运用Luminex系统和传统检测方法分析妊高征母婴相关因子
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摘要
目的和意义：
宫内发育迟缓(IUGR)如果没有正确诊断和处理，则新生儿出生时的早产、窒息、低体重的发病率和死亡率明显升高。IUGR的主要原因是由于母体-胎儿循环血量不足，其他病因包括巨细胞病毒和风疹病毒的宫内感染，以及先天性21三体和18三体异常等。
孕母高血压是引起宫内发育迟缓最常见的因素，妊高征(PIH)合并IUGR的发生率为20％～30％，其围产儿死亡率是正常出生体重儿的6～9倍，PIH母亲分娩的IUGR的围产期死亡率是非妊高征母亲娩出的IUGR的3倍。特别是重度PIH有合并症者，IUGR的发生率更高，并直接影响围产儿预后，围产儿死亡率亦升高。PIH增加了低出生体重的风险，而另一方面，IUGR对于PIH后果：胎儿发病率和死亡率、孕母的处理和治疗效果来说也是一个决定因素。
PIH是妊娠20周后出现的特发性综合征，是导致围产期母儿发病率和死亡率的重要原因之一，其病因及发病机理仍未完全阐明，尚无有效的预防及治疗方法。目前研究显示其发病机理及其导致IUGR的原因可能与下列因素有关：
1．Th1／Th2失衡1991年Romagnani证实人体CD~(4+)／h细胞(辅助T淋巴细胞)可分为Th1和／h2两个亚型。Th1细胞主要分泌IL-2(白细胞介素-2)、IFN-r(r-干扰素)、TNF-a(肿瘤坏死因子-a)，介导细胞免疫，Th2细胞主要分泌IL-4、IL-5、IL-6和IL-10、IL-13等，介导体液免疫。Th1和／h2细胞来源于共同的前体细胞-ThO细胞，IL-12和IL-4分别是Th0向Th1和／h2细胞分化的主要诱导因子。大量研究证实，Th1和Th2细胞为一对重要的调节因子，同时又互为抑制因子，Th1细胞分泌的IFN-r可抑制Th2细胞的分化功能，Th2细胞分泌的IL-10和TGF-β(转化生长因子-β)又可抑制Th1细胞的分化和功能。
近年来研究表明PIH患者Th1／Th2比率趋向于升高，Th1／Th2失衡是妊高征发病的一个重要因素。
Saito等报道PIH患者血清IL-2、TNF-a和IFN-r水平均高于正常妊娠者，IL-4则明显低于正常妊娠者，IL-2／IL-4、IFN-r／IL-4比率均高于正常妊娠，且血压与Th1型细胞因子的水平明显正相关。正常妊娠中，Th1细胞百分比在孕晚期显著下降，Th1／Th2比率比非孕妇女也明显降低；而先兆子痫患者体内Th1细胞百分比及Th1／Th2比率高于正常晚孕妇女，Th2百分比下降，Th1、Th2细胞百分比及Th1／Th2比率的变化与细胞因子分泌水平有关，说明先兆子痫患者体内Th1免疫占优势。Th1细胞分泌的另一种因子TNF-a可诱导血管内皮细胞激活，刺激血小板衍生生长因子的产生和有丝分裂活性并导致肾小球内皮细胞破坏，还可抑制血管扩张剂NO的产生并减轻各类细胞对氧自由基毒性的敏感性。
胎盘是母体与胎儿间物质交换的器官，也是一种复杂的局部免疫器官，可分泌多种因子，也有学者认为母体对滋养层的免疫保护被损害是PIH发病的起因。Th1型细胞因子IL-2可激活外周血和蜕膜NK细胞，被激活的NK细胞可攻击滋养层。
2．血管内皮生长因子(VEGF)表达异常VEGF是一种特异性的内皮细胞有丝分裂原，具有强烈的促血管生成及增加微血管通透性的作用，为人体生理性新生血管网形成的重要因子；胎盘VEGF主要分布于细胞滋养层细胞、合体滋养层细胞、血管内皮细胞和绒毛间质细胞，可特异性作用于内皮细胞，促进血管形成，改善血液供应。VEGF的主要功能是促进内皮细胞的分化、增生、迁移侵润，调控血管发生和维持血管内皮细胞功能。血管发生和血管化是胎盘构建蜕膜毛细血管网络以及胚胎生长发育的基础。正常胎盘蜕膜组织切片中VEGF表达十分丰富，其中合体滋养细胞和蜕膜内侵润性滋养细胞均呈强阳性染色。而妊高征患者外周血VEGF水平显著降低，胎儿脐静脉血VEGF也呈下降趋势。
3．胎盘肾素-血管紧张素(RAS)系统研究表明PIH患者绒毛滋养层组织中活性肾素浓度增加。Takimoto等将转入人血管紧张素原基因的雌鼠与转入人肾素基因雄鼠交配，导致怀孕母鼠出现PIH，孕鼠胎盘产生的肾素进入到母体循环血中，使母血中肾素浓度升高，组织切片显示胎盘水肿并出现组织坏死，显示PIH发生与RAS系统有关。
血管紧张素Ⅱ(AngⅡ)增加血管通透性并且刺激血管新生，也参与对胎盘血流量的调节。妊娠期间子宫-胎盘血管阻力与血流的调节是非常复杂的，AngⅡ通过刺激舒血管物质前列腺素E和I的合成，维持血管压力稳定，保证妊娠时子宫-胎盘的血液供应。给予低剂量的AngⅡ，由于刺激舒血管物质生成，或子宫血流量增加，高剂量AngⅡ则使子宫-胎盘血流量减少。
本课题采用多功能液相芯片分析系统(luminex 200)，结合传统ELISA、免疫组化及荧光技术，检测了PIH相关因素(IL-2、4、6、10、12，TNF-a，IFN-r，ANGⅡ，VEGF)在母血、脐带血以及胎盘母面和子面的水平，全面分析其对母婴的影响，探索PIH发病因素与IUGR发生的机制，为减少IUGR的发病率和更好地防治IUGR、PIH奠定了一定的基础。
方法：
1．标本选择及处理：①选取妊高征组及正常孕妇组各30人，均为初产妇，两组孕妇年龄、孕周及体重无显著差异；②母亲外周静脉血及新生儿脐带血各4ml，不抗凝，2000r／min离心后取上清液置于-70℃冰箱保存；③取胎盘母面及子面各两块，约1g左右，清除残留血液，其中各一块置于多聚甲醛中固定，另一块称取0.5g捣碎研磨后加生理盐水1ml，3000r／min离心后取上清夜置于-70℃冰箱冷冻保存。
2．经典ELISA法检测母血、脐带血、胎盘母面及子面组织匀浆中的ANGⅡ和VEGF含量。
3．多聚甲醛固定的胎盘经石蜡切片后进行免疫组化染色及荧光染色镜检。
4．运用多功能液相芯片分析系统(luminex 200)同时检测母血、脐带血、胎盘母面及子面组织匀浆中的IL-2、IL-4、IL-6、IL-10、IL-12、IFN-r、TNF-α的含量。
5．统计分析：实验结果采用Excel 2003软件进行两独立样本的t检验和Pearson相关分析。
结果：
1．ANGⅡ：①免疫组化染色显示妊高征胎盘子面强阳性；②荧光染色也显示妊高征胎盘子面强阳性；③ELISA结果妊高征胎盘母面(8.51±4.01pg／ml)，正常妊娠胎盘母面(7.76±3.47pg／ml)，两者差别没有统计学意义(P＞0.05)：妊高征胎盘子面(11.82±3.92pg／ml)，正常妊娠胎盘子面(9.64±2.63pg／ml)，两者差别有统计学意义(P＜0.05)：妊高征母亲外周血(46.44±8.48pg／ml)，正常产妇外周血(32.43±5.87pg／ml)，两者差别有统计学意义(P＜0.001)：妊高征脐带血(68.83±8.68pg／ml)，正常产妇脐带血(72.47±8.51pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征脐带血ANGⅡ水平与母亲外周血水平相关系数为0.7379，P＜0.05。
2．VEGF：①免疫组化染色显示正常产妇胎盘子面强阳性；②荧光染色也显示正常产妇胎盘子面强阳性；③ELISA结果妊高征胎盘母面(262.88±123.00pg／ml)，正常妊娠胎盘母面(280.03±130.98pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(200.11±110.32pg／ml)，正常妊娠胎盘子面(261.52±82.84pg／ml)，两者差别有统计学意义(P＜0.05)；妊高征母亲外周血(27.82±11.14pg／ml)，正常产妇外周血(23.54±12.05pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征脐带血(291.62±126.36pg／ml)，正常产妇脐带血(209.27±141.21pg／ml)，两者差别有统计学意义(P＜0.05)。
3．TNF-a：妊高征胎盘母面(17.40±8.08pg／ml)，正常妊娠胎盘母面(10.70±5.55pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(10.64±2.92pg／ml)，正常妊娠胎盘子面(5.94±3.42pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征母亲外周血(13.34±6.50pg／ml)，正常产妇外周血(7.78±4.73pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征脐带血(4.50±2.12pg／ml)，正常产妇脐带血(5.36±2.13pg／ml)，两者差别没有统计学意义(P＞0.05)。
4．IFN-r：妊高征胎盘母面(39.88±18.15pg／ml)，正常妊娠胎盘母面(21.38±9.39pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(20.99±6.60pg／ml)，正常妊娠胎盘子面(22.99±7.85pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(19.93±6.47pg／ml)，正常产妇外周血(15.65±5.80pg／ml)，两者差别有统计学意义(P＜0.01)；妊高征脐带血(15.32±6.31pg／ml)，正常产妇脐带血(10.70±4.34pg／ml)，两者差别有统计学意义(P＜0.01)。
5．IL-2：妊高征胎盘母面(8.27±1.55pg／ml)，正常妊娠胎盘母面(5.84±2.21pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征胎盘子面(5.24±2.45pg／ml)，正常妊娠胎盘子面(5.49±2.35pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(9.10±5.55pg／ml)，正常产妇外周血(3.22±2.41pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(4.18±2.51pg／ml)，正常产妇脐带血(3.98±1.71pg／ml)，两者差别没有统计学意义(P＞0.05)。
6．IL-4：妊高征胎盘母面(3.03±2.06pg／ml)，正常妊娠胎盘母面(2.87±1.69pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(2.11±0.77pg／ml)，正常妊娠胎盘子面(2.26±1.29pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(2.37±1.06pg／ml)，正常产妇外周血(5.60±4.81pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征脐带血(2.15±1.33pg／ml)，正常产妇脐带血(1.66±0.59pg／ml)，两者差别没有统计学意义(P＞0.05)。
7．IL-6：妊高征胎盘母面(33.02±12.57pg／ml)，正常妊娠胎盘母面(47.51±25.95pg／ml)，两者差别有统计学意义(P＜0.01)；妊高征胎盘子面(45.33±17.96pg／ml)，正常妊娠胎盘子面(42.31±15.57pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(5.41±0.79pg／ml)，正常产妇外周血(2.77±1.11pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(2.65±0.53pg／ml)，正常产妇脐带血(1.52±0.92pg／ml)，两者差别有统计学意义(P＜0.001)。
