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FADD、Livin在稽留流产绒毛组织中的表达及其意义

FADD、Livin in the Missed Abortion Villi Tissue Expression and Its Significance

作者: 专业:妇产科学 导师:杨玲竹 年度:2010 学位:硕士  院校: 郑州大学

Keywords

missed abortion, FADD, Livin, Immunohistochemistry, RT-PCR

        背景与目的稽留流产(missed abortion)又称过期流产。若死亡的妊娠物在宫腔内滞留过久,,可发生严重的凝血功能障碍。且胚胎死亡后滞留宫腔,易于子宫肌壁粘连,清宫困难。发生稽留流产的原因较为复杂。本研究主要是检测Fas相关死亡结构域蛋白(Fas-associated protein with death domain, FADD), Livin是凋亡抑制蛋白家族(inhibitor of apoptosis proteins, IAPs)中的成员,两者在稽留流产患者绒毛组织中的表达情况,以研究FADD、Livin与稽留流产的关系。FADD是Chinnaiyan等克隆出的一种能与Fas相互作用而诱导细胞凋亡的蛋白,是凋亡通路上的关键信号转导因子。FADD是公认的凋亡基因位于人11q13.3,其主要的凋亡传导途径为:Fas触发凋亡是通过FADD来募集并激活caspase-8来实现的。细胞受Fas系统作用后,FADD蛋白移至质膜,FADD的死亡结构域(death domains, DD)与死亡受体Fas的DD部位结合,进而FADD与caspase-8前体的死亡效应结构域(death effector domains, DED)结合。Caspase-8前体经过加工,以活性形式从死亡诱导信号复合体(death-inducing signaling complex, DISC)中释放出来,进而使其他效应子caspase (caspase-3、6、7等)裂解、激活,激活caspase级联反应,分解细胞蛋白,触发细胞凋亡。对FADD进行了深入的研究后,发现FADD不仅在凋亡信号传导通路中起重要作用,而且在胚胎的发育和免疫系统等方面也有一定的作用。Livin是2000年被发现的,它属于抑制细胞凋亡蛋白(inhibitor of apoptosis protein, IAP)家族的新成员。杆状病毒IAP重复序列(baculovirus IAP repeat,BIR)结构是IAPs蛋白家族发挥抗凋亡作用的关键,而BIR功能区可与半胱天冬酶(caspase)结合。Livin能有效阻断由FADD等多种促凋亡因子诱导的细胞凋亡。它阻断凋亡受体和以线粒体为基础的凋亡途径是通过抑制caspase的活性,尤其是caspase-3/-7和caspase-9的活性达到的。目前研究表明Livin主要在胎儿发育过程中多种组织高表达,如胎儿的脑、胸腺、胎盘、肾和肝,而正常成年组织未见表达(除胎盘)。Hakhyun等采用RT-PCR技术检测到LivinmRNA在胎盘的滋养层细胞中有表达,主要在在绒毛膜细胞滋养层的细胞浆中有表达,在合体滋养层细胞中表达较低,在胚胎发育中起调控作用。本研究采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法和免疫组化SP法,检测稽留流产绒毛组织及正常早孕绒毛组织中FADD与Livin的表达情况,分析FADD与Livin与稽留流产的关系,及两者之间的相关性。探讨两者在稽留流产发生发展中的作用,进一步揭示稽留流产可能的分子生物学发病机制。材料与方法1临床病例资料标本选自2008年7月至2008年11月在郑州大学第一附属医院和郑州大学第三附属医院妇产科的稽留流产患者30例作为实验组,稽留流产诊断标准为:以第7版妇产科教材为主。以同期无合并症的健康人群行人工流产的早孕妇女30例为对照组。所有研究对象无合并症,无全身感染性疾病,月经周期规律,流产前无用药史。两组样本的基本临床资料如年龄、孕周数无统计学意义。均为早期流产(孕周<12周),平均年龄27.6岁(23-37岁)。2实验方法运用RT-PCR方法从基因水平分别检测正常妊娠与稽留流产患者胎盘绒毛组织中FADDmRNA及Livin mRNA的表达。应用免疫组化SP法从蛋白水平分别检测正常妊娠与稽留流产患者胎盘绒毛组织中FADD蛋白及Livin蛋白的表达。3统计学分析采用SPSS13.0统计软件分析,计数资料进行χ2检验和Fisher精确概率检验。计量资料进行t检验。以α=0.05为检验水准。结果免疫组化结果发现FADD蛋白在稽留流产组绒毛组织中表达的阳性率为86.67%(26/30),其在正常妊娠组绒毛组织中表达的阳性率为60.0%(18/30)。FADD蛋白的表达稽留流产组高于正常妊娠组,差异具有统计学意义(χ2=5.455,P=0.020)。而Livin蛋白在稽留流产组绒毛组织中表达的阳性率为63.33%(19/30),其在正常妊娠组绒毛组织中表达的阳性率为90.0%(27/30)。Livin蛋白的表达稽留流产组低于正常妊娠组,差异具有统计学意义(χ2=5.963,P=0.015)。RT-PCR结果为稽留流产组绒毛组织中FADDmRNA表达高于正常早孕组(t=-7.220,P=0.002),而稽留流产组绒毛中LivinmRNA的表达则低于正常早孕组(t=10.661,P=0.000)。而进行FADD、Livin在稽留流产组胎盘滋养细胞中表达的相关性比较后发现,两者之间不存在关联性。结论1 FADD蛋白及FADDmRNA在稽留流产组绒毛组织中的表达显著高于正常早孕组,提示其高表达在稽留流产的发生发展中可能有一定作用。2 Livin蛋白及LivinmRNA在稽留流产组绒毛组织中的表达显著低于正常早孕组,提示其低表达与稽留流产关系密切。3 FADD在稽留流产胎盘滋养细胞中的高表达和Livin在稽留流产胎盘滋养细胞中的低表达,提示其可能参与稽留流产的发生与发展。可望为稽留流产的预防、基因诊断和治疗提供新的切入点。
