【作者】 郭辉；

【导师】 尹洁；

【作者基本信息】 河北医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的:妊娠期高血压疾病是妊娠期特有的疾病,是导致孕产妇及围产儿死亡率升高的主要原因之一,迄今明确的发病机制尚未完全阐明。目前一般认为与血管内皮细胞受损、异常滋养层细胞侵入子宫肌层、免疫失调或免疫不匹配等因素密切相关。其中血管内皮细胞损伤是妊娠期高血压疾病病理变化的中心环节。磷酸化丝裂原蛋白活化激酶p38(phosphorylated p38 mitogen-activated protein kinasep,P-p38MAPK)在调控细胞因子产生中起着“分子开关”的作用,其介导中性粒细胞的炎症反应从而造成血管内皮的损伤,影响蛋白合成、细胞表面受体表达、细胞骨架重构,最终导致细胞的炎症、分化和凋亡及多种细胞反应。肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)是一种重要的炎症介质和免疫调节因子,具有广泛的生物学活性,参与了体内许多疾病的病理生理过程。核因子-κB (nuclear factor-kappaB,NF-κB)是1986年发现的转录因子,包括NF-κB/P65及NF-κB/P50,在机体的免疫应答、炎症反应及细胞生长发育、凋亡等多种生理、病理过程中发挥重要作用。本文旨在通过研究P-p38MAPK、TNF-α、NF-κBp65在病例-对照组胎盘组织中的表达情况及其相关性,探讨其在妊娠期高血压疾病发病中的作用,并为临床治疗提供实验性理论依据。方法:1选择2007年9月-2009年9月晚孕期正常孕妇、子痫前期轻度、子痫前期重度患者作为对照组和研究组(三组均除外患有其他疾病的孕妇)。临产前行剖宫产术或经阴道顺娩的晚孕期正常组孕妇30例,子痫前期轻度组35例,子痫前期重度组35例。胎盘娩出后30分钟内取胎盘母面中央绒毛组织块约l×l×lcm,避开钙化区及坏死区,留取胎盘组织。生理盐水充分清洗,10%中性福尔马林溶液固定24小时以上,常规石蜡包埋,4um连续切片。2应用免疫组织化学(immunohistochemistry,IHC)S-P法检测胎盘组织中P-p38MAPK、TNF-α、NF-κBp65的表达强度,根据细胞内染色强度和阳性细胞数分级。3应用SPSS13.0进行统计学分析,临床一般资料为计量资料采用单因素方差分析;计数资料采用Kruskal-wallis H检验,Wilcoxon秩和检验及Fisher’s精确概率计算;两变量间的相关分析用Spearman等级相关分析。采用双侧检验,以P≤0.05为差异在统计学上有显著性意义,P≤0.01为差异在统计学上有非常显著性意义。结果:1临床一般资料比较:各组间年龄、孕周的差异均无统计学意义(P>0.05)。2 P-p38MAPK的表达2.1 P-p38MAPK蛋白阳性染色主要位于细胞核,同时细胞质也有表达,胎盘绒毛滋养细胞(尤其为合体滋养细胞)及毛细血管内皮细胞可见P-p38MAPK棕黄色阳性颗粒。依据前述评分标准,将细胞着色分别分为(-~+++)四个等级。P-p38MAPK在子痫前期轻度组及子痫前期重度组胎盘中表达的阳性率分别为80%和94.29%,高于其在对照组中表达的阳性率30%,统计学上两者有显著性差异(P=0.000<0.05, P=0.000<0.05)。2.2 P-p38MAPK在对照组,子痫前期轻度组及子痫前期重度组三组间均有不同程度的表达,但表达强度不同,三者相比具有显著性差异(p=0.000<0.05)。妊娠期高血压疾病组胎盘中P-p38MAPK的表达较对照组明显升高,进一步两两比较:子痫前期轻度组P-p38MAPK的表达较对照组明显升高(P=0.000<0.05) ;子痫前期重度组较对照组明显升高(p=0.000<0.05)。且随疾病加重,P-p38MAPK的表达逐渐增加:子痫前期重度组P-p38MAPK的表达较子痫前期轻度组增加(p=0.021<0.05)。3 TNF-α的表达3.1 TNF-α主要表达于胞浆和胞膜,胎盘合体滋养细胞内可见TNF-α棕黄色阳性颗粒,其次还可见于蜕膜细胞、血管平滑肌细胞、内皮细胞和绒毛间质细胞等。TNF-α在子痫前期轻度组胎盘中表达的阳性率为82.86%,在子痫前期重度组胎盘中表达的阳性率为97.14%,二者均高于其在对照组中表达的阳性率56.67%,统计学上两者有显著性差异(p=0.029<0.05, p=0.000<0.05)。3.2 TNF-α在对照组,子痫前期轻度组及子痫前期重度组三组间均有不同程度的表达,但其表达强度互不相同,三者相比具有显著性差异(p=0.000<0.05)。妊娠期高血压疾病组胎盘中TNF-α的表达较对照组明显升高,进一步两两比较:子痫前期轻度组TNF-α的表达较对照组明显升高(p=0.002<0.05);子痫前期重度组较对照组明显升高(p=0.000<0.05)。且随疾病加重, TNF-α的表达逐渐增加:子痫前期重度组TNF-α的表达较子痫前期轻度组增加(p=0.037<0.05)。4 NF-κBp65的表达4.1 NF-κBp65在胎盘的合体滋养细胞、血管内皮的细胞浆和细胞核中可见NF-κBp65棕黄色阳性颗粒,阳性染色主要位于细胞核。NF-κBp65在子痫前期轻度组胎盘中表达的阳性率为80%。NF-κBp65在子痫前期重度组胎盘中表达的阳性率约为100%,二者均高于其在对照组中表达的阳性率33.33%,统计学上两者有显著性差异(p=0.000<0.05, p=0.000<0.05)。4.2 NF-κBp65在对照组,子痫前期轻度组及子痫前期重度组三组间均有不同程度的表达,三者相比具有显著性差异(p=0.000<0.05)。妊娠期高血压疾病组胎盘中NF-κBp65的表达较对照组明显升高,进一步两两比较:子痫前期轻度组NF-κBp65的表达较对照组明显升高(p=0.000<0.05);子痫前期重度组NF-κBp65的表达较对照组明显升高(p=0.000<0.05)。且随疾病加重, NF-κBp65的表达逐渐增加:子痫前期重度组NF-κBp65的表达较子痫前期轻度组增加(p=0.019<0.05)。5 P-p38MAPK、TNF-α、NF-κBp65三者间的激活程度两两互成正相关(p<0.05)。结论:1 P-p38MAPK、TNF-α、NF-κBp65蛋白在正常妊娠对照组,子痫前期轻度组及子痫前期重度组均有不同程度的表达。2 P-p38MAPK、TNF-α、NF-κBp65在对照组,子痫前期轻度组及子痫前期重度组的表达程度不同,随妊娠期高血压疾病病情进展,三者的表达分别逐渐升高,可能为该病发病的重要原因3病理性妊娠患者的胎盘中大量的TNF-α表达,激活p38MAPK,从而形成P-p38MAPK信号通路,调控多种细胞反应,并刺激更多的炎性因子释放加重血管内皮损伤,并和作为炎症介质和免疫调节因子NF-κBp65共同参与妊娠期高血压疾病发生及胎盘的病理改变。4 P-p38MAPK、TNF-α、NF-κBp65两两成正相关,三者紧密联系,相互影响,共同在妊娠期高血压疾病中发挥作用。因此从分子生物学水平上的进一步研究,有望揭示妊娠期高血压疾病的确切机制,从而为该病的早期预防、早期治疗提供有力的理论依据。更多还原

【Abstract】 Objective: Hypertensive disorders in pregnancy is the peculiar disease in pregnancy, it is one of the main causes for the death of pregnancies and neonates, however, up to now, the conclusive etiology of hypertensive disorders in pregnancy remains unclear. At present, injury of maternal vascular endothelium, the muscle of uterus was invasived by abnormality trophoblast, immunity mechanism are thought the important reasons. However ,the injury of maternal vascular endothelium is the core link of the pathological change of the hypertensive disorders in pregnancy. Phosphorylated p38 mitogen-activated protein kinasep(P-p38MAPk) is used as "on-and-off" switch on the regulation of