml血清同等体积binding buffer稀释后，使用注射器加入柱体中，然后用7ml binding buffer冲洗，5ml洗脱液进行洗脱，洗脱下来的蛋白液保存于加有中性液(1M Tris)的EP管中，保持蛋白活性，至少用5ml bindingbuffer对亲和柱进行复性，然后进行下一轮操作。
(2)蛋白定性及定量测定：PAGE胶凝胶电泳检测其纯度后，按照BCA试剂盒步骤要求测定样品蛋白含量。
对阳性血清中的抗体IgG进行提取后剩余的IgG为nsIgG(nonspecific IgG)，作为阴性对照；从正常孕妇阴性血清中提取的IgG为npIgG(non-preeclamptic IgG)，也作为阴性对照。
2．大鼠胸主动脉环制备：取雌性Wistar大鼠，体重240-260g，击头致昏，立即开胸取胸主动脉置于O_2饱和的4℃营养液PSS中。(PSS成分如下(mmol／L)：NaCl154．7；KCl5．4；Glucose 11．0；CaCl_2 2．5；Triss 6．0。无钙PSS成分为(mmol／L)：NaCl 154．7；KCl 5．4：Glucose 11．0；EGTA 0．5；Triss 6．0。用HCl调pH至7．4。)剔除脂肪及周围结缔组织后，剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔，水平悬挂在浴管内，下方固定，上方以一细钢丝连于张力换能器(JH-2，北京)。经MS4000计算机生物信号采集分析系统记录血管的张力变化。浴管内含有通以100％O_2、37℃的PSS液10 ml。每个血管环悬挂在浴管后，基础张力调至2g，平衡1h，其间不断调整张力，使之维持在2g左右，每15 min换一次PSS。所有动脉环用60 mmol／L KCl多次刺激，当标本对刺激稳定时，即连续2次同样的刺激所引起的收缩幅度差别<5％时，开始正式实验。本实验所述浓度均为10ml浴管内试剂的终浓度。
3．AT1-AA对大血管收缩功能的影响：在大鼠离体胸主动脉血管环标本上，观察AT_1-AA对于大血管平滑肌活动的影响。分组为自身抗体组(preeclamptic IgG组)，血管紧张素Ⅱ(AngⅡ)阳性对照组，nsIg和npIgG阴性对照组，以及preeclamptic IgG+losanrtan(AT_1受体拮抗剂)组和preeclamptic IgG+SEL-AT1R(AT1受体细胞外第二环表位肽段)组。
4．大鼠肠系膜动脉、肾动脉、大脑中动脉、冠状动脉和子宫动脉血管环的制备：大鼠脱臼后，立即取出肠系膜、肾脏、心脏、大脑和子宫，浸入4℃的PSS液中。肠系膜动脉环的制备：将肠系膜浸入含有PSS液的平皿，用大头针固定动脉主干和肠管，分离动脉周围的脂肪组织，选取3级分支，剪取2mm左右的血管环。肾动脉环的制备：先用大头针固定肾脏于盛有PSS液的平皿，肾组织中动脉较韧，可用镊子直接剥碎肾组织暴露肾动脉各级分支，沿肾动脉剥离动脉的三级分支，分离干净周围的组织，剪取2mm左右的血管环。大脑中动脉环的制备：用骨钳揭开颅骨，取出脑组织，将脑组织贴近颅底面向上固定于盛有PSS液的平皿，可清楚的看到颈内动脉的延续-大脑中动脉，剥离大脑膜，钝性分离大脑中动脉，在靠近内侧处剪取2mm左右的血管环。冠状动脉血管环的制备：用大头针分别固定心脏的心尖端和升主动脉端于平皿，在左心耳下面寻找冠状动脉，顺着冠状动脉的走向钝性分离，在前降支处剪取2mm的血管环。子宫动脉血管环的制备：用大头针分别固定子宫的两端于平皿，在子宫的外侧可清晰的看到子宫动脉的走形，顺着子宫动脉的走向钝性分离，剪取2mm的血管环。将两根直径为40μm的钨丝传入管腔，固定血管环在Multi Myograph System-610M浴漕内传感器上，持续通以100％O_2。浴漕温度恒定在37℃，平衡60min后开始实验，平衡期间每隔15min用预热的PSS液(37℃)更换浴漕内液体一次。血管环的张力变化通过DMT的换能系统采集，并用Chart 5．3记录在微机上。
5．微血管环标准化：为了使血管环处于最佳反应状态，分别调整肠系膜动脉、肾动脉、大脑中动脉、冠状动脉和子宫动脉的跨壁压使其保持在100mmHg、80mmHg、60mmHg、80mmHg和80mmHg基础压力状态，在实验前对血管环进行标准化，操作过程概括如下：挂血管环时使两根钨丝平行零距离，温育60min后，通过逆时针旋转Myograph的螺旋测微尺逐步牵拉血管环，得出每个直径时血管环的张力值，从而得出其有效跨壁压P_i。P_i=2~πT_i／IC_iT_i=F_i／2LIC_i=205．6+2χ_iP_i(kPa)：有效跨壁压T_i(mN／mm)：单位长度血管环张力IC_i(μm)：血管环内周长F_i(mN)：血管环总张力χ_1(μm)：两根钨丝间的距离，可由Myograph螺旋测微尺读出L(mm)：血管环长度，可用解剖纤维镜上的目镜测微尺测出
以肠系膜动脉环为例，每隔2min牵拉一次血管环，当Pi超过血管环的基础压力后，可绘制横坐标为IC_i，纵坐标为T_i的指数曲线图，得出血管环内径为90％IC_(100)时的螺旋测微尺的读数，顺时针旋转螺旋测微尺使其恢复到此位置(图31、32和33)。
6．AT1-AA对不同部位微血管收缩功能的影响：用120mmol／L的KCl预收缩，用Ach(1×10~(-5)mol／L)舒张，连续三次，收缩幅度差别不大于5％，然后开始正式实验。分组同大血管环，在离体微血管环上观察AT1-AA对不同部位血管收缩活动的影响。
结果
1．AT1-AA剂量依赖性的引起大鼠胸主动脉的收缩：AT_1-AA能浓度依赖性地增强大鼠胸主动脉血管环的最大收缩张力，0．01、0．1和1．0μmol／L的AT_1-AA可使大鼠胸主动脉血管环的最大收缩张力从对照组的1．02±0．04(g)分别增加到1．06±0．05(g)、1．28±0．09(g)和1．72±0．17(g)，其作用与AngⅡ相似；1．0μmol／L的losartan可以有效地拮抗AT_1受体抗体和Ang-Ⅱ对血管环收缩张力的影响；1．0μmol／L的SEL-ATlR预孵后能有效的中和AT1-AA和Ang-Ⅱ的作用(图5，n=6)。nsIgG和npIgG对胸主动脉的收缩无影响(图6，n=8)。
2．AT1-AA引起不同部位微血管收缩的影响
(1)AT1-AA浓度依赖性收缩大脑中动脉和冠状动脉，1．0μmol／L的AT_1-AA可使大鼠大脑中动脉和冠状动脉的最大收缩张力从对照组的0．03±0．02(mN)分别增加到0．82±0．09(mN)和2．51±0．19(mN)，Ang-Ⅱ的作用和AT1-AA相似。(图7，n=6．10)。
(2)AT1-AA对肾动脉、肠系膜动脉和子宫动脉的收缩作用不明显。而这些血管上均有AT1R的表达和分布，对于这一矛盾，我们分析可能由以下几种原因所致：(1)AT1-AA对不同血管造成不同影响；(2)正常未孕时肾动脉、子宫动脉和肠系膜动脉等小血管AT1R处于低表达状态；(3)AT1-AA对血管造成了其他影响而不表现为收缩效应。那么，究竟是其中的一种，抑或是几种原因都需要进一步证实。
(3)AT1-AA对大脑中动脉和冠状动脉的收缩作用可以被1．0μmol／L的losartan或AT1受体细胞外第二环表位肽段(SEL-AT1R)中和。1．0μmol／L的losartan或SEL-AT1R可以使1．0μmol／L的AT_1受体抗体引起的大脑中动脉的收缩从0．82±0．09(mN)降低到0．06±0．03(mN)，1．0μmol／L的losartan或SEL-AT1R可以使1．0μmol／L的AT_1受体抗体引起的冠状动脉的收缩从2．51±0．19(mN)降低到0．05±0．01(mN)，(图8，n=6)。
(4)nsIgG和npIgG对各部位的血管均无明显的收缩作用(图9，n=5)。
结论
1．AT1-AA浓度依赖性的增强大鼠胸主动脉的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节；
2．AT1-AA通过激活AT1R细胞外第二环，浓度依赖性地增强大鼠大脑中动脉和冠状动脉的收缩，但对肾动脉、肠系膜动脉和子宫动脉的收缩作用不明显。
三．血管紧张素ⅡAT1受体自身抗体对血管内皮结构和功能的影响
目的
本实验主要目的是：1)研究AT1-AA对离体培养的内皮细胞的损伤效应及其致内皮细胞损伤的途径；2)使用人工合成的AT1R-ECⅡ肽段主动免疫正常大鼠，获得免疫动物模型，观察该抗体长期作用下引起在体内皮细胞损伤情况，阐明AT1-AA致血管类动脉粥样硬化改变的可能性及其防治的意义。
方法
1．内皮细胞培养：脐静脉内皮细胞株(sciencell公司，USA)培养在6孔培养皿内，倒置显微镜下内皮细胞呈梭形或多边形，细胞生长融合成单层后呈鹅卵石或铺路石镶嵌排列(图30)，待细胞达到80％融合时弃原培养液，D-Hanks液清洗2次，0．125％胰蛋白酶消化，倒置显微镜下见细胞收缩变圆，细胞间隙增宽后，弃消化液，按1：3比例传代。传至第4代时，分别给予AngⅡ、preeclamptic IgG、nsIgG、npIgG、losartan和SEL-AT1R处理后在37℃，5%CO_2培养箱内孵育，并于24h、48h和72h收集细胞，用比色法测定LDH、caspase3、8和9的活性。
2．Caspase-3、caspase-8、caspase-9的活性测定：利用caspase-3、caspase-8、caspase-9
Apoptosis Detection Colorimetric Biogene试剂盒进行检测。每孔细胞内加入80μl裂解液进行裂解，之后置4℃、10,000 rpm离心10分钟。将上清转移至新的Eppendorf管，用考马斯亮兰试剂盒测定蛋白浓度。取30μl上清，65μl反应缓冲液，5μl底物(caspase 3、caspase 8、caspase 9的底物分别为DEVD-pNA、IETD-pNA和LEHD-pNA)加至96孔ELISA板中；背景对照除不加底物外，余同实验孔，用反应液将每孔的液体补齐至100μl。37℃水浴1．5小时后，在405 nn3波长下测定光密度值。
3．抗AT1R自身抗体阴性、体重180-220g的健康Wistar雌性大鼠随机分成四组：
(1)AT1R-ECⅡ免疫组(Immunization group)，将抗原β_1-AR-ECⅡ(按照大鼠AT1受体细胞外第二环功能表位肽段序列165-191位，见表2，由西安联美生物科技有限公司合成，肽段合成的质量报告单见附录。注射剂量：0．4μg/g体重)溶于生理盐水溶液中，浓度为1mg／ml。首次免疫采用背部皮下多点注射法，给予抗原溶液和等体积福式完全佐剂的混合物，以增加机体的细胞免疫功能。1周后采用背部皮下一点注射法进行第一次加强免疫，同时采用福式不完全佐剂以增加机体的体液免疫功能，抗原和佐剂的剂量与首次免疫相同。此后每隔两周用同样的方法加强免疫一次，共免疫12周。
(2)伪免疫组(Sham Immunization group)，用等量生理盐水溶液替代抗原溶液，给药方法、免疫程序以及免疫增强剂的使用均同免疫组。
(3)内皮素抑制组(Immunization+Bosentan group)，免疫处理方法同免疫组，在免疫开始即给予广谱内皮素抑制剂Bosentan。由于动物体内抗体产生是一个相对较长的过程，同时产生的抗体对内皮细胞造成影响是一种长期效应的累加，我们将Bosentan置于渗透泵中，再将泵植入动物腹腔内。这种给药方式适合于需长期药物处理的慢性动物实验，保证药物恒量释放，血药浓度维持在恒定水平，减少不同时间给药处理造成的系统误差。具体方法为，大鼠称重，10％水合氯醛(3 ml／kg)腹腔注射麻醉，仰卧位固定于手术台上，腹部皮肤去毛，碘伏消毒，铺消毒巾，肋弓下2-2．5cm处沿腹正中线纵行切开皮肤，钝性分离腹部肌肉，将已充满Bosentan溶液的渗透泵植入腹腔后逐层缝合肌肉、皮肤。渗透泵的额定工作时间为14天，每隔两周更换一次。
(4)内皮素抑制剂对照组(Immunization+Vehicle group)，大鼠处理同内皮素抑制组，DMSO中不含Bosentan。
4．血清内皮素-1的测定：用定量EIA试剂盒进行检测。主要技术流程如下：建立标准曲线→加入大鼠血清→加入酶标抗体工作液→加入底物工作液→加入终止液→在酶标仪上(Molecular Devices Corp，Menio Park，CA，USA)测定反应物的光密度值(OD)，根据样品OD值在标准曲线图上查出相应内皮素-1的含量。
5．iNOS免疫组织化学染色及结果半定量：分离大鼠血管后用4％福尔马林固定48h，然后切片包埋，采用SP法进行免疫组织化学染色。主要流程如下：切片脱蜡水化，3％H_2O_2处理15min，胰酶消化10min，热抗原修复10min，山羊血清封闭，滴加1：100iNOS一抗(iNOS多克隆抗体均由美国Santa Cruz公司提供)，4℃过夜，滴加二抗(中山生物技术有限公司)37℃孵育10min，DAB显色，阴性对照以PBS代替一抗。采用Olympus-BH2显微系统摄像，成都泰盟BI2000医学图像分析系统测定阳性细胞所占的面积。
结果
1．不同剂量AT1受体自身抗体对内皮细胞损伤的影响
(1)不同剂量AT1-AA引起内皮细胞LDH释放的情况：我们的实验首先检测了乳酸脱氢酶(Lactate Dehydrogenase，LDH)漏出量。结果表明，1．0μmol／L的AT1-AA与内皮细胞孵育48h后显著的引起LDH活性的增高(与阴性对照组相比约增加了两倍，P<0．01)，但0．01μmol／L和0．1μmol／L的AT1-AA对LDH的活性无显著性的影响(图10，表1，n=8)。由于LDH是一种稳定的蛋白质，存在于正常细胞的胞质中，只有当细胞膜受损后才被释放到细胞外，而一般认为，坏死表现为细胞膜破裂，内含物外泄。因此，我们推测，1．0μmol／L的AT1-AA能够导致内皮细胞坏死。
(2)不同剂量AT1-AA引起内皮细胞caspase释放的情况：除坏死外，凋亡是细胞损伤的另一方式，根据发生过程中是否有caspase(cysteiny aspartate specific protease，半胱氨酰天冬氨酸特异性蛋白酶)参与，分为caspase依赖性途径和caspase非依赖性途径，其中以前者为主。目前己证实的caspases活化途径主要有三条：一是经细胞表面受体途径(死亡受体途径或外源性途径)引起caspase-8(和／或某些类型细胞中的caspase-10)活化；二是经线粒体途径(内源性途径)释放多种致凋亡因子引起caspase-9活化；第三是经内质网途径引起caspase-12活化。这三条途径最终主要激活凋亡效应酶即caspase-3，进而通过水解多种重要的蛋白而导致细胞凋亡。为进一步观察AT1-AA对内皮细胞的作用，我们检测了AT1-AA作用于离体培养的内皮细胞后多种caspase的活性。结果表明，1．0μmol／L抗AT1R-EC_Ⅱ抗体作用内皮细胞48h后，caspase3和caspase8的活性均增高，与npIgG组相比均有显著性差异(caspase3和8的活性分别为，0．63±0．05 mmol／h／mg protein vs．0．32±0．02mmol／h／mg protein和2．98±0．3 mmol／h／mg protein vs．1．53±0．21 mmol／h／mg protein，p<0．01)，AT1-AA引起的caspase活性的增高可以通过预先用losartan或AT1受体细胞外第二环特异性肽段孵育而消失(图10和11，表4，n=8．10)；0．01μmol／L和0．1μmol／L的AT1-AA对caspase3和8的活性无显著性影响；AT1-AA对caspase9的活性却无明显的作用(图13，n=8)。提示1．0μmol／L的AT1-AA通过激活AT1受体细胞外第二环特异性肽段除能引起坏死外，还可能通过凋亡(尤其是外源性途径)造成内皮细胞损伤。
2．1．0μmol／L的AT1受体自身抗体在不同时间点引起内皮细胞损伤的研究
(1)1．0μmol／L的AT1-AA可以时间依赖性的引起内皮细胞的坏死：我们的研究已经证实AT1-AA在1．0μmol／L的浓度可以引起细胞的损伤，因此，我们采用这个浓度来观察不同时间点AT1-AA对细胞损伤的影响。结果表明，LDH的活性在48h开始升高，且呈时间依赖性，72h时达到最高点，与24h相比，LDH在72h时的活性增加了约3．5倍(图14，表5，n=8)，并且此种效应能够通过与losartan以及细胞外第二环的特异性肽段孵育而消失。
(2)1．0μmol／L的AT1-AA在不同时间点对caspase活性的影响：Caspase3在24h开始增高(从0．29±0．05 nmol／h／mg protein增加到0．45±0．06 nmol／h／mg protein，P<0．05)，48h达到高峰(从0．29±0．05 nmol／h／mg protein增加到0．63±0．06 nmol／h／mg protein，P<0．01)且可以维持到72h(图15，表6，n=6)；caspase8从24h开始增高(从1．39±0．05 nmol／h／mg protein增加到2．02±0．06 nmol／h／mg protein，P<0．05)，48h达到峰值(从1．39±0．05 nmol／h／mg protein增加到2．96±0．07 nmol／h／mg protein，P<0．01)，随后即开始下降，但在72h时仍高于阴性对照组(从1．39±0．05 nmol／h／mg protein增加到2．05±0．07 nmol／h／mg protein，P<0．05)(图16，表7，n=6)：AT1-AA引起caspase3和8活性增高的效应能够通过与losartan或AT1R细胞外第二环的特异性肽段孵育而消失；caspase9的活性在各组之间均无明显的差异(图17，表8，n=5)。
