Gadd45α对滋养细胞功能的调控及其在子痫前期发病中的作用机制研究

发布时间：2018-06-30 17:10

本文选题：Gadd45α + 绒毛外滋养细胞　；参考：《重庆医科大学》2014年博士论文

【摘要】：目的：检测生长阻滞和DNA损伤45α（Growth arrest and DNAdamage45alpha，Gadd45α）在正常早孕期绒毛组织、蜕膜组织的表达和定位；研究Gadd45α在调控人滋养细胞迁移和侵袭中的作用和可能的机制；采用缺氧复氧体外构建绒毛外滋养细胞的氧化应激损伤模型，探讨Gadd45α及其下游信号通路p38MAPK在滋养细胞功能损伤和子痫前期发病中的可能作用，进一步明确相关分子机制，为子痫前期的治疗提供新的靶点和理论依据。方法：（1）免疫组化法检测Gadd45α蛋白在人类早孕期绒毛和蜕膜组织中的表达和定位。(2)通过转染慢病毒颗粒，干扰绒毛外滋养细胞HTR8/SVneo和早孕绒毛外植体的Gadd45α基因表达。(3)采用PCR和western blotting检测干扰效率。(4)Transwell迁移实验和Matrigel侵袭实验检测干扰Gadd45α对HTR8/SVneo细胞迁移和侵袭能力的影响。(5)采用早孕绒毛外植体培养模型研究Gadd45α对绒毛外滋养细胞外生性迁移的影响。(6)明胶酶谱法检测外植体和细胞培养上清液中MMPs的活性，western blotting检测MMPs的组织抑制因子TIMPs的蛋白表达水平。(7)体外通过缺氧复氧构建绒毛外滋养细胞氧化应激损伤模型，检测细胞内活性氧的含量、细胞凋亡率，及绒毛外滋养细胞体外迁移、侵袭及管腔成型的能力及细胞培养上清液中MMPs的活性和sFlt-1sEng的分泌，并观察干扰Gadd45α基因及阻断其经典下游信号通路p38MAPK对缺氧/复氧所致的绒毛外滋养细胞生物学功能的影响，，进一步明确Gadd45α调控滋养细胞功能在子痫前期发病中的作用及可能机制。结果：（1）正常早孕期绒毛组织和蜕膜组织中均有Gadd45α蛋白表达，在绒毛组织主要定位于滋养细胞柱和合体滋养细胞；在蜕膜组织的腺上皮细胞和EVT中高表达，蜕膜间质细胞低表达。Gadd45α的表达在早孕期各孕周间无统计学差异。（2）敲减Gadd45α可以促进绒毛外滋养细胞迁移和侵袭能力，而对细胞增殖和凋亡无明显影响。（3）Gadd45α抑制滋养细胞的迁移和侵袭可能是通过降低MMP2和MMP9的活性，增加TIMP1和2的表达来实现的。（4）缺氧/复氧处理可以导致绒毛外滋养细胞迁移、侵袭和管腔形成能力下降，同时HTR8/SVneo细胞中Gadd45α的表达上调、p38MAPK磷酸化增加，伴有上清液中MMPs活性的下降及sFlt-1sEng的分泌增加，为研究Gadd45α参与子痫前期发病的机制研究提供了简便、可靠的体外模型。（5）转染慢病毒干扰Gadd45α的基因表达和p38MAPK特异性的抑制剂SB203580阻断p38信号通路均可以有效地促进缺氧/复氧损伤所致的绒毛外滋养细胞的体外迁移/侵袭和管腔成型能力，减少抗血管生成因子sFlt-1sEng的分泌，这可能是通过调控MMP2和MMP9的活性、减少氧化应激来实现的。结论：(1) Gadd45α作为一个负性调节因子在滋养细胞侵袭和早期胎盘发育过程中发挥着重要的作用，这可能是通过直接或间接地调控基质金属蛋白酶MMP2和MMP9的活性来实现的。(2)在绒毛外滋养细胞中存在着这样一条信号通路：氧化应激损伤—Gadd45α↑—p38MAPK磷酸化↑—滋养细胞功能损伤和sFlt-1sEng的分泌↑，证实了Gadd45α通过p38MAPK信号通路参与子痫前期胎盘形成过程中的滋养细胞侵袭和血管重铸。(3) Gadd45α的RNAi和p38MAPK特异性抑制剂SB253580可促进H/R下滋养细胞的迁移、侵袭能力及血管生成能力，从而改善子痫前期胎盘浅着床和螺旋动脉重铸异常情况，为子痫前期的治疗提供了新的思路。
[Abstract]:Objective: to detect the expression and localization of 45 alpha (Growth arrest and DNAdamage45alpha, Gadd45 a) in the villus and decidua in normal early pregnancy, and to study the role and possible mechanism of Gadd45 alpha in regulating the migration and invasion of human trophoblastic cells; the oxidation of oxygen deficient oxygen in vitro to construct the oxidation of villous trophoblast cells in vitro The stress damage model is used to explore the possible role of Gadd45 alpha and its downstream signal pathway p38MAPK in the functional damage of trophoblast and preeclampsia, and further clarify the molecular mechanism to provide new targets and theoretical basis for the treatment of preeclampsia.
Methods: (1) immunohistochemical method was used to detect the expression and localization of Gadd45 alpha protein in the villi and decidual tissues of human early pregnancy. (2) by transfecting lentivirus particles, interfering with Gadd45 alpha gene expression of HTR8/SVneo and early trimester villi explants. (3) PCR and Western blotting were used to detect interference efficiency. (4) Transwell migration test The effect of Gadd45 alpha on the migration and invasion ability of HTR8/SVneo cells. (5) the effect of Gadd45 alpha on the external migration of outer villous trophoblast cells was studied by early pregnancy villous explant culture. (6) the activity of MMPs in explants and cell culture supernatants was detected by gelatin zymogram, and Western blotting was used to detect MMPs. (5) Western blotting The protein expression level of the tissue inhibitory factor TIMPs. (7) to construct the oxidative stress damage model of the villous trophoblast by anoxic reoxygenation