production of cytokines. Neutrophil inflammatory response mediated by P-p38MAPk affects the protein synthesis, cell surface receptors and cytoskeleton remodeling,which makes the injury of maternal vascular endothelium.At last, a variety of cellular reactions appears, including cellular inflammation, differentiation and apoptosis.Tumor necrosis factor-alpha(TNF-α) is an important inflammatory mediators and immune regulatory factors, which be with a wide range of biological activity,and it also plays an important role in patho-physiological course of some disease. Activation of transcription factor nuclear factor kappa B (NF-κB) which includes NF-κB/P65 and NF-κB/P50 was found in 1986. It has been proved to play an important role in immunity, Burning disease reaction, growth of cell and apoptosis and so on. Through studying characteristics and relativity among P-p38MAPK TNF-αand NF-κBp65 in placenta of hypertensive disorders in pregnancy, we hope provide the evidence of experiment theories for clinical treatment.Methods:1 70 women with hyptensive disorders in pregnancy(35 light preeclampsia, 35 severe preeclampsia)and 35 women with normal pregnancy were recruited the fourth affiliated hospital of Hebei medical university as study group and control group from 2007 to 2009. Respectively, the two groups excepted other disease were maternal age, party gestational age. Sample collecting: About l×l×lcm placental tissues were collected from the pregnant women after the placenta was deliveried in 30 minutes. Use axenic 0.9% sodium chloride to wash it and fix it with formaldehyde.2 The expression of P-p38MAPK TNF-αand NF-κBp65 in placenta was detected by immuno - histochemical SP method .3 The data was analyzed by SPSS13.0 software wrap, Kruskal-wallis H Test, Wilcoxon signed-rank test, Faisher probabilities in 2×2 table and Spearman grade relation analysis are involved. Statistically significant level was considered as“alpha equals 0.05”and“P<0.01”indicating very significant difference.Results:1 there were no statistical difference between control and preeclampsia group in age and gestational age(p>0.05).2 The expression of P-p38MAPK protein2.1 Immunohistochemical staining for P-p38MAPK protein was located at the nucleus and cytoplasm of the cytotrophoblast as well as extra villous trophoblastic cells, some weak signals were showed in the nucleus and cytoplasm of the vascular endothelium. Immunohistochemical staining in the cytotrophpblast showed weaker than syneytiotrophoblast. Also,immunohis- tochemical staining in the nucleus showed stronger than in the cytoplasm. The expression of P-p38MAPK protein in trophocyte showed significant higher in light preeclampsia group (positive rate=80%) than in control group(positive rate=30%), P<0.05. The expression of P-p38MAPK protein in trophocyte showed significant higher in evere preeclampsia group (positive rate=94.29%) than in control group(positive rate=30%), P<0.05.2.2 The expression of P-p38MAPK is different from control group to light preeclampsia group and evere preeclampsia group , there is statistic significance(p=0.000<0.05).The expression in light preeclampsia group is higher than that in control group(P=0.000<0.05). The expression in severe preeclampsia group is higher than that in control group(P=0.000<0.05).The expression of P-p38MAPK protein showed significances correlated with the degree of differentiation: The expression in light preeclampsia group is lower than that in severe preeclampsia group(P=0.021<0.05).3 The expression of TNF-αprotein3.1 Immunohistochemical staining for TNF-αprotein was located at the cytoplasm of the cytotrophoblast as well as extra villous trophoblastic cells, some weak signals were showed in the cytoplasm of the vascular endothelium decidualcells、hemal