【作者】 罗健英；

【导师】 乔福元；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 博士

【摘要】 研究背景子痫前期(preeclampsia, PE)是人类妊娠所特有的多系统受累的疾病,其病因有多种理论学说。由于该病在临床有一定的发病率,且可发生严重并发症威胁母体和围生儿的生命,因此一直是病理产科防治的重点对象。虽然对其病因和预防的研究已取得一定的进展,但临床处理除了常规的解痉、降压、终止妊娠等方法外还未有最有效的预防和治疗手段。目前普遍公认其发病的中心点是胎盘“浅着床”。正常妊娠过程中子宫螺旋动脉被浸润的滋养细胞改建成高容量、低阻抗的血管以有利血流量的增加和满足胎儿营养的需要。然而滋养细胞重建子宫螺旋动脉是在孕期的什么时候发生?又是在孕期的什么时候终止?这个精确的时间还不是十分清楚,但总的研究表明是在孕早期和中期,一般认为是孕20周前。因此,妊娠20周前的滋养细胞浸润能力减弱致子宫螺旋动脉胎盘床血管重铸障碍可能是子痫前期发生的关键因素。近年来MMP9在滋养细胞侵入和胎盘血管重塑中的作用日益受到重视。实验证明MMP9的表达或激活是滋养细胞侵袭的关键和限速步骤；敲除MMP9基因的小鼠生殖可受到严重影响。内源性的TIMP1是MMP9的重要抑制剂。研究表明使用TIMP1和MMP9的特异性抗体能完全抑制细胞滋养细胞的迁移,因而认为MMP9／TIMP1在滋养细胞侵蚀过程中起重要作用。MMP9/TIMP1在滋养细胞的表达或调控与孕期胎盘氧含量密切相关。其调控失衡可能引起胎盘浅着床致子痫前期,但目前仅限于组织水平的研究。本实验模拟妊娠过程中细胞滋养细胞分别在早孕时生理性低氧和中孕时因血管内皮受损的病理缺氧状态下,从基因水平研究MMP9基因在滋养细胞方面的作用,同时探讨受损的血管内皮细胞对细胞滋养细胞侵袭力的影响,为子痫前期的病因研究以及在体外实验模型建立的探讨,提供新的思路。本研究内容包括三部分,现概述如下：第一部分SiRNA沉默MMP9基因对滋养细胞生物学行为影响的研究目的：利用siRNA抑制人细胞滋养细胞系MMP9的表达,从基因水平探讨MMP9对滋养细胞生物学行为的直接影响,探讨子痫前期胎盘“浅着床”与其之间的关系。方法：体外化学合成靶向MMP9的有效双链RNA(siRNA)与脂质体lipofectamineTM2000结合后,转染体外培养的人细胞滋养细胞系TEV-1, FQ-PCR和western blot分别观察MMP9mRNA和其蛋白的表达、MTT法检测滋养细胞的生长和TFranswell了解细胞侵袭力的变化。结果：(1) lipofectamine TM2000转染效率达81.53%(大于70%的标准)；(2) FQ-PCR显示MMP9mRNA的表达：转染组(1.29±0.31)明显低于正常细胞组(24.77±4.16); (3) western blot显示MMP9蛋白的表达：转染组(131.02±11.01)明显低于正常细胞组(305.93±34.31)；(4)MTT法检测转染组细胞生长在12h、24h、48h、72h均受到抑制；(5) Transwel1测定细胞侵袭力：转染组穿透Matrigel膜细胞数(16.2±5.2)明显少于正常细胞组(33.0±3.4)(p<0.01)。结论：MMP9基因受到si RNA干扰后可抑制滋养细胞的生长和侵袭力,增加滋养细胞MMP9的表达,可能对预防及治疗子痫前期有一定作用。第二部分缺氧对细胞滋养细胞MMP9/TIMP1基因表达及其侵袭力的影响目的：探讨缺氧对细胞滋养细胞分泌基质金属蛋白酶9(MMP9)及其抑制物1(TIMP1)的mRNA和二者的蛋白表达及其侵袭能力的影响。方法：将永生化的早孕细胞滋养细胞TEV-1进行化学缺氧(150μmol/L CoCL2),用免疫组化、荧光定量PCR、Western blot等方法检测正常和缺氧细胞滋养细胞系的MMP9/TIMP1基因的表达,应用Transwell侵袭模型检测缺氧对细胞滋养细胞侵袭能力的影响。结果：(1)免疫细胞化学染色显示, MMP9和TIMP1的阳性表达主要定位于细胞质,为淡黄色或棕黄色颗粒。缺氧组MMP9和TIMP1的蛋白PEI阳性表达分别为(1.15±0.15)、(1.12±0.11),而对照组的分别为(0.54±0.09)、(0.51±0.04),缺氧组与对照组比较均较高有明显差异(P<0.05)；(2)缺氧组的细胞滋养细胞分泌MMP9、TIMP1 mRNA,及二者的蛋白表达在24h、48h、72h均升高(P<0.05), MMP9/TIMP1比值升高,且与时间成正相关。(3)缺氧组的细胞滋养细胞侵袭力在24h、48h、72h较对照组的亦均增强(P<0.01)。结论：在生理性缺氧条件下,早孕细胞滋养细胞的侵袭力是增加的,并与MMP9/TIMP1比值的升高有关；同时TIMP1除了调节MMP9外,很可能还促进细胞滋养细胞的增殖与分化。第三部分缺氧共培养血管内皮细胞产生的FGF2对细胞滋养细胞MMP9、TIMP1的表达及其侵袭力的影响目的：探讨血管内皮细胞与细胞滋养细胞在缺氧共培养条件下,血管内皮细胞产生的FGF2对细胞滋养细胞MMP9、TIMP1的表达及其侵袭力的影响。方法：(1)细胞滋养细胞与血管内皮细胞用Transwell小室建立缺氧(150μmol/L CoCL2)共培养模型,应用荧光定量PCR、western-Blot等方法分别在缺氧共培养24h、48h、72h检测血管内皮细胞FGF2以及细胞滋养细胞MMP9、TIMP1基因的表达；(2)应用Transwell侵袭模型检测缺氧共培养72h下细胞滋养细胞侵袭力的影响。结果：(1)缺氧共培养组中上室的细胞滋养细胞在24h、48h、72h表达的MMP9mRNA分别为(2.659±0.028)、(1.962±0.041)、(1.171±0.196),对照组为(5.60±0.143)、(3.860±0.201)、(2.823±0.017)；其蛋白分别为(204.61±19.79)、(154.46±10.63)、(97.65±3.98),对照组为(390.19±22.66)、(294.13±8.46)、(238.58±4.71)；与对照组比较两者均明显降低(P<0.01)。(2)缺氧共培养组中上室的细胞滋养细胞在24h、48h、72h表达的TIMP1 mRNA分别为(1.304±0.061)、(1.867±0.120)、(2.501±0.128),对照组为(1.068±0.082)、(1.256±0.140)、(1.273±0.027)；其蛋白分别为(136.87±7.95)、(193.91±12.22)、(246.90±19.54),对照组为(107.35±2.86)、(117.76±2.73)、(121.11±1.37)；与对照组比较两者均明显明显上升(P<0.05)。(3)缺氧共培养组中下室的血管内皮细胞在24h、48h、72h表达的FGF2 mRNA分别为(2.588±0.128)、(2.809±0.016)、(3.550±0.294),对照组为(1.031±0.051)、(1.687±0.037)、(2.327±0.111)；其蛋白分别为(328.42±17.16)、(352.67±24.38)、(394.11±3.98),对照组为(125.40±2.93)、(201.58±3.65)、(270.32±2.76)；与对照组比较两者均明显明显上升(P<0.05)；且缺氧组MMP9/TIMP1的蛋白比值与缺氧组FGF2蛋白呈负相关(p<0.01)(4)缺氧共培养组穿透Matrigel膜滋养细胞数为(29.6±3.91)明显少于对照组(50.8±4.52) (P<0.01)。结论：血管内皮细胞与细胞滋养细胞缺氧共培养条件下,细胞滋养细胞表达MMP9下降、而TIMP1升高致细胞滋养细胞的侵袭力下降有关,且血管内皮细胞产生的FGF2可能参与了该调控,但具体调节机制还不清楚,需进一步探讨。更多还原

