【作者】 刘文萃；

【导师】 郝淑维； 崔澂；

【作者基本信息】 河北医科大学 ， 妇产科学， 2004， 硕士

【摘要】 目的：妊娠高血压综合征（妊高征）是产科常见的严重并发症，是导致母儿病率及死亡率的主要原因之一，其病因及发病机理尚未完全阐明。近年来关于妊高征免疫学发病机制研究较多，其中细胞因子(CK)与疾病的关系引起越来越多的关注，研究结果表明妊高征的发生与机体免疫失衡密切相关。本实验通过对正常妊娠妇女与妊高征患者外周血及蜕膜局部免疫相关细胞形态学观察以及免疫细胞诱生CK活性检测，了解外周血及蜕膜局部诱生CK活性的变化，进一步摸索两者免疫功能指标之间是否具有相关性，以探讨妊高征的免疫学发病机制，并为临床预测及防治提供实验室依据。方法：排除原发性高血压、糖尿病、慢性肾炎、肝炎、甲亢、感染等疾病以及产次、产程的影响，随机抽取妊高征组和正常妊娠组各20例，于入院后未进入产程且未使用任何药物时，抽取肘前静脉血10ml；再于剖宫产或阴道分娩胎盘娩出后，立即无菌刮取贴近胎盘边缘的蜕膜组织。自外周血及蜕膜中分别采用酶消化法及密度梯度离心法分离纯化淋巴细胞及单核/巨噬细胞，其中淋巴细胞用ConA刺激72h，光镜下观察淋巴细胞转化情况。淋巴细胞及单核/巨噬细胞悬液分别用不同种类及不同浓度的有丝分裂原刺激培养，采用酶联免疫吸附试验测定培养上清液中诱生CK活性。实验数据使用SPSS10.0软件中的t检验、方差分析、直线回归及直线相关进行统计学处理。 <WP=4>结果：1 蜕膜组织中各种类型免疫相关细胞的光镜观察：与对照组相比，实验组蜕膜组织中大颗粒淋巴细胞（LGL）数量显著增多，且与病情严重程度有关；与对照组相比，轻度PIH组蜕膜组织LGL数量未见明显变化（P＞0.05），中、重度PIH组显著升高，实验组组间相比差异亦显著（均P＜0.01）。蜕膜组织中的其他类型细胞，如蜕膜基质细胞、蜕膜巨噬细胞及小淋巴细胞百分率在各组间无统计学意义（P＞0.05）。2 外周血及蜕膜组织中非贴壁细胞淋巴细胞转化率（LT）的观察：实验组各组与对照组相比，外周血LT无统计学意义，实验组组间相比亦无显著差异（均P＞0.05）；蜕膜组织中实验组LT比对照组显著升高，且随病情严重程度的加剧而呈现上升趋势，其中对照组与轻度PIH组之间无统计学意义（P＞0.05），与中、重度PIH组相比差异显著，实验组组间相比差异亦显著（均P＜0.01）。3 外周血及蜕膜组织中非贴壁细胞诱生的各种类型CK活性变化：实验组外周血中IL-2、IFN-γ诱生活性较对照组相比显著升高，IL-4、IL-10诱生活性则显著下降，且与病情严重程度有关，对照组外周血中上述几种CK诱生活性分别与轻度PIH组相比无统计学意义（均P＞0.05），与中、重度PIH组相比差异显著，实验组组间相比差异亦显著（均P＜0.01）。实验组蜕膜组织中非贴壁细胞诱生的上述几种CK除IL-4外，其变化趋势及统计学分析与外周血中相似，而IL-4的诱生活性则在对照组与实验组各组之间均无统计学意义（P＞0.05）。4 外周血及蜕膜组织中贴壁细胞诱生的各种类型CK活性变化：实验组外周血及蜕膜组织中TNF-α、IL-6诱生活性均较对照组相比显著升高，且与病情严重程度有关，与对照组相比，<WP=5>轻度PIH组诱生活性无显著变化（均P＞0.05），而中、重度PIH组诱生的TNF-α、IL-6活性显著升高，实验组组间相比差异亦显著（均P＜0.01）。5 妊高征患者外周血及蜕膜组织中诱生CK相关性比较：实验组外周血与蜕膜组织中诱生的IL-2、IFN-γ、IL-4、IL-10、TNF-α、IL-6活性之间均呈现显著正相关，对相关系数进行假设检验均有统计学意义（P＜0.01＝。结论：1 妊高征患者蜕膜组织中LGL百分率及LT均显著增高，提示患者蜕膜局部由LGL介导的非特异性免疫及T淋巴细胞介导的特异性免疫功能可能增强，也许存在LGL自然杀伤活性被激活的状态，造成蜕膜局部血管内皮的损伤，从而引发妊高征的一系列临床病理变化。2 妊高征患者与正常妊娠妇女外周血LT均呈下降趋势，且两者间无明显差异，表明患者外周血LT的降低可能是由于妊娠时机体免疫功能下降与妊高征发病免疫排斥作用增强之间相互作用的结果，妊娠时机体免疫功能下降也许更占优势，因此外周血LT的变化能否作为评价疾病严重程度的指标还有待于进一步确定。3 妊高征患者外周血及蜕膜组织中非贴壁细胞诱生的IL-2、IFN-γ活性显著升高，而IL-4、IL-10 的活性明显下降，提示患者Th1/Th2免疫失衡，Th1介导的细胞免疫占优势，而Th2所介导的体液免疫受到抑制，从而有利于机体免疫系统对胎儿发挥免疫攻击。IL-4的变化与其它几种CK略有不同，在蜕膜中无大量表达，且在实验组与对照组之间无显著差异，提示IL-4有可能主要在全身发挥作用，局部的作用并不显著。4 妊高征患者外周血及蜕膜组织中贴壁细胞诱生的TNF-α、IL-6活性显著升高，且两者间差异非<WP=6>常明显，表明炎性CK在妊高征发病中占有极其重要的地位，有可能在全身及局部参与妊高征的病理生理变化。5 妊高征患者外周血与蜕膜局部CK活性变化之间存在显著正相关，表明全身与局部免疫功能变化之间存在相关性，CK活性变化也许可以作为反映患者局部及全身免疫状态变化的指标，在妊高征的预测、疗效观察、终止妊娠时机的选择等临床应用中发挥理论指导作用。更多还原