endothelial cells and mediate cells. The expression of TNF-αprotein in trophocyte showed significant higher in light preeclampsia group (positive rate=82.86%) than in control group(positive rate=56.67%), P<0.05. The expression of P-p38MAPK protein in trophocyte showed significant higher in evere preeclampsia group (positive rate=97.14%) than in control group(positive rate=56.67%), P<0.05.3.2 The expression of TNF-αis different in control group,light preeclampsia group and evere preeclampsia group, there is statistic significance(p=0.000<0.05).The expression in light preeclampsia group is higher than that in control group(P=0.002<0.05). The expression in severe preeclampsia group is higher than that in control group(P=0.000<0.05).The expression of P-p38MAPK protein showed significances correlated with the degree of differentiation: The expression in light preeclampsia group is lower than that in severe preeclampsia group(P=0.037<0.05).4 The expression of NF-κBp65 protein4.1 Immunohistochemical staining for NF-κBp6 protein was located at the cytoplasm and nucleus of the cytotrophoblast as well as extra villous trophoblastic cells, some weak signals were showed in the cytotrophoblast. The expression of NF-κBp6 protein in trophocyte showed significant higher in light preeclampsia group (positive rate=80%) than in control group(positive rate=33.33%), P<0.05. The expression of NF-κBp6 protein in trophocyte showed significant higher in evere preeclampsia group (positive rate=100%) than in control group(positive rate=33.33%), P<0.05.4.2 The expression of NF-κBp6 is different from control group to light preeclampsia group and evere preeclampsia group , there is statistic significance(p=0.000<0.05).The expression in light preeclampsia group is higher than that in control group(P=0.000<0.05). The expression in severe preeclampsia group is higher than that in control group(P=0.000<0.05).The expression of P-p38MAPK protein showed significances correlated with the degree of differentiation: The expression in light preeclampsia group is lower than that in severe preeclampsia group(P=0.019<0.05).5 The P-p38MAPK TNF-αand NF-κBp65 in the placental was activated. At the same time, the expression degree of P-p38MAPK TNF-αand NF-κBp65 was positive correlate to the degree of hypertensive disorders in pregnancy. In addition, they were positive correlate to each other.Conclusion:1 There are different expressions of P-p38MAPK TNF-αand NF-κBp65 protein in control group,light preeclampsia group and evere preeclampsia group.2 The abnormal expression of P-p38MAPK TNF-αand NF-κBp65 were more severity accompanied with the progress of hypertensive disorders in pregnancy, which was positive correlate to pathogenetic condition degree of the disease and was the importance season possibility.3 High expression of TNF-αin placenta of hypertensive disorders in pregnancy may make P-p38MAPK express highly. then results in the activation of the P-p38MAPK signal transduction pathway ,through which extracellular signal can resulnuclear reaction . Hemal wall gets of inflammation cell infiltrate and bring about injury of hemal endodermis sell,through the high expression of P-p38MAPK signal transduction pathway.P-p38MAPK TNF-αand NF-κBp65 which is an important inflammatory mediators and immune regulatory factors are together related to the pathogenesis of hypertensive disorders in pregnancy and typical pathological change in placenta. 4 Two of P-p38MAPK TNF-αand NF-κBp65 are positive correlate to each other, which contact closely and effect each other. We will get the accurate mechanism of hypertensive disorders in pregnancy by further research in the molecular biology level, which will provide theories evidence of earlier period prevention and treatment.更多还原