IL-33对母-胎界面滋养细胞生物学功能的调控作用

发布时间：2018-05-09 14:10

本文选题：母-胎界面 + IL-33　；参考：《复旦大学》2014年硕士论文

【摘要】：妊娠是胚胎(胎儿)在母体内生长发育的全过程,即从胚胎形成到胎儿及其附属物从母体排出的全过程。这是一个由多种因素参与精密调控的复杂生理过程。而这其中所蕴含的大自然赋予的科学真理值得深入研究。同时,母-胎界面的生物学研究对器官移植和肿瘤研究领域都有着重要的推动作用。母-胎界面主要由滋养细胞、蜕膜基质细胞、蜕膜腺上皮细胞以及免疫细胞等多种细胞及细胞外环境中的多种细胞因子共同构成。正常妊娠时母-胎界面以Th2型免疫占优势,形成免疫耐受的微环境,以利于同种异体的胚胎在子宫内生长发育。胎盘的形成是母-胎界面的重要生物学事件,其成功与否直接影响妊娠的结局。滋养细胞增殖与侵袭对于囊胚植入、胎盘形成,并建立适当的母胎关系至关重要。母-胎界面的滋养细胞是胚胎种植、胎盘形成过程中一种特殊的上皮细胞,具有独特的高增殖高侵袭的能力。滋养细胞首先黏附于蜕膜化的子宫内膜,而后侵入蜕膜基质,与母体子宫螺旋小动脉接触,继而侵入血管腔,沿着血管内皮细胞逆行,向蜕膜深部及子宫肌层的浅1/3-1/2浸润,分化并替代血管内皮细胞,形成胎盘的胎儿部分,最终建立母胎循环,促成血管低阻高流量的形态学变化,使大量血液供向胎儿,以确保胎儿生长发育的需要。以往研究发现滋养细胞增殖与侵袭障碍是子痫前期、胎儿宫内生长受限、自然流产等妊娠相关疾病的主要原因。相反,滋养细胞的增生和侵袭如果失去了限制,则表现出肿瘤细胞的特性,导致妊娠滋养细胞疾病的发生。滋养细胞侵入子宫蜕膜受母-胎界面微环境细胞与分子间相互作用的调控。正常情况下,滋养细胞极少发生无限增殖和远处转移,提示滋养细胞的黏附和侵袭可能直接或间接受到某些细胞和细胞间分子的调控,使黏附侵袭的促进和抑制维持在生理性动态平衡。因此,母-胎界面参与调控滋养细胞侵袭力的相关分子,直接影响胚泡的正常植入和生长。白细胞介素(interleukin, IL)是一类参与多种重要生物学功能调节的生物活性分子,参与细胞间信息的传递,激活免疫细胞,介导T、B淋巴细胞的活化、增殖与分化,非特异性的调节机体的免疫反应并在炎症反应中发挥重要作用。白细胞介素-33(IL-33)是IL-1家族的新成员,是炎症反应和免疫偏倚的重要调节因子之一,主要诱导Th2型免疫反应。IL-33与免疫性疾病、炎症性疾病、心血管疾病、生殖系统疾病等多种疾病的发生发展有关。IL-33可降低细胞与细胞之间钙粘蛋白的表达,促进肿瘤细胞的脱落、转移。本课题组前期研究已证实IL-33及其受体ST2在早孕绒毛组织及蜕膜组织中均有表达。并且研究证实IL-33可以通过NF-κB和ERK1/2信号通路上调CCL2/CCR2促进蜕膜基质细胞的增殖和侵袭功能。其他研究还发现胎盘的IL-33主要来自母-胎界面的巨噬细胞,通过旁分泌途径激活滋养细胞AKT和ERK1/2信号通路,促进滋养细胞的增殖,对胎盘的形成具有重要作用。而IL-33对滋养细胞的重要生物学功能——黏附和侵袭是否有影响,目前尚未见报道。进一步解析IL-33在母-胎界面的作用机制,将有助于阐明生理状态下胚泡植入和病理性滋养细胞疾病发生的机理,对治疗复发性流产、子痫前期、胎儿宫内生长受限及滋养细胞相关疾病等亦具有潜在的临床价值。本课题在以往研究的基础上,进一步关注IL-33对滋养细胞黏附和侵袭功能的影响,并解析IL-33在母-胎界面对滋养细胞生物学功能调控的可能机制。第一部分IL-33及其受体ST2在母-胎界面的表达目的：分析孕晚期IL-33及其受体ST2在母-胎界面的表达情况,了解IL-33及受体ST2在母-胎界面的表达特征。方法：选取2013年在复旦大学附属妇产科医院正规产检并足月妊娠行选择性剖宫产的正常孕妇8例。于剖宫产手术时收集新鲜胎盘组织。石蜡包埋切片。采用免疫组织化学技术,检测母-胎界面IL-33及其受体ST2的表达情况。结果：正常足月妊娠母-胎界面共表达细胞因子IL-33及其受体ST2。免疫组织化学结果显示正常足月妊娠分娩的胎盘组织中可见IL-33及ST2表达,IL-33主要表达于滋养细胞的细胞核中,ST2则主要表达于滋养细胞和基质细胞,以胞膜胞浆表达为主。结论：IL-33及其受体ST2共表达于母-胎界面。提示：IL-33/ST2轴在正常妊娠的胎盘形成及功能维持中可能发挥作用。第二部分IL-33对滋养细胞黏附和侵袭功能的影响目的：明确滋养细胞系细胞表面IL-33受体ST2表达情况；探讨IL-33对滋养细胞黏附功能和侵袭功能的调控作用。方法：采用流式细胞术检测滋养细胞系JEG-3、JAR、BeWo、HTR-8细胞表面ST2的表达情况。利用多基质黏附试剂盒检测不同浓度IL-33作用48小时后,滋养细胞对基质Fibronectin、Laminin Ⅰ、Fibrinogen、Collagen Ⅰ和CollagenⅣ的黏附能力的改变。通过建立Trans well体系,检测不同浓度IL-33作用48小时后滋养细胞的侵袭能力的改变。结果：流式细胞术检测结果显示JAR、BeWo、JEG-3和HTR8细胞均表达IL-33受体ST2,其中以BeWo细胞表达最高,而HTR8细胞最低。选择对子宫内膜黏附和侵袭能力最高的JEG-3细胞进行后续实验。黏附实验结果显示,外源性重组人IL-33(rhIL-33)作用JEG-3细胞48小时后,JEG-3细胞对基质Fibronectin、 Laminin Ⅰ、Collagen Ⅳ和Fibrinogen的黏附能力减弱,且差异具有统计学意义。IL-33对滋养细胞系JEG-3细胞与基质Collagen Ⅰ的黏附能力没有明显影响。侵袭结果显示,rhIL-33作用JEG-3细胞48小时后,JEG-3细胞对Matrigel的穿透能力下降即JEG-3细胞的侵袭力减弱。结论：外源性重组人IL-33明显抑制滋养细胞对基质Fibronectin、Laminin Ⅰ、 Fibrinogen和Collagen Ⅳ的黏附能力,并且抑制滋养细胞的侵袭功能。此部分结果提示在母-胎界面中,IL-33通过调节滋养细胞的黏附和侵袭能力,有利于控制滋养细胞对母体组织的侵入深度,防止过度侵袭,维持母-胎界面生理性动态平衡,有利于正常胎盘的形成,从而有助于正常妊娠的维持。第三部分IL-33对滋养细胞表面黏附分子表达的调节目的：探讨IL-33调节滋养细胞黏附能力的机制,即IL-33通过何种方式实现对滋养细胞黏附功能的调控。方法：以滋养细胞系JEG-3细胞为研究对象,重组人IL-33作用48小时后,采用流式细胞术分析JEG-3细胞表面黏附和侵袭相关分子(integrin α3β1、integrin α4β1、integrin α5β1、integrin α6β1、integrin αvβ3、E-cadherin、CD62L、CD44)的表达变化。