3．抗体滴度的检测结果：AT1R-ECⅡ首次免疫1周内，各组大鼠血清中AT1-AA的平均抗体滴度小于1：10，加强免疫后AT1R-ECⅡ免疫组(n=50)、AT1R-ECⅡ+Vehicle(n=48)组和AT1R-ECⅡ+Bosentan(n=48)组的抗体滴度开始升高，并在免疫后8周达峰值，其抗体滴度分别为1：17056±3．32，1：198413±3．58和1：16054±2．98；伪免疫组(n=46)大鼠血清中AT1-AA的平均抗体滴度始终保持在1：10以下(图18，表9)。
4．为了验证我们的假设，即AT1-AA长期作用下，可以导致内皮细胞的损伤，我们检测了免疫大鼠血清内皮素-1(ET-1)的浓度，结果证实，AT1R-ECⅡ免疫组大鼠免疫后第3周开始血清中ET-1含量增高，第9周达高峰，与对照组相比有显著性差异(38．2％±2．8vs．1．5％±0．01，P<0．01)(图19，表10)。ET-1由内皮细胞合成分泌，存在于正常的组织中，但在内皮受损时，ET-1的合成和分泌增加，因此，ET-1水平的升高在很大程度上反应了内皮受损的程度。因此，我们推测，AT1-AA能引起在体血管内皮的损伤。
5．为了进一步证实我们的假设，我们采用大血管环及微血管环技术检测了免疫大鼠胸主动脉和各组小动脉的收缩及舒张功能，结果显示：免疫12周时，各组血管内皮依赖性舒张功能均显著下降，与同期伪免疫组相比有显著性差异(P<0．01)(图21-24，表11-16)；但各组大鼠的血管收缩功能无显著差异(图25-28)。这一结果更加确证了我们的假设：AT1-AA长期作用下，血管内皮细胞的结构和功能均受到严重影响。
6．免疫组化检测的结果显示，AT1R-ECⅡ免疫组大鼠免疫后8周血管内皮可见典型的iNOS染色，而伪免疫组为阴性，AT1R-ECⅡ免疫组大鼠iNOS的表达是阴性对照组的3．16倍(图29，表17)。研究表明，由iNOS产生的病理浓度NO可以通过生成活性氮代谢物(ReacticeNitrogen Species，RNS)，尤其是硝基化作用很强的过氧亚硝基(peroxynitrite，ONOO~-)分子导致硝基化应激(nitrative stress)和组织损伤。因此，我们推测，AT1-AA长期存在的情况下，可能通过引起iNOS的释放，从而产生病理浓度的NO，最终导致内皮细胞的损伤。
结论
1.AT1-AA可以通过坏死和凋亡(尤其是外源性途径)两种方式，直接引起血管内皮细胞的损伤；
2．在体情况下，AT1-AA长期作用可以引起大鼠内皮细胞的损伤，并使大鼠内皮依赖性舒张血管功能显著降低；但对大鼠血管收缩功能无明显影响；
3．AT1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示AT1-AA致血管内皮损伤的机制可能与NO生理功能异常有关。
Preeclampsia is a serious pathologic complication during pregnancy and its pathogenesis remains poorly understood. Recent studies have demonstrated that autoantibody against angiotensinⅡtype 1A receptor (AT1-AA) are present in women with preeclampsia and the possible role of this autoantibody in the pathogenesis of preeclampsia has been paid increasingly more attention. In order to evaluate correctly the pathological significance of this autoantibody detection in sera from patients, it is necessary to understand the characteristic distribution of this autoantibody and the relationship between the AT1-AA level and the systolic systolic blood pressure (SBP), proteinuria or platelet in preeclamptic patients. The peptide corresponding to the sequences of the second extracellular loop of the human AT1 receptor is used as antigen in an enzyme immunoassay to screen the sera from preeclamptic patients. Positively was defined as P/N≥2.1; antibody titer was expressed as the maximum dilution at P/N≥2.1. Compared with the normal pregnant women and the healthy nonpregnant women, frequency of AT1-AA positive samples was markedly increased in preeclamptic patients (69.0%±2.9% vs. 29.0%±2.1%, 69.0%±2.9% vs. 8.3%±0.9%, respectively, P<0.01). The antibody titer of the positive sera from preeclamptic patients was in high level with the geometric mean titer being 1:168.6±6.7 for anti-AT1 receptor antibody. Notably, the dependability was observed between the AT1-AA level and the systolic blood pressure in preeclamptic patients. Nevertheless, there was no dependability between the AT1-AA level and the proteinuria or platelet in preeclamptic patients. For comparison, the characteristics of this autoantibody in sera from 56 normal pregnancies and in sera from 48 normal nonpregnancies were also studied.
In conclusion, compared with the normal pregnant women and the healthy nonpregant women, the autoantibody against AT1 receptor in sera from preeclamptic patients is characterized by high frequency and high titer with the positively being increased with blood pressure. These results suggest that overproduction of AT1-AA is a novel risk factor in pregnant women and may play a causative role in the development of preeclamptic patients.
Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. Recent studies have shown that autoantibodies against angiotensinⅡAT_1 receptors (AT1-AA) develop in the sera of patients with preeclampsia and may be involved in the pathogenesis of the disorder. In view of the vascular damages play a crucial role in the pathogenetic feature of preeclampsia, the aim of this study was to clarify the fundamental alterations in vasculature induced by AT1-AA. Vasoconstrictive effect of purified IgG from the sera of either preeclamptic patients or normal pregnant women was determined in isolated rat thoracic aorta, middle cerebral artery, coronary artery, renal artery, mesenteric artery and uterine artery. Our results showed that AT_1-AA induced a marked dose-dependent vasoconstriction in aortic vascular rings. However, curiously enough, AT_1-AA showed variant effects on small resistant vascular rings isolated from different organs. Of these vessels, middle cerebral artery and coronary artery were remarkably constricted by AT_1-AA, but mesenteric, renal and uterine arteries were essentially nonresponsive to AT_1-AA. The effects of AT_1-AA were neutralized by the antagonist Losartan and the peptide corresponding to the second extracellular loop of the AT_1 receptor. These data demonstrated that the AT1-AA caused marked vasoconstriction in some arteries through activate the peptide corresponding to the second extracellular loop of the AT_1 receptor and thus can account for the increased arterial tone in these vascular beds. These findings suggest that AT_1-AA is a novel risk factor in pregnant women and may play a causative role in the development of preeclampsia.
Background: In view of the endothelial damages play a crucial role in the pathogenetic feature of preeclampsia, the aim of this study was to clarify the structural and functional damage of endothelium induced by AT1-AA. The present study investigated whether AT1-AA may induce endothelial structural dysfunction in vitro, and if so, to determine whether long-term active immunization with synthetic peptides corresponding to the second extracellular loop of the AT1-receptor (AT1R) may induce endothelial functional injury in vivo.
Methods and Results: In this study, we investigated the pro-apoptotic and cytotoxic effect of AT1-AA on endothelial cell in vitro. The result demonstrated that AT1-AA was able to significantly increase the release of LDH and caspase 3 from endothelial cell. In addition, Adult female Wistar rats were immunized with synthetic peptides. Anti-ATIR autoantibody, endothelial injury (production of endothelin-1) and vascular function (systolic and diastolic function of thoracic aorta ring and small resistant vascular rings) were determined after immunization. In the sham immunization group, the antibody titer remained below 1:10 and no endothelial injury and vascular functional change were observed. However, in the immunized rats, a significant increase in anti-AT1-R autoantibody occurred 2 weeks after the immunization and peaked at week 8. A significant increase in production of endothelin-1 (ET-1) occurred at week 3 and peaked at week 9. A significant increase of iNOS production occurred 8 weeks after immunization, and severe vascular dysfunction occurred 12 weeks after immunization. Treatment with Bosentan (pan-ET-receptor antagonist) abolished endothelial injury and significantly improved vascular function in the immunized rats.
Conclusion: Our results demonstrated for the first time that AT_1-AA was able to induce endothelial injury in vitro, and also suggested that long-term stimulation with the autoantibody against the second extracellular loop of the AT1R induces endothelial injury in vivo. These results suggest that AT1-AA may be a key role in vascular dysfunction in preeclampsia and the treatments of protecting endothelium may be a novel strategy in reducing multiple organ injury associated with autoimmune response.