8．IL-10：妊高征胎盘母面(2.09±1.46pg／ml)，正常妊娠胎盘母面(3.08±1.05pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(3.07±0.99pg／ml)，正常妊娠胎盘子面(3.35±1.24pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(3.65±1.09pg／ml)，正常产妇外周血(2.52±0.90pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(2.66±0.58pg／ml)，正常产妇脐带血(2.71±1.31pg／ml)，两者差别没有统计学意义(P＞0.05)。
9．IL-12：妊高征胎盘母面(18.56±7.71pg／ml)，正常妊娠胎盘母面(16.09±8.19pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(10.14±3.69pg／ml)，正常妊娠胎盘子面(10.40±3.21pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(6.57±2.35pg／ml)，正常产妇外周血(4.81±0.80pg／ml)，两者差别有统计学意义(P＜0.05)；妊高征脐带血(4.25±1.72pg／ml)，正常产妇脐带血(3.73±1.46pg／ml)，两者差别没有统计学意义(P＞0.05)。
结论：
1．妊高征患者胎盘子面ANGⅡ水平升高、VEGF水平下降，使绒毛膜血管收缩，血管生成障碍和血管内皮细胞受损，减少胎盘子面血流量，影响胎儿血液供应，出现慢性缺氧，是导致IUGR的重要原因；妊高征患者血清中ANGⅡ水平升高可加重PIH，且其脐带血中的ANGⅡ水平与之正相关；妊高征脐带血中VEGF水平升高为胎儿缺氧刺激导致体内生成增加。
2．妊高征母血中TNF-a、IFN-r、IL-2、IL-12、IL-6、IL-10升高，IL-4降低；妊高征胎盘母面中TNF-a、IFN-r、IL-2升高，IL-10、IL-6降低；妊高征胎盘子面中TNF-a升高；妊高征脐带血中IL-6、IFN-r升高。说明Th1／Th2失衡是导致PIH和IUGR的重要原因。
Objective and meaning:
If intrauterine growth restriction (IUGR) don't get correct diagnosis and management, the morbidity and mortality of preterm delivery, asphyxia, low birth weight may be increased significantly. The important reason for IUGR is the insufficient uteroplacental and fetoplacental blood flow. There are also other reasons, such as the intrauterine infection of Cytomegalic virus and Rubella virus, as well as the Down's and Edward syndrome.
Gestation hypertension is the most common factor for IUGR. The morbidity of pregnancy induced hypertension (PIH) complicated with IUGR is 20%-30% and the perinatal mortality of it is 6-to 9- fold than that of the normal birth weight newborns. The perinatal mortality of the IUGR babies delivered by mother with PIH is 3 times than by mother without PIH. Especially, the severe PIH can induce higher risk for IUGR, increase the perinatal mortality, and impact directly the prognosis of the fetus and newborns. Also, PIH is a risk factor for low birth weight; on the other hand, IUGR is a determinant factor for the outcomes of PIH. As follows: perinatal morbidity and mortality of the fetus, the management and treatment efficacy of the mother.
PIH whose etiologic factor and mechanism still need further evaluation is the idiopathic syndrome at＞20 weeks' gestation and one of the important reasons for perinatal morbidity and mortality. Further, there are no effective prevention and treatment for it. Studies to date showed that the etiopathology of PIH and it's reasons of inducing IUGR may have relationship with the following factors:
1. The imbalance of Th1/Th2 Romagnani confirmed that the Human CD~(4+) Th cells (assist T-lymphocytes) can be divided into Thl and Th2 subtypes in 1991. The Thl cells mostly secreted IL-2(interleukin-2), IFN-r(interferon-r), TNF-a(tumor necrosis factor-a), and mediate the cell immunity; The Th2 cells mostly secreted IL-4, IL-5, IL-6, IL-10 and i1-13, and mediate the humoral immunity. The Th1 and Th2 cells shared a common precursor cell-Th0 cell. The important inducer of the differentiation fromTh0 to Thl and Th2 is IL-12 and IL-4, respective. Lots of studies have confirmed that Thl and Th2 cell are one couple regulators. At the same time, they are the inhibitors to each other. IFN-r secreted by Th1 cells can prevent the differentiation and function of the Th2, and IL-10 and TGF-b secreted by Th2 can prevent the differentiation and function of the Th1.
Todate, the studies showed that the ratio of Th1/Th2 which belonged to the patients with PIH was likely to increase. The imbalance of Th1 and Th2 was a important etiologic factor for PIH.
Saito has reported that there were more IL-2, TNF-a and IFN-r and significant less IL-4 in the blood plasma of the mothers complicated PIH than of the normal mothers, also that the ratios of IL-2/IL-4, IFN-r/IL-4 were both higher in comparison with the normal pregnancy. Further, the blood pressure was significantly relative to the level of cell factors of the Thl cells. The proportion of the Thl cells significantly decreased in the late trimester of normal pregnancy and the ratio of Th1/Th2 also obviously fell in comparison with women without pregnancy. However, the proportion of Thl cells and the ratio of Th1/Th2 were higher in the women complicated with preeclampsia than the normal pregnant women. The reductive proportion of the Th2 cells, also the changes of the proportion of Thl, Th2 cells and changes of the ratio of Th1/Th2 had a relationship with the level of the cell factors. All aboved showed that the Thlimmunity had a advantage in the patients with preeclampsia. Another factor TNF-a which secreted by the Thl cells can induce the activation of vascular endothelial cells, stimulate the product of platelet-derived growth factor and mitotic activity, result in the break of renal endothelial cells, as well as prevent the product of vasodilator NO and reduce the sensitivity of any cell to the toxicity of oxygen free radicals.
