【作者】 张冬丽；

【导师】 杨雪峰；

【作者基本信息】 郑州大学 ， 妇产科学， 2009， 硕士

【摘要】 复发性流产(recurrent spontaneous abortion,RSA)是指与同一性伴侣连续发生3次或3次以上自然流产,RSA病因复杂,现已明确的因素有:遗传基因缺陷、母体生殖结构畸形、感染、内分泌异常、环境理化与机械因素,免疫因素方面已明确的病因有父母血型不合、父母双方抗精子抗体的存在、母体抗父方淋巴细胞的细胞毒抗体不足、母体抗子宫内膜抗体以及孕妇抗磷脂抗体产生过多等。除此之外,约50%RSA患者不存在上述病因,称为原因不明复发性流产(URSA)。信号转导和转录激活因子(signal transducersand activators of transcription,STATs)是一类隐藏在胞浆中,能被众多的细胞外多肽类分子激活,参与基因转录调控的蛋白质家族。STAT6是该家族成员之一,它通过JAK-STAT信号转导途径介导细胞因子IL-4、IL-13等的基因表达,发挥多种生物学功能。当IL-4同膜受体(IL-4Rα)发生作用,引起酪氨酸系统(JAKs)的激活,进而激活胞浆内STAT6,引起该蛋白Y641位点上的酪氨酸残基磷酸化,激活的SH2区域发生同源或异源二聚化而形成二聚体;二聚体进入细胞核内与特定的DNA位点结合,引起IL-4相关基因的表达。STAT6激活后的多种生物学功能包括调节TH2优势应答等在RSA的发病中起重要作用。STAT4基因位于染色体2q32.2-q32.3,它是调节IL-12和T细胞分化的必不可少的因素,以往的研究表明IL-12介导STAT家族的激活,激活后的STAT4通过IL-12的信号通路调节Th1的细胞分化,如果STAT4表达下降,会明显抑制IL-12介导的Th1反应。敲除IL-12或STAT4导致TH1细胞反应显著降低,IFN-γ主要诱导Th1应答,抑制IL-4介导的Th2反应,在RSA的发病中同样起着重要的作用。STAT4、STAT6与Th1、Th1的平衡关系密切,STAT4可激活诱导Th1的极化及一系列的基因,实验发现野生型和STAT4缺陷小鼠表现Th2免疫应答优势,STAT6缺陷小鼠表现Th1免疫优势,因此STAT4途径是细胞因子调节免疫细胞向Th1分化的最主要的信号传导通路,而STAT6途径是细胞因子调节免疫细胞向Th2分化的最主要的信号传导通路。我们采用免疫组织化学方法研究RSA患者蜕膜和绒毛组织STAT4、STAT6的表达强度提供了方法学依据。STAT4和STAT6与Th1,Th2的关系研究较多,其在RSA患者蜕膜和绒毛组织中表达是否也存在这种关系?STAT4和STAT6的表达变化是否与RSA有关?二者是否存在相关性?国内尚未见报道。目的1.研究RSA患者蜕膜和绒毛组织中STAT4和STAT6表达情况。探讨蜕膜和绒毛组织STAT4和STAT6与Th1,Th2的关系,以及在RSA患者蜕膜、绒毛中的相关性。2.将STAT4和STAT6从信号转导通路方面导入RSA的发病机制研究领域。材料和方法1.研究对象及分组①RSA组选择2007年11月至2008年5月就诊于郑州大学第三附属医院妇科门诊妊娠12周以内行清宫术的URSA患者20例,年龄26—35岁,平均29岁,自然流产次数≥3(包括本次),现已妊娠,本次B超提示胚胎停止发育。排除染色体、解剖、内分泌及感染、自身免疫性疾病。②对照组:选择同期就诊于我院妇科门诊、妊娠12周以内要求人工流产的正常早期妊娠妇女20例,年龄24～35岁,平均28岁;既往无自然流产、死胎、死产史、无染色体、解剖、内分泌方面的异常及感染、自身免疫性疾病史,均有1胎以上正常分娩史,本次妊娠无阴道流血、腹痛等先兆流产症状和体征;B超检查证实胚胎发育正常。2.方法收集两组孕妇新鲜蜕膜和绒毛组织,经10%中性甲醛固定,石蜡包埋。采用免疫组化进行定性及半定量测定蜕膜和绒毛组织中STAT4和STAT6的表达情况。3.统计学分析所有数据均运用SPSS10.0软件进行统计学分析。α=0.05作为检验水准。结果1.研究对象一般情况比较:RSA组和对照组相比,年龄和孕周差异无统计学意义(p＞0.01).2.STAT4在两组绒毛组织中的表达:STAT4在两组绒毛组织均呈阳性表达,主要表达在绒毛滋养细胞的胞浆。RSA组表达水平高于对照组。(p＜0.01)3.STAT4在两组蜕膜组织中的表达:主要表达在蜕膜组织中间质细胞胞浆。STAT4在两组蜕膜组织中的表达水平均低于同组绒毛组织(p＜0.01)。4.STAT6在两组绒毛组织中的表达:STAT6主要表达在绒毛滋养细胞的胞浆。RSA组低于对照组表达水平。(p＜0.01)5.RSA组母胎界面上STAT4和STAT6的表达水平呈负相关。(r=-0.789,P＜0.01)结论1.RSA组绒毛和蜕膜组织中STAT4高表达,STAT6低表达,可能与RSA的发生有关。2.RSA组母胎界面上STAT4和STAT6的表达水平呈负相关,提示二者可能协同作用导致RSA的发生,为进一步从分子水平研究RSA的发病机制提供依据。更多还原

【Abstract】 Recurrent spontaneous abortion (RSA) ,partner with the identity refers to a succession of 3 or more than 3 spontaneous abortion, RSA complex etiology, there is now specific factors: genetic defects in the structure of maternal reproductive malformations、infections、endocrine abnormalities、environmental physico chemical and mechanical factors, immune factors already have specific causes of ABO Parent, both parents of the existence of anti-sperm antibodies, anti-mother-father antibody cytotoxic lymphocytes lack of maternal endometrial antibody and antiphospholipid antibodies in pregnant women, such as excessive. In addition, about 50% RSA patients with non-existence of these causes, referred to as unexplained recurrent spontaneous abortion (URSA)。Reproductive immunology Considers, pregnancy similar to allografts, the success of pregnancy depends on the pregnant women of fetal immune tolerance shown. Abortion allografts were considered failures. With the progress of the study of immunology, and gradually realize that embryos carrying 1 / 2 is different from the mother’s tissue antigens, but not maternal rejection of the substance of the immune system are dependent on relations between the maternal-fetal immune tolerance. Once this pattern of immune tolerance is broken, will lead to the occurrence of RSA.In the above-mentioned mechanism of immune tolerance "Th1/Th2 type cytokine balance" dominant doctrine. Normal pregnancy between Th1/Th2 type cytokines to maintain dynamic balance to the Th2-type cytokines dominated by the so-called "Th2 deviated from." Th1-type cytokines, mainly through the release of IL-2, IFN-γ, IFN-β, and TNF-αinhibiting T cell