    Background and ObjectiveMissed abortion is a special case of abortion, refers to an embryo or fetus died stranded intrauterine failed to natural discharges. If the dead materials in intrauterine pregnancy stay too long, they can cause severe coagulation dysfunction. Intrauterine fetal death and retention is easy to adhersed with uterine muscle wall, and they can not be cleared easily. The reason for missed abortion is not clear. This study was detected two materials expression in missed abortion tissues:Fas-associated protein with death domain (FADD) and Livin (a member of the inhibitor of apoptosis proteins family, IAPs). To explore the relationships of FADD, Livin and missed abortion.FADD was cloned by Chinnaiyan, etal. It is a Fas-induced apoptosis protein and a critical signal transduction factor in the apoptosis pathway. FADD is recongnized as apoptosis gene in the human 11q13.3.Its main pathway:Fas triggers apoptosis through FADD, and caspase-8 is raised and activated. After the effects of the Fas system, FADD protein moves to the plasma membrane. FADD’s death domain of Fas death receptor DD bines the DD part of integration, then FADD bines with the caspase-8 precursors of the death effector domain (DED). Active Caspase-8 precursor is released from the DISC, then other effectors caspase (caspase-3\cspase-6\caspase-7, etal) are cleavaged and activated. Lastly the caspase cascade is activated, and cells apoptosis occurs. After Pairs of FADD in-depth studys, FADD plays impotant role not only in apoptosis signal transduction pathway, but also in embryonic development and immune system.Livin was found in 2000, it belongs to inhibitor of apoptosis proteins (inhibitor of apoptosis protein, IAP) family. It is a new member of BIR proteins family structure and IAPs play a critical role in anti-apoptosis. BIR domains can bind with caspase enzymes. Livin can blockd apaptosis induced by FADD, etal. It blocks apoptosis receptors and the mitochondriabased apoptotic pathway by inhibiting caspase activity, especially caspase-3/-7 and caspase-9.Current research shows that Livin expresses in variety of organizations of fetal development, such as the fetal brain, thymus, placenta, kidney and liver, while no expression in normal adult organizations (except the placenta). Hakhyun uses RT-PCR technique to detect Livin mRNA in placental trophoblast cells to express. They found that expressed mainly in the chorionic trophoblast cells of the cytoplasm. It played a regulatory role in embryonic development.Using RT-PCR method and SP immunohistochemistry, To detect the expressions of FADD and Livin in missed abortion and normal pregnancy chorionic villi. Analysis their relationships, and the correlation with missed abortion, further to reveal the possible missed abortion molecular pathogenesis.Materials and Methods1 Clinical cases dateSamples take from July 2008 to November 2008 in the First Affiliated Hospital of Zhengzhou University and the Third Affiliated Hospital of Zhengzhou University, Obstetrics and Gynecology.30 patients with missed abortion are research objects (experimental group). Missed