结果：外源性重组人的IL-33降低滋养细胞系JEG-3细胞表面黏附侵袭相关分子的表达。外源性加入重组人IL-33培养JEG-3细胞48小时后,流式细胞术分析滋养细胞JEG-3细胞表面黏附侵袭相关分子表达的情况。结果显示,滋养细胞系JEG-3细胞表面高表达inte grin α3β1、integrin α5β1、integrin α6β1、E-cadherin,低表达integrin α4β1、CD62L,不表达CD44、integrin αvβ3分子。在细胞培养液中加入rhIL-33后,与对照组相比,实验组integrin α4β1、CD62L的表达下调,且差异具有统计学意义。其他分子表达变化无显著性差异。结论：IL-33通过下调滋养细胞表面的黏附侵袭相关分子的表达,实现对滋养细胞黏附和侵袭功能的调节。综上所述,本研究发现妊娠母-胎界面滋养细胞共表达IL-33及其受体ST2,提示IL-33/ST2可能通过自分泌或者旁分泌方式参与滋养细胞生物学功能的调节。以表达受体ST2且对子宫内膜黏附和侵袭力较强的滋养细胞系JEG-3细胞代替母-胎界面滋养细胞进行本研究。结果显示,IL-33可能通过下调细胞表面黏附侵袭相关分子integrin α4β1、CD62L的表达,从而抑制滋养细胞黏附和侵袭功能,参与滋养细胞生物学行为的精密调控。本研究为完善滋养细胞黏附和侵袭调控机制,及临床治疗反复自然流产、妊娠滋养细胞疾病等妊娠相关疾病提供了新策略与新思路。
[Abstract]:Pregnancy is the whole process of growth and development of the embryo (fetus) in the mother body, from embryo formation to the whole process of the fetus and its appendages discharged from the mother body. This is a complex physiological process involved in a variety of factors, and the scientific truth endowed by nature deserves in-depth study. Studies have played an important role in the field of organ transplantation and cancer research. The mother fetal interface is mainly composed of a variety of cells, such as trophoblastic cells, decidual stromal cells, decidual epithelial cells and immune cells, and a variety of cytokines in the extracellular environment. The mother fetal interface is dominated by Th2 type immunization during normal pregnancy. The microenvironment of immune tolerance is beneficial to the growth and development of allogenic embryos in the uterus. The formation of the placenta is an important biological event of the mother fetal interface. Its success directly affects the outcome of pregnancy. The proliferation and invasion of trophoblastic cells are essential to the implantation of blastocysts, the formation of placenta, and the establishment of a proper maternal fetal relationship. Trophoblast is a special epithelial cell in the process of embryo cultivation. It has a unique high proliferation and high invasion ability. The trophoblastic cells first adhered to the deciduated endometrium and then intruded into the decidua matrix, exposed to the uterine spiral arterioles of the mother's uterus, then intruded into the blood tube cavity, and followed the vascular endothelial cells retrograde to the decidua. The shallow 1/3-1/2 infiltration of the Ministry and the myometrium of the uterus, differentiating and replacing the vascular endothelial cells, forming the fetal part of the placenta, finally establishing the fetal cycle, contributing to the morphological changes of the low resistance and high flow of blood vessels, making a large amount of blood supply to the fetus to ensure the growth and development of the fetus. The main causes of pregnancy related diseases such as fetal intrauterine growth restriction and spontaneous abortion. On the contrary, if the proliferation and invasion of the trophoblastic cells are lost, it shows the