子痫前期(Preeclampsia，PE)是妊娠期特发的全身性疾病，多在妊娠20周以后发病。临床表现以高血压、蛋白尿为特征，并伴有全身多脏器的损害，其基本病理生理改变是全身小动脉的痉挛性收缩。流行病学调查结果显示，子痫前期的发病率为5-10％，是母婴发病率及死亡率较高的主要原因。子痫前期对孕产妇的影响主要表现为胎盘早剥、肺水肿、凝血功能障碍、产后出血及产后血循环衰竭等并发症，严重者可致死亡：对胎儿多是由于胎盘供血不足，胎盘功能减退导致胎儿窘破、胎儿发育迟缓、死胎、死产或新生儿死亡。目前，对子痫前期普遍认同的病因学说主要有免疫学说、子宫-胎盘缺血学说、血管调节物质异常、遗传学说等，但是相关治疗措施虽能改善病人急性和慢性期的症状，但不能从根本上逆转疾病的进展和最终防止子痫前期的发生，提示在子痫前期发生发展过程中可能还有其它未知因素的参与。
20世纪90年代早期，Hanssens和Haller等先后发现并证实，子痫前期患者血液中存在一种作用类似血管紧张素Ⅱ(AngiotensinⅠ，Ang-Ⅱ)的循环因子，使血管紧张素Ⅱ-1型受体(AngiotensinⅡType 1 Receptor，AT1R)功能增强。1999年，Wallukat等证实这种类Ang-Ⅱ因子是抗ATlR自身抗体(AngiotensinⅡtype 1 Receptor Autoantibodies，AT1-AA)。随后的研究也相继证实AT1-AA存在于子痫前期患者的血清中，但是，研究的标本数较少，此外，没有该抗体存在与子痫前期体征关系的分析，即缺乏AT1-AA是否与子痫前期有关的分析资料，而这一问题的解决是研究AT1-AA在子痫前期发病中作用的前提。
随后的研究表明，这种自身抗体通过专一识别AT1R的细胞外第二环功能表位肽段(thesecond extracellular loop of the angiotensinⅡtype 1 receptor，AT1R-EC_Ⅱ，人鼠同源性为99％)促进血管平滑肌细胞增殖、NADPH氧化酶活化、ROS生成、细胞浆内Ca~(2+)浓度增加和激活纤溶酶原激活物抑制因子1。这些研究结果均提示，AT1-AA的过度活动与许多重要的病理生理改变有关，可能在子痫前期的发病中起着重要作用。由于子痫前期危害性极大而病因又极其诡异，因而这一成果的重要性和亟待被深入研究的必要性被国内的学者迅速而敏锐地捕捉到，并对此展开了广泛的研究。其中尤以廖玉华实验室的研究最为深入，他们的研究证实，AT1-AA通过激活AT1受体诱导大鼠。肾脏和心肌组织的免疫损伤并促进大鼠血管平滑肌细胞增殖。我实验室的前期研究也发现，抗AT1R-EC_Ⅱ抗体通过作用AT1R增强离体灌流心脏的心功能、升高心肌细胞内游离钙含量、增加心肌细胞I_(Ca-L)电流、激动I_(Na／Ca)电流和减小I_(k1)电流等。这些结果进一步提示，AT1-AA具有AT1受体类激动剂样效应。
虽然AT1R广泛分布于人体各组织器官，介导了目前已知Ang-Ⅱ的所有作用(如收缩血管、促进醛固酮分泌、刺激血管平滑肌细胞增殖和水钠潴留等)，但AT1R对脉管系统的作用尤为突出。因此我们推测，具有AT1受体激动剂样效应的AT1-AA是否也具有同样的血管效应呢?如果是，那么AT1-AA是否象AT1受体激动剂那样，也具有致血管病变的病理意义呢?但是到目前为止，已有的研究报道(包括我们的前期工作)仅局限于通过急性实验来探讨AT1-AA作用的微观机制，但缺乏AT1-AA对各类血管(尤其是阻力血管)作用的直接证据，而对这一问题的阐明是认识AT1-AA在子痫前期发生发展中作用和研究AT1-AA作用机制的关键。
如前所述，AT1R广泛分布于人体各组织器官。在血管系统，AT1R除分布在血管平滑肌外，还广泛分布在血管内皮细胞，后者在调节血管收缩舒张功能中具有重要的作用。如果存在于血液中的AT1-AA随血液循环在血管内流动时，由于其具有类AT1受体类激动剂样效应，那么AT1-AA应该可以和血管内皮细胞上的AT1R结合。但是，这一推测需要实验来证实。众所周知，在体情况下血管内皮损伤后，会导致流经损伤部位的血液中诸多血管活性物质的聚集(如血小板、凝血因子等)并引发随后的释放反应，这些病理过程都有利于动脉粥样硬化斑块的形成。在子痫前期患者发病过程中，一个典型的病理改变是子宫螺旋小动脉存在“类粥样硬化样”改变，因此导致胎盘血流量下降，直接危害到胎儿的健康；此外，另一个典型病理改变是肾小球血管内皮下大量纤维样物质沉积，进而造成血管管腔狭窄、肾脏血液灌注减少和肾小球滤过率下降等后果。但是，多年以来，导致这种血管内皮细胞病理改变的机制一直不清。因此，如果能够通过直接的实验证据说明AT1-AA是导致“类粥样硬化样”病变的原因将有助于认识子痫前期血管病变的机制。
综上所述，我们设计了本研究课题：(1)通过临床流行病学调查，追踪子痫前期患者血清AT1-AA的变化，观察该抗体水平和病情相关性，明确该抗体的临床意义。(2)在此基础上开展实验室研究，首先以人工合成的AT1R-EC_Ⅱ主动免疫大鼠制备抗AT1R-EC_Ⅱ抗体，然后通过离体大血管和微血管环技术明确该抗体对大鼠胸主动脉、主要脏器的小动脉(包括冠状动脉、大脑中动脉和肾动脉)、主要阻力血管(肠系膜动脉)和反应子宫-胎盘血流量的血管(子宫动脉)等不同类型小动脉的直接作用。(3)以血管内皮的结构和功能为切入点，通过离体细胞培养技术研究抗AT1R-EC_Ⅱ抗体对内皮细胞的损伤效应和形式；并将抗AT1R-EC_Ⅱ抗体转移进入正常大鼠体内建立大鼠被动免疫模型，然后通过检测血清中内皮素的含量来观察抗AT1R-EC_Ⅱ抗体对在体血管内皮的损伤作用；通过大血管和微血管环技术明确该抗体在在体情况对血管活动的影响。以期阐明AT1-AA致血管类动脉粥样硬化改变的可能性及其防治的意义。
本课题拟验证的假说：子痫前期患者血清中存在的AT1-AA，有可能通过影响血管结构和功能而与子痫前期发生发展相关。(1)在子痫前期患者血清中的AT1-AA与该病的某些特殊指征呈一定的相关性；(2)AT1-AA对大血管具有直接的收缩效应，但对不同小动脉可能表现各不相同；(3)AT1-AA通过作用于AT1R细胞外第二环，引起血管内皮细胞损伤，或许可以进而诱发血管“类动脉粥样硬化”改变。所有这些结构和功能变化可能与子痫前期患者所表现的血压升高、组织器官血流量下降以及和其它病理学改变密切相关。
一、子痫前期患者血清中血管紧张素ⅡAT1受体自身抗体的检测及其免疫学特征分析
目的
本工作首次报道了AT1-AA在子痫前期患者血清中的分布规律并分析了该抗体与子痫前期患者典型体征的关系。
方法
1．检测对象：子痫前期患者82例，年龄26-33岁，正常孕妇56例，年龄23-34岁，未孕健康妇女48例，年龄24-23岁。子痫前期的诊断符合ISSHP2001年标准，即妊娠20周以后出现，血压≥140／90mmHg，且尿蛋白≥0．3g／24h或(+)，可伴有上腹部不适、头痛、视力模糊等症状(见表1)。以上三组均排除内分泌及自身免疫性疾病。
2．AT1受体抗原多肽的合成：按照人AT1受体细胞外第二环功能表位肽段序列(165-191位，见表2)，由西安联美生物科技有限公司合成(肽段合成的质量报告单见附录)。用于血清抗体的定性与定量测定、血清抗体的功能测定。
3．血清自身抗体的测定：用间接SA-ELISA(Streptavidin-ELISA)方法，抗原包被用量为AT1抗原10μg/ml。主要技术流程如下：包被合成的抗原多肽→加入人的血清→加入标记有生物素的抗人IgG抗体→加入标记有辣根过氧化物酶的亲和素→加入辣根过氧化物酶酶的底物→在酶标仪上(Molecular Devices Corp，Menio Park，CA，USA)测定反应物的光密度值(OD)，以OD值的大小代表人血清中抗多肽抗体的量。
4．结果判定：以P／N≥2．1为阳性，P／N≤1．5为阴性，P／N=(标本OD值-空白对照OD值)／(阴性对照OD值-空白对照OD值)；将血清标本从1：20起连续倍比稀释，以出现P／N≥2．1的最高稀释度作为该标本的效价。
5．统计处理：抗AT1R抗体在不同组间的分布用阳性率表示；每组抗体水平的平均值用几何平均数(Geometric mean)来表示；用有序2×C表资料直线回归的显著性检验进行抗体阳性率年龄趋势的检验；用U检验进行两样本阳性率的差别检验；两几何均数之间的差别检验用t检验：率的检验用经平方根反正弦变换后的数据进行；几何均数的检验用经对数转换后的数据进行。P值小于0．05认为有显著差异。
结果
1．82例子痫前期患者的血清中，抗AT1受体自身抗体的阳性率为69％±2．9％，显著高于56例正常孕妇和48例未孕健康妇女血清中AT1受体自身抗体的阳性率(分别为29％±2．1％和8.3％±0．9％，P<0．01)，正常孕妇和未孕健康妇女血清中AT1-AA的阳性率相比也有统计学差异(29％±2．1％vs．8．3％±0．9％，P<0．05)(见图1、图2)。
2．子痫前期患者抗体阳性血清的抗体效价为1：168．6±6．7，远高于正常孕妇和未孕健康妇女抗体阳性血清的抗体效价(分别为1：52．9±2．1和1：16．2±1．8，p<0．01)(图3)。
3．子痫前期患者血清中AT1受体自身抗体的滴度随孕妇收缩压的增高而呈上升趋势，(图4，r=0．856)。
4．子痫前期患者血清中AT1受体自身抗体的滴度和孕妇蛋白尿程度及血小板计数无相关(r=-0．013，r=0．205)。
结论
1．本研究结果证实，子痫前期患者血清中抗AT1-AA的分布特征是高阳性率、高抗体滴度，提示AT1-AA和子痫前期有着密不可分的关系；正常孕妇血清中AT1-AA的阳性率也明显高于同龄未孕健康妇女，提示怀孕过程中有免疫机制的参与；
2．子痫前期时AT1-AA滴度随孕妇收缩压的增高而呈上升趋势，提示AT1-AA可能与子痫前期患者血压增高有关；
3．AT1-AA滴度和子痫前期患者蛋白尿程度及血小板计数无相关性，这个结果提示：(1)可能样本例数较少，还不足以揭示AT1-AA滴度和蛋白尿程度及血小板计数之间真正的关系：(2)AT1-AA可能通过其他的途径导致了子痫前期患者肾脏的损害和凝血功能的障碍，而非直接引起蛋白尿的生成和血小板的异常。
二．血管紧张素ⅡAT1受体自身抗体对血管功能的影响
目的
本实验主要目的是：1)采用大血管环技术，观察AT1-AA对大鼠容量血管收缩功能的作用，分析该自身抗体对患者血压的影响；2)采用微血管环技术，观察AT1-AA对主要脏器的小血管(包括冠状动脉、大脑中动脉和肾动脉)的直接作用，明确该自身抗体对患者主要脏器的影响；3)采用微血管环技术，观察AT1-AA对子宫动脉的作用，阐明该抗体对胎盘血流量的影响；4)采用微血管环技术，观察AT1-AA对肠系膜动脉的作用，证实该抗体与外周阻力血管功能改变之间的关系。
方法
1．AT1-AA的制备：利用MAb Trap Kit试剂盒对子痫前期患者抗体阳性者血清中的抗体IgG进行提取和纯化。纯化后的抗体经PAGE胶凝胶电泳检测其纯度。具体方法如下：
(1)特异性亲和柱试剂盒提纯抗体：将合成的人ATlR-EC_Ⅱ抗原肽段连于亲和柱内，至少3ml结合缓冲液平衡亲和柱(流速0．5ml／min)，0．5ml血清同等体积binding buffer稀释后，使用注射器加入柱体中，然后用7ml binding buffer冲洗，5ml洗脱液进行洗脱，洗脱下来的蛋白液保存于加有中性液(1M Tris)的EP管中，保持蛋白活性，至少用5ml bindingbuffer对亲和柱进行复性，然后进行下一轮操作。
(2)蛋白定性及定量测定：PAGE胶凝胶电泳检测其纯度后，按照BCA试剂盒步骤要求测定样品蛋白含量。
对阳性血清中的抗体IgG进行提取后剩余的IgG为nsIgG(nonspecific IgG)，作为阴性对照；从正常孕妇阴性血清中提取的IgG为npIgG(non-preeclamptic IgG)，也作为阴性对照。
2．大鼠胸主动脉环制备：取雌性Wistar大鼠，体重240-260g，击头致昏，立即开胸取胸主动脉置于O_2饱和的4℃营养液PSS中。(PSS成分如下(mmol／L)：NaCl154．7；KCl5．4；Glucose 11．0；CaCl_2 2．5；Triss 6．0。无钙PSS成分为(mmol／L)：NaCl 154．7；KCl 5．4：Glucose 11．0；EGTA 0．5；Triss 6．0。用HCl调pH至7．4。)剔除脂肪及周围结缔组织后，剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔，水平悬挂在浴管内，下方固定，上方以一细钢丝连于张力换能器(JH-2，北京)。经MS4000计算机生物信号采集分析系统记录血管的张力变化。浴管内含有通以100％O_2、37℃的PSS液10 ml。每个血管环悬挂在浴管后，基础张力调至2g，平衡1h，其间不断调整张力，使之维持在2g左右，每15 min换一次PSS。所有动脉环用60 mmol／L KCl多次刺激，当标本对刺激稳定时，即连续2次同样的刺激所引起的收缩幅度差别<5％时，开始正式实验。本实验所述浓度均为10ml浴管内试剂的终浓度。
3．AT1-AA对大血管收缩功能的影响：在大鼠离体胸主动脉血管环标本上，观察AT_1-AA对于大血管平滑肌活动的影响。分组为自身抗体组(preeclamptic IgG组)，血管紧张素Ⅱ(AngⅡ)阳性对照组，nsIg和npIgG阴性对照组，以及preeclamptic IgG+losanrtan(AT_1受体拮抗剂)组和preeclamptic IgG+SEL-AT1R(AT1受体细胞外第二环表位肽段)组。
4．大鼠肠系膜动脉、肾动脉、大脑中动脉、冠状动脉和子宫动脉血管环的制备：大鼠脱臼后，立即取出肠系膜、肾脏、心脏、大脑和子宫，浸入4℃的PSS液中。肠系膜动脉环的制备：将肠系膜浸入含有PSS液的平皿，用大头针固定动脉主干和肠管，分离动脉周围的脂肪组织，选取3级分支，剪取2mm左右的血管环。肾动脉环的制备：先用大头针固定肾脏于盛有PSS液的平皿，肾组织中动脉较韧，可用镊子直接剥碎肾组织暴露肾动脉各级分支，沿肾动脉剥离动脉的三级分支，分离干净周围的组织，剪取2mm左右的血管环。大脑中动脉环的制备：用骨钳揭开颅骨，取出脑组织，将脑组织贴近颅底面向上固定于盛有PSS液的平皿，可清楚的看到颈内动脉的延续-大脑中动脉，剥离大脑膜，钝性分离大脑中动脉，在靠近内侧处剪取2mm左右的血管环。冠状动脉血管环的制备：用大头针分别固定心脏的心尖端和升主动脉端于平皿，在左心耳下面寻找冠状动脉，顺着冠状动脉的走向钝性分离，在前降支处剪取2mm的血管环。子宫动脉血管环的制备：用大头针分别固定子宫的两端于平皿，在子宫的外侧可清晰的看到子宫动脉的走形，顺着子宫动脉的走向钝性分离，剪取2mm的血管环。将两根直径为40μm的钨丝传入管腔，固定血管环在Multi Myograph System-610M浴漕内传感器上，持续通以100％O_2。浴漕温度恒定在37℃，平衡60min后开始实验，平衡期间每隔15min用预热的PSS液(37℃)更换浴漕内液体一次。血管环的张力变化通过DMT的换能系统采集，并用Chart 5．3记录在微机上。
5．微血管环标准化：为了使血管环处于最佳反应状态，分别调整肠系膜动脉、肾动脉、大脑中动脉、冠状动脉和子宫动脉的跨壁压使其保持在100mmHg、80mmHg、60mmHg、80mmHg和80mmHg基础压力状态，在实验前对血管环进行标准化，操作过程概括如下：挂血管环时使两根钨丝平行零距离，温育60min后，通过逆时针旋转Myograph的螺旋测微尺逐步牵拉血管环，得出每个直径时血管环的张力值，从而得出其有效跨壁压P_i。P_i=2~πT_i／IC_iT_i=F_i／2LIC_i=205．6+2χ_iP_i(kPa)：有效跨壁压T_i(mN／mm)：单位长度血管环张力IC_i(μm)：血管环内周长F_i(mN)：血管环总张力χ_1(μm)：两根钨丝间的距离，可由Myograph螺旋测微尺读出L(mm)：血管环长度，可用解剖纤维镜上的目镜测微尺测出
以肠系膜动脉环为例，每隔2min牵拉一次血管环，当Pi超过血管环的基础压力后，可绘制横坐标为IC_i，纵坐标为T_i的指数曲线图，得出血管环内径为90％IC_(100)时的螺旋测微尺的读数，顺时针旋转螺旋测微尺使其恢复到此位置(图31、32和33)。
6．AT1-AA对不同部位微血管收缩功能的影响：用120mmol／L的KCl预收缩，用Ach(1×10~(-5)mol／L)舒张，连续三次，收缩幅度差别不大于5％，然后开始正式实验。分组同大血管环，在离体微血管环上观察AT1-AA对不同部位血管收缩活动的影响。
结果
1．AT1-AA剂量依赖性的引起大鼠胸主动脉的收缩：AT_1-AA能浓度依赖性地增强大鼠胸主动脉血管环的最大收缩张力，0．01、0．1和1．0μmol／L的AT_1-AA可使大鼠胸主动脉血管环的最大收缩张力从对照组的1．02±0．04(g)分别增加到1．06±0．05(g)、1．28±0．09(g)和1．72±0．17(g)，其作用与AngⅡ相似；1．0μmol／L的losartan可以有效地拮抗AT_1受体抗体和Ang-Ⅱ对血管环收缩张力的影响；1．0μmol／L的SEL-ATlR预孵后能有效的中和AT1-AA和Ang-Ⅱ的作用(图5，n=6)。nsIgG和npIgG对胸主动脉的收缩无影响(图6，n=8)。
2．AT1-AA引起不同部位微血管收缩的影响
(1)AT1-AA浓度依赖性收缩大脑中动脉和冠状动脉，1．0μmol／L的AT_1-AA可使大鼠大脑中动脉和冠状动脉的最大收缩张力从对照组的0．03±0．02(mN)分别增加到0．82±0．09(mN)和2．51±0．19(mN)，Ang-Ⅱ的作用和AT1-AA相似。(图7，n=6．10)。
(2)AT1-AA对肾动脉、肠系膜动脉和子宫动脉的收缩作用不明显。而这些血管上均有AT1R的表达和分布，对于这一矛盾，我们分析可能由以下几种原因所致：(1)AT1-AA对不同血管造成不同影响；(2)正常未孕时肾动脉、子宫动脉和肠系膜动脉等小血管AT1R处于低表达状态；(3)AT1-AA对血管造成了其他影响而不表现为收缩效应。那么，究竟是其中的一种，抑或是几种原因都需要进一步证实。