abortion diagnostic criteria are referenced from the seventh edition textbook to obstetrics and gynecology. Compared group are 30 patients requested termination of pregnancy. All the subjects have no pregnancy complications or infectious diseases. They have normal menstruation.The former did not use any steroid hormone drugs. Two groups of pregnant women’s age and the number of days of pregnancy have no statistically significant differences.They are all early abortions (gestational age<12weeks), with an average age of 27.6 years (23-37years).2 Experimental methodsFADDmRNA and LivinmRNA was detected of the placental villi in patients with normal pregnancy and missed abortion using RT-PCR method. Livin protein and’ FADD protein were detected in the placental villi of patients with normal pregnancy and missed abortion by immunohistochemical.3 Statistical analysisAll datas are analysised by SPSS13.0 statistical software. Analysis of attribute dates comparison used contingency table chi-square test and Fisher’s exact test.The statistical methods of measurement dates used the t-test. The test standard wasα=0.05ResultsImmunohistochemistry results:FADD protein in missed abortion group Chorion positive rate was 86.67%(26/30),60.0%(18/30) in normal chorionic villi. The difference was significant. (χ2=5.455, P=0.020). Livin protein in missed abortion villi, the positive rate was 63.33%(19/30), and 90.0%(27/30) in normal villi. The difference was significant (χ2=5.963, P=0.015)RT-PCR results:In missed abortion group villi FADD mRNA expression is higher than the normal pregnancy group, (t=7.220, P=0.002). And the missed abortion group Livin mRNA expression villi was lower than the normal group (t=10.661,P=0.000).FADD and Livin in the missed abortion group placenta expression has no correlation.Conclusions 1 FADD protein and FADDmRNA in villi of the missed abortion group was significantly higher than in normal pregnancy group. FADD may play a role in the development of missed abortion.2 Livin protein and Livin mRNA in villi of missed abortion group was significantly lower in early pregnancy. This indicated that low expression of Livin may have close relationship with missed abortion. 3 FADD in missed abortion placenta and the high expression of Livin in the missed abortion placenta low expression may involve in the occurrence and development of missed abortion. The two factors are probably expected as new entry points in missed abortion prevention, gene diagnosis and treatment.
        

FADD、Livin在稽留流产绒毛组织中的表达及其意义

摘要4-7
Abstract7-10
英文缩略语表12-14
正文部分 FADD、Livin在稽留流产绒毛组织中的表达及其意义14-41
    引言14-16
    材料与方法16-25
    结果25-30
    讨论30-35
    结论35-36
    参考文献36-39
    附图39-41
综述部分 FADD与Livin及凋亡相关因子与稽留流产关系的研究进展41-58
    参考文献53-58
附录部分58-60
    个人简历、硕士在读期间发表的论文58-59
    致谢59-60
        下载全文需50


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