characteristics of the tumor cells and leads to the occurrence of gestational trophoblastic diseases. Regulation. Under normal conditions, the trophoblastic cells rarely occur unlimited proliferation and distant metastasis, suggesting that the adhesion and invasion of trophoblast may directly or indirectly accept the regulation of certain cells and intercellular molecules, so that the promotion and inhibition of adhesion invasion are maintained in the physiological dynamic balance. Therefore, the mother fetal interface participates in the regulation of the invasiveness of trophoblast cells. Related molecules, directly affecting the normal implantation and growth of the blastocyst. Interleukin (IL) is a class of bioactive molecules involved in a variety of important biological functions. It participates in the transmission of information between cells, activates the immune cells, mediates the activation, proliferation and differentiation of T, B lymphocytes, and the nonspecific immune response to the body. Interleukin -33 (IL-33), a new member of the IL-1 family, is one of the important regulators of the inflammatory response and immune bias. The main inducement of the Th2 type immune response.IL-33 is related to the development of a variety of diseases such as immune diseases, inflammatory diseases, cardiovascular diseases, and biological diseases. Reducing the expression of cadherin between cells and cells and promoting the Exfoliative and metastasis of tumor cells. Previous studies have confirmed that IL-33 and its receptor ST2 are expressed in the villi and decidua tissues of early pregnancy. And it has been proved that IL-33 can promote the proliferation of decidual stromal cells through the NF- kappa B and ERK1/2 signaling pathways. Other studies have also found that the IL-33 of the placenta mainly comes from the macrophages of the mother fetal interface, activating the AKT and ERK1/2 signaling pathways through paracrine pathways, promoting the proliferation of trophoblastic cells and the important role of the placenta, and the important biological function of IL-33 to the trophoblastic cells: whether or not the adhesion and invasion of trophoblast are available. No reports have been reported at present. Further analysis of the mechanism of IL-33 in the mother fetal interface will help to elucidate the mechanism of blastocyst implantation and pathological trophoblastic disease in physiological state. It is also of potential clinical value for the treatment of recurrent abortion, preeclampsia, fetal intrauterine growth restriction and trophoblast related diseases. On the basis of previous studies, we further pay attention to the effect of IL-33 on the adhesion and invasion of trophoblastic cells, and analyze the possible mechanism of IL-33 in the control of the biological function of trophoblast in the mother fetal boundary. The first part of the expression of