LAMA4基因在子痫前期发病机制中的功能研究

发布时间：2018-03-17 05:37

本文选题：laminin　切入点：α　出处：《重庆医科大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：妊娠期高血压疾病是妊娠期特有的全身系统性疾病,子痫前期(preeclampsia)是妊娠期高血压疾病中最常见的类型,发病率约为5-10%,严重威胁母婴健康。子痫前期的病因及发生发展过程还不清楚,但大多数学者认为,其发病原因主要与胎盘滋养细胞缺血、胎盘浅着床、血管内皮细胞损伤和免疫失衡等机制相关。层黏连蛋白(laminin)是细胞基底膜重要的构成成分,层黏连蛋白α4蛋白(LAMA4)可调控多种细胞的迁移、增殖和凋亡功能。但目前国内外尚未见到关于LAMA4在滋养细胞中的功能的相关报道。因此,我们课题组的研究目的为：首先检测在正常妊娠妇女早孕期绒毛组织、蜕膜组织、足月胎盘和子痫前期患者胎盘组织中,LAMA4的定位及表达情况；在人类绒毛外滋养细胞株HTR-8/Svneo和脐静脉内皮细胞株HUVEC中探究LAMA4的表达及对这两种细胞生物学功能的作用和影响；采用缺氧/复氧处理,模拟子痫前期胎盘组织中的氧化应激损伤,结合p38 MAPK信号通路特异性抑制剂SB203580,探究LAMA4基因在MAPK信号通路中对滋养细胞和内皮细胞的生物学功能的调控和在子痫前期发病中的可能的相关分子机制,为子痫前期的病因机制及诊断提供理论依据和新的思路。方法：1.通过免疫组织化学法检测在人类妊娠不同时期绒毛、蜕膜和胎盘组织及子痫前期患者胎盘组织中LAMA4的表达位置。2.采用蛋白免疫印迹和teal time qRT-PCR对照研究正常足月胎盘组织和重度子痫前期胎盘组织中LAMA4表达差异。3.通过质粒将LAMA4小分子干扰RNA (si-LAMA4)转染人类绒毛外滋养细胞株HTR-8/Svneo细胞和脐静脉内皮细胞株HUVEC细胞,干扰HTR-8/SVneo细胞、HUVEC细胞和早孕绒毛外植体中的LAMA4蛋白量的表达,并采用western blotting、细胞免疫荧光和Real time qRT-PCR检测并验证干扰效率。4.干扰LAMA4的表达后,运用细胞迁移和侵袭实验检测HTR-8/SVneo细胞生物学功能的改变。5.干扰LAMA4的表达后,运用细胞迁移实验和管腔成型实验检测HUVEC细胞生物学功能的改变。6.在体外早孕绒毛外植体培养模型中干扰LAMA4的表达,检测绒毛外滋养细胞外生性迁移情况。7.干扰LAMA4蛋白表达后,检测MMPs(MMP2和MMP9)及其特异性抑制因子TIMPs (TIMP1和TIMP2)的蛋白水平。8.模拟子痫前期氧化应激损伤,检测缺氧／复氧损伤对HTR-8/SVneo细胞和HUVEC细胞生物学功能的影响及对LAMA4表达的调控。9.采用p38MAPK信号通路特异性抑制剂SB203580,观察MAPK通路在缺氧／复氧条件下对HTR-8/SVneo细胞和HUVEC细胞生物学功能的调控,明确LAMA4基因参与调控滋养细胞和内皮细胞的生物学功能及其在子痫前期发病中的作用及机制。结果：1.LAMA4在正常早孕期绒毛、蜕膜组织和正常足月胎盘中均有表达,主要定位于滋养层细胞及血管内皮细胞；LAMA4蛋白在早孕期蜕膜组织的EVT细胞和蜕膜间质细胞中高表达,然而,LAMA4蛋白在子痫前期胎盘组织中表达较低。2.干扰LAMA4蛋白的表达可抑制HTR-8/SVneo细胞和HUVEC细胞生物学功能,而对细胞增殖和凋亡功能无明显影响。3.干扰LAMA4的表达可降低MMP2和MMP9的表达,增加TIMP1和2的表达。4.缺氧/复氧处理可导致HTR-8/SVneo细胞和HUVEC细胞生物学功能下降,并下调HTR-8/SVneo细胞及HUVEC细胞中LAMA4蛋白的表达。同时,缺氧/复氧处理可增强HTR-8/SVneo细胞及HUVEC细胞中MAPK通路发生磷酸化活化,伴随MMP2和MMP9的表达下降,TIMP1和TIMP2的表达增加。结论：1.LAMA4基因参与调控整个妊娠期滋养细胞和内皮细胞的生物学功能,其表达呈动态变化。2.LAMA4在子痫前期胎盘组织中的表达明显低于正常孕晚期胎盘组织。3.LAMA4表达水平的下降可以抑制滋养细胞的侵袭和迁移能力,降低内皮细胞迁移及管腔形成等生物学功能。4.LAMA4基因在滋养细胞及内皮细胞中的功能受MAPK通路调控及氧化应激的影响,其表达的下调可能对子痫前期的发生发展过程具有促进作用。
[Abstract]:Objective: hypertensive disorders in pregnancy is a systemic disease pregnancy specific, preeclampsia (preeclampsia) is the most common type of hypertensive disorders in pregnancy, the incidence rate is about 5-10%, a serious threat to the health of mother and infant. The process and cause of preeclampsia development is unclear, but most scholars believe that the main causes with placenta ischemia, shallow placental implantation, vascular endothelial cell injury and immune imbalance mechanism. Laminin (laminin) is an important component of cell basement membrane, laminin alpha 4 protein (LAMA4) can regulate a variety of cell migration, proliferation and apoptosis. But not at home and abroad see related reports about the function of LAMA4 in trophoblast cells. Therefore, our research objective is: first detected in the villi of early pregnancy women with normal pregnancy, gestational Decidua Tissue. And in the placenta of patients with preeclampsia, the expression of LAMA4 and localization; explore the expression of LAMA4 in human extravillous trophoblast cell line HTR-8/Svneo and human umbilical vein endothelial cell line HUVEC and on the two kinds of cell biology function and effect; the hypoxia / reoxygenation injury, oxidative stress simulation of preeclampsia placenta the combination of p38 MAPK signaling pathway inhibitor SB203580 and related molecular mechanism of regulation of LAMA4 gene on the biological function of trophoblast cells and endothelial cells in the MAPK signaling pathway and in the pathogenesis of preeclampsia may, to provide a theoretical basis and new ideas for the pathogenesis of preeclampsia and diagnosis. Methods: 1. by immunohistochemical staining in human villi during different periods of pregnancy, the expression of.2. LAMA4 in decidua and