Placenta is the organ for substantial exchange between mothers and fetus, also a complex partial immune organs. Furture, placenta can secrete many kinds of cell factors. Some scholars believed that the damage of the immune protection which was from mother to trophoblast is the cause to PIH. IL-2 which secreted by Thl cells can active the NK cells of peripheral blood and the decidua, and the actived NK cells can attack the trophoblast.
2. Vascular Endothelial Growth Factor(VEGF) express anormaly VEGF is specific mitogen coming endotheliocyte, which have facilitative intensively function to angiogenesis and can increase microvascular permeability, and play an important role in the formation of physiology new vessels net. Placenta VEGF distributed mainly over cytotroblast, syncytiotrophoblast, vassular endotheliocyte, villose mesenchymocyte. VEGF can effect particulaly endotheliocyte, promote blood vessel formation, and improve blood provision. It's main functions are follows: promoting endotheliocyte differentiation, accrementition, immigration, infiltrate;regulation vasculogenesis and maintaining the function of vascular endothelial cell. Establishment caduca capillary network and embryo's growth and development are based on angiogenesis and vascularize. Normal deciduate placenta histological sectiones are riched of VEGF, they express obviously at syneytiotrophoblast and caduca infiltrate trophocyte. However, the blood concentration of VEGF in patients suffered from PIH are very low, so did fetal umbilical vein.
3.Placental Renin Angiotensin System(RAS) Some researches indicated that the patients suffering from PIH have more renin in villous trophoblast tissue. Takimoto make female rats which have been transferred angiotensinogen gene mate with male rats which have been transferred rennin gene, they discovered that the pregnant rats suffered from PIH, the renin coming from placenta enter maternal blood, higher renin's concentration in maternal plasma, and the placentas edema and organisms necrosis in histological sectiones. Above the all indicate that PIH is relate to RAS.
AngⅡcan increase vasopermeability, stimulate vascellum neogenesis, and play an important role in adjustment placental blood flow, too. Uterus-placenta vascular resistance and adjustment of blood flow is very sophisticated during pregnancy. Ang-Ⅱcan keep blood pressure stabilization and sustain uterus-placenta blood supply by stimulating vasodilator synthesis that prostaglandin-E,Ⅰ. So, low dose AngⅡcan increase uterine blood flow by stimulating vasodilator synthesis,high dose AngⅡproduce inverse role.
The study use multifunction liquid-phase analytical system(Luminex 200), traditional ELISA, immunohistochemistry and fluorescent technique, detected PIH correlation factors (IL-2、4、6、10、12,TNF-a, IFN-r, ANGⅡ,VEGF) expressing in maternal blood, cord blood, matemal surface, fetal surface, respectively. To analysis how they effect to pregnant mother and fetal, to explore etiopathogenisis of PIH and mechanism of IUGR, in order to decrease morbidity of IUGR and improve prognosises of IUGR and PTH.
Methods:
1.The selection and handling of samples:①There are 30 peoples all of them are primipara both in pregnancy-induced hypertension syndrome group and in normal pregnant women group. Their aetas, gestational weeks and weight were not significant deviation;②peripheral vein blood of mothers and cord blood of newborn infants were selected 4ml, not anticoagulation, 2000r/min centrifulged, then supernate fluid was obtained and stored in refrigerator-70℃;③Two tissues weighting about lg both from maternal surface and child surface were taken down,residual blood was removed. Both the one fixed in paraformaldehyde, and the other weighed 0.5g and breakdown milled, then added lml normal sodium, 3000r/min centrifulged, and then supernate fluid was obtained and stored in refrigerator -70℃.
2.To detect the contents of ANG II and VEGF in motermal blood, cord blood, maternal surface and child surface by classic ELISA.
3. The placenta fixed by paraformaldehyde were maken into paraffin section to carry out immunohistochemistry stain and fluorescein stain.
4. The contents of IL-2、IL-4、IL-6、IL-10、IL-12、IFN-r、TNF-a in motermal blood, cord blood, maternal surface and child surface by multipurpose liquid phase array analytical system (Luminex 200).
5. Statistical analysis: experimental results were applied two independent-sample T test and Pearson correlation analysis with Excel 2003 software.
Results:
1.ANGⅡ:①It's strong positive at placental fetal surface of PIH by immunohistochemistry;②It's so by fluorescein stain.③By ELISA, Maternal surface, PIH: 8.51±4.01pg/ml, normal pregnancy: 7.76±3.47pg/ml, no statistical significance (P＞0.05); placental fetal surface, PIH: 11.82±3.92pg/ml, normal pregnancy: 9.64±2.63pg/ml, the difference has statistical significance (P＜0.005); peripheral blood,PIH: 46.44±8.48pg/ml, normal pregnancy: 32.43±5.87pg/ml, the difference hasstatistical significance(P＜0.005); cord blood, PIH: 68.83±8.68pg/ml, normalpregnancy: 72.47±8.51pg/ml, no statistical significance(P＞0.05). The coefficient ofcorrelation between cord blood and peripheral blood of PIH is 0.7379, P＜0.005.
2.VEGF:①It's strong positive at placental fetal surface of normal pregnancy byimmunohistochemistry;②It's so by fluorescein stain.③By ELISA, Maternal surface,PIH: 262.88±123.00pg/ml, normal pregnancy: 280.03±130.98pg/ml, no statisticalsignificance(P＞0.05); placental fetal surface, PIH: 200.11±110.32pg/ml, normalpregnancy: 261.52±82.84pg/ml, no statistical significance(P＞0.05); peripheral blood,PIH: 27.82±11.14pg/ml, normal pregnancy: 23.54±12.05pg/ml, no statisticalsignificance(P＞0.05); cord blood, PIH: 291.62±126.36pg/ml, normal pregnancy:209.27±141.21pg/ml, the difference has statistical significance(P＜0.005).
3.TNF-a: maternal surface, the patients suffered from PIH: 17.40±8.08pg/ml,normal pregnancy: 10.70±5.55pg/ml, the difference has statistical significance(P＜0.005); placental fetal surface, PIH: 10.64±2.92pg/ml, normal pregnancy:5.94±3.42pg/ml, the difference has statistical significance (P＜0.005); peripheral blood,PIH: 13.34±6.50pg/ml, normal pregnancy: 7.78±4.73pg/ml, the difference hasstatistical significance (P＜0.005); cord blood, PIH: 4.50±2.12pg/ml, normalpregnancy: 5.36±2.13pg/ml, no statistical significance(P＞0.05).
4.IFN-r: maternal surface, the patients suffered from PIH: 39.88±18.15pg/ml,normal pregnancy: 21.38±9.39pg/ml, the difference has statistical significance(P＜0.005); placental fetal surface, PIH: 20.99±6.60pg/ml, normal pregnancy:22.99±7.85pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH:19.93±6.47pg/ml, normal pregnancy: 15.65±5.80pg/ml, the difference has statisticalsignificance (P＜0.01); cord blood, PIH: 15.32±6.31pg/ml, normal pregnancy: 10.70±4.34pg/ml, the difference has statistical significance (P＜0.01).
5.IL-2: maternal surface, the patients suffered from PIH: 8.27±1.55pg/ml, normal pregnancy: 5.84±2.21pg/ml, the difference has statistical significance (P＜0.001); placental fetal surface, PIH: 5.24±2.45pg/ml, normal pregnancy: 5.49±2.35pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 9.10±5.55pg/ml, normal pregnancy: 3.22±2.41pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 4.18±2.51pg/ml, normal pregnancy: 3.98±1.71pg/ml, no statistical significance(P＞0.05).
6.IL-4: maternal surface, the patients suffered from PIH: 3.03±2.06pg/ml, normal pregnancy: 2.87±1.69pg/ml, they have no statistical significance(P＞0.05); placental fetal surface, PIH: 2.11±0.77pg/ml, normal pregnancy: 2.26±1.29pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 2.37±1.06pg/ml, normal pregnancy: 5.60±4.81pg/ml, the difference has statistical significance (P＜0.005); cord blood, PIH: 2.15±1.33pg/ml, normal pregnancy: 1.66±0.59pg/ml, no statistical significance(P＞0.05).