IL-33 and its receptor ST2 at the mother fetal interface: analysis of IL-33 and its receptor ST2 at the mother fetal interface in the late pregnancy To understand the expression of IL-33 and receptor ST2 at the mother fetal interface. Methods: 8 normal pregnant women who were selected in 2013 at the Department of Obstetrics and Gynecology, affiliated to the Fudan University, were selected for regular pregnancy and full term pregnancy. The fresh placenta tissue was collected during cesarean section. The paraffin embedded section was examined by immunohistochemistry. The expression of IL-33 and its receptor ST2 at the mother fetal interface. Results: the immunohistochemical results of the co expression of cytokine IL-33 and its receptor ST2. in the mother fetal interface of normal term pregnancy showed that the expression of IL-33 and ST2 was found in the placental tissue of normal term pregnancy, and IL-33 was mainly expressed in the nucleus of trophoblastic cells and ST2 was mainly expressed in the trophoblast. IL-33 and its receptor ST2 are co expressed in the mother fetal interface. It is suggested that the IL-33/ST2 axis may play a role in the formation and function maintenance of placenta in normal pregnancy. Second the effect of part IL-33 on the adhesion and invasion of trophoblastic cells: the clear cell line of the trophoblastic cell line The expression of IL-33 receptor ST2 and the regulation of IL-33 on the adhesion and invasion of trophoblastic cells. Methods: flow cytometry was used to detect the expression of ST2 in the trophoblastic line JEG-3, JAR, BeWo, HTR-8 cells. 48 hours after the detection of different concentration IL-33 activity by multi matrix adhesion kit, the trophoblastic cell to matrix Fibr. Changes in the adhesion of onectin, Laminin I, Fibrinogen, Collagen I and Collagen IV. By establishing a Trans well system, the invasion ability of the trophoblastic cells was detected after 48 hours of action of different concentrations of IL-33. Results: the results of flow cytometry showed that JAR, BeWo, JEG-3, and HTR8 cells were all expressed. The cell expression was the highest and the HTR8 cell was the lowest. The JEG-3 cells with the highest adhesion and invasion ability of the endometrium were selected for follow-up experiments. The adhesion test showed that the adhesion ability of JEG-3 cells to matrix Fibronectin, Laminin I, Collagen IV and Fibrinogen was weakened after 48 hours of exogenous recombinant human IL-33 (rhIL-33). The difference had statistical significance.IL-33 on the adhesion ability of the trophoblast line JEG-3 cells to the matrix Collagen I. The invasion results showed that the penetration ability of JEG-3 cells to Matrigel decreased after 48 hours of rhIL-33 action, that is, the invasion ability of JEG-3 cells was weakened. Cell adhesion to matrix Fibronectin, Laminin I, Fibrinogen and Collagen IV, and inhibition of the