(3)AT1-AA对大脑中动脉和冠状动脉的收缩作用可以被1．0μmol／L的losartan或AT1受体细胞外第二环表位肽段(SEL-AT1R)中和。1．0μmol／L的losartan或SEL-AT1R可以使1．0μmol／L的AT_1受体抗体引起的大脑中动脉的收缩从0．82±0．09(mN)降低到0．06±0．03(mN)，1．0μmol／L的losartan或SEL-AT1R可以使1．0μmol／L的AT_1受体抗体引起的冠状动脉的收缩从2．51±0．19(mN)降低到0．05±0．01(mN)，(图8，n=6)。
(4)nsIgG和npIgG对各部位的血管均无明显的收缩作用(图9，n=5)。
结论
1．AT1-AA浓度依赖性的增强大鼠胸主动脉的收缩，提示该抗体可能通过增加心肌收缩后负荷，进而影响心脏泵血功能的调节；
2．AT1-AA通过激活AT1R细胞外第二环，浓度依赖性地增强大鼠大脑中动脉和冠状动脉的收缩，但对肾动脉、肠系膜动脉和子宫动脉的收缩作用不明显。
三．血管紧张素ⅡAT1受体自身抗体对血管内皮结构和功能的影响
目的
本实验主要目的是：1)研究AT1-AA对离体培养的内皮细胞的损伤效应及其致内皮细胞损伤的途径；2)使用人工合成的AT1R-ECⅡ肽段主动免疫正常大鼠，获得免疫动物模型，观察该抗体长期作用下引起在体内皮细胞损伤情况，阐明AT1-AA致血管类动脉粥样硬化改变的可能性及其防治的意义。
方法
1．内皮细胞培养：脐静脉内皮细胞株(sciencell公司，USA)培养在6孔培养皿内，倒置显微镜下内皮细胞呈梭形或多边形，细胞生长融合成单层后呈鹅卵石或铺路石镶嵌排列(图30)，待细胞达到80％融合时弃原培养液，D-Hanks液清洗2次，0．125％胰蛋白酶消化，倒置显微镜下见细胞收缩变圆，细胞间隙增宽后，弃消化液，按1：3比例传代。传至第4代时，分别给予AngⅡ、preeclamptic IgG、nsIgG、npIgG、losartan和SEL-AT1R处理后在37℃，5%CO_2培养箱内孵育，并于24h、48h和72h收集细胞，用比色法测定LDH、caspase3、8和9的活性。
2．Caspase-3、caspase-8、caspase-9的活性测定：利用caspase-3、caspase-8、caspase-9
Apoptosis Detection Colorimetric Biogene试剂盒进行检测。每孔细胞内加入80μl裂解液进行裂解，之后置4℃、10,000 rpm离心10分钟。将上清转移至新的Eppendorf管，用考马斯亮兰试剂盒测定蛋白浓度。取30μl上清，65μl反应缓冲液，5μl底物(caspase 3、caspase 8、caspase 9的底物分别为DEVD-pNA、IETD-pNA和LEHD-pNA)加至96孔ELISA板中；背景对照除不加底物外，余同实验孔，用反应液将每孔的液体补齐至100μl。37℃水浴1．5小时后，在405 nn3波长下测定光密度值。
3．抗AT1R自身抗体阴性、体重180-220g的健康Wistar雌性大鼠随机分成四组：
(1)AT1R-ECⅡ免疫组(Immunization group)，将抗原β_1-AR-ECⅡ(按照大鼠AT1受体细胞外第二环功能表位肽段序列165-191位，见表2，由西安联美生物科技有限公司合成，肽段合成的质量报告单见附录。注射剂量：0．4μg/g体重)溶于生理盐水溶液中，浓度为1mg／ml。首次免疫采用背部皮下多点注射法，给予抗原溶液和等体积福式完全佐剂的混合物，以增加机体的细胞免疫功能。1周后采用背部皮下一点注射法进行第一次加强免疫，同时采用福式不完全佐剂以增加机体的体液免疫功能，抗原和佐剂的剂量与首次免疫相同。此后每隔两周用同样的方法加强免疫一次，共免疫12周。
(2)伪免疫组(Sham Immunization group)，用等量生理盐水溶液替代抗原溶液，给药方法、免疫程序以及免疫增强剂的使用均同免疫组。
(3)内皮素抑制组(Immunization+Bosentan group)，免疫处理方法同免疫组，在免疫开始即给予广谱内皮素抑制剂Bosentan。由于动物体内抗体产生是一个相对较长的过程，同时产生的抗体对内皮细胞造成影响是一种长期效应的累加，我们将Bosentan置于渗透泵中，再将泵植入动物腹腔内。这种给药方式适合于需长期药物处理的慢性动物实验，保证药物恒量释放，血药浓度维持在恒定水平，减少不同时间给药处理造成的系统误差。具体方法为，大鼠称重，10％水合氯醛(3 ml／kg)腹腔注射麻醉，仰卧位固定于手术台上，腹部皮肤去毛，碘伏消毒，铺消毒巾，肋弓下2-2．5cm处沿腹正中线纵行切开皮肤，钝性分离腹部肌肉，将已充满Bosentan溶液的渗透泵植入腹腔后逐层缝合肌肉、皮肤。渗透泵的额定工作时间为14天，每隔两周更换一次。
(4)内皮素抑制剂对照组(Immunization+Vehicle group)，大鼠处理同内皮素抑制组，DMSO中不含Bosentan。
4．血清内皮素-1的测定：用定量EIA试剂盒进行检测。主要技术流程如下：建立标准曲线→加入大鼠血清→加入酶标抗体工作液→加入底物工作液→加入终止液→在酶标仪上(Molecular Devices Corp，Menio Park，CA，USA)测定反应物的光密度值(OD)，根据样品OD值在标准曲线图上查出相应内皮素-1的含量。
5．iNOS免疫组织化学染色及结果半定量：分离大鼠血管后用4％福尔马林固定48h，然后切片包埋，采用SP法进行免疫组织化学染色。主要流程如下：切片脱蜡水化，3％H_2O_2处理15min，胰酶消化10min，热抗原修复10min，山羊血清封闭，滴加1：100iNOS一抗(iNOS多克隆抗体均由美国Santa Cruz公司提供)，4℃过夜，滴加二抗(中山生物技术有限公司)37℃孵育10min，DAB显色，阴性对照以PBS代替一抗。采用Olympus-BH2显微系统摄像，成都泰盟BI2000医学图像分析系统测定阳性细胞所占的面积。
结果
1．不同剂量AT1受体自身抗体对内皮细胞损伤的影响
(1)不同剂量AT1-AA引起内皮细胞LDH释放的情况：我们的实验首先检测了乳酸脱氢酶(Lactate Dehydrogenase，LDH)漏出量。结果表明，1．0μmol／L的AT1-AA与内皮细胞孵育48h后显著的引起LDH活性的增高(与阴性对照组相比约增加了两倍，P<0．01)，但0．01μmol／L和0．1μmol／L的AT1-AA对LDH的活性无显著性的影响(图10，表1，n=8)。由于LDH是一种稳定的蛋白质，存在于正常细胞的胞质中，只有当细胞膜受损后才被释放到细胞外，而一般认为，坏死表现为细胞膜破裂，内含物外泄。因此，我们推测，1．0μmol／L的AT1-AA能够导致内皮细胞坏死。
(2)不同剂量AT1-AA引起内皮细胞caspase释放的情况：除坏死外，凋亡是细胞损伤的另一方式，根据发生过程中是否有caspase(cysteiny aspartate specific protease，半胱氨酰天冬氨酸特异性蛋白酶)参与，分为caspase依赖性途径和caspase非依赖性途径，其中以前者为主。目前己证实的caspases活化途径主要有三条：一是经细胞表面受体途径(死亡受体途径或外源性途径)引起caspase-8(和／或某些类型细胞中的caspase-10)活化；二是经线粒体途径(内源性途径)释放多种致凋亡因子引起caspase-9活化；第三是经内质网途径引起caspase-12活化。这三条途径最终主要激活凋亡效应酶即caspase-3，进而通过水解多种重要的蛋白而导致细胞凋亡。为进一步观察AT1-AA对内皮细胞的作用，我们检测了AT1-AA作用于离体培养的内皮细胞后多种caspase的活性。结果表明，1．0μmol／L抗AT1R-EC_Ⅱ抗体作用内皮细胞48h后，caspase3和caspase8的活性均增高，与npIgG组相比均有显著性差异(caspase3和8的活性分别为，0．63±0．05 mmol／h／mg protein vs．0．32±0．02mmol／h／mg protein和2．98±0．3 mmol／h／mg protein vs．1．53±0．21 mmol／h／mg protein，p<0．01)，AT1-AA引起的caspase活性的增高可以通过预先用losartan或AT1受体细胞外第二环特异性肽段孵育而消失(图10和11，表4，n=8．10)；0．01μmol／L和0．1μmol／L的AT1-AA对caspase3和8的活性无显著性影响；AT1-AA对caspase9的活性却无明显的作用(图13，n=8)。提示1．0μmol／L的AT1-AA通过激活AT1受体细胞外第二环特异性肽段除能引起坏死外，还可能通过凋亡(尤其是外源性途径)造成内皮细胞损伤。
2．1．0μmol／L的AT1受体自身抗体在不同时间点引起内皮细胞损伤的研究
(1)1．0μmol／L的AT1-AA可以时间依赖性的引起内皮细胞的坏死：我们的研究已经证实AT1-AA在1．0μmol／L的浓度可以引起细胞的损伤，因此，我们采用这个浓度来观察不同时间点AT1-AA对细胞损伤的影响。结果表明，LDH的活性在48h开始升高，且呈时间依赖性，72h时达到最高点，与24h相比，LDH在72h时的活性增加了约3．5倍(图14，表5，n=8)，并且此种效应能够通过与losartan以及细胞外第二环的特异性肽段孵育而消失。
(2)1．0μmol／L的AT1-AA在不同时间点对caspase活性的影响：Caspase3在24h开始增高(从0．29±0．05 nmol／h／mg protein增加到0．45±0．06 nmol／h／mg protein，P<0．05)，48h达到高峰(从0．29±0．05 nmol／h／mg protein增加到0．63±0．06 nmol／h／mg protein，P<0．01)且可以维持到72h(图15，表6，n=6)；caspase8从24h开始增高(从1．39±0．05 nmol／h／mg protein增加到2．02±0．06 nmol／h／mg protein，P<0．05)，48h达到峰值(从1．39±0．05 nmol／h／mg protein增加到2．96±0．07 nmol／h／mg protein，P<0．01)，随后即开始下降，但在72h时仍高于阴性对照组(从1．39±0．05 nmol／h／mg protein增加到2．05±0．07 nmol／h／mg protein，P<0．05)(图16，表7，n=6)：AT1-AA引起caspase3和8活性增高的效应能够通过与losartan或AT1R细胞外第二环的特异性肽段孵育而消失；caspase9的活性在各组之间均无明显的差异(图17，表8，n=5)。
3．抗体滴度的检测结果：AT1R-ECⅡ首次免疫1周内，各组大鼠血清中AT1-AA的平均抗体滴度小于1：10，加强免疫后AT1R-ECⅡ免疫组(n=50)、AT1R-ECⅡ+Vehicle(n=48)组和AT1R-ECⅡ+Bosentan(n=48)组的抗体滴度开始升高，并在免疫后8周达峰值，其抗体滴度分别为1：17056±3．32，1：198413±3．58和1：16054±2．98；伪免疫组(n=46)大鼠血清中AT1-AA的平均抗体滴度始终保持在1：10以下(图18，表9)。
4．为了验证我们的假设，即AT1-AA长期作用下，可以导致内皮细胞的损伤，我们检测了免疫大鼠血清内皮素-1(ET-1)的浓度，结果证实，AT1R-ECⅡ免疫组大鼠免疫后第3周开始血清中ET-1含量增高，第9周达高峰，与对照组相比有显著性差异(38．2％±2．8vs．1．5％±0．01，P<0．01)(图19，表10)。ET-1由内皮细胞合成分泌，存在于正常的组织中，但在内皮受损时，ET-1的合成和分泌增加，因此，ET-1水平的升高在很大程度上反应了内皮受损的程度。因此，我们推测，AT1-AA能引起在体血管内皮的损伤。
5．为了进一步证实我们的假设，我们采用大血管环及微血管环技术检测了免疫大鼠胸主动脉和各组小动脉的收缩及舒张功能，结果显示：免疫12周时，各组血管内皮依赖性舒张功能均显著下降，与同期伪免疫组相比有显著性差异(P<0．01)(图21-24，表11-16)；但各组大鼠的血管收缩功能无显著差异(图25-28)。这一结果更加确证了我们的假设：AT1-AA长期作用下，血管内皮细胞的结构和功能均受到严重影响。
6．免疫组化检测的结果显示，AT1R-ECⅡ免疫组大鼠免疫后8周血管内皮可见典型的iNOS染色，而伪免疫组为阴性，AT1R-ECⅡ免疫组大鼠iNOS的表达是阴性对照组的3．16倍(图29，表17)。研究表明，由iNOS产生的病理浓度NO可以通过生成活性氮代谢物(ReacticeNitrogen Species，RNS)，尤其是硝基化作用很强的过氧亚硝基(peroxynitrite，ONOO~-)分子导致硝基化应激(nitrative stress)和组织损伤。因此，我们推测，AT1-AA长期存在的情况下，可能通过引起iNOS的释放，从而产生病理浓度的NO，最终导致内皮细胞的损伤。
结论
1.AT1-AA可以通过坏死和凋亡(尤其是外源性途径)两种方式，直接引起血管内皮细胞的损伤；
2．在体情况下，AT1-AA长期作用可以引起大鼠内皮细胞的损伤，并使大鼠内皮依赖性舒张血管功能显著降低；但对大鼠血管收缩功能无明显影响；
3．AT1-AA长期作用下，还可以使大鼠血管内皮iNOS的表达显著增强，提示AT1-AA致血管内皮损伤的机制可能与NO生理功能异常有关。
Preeclampsia is a serious pathologic complication during pregnancy and its pathogenesis remains poorly understood. Recent studies have demonstrated that autoantibody against angiotensinⅡtype 1A receptor (AT1-AA) are present in women with preeclampsia and the possible role of this autoantibody in the pathogenesis of preeclampsia has been paid increasingly more attention. In order to evaluate correctly the pathological significance of this autoantibody detection in sera from patients, it is necessary to understand the characteristic distribution of this autoantibody and the relationship between the AT1-AA level and the systolic systolic blood pressure (SBP), proteinuria or platelet in preeclamptic patients. The peptide corresponding to the sequences of the second extracellular loop of the human AT1 receptor is used as antigen in an enzyme immunoassay to screen the sera from preeclamptic patients. Positively was defined as P/N≥2.1; antibody titer was expressed as the maximum dilution at P/N≥2.1. Compared with the normal pregnant women and the healthy nonpregnant women, frequency of AT1-AA positive samples was markedly increased in preeclamptic patients (69.0%±2.9% vs. 29.0%±2.1%, 69.0%±2.9% vs. 8.3%±0.9%, respectively, P<0.01). The antibody titer of the positive sera from preeclamptic patients was in high level with the geometric mean titer being 1:168.6±6.7 for anti-AT1 receptor antibody. Notably, the dependability was observed between the AT1-AA level and the systolic blood pressure in preeclamptic patients. Nevertheless, there was no dependability between the AT1-AA level and the proteinuria or platelet in preeclamptic patients. For comparison, the characteristics of this autoantibody in sera from 56 normal pregnancies and in sera from 48 normal nonpregnancies were also studied.
