本文選題：Gadd45α + 絨毛外滋養(yǎng)細胞�。� 參考：《重慶醫(yī)科大學》2014年博士論文

【摘要】：目的：檢測生長阻滯和DNA損傷45α（Growth arrest and DNAdamage45alpha，Gadd45α）在正常早孕期絨毛組織、蛻膜組織的表達和定位；研究Gadd45α在調(diào)控人滋養(yǎng)細胞遷移和侵襲中的作用和可能的機制；采用缺氧復氧體外構(gòu)建絨毛外滋養(yǎng)細胞的氧化應(yīng)激損傷模型，探討Gadd45α及其下游信號通路p38MAPK在滋養(yǎng)細胞功能損傷和子癇前期發(fā)病中的可能作用，進一步明確相關(guān)分子機制，為子癇前期的治療提供新的靶點和理論依據(jù)。 方法：（1）免疫組化法檢測Gadd45α蛋白在人類早孕期絨毛和蛻膜組織中的表達和定位。(2)通過轉(zhuǎn)染慢病毒顆粒，干擾絨毛外滋養(yǎng)細胞HTR8/SVneo和早孕絨毛外植體的Gadd45α基因表達。(3)采用PCR和western blotting檢測干擾效率。(4)Transwell遷移實驗和Matrigel侵襲實驗檢測干擾Gadd45α對HTR8/SVneo細胞遷移和侵襲能力的影響。(5)采用早孕絨毛外植體培養(yǎng)模型研究Gadd45α對絨毛外滋養(yǎng)細胞外生性遷移的影響。(6)明膠酶譜法檢測外植體和細胞培養(yǎng)上清液中MMPs的活性，western blotting檢測MMPs的組織抑制因子TIMPs的蛋白表達水平。(7)體外通過缺氧復氧構(gòu)建絨毛外滋養(yǎng)細胞氧化應(yīng)激損傷模型，檢測細胞內(nèi)活性氧的含量、細胞凋亡率，及絨毛外滋養(yǎng)細胞體外遷移、侵襲及管腔成型的能力及細胞培養(yǎng)上清液中MMPs的活性和sFlt-1sEng的分泌，并觀察干擾Gadd45α基因及阻斷其經(jīng)典下游信號通路p38MAPK對缺氧/復氧所致的絨毛外滋養(yǎng)細胞生物學功能的影響，，進一步明確Gadd45α調(diào)控滋養(yǎng)細胞功能在子癇前期發(fā)病中的作用及可能機制。 結(jié)果：（1）正常早孕期絨毛組織和蛻膜組織中均有Gadd45α蛋白表達，在絨毛組織主要定位于滋養(yǎng)細胞柱和合體滋養(yǎng)細胞；在蛻膜組織的腺上皮細胞和EVT中高表達，蛻膜間質(zhì)細胞低表達。Gadd45α的表達在早孕期各孕周間無統(tǒng)計學差異。（2）敲減Gadd45α可以促進絨毛外滋養(yǎng)細胞遷移和侵襲能力，而對細胞增殖和凋亡無明顯影響。（3）Gadd45α抑制滋養(yǎng)細胞的遷移和侵襲可能是通過降低MMP2和MMP9的活性，增加TIMP1和2的表達來實現(xiàn)的。（4）缺氧/復氧處理可以導致絨毛外滋養(yǎng)細胞遷移、侵襲和管腔形成能力下降，同時HTR8/SVneo細胞中Gadd45α的表達上調(diào)、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，為研究Gadd45α參與子癇前期發(fā)病的機制研究提供了簡便、可靠的體外模型。（5）轉(zhuǎn)染慢病毒干擾Gadd45α的基因表達和p38MAPK特異性的抑制劑SB203580阻斷p38信號通路均可以有效地促進缺氧/復氧損傷所致的絨毛外滋養(yǎng)細胞的體外遷移/侵襲和管腔成型能力，減少抗血管生成因子sFlt-1sEng的分泌，這可能是通過調(diào)控MMP2和MMP9的活性、減少氧化應(yīng)激來實現(xiàn)的。 結(jié)論：(1) Gadd45α作為一個負性調(diào)節(jié)因子在滋養(yǎng)細胞侵襲和早期胎盤發(fā)育過程中發(fā)揮著重要的作用，這可能是通過直接或間接地調(diào)控基質(zhì)金屬蛋白酶MMP2和MMP9的活性來實現(xiàn)的。(2)在絨毛外滋養(yǎng)細胞中存在著這樣一條信號通路：氧化應(yīng)激損傷—Gadd45α↑—p38MAPK磷酸化↑—滋養(yǎng)細胞功能損傷和sFlt-1sEng的分泌↑，證實了Gadd45α通過p38MAPK信號通路參與子癇前期胎盤形成過程中的滋養(yǎng)細胞侵襲和血管重鑄。(3) Gadd45α的RNAi和p38MAPK特異性抑制劑SB253580可促進H/R下滋養(yǎng)細胞的遷移、侵襲能力及血管生成能力，從而改善子癇前期胎盤淺著床和螺旋動脈重鑄異常情況，為子癇前期的治療提供了新的思路。
[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.
【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】：2014
【分類號】：R714.244

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