invasion function of trophoblastic cells. This part suggests that in the mother fetal interface, IL-33 can control the invasion depth of the trophoblastic cells by regulating the adhesion and invasion of trophoblastic cells, preventing excessive invasion and maintenance. The physiological dynamic balance of mother fetal interface is beneficial to the formation of normal placenta and helps to maintain normal pregnancy. Third part IL-33 regulates the expression of adhesion molecules on the surface of trophoblastic cells: the mechanism of IL-33 regulating the adhesion of trophoblastic cells, that is, how IL-33 regulates the adhesion function of trophoblastic cells. Methods: the expression changes of JEG-3 cell adhesion and invasion related molecules (integrin alpha 3 beta 1, integrin alpha 4 beta 1, integrin a 5 beta 1, integrin a 6 beta 1, integrin a V beta 3, E-cadherin, CD62L, CD44) were analyzed by flow cytometry after 48 hours of recombinant human IL-33. Group human IL-33 reduced the expression of adhesion and invasion related molecules on the surface of trophoblast cell line JEG-3. After exogenous IL-33 was added to JEG-3 cells for 48 hours, flow cytometry was used to analyze the expression of adhesion and invasion related molecules on the surface of trophoblast JEG-3 cells. The results showed that the surface of the trophoblastic line JEG-3 cells expressed inte grin on the surface of the cell line. Alpha 3 beta 1, integrin alpha 5 beta 1, integrin alpha 6 beta 1, E-cadherin, low expression of integrin alpha 4 beta 1, CD62L, did not express CD44, integrin alpha v beta 3. After adding rhIL-33 to the cell culture solution, the expression of integrin alpha 4 beta 1, CD62L was down, and the difference was of no significant difference between the other molecules. IL-33 can regulate the adhesion and invasion function of trophoblastic cells by lowering the expression of adhesion and invasion related molecules on the surface of trophoblastic cells. To sum up, this study found that the pregnancy mother fetal interface trophoblastic cells co expressed IL-33 and its receptor ST2, suggesting that IL-33/ST2 may participate in trophoblast through autocrine or paracrine ways. The regulation of physical function. This study was conducted to express receptor ST2 and to replace the mother fetal interface trophoblast cell line JEG-3 cells with strong endometrium adhesion and invasiveness. The results showed that IL-33 may inhibit the adhesion and invasion of trophoblastic cells by regulating the adhesion and invasion of integrin alpha 4 beta 1, the expression of CD62L. This study provides new strategies and new ideas for improving the regulation mechanism of trophoblast adhesion and invasion, and the clinical treatment of recurrent spontaneous abortion, gestational trophoblastic disease and other pregnancy related diseases.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714

【共引文献】