7.IL-6: maternal surface, the patients suffered from PIH: 33.02±12.57pg/ml, normal pregnancy: 47.51±25.95pg/ml, the difference has statistical significance (P＜0.01); placental fetal surface, PIH: 45.33±17.96pg/ml, normal pregnancy: 42.31±15.57pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 5.41±0.79pg/ml, normal pregnancy: 2.77±1.11pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 2.65±0.53pg/ml, normal pregnancy: 1.52±0.92pg/ml, the difference has statistical significance (P＜0.001).
8.IL-10: maternal surface, the patients suffered from PIH: 2.09±1.46pg/ml, normal pregnancy: 3.08±1.05pg/ml, the difference has statistical significance (P＜0.005); placental fetal surface, PIH: 3.07±0.99pg/ml, normal pregnancy: 3.35±1.24pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 3.65±1.09pg/, normal pregnancy: 2.52±0.90pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 2.66±0.58pg/ml, normal pregnancy: 2.71+1.31pg/ml, no statistical significance(P＞0.05).
9.IL-12: maternal surface, the patients suffered from PIH: 18.56±7.71pg/ml, normal pregnancy: 16.09±8.19pg/ml, they have no statistical significance(P＞0.05); placental fetal surface, PIH: 10.14±3.69pg/ml, normal pregnancy: 10.40±3.21pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 6.57±2.35pg/ml, normal pregnancy: 4.81±0.80pg/ml, the difference has statistical significance (P＜0.05); cord blood, PIH: 4.25±1.72pg/ml, normal pregnancy: 3.73±1.46pg/ml, no statistical significance(P＞0.05).
Conclusion:
1.ANGⅡlevel of placenta closed with infants in PIH is increased while VEGF level decline. And which lead to chorionic vasoconstriction,Angiogenesis and vascular endothelial cell damaged, blood flow of placental closed with infants reduced.The impacts of fetal blood supply lead to Chronic hypoxia, which is the result of the important causes of IUGR. The increased ANGⅡlevels of PIH serum can add to PIH. Meanwhile, ANGⅡlevels of the cord blood are associated with them. The increased VEGF levels of cord blood in PIH are results of fetal hypoxia which stimulate the body to increase production.
2.TNF-a、IFN-r、IL-2、IL-12、IL-6、IL-10 are increased and IL-4 is decreased in maternal blood of PIH. TNF-a、IFN-r、IL-2 increased and 1L-10、IL-6 decreasd in placenta closed with pregnant of PIH. IL-6、IFN-r rise in cord blood of PIH.The conclusions suggest that Th1/Th2 imbalance is the main reason of PIH and IUGR.
宫内发育迟缓(IUGR)如果没有正确诊断和处理，则新生儿出生时的早产、窒息、低体重的发病率和死亡率明显升高。IUGR的主要原因是由于母体-胎儿循环血量不足，其他病因包括巨细胞病毒和风疹病毒的宫内感染，以及先天性21三体和18三体异常等。
孕母高血压是引起宫内发育迟缓最常见的因素，妊高征(PIH)合并IUGR的发生率为20％～30％，其围产儿死亡率是正常出生体重儿的6～9倍，PIH母亲分娩的IUGR的围产期死亡率是非妊高征母亲娩出的IUGR的3倍。特别是重度PIH有合并症者，IUGR的发生率更高，并直接影响围产儿预后，围产儿死亡率亦升高。PIH增加了低出生体重的风险，而另一方面，IUGR对于PIH后果：胎儿发病率和死亡率、孕母的处理和治疗效果来说也是一个决定因素。
PIH是妊娠20周后出现的特发性综合征，是导致围产期母儿发病率和死亡率的重要原因之一，其病因及发病机理仍未完全阐明，尚无有效的预防及治疗方法。目前研究显示其发病机理及其导致IUGR的原因可能与下列因素有关：
1．Th1／Th2失衡1991年Romagnani证实人体CD~(4+)／h细胞(辅助T淋巴细胞)可分为Th1和／h2两个亚型。Th1细胞主要分泌IL-2(白细胞介素-2)、IFN-r(r-干扰素)、TNF-a(肿瘤坏死因子-a)，介导细胞免疫，Th2细胞主要分泌IL-4、IL-5、IL-6和IL-10、IL-13等，介导体液免疫。Th1和／h2细胞来源于共同的前体细胞-ThO细胞，IL-12和IL-4分别是Th0向Th1和／h2细胞分化的主要诱导因子。大量研究证实，Th1和Th2细胞为一对重要的调节因子，同时又互为抑制因子，Th1细胞分泌的IFN-r可抑制Th2细胞的分化功能，Th2细胞分泌的IL-10和TGF-β(转化生长因子-β)又可抑制Th1细胞的分化和功能。
近年来研究表明PIH患者Th1／Th2比率趋向于升高，Th1／Th2失衡是妊高征发病的一个重要因素。
Saito等报道PIH患者血清IL-2、TNF-a和IFN-r水平均高于正常妊娠者，IL-4则明显低于正常妊娠者，IL-2／IL-4、IFN-r／IL-4比率均高于正常妊娠，且血压与Th1型细胞因子的水平明显正相关。正常妊娠中，Th1细胞百分比在孕晚期显著下降，Th1／Th2比率比非孕妇女也明显降低；而先兆子痫患者体内Th1细胞百分比及Th1／Th2比率高于正常晚孕妇女，Th2百分比下降，Th1、Th2细胞百分比及Th1／Th2比率的变化与细胞因子分泌水平有关，说明先兆子痫患者体内Th1免疫占优势。Th1细胞分泌的另一种因子TNF-a可诱导血管内皮细胞激活，刺激血小板衍生生长因子的产生和有丝分裂活性并导致肾小球内皮细胞破坏，还可抑制血管扩张剂NO的产生并减轻各类细胞对氧自由基毒性的敏感性。
胎盘是母体与胎儿间物质交换的器官，也是一种复杂的局部免疫器官，可分泌多种因子，也有学者认为母体对滋养层的免疫保护被损害是PIH发病的起因。Th1型细胞因子IL-2可激活外周血和蜕膜NK细胞，被激活的NK细胞可攻击滋养层。
2．血管内皮生长因子(VEGF)表达异常VEGF是一种特异性的内皮细胞有丝分裂原，具有强烈的促血管生成及增加微血管通透性的作用，为人体生理性新生血管网形成的重要因子；胎盘VEGF主要分布于细胞滋养层细胞、合体滋养层细胞、血管内皮细胞和绒毛间质细胞，可特异性作用于内皮细胞，促进血管形成，改善血液供应。VEGF的主要功能是促进内皮细胞的分化、增生、迁移侵润，调控血管发生和维持血管内皮细胞功能。血管发生和血管化是胎盘构建蜕膜毛细血管网络以及胚胎生长发育的基础。正常胎盘蜕膜组织切片中VEGF表达十分丰富，其中合体滋养细胞和蜕膜内侵润性滋养细胞均呈强阳性染色。而妊高征患者外周血VEGF水平显著降低，胎儿脐静脉血VEGF也呈下降趋势。
3．胎盘肾素-血管紧张素(RAS)系统研究表明PIH患者绒毛滋养层组织中活性肾素浓度增加。Takimoto等将转入人血管紧张素原基因的雌鼠与转入人肾素基因雄鼠交配，导致怀孕母鼠出现PIH，孕鼠胎盘产生的肾素进入到母体循环血中，使母血中肾素浓度升高，组织切片显示胎盘水肿并出现组织坏死，显示PIH发生与RAS系统有关。
血管紧张素Ⅱ(AngⅡ)增加血管通透性并且刺激血管新生，也参与对胎盘血流量的调节。妊娠期间子宫-胎盘血管阻力与血流的调节是非常复杂的，AngⅡ通过刺激舒血管物质前列腺素E和I的合成，维持血管压力稳定，保证妊娠时子宫-胎盘的血液供应。给予低剂量的AngⅡ，由于刺激舒血管物质生成，或子宫血流量增加，高剂量AngⅡ则使子宫-胎盘血流量减少。
本课题采用多功能液相芯片分析系统(luminex 200)，结合传统ELISA、免疫组化及荧光技术，检测了PIH相关因素(IL-2、4、6、10、12，TNF-a，IFN-r，ANGⅡ，VEGF)在母血、脐带血以及胎盘母面和子面的水平，全面分析其对母婴的影响，探索PIH发病因素与IUGR发生的机制，为减少IUGR的发病率和更好地防治IUGR、PIH奠定了一定的基础。