In conclusion, compared with the normal pregnant women and the healthy nonpregant women, the autoantibody against AT1 receptor in sera from preeclamptic patients is characterized by high frequency and high titer with the positively being increased with blood pressure. These results suggest that overproduction of AT1-AA is a novel risk factor in pregnant women and may play a causative role in the development of preeclamptic patients.
Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. Recent studies have shown that autoantibodies against angiotensinⅡAT_1 receptors (AT1-AA) develop in the sera of patients with preeclampsia and may be involved in the pathogenesis of the disorder. In view of the vascular damages play a crucial role in the pathogenetic feature of preeclampsia, the aim of this study was to clarify the fundamental alterations in vasculature induced by AT1-AA. Vasoconstrictive effect of purified IgG from the sera of either preeclamptic patients or normal pregnant women was determined in isolated rat thoracic aorta, middle cerebral artery, coronary artery, renal artery, mesenteric artery and uterine artery. Our results showed that AT_1-AA induced a marked dose-dependent vasoconstriction in aortic vascular rings. However, curiously enough, AT_1-AA showed variant effects on small resistant vascular rings isolated from different organs. Of these vessels, middle cerebral artery and coronary artery were remarkably constricted by AT_1-AA, but mesenteric, renal and uterine arteries were essentially nonresponsive to AT_1-AA. The effects of AT_1-AA were neutralized by the antagonist Losartan and the peptide corresponding to the second extracellular loop of the AT_1 receptor. These data demonstrated that the AT1-AA caused marked vasoconstriction in some arteries through activate the peptide corresponding to the second extracellular loop of the AT_1 receptor and thus can account for the increased arterial tone in these vascular beds. These findings suggest that AT_1-AA is a novel risk factor in pregnant women and may play a causative role in the development of preeclampsia.
Background: In view of the endothelial damages play a crucial role in the pathogenetic feature of preeclampsia, the aim of this study was to clarify the structural and functional damage of endothelium induced by AT1-AA. The present study investigated whether AT1-AA may induce endothelial structural dysfunction in vitro, and if so, to determine whether long-term active immunization with synthetic peptides corresponding to the second extracellular loop of the AT1-receptor (AT1R) may induce endothelial functional injury in vivo.
Methods and Results: In this study, we investigated the pro-apoptotic and cytotoxic effect of AT1-AA on endothelial cell in vitro. The result demonstrated that AT1-AA was able to significantly increase the release of LDH and caspase 3 from endothelial cell. In addition, Adult female Wistar rats were immunized with synthetic peptides. Anti-ATIR autoantibody, endothelial injury (production of endothelin-1) and vascular function (systolic and diastolic function of thoracic aorta ring and small resistant vascular rings) were determined after immunization. In the sham immunization group, the antibody titer remained below 1:10 and no endothelial injury and vascular functional change were observed. However, in the immunized rats, a significant increase in anti-AT1-R autoantibody occurred 2 weeks after the immunization and peaked at week 8. A significant increase in production of endothelin-1 (ET-1) occurred at week 3 and peaked at week 9. A significant increase of iNOS production occurred 8 weeks after immunization, and severe vascular dysfunction occurred 12 weeks after immunization. Treatment with Bosentan (pan-ET-receptor antagonist) abolished endothelial injury and significantly improved vascular function in the immunized rats.
Conclusion: Our results demonstrated for the first time that AT_1-AA was able to induce endothelial injury in vitro, and also suggested that long-term stimulation with the autoantibody against the second extracellular loop of the AT1R induces endothelial injury in vivo. These results suggest that AT1-AA may be a key role in vascular dysfunction in preeclampsia and the treatments of protecting endothelium may be a novel strategy in reducing multiple organ injury associated with autoimmune response.
引文
1. Boulanger H, Flamant M. New insights in the pathophysiology of preeclampsia and potential therapeutic implications. Nephrol Ther. 2007 Dec; 3(7):437-48.
2. Stepan H, Heihoff-Klose A, Faber R. Reduced antioxidant capacity in second-trimester pregnancies with pathological uterine perfusion. Ultrasound Obstet Gynecol 2004; 23(6):579-583.
3. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, et al. Ptients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest 1999; 103(7):945-952.
4. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation 2004; 110(12): 1612-1619.
5. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, et al. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation 2003; 107(12):1632-1639.
6. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001; 20(1):IX-XIV.
7. Merrifield. R.B, Solid phase peptide synthesis. J. Am. Chem. Soc.85,2149-2154,1963.
8. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity 1999; 29(1):43-51.
9. Fu LX, Magnusson Y, Bergh C, et al. Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in-patients with idiopathic dilated cardiopathy. J. Clin Invest 91:1964-1968, 1993.
10. Weinberger MH, Kramer NJ, Petersen LP, Cleary RE, Young PC. Sequential changes in the renin--angiotensin--aldosterone systems and plasma progesterone concentration in normal and abnormal human pregnancy. Perspect Nephrol Hypertens 1976; 5:263-269.
11. Cooper AC, Robinson G, Vinson GP, Cheung WT, Broughton PF. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancy. Placenta 1999; 20(5-6):467-474.
12. Gant NF, Daley GL, Chand S, Whalley PJ, MacDonald PC. A study of angiotensin II pressor response throughout primigravid pregnancy. J Clin Invest 1973; 52(11):2682-2689.
13. Kyle PM, Buckley D, Kissane J, de SM, Redman CW. The angiotensin sensitivity test and low-dose aspirin are ineffective methods to predict and prevent hypertensive disorders in nulliparous pregnancy. Am J Obstet Gynecol 1995; 173(3 Pt 1):865-872.
14. Davison JM, Homuth V, Jeyabalan A, Conrad KP, Karumanchi SA, Quaggin S, Dechend R, et al. New aspects in the pathophysiology of preeclampsia. J Am Soc Nephrol 2004; 15(9):2440-2448.
15. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation 2000; 101(20):2382-2387.
16. Dechend R, Homuth V, Wallukat G, Miiller DN, Krause M, Dudenhausen J, et al. Agonistic antibodies directed at the angiotensin II, AT1 receptor in preeclampsia. J Soc Gynecol Investig 2006;13(2):79-86.
17. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig 2003; 10(2):82-93.
18. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998; 179(5):1359-1375.
19. Bobst SM, Day MC, Gilstrap LC, III, Xia Y, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion. Am J Hypertens 2005; 18(3):330-336.
1. Boulanger H, Flamant M. New insights in the pathophysiology of preeclampsia and potential therapeutic implications. Nephrol Ther. 2007 Dec; 3(7):437-48.
2. Gant NF, Daley GL, Chand S, et al. A study of angiotensin II pressor response throughout primigravid pregnancy. J Clin Invest. 1973; 52:2682-2689.
3. Baker PN, Broughton Pipkin F, Symonds EM. Comparative study of platelet angiotensin II binding and the angiotensin II sensitivity test as predictors of pregnancy-induced hypertension. Clin Sci (Lond). 1992 Jul; 83(1):89-95.
4. Chesley LC. The renin-angiotensin system in pregnancy. J Reprod Med. 1975 Nov; 15(5): 173-80.
5. Weinberger MH, Kramer NJ, Petersen LP, Cleary RE, Young PC. Sequential changes in the renin-angiotensin-aldosterone systems and plasma progesterone concentration in normal and abnormal human pregnancy. Perspect Nephrol Hypertens. 1976; 5: 263-269.
6. Kyle PM, Buckley D, Kissane J, et al. The angiotensin sensitivity test and low-dose aspirin are ineffective methods to predict and prevent hypertensive disorders in nulliparous pregnancy. Am J Obstet Gynecol. 1995; 173: 865-872.
7. Cooper AC, Robinson G, Vinson GP, Cheung WT, Broughton Pipkin F. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancy. Placenta. 1999; 20: 467-474.
8. Haller H, Oeney T, Hauck U, Distler A, Philipp T. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women. Am J Hypertens. 1989 Apr; 2(4):238-43.
9. Davison JM, Homuth V, Jeyabalan A, Conrad KP, Karumanchi SA, Quaggin S, Dechend R, Luft FC. New aspects in the pathophysiology of preeclampsia. J Am Soc Nephrol. 2004; 9:2440-2448.
10. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 103: 945-952.
11. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, Juepner A, Gulba DC, Mackman N, Haller H, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation. 2000; 101: 2382-2387.
12. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003; 10: 82-93.
13. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107:1632-1639.
14. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap 3rd LC, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation. 2004; 110:1612-1619.
15. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001; 20(1): IX-XIV.
16. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity 1999; 29(1):43-51.
17. Leung DW, Parent AS, Cachianes G, Esch F, Coulombe JN, Nikolics K, Eckenstein FP, Nishi R. Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons. Neuron. 1992; Jun; 8(6):1045-53.
18. Liu HR, Tao L, Gao E, Lopez BL, Christopher TA, Willette RN, Ohlstein EH, Yue TL and Ma XL. Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion. Cardiovasc Res 2004; 62: 135-144.
19. Kezic A, Sparic R, Stojimirovic B, Milenkovic V. Multiorgan dysfunction in a gravid woman with placental abruption and disseminated intravascular coagulation. Srp Arh Celok Lek. 2007 Jul-Aug; 135(7-8):465-7.
20. Dechend R, Homuth V, Wallukat G, Muller DN, Krause M, Dudenhausen J, et al. Agonistic antibodies directed at the angiotensin II, AT1 receptor in preeclampsia. J Soc Gynecol Investing 2006; 13(2):79-86.
21. Bobst SM, Day MC, Gilstrap LC, III, Xia Y, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion. Am J Hypertens 2005; 18(3):330-336.
22. Luther HP, Homuth V, Wallukat G. Alpha 1-adrenergic receptor antibodies in patients with primary hypertension. Hypertension 1997; 29(2):678-682.
23. Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van AA. A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 1994; 101(8):669-674.
24. Barden A. Pre-eclampsia: contribution of maternal constitutional factors and the consequences for cardiovascular health. Clin Exp Pharmacol Physiol 2006; 33(9):826-830.
25. Funai EF, Friedlander Y, Paltiel O, Tiram E, Xue X, Deutsch L, Harlap S. Long-term mortality after preeclampsia. Epidemiology 2005; 16(2):206-215.
26. Irgens HU, Reisaeter L, Irgens LM, Lie RT. Long term mortality of mothers and fathers after pre-eclampsia: population based cohort study. BMJ 2001; 323(7323):1213-1217.
27. Van Geel PP , Pinto YM , Voors AA, et al. Angiotensin Ⅱ type 1 receptor A1166/ C gene polymorphism is associated with an increased response to angiotensin Ⅱ in human arteries. Hypertension. 2000; 35 (3): 717-721
28. Konopleva M, Zhao S, Xie Z, Segall H, Younes A, Claxton DF, Estrov Z, Kornblau SM, Andreeff M. Apoptosis. Molecules and mechanisms. Adv Exp Med Biol. 1999; 457:217-36.
29. Brenner C, Kroemer G. Apoptosis. Mitochondria-the death signal integrators. Science. 2000 Aug 18; 289(5482): 1150-1.
1. Cunningham FG, Lindheimer MD. Hypertension in pregnancy. N Engl J Med. 1992;326:927-932.
2. Roberts JM, Taylor RN, Goldfien A. Endothelial cell activation as a pathogenetic factor in preeclampsia. Semin Perinatol. 1991; 15:86-93.
3. Pascoal IF, Lindheimer MD, Brandt CN, et al. Preeclampsia selectively impairs endothelium-dependent relaxation and leads to oscillatory activity in small omental arteries. J Clin Invest. 1998; 101:464-470.
4. Friedman SA, Taylor RN, Roberts JM. Pathophysiology of preeclampsia. Clin Perinatol. 1991; 18:661-682.
5. Baker PN, Davidge ST, Barankiewicz J, et al. Plasma of preeclamptic women stimulates and then inhibits endothelial prostacyclin. Hypertension. 1996; 27:56-61.
6. Baker PN, Davidge ST, Roberts JM. Plasma from women with preeclampsia increases endothelial cell nitric oxide production. Hypertension. 1995; 26:244 -248.
7. Taylor RN, Musci TJ, Rodgers GM, et al. Preeclamptic sera stimulate increased platelet-derived growth factor mRNA and protein expression by cultured human endothelial cells. Am J Reprod Immunol. 1991; 25: 105-108.
8. Taylor RN, Casal DC, Jones LA, et al. Selective effects of preeclamptic sera on human endothelial cell procoagulant protein expression. Am J Obstet Gynecol. 1991; 165:1705-1710.
9. Mills JL, DerSimonian R, Raymond E, et al. Prostacyclin and thromboxane changes predating clinical onset of preeclampsia. JAMA. 1999; 282:356 -362.
10. Wallukat G, Homuth V, Fischer T, et al. Patients with preeclampsia develop agonistic antibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 1103:945-952.
11. Dechend R, Muller DN, Wallukat G. AT1 receptor agonistic antibodies, hypertension and preeclampsia. Semin Nephrol 2004; 24:571-79.
12. Sun YX, Zhang HY, Wei YM, Zhu F, Wang M, Liao YH. The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension. Chin Med J (Engl). 2008; 121(1):43-8.
13. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107(12):1632-9.
14. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation. 2004; 110(12):1612-9.
15. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003;10(2):82-93.
16. Liu HR. Effects of long-term immunization with synthesized receptor peptide on in vivo cardiac activity in rats. J Shanxi Med Univ. 2001; 32(suppl): 93-9.
17. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac β_1-adrenoceptors and M_2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity. 1999; 29(1):43-51.
18. Ma XL, Gao F, Nelson AH, et al. Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats. J Pharmacol Exp Ther 2001;298:879-85.
19. Staudt A, Mobini R, Fu M, Grosse Y, Stangl V, Stangl K, Thiele A, Baumann G, Felix SB. β_1-adrenoceptor antibodies induce positive inotropic response in isolated cardiomyocytes. Eur J Pharmacol. 2001; 423(2-3): 115-9.
20. Arstall MA, Sawyer DB, Fukazawa R, and Kelly RA: Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ Res 1999,85:829-40
21. Li J, Baud O, Vartanian T, Volpe JJ, and Rosenberg PA: Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci USA 2005,102:9936-41
22. Ohkita M, Takaoka M, Matsumura Y. Endothelin-1 production and its involvement in cardiovascular diseasesYakugaku Zasshi. 2007 Sep; 127(9): 1319-29.