【Abstract】 Background Preeclampsia(PE) is a specific complication during the late pregnancy and the exact cause and pathogenesis of PE have not been clear out. A currently accepted hypothese is the placenta shallow implantation that the uterine spiral arteries remodeling has been obstacled by defective invasion of cytotrophoblasts. It might cause the ischemia and hypoxia of trophoblasts and increase local cellular immune response to placental oxidative stress with manifesting as lipid peroxidation and releasing of oxygen free radicals. Then released inflammatory factors and activated neutrophils directly or indirectly might cause maternal vascular endothelial damaged and accompany systemic small artery spasm, ultimately developing hypertension, proteinuria, edema, and eclampsia. Therefore, It may be a key factor in the pathogenesis of preeclampsia that defective invasion of cytotrophoblasts would hinder placental bed vascular remodeling.Placental angiogenesis and the physiological rebuilding of uterine spiral arteries depend on the proliferation and differentiation of cytotrophoblastes and the invasion of cytotrophoblastes to the uterine decidua and spiral arteries. The process includes the trophoblast adhesion, matrix degradation and cell migration and involves the related proteases, adhesion molecules, extracellular matrix and decidua inhibitory factor etc. The degradation of extracellular matrix is an important part of the process. Matrix metalloproteinase family (MMPs) is one of the most important group of proteases, which can degrade almost all of the matrix elements. Tissue inhibitor of metalloproteinases(TIMPs) is a natural inhibitor of MMPs and both of MMPs and TIMPs play an important role in the maintenance of ECM homeostasis and structural integrity. In recent years,MMP9 which is one of currently known species of more than 20 MMPs is paid more attention to the cytotrophoblastic invasion and placental vascular remodeling. Experiments have shown that TIMP1 and MMP9 specific antibodies can completely inhibit the invasion of trophoblast. Therefore, The balance of MMP9/TIMP1 is particularly important in the invasive processes of cytotrophoblast.The imbalance of MMP9/TIMP1 may lead to the placenta shallow implantation that might initiate preeclampsia, but the study is mainly in the level of the organization now. This experiment simulated the process of cytotrophoblast at the stage of early pregnancy when the pregnancy was physiological hypoxia condition and at the stage of late pregnancy when the damaged vascular endothelial was due to pathological hypoxia condition. We will further research the role of MMP9 gene in the level of the cell and also investigat the effect of the damaged vascular endothelial cells on the invasion of cytotrophoblasts.we hope to provide new ideas for a model in vitro of preeclampsia to study the pathogenesis.This study is divided into three parts as follows:Part I Inhibitory effects of siRNA-silencing MMP9 gene on CytotrophoblastObjective To confirm the relationship between mmp9 and plancenta-shallow implanation of preeclampsia by means of siRAN-silencing mmp9 gene that inhibit the invasion of cytotrophoblast. Methods Effective siRNA of mmp9 gene was synthesized in vitro.The siRNA was transfected into cytotrophoblast by lipofectamine TM 2000.Then mmp9mRNA and protein expressions in the transfected cytotrophoblast were detected by fluorescence quantitative reverse transcriptase polymerase chain reaction (FQ-PCR) and Western blotting. The biological effects of the cytotrophoblast were evaluated through the detection of their anchorage-in-dependent growth and in vitro invasion by cell migration assay and