方法：
1．标本选择及处理：①选取妊高征组及正常孕妇组各30人，均为初产妇，两组孕妇年龄、孕周及体重无显著差异；②母亲外周静脉血及新生儿脐带血各4ml，不抗凝，2000r／min离心后取上清液置于-70℃冰箱保存；③取胎盘母面及子面各两块，约1g左右，清除残留血液，其中各一块置于多聚甲醛中固定，另一块称取0.5g捣碎研磨后加生理盐水1ml，3000r／min离心后取上清夜置于-70℃冰箱冷冻保存。
2．经典ELISA法检测母血、脐带血、胎盘母面及子面组织匀浆中的ANGⅡ和VEGF含量。
3．多聚甲醛固定的胎盘经石蜡切片后进行免疫组化染色及荧光染色镜检。
4．运用多功能液相芯片分析系统(luminex 200)同时检测母血、脐带血、胎盘母面及子面组织匀浆中的IL-2、IL-4、IL-6、IL-10、IL-12、IFN-r、TNF-α的含量。
5．统计分析：实验结果采用Excel 2003软件进行两独立样本的t检验和Pearson相关分析。
结果：
1．ANGⅡ：①免疫组化染色显示妊高征胎盘子面强阳性；②荧光染色也显示妊高征胎盘子面强阳性；③ELISA结果妊高征胎盘母面(8.51±4.01pg／ml)，正常妊娠胎盘母面(7.76±3.47pg／ml)，两者差别没有统计学意义(P＞0.05)：妊高征胎盘子面(11.82±3.92pg／ml)，正常妊娠胎盘子面(9.64±2.63pg／ml)，两者差别有统计学意义(P＜0.05)：妊高征母亲外周血(46.44±8.48pg／ml)，正常产妇外周血(32.43±5.87pg／ml)，两者差别有统计学意义(P＜0.001)：妊高征脐带血(68.83±8.68pg／ml)，正常产妇脐带血(72.47±8.51pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征脐带血ANGⅡ水平与母亲外周血水平相关系数为0.7379，P＜0.05。
2．VEGF：①免疫组化染色显示正常产妇胎盘子面强阳性；②荧光染色也显示正常产妇胎盘子面强阳性；③ELISA结果妊高征胎盘母面(262.88±123.00pg／ml)，正常妊娠胎盘母面(280.03±130.98pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(200.11±110.32pg／ml)，正常妊娠胎盘子面(261.52±82.84pg／ml)，两者差别有统计学意义(P＜0.05)；妊高征母亲外周血(27.82±11.14pg／ml)，正常产妇外周血(23.54±12.05pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征脐带血(291.62±126.36pg／ml)，正常产妇脐带血(209.27±141.21pg／ml)，两者差别有统计学意义(P＜0.05)。
3．TNF-a：妊高征胎盘母面(17.40±8.08pg／ml)，正常妊娠胎盘母面(10.70±5.55pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(10.64±2.92pg／ml)，正常妊娠胎盘子面(5.94±3.42pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征母亲外周血(13.34±6.50pg／ml)，正常产妇外周血(7.78±4.73pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征脐带血(4.50±2.12pg／ml)，正常产妇脐带血(5.36±2.13pg／ml)，两者差别没有统计学意义(P＞0.05)。
4．IFN-r：妊高征胎盘母面(39.88±18.15pg／ml)，正常妊娠胎盘母面(21.38±9.39pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(20.99±6.60pg／ml)，正常妊娠胎盘子面(22.99±7.85pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(19.93±6.47pg／ml)，正常产妇外周血(15.65±5.80pg／ml)，两者差别有统计学意义(P＜0.01)；妊高征脐带血(15.32±6.31pg／ml)，正常产妇脐带血(10.70±4.34pg／ml)，两者差别有统计学意义(P＜0.01)。
5．IL-2：妊高征胎盘母面(8.27±1.55pg／ml)，正常妊娠胎盘母面(5.84±2.21pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征胎盘子面(5.24±2.45pg／ml)，正常妊娠胎盘子面(5.49±2.35pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(9.10±5.55pg／ml)，正常产妇外周血(3.22±2.41pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(4.18±2.51pg／ml)，正常产妇脐带血(3.98±1.71pg／ml)，两者差别没有统计学意义(P＞0.05)。
6．IL-4：妊高征胎盘母面(3.03±2.06pg／ml)，正常妊娠胎盘母面(2.87±1.69pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(2.11±0.77pg／ml)，正常妊娠胎盘子面(2.26±1.29pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(2.37±1.06pg／ml)，正常产妇外周血(5.60±4.81pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征脐带血(2.15±1.33pg／ml)，正常产妇脐带血(1.66±0.59pg／ml)，两者差别没有统计学意义(P＞0.05)。
7．IL-6：妊高征胎盘母面(33.02±12.57pg／ml)，正常妊娠胎盘母面(47.51±25.95pg／ml)，两者差别有统计学意义(P＜0.01)；妊高征胎盘子面(45.33±17.96pg／ml)，正常妊娠胎盘子面(42.31±15.57pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(5.41±0.79pg／ml)，正常产妇外周血(2.77±1.11pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(2.65±0.53pg／ml)，正常产妇脐带血(1.52±0.92pg／ml)，两者差别有统计学意义(P＜0.001)。
8．IL-10：妊高征胎盘母面(2.09±1.46pg／ml)，正常妊娠胎盘母面(3.08±1.05pg／ml)，两者差别有统计学意义(P＜0.005)；妊高征胎盘子面(3.07±0.99pg／ml)，正常妊娠胎盘子面(3.35±1.24pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(3.65±1.09pg／ml)，正常产妇外周血(2.52±0.90pg／ml)，两者差别有统计学意义(P＜0.001)；妊高征脐带血(2.66±0.58pg／ml)，正常产妇脐带血(2.71±1.31pg／ml)，两者差别没有统计学意义(P＞0.05)。
9．IL-12：妊高征胎盘母面(18.56±7.71pg／ml)，正常妊娠胎盘母面(16.09±8.19pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征胎盘子面(10.14±3.69pg／ml)，正常妊娠胎盘子面(10.40±3.21pg／ml)，两者差别没有统计学意义(P＞0.05)；妊高征母亲外周血(6.57±2.35pg／ml)，正常产妇外周血(4.81±0.80pg／ml)，两者差别有统计学意义(P＜0.05)；妊高征脐带血(4.25±1.72pg／ml)，正常产妇脐带血(3.73±1.46pg／ml)，两者差别没有统计学意义(P＞0.05)。
结论：
1．妊高征患者胎盘子面ANGⅡ水平升高、VEGF水平下降，使绒毛膜血管收缩，血管生成障碍和血管内皮细胞受损，减少胎盘子面血流量，影响胎儿血液供应，出现慢性缺氧，是导致IUGR的重要原因；妊高征患者血清中ANGⅡ水平升高可加重PIH，且其脐带血中的ANGⅡ水平与之正相关；妊高征脐带血中VEGF水平升高为胎儿缺氧刺激导致体内生成增加。
2．妊高征母血中TNF-a、IFN-r、IL-2、IL-12、IL-6、IL-10升高，IL-4降低；妊高征胎盘母面中TNF-a、IFN-r、IL-2升高，IL-10、IL-6降低；妊高征胎盘子面中TNF-a升高；妊高征脐带血中IL-6、IFN-r升高。说明Th1／Th2失衡是导致PIH和IUGR的重要原因。
Objective and meaning:
If intrauterine growth restriction (IUGR) don't get correct diagnosis and management, the morbidity and mortality of preterm delivery, asphyxia, low birth weight may be increased significantly. The important reason for IUGR is the insufficient uteroplacental and fetoplacental blood flow. There are also other reasons, such as the intrauterine infection of Cytomegalic virus and Rubella virus, as well as the Down's and Edward syndrome.
