【作者】 冯冲；

【导师】 尚涛；

【作者基本信息】 中国医科大学 ， 妇产科学， 2009， 博士

【摘要】 目的妊娠期高血压疾病是产科最常见的合并症,在我国发病率为9.40%,是导致孕产妇和围生儿病率及死亡率升高的主要原因。子痫前期和子痫是妊娠期高血压疾病的严重阶段,至今病因和发病机理不明,给预防和治疗带来困难。近年来,国内国外的专家学者对其病因和发病机理作了大量的研究工作,提出了许多学说,目前比较公认的发病机制是免疫学说、胎盘或滋养叶细胞缺血学说、遗传学说、血管活性物质学说、钙平衡失调学说、血管内皮损伤学说,但仍无一种理论能满意、全面地解释其病因。现又有学者提出了一种新的理论,炎症刺激可能参与了妊娠期高血压疾病的发病过程,并且已有学者用内毒素刺激成功地建立了子痫前期的动物模型,且抗炎治疗有效。说明感染可导致母胎免疫平衡失调,引起免疫损伤,滋养细胞缺血及胎盘植入异常,加重胎盘缺血缺氧,血浆毒性因子释放,引起全身的血管内皮损伤,最终导致子痫前期和子痫。胎盘是一个重要的免疫器官,内含大量单核巨噬细胞,被认为是子痫前期患者体内“毒性因子”的来源,胎盘滋养细胞的作用颇受关注。有学者认为妊娠期滋养细胞也是一种免疫细胞,是天然免疫系统的组成部分,影响母儿的免疫应答。滋养细胞和巨噬细胞具有某些共同特性:吞噬、形成合体细胞、组织浸润功能;表达CD4、CD14、IgG受体;分泌白介素-1、2和6(IL-1,IL-2,IL-6),干扰素-γ(INF-γ)和肿瘤坏死因子-α(TNF-α)等多种细胞因子,并表达它们的受体。近年来,钙卫蛋白(Calprotectin,Cal)在妊娠期高血压疾病中的作用引起了许多学者的关注。钙卫蛋白是近年来发现的一种新的与炎症反应密切相关的钙结合蛋白,属于S100蛋白家族的成员,是由S100A8和S100A9构成的异二聚体,主要表达于中性粒细胞、单核-巨噬细胞,具有抑制细胞增殖、诱导细胞凋亡、调节免疫、参与炎症反应等多种生物学功能,在许多急慢性炎症疾病中发现其表达升高,有提出钙卫蛋白可以在一定程度上反映炎症疾病的严重程度。糖基化终末产物受体(RAGE)是钙卫蛋白的受体,可与钙卫蛋白结合后启动若干信号通路,介导多种炎症病理过程。TNF-α是一种促炎性细胞因子,在妊娠组织中通过自分泌或旁分泌作用调节局部的子宫活动,从而参与许多生理和病理过程。目前,钙卫蛋白在子痫前期母血中的表达国外仅有少量报道,国内尚无报道。在母血、胎盘、滋养细胞中的研究国内外至今无报道。为探讨钙卫蛋白对滋养细胞的致炎作用及妊娠期高血压疾病的发病机制,拟检测钙卫蛋白及其受体RAGE和TNF-α在妊娠期高血压疾病中的表达,研究钙卫蛋白对滋养细胞表达炎性介质的诱导作用及NF-κB、p38 MAPK、ERK信号传导通路对其的调控作用。以寻找一种药物能够抑制钙卫蛋白的上调,进而抑制炎性介质的释放,及是否需联合应用抗生素来治疗妊娠期高血压疾病开辟一条新路。研究方法一、钙卫蛋白及其受体RAGE和TNF-α在妊娠期高血压疾病中的表达采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(SP)法检测妊娠期高血压疾病患者胎盘组织中钙卫蛋白、RAGE和TNF-α的表达,采用酶联免疫吸附(ELISA)法,检测妊娠期高血压疾病患者血清中钙卫蛋白和TNF-α的表达。二、钙卫蛋白对人滋养细胞表达TN-α的诱导作用采用人钙卫蛋白作用于原代培养的早孕期细胞滋养细胞,以MTT法检测钙卫蛋白对滋养细胞的增殖活性的影响,透射电镜下观察钙卫蛋白作用后滋养细胞超微结构的改变,以ELISA法、Real Time PCR法检测钙卫蛋白对滋养细胞释放TNF-α在不同的时间效应和剂量效应下的影响。三、钙卫蛋白诱导人滋养细胞表达TNF-α的细胞信号传导通路探讨体外培养的人原代滋养细胞分别与ERK抑制剂PD98059、p38MAPK抑制剂SB203580和NF-κB抑制剂BAY11-7082,以及PD98059联合SB203580共孵育2h后,培养基中分别加入重组人钙卫蛋白作用6h。采用ELISA法测定培养上清中TNF-α的含量,用Real Time PCR法检测培养细胞中TNF-αmRNA表达。结果一、钙卫蛋白及其受体RAGE和TNF-α在妊娠期高血压疾病中的表达妊娠期高血压疾病组血清钙卫蛋白和TNF-α水平明显高于对照组,两组比较,差异有统计学意义(P＜0.01);重度子痫前期患者较轻度子痫前期患者明显升高(P＜0.01),而轻度子痫前期患者明显高于妊娠期高血压患者(P＜0.05),妊娠期高血压患者高于对照组,但差异无统计学意义(P＞0.05)。两组孕妇绒毛间质巨噬细胞和中性粒细胞中均有钙卫蛋白阳性表达,主要位于细胞膜及细胞质中。妊娠期高血压疾病组胎盘组织中钙卫蛋白阳性表达率明显高于对照组,两组比较,差异有统计学意义(P＜0.01);轻、重度子痫前期患者明显高于对照组(P＜0.05,P＜0.01),且轻、重度子痫前期患者间相比,差异有统计学意义(P＜0.05);妊娠期高血压患者与对照组比较,差异无统计学意义(P＞0.05)。两组孕妇胎盘绒毛合体滋养细胞及部分蜕膜细胞中均有RAGE、TNF-α阳性表达,位于主要在细胞膜和细胞质。妊娠期高血压疾病组胎盘组织中RAGE、TNF-α阳性表达率明显高于对照组,两组比较,差异有统计学意义(P＜0.01);妊娠期高血压和轻、重度子痫前期患者呈逐渐升高趋势,轻、重度间比较,差异有统计学意义(P＜0.05)。妊娠期高血压疾病组血清钙卫蛋白水平与TNF-α水平呈正相关(r=0.359,P＜0.05);对照组血清钙卫蛋白水平与TNF-α水平无相关性(r=0.184,P＞0.05)。妊娠期高血压疾病组胎盘组织钙卫蛋白水平与TNF-α水平呈正相关(r=0.383,P＜0.01);对照组胎盘组织钙卫蛋白水平与TNF-α水平无相关性(r=0.130,P＞0.05)。妊娠期高血压疾病组及对照组血清钙卫蛋白表达与胎盘组织中钙卫蛋白的阳性表达均呈正相关性(r=0.365,P＜0.01;r=0.675,P＜0.01)。二、钙卫蛋白对人滋养细胞表达TNF-α的诱导作用钙卫蛋白作用24小时后药物浓度与细胞滋养细胞增殖活性呈负相关(r=-0.735,P＜0.01),即随着钙卫蛋白药物浓度的增加,细胞生长受到抑制。在800ng/ml及其以上组表现更为明显(P＜0.01)。但是,在小剂量药物浓度(＜0.39ng/ml)刺激时,钙卫蛋白可刺激细胞滋养细胞的增殖,特别是在浓度0.20ng/ml处更为明显(P＜0.01)。作用时间为48小时,钙卫蛋白与细胞滋养细胞增殖活性的上述关系更为明显。透射电镜下可见,经钙卫蛋白处理过的细胞滋养细胞的细胞核小,核膜模糊,核内异染色质边集,呈半月状,呈凋亡改变,胞质内线粒体多有嵴减少,个别呈空泡样变,胞膜多溶解。