proliferation and IL-4 in T cell signal transduction and promoting cell-mediated immunity, involving in the occurrence of pathological pregnancy . Th2-type cytokines through the release of IL-4, IL-5, IL-10, IL-13 and other cytokines and chemokines and promotes the formation of IgE, mainly involved in humoral immunity, and protection of the role of nourishing the fetus. At the same time, many cells in the body and influence the interaction between factors, a common intervention and adjustment of the ratio of Thl/Th2 Change.The imbalance between Th1 and Th2 recurrent spontaneous abortion are an important feature. To explore the same precursor cells divide to different subtypes will help clarify the molecular mechanism of the nature of recurrent spontaneous abortion. Extracellular signaling molecules, intracellular signaling, nuclear transcription factor-control aspect of the three T cell differentiation, including transcription factor T-bet, GATA-3, extracellular signaling molecules IL-12, IL-4 and intracellular signaling molecule STAT4, STAT6 plays a key role.Th1 and Th2 cells are from a common naive precursor cells from. What mechanisms cause the same precursor cells divide to different types of people attracted great interest, making the impact of T cell differentiation factors and intracellular signal transduction pathway has become the research hot spots. Therefore, to explore the mechanism of T cell differentiation clear recurrent spontaneous abortion for the pathogenesis and to explore new intervention on the treatment aspect of great significance。Th2 differentiation, connected with the extracellular cytokine transcription factor Th differentiation between the intracellular signal transduction, differentiation induced by Th2 cytokines are the key IL-4, it is to play a role through the STAT6. Th1 differentiation induced by cytokines are the key IL-12, it is through the role of STAT4.Signal transducer and activator of transcription factor are a class of hidden in the cytoplasm, can be a large number of activated extracellular peptide molecules involved in regulation of gene transcription protein family. STAT6 is a member of the family, through the JAK-STAT signal transduction pathway mediated by cytokines IL-4, IL-13 gene expression, etc., play a variety of biological functions. When IL-4 with the membrane receptor (IL-4Rα) effect, caused by tyrosine System (JAKs) activation, and activation of cytoplasmic STAT6, caused by the Y641 site on protein tyrosine phosphorylation of residues, activation of the SH2 region homologous or heterologous dimerization and the formation of dimers; dimer into the nucleus with a specific DNA binding sites, caused by IL-4 gene expression. STAT6 activation of a variety of biological functions, including regulation of TH2 response, such as advantages in the pathogenesis of RSA play an important role. STAT4 gene located on chromosome 2q32.2-q32.3, it is the regulation of IL-12 and T cell differentiation of the essential factors, previous studies have shown that IL-12 mediated activation of STAT family, through the activation of STAT4 after IL-12 of the signaling pathway regulating Th1 cell differentiation, if the decline in the expression of STAT4, will significantly inhibited IL-12-mediated Th1 response. IL-12 knockout or TH1 cells STAT4 cause a significant reduction in the response, IFN-γinduction of Th1 response mainly inhibit IL-4-mediated Th2 response, in the pathogenesis of RSA play an important role in same.STAT4、STAT6 and Th1、Th1 closely related to the balance , Can be induced STAT4 activation and Th1 polarization of a series of genes, the experiment found that wild-type and STAT4-deficient mice demonstrated the advantages Th2 immune response, STAT6-deficient mice show Th1 immune advantage through STAT4 cytokine regulation of immune