Gestation hypertension is the most common factor for IUGR. The morbidity of pregnancy induced hypertension (PIH) complicated with IUGR is 20%-30% and the perinatal mortality of it is 6-to 9- fold than that of the normal birth weight newborns. The perinatal mortality of the IUGR babies delivered by mother with PIH is 3 times than by mother without PIH. Especially, the severe PIH can induce higher risk for IUGR, increase the perinatal mortality, and impact directly the prognosis of the fetus and newborns. Also, PIH is a risk factor for low birth weight; on the other hand, IUGR is a determinant factor for the outcomes of PIH. As follows: perinatal morbidity and mortality of the fetus, the management and treatment efficacy of the mother.
PIH whose etiologic factor and mechanism still need further evaluation is the idiopathic syndrome at＞20 weeks' gestation and one of the important reasons for perinatal morbidity and mortality. Further, there are no effective prevention and treatment for it. Studies to date showed that the etiopathology of PIH and it's reasons of inducing IUGR may have relationship with the following factors:
1. The imbalance of Th1/Th2 Romagnani confirmed that the Human CD~(4+) Th cells (assist T-lymphocytes) can be divided into Thl and Th2 subtypes in 1991. The Thl cells mostly secreted IL-2(interleukin-2), IFN-r(interferon-r), TNF-a(tumor necrosis factor-a), and mediate the cell immunity; The Th2 cells mostly secreted IL-4, IL-5, IL-6, IL-10 and i1-13, and mediate the humoral immunity. The Th1 and Th2 cells shared a common precursor cell-Th0 cell. The important inducer of the differentiation fromTh0 to Thl and Th2 is IL-12 and IL-4, respective. Lots of studies have confirmed that Thl and Th2 cell are one couple regulators. At the same time, they are the inhibitors to each other. IFN-r secreted by Th1 cells can prevent the differentiation and function of the Th2, and IL-10 and TGF-b secreted by Th2 can prevent the differentiation and function of the Th1.
Todate, the studies showed that the ratio of Th1/Th2 which belonged to the patients with PIH was likely to increase. The imbalance of Th1 and Th2 was a important etiologic factor for PIH.
Saito has reported that there were more IL-2, TNF-a and IFN-r and significant less IL-4 in the blood plasma of the mothers complicated PIH than of the normal mothers, also that the ratios of IL-2/IL-4, IFN-r/IL-4 were both higher in comparison with the normal pregnancy. Further, the blood pressure was significantly relative to the level of cell factors of the Thl cells. The proportion of the Thl cells significantly decreased in the late trimester of normal pregnancy and the ratio of Th1/Th2 also obviously fell in comparison with women without pregnancy. However, the proportion of Thl cells and the ratio of Th1/Th2 were higher in the women complicated with preeclampsia than the normal pregnant women. The reductive proportion of the Th2 cells, also the changes of the proportion of Thl, Th2 cells and changes of the ratio of Th1/Th2 had a relationship with the level of the cell factors. All aboved showed that the Thlimmunity had a advantage in the patients with preeclampsia. Another factor TNF-a which secreted by the Thl cells can induce the activation of vascular endothelial cells, stimulate the product of platelet-derived growth factor and mitotic activity, result in the break of renal endothelial cells, as well as prevent the product of vasodilator NO and reduce the sensitivity of any cell to the toxicity of oxygen free radicals.
Placenta is the organ for substantial exchange between mothers and fetus, also a complex partial immune organs. Furture, placenta can secrete many kinds of cell factors. Some scholars believed that the damage of the immune protection which was from mother to trophoblast is the cause to PIH. IL-2 which secreted by Thl cells can active the NK cells of peripheral blood and the decidua, and the actived NK cells can attack the trophoblast.
2. Vascular Endothelial Growth Factor(VEGF) express anormaly VEGF is specific mitogen coming endotheliocyte, which have facilitative intensively function to angiogenesis and can increase microvascular permeability, and play an important role in the formation of physiology new vessels net. Placenta VEGF distributed mainly over cytotroblast, syncytiotrophoblast, vassular endotheliocyte, villose mesenchymocyte. VEGF can effect particulaly endotheliocyte, promote blood vessel formation, and improve blood provision. It's main functions are follows: promoting endotheliocyte differentiation, accrementition, immigration, infiltrate;regulation vasculogenesis and maintaining the function of vascular endothelial cell. Establishment caduca capillary network and embryo's growth and development are based on angiogenesis and vascularize. Normal deciduate placenta histological sectiones are riched of VEGF, they express obviously at syneytiotrophoblast and caduca infiltrate trophocyte. However, the blood concentration of VEGF in patients suffered from PIH are very low, so did fetal umbilical vein.
3.Placental Renin Angiotensin System(RAS) Some researches indicated that the patients suffering from PIH have more renin in villous trophoblast tissue. Takimoto make female rats which have been transferred angiotensinogen gene mate with male rats which have been transferred rennin gene, they discovered that the pregnant rats suffered from PIH, the renin coming from placenta enter maternal blood, higher renin's concentration in maternal plasma, and the placentas edema and organisms necrosis in histological sectiones. Above the all indicate that PIH is relate to RAS.
AngⅡcan increase vasopermeability, stimulate vascellum neogenesis, and play an important role in adjustment placental blood flow, too. Uterus-placenta vascular resistance and adjustment of blood flow is very sophisticated during pregnancy. Ang-Ⅱcan keep blood pressure stabilization and sustain uterus-placenta blood supply by stimulating vasodilator synthesis that prostaglandin-E,Ⅰ. So, low dose AngⅡcan increase uterine blood flow by stimulating vasodilator synthesis,high dose AngⅡproduce inverse role.
The study use multifunction liquid-phase analytical system(Luminex 200), traditional ELISA, immunohistochemistry and fluorescent technique, detected PIH correlation factors (IL-2、4、6、10、12,TNF-a, IFN-r, ANGⅡ,VEGF) expressing in maternal blood, cord blood, matemal surface, fetal surface, respectively. To analysis how they effect to pregnant mother and fetal, to explore etiopathogenisis of PIH and mechanism of IUGR, in order to decrease morbidity of IUGR and improve prognosises of IUGR and PTH.
Methods:
1.The selection and handling of samples:①There are 30 peoples all of them are primipara both in pregnancy-induced hypertension syndrome group and in normal pregnant women group. Their aetas, gestational weeks and weight were not significant deviation;②peripheral vein blood of mothers and cord blood of newborn infants were selected 4ml, not anticoagulation, 2000r/min centrifulged, then supernate fluid was obtained and stored in refrigerator-70℃;③Two tissues weighting about lg both from maternal surface and child surface were taken down,residual blood was removed. Both the one fixed in paraformaldehyde, and the other weighed 0.5g and breakdown milled, then added lml normal sodium, 3000r/min centrifulged, and then supernate fluid was obtained and stored in refrigerator -70℃.
2.To detect the contents of ANG II and VEGF in motermal blood, cord blood, maternal surface and child surface by classic ELISA.
3. The placenta fixed by paraformaldehyde were maken into paraffin section to carry out immunohistochemistry stain and fluorescein stain.
4. The contents of IL-2、IL-4、IL-6、IL-10、IL-12、IFN-r、TNF-a in motermal blood, cord blood, maternal surface and child surface by multipurpose liquid phase array analytical system (Luminex 200).
5. Statistical analysis: experimental results were applied two independent-sample T test and Pearson correlation analysis with Excel 2003 software.
Results:
1.ANGⅡ:①It's strong positive at placental fetal surface of PIH by immunohistochemistry;②It's so by fluorescein stain.③By ELISA, Maternal surface, PIH: 8.51±4.01pg/ml, normal pregnancy: 7.76±3.47pg/ml, no statistical significance (P＞0.05); placental fetal surface, PIH: 11.82±3.92pg/ml, normal pregnancy: 9.64±2.63pg/ml, the difference has statistical significance (P＜0.005); peripheral blood,PIH: 46.44±8.48pg/ml, normal pregnancy: 32.43±5.87pg/ml, the difference hasstatistical significance(P＜0.005); cord blood, PIH: 68.83±8.68pg/ml, normalpregnancy: 72.47±8.51pg/ml, no statistical significance(P＞0.05). The coefficient ofcorrelation between cord blood and peripheral blood of PIH is 0.7379, P＜0.005.