transwell chamber assay.Results The expression of mmp9 in transfected cytotrophoblasts was markedly depressed at both mRNA and protein levels as compared with the controlled group Anchorage-in-dependent growth assay showed the growth of transfected cells was retarded. Transwell chamber assay showed the invasion ability of transfected cells was inhibited significantly (p<0.01).Conclusion Effective siRNA can be successfully transfected into Cytotrophoblast and can effectively inhibit the expression of mmp9mRNA and protein. Therefore, the proliferation and invasion of transfected cells are inhibited. Increasing the expression of mmp9 may have a potential role in prevention and treatment of pregnancy-induced hypertension.PartⅡHypoxia Alters Matrix metalloproteinases9/Tissue inhibitor of metalloproteinases1 and promotes invasion of cytotrophoblast in VitroObjective To investigate the effection of Hypoxia on expression of matrix metallo-proteinases9/Tissue inhibitor of metalloproteinasesl and invasion activity in vitro cytotrophoblast.Methods immortalized human extravillous trophoblast cell line were induced in hypoxia circumstance by 150μmol/L CoCL2. Immunocytochemistry、fluorescence quantitative real-time PCR and Western blotting were used to determine the gene expression of MMP9/TIMP1 in cytotrophoblast. The invasion of hypoxia-induced cytotrophoblast were assessed by Transwell chamber assay.Results The mRNA of MMP9 and TIMP1 and the protein expression of both were up-regulated in hypoxia group(P<0.05),and the ratio of MMP9/TIMP1 was increased with prolonged time. The invasion of hypoxia group was more enhanced than the controlled group(P<0.01).Conclusion Physiological hypoxia could enhance cytotrophoblast invasion during early pregnancy with the increase of the ratio of MMP9/TIMP1,and TIMP1 may promote cytotrophoblast proliferation and differentiation besides regulating MMP9.PartⅢHypoxia induces FGF2 production by co-cultured vascular endothelial cells and alters MMP9 and TIMP1 expression in extravillous trophoblasts and their invasivenessObjective To investigate the role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during co-cultured hypoxic cytotrophoblast and the effection of FGF2 on expression of matrix metallo-proteinases9/Tissue inhibitor of metalloproteinasesl and invasion activity in vitro cytotrophoblast.Methods The human extravillous cytotrophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were co-cultured under normal and hypoxic(150μmol/L CoCL2)conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells was analyzed using fluorescence quantitative RT-PCR and Western blotting analyses. TEV-1 cell invasion was also examined by Transwell chamber assay.Results The mRNA and the protein expression of FGF2 in HUVE-12 cells co-cultured with TEV-1 cells was significantly increased under hypoxic conditions (P < 0.05). In TEV-1 cells co-cultured with HUVE-12, hypoxia reduced the mRNA and the protein expression of MMP9 (P<0.01)and increased the mRNA and the protein expression of TIMP1(P< 0.05); The invasion of co-cultured hypoxic group was more reduced than the controlled group(P<0.01). Conclusion FGF2 release by stressed endothelial cells of uterine spiral arteries might decrease MMP9 and increase TIMP1 production in extravillous cytotrophoblast in response to stress, resulting in reduced extravillous cytotrophoblast invasion and possibly shallow implantation of the placenta, but further study need be continued as the exact regulatory mechanism is unclear.更多还原