将人钙卫蛋白加入到原代培养的早孕期细胞滋养细胞培养液中能明显刺激其TNF-α的分泌。在0～500ng/ml范围内,ELISA、Real Time PCR法检测钙卫蛋白诱导滋养细胞的TNF-α分泌均增加,均呈明显的剂量依赖性关系,在1000ng/ml钙卫蛋白对滋养细胞分泌TNF-α的诱导作用减弱。钙卫蛋白诱导滋养细胞的TNF-α分泌在3～48h间均呈双峰形,在6h和24h分泌TNF-α增强。三、钙卫蛋白诱导人滋养细胞表达TNF-α的细胞信号传导通路探讨ELISA法检测钙卫蛋白诱导人滋养细胞表达TNF-α增加,PD98059、SB203580及BAY11-7082均可显著下调钙卫蛋白诱导人滋养细胞产生TNF-α(P＜0.05),同时使用PD98059和SB203580可增强抑制TNF-α的分泌水平(P＜0.05)。Real Time PCR法检测钙卫蛋白诱导人滋养细胞表达TNF-αmRNA增加,PD98059、SB203580或BAY11-7082均可显著下调钙卫蛋白诱导人滋养细胞表达TNF-αmRNA(P＜0.05),PD98059和SB203580具有协同抑制作用(P＜0.05)。结论1、钙卫蛋白、RAGE、TNF-α蛋白在妊娠期高血压疾病病情加重进展中表达逐渐上调,有望成为子痫前期严重程度评估、疾病预测和判断预后的有效指标。2、高浓度的钙卫蛋白对体外培养的早孕期细胞滋养细胞有明显的抗增殖和诱导凋亡作用,钙卫蛋白能诱导滋养细胞TNF-α的分泌,证实钙卫蛋白在子痫前期的病理过程中发挥作用。3、信号分子ERK、p38MAPK和NF-κB均参与了钙卫蛋白诱导早孕期细胞滋养细胞表达TNF-α。更多还原

【Abstract】 ObjectionHypertensive disorder complicating Pregnancy is the most common obstetric complications,the incidence rate is 9.40%in our country,which is the main reason of maternal perinatal morbidity and mortality.Pre-eclampsia and eclampsia are serious stage of pregnancy-induced hypertension,so far that etiology and pathogenesis is unknown make it difficult to prevent and treat.In recent years,experts scholars at home and abroad have done many studies for its etiology and pathogenesis,and raised a lot of theory.Theory of pathogens that now is commonly accepted is immune,or ischemic in placental trophoblast cells leaf theory,genetics,vasoactive substance doctrine,the theory of calcium imbalance,vascular endothelial injury theory,but none of them can fully satisfactied explain its cause.Scholars now have put forward a new theory, stimulate inflammation may be involved in the pathogenesis of preeclampsia,and there were scholars successfully established an animal model of hypertensive disorder complicating Pregnancyby endotoxin,and anti-inflammatory treatment is effective. This proved that infection can lead to maternal-fetal immune imbalance,immune injury, ischemia and trophoblastic abnormal placenta accreta,increased placental ischemia,the release of plasma toxic factors,arising from systemic vascular endothelial damage, eventually leading to pre-eclampsia and eclampsia.Placenta is an important immune organ,containing a large number of mononuclear cells,which is considered sources of "toxicity factor" in patients of pre-eclampsia in vivo,and the role of placental trophoblast is popular concerned.Some scholars believe that the trophoblast cells during pregnancy is also a kind of immune cells,are natural components of the immune system,impact immune response of maternal-fetal. Trophoblast cells and macrophages have certain common characteristics:phagocytosis, the formation of syncytial cell,tissue infiltration function;the expression of CD4, CD14,IgG receptor;secretion of IL--1,2,6(IL-1,IL-2,IL-6),interferon-γ(INF-γ) and tumor necrosis factorα(TNF-α) and other cytokines,and