本文選題：laminin　切入點：α　出處：《重慶醫(yī)科大學》2015年博士論文　論文類型：學位論文

【摘要】：目的：妊娠期高血壓疾病是妊娠期特有的全身系統(tǒng)性疾病,子癇前期(preeclampsia)是妊娠期高血壓疾病中最常見的類型,發(fā)病率約為5-10%,嚴重威脅母嬰健康。子癇前期的病因及發(fā)生發(fā)展過程還不清楚,但大多數(shù)學者認為,其發(fā)病原因主要與胎盤滋養(yǎng)細胞缺血、胎盤淺著床、血管內(nèi)皮細胞損傷和免疫失衡等機制相關(guān)。層黏連蛋白(laminin)是細胞基底膜重要的構(gòu)成成分,層黏連蛋白α4蛋白(LAMA4)可調(diào)控多種細胞的遷移、增殖和凋亡功能。但目前國內(nèi)外尚未見到關(guān)于LAMA4在滋養(yǎng)細胞中的功能的相關(guān)報道。因此,我們課題組的研究目的為：首先檢測在正常妊娠婦女早孕期絨毛組織、蛻膜組織、足月胎盤和子癇前期患者胎盤組織中,LAMA4的定位及表達情況；在人類絨毛外滋養(yǎng)細胞株HTR-8/Svneo和臍靜脈內(nèi)皮細胞株HUVEC中探究LAMA4的表達及對這兩種細胞生物學功能的作用和影響；采用缺氧/復氧處理,模擬子癇前期胎盤組織中的氧化應激損傷,結(jié)合p38 MAPK信號通路特異性抑制劑SB203580,探究LAMA4基因在MAPK信號通路中對滋養(yǎng)細胞和內(nèi)皮細胞的生物學功能的調(diào)控和在子癇前期發(fā)病中的可能的相關(guān)分子機制,為子癇前期的病因機制及診斷提供理論依據(jù)和新的思路。方法：1.通過免疫組織化學法檢測在人類妊娠不同時期絨毛、蛻膜和胎盤組織及子癇前期患者胎盤組織中LAMA4的表達位置。2.采用蛋白免疫印跡和teal time qRT-PCR對照研究正常足月胎盤組織和重度子癇前期胎盤組織中LAMA4表達差異。3.通過質(zhì)粒將LAMA4小分子干擾RNA (si-LAMA4)轉(zhuǎn)染人類絨毛外滋養(yǎng)細胞株HTR-8/Svneo細胞和臍靜脈內(nèi)皮細胞株HUVEC細胞,干擾HTR-8/SVneo細胞、HUVEC細胞和早孕絨毛外植體中的LAMA4蛋白量的表達,并采用western blotting、細胞免疫熒光和Real time qRT-PCR檢測并驗證干擾效率。4.干擾LAMA4的表達后,運用細胞遷移和侵襲實驗檢測HTR-8/SVneo細胞生物學功能的改變。5.干擾LAMA4的表達后,運用細胞遷移實驗和管腔成型實驗檢測HUVEC細胞生物學功能的改變。6.在體外早孕絨毛外植體培養(yǎng)模型中干擾LAMA4的表達,檢測絨毛外滋養(yǎng)細胞外生性遷移情況。7.干擾LAMA4蛋白表達后,檢測MMPs(MMP2和MMP9)及其特異性抑制因子TIMPs (TIMP1和TIMP2)的蛋白水平。8.模擬子癇前期氧化應激損傷,檢測缺氧／復氧損傷對HTR-8/SVneo細胞和HUVEC細胞生物學功能的影響及對LAMA4表達的調(diào)控。9.采用p38MAPK信號通路特異性抑制劑SB203580,觀察MAPK通路在缺氧／復氧條件下對HTR-8/SVneo細胞和HUVEC細胞生物學功能的調(diào)控,明確LAMA4基因參與調(diào)控滋養(yǎng)細胞和內(nèi)皮細胞的生物學功能及其在子癇前期發(fā)病中的作用及機制。結(jié)果：1.LAMA4在正常早孕期絨毛、蛻膜組織和正常足月胎盤中均有表達,主要定位于滋養(yǎng)層細胞及血管內(nèi)皮細胞；LAMA4蛋白在早孕期蛻膜組織的EVT細胞和蛻膜間質(zhì)細胞中高表達,然而,LAMA4蛋白在子癇前期胎盤組織中表達較低。2.干擾LAMA4蛋白的表達可抑制HTR-8/SVneo細胞和HUVEC細胞生物學功能,而對細胞增殖和凋亡功能無明顯影響。3.干擾LAMA4的表達可降低MMP2和MMP9的表達,增加TIMP1和2的表達。4.缺氧/復氧處理可導致HTR-8/SVneo細胞和HUVEC細胞生物學功能下降,并下調(diào)HTR-8/SVneo細胞及HUVEC細胞中LAMA4蛋白的表達。同時,缺氧/復氧處理可增強HTR-8/SVneo細胞及HUVEC細胞中MAPK通路發(fā)生磷酸化活化,伴隨MMP2和MMP9的表達下降,TIMP1和TIMP2的表達增加。結(jié)論：1.LAMA4基因參與調(diào)控整個妊娠期滋養(yǎng)細胞和內(nèi)皮細胞的生物學功能,其表達呈動態(tài)變化。2.LAMA4在子癇前期胎盤組織中的表達明顯低于正常孕晚期胎盤組織。3.LAMA4表達水平的下降可以抑制滋養(yǎng)細胞的侵襲和遷移能力,降低內(nèi)皮細胞遷移及管腔形成等生物學功能。4.LAMA4基因在滋養(yǎng)細胞及內(nèi)皮細胞中的功能受MAPK通路調(diào)控及氧化應激的影響,其表達的下調(diào)可能對子癇前期的發(fā)生發(fā)展過程具有促進作用。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R714.244

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