【Abstract】 Objective: Pregnancy-induced hypertension syndrome(PIH), is a common and severe obstetric syndrome, and is a major cause of maternal and fetal morbidity and mortality, whose etiology and pathophysiology are still understanded. Recent data suggest that the immune imbalance has been implicated in the pathophysiology of PIH, with modification of the maternal cellular immunity. We observed peripheral blood and decidual immuno-associated cells and investigated the changes of the activities of induced cytokines, in order to know the correlation of the peripheral blood and decidual immunological factors, discuss the mechanisms of PIH and offer laboratorial evidences for preveneion and therapy of this disease. Methods: The study group consisted of 20 women with PIH, the control group consisted of 20 women with uncomplicated pregnancies. Women with the previous history of underlying hypertension, diabetes, chronic renal disease, platelet disorder, infection were excluded from the study. Two groups were matched for age and parity. Peripheral blood samples were obtained before antihypertensive treatment and labor. 10 milliliters of blood were taken by venepuncture in sterile <WP=8>conditions, and collected in sterile heparinized tubes. Maternal decidual tissues were obtained during elective caesarean sections and vaginal deliveries. Lymphocyte and monocyte/macrophage were isolated from peripheral blood and decidual tissue by centrifuge and enzyme digestion. Then lymphocyte was stimulated with ConA for 72h and calculated the ratio of lymphocyte transforming(LT). Lymphocyte and monocyte/macrophage were respectively cultured and stimulated with different kinds and concentrations of mitogens, then determined the activities of induced cytokines in the culture supernatants by enzyme-linked immunosorbent assay(ELISA). All data were analyzed with the SPSS 10.0 software, including t-test, ANOVA, linear regression and linear correlation.Results: 1 The changes of all kinds of decidual cells :Compared with control group, the number of large granulated lymphocyte(LGL) obviously increased in decidual tissue of patients with middle and severe PIH groups(P＜0.01). In mild PIH group, it was not higher (P＞0.05).2 The comparison of LT in peripheral blood or decidual tissue: Compared with control group, peripheral blood LT in patients with PIH was not higher(P＞0.05). Decidual tissue LT in mild PIH group was not higher than in control group(P＞0.05). But it was obviously higher in middle and severe PIH groups than control group(P＜0.01).3 The comparison of activities of induced cytokines in peripheral blood and decidual tissue <WP=9>lymphocyte: Compared with control group, the activities of IL-2, IFN-γin mild PIH group were not higher (P＞0.05), but in middle and severe PIH groups, they were obviously higher(P＜0.01). The activities of IL-4, IL-10 from peripheral blood lymphocyte decreased in patients with PIH. In mild PIH group, the activities of IL-4, IL-10 were not lower(P＞0.05), but in middle and severe PIH groups, they were obviously lower than control group(P＜0.01). Compared with control group, the activities of IL-2, IFN-γfrom decidual tissue lymphocyte in mild PIH group were not higher(P＞0.05), but in middle and severe PIH groups, they were obviously higher(P＜0.01). The levels of IL-4 from decidual tissue lymphocyte in PIH group were lower than control group, but had no significant statistical difference(P＞0.05). Compared with control group, The levels of IL-10 from decidual tissue lymphocyte in mild PIH group were not lower(P＞0.05),but in middle and severe PIH groups, it were obviously lower(P＜0.01).4 The comparison of the activities of induced cytokines in peripheral blood monocyte and decidual macrophage: Compared with control group, the activities of cytokines induced from peripheral blood monocyte and decidual macrophage, such as TNF-α, IL-6, were not higher in mild PIH group (P＞0.05), but in middle and severe PIH groups were obviously higher(P＜0.01). 5 The correlation of cytokines between peripheral blo更多还原