express their receptors.In recent years,the role of calprotectin(Cal) in hypertensive disorder complicating Pregnancydisease caused a lot of the attention of scholars.Calprotectin are found in recent years as a new calcium-binding protein,which has close relationship with the inflammatory response,belongs to the S100 protein family,heterodimers consisting S100A8 and S100A9,mainly expressed in neutrophils,single nuclear - macrophages, can inhibit cell proliferation and induced apoptosis,immune regulation,inflammatory response,and so on,participation in a variety of biological functions,and in many acute and chronic inflammatory diseases was found increased expression,so calprotectin has raised to a certain extent reflect the severity of inflammatory diseases. Advanced glycation end products receptor(RAGE),a receptor of calprotectin,may be combined calprotectin to start a number of signaling pathways,mediate a variety of inflammation pathological process.Tumor necrosis factor-α(TNF-α) is a proinflammatory cytokine,regulating the local activity of the uterus in the pregnancy tissue through autocrine or paracrine,and then participate in many physiological and pathological processes.At present,the expression of calprotectin at maternal pre-eclampsia was reported only a little in foreign,there are no reports in domestic.So far no study about it in maternal blood,placenta,trophoblast cells have reported at home and abroad.In order to investigate proinflammatory role of calprotectin in trophoblast cells and the pathogenesis of preeclampsia,we intend to detected expression of calprotectin and RAGE in pregnancy-induced hypertension,induction role of calprotectin on trophoblast cells to produce inflammatory mediators and regulation of NF-kB,p38 MAPK,ERK signal transduction pathway in vitro.Building a new way to find a drug inhibiting calprotectin increase,thereby inhibiting the release of inflammatory mediators,and whether or not it is need to combined with antibiotics to treat hypertensive disorder complicating Pregnancydiseases.Methods1.Expression of calprotectin and its receptor,RAGE and TNF-αin pregnancy-induced hypertension. By immunohistochemical streptavidin-biotin-protein - enzyme-linked peroxidase (SP) detection Cal,RAGE and TNF-α’s expression in placenta of patients with pregnancy-induced hypertension,by enzyme-linked immunosorbent assay(ELISA) method to detect expression of Cal and TNF-αin pregnancy serum of patients with hypertensive disease.2.Induction of calprotectin to human trophoblast cells of expressing TNF-αCalprotectin treat early primary culture of trophoblast cells,MTT assay the role of calprotectin on proliferation of trophoblastic activity,observed by transmission electron microscope ultrastructural changes of trophoblastic after treated with calprotectin, ELISA,Real Time PCR detect the