37
1. Meng H, Zhu FF, Wang CH, Xiao GX. Role of platelet-derived growth factor in the pathogenesis of preeclampsia. Nan Fang Yi Ke Da Xue Xue Bao. 2007;27(8): 1274-6.
2. Miitze S, Rudnik-Schoneborn S, Zerres K, Rath W. Genes and the preeclampsia syndrome. J??Perinat Med. 2008; 36(1):38-58.
3. Harskamp RE, Zeeman GG. Preeclampsia: at risk for remote cardiovascular disease. Am J Med Sci. 2007; 34(4):291-5.
4. Arngrimsson R, Siguroardottir S, Frigge ML, et al. A genome2wide scan reveals a maternal susceptibility locus for pre2eclampsia on chromosome 2p13. Hum Molec Genet, 1999, 8: 179921805.
5. Moses EK, Lade JA, Guo G, et al. A genome scan in families from Australia and New Zealand confirms the presence of a maternal susceptibility locus for preeclampsia, on chromosome 2. Am J Hum Genet, 2000, 67: 158121585.
6. Hoff C, Peevy K. Maternal fetal HLA DR 4 relationships and pregnancy induced hypertension [J]. Obset Gynecol, 1992, 80 (3): 1007.
7. Jain A, Rohatgi P, Singh VK, et al. Circulating immune complexes in preeclampsia[J] . J Indian Med Assoc, 1991, 89(2): 251.
8. Bobik A, Scott JA. Growth factor activity of endothelin on vascular smooth muscle cells [J]. AmJ Physiol, 1990, 258(2): 408.
9．吕兴东，耿洪亚，邓仁爱等。妇产科病最新治疗[M]，天津：天津科技出版社，1995．360．
10．徐望明，杨菁，王彩霞。肿瘤坏死因子对妊高征致病作用与内皮素的关系[J]．中国 实用妇科与产科杂志，1998，14(1)：25227．
11. Hanssens M, Keirse MJ, Spitz B, Van Assche FA. Measurement of individual plasma angiotensins in normal pregnancy and pregnancy-induced hypertension. J Clin Endocrinol Metab. 1991;73(3):489-94.
12. Haller H, Ziegler E-M, Homuth V, Eichhorn J, Nagy Z, Luft FC: Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension. 1997;29(1 Pt 2): 291-96.
13. Haller H, Hempel A, Homuth V, Mandelkow A, Maasch C, Drab M, Lindschau C, Vetter K, Dudenhausen J, Luft FC: Endothelial cell permeability and protein kinase C in preeclampsia. Lancet. 1998;351 (9107): 945-949.
14. Wallukat G, Homuth V, Fisher T, Lindschau C, Horstkamp B, Jupner A, Bauer E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin ATI receptor. J Clin Invest. 1999; 103 (7):945-52.
15. Dechend R, Muller DN, Wallukat G. AT1 receptor agonistic antibodies, hypertension and preeclampsia. Semin Nephrol 2004;24:571-79.
16. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation 2000; 101(20):2382-2387.
17. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003;107(12):1632-9.
18. Sun YX, Zhang HY, Wei YM, Zhu F, Wang M, Liao YH. The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension. Chin Med J (Engl). 2008;121(1):43-8.
19.Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca~(2+) mobilization through angiotensin receptor activation. Circulation. 2004;110(12):1612-9.
20. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003;10(2):82-93.
21. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA: Excess placental soluble fmslike tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 111: 649-658, 2003.
22. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, Takeda S, Yano T, Tsutsumi O, Taketani Y: Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab 88: 2348-2351, 2003.
23. Tsatsaris V, Goffin F, Munaut C, Brichant JF, Pignon MR, Noel A, Schaaps JP, Cabrol D, Frankenne F, Foidart JM: Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: Pathophysiological consequences. J Clin Endocrinol Metab 88: 5555-5563, 2003.
24. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE: Glomerularspecific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111: 707-716, 2003.
25. Dvorak HF: Vascular permeability factor/vascular endothelial growth factor: A critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 20: 4368-4380, 2002.
26. Kendall RL, Thomas KA: Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A 90: 10705-10709,1993.
27. He Y, Smith SK, Day KA, Clark DE, Licence DR, Charnock-Jones DS: Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol 13: 537-545, 1999.
28. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA: Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 350: 672-683, 2004.
1. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet.2005; 365:785-799.
2. Robertson WB, Brosens I, Dixon HG. The pathological response of the vessels of the placental bed to hypertensive pregnancy. J Pathol Bacteriol. 1967 Apr; 93(2): 581-92.
3. Lucilla Poston. Endothelial dysfunction in pre-eclampsia. Pharmacological Reports. 2006; 58: 67-74.
4. Taylor RN. Immunhiology of preeclampsia. Am J Raprod Immunol. 1997; 37(1): 79.
5. Robertson L, Wu 0, Langhorne P, Twaddle S, Clark P, Lowe GD, Walker ID, Greaves M, Brenkel I, Regan L, Greer IA; The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) Study. Thrombophilia in pregnancy: a systematic review. Br J Haematol. 2006 Jan; 132(2): 171-96.
6. Roberts JM, Pearson G, Cutler J, Lindheimer M. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension. 2003 Mar; 41(3): 437-45.
7. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 103: 945-952.
8. James M., Roberts, Gail Pearson, Jeff Cutler Marshall Lindheimer. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension.2003; 41:437 -445.
9. Lucilla Poston. Endothelial dysfunction in pre-eclampsia. Pharmacological Reports.2006; 58: 69-74.
10. Roberts JM, Hubei CA. Is oxidative stress the link in the two-stage model of pre-eclampsia? Lancet. 1999;354:788-789. Letter and comment.
11. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA et al. Excess placental soluble fms-like tyrosine kinase 1 (sFltl) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest, 2003; 111, 649-658.
12. Pipkin FB. What is the place of genetics in the pathogenesis of preeclampsia? Biol Neonate. 1999; 76:325-330.
13. James M. Roberts, Judith L. Balk, Lisa M. Bodnar, Jose' M. Beliza' n, yEduardo Bergely and Anibal Martinez. Nutrient Involvement in Preeclampsia. J. Nutr. 2003; 133.1684S-1692S.
14. Ward K, Hata A, Jeunemaitre X, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet.1993; 4:59- 61.
15. Shokei Kim, Hiroshi Iwao. Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases. Pharmacol Reviews. 2000; 52(1):11-34.
16. Haller, H., Oeney, T., Hauck, U., Distler, A., and Philipp, T. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women. Am. J. Hypertens.1989; 2:238-243.
17. Pipkin, F.B. 1988. The renin-angiotensin system in normal and hypertensive pregnancies. In Handbook of hypertension. Vol. 10. P.C. Rubin, editor. Elsevier Science Publishers. Amsterdam, the Netherlands. 118-151.
18. Hanssens, M., Keirse, M., Spitz, B., and Van Assche, F.A. 1991. Angiotensin II levels in hypertensive and normotensive pregnancies. Br. J. Obstet. Gynaecol. 98:155-161.
19. Hannsens, M., Keirse, M., Spitz, B., and Van Assche, F.A. 1991. Measurement
20. Hannsens, M., Keirse, M, Spitz, B., and Van Assche, F.A. Measurement of individual plasma angiotensins in normal pregnancy and pregnancy- induced hypertension. J. Clin. Endocrinol. Metab.1991;73:489-494.
21.Haller H, Ziegler E-M Homuth V, Eichhorn J, Nagy Z, Luft FC: Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension. 1997;29: 291-296.
22. Haller H, Hempel A, Homuth V, Mandelkow A, Maasch C, Drab M, Lindschau C, Vetter K, Dudenhausen J, Luft FC: Endothelial cell permeability and protein kinase C in preeclampsia. Lancet.1998;351: 945-949.
23. Matsui S, Fu ML. Dilated cardiomyopathy defines serum autoantibodies against G- protein-coupled cardiovascular receptors. Autoimmunity.1995;21(2):85-8.
24. Iwata M, Yoshikawa T, Baba A et al. Autoantibodies against the second extracellular loop of beta1-adrenergic receptors predict ventricular tachycardia and sudden death in patients with idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 2001;37:418-424.
25. Zhang L, Hu D, Li J et al. Autoantibodies against the myocardial betal-adrenergic and M2-muscarinic receptors in patients with congestive heart failure. Chin. Med. J. 2002; 115: 1127-1131.
26. Conrad KP, Lindheimer MD: Renal and cardiovascular alterations, in Lindheimer MD, Roberts JM, Cunningham FG (eds): Chesley's HypertensiveDisorders in Pregnancy (2nd ed). Stamford, Appleton and Lange.1999;pp 263-326.
27. 刘虹, 杨宇, 陈珑珑. AT1受体自身抗体与原发性高血压的关系[J].湖南医科大学学报, 2003; 28(3): 255—258.
28. Ralf Dechend, Dominik N. Muller, Gerd Wallukat, Volker Homuth, Manya Krause, Joachim Dudenhausen, and Friedrich C. Luft AT1 Receptor Agonistic Antibodies,Hypertension, and Preeclampsia. Elsevier Inc.2004; 24:571-579 .
29. Robillard PY, Dekker GA, Hulsey TC. Primipaternities in families: is the incidence of pregnancy-induced hypertensive disorders in multigravidas an anthropological marker of reproduction? Aust N Z j Obstet Gynaecol.1998; 38:284 -287. Review.
30. Almus FE, Rao LV, Fleck RA, Rapaport SI. Properties of factor VIIa/tissue factor complexes in an umbilical vein model.Blood; 1990 Jul 15;76(2):354-60.
31. Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol. 1989 May; 134(5): 1087-97.
32．熊石龙，王前，郑磊，李俊玲，文志斌，贺石林．．急性心肌梗死及脑梗死患者组织因 子、组织因子途径抑制物、因子Ⅶ的检测及临床意义．南方医科大学学报，2007；27(12)： 1821-1823．
33. Roque M, Reis ED, Fuster V, Padureau A, Fallon JT, Taubman MB, et al. Inhibition of tissue factor reduces thrombus formation and intinal hyperplasia after porcine coronary angioplasty. J Am Coll cardiol.2000; 36(7): 2 303-310.
34. Wada H, Mori Y, Okabayahi K, Gabazza EC, Kushiya F, Watanabe M, Nishikawa M, Shiku H, Nobori T: High plasma fibrinogen level is associated with poor clinical outcome in DIC patients. Am J Hematol.2003; 72:1-7.
35. De Boer K, Lecander I, ten Cate H, Borm JJ, Treffers PE: Placentaltype plasminogen activator inhibitor in preeclampsia. Am J Obstet Gynecol.1988; 158:518 -522.
36. Lorentzen B, Henriksen T. Plasma lipids and vascular dysfunction in preeclampsia. Semin Reprod Endocrinol. 1998; 16(l):33-9.
37. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. J Clin Invest.1997; 99:2152-2164.
38. Udagawa K, Miyagi Y, Hirahara F, et al. Specific expression of PP5/TFPI2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization. Placenta. 1998; 19:217-223.
39. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, Juepner A, Gulba DC, Mackman N, Haller H, Luft FC: AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation.2000; 101: 2382-2387.
40．王冰，吴燕红，杨志，张舒．MAPK／ERK信号转导通路的分子生物学特征及生物效应．第 四军医大学学报(J Fourth Mil Med Univ)2005；26(12)：18-21．
41. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med. 1999; 222:222-235.
42. Walsh SW, Vaughan JE, Wang Y, et al. Placental isoprostane is significantly increased in preeclampsia. FASEB J. 2000; 14:1289-1296.
43. Many A, Hubel CA, Fisher SJ. Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am J Pathol. 2000;156:321-331.
44. Raijmakers MT, Dechend R, Poston L. Oxidative stress and preeclampsia eationale for antioxident clinical trials f J I. Hypertension.2004; 44(4): 374-380.
45. Sikkema JM, van Rijn BB, Franx A, et al. Placental superoxide is increased in pre-eclampsia. Placenta. 2001; 22:304-308.
46. De Wolf F, Robertson WB, Brosens I. The ultrastructure of acute atherosis in hypertensive pregnancy. Am J Obstet Gynecol.1975; 123:164-174.
47．朱梅仙．NADPH氧化酶与子痫前期关系的研究进展．国外医学：妇产科学分册．2007； 34(1)： 18-21．
48. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC: ATI receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107: 1632-1639.
49. Engeli S, Negrel R, Sharma AM. Physiology and pathophysiology of the adipose tissue renin-angiotensin system. Hypertension. 2000; 35:1270-1277.
50. Zatisev SV, AppolskogIB, etal. Imodazoline compounds protect againstin- terleukin 1 beta-induced beta-cell apoptosis[J]. Diabetes.2001; 50(1): 70-76.
51. Szabo, et al. Inhibition of terminal calcium overload protects against peroxynirtite induced cellular injury in macrophages [J]. Immunol Lett, 1996, 51(3): 163-167.
52．高利平．钙超载与细胞损伤．钙超载与细胞损伤．四川医学．2004；25(9)：1039-1041．
53. Pridjian G, Puschett JB. Preeclampsia: part 2: experimental and genetic considerations. Obstet Gynecol Surv. 2002; 57:619-640.
54. Ray J, Vasishta K, Kaur S, Majumdar S, Sawhney H. Calcium metabolism in pre-eclampsia. Int J Gynaecol Obstet. 1999; 66:245-250.
55. Haller H, Oeney T, Hauck U, Distler A, Philipp T. Increased intracellular free calcium and sensitivity to angiotensin Ⅱ in platelets of preeclamptic women. Am J Hypertens. 1989;2:238-243.
56. Hojo M, Suthanthiran M, Helseth G, August P. Lymphocyte intracellular free calcium concentration is increased in preeclampsia. Am J Obstet Gynecol. 1999; 180:1209 -1214.
57. Sowers JR, Zemel MB, Bronsteen RA, Zemel PC, Walsh MF, Standley PR, Sokol RJ. Erythrocyte cation metabolism in preeclampsia. Am J Obstet Gynecol. 1989; 161:441- 445.
58. Hanssens M, Keirse MJ, Spitz B, van Assche FA. Angiotensin Ⅱ levels in hypertensive and normotensive pregnancies. Br J Obstet Gynaecol. 1991; 98:155-161.
59. Gant NF, Daley GL, Chand S, Whalley PJ, MacDonald PC. A study of angiotensin II??pressor response throughout primigravid pregnancy. J Clin Invest. 1973; 52:2682-2689.
60. Theingi M. Thway, Sergiy G. Shlykov, Mary-Clare Day, Barbara M. Sanborn, LarryC. Gilstrap, Ⅲ, Yang Xia and Rodney E. Kellems. Antibodies From Preeclamptic Patients Stimulate Increased Intracellular Ca~(2+) Mobilization Through Angiotensin Receptor Activation. Circulation. 2004; 110:1612-1619.
61．陈燕．钙调神经磷酸酶和NFAT在T细胞活化中的作用．国外医学免疫学分册1999；22 (4)： 225-228．
62. Xia Y, Wen H, Bobst S, Day MC, Kellems RE: Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig 2003; 10:82-93.
63．慕澜，魏连波，李学．白介素6的研究进展．深圳中西医结合杂志．2003；13(3)：185-188．
64. Moriyama T, Fujibayashi M, Fujiwara Y, Kaneko T, Xia C, Imai E, Kamada T, Ando A, Ueda N: Angiotensin Ⅱ stimulates interleukin-6 release from cultured mouse mesangial cells. J Am Soc Nephrol.1995; 6:95-101.