in vitro, to detect the content of intracellular reactive oxygen species, the rate of cell apoptosis, the in vitro migration of villous trophoblast cells, the energy of invasion and lumen molding and the activity of MMPs in the cell culture supernatant and sFlt-1sEng The effects of interfering Gadd45 alpha gene and blocking the classical downstream signal pathway p38MAPK on the biological function of the extracellular trophoblast induced by hypoxia / reoxygenation were observed, and the role of Gadd45 alpha to regulate the function of trophoblast in preeclampsia and its possible mechanism were further clarified.
Results: (1) the expression of Gadd45 alpha protein in the villi and decidua tissues in the normal early pregnancy, the villous tissue mainly located in the trophoblast column and the syncytial trophoblast, the high expression of the gland epithelial cells and EVT in the decidua tissue. The expression of the low expression of.Gadd45 a in the decidua stromal cells was not statistically different between the gestational weeks in the early pregnancy. (2) Reduction of Gadd45 a can promote the migration and invasion of villous trophoblast cells, but has no obvious effect on cell proliferation and apoptosis. (3) Gadd45 alpha inhibition of trophoblast migration and invasion may be achieved by reducing the activity of MMP2 and MMP9 and increasing the expression of TIMP1 and 2. (4) oxygen deficiency / reoxygenation can lead to the migration of villous trophoblast cells, The ability of invasion and lumen formation decreased, and the expression of Gadd45 alpha in HTR8/SVneo cells increased, p38MAPK phosphorylation increased, with the decrease of MMPs activity in the supernatant and the increase of sFlt-1sEng secretion, which provided a simple and reliable model for studying the mechanism of Gadd45 alpha involved in preeclampsia. (5) transfection of lentivirus to Gadd45 alpha Gene expression and p38MAPK specific inhibitor SB203580 blocking the p38 signaling pathway can effectively promote the migration / invasion and lumen formation of extracellular trophoblast induced by hypoxia / reoxygenation injury, and reduce the secretion of anti angiogenic factor sFlt-1sEng. This may be by regulating the activity of MMP2 and MMP9 and reducing the oxidative stress. Come true.
Conclusions: (1) Gadd45 alpha plays an important role in the invasion of trophoblast and early placental development as a negative regulatory factor. This may be achieved by direct or indirect regulation of the activity of matrix metalloproteinase MMP2 and MMP9. (2) there is such a signal pathway in the villous trophoblast cells: oxidation should Stimulated damage - the functional damage of Gadd45 alpha p38MAPK phosphorylated trophoblast and the secretion of sFlt-1sEng, confirmed that Gadd45 alpha participates in the invasion of trophoblast and vascular recasting in the process of placenta formation in preeclampsia through p38MAPK signaling pathway. (3) Gadd45 alpha RNAi and p38MAPK specific inhibitor SB253580 can promote the trophoblast of H/R Migration, invasiveness and angiogenesis can improve the abnormal condition of placental superficial implantation and spiral artery recasting in preeclampsia, and provide new ideas for the treatment of preeclampsia.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244

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