influence under different times and dose effect of calprotectin to release of TNF-αfor trophoblast cells.3.Human calprotectin-induced trophoblast expression of TNF-αsignal transduction pathwayCultured human trophoblast cells in primary separately treat with the ERK inhibitor PD98059,p38MAPK inhibitor SB203580 and NF-κB inhibitor BAY11-7082, United SB203580 and PD98059,incubated 2h,the medium of recombinant human separately add calprotectin,incubated 6h.ELISA detect TNF-αcontent in the culture supernatant,Real Time PCR detect of mRNA expression of TNF-α.Results1.Expression of calprotectin and its receptor,RAGE and TNF-αin pregnancy-induced hypertension.The calprotectin and TNF-αlevels of serum in hypertensive disorder complicating Pregnancywas significantly higher than control group,the two groups has statistical significance difference(P＜0.01);patients with severe pre-eclampsia in patients was significantly increased compared with less severe pre-eclampsia(P＜0.01),while patients with mild preeclampsia was significantly higher than that in patients with hypertensive disorder complicating Pregnancy(P＜0.05),hypertensive disorder complicating Pregnancywas higher than control group,but the difference has not statistically significant(P＞0.05). In two groups of pregnant women,villous stromal macrophagocyte and neutrophils was both positive expression of calprotectin,mainly located in the cell membrane and cytoplasm.Hypertensive group was higher than control group,the difference has statistical significance(P＜0.01);light,severe pre-eclampsia patients was significantly higher than control group(P＜0.05,P＜0.01),and the difference between mild and severe pre-eclampsia patients has statistically significant(P＜0.05);there was no significant difference between hypertensive disorder complicating Pregnancypatients and control group(P＞0.05).Two groups of pregnant women placental of villi syncytiotrophoblast cells and decidual cells have RAGE,TNF-αexpression,located mainly in the cell membrane and cytoplasm.Hypertensive group pregnancy placenta RAGE,TNF-αexpression was significantly higher than control group,the difference has statistical significance in two groups(P＜0.01);pregnancy-induced hypertension, light,severe pre-eclampsia patients was gradually increasing,and the difference between mild and severe pre-eclampsia patients has statistically significant(P＜0.05).The calprotectin serum levels of hypertensive disorder complicating Pregnancyhad positively correlated with that of TNF-α(r=0.359,P＜0.05);control group had no(r= 0.184,P＞0.05).Calprotectin pregnancy placenta levels of hypertensive group was positively correlated with thai of TNF-α(r=0.383,P＜0.01);control group had not.