2.VEGF:①It's strong positive at placental fetal surface of normal pregnancy byimmunohistochemistry;②It's so by fluorescein stain.③By ELISA, Maternal surface,PIH: 262.88±123.00pg/ml, normal pregnancy: 280.03±130.98pg/ml, no statisticalsignificance(P＞0.05); placental fetal surface, PIH: 200.11±110.32pg/ml, normalpregnancy: 261.52±82.84pg/ml, no statistical significance(P＞0.05); peripheral blood,PIH: 27.82±11.14pg/ml, normal pregnancy: 23.54±12.05pg/ml, no statisticalsignificance(P＞0.05); cord blood, PIH: 291.62±126.36pg/ml, normal pregnancy:209.27±141.21pg/ml, the difference has statistical significance(P＜0.005).
3.TNF-a: maternal surface, the patients suffered from PIH: 17.40±8.08pg/ml,normal pregnancy: 10.70±5.55pg/ml, the difference has statistical significance(P＜0.005); placental fetal surface, PIH: 10.64±2.92pg/ml, normal pregnancy:5.94±3.42pg/ml, the difference has statistical significance (P＜0.005); peripheral blood,PIH: 13.34±6.50pg/ml, normal pregnancy: 7.78±4.73pg/ml, the difference hasstatistical significance (P＜0.005); cord blood, PIH: 4.50±2.12pg/ml, normalpregnancy: 5.36±2.13pg/ml, no statistical significance(P＞0.05).
4.IFN-r: maternal surface, the patients suffered from PIH: 39.88±18.15pg/ml,normal pregnancy: 21.38±9.39pg/ml, the difference has statistical significance(P＜0.005); placental fetal surface, PIH: 20.99±6.60pg/ml, normal pregnancy:22.99±7.85pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH:19.93±6.47pg/ml, normal pregnancy: 15.65±5.80pg/ml, the difference has statisticalsignificance (P＜0.01); cord blood, PIH: 15.32±6.31pg/ml, normal pregnancy: 10.70±4.34pg/ml, the difference has statistical significance (P＜0.01).
5.IL-2: maternal surface, the patients suffered from PIH: 8.27±1.55pg/ml, normal pregnancy: 5.84±2.21pg/ml, the difference has statistical significance (P＜0.001); placental fetal surface, PIH: 5.24±2.45pg/ml, normal pregnancy: 5.49±2.35pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 9.10±5.55pg/ml, normal pregnancy: 3.22±2.41pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 4.18±2.51pg/ml, normal pregnancy: 3.98±1.71pg/ml, no statistical significance(P＞0.05).
6.IL-4: maternal surface, the patients suffered from PIH: 3.03±2.06pg/ml, normal pregnancy: 2.87±1.69pg/ml, they have no statistical significance(P＞0.05); placental fetal surface, PIH: 2.11±0.77pg/ml, normal pregnancy: 2.26±1.29pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 2.37±1.06pg/ml, normal pregnancy: 5.60±4.81pg/ml, the difference has statistical significance (P＜0.005); cord blood, PIH: 2.15±1.33pg/ml, normal pregnancy: 1.66±0.59pg/ml, no statistical significance(P＞0.05).
7.IL-6: maternal surface, the patients suffered from PIH: 33.02±12.57pg/ml, normal pregnancy: 47.51±25.95pg/ml, the difference has statistical significance (P＜0.01); placental fetal surface, PIH: 45.33±17.96pg/ml, normal pregnancy: 42.31±15.57pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 5.41±0.79pg/ml, normal pregnancy: 2.77±1.11pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 2.65±0.53pg/ml, normal pregnancy: 1.52±0.92pg/ml, the difference has statistical significance (P＜0.001).
8.IL-10: maternal surface, the patients suffered from PIH: 2.09±1.46pg/ml, normal pregnancy: 3.08±1.05pg/ml, the difference has statistical significance (P＜0.005); placental fetal surface, PIH: 3.07±0.99pg/ml, normal pregnancy: 3.35±1.24pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 3.65±1.09pg/, normal pregnancy: 2.52±0.90pg/ml, the difference has statistical significance (P＜0.001); cord blood, PIH: 2.66±0.58pg/ml, normal pregnancy: 2.71+1.31pg/ml, no statistical significance(P＞0.05).
9.IL-12: maternal surface, the patients suffered from PIH: 18.56±7.71pg/ml, normal pregnancy: 16.09±8.19pg/ml, they have no statistical significance(P＞0.05); placental fetal surface, PIH: 10.14±3.69pg/ml, normal pregnancy: 10.40±3.21pg/ml, no statistical significance(P＞0.05); peripheral blood, PIH: 6.57±2.35pg/ml, normal pregnancy: 4.81±0.80pg/ml, the difference has statistical significance (P＜0.05); cord blood, PIH: 4.25±1.72pg/ml, normal pregnancy: 3.73±1.46pg/ml, no statistical significance(P＞0.05).
Conclusion:
1.ANGⅡlevel of placenta closed with infants in PIH is increased while VEGF level decline. And which lead to chorionic vasoconstriction,Angiogenesis and vascular endothelial cell damaged, blood flow of placental closed with infants reduced.The impacts of fetal blood supply lead to Chronic hypoxia, which is the result of the important causes of IUGR. The increased ANGⅡlevels of PIH serum can add to PIH. Meanwhile, ANGⅡlevels of the cord blood are associated with them. The increased VEGF levels of cord blood in PIH are results of fetal hypoxia which stimulate the body to increase production.
2.TNF-a、IFN-r、IL-2、IL-12、IL-6、IL-10 are increased and IL-4 is decreased in maternal blood of PIH. TNF-a、IFN-r、IL-2 increased and 1L-10、IL-6 decreasd in placenta closed with pregnant of PIH. IL-6、IFN-r rise in cord blood of PIH.The conclusions suggest that Th1/Th2 imbalance is the main reason of PIH and IUGR.
引文
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18. Giardina JB, Green GM, Cockrell KL, et al. TNF-alpha enhances contration and inhibits endothelial No-cGMP relaxation in systemic vessels of pregnant rats[J]. Am J Physiol Regul Comp Physiol, 2002, 283(1):R130-R143.
19. Benian A, Madazh R, Aksu F, etal. Plasma and placental levels of interleukin-10, transforming growth factor-B1, andepithelial cadherin in preeclampsia[J]. Obstet Gynecol, 2002, 100(2), 327-331.
20. Granger JP, Alexander BT, Linas MT, et al. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction[J]. Hypertension, 2001, 38(3Pt2): 718-722.
21. Vince GS, Starkey PM. Interleukin-6, tumour necrosis factors and soluble tumour necrosis factor receptors in women with preeclampsia. Br J Obstet Gynecol 1995; 102: 20.
22．吕秀萍，张凤华，郭宝强等．妊高征患者血浆巨噬细胞集落刺激因子的测定．中华妇产科杂志1999；34(6)：366．
23. Lechner WE, Bergant A, Marth C, et al. Effect of IL-6 on Uterine contractility in the human in Vivo. Z Geburtshilfe Neonatol 1998; 202: 10.
24．王子芬，宋海翔，石少兰等．妊高征患者白细胞介素-6变化的意义．中华妇产科杂志．1999；34(4)：220．
25. Dammann O, Leviton A. Maternal intrauterine infecttion, Cytokines and brain damage in the preterm new-born. Pediatr Res 1997; 42(1): 1.
01. Lenz T, Sealey JE, Tewksbury DA. Regional distribution of the angiotensinogens in human placentae.Placenta. 1993 Nov-Dec; 14(6): 695-9.
02. Kalenga MK, De Hertogh R, Whitebread S, et al. Distribution of the concentrations of angiotensin II (A II), A II receptors, hPL, prolactin, and steroids in human fetal membranes]Rev Fr Gynecol Obstet. 1991 Oct;86(10):585-91.
03. Schauser KH, Nielsen AH, WintherH, et al. Autoradiographic localization and characterization of angiotensin II receptors in the bovine placenta and fetal membranes.Biol Reprod. 1998 Sep;59(3): 684—92.
04. Hagemann A, Nilsen AH, Poulsen K. The uteroplacental renin-angiotensin system: a review.Exp Clin Endocrinol. 1994;102(3):252-61.