cells to Th1 differentiation The most important signal transduction pathways, and STAT6 through cytokine regulation of immune cells to the Th2 differentiation of the most important signal transduction pathway.The use of our method of imrnunohistochemistry in patients with RSA decidual and villous tissues of STAT4, STAT6 expression method provides a basis for study. STAT6 and STAT4 and Th1, Th2 study the relationship between the more RSA patients in the decidua and chorionic villi tissues whether such a relationship exist? The expression of STAT4 and STAT6 with RSA on the Change? Whether or not there is correlation between the two? Home has not been reported.Purpose 1. Research RSA patients decidual and villous tissues the expression of STAT4 and STAT6. Explore the decidual and villous STAT6 and STAT4 and Th1, Th2, as well as in the decidua of patients with RSA, the relevance of villi.2. STAT4 and STAT6 signal transduction pathway from the aspects of the pathogenesis of RSA into fields of study.Materials and methods1.①RSA study group Choice 2007年11 to 2008 in the 5 month treatment Third Affiliated Hospital of Zhengzhou University gynecology outpatient visit within 12 weeks of pregnancy Qinggong URSA patients 20 cases of patients, aged 26-35 years old, average 29-year-old, natural Abortion number≥2, the B-prompt cessation of embryonic development. Exclude chromosomal, anatomical, endocrine, and infections, autoimmune diseases.②normal pregnancy group: select the same period of treatment in our hospital out-patient gynecology, pregnancy within 12 weeks of the normal requirements of the early abortion 20 cases of pregnant women, aged 24～35 years old, average 28-year-old; past without spontaneous abortion, stillbirth, stillbirth history No chromosomal, anatomical, endocrine abnormalities and infection area, the history of autoimmune diseases, there is one child more than the history of normal birth, this pregnancy without vaginal bleeding, abdominal pain and other symptoms and signs of threatened abortion; B ultrasonography confirmed normal embryonic development.2. Ways to collect two sets of maternal decidua and villus fresh, with 10% neutral formalin-fixed, paraffin-embedded. By immunohistochemistry for qualitative and semi-quantitative measurement of decidual and villous tissues of STAT4 and STAT6 expression.3. All data were statistically analyzed using statistical analysis SPSS10.0 software. A = 0.05 as there is no significant difference test. Results1. Study the general situation is: RSA group and the control group, gestational age and there was no significant difference (p> 0.01).2. STAT4 villi in the two tissues: STAT4 in the two groups showed a positive expression of villi, the main expression in the cytoplasm of trophoblast cells. Higher than the expression level of RSA group. (P <0.01)3. STAT4 in the two decidual tissues: The expression of quality in the middle of decidual cells. STAT4 in the two decidual tissues lower than the same group villi (p4. STAT6 villi in the two tissues: STAT6 main expression in the cytoplasm of trophoblast cells. The expression level of the control group than the RSA group. (P5. STAT6 in the two decidual tissues: The expression in the middle of decidual stromal cells. STAT6 in the two decidual tissues lower than the same group villi (p6. RSA group of maternal-fetal interface of STAT4 and STAT6 expression level was negatively correlated. (R =- 0.789, P <0.01)Conclusion1. RSA Group villi and decidua in STAT4 expression, STAT6 decreased expression may be related to the occurrence of RSA.2. RSA group on maternal-fetal interface of STAT4 and STAT6 expression level was negatively correlated, suggesting possible synergy between the two cause the occurrence of RSA in order to further study the molecular pathogenesis of RSA to provide a basis.更多还原