(r =0.130,P＞0.05).The calprotectin serum and placenta level of Pregnancy-induced hypertension group and control group were positively correlated(r=0.365,P＜0.01;r =0.675,P＜0.01).2.Induction of calprotectin to human trophoblast cells of expressing TNF-αAfter treated with calprotectin for 24 hours,the drug concentration was a negative correlation with the proliferative activity of cytotrophoblasts,namely,as calprotectin increase,cell growth was inhibited.Especially at 800 ng/ml and the more higher the more obvious(P＜0.01).However,at low dose the concentration of drug(＜0.39 ng/ml) stimulated,calprotectin can stimulate cytotrophoblast proliferation,especially in the concentration of 0.20 ng/ml more evident Department(P＜0.01).Time for 48 hours, proliferation and activity of these relations between calprotectin and cytotrophoblast cell becomes more apparent.By transmission electron microscope,after calprotectin-treated cytotrophoblast nuclear can be seen small nuclear ambiguity,nuclear heterochromatin margination,was like a half months,showing apoptosis changes,mitochondria’s cristae reduced in cytoplasm,some vacuolar degeneration,membrane dissolved.Add calprotectin to to the primary cultured trophoblast cells during early culture medium significantly stimulated the secretion of TNF-α.At the scope of 0～500ng/ml, ELISA,Real Time PCR detected that calprotectin-induced trophoblast secretion of TNF-αwere increased,showing a dose-dependent relationship,in1000ng/ml induction of calprotectin to TNF-αsecretion reduced.Calprotectin Group trophoblastic secretion of TNF-αat 3～48h showed a bimodal shape,in 6h and 24h the secretion of TNF-αenhanced.3.Human calprotectin-induced trophoblast expression of TNF-αsignal transduction pathwayELISA assay calprotectin induced human trophoblast TNF-αincreased,PD98059, SB203580 and BAY11-7082 can be significantly reduced TNF-αinduced by calprotectin in human trophoblast cells(P＜0.05),while the use of PD98059 and SB203580 could enhance the inhibition of the secretion of TNF-αlevels(P＜0.05).Real Time PCR detect mRNA xpression of TNF-αincreased by calprotectin-induced,PD98059,SB203580 or BAY11-7082 can be significantly reduced TNF-αmRNA(P＜0.05),PD98059 and SB203580 has a synergistic inhibitory effect(P＜0.05).Conclusion1.The expression of calprotectin,RAGE,TNF-αprotein in hypertensive disorder complicating Pregnancyin progress is increased as the disease gradually severe,which would be expected to be the assessment target of the severity of pre-eclampsia,disease prognosis and prediction of the effective.2.High doses of calprotectin in vitro has a clear anti-proliferation and induction of apoptosis to cytotrophoblast early pregnancy,calprotectin can induce trophoblastic secretion of TNF-α,it was confirmed that calprotectin play a role in the pathological process of pre-eclampsia.3.Signaling molecules ERK,p38MAPK and NF-κB are involved in expression of TNF-αby calprotectin-induced early pregnancy cytotrophoblast.α更多还原