65. Theuer J, Dechend R, Muller DN, Park JK, Fiebeler A, Barta P, Ganten D, Haller H, Dietz R, Luft FC: Angiotensin Ⅱ induced inflammation in the kidney and in the heart of double transgenic rats. BioMed Central Cardiovasc Disord 2002;2:3.
66. Bluethmann H, Rothe J, Schultze N, Tkachuk M, Koebel P: Establishment of the role of IL-6 and TNF receptor 1 using gene knockout mice. J Leukoc Biol 1994; 56:565-570.
67．傅勤，林建华．正常妊娠妇女与重度子痫前期患者止凝血功能的研究．现代妇产科进 展2007：16(9)：657-660．
68. Phillips MI, Kagiyama S: Angiotensin Ⅱ as a pro-inflammatory mediator. Curr Opin Invest Drugs 2002; 4:569 -577.
69. Wilson HM, Haites NE, Booth NA: Effect of angiotensin Ⅱ on plasminogen activator inhibitor-1 production by cultured human mesangial cells. Nephron 1997;77:197-204.
70. Sol M. Bobst, Mary-Clare Day, Larry C. Gilstrap Ⅲ, Yang Xia, and Rodney E. Kellems. Maternal Autoantibodies From Preeclamptic Patients Activate Angiotensin Receptors on Human MesangialCells and Induce Interleukin-6 and Plasminogen Activator Inhibitor-1 Secretion. AJH2005; 18:330-336.
71. Lyall F, Simpson H, Nicola BJ, et al. Transforming growth factor-beta expression in human placenta and placental bed in third trimester norm al pregnancy, preeclampsia. and fetal growth restriction. Am J Pathol. 2001; 159(5): 1827-1838.
72. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003 Feb; 10(2):82-93.
73. Bottazano GF, Doniach D: Autoimmune thyroid disease. Annu Rev Med 1986;37:353-359.
74. Chistiakov DA: Thyroid-stimulating hormone receptor and its role in Graves' disease. Mol Genet Metab 2003; 80:377-388.
75. Jahns R, Boivin V, Siegmund C, Inselmann G, Lohse MJ, Boege F: Autoantibodies activating human beta 1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation 1999; 99:649-654.
2. Stepan H, Heihoff-Klose A, Faber R. Reduced antioxidant capacity in second-trimester pregnancies with pathological uterine perfusion. Ultrasound Obstet Gynecol 2004; 23(6):579-583.
3. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, et al. Ptients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest 1999; 103(7):945-952.
4. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation 2004; 110(12): 1612-1619.
5. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, et al. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation 2003; 107(12):1632-1639.
6. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001; 20(1):IX-XIV.
7. Merrifield. R.B, Solid phase peptide synthesis. J. Am. Chem. Soc.85,2149-2154,1963.
8. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity 1999; 29(1):43-51.
9. Fu LX, Magnusson Y, Bergh C, et al. Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in-patients with idiopathic dilated cardiopathy. J. Clin Invest 91:1964-1968, 1993.
10. Weinberger MH, Kramer NJ, Petersen LP, Cleary RE, Young PC. Sequential changes in the renin--angiotensin--aldosterone systems and plasma progesterone concentration in normal and abnormal human pregnancy. Perspect Nephrol Hypertens 1976; 5:263-269.
11. Cooper AC, Robinson G, Vinson GP, Cheung WT, Broughton PF. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancy. Placenta 1999; 20(5-6):467-474.
12. Gant NF, Daley GL, Chand S, Whalley PJ, MacDonald PC. A study of angiotensin II pressor response throughout primigravid pregnancy. J Clin Invest 1973; 52(11):2682-2689.
13. Kyle PM, Buckley D, Kissane J, de SM, Redman CW. The angiotensin sensitivity test and low-dose aspirin are ineffective methods to predict and prevent hypertensive disorders in nulliparous pregnancy. Am J Obstet Gynecol 1995; 173(3 Pt 1):865-872.
14. Davison JM, Homuth V, Jeyabalan A, Conrad KP, Karumanchi SA, Quaggin S, Dechend R, et al. New aspects in the pathophysiology of preeclampsia. J Am Soc Nephrol 2004; 15(9):2440-2448.
15. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation 2000; 101(20):2382-2387.
16. Dechend R, Homuth V, Wallukat G, Miiller DN, Krause M, Dudenhausen J, et al. Agonistic antibodies directed at the angiotensin II, AT1 receptor in preeclampsia. J Soc Gynecol Investig 2006;13(2):79-86.
17. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig 2003; 10(2):82-93.
18. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998; 179(5):1359-1375.
19. Bobst SM, Day MC, Gilstrap LC, III, Xia Y, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion. Am J Hypertens 2005; 18(3):330-336.
1. Boulanger H, Flamant M. New insights in the pathophysiology of preeclampsia and potential therapeutic implications. Nephrol Ther. 2007 Dec; 3(7):437-48.
2. Gant NF, Daley GL, Chand S, et al. A study of angiotensin II pressor response throughout primigravid pregnancy. J Clin Invest. 1973; 52:2682-2689.
3. Baker PN, Broughton Pipkin F, Symonds EM. Comparative study of platelet angiotensin II binding and the angiotensin II sensitivity test as predictors of pregnancy-induced hypertension. Clin Sci (Lond). 1992 Jul; 83(1):89-95.
4. Chesley LC. The renin-angiotensin system in pregnancy. J Reprod Med. 1975 Nov; 15(5): 173-80.
5. Weinberger MH, Kramer NJ, Petersen LP, Cleary RE, Young PC. Sequential changes in the renin-angiotensin-aldosterone systems and plasma progesterone concentration in normal and abnormal human pregnancy. Perspect Nephrol Hypertens. 1976; 5: 263-269.
6. Kyle PM, Buckley D, Kissane J, et al. The angiotensin sensitivity test and low-dose aspirin are ineffective methods to predict and prevent hypertensive disorders in nulliparous pregnancy. Am J Obstet Gynecol. 1995; 173: 865-872.
7. Cooper AC, Robinson G, Vinson GP, Cheung WT, Broughton Pipkin F. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancy. Placenta. 1999; 20: 467-474.
8. Haller H, Oeney T, Hauck U, Distler A, Philipp T. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women. Am J Hypertens. 1989 Apr; 2(4):238-43.
9. Davison JM, Homuth V, Jeyabalan A, Conrad KP, Karumanchi SA, Quaggin S, Dechend R, Luft FC. New aspects in the pathophysiology of preeclampsia. J Am Soc Nephrol. 2004; 9:2440-2448.
10. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 103: 945-952.
11. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, Juepner A, Gulba DC, Mackman N, Haller H, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation. 2000; 101: 2382-2387.
12. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003; 10: 82-93.
13. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107:1632-1639.
14. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap 3rd LC, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation. 2004; 110:1612-1619.
15. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001; 20(1): IX-XIV.
16. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity 1999; 29(1):43-51.
17. Leung DW, Parent AS, Cachianes G, Esch F, Coulombe JN, Nikolics K, Eckenstein FP, Nishi R. Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons. Neuron. 1992; Jun; 8(6):1045-53.
18. Liu HR, Tao L, Gao E, Lopez BL, Christopher TA, Willette RN, Ohlstein EH, Yue TL and Ma XL. Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion. Cardiovasc Res 2004; 62: 135-144.
19. Kezic A, Sparic R, Stojimirovic B, Milenkovic V. Multiorgan dysfunction in a gravid woman with placental abruption and disseminated intravascular coagulation. Srp Arh Celok Lek. 2007 Jul-Aug; 135(7-8):465-7.
20. Dechend R, Homuth V, Wallukat G, Muller DN, Krause M, Dudenhausen J, et al. Agonistic antibodies directed at the angiotensin II, AT1 receptor in preeclampsia. J Soc Gynecol Investing 2006; 13(2):79-86.
21. Bobst SM, Day MC, Gilstrap LC, III, Xia Y, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion. Am J Hypertens 2005; 18(3):330-336.
22. Luther HP, Homuth V, Wallukat G. Alpha 1-adrenergic receptor antibodies in patients with primary hypertension. Hypertension 1997; 29(2):678-682.
23. Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van AA. A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 1994; 101(8):669-674.
24. Barden A. Pre-eclampsia: contribution of maternal constitutional factors and the consequences for cardiovascular health. Clin Exp Pharmacol Physiol 2006; 33(9):826-830.
25. Funai EF, Friedlander Y, Paltiel O, Tiram E, Xue X, Deutsch L, Harlap S. Long-term mortality after preeclampsia. Epidemiology 2005; 16(2):206-215.
26. Irgens HU, Reisaeter L, Irgens LM, Lie RT. Long term mortality of mothers and fathers after pre-eclampsia: population based cohort study. BMJ 2001; 323(7323):1213-1217.
27. Van Geel PP , Pinto YM , Voors AA, et al. Angiotensin Ⅱ type 1 receptor A1166/ C gene polymorphism is associated with an increased response to angiotensin Ⅱ in human arteries. Hypertension. 2000; 35 (3): 717-721
28. Konopleva M, Zhao S, Xie Z, Segall H, Younes A, Claxton DF, Estrov Z, Kornblau SM, Andreeff M. Apoptosis. Molecules and mechanisms. Adv Exp Med Biol. 1999; 457:217-36.
29. Brenner C, Kroemer G. Apoptosis. Mitochondria-the death signal integrators. Science. 2000 Aug 18; 289(5482): 1150-1.
1. Cunningham FG, Lindheimer MD. Hypertension in pregnancy. N Engl J Med. 1992;326:927-932.
2. Roberts JM, Taylor RN, Goldfien A. Endothelial cell activation as a pathogenetic factor in preeclampsia. Semin Perinatol. 1991; 15:86-93.
3. Pascoal IF, Lindheimer MD, Brandt CN, et al. Preeclampsia selectively impairs endothelium-dependent relaxation and leads to oscillatory activity in small omental arteries. J Clin Invest. 1998; 101:464-470.
4. Friedman SA, Taylor RN, Roberts JM. Pathophysiology of preeclampsia. Clin Perinatol. 1991; 18:661-682.
5. Baker PN, Davidge ST, Barankiewicz J, et al. Plasma of preeclamptic women stimulates and then inhibits endothelial prostacyclin. Hypertension. 1996; 27:56-61.
6. Baker PN, Davidge ST, Roberts JM. Plasma from women with preeclampsia increases endothelial cell nitric oxide production. Hypertension. 1995; 26:244 -248.
7. Taylor RN, Musci TJ, Rodgers GM, et al. Preeclamptic sera stimulate increased platelet-derived growth factor mRNA and protein expression by cultured human endothelial cells. Am J Reprod Immunol. 1991; 25: 105-108.
8. Taylor RN, Casal DC, Jones LA, et al. Selective effects of preeclamptic sera on human endothelial cell procoagulant protein expression. Am J Obstet Gynecol. 1991; 165:1705-1710.
9. Mills JL, DerSimonian R, Raymond E, et al. Prostacyclin and thromboxane changes predating clinical onset of preeclampsia. JAMA. 1999; 282:356 -362.
10. Wallukat G, Homuth V, Fischer T, et al. Patients with preeclampsia develop agonistic antibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 1103:945-952.
11. Dechend R, Muller DN, Wallukat G. AT1 receptor agonistic antibodies, hypertension and preeclampsia. Semin Nephrol 2004; 24:571-79.
12. Sun YX, Zhang HY, Wei YM, Zhu F, Wang M, Liao YH. The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension. Chin Med J (Engl). 2008; 121(1):43-8.
13. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107(12):1632-9.
14. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation. 2004; 110(12):1612-9.
15. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003;10(2):82-93.
16. Liu HR. Effects of long-term immunization with synthesized receptor peptide on in vivo cardiac activity in rats. J Shanxi Med Univ. 2001; 32(suppl): 93-9.
17. Liu HR, Zhao RR, Zhi JM, Wu BW, Fu ML. Screening of serum autoantibodies to cardiac β_1-adrenoceptors and M_2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages. Autoimmunity. 1999; 29(1):43-51.
18. Ma XL, Gao F, Nelson AH, et al. Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats. J Pharmacol Exp Ther 2001;298:879-85.
19. Staudt A, Mobini R, Fu M, Grosse Y, Stangl V, Stangl K, Thiele A, Baumann G, Felix SB. β_1-adrenoceptor antibodies induce positive inotropic response in isolated cardiomyocytes. Eur J Pharmacol. 2001; 423(2-3): 115-9.
20. Arstall MA, Sawyer DB, Fukazawa R, and Kelly RA: Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ Res 1999,85:829-40
21. Li J, Baud O, Vartanian T, Volpe JJ, and Rosenberg PA: Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci USA 2005,102:9936-41
22. Ohkita M, Takaoka M, Matsumura Y. Endothelin-1 production and its involvement in cardiovascular diseasesYakugaku Zasshi. 2007 Sep; 127(9): 1319-29.
37
1. Meng H, Zhu FF, Wang CH, Xiao GX. Role of platelet-derived growth factor in the pathogenesis of preeclampsia. Nan Fang Yi Ke Da Xue Xue Bao. 2007;27(8): 1274-6.
2. Miitze S, Rudnik-Schoneborn S, Zerres K, Rath W. Genes and the preeclampsia syndrome. J??Perinat Med. 2008; 36(1):38-58.
3. Harskamp RE, Zeeman GG. Preeclampsia: at risk for remote cardiovascular disease. Am J Med Sci. 2007; 34(4):291-5.
4. Arngrimsson R, Siguroardottir S, Frigge ML, et al. A genome2wide scan reveals a maternal susceptibility locus for pre2eclampsia on chromosome 2p13. Hum Molec Genet, 1999, 8: 179921805.
5. Moses EK, Lade JA, Guo G, et al. A genome scan in families from Australia and New Zealand confirms the presence of a maternal susceptibility locus for preeclampsia, on chromosome 2. Am J Hum Genet, 2000, 67: 158121585.
6. Hoff C, Peevy K. Maternal fetal HLA DR 4 relationships and pregnancy induced hypertension [J]. Obset Gynecol, 1992, 80 (3): 1007.
7. Jain A, Rohatgi P, Singh VK, et al. Circulating immune complexes in preeclampsia[J] . J Indian Med Assoc, 1991, 89(2): 251.
8. Bobik A, Scott JA. Growth factor activity of endothelin on vascular smooth muscle cells [J]. AmJ Physiol, 1990, 258(2): 408.
9．吕兴东，耿洪亚，邓仁爱等。妇产科病最新治疗[M]，天津：天津科技出版社，1995．360．
10．徐望明，杨菁，王彩霞。肿瘤坏死因子对妊高征致病作用与内皮素的关系[J]．中国 实用妇科与产科杂志，1998，14(1)：25227．
11. Hanssens M, Keirse MJ, Spitz B, Van Assche FA. Measurement of individual plasma angiotensins in normal pregnancy and pregnancy-induced hypertension. J Clin Endocrinol Metab. 1991;73(3):489-94.
12. Haller H, Ziegler E-M, Homuth V, Eichhorn J, Nagy Z, Luft FC: Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension. 1997;29(1 Pt 2): 291-96.
13. Haller H, Hempel A, Homuth V, Mandelkow A, Maasch C, Drab M, Lindschau C, Vetter K, Dudenhausen J, Luft FC: Endothelial cell permeability and protein kinase C in preeclampsia. Lancet. 1998;351 (9107): 945-949.