05. Mackenzie LW, Word RA, Casey ML, et al. Myosin light chain phosphorylation in human myometrial smooth muscle cells.Am J Physiol. 1990 Jan;258(1 Pt 1):C92-8.
06. Moeller I, Chai SY, MacGregor DP,et al. Localization and quantitation of angiotensin AT1 and AT2 receptors in the pregnant and non-pregnant sheep uterus.Regul Pept. 1996 Mar 22;61(3):213-8.
07. Takimoto E, Ishida J, Sugiyama F, et al.Hypertension induced in pregnant mice by placental renin and maternal angiotensinogen.Science. 1996 Nov 8;274(5289):995-8.
08. Lumbers ER, Kingsford NM, Menzies RI, et al. Acute effects of captopril, an angiotensin-converting enzyme inhibitor, on the pregnant ewe and fetus.Am J Physiol. 1992 May;262(5 Pt 2):R754-60.
09. Mattei MG, Brog JP,Rosnet O,et al. Assiginment of vascular endothelial growth factor(VEGF) and placenta growth factor(PLGF) genes to human chromosome 6p12- 6p21 and 14q24- 14q31 regions, respectively[J]. Genomies,1996,32:168- 169. 10. Shirashi S, Nakagawa K, Kinukawa N, et al. Immunohitochemical localization of vascular endothelial growth factor in human placenta[J]. Placenta,1996,17:111-121.
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01. Chaouat G, Ledee-Bataille N, Dubanchet S, et al. TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm. Int Arch Allergy Immunol. 2004 Jun;134(2):93-119.
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16. Sakai M,Tsuda H,Tanebe K,etal.Interleukin-12 secretion by peripheral blood mononuclear cells is decreased in normal pregnant subjects and increased in preeclamptic patients[J].AJRI, 2002,47:91-97.
17. Pijnenborg R, Mclaughlin PJ, Vercruyssel L, et al. Immunolocalization of tumor necrosis factor-alpha(TNF-a) in the placental bed of normotension and hypertensive human pregnancies[J]. Placenta, 1998,19(4):231-239.
18. Giardina JB, Green GM, Cockrell KL, et al. TNF-alpha enhances contration and inhibits endothelial No-cGMP relaxation in systemic vessels of pregnant rats[J]. Am J Physiol Regul Comp Physiol, 2002, 283(1):R130-R143.
19. Benian A, Madazh R, Aksu F, etal. Plasma and placental levels of interleukin-10, transforming growth factor-B1, andepithelial cadherin in preeclampsia[J]. Obstet Gynecol, 2002, 100(2), 327-331.
20. Granger JP, Alexander BT, Linas MT, et al. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction[J]. Hypertension, 2001, 38(3Pt2): 718-722.
21. Vince GS, Starkey PM. Interleukin-6, tumour necrosis factors and soluble tumour necrosis factor receptors in women with preeclampsia. Br J Obstet Gynecol 1995; 102: 20.
22．吕秀萍，张凤华，郭宝强等．妊高征患者血浆巨噬细胞集落刺激因子的测定．中华妇产科杂志1999；34(6)：366．
23. Lechner WE, Bergant A, Marth C, et al. Effect of IL-6 on Uterine contractility in the human in Vivo. Z Geburtshilfe Neonatol 1998; 202: 10.
24．王子芬，宋海翔，石少兰等．妊高征患者白细胞介素-6变化的意义．中华妇产科杂志．1999；34(4)：220．
25. Dammann O, Leviton A. Maternal intrauterine infecttion, Cytokines and brain damage in the preterm new-born. Pediatr Res 1997; 42(1): 1.
01. Lenz T, Sealey JE, Tewksbury DA. Regional distribution of the angiotensinogens in human placentae.Placenta. 1993 Nov-Dec; 14(6): 695-9.
02. Kalenga MK, De Hertogh R, Whitebread S, et al. Distribution of the concentrations of angiotensin II (A II), A II receptors, hPL, prolactin, and steroids in human fetal membranes]Rev Fr Gynecol Obstet. 1991 Oct;86(10):585-91.
03. Schauser KH, Nielsen AH, WintherH, et al. Autoradiographic localization and characterization of angiotensin II receptors in the bovine placenta and fetal membranes.Biol Reprod. 1998 Sep;59(3): 684—92.
04. Hagemann A, Nilsen AH, Poulsen K. The uteroplacental renin-angiotensin system: a review.Exp Clin Endocrinol. 1994;102(3):252-61.
05. Mackenzie LW, Word RA, Casey ML, et al. Myosin light chain phosphorylation in human myometrial smooth muscle cells.Am J Physiol. 1990 Jan;258(1 Pt 1):C92-8.
06. Moeller I, Chai SY, MacGregor DP,et al. Localization and quantitation of angiotensin AT1 and AT2 receptors in the pregnant and non-pregnant sheep uterus.Regul Pept. 1996 Mar 22;61(3):213-8.
07. Takimoto E, Ishida J, Sugiyama F, et al.Hypertension induced in pregnant mice by placental renin and maternal angiotensinogen.Science. 1996 Nov 8;274(5289):995-8.
08. Lumbers ER, Kingsford NM, Menzies RI, et al. Acute effects of captopril, an angiotensin-converting enzyme inhibitor, on the pregnant ewe and fetus.Am J Physiol. 1992 May;262(5 Pt 2):R754-60.
09. Mattei MG, Brog JP,Rosnet O,et al. Assiginment of vascular endothelial growth factor(VEGF) and placenta growth factor(PLGF) genes to human chromosome 6p12- 6p21 and 14q24- 14q31 regions, respectively[J]. Genomies,1996,32:168- 169. 10. Shirashi S, Nakagawa K, Kinukawa N, et al. Immunohitochemical localization of vascular endothelial growth factor in human placenta[J]. Placenta,1996,17:111-121.
11.Lyall F, Greer IA, Boswell F, et al. Suppression of serum vascular endothieal growth factor immunoreactivity in normal pregnancy and preeclamsia[J]. Br J Obstet Gynaecol,1997,104:223-228.
12. Brockelsby J, Hayman R, Ahmed A, et al. VEGF via VEGF receptor-1 (flt-1) mimics preeclamptic plasma in inhibiting uterine blood vessel relaxation in pregnancy: implication in the pathogenesis of preeclampsia[J]. Lab Invest, 1999, 79(9): 1101-1111.
13. Lyall F, Young A, Boswell F, et al. Placental expression of vascular endothelial growth factor in placentae from pregnancies complicated dy preeclampsia and interine growth restriction does not support placental hypoxia at delivery[J]. Placenta,1997,18:269-276.
14. Cooper JC, Sharkey AM, Jones DSC, et al. VEGF mRNA levels in placentae from pregnancies complicated by preeclampsia[J]. Br J Obstet Gynaecol, 1996, 103: 1191-1196.
15. Hunter A, Aitkenhead M,Calewell C, et al. Serum levels of vascular endothelial growth factor in preeclamptic and normotensive pregnancy[J]. Hypertension, 2002, 36: 965-969.
16. Saito S, Umekage H, Sakamoto Y, et al. Increased T-helper-1-type immunity and decreased T-helper-2-type immunity in patients with preeclampsia[J]. Am J Reprod Immunol,1999,41(5):297-306.
17. Saito S, Sakai M, Sasaki Y, et al. Quantitative analysis of peripheral blood Th0,Th1,Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia[J]. Clin Exp Immunol, 1999, 117(3): 550～555.
18. Vizi ES, Szelenyi J, Selmeczy ZS, et al. Enhanced tumor necrosis factor-alpha-specific and decreased interleukin-10-specific immune responses to LPS during the third trimester of pregnancy in mice[J]. J Endocrinol, 2001, 171(2): 355～361.
19. Rinehart BK, Terrone DA, Lagoo-Deenadayalan S, et al. Expression of the placental cytokines tumor necrosis factor alpha, interleukin-1 beta and interleukin-10 is increased in preeclampsia[J]. Am J Obstet Gynecol, 1999, 181(4): 915～920.
20. Benyo DF, Miles TM, Conrad KP. Hypoxia stimulates cytokine production by villous explants from human placenta[J]. J Clin Endocrinol Metab, 1997, 82(5): 1582～1588.
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