placenta position and in placenta in preeclampsia patients Western blot and teal time qRT-PCR control study of normal term placenta and preeclampsia placenta tissue LAMA4.3. expression of LAMA4 small interfering RNA (si-LAMA4) by plasmid transfection of human extravillous trophoblast cell line HTR-8/Svneo cells and human umbilical vein endothelial cell line HUVEC cells, the interference of HTR-8/SVneo cells, the expression level of LAMA4 protein in HUVEC cells and in villous explants, and the expression of Western blotting, cell immunofluorescence and Real time detection of qRT-PCR and verify the interference efficiency after LAMA4.4. interference, the change of expression of.5. by HTR-8/SVneo interference LAMA4 cell biology function to detect the invasion and migration of experimental cells after using HUVEC cell migration assay and detection of cell biological function the change of.6. lumen forming experiment in vitro villous explant culture expression LAMA4 interference model, inspection Measurement of extravillous trophoblast migration exogenous expression of.7. protein after LAMA4 interference, the detection of MMPs (MMP2 and MMP9) and its specific inhibitor TIMPs (TIMP1 and TIMP2) of the.8. protein level in simulated preeclampsia oxidative stress injury, detection hypooxide oxygen damage effect on regulation of.9. cells and HUVEC cells and the biological function of HTR-8/SVneo the expression of LAMA4 by p38MAPK signaling pathway inhibitor SB203580, observe the MAPK pathway in anoxia / reoxygenation conditions on the biological function of HTR-8/SVneo cell and HUVEC cell regulation, biological function of clear LAMA4 genes involved in the regulation of trophoblast and endothelial cells and its role in the pathogenesis of preeclampsia and its mechanism. Results: 1.LAMA4 in normal pregnancy during the period of villi, expressed in decidual tissues and normal term placenta, mainly located in trophoblast cells and vascular endothelial cells; LAMA4 protein In EVT cells and decidual tissues in early pregnancy decidual stromal cells in high expression, however, the expression of LAMA4 protein in placenta of pre eclampsia lower expression of.2. interference LAMA4 protein can inhibit the biological function of HTR-8/SVneo cells and HUVEC cells, while the expression of cell proliferation and apoptosis had no obvious effect of.3. interference of LAMA4 can reduce the expression of MMP2 and MMP9, increase TIMP1 and 2.4. expression of hypoxia / reoxygenation can lead to the biological function of HTR-8/SVneo cells and HUVEC cells decreased, and the expression of LAMA4 HTR-8/SVneo cells and HUVEC cells. At the same time, hypoxia / reoxygenation treatment can enhance the HTR-8/SVneo cells and HUVEC cells in the MAPK pathway phosphorylation activation, accompanied by the expression of MMP2 and MMP9 decreased, increased expression of TIMP1 and TIMP2. Conclusion: 1.LAMA4 gene is involved in the regulation of the whole period of trophoblast cells and endothelial cells of the biological function of the pregnancy. The expression is the migration and invasion of changes in the expression of.2.LAMA4 in placenta of pre eclampsia was significantly lower than that in normal placenta tissue.3.LAMA4 expression can inhibit the decline of trophoblast cells, reduce the migration and tube formation of endothelial cells of the biological function of.4.LAMA4 gene in trophoblast cell and endothelial cell function in regulated by oxidative stress and MAPK the pathway, down-regulation of pre eclampsia occurrence and development process of its expression has a role in promoting.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.244

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