14. Wallukat G, Homuth V, Fisher T, Lindschau C, Horstkamp B, Jupner A, Bauer E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin ATI receptor. J Clin Invest. 1999; 103 (7):945-52.
15. Dechend R, Muller DN, Wallukat G. AT1 receptor agonistic antibodies, hypertension and preeclampsia. Semin Nephrol 2004;24:571-79.
16. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation 2000; 101(20):2382-2387.
17. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003;107(12):1632-9.
18. Sun YX, Zhang HY, Wei YM, Zhu F, Wang M, Liao YH. The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension. Chin Med J (Engl). 2008;121(1):43-8.
19.Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC 3rd, Xia Y, Kellems RE. Antibodies from preeclamptic patients stimulate increased intracellular Ca~(2+) mobilization through angiotensin receptor activation. Circulation. 2004;110(12):1612-9.
20. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003;10(2):82-93.
21. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA: Excess placental soluble fmslike tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 111: 649-658, 2003.
22. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, Takeda S, Yano T, Tsutsumi O, Taketani Y: Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab 88: 2348-2351, 2003.
23. Tsatsaris V, Goffin F, Munaut C, Brichant JF, Pignon MR, Noel A, Schaaps JP, Cabrol D, Frankenne F, Foidart JM: Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: Pathophysiological consequences. J Clin Endocrinol Metab 88: 5555-5563, 2003.
24. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE: Glomerularspecific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111: 707-716, 2003.
25. Dvorak HF: Vascular permeability factor/vascular endothelial growth factor: A critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 20: 4368-4380, 2002.
26. Kendall RL, Thomas KA: Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A 90: 10705-10709,1993.
27. He Y, Smith SK, Day KA, Clark DE, Licence DR, Charnock-Jones DS: Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol 13: 537-545, 1999.
28. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA: Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 350: 672-683, 2004.
1. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet.2005; 365:785-799.
2. Robertson WB, Brosens I, Dixon HG. The pathological response of the vessels of the placental bed to hypertensive pregnancy. J Pathol Bacteriol. 1967 Apr; 93(2): 581-92.
3. Lucilla Poston. Endothelial dysfunction in pre-eclampsia. Pharmacological Reports. 2006; 58: 67-74.
4. Taylor RN. Immunhiology of preeclampsia. Am J Raprod Immunol. 1997; 37(1): 79.
5. Robertson L, Wu 0, Langhorne P, Twaddle S, Clark P, Lowe GD, Walker ID, Greaves M, Brenkel I, Regan L, Greer IA; The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) Study. Thrombophilia in pregnancy: a systematic review. Br J Haematol. 2006 Jan; 132(2): 171-96.
6. Roberts JM, Pearson G, Cutler J, Lindheimer M. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension. 2003 Mar; 41(3): 437-45.
7. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest. 1999; 103: 945-952.
8. James M., Roberts, Gail Pearson, Jeff Cutler Marshall Lindheimer. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension.2003; 41:437 -445.
9. Lucilla Poston. Endothelial dysfunction in pre-eclampsia. Pharmacological Reports.2006; 58: 69-74.
10. Roberts JM, Hubei CA. Is oxidative stress the link in the two-stage model of pre-eclampsia? Lancet. 1999;354:788-789. Letter and comment.
11. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA et al. Excess placental soluble fms-like tyrosine kinase 1 (sFltl) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest, 2003; 111, 649-658.
12. Pipkin FB. What is the place of genetics in the pathogenesis of preeclampsia? Biol Neonate. 1999; 76:325-330.
13. James M. Roberts, Judith L. Balk, Lisa M. Bodnar, Jose' M. Beliza' n, yEduardo Bergely and Anibal Martinez. Nutrient Involvement in Preeclampsia. J. Nutr. 2003; 133.1684S-1692S.
14. Ward K, Hata A, Jeunemaitre X, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet.1993; 4:59- 61.
15. Shokei Kim, Hiroshi Iwao. Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases. Pharmacol Reviews. 2000; 52(1):11-34.
16. Haller, H., Oeney, T., Hauck, U., Distler, A., and Philipp, T. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women. Am. J. Hypertens.1989; 2:238-243.
17. Pipkin, F.B. 1988. The renin-angiotensin system in normal and hypertensive pregnancies. In Handbook of hypertension. Vol. 10. P.C. Rubin, editor. Elsevier Science Publishers. Amsterdam, the Netherlands. 118-151.
18. Hanssens, M., Keirse, M., Spitz, B., and Van Assche, F.A. 1991. Angiotensin II levels in hypertensive and normotensive pregnancies. Br. J. Obstet. Gynaecol. 98:155-161.
19. Hannsens, M., Keirse, M., Spitz, B., and Van Assche, F.A. 1991. Measurement
20. Hannsens, M., Keirse, M, Spitz, B., and Van Assche, F.A. Measurement of individual plasma angiotensins in normal pregnancy and pregnancy- induced hypertension. J. Clin. Endocrinol. Metab.1991;73:489-494.
21.Haller H, Ziegler E-M Homuth V, Eichhorn J, Nagy Z, Luft FC: Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension. 1997;29: 291-296.
22. Haller H, Hempel A, Homuth V, Mandelkow A, Maasch C, Drab M, Lindschau C, Vetter K, Dudenhausen J, Luft FC: Endothelial cell permeability and protein kinase C in preeclampsia. Lancet.1998;351: 945-949.
23. Matsui S, Fu ML. Dilated cardiomyopathy defines serum autoantibodies against G- protein-coupled cardiovascular receptors. Autoimmunity.1995;21(2):85-8.
24. Iwata M, Yoshikawa T, Baba A et al. Autoantibodies against the second extracellular loop of beta1-adrenergic receptors predict ventricular tachycardia and sudden death in patients with idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 2001;37:418-424.
25. Zhang L, Hu D, Li J et al. Autoantibodies against the myocardial betal-adrenergic and M2-muscarinic receptors in patients with congestive heart failure. Chin. Med. J. 2002; 115: 1127-1131.
26. Conrad KP, Lindheimer MD: Renal and cardiovascular alterations, in Lindheimer MD, Roberts JM, Cunningham FG (eds): Chesley's HypertensiveDisorders in Pregnancy (2nd ed). Stamford, Appleton and Lange.1999;pp 263-326.
27. 刘虹, 杨宇, 陈珑珑. AT1受体自身抗体与原发性高血压的关系[J].湖南医科大学学报, 2003; 28(3): 255—258.
28. Ralf Dechend, Dominik N. Muller, Gerd Wallukat, Volker Homuth, Manya Krause, Joachim Dudenhausen, and Friedrich C. Luft AT1 Receptor Agonistic Antibodies,Hypertension, and Preeclampsia. Elsevier Inc.2004; 24:571-579 .
29. Robillard PY, Dekker GA, Hulsey TC. Primipaternities in families: is the incidence of pregnancy-induced hypertensive disorders in multigravidas an anthropological marker of reproduction? Aust N Z j Obstet Gynaecol.1998; 38:284 -287. Review.
30. Almus FE, Rao LV, Fleck RA, Rapaport SI. Properties of factor VIIa/tissue factor complexes in an umbilical vein model.Blood; 1990 Jul 15;76(2):354-60.
31. Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol. 1989 May; 134(5): 1087-97.
32．熊石龙，王前，郑磊，李俊玲，文志斌，贺石林．．急性心肌梗死及脑梗死患者组织因 子、组织因子途径抑制物、因子Ⅶ的检测及临床意义．南方医科大学学报，2007；27(12)： 1821-1823．
33. Roque M, Reis ED, Fuster V, Padureau A, Fallon JT, Taubman MB, et al. Inhibition of tissue factor reduces thrombus formation and intinal hyperplasia after porcine coronary angioplasty. J Am Coll cardiol.2000; 36(7): 2 303-310.
34. Wada H, Mori Y, Okabayahi K, Gabazza EC, Kushiya F, Watanabe M, Nishikawa M, Shiku H, Nobori T: High plasma fibrinogen level is associated with poor clinical outcome in DIC patients. Am J Hematol.2003; 72:1-7.
35. De Boer K, Lecander I, ten Cate H, Borm JJ, Treffers PE: Placentaltype plasminogen activator inhibitor in preeclampsia. Am J Obstet Gynecol.1988; 158:518 -522.
36. Lorentzen B, Henriksen T. Plasma lipids and vascular dysfunction in preeclampsia. Semin Reprod Endocrinol. 1998; 16(l):33-9.
37. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. J Clin Invest.1997; 99:2152-2164.
38. Udagawa K, Miyagi Y, Hirahara F, et al. Specific expression of PP5/TFPI2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization. Placenta. 1998; 19:217-223.
39. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, Juepner A, Gulba DC, Mackman N, Haller H, Luft FC: AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation.2000; 101: 2382-2387.
40．王冰，吴燕红，杨志，张舒．MAPK／ERK信号转导通路的分子生物学特征及生物效应．第 四军医大学学报(J Fourth Mil Med Univ)2005；26(12)：18-21．
41. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med. 1999; 222:222-235.
42. Walsh SW, Vaughan JE, Wang Y, et al. Placental isoprostane is significantly increased in preeclampsia. FASEB J. 2000; 14:1289-1296.
43. Many A, Hubel CA, Fisher SJ. Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am J Pathol. 2000;156:321-331.
44. Raijmakers MT, Dechend R, Poston L. Oxidative stress and preeclampsia eationale for antioxident clinical trials f J I. Hypertension.2004; 44(4): 374-380.
45. Sikkema JM, van Rijn BB, Franx A, et al. Placental superoxide is increased in pre-eclampsia. Placenta. 2001; 22:304-308.
46. De Wolf F, Robertson WB, Brosens I. The ultrastructure of acute atherosis in hypertensive pregnancy. Am J Obstet Gynecol.1975; 123:164-174.
47．朱梅仙．NADPH氧化酶与子痫前期关系的研究进展．国外医学：妇产科学分册．2007； 34(1)： 18-21．
48. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC: ATI receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation. 2003; 107: 1632-1639.
49. Engeli S, Negrel R, Sharma AM. Physiology and pathophysiology of the adipose tissue renin-angiotensin system. Hypertension. 2000; 35:1270-1277.
50. Zatisev SV, AppolskogIB, etal. Imodazoline compounds protect againstin- terleukin 1 beta-induced beta-cell apoptosis[J]. Diabetes.2001; 50(1): 70-76.
51. Szabo, et al. Inhibition of terminal calcium overload protects against peroxynirtite induced cellular injury in macrophages [J]. Immunol Lett, 1996, 51(3): 163-167.
52．高利平．钙超载与细胞损伤．钙超载与细胞损伤．四川医学．2004；25(9)：1039-1041．
53. Pridjian G, Puschett JB. Preeclampsia: part 2: experimental and genetic considerations. Obstet Gynecol Surv. 2002; 57:619-640.
54. Ray J, Vasishta K, Kaur S, Majumdar S, Sawhney H. Calcium metabolism in pre-eclampsia. Int J Gynaecol Obstet. 1999; 66:245-250.
55. Haller H, Oeney T, Hauck U, Distler A, Philipp T. Increased intracellular free calcium and sensitivity to angiotensin Ⅱ in platelets of preeclamptic women. Am J Hypertens. 1989;2:238-243.
56. Hojo M, Suthanthiran M, Helseth G, August P. Lymphocyte intracellular free calcium concentration is increased in preeclampsia. Am J Obstet Gynecol. 1999; 180:1209 -1214.
57. Sowers JR, Zemel MB, Bronsteen RA, Zemel PC, Walsh MF, Standley PR, Sokol RJ. Erythrocyte cation metabolism in preeclampsia. Am J Obstet Gynecol. 1989; 161:441- 445.
58. Hanssens M, Keirse MJ, Spitz B, van Assche FA. Angiotensin Ⅱ levels in hypertensive and normotensive pregnancies. Br J Obstet Gynaecol. 1991; 98:155-161.
59. Gant NF, Daley GL, Chand S, Whalley PJ, MacDonald PC. A study of angiotensin II??pressor response throughout primigravid pregnancy. J Clin Invest. 1973; 52:2682-2689.
60. Theingi M. Thway, Sergiy G. Shlykov, Mary-Clare Day, Barbara M. Sanborn, LarryC. Gilstrap, Ⅲ, Yang Xia and Rodney E. Kellems. Antibodies From Preeclamptic Patients Stimulate Increased Intracellular Ca~(2+) Mobilization Through Angiotensin Receptor Activation. Circulation. 2004; 110:1612-1619.
61．陈燕．钙调神经磷酸酶和NFAT在T细胞活化中的作用．国外医学免疫学分册1999；22 (4)： 225-228．
62. Xia Y, Wen H, Bobst S, Day MC, Kellems RE: Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig 2003; 10:82-93.
63．慕澜，魏连波，李学．白介素6的研究进展．深圳中西医结合杂志．2003；13(3)：185-188．
64. Moriyama T, Fujibayashi M, Fujiwara Y, Kaneko T, Xia C, Imai E, Kamada T, Ando A, Ueda N: Angiotensin Ⅱ stimulates interleukin-6 release from cultured mouse mesangial cells. J Am Soc Nephrol.1995; 6:95-101.
65. Theuer J, Dechend R, Muller DN, Park JK, Fiebeler A, Barta P, Ganten D, Haller H, Dietz R, Luft FC: Angiotensin Ⅱ induced inflammation in the kidney and in the heart of double transgenic rats. BioMed Central Cardiovasc Disord 2002;2:3.
66. Bluethmann H, Rothe J, Schultze N, Tkachuk M, Koebel P: Establishment of the role of IL-6 and TNF receptor 1 using gene knockout mice. J Leukoc Biol 1994; 56:565-570.
67．傅勤，林建华．正常妊娠妇女与重度子痫前期患者止凝血功能的研究．现代妇产科进 展2007：16(9)：657-660．
68. Phillips MI, Kagiyama S: Angiotensin Ⅱ as a pro-inflammatory mediator. Curr Opin Invest Drugs 2002; 4:569 -577.
69. Wilson HM, Haites NE, Booth NA: Effect of angiotensin Ⅱ on plasminogen activator inhibitor-1 production by cultured human mesangial cells. Nephron 1997;77:197-204.
70. Sol M. Bobst, Mary-Clare Day, Larry C. Gilstrap Ⅲ, Yang Xia, and Rodney E. Kellems. Maternal Autoantibodies From Preeclamptic Patients Activate Angiotensin Receptors on Human MesangialCells and Induce Interleukin-6 and Plasminogen Activator Inhibitor-1 Secretion. AJH2005; 18:330-336.
71. Lyall F, Simpson H, Nicola BJ, et al. Transforming growth factor-beta expression in human placenta and placental bed in third trimester norm al pregnancy, preeclampsia. and fetal growth restriction. Am J Pathol. 2001; 159(5): 1827-1838.
72. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003 Feb; 10(2):82-93.
73. Bottazano GF, Doniach D: Autoimmune thyroid disease. Annu Rev Med 1986;37:353-359.
74. Chistiakov DA: Thyroid-stimulating hormone receptor and its role in Graves' disease. Mol Genet Metab 2003; 80:377-388.
75. Jahns R, Boivin V, Siegmund C, Inselmann G, Lohse MJ, Boege F: Autoantibodies activating human beta 1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation 1999; 99:649-654.