【摘要】：第一部分SGK1在早期流產(chǎn)子宮蛻膜組織中的表達(dá)改變目的:通過(guò)測(cè)定妊娠早期蛻膜組織中血清和糖皮質(zhì)激素調(diào)節(jié)蛋白激酶1(SGK1)的表達(dá),比較早期自然流產(chǎn)和正常妊娠蛻膜組織中SGK1表達(dá)的差異,探討SGK1在妊娠早期母胎界面微環(huán)境中的生物學(xué)意義。材料及方法:1.選擇妊娠早期不明原因自然流產(chǎn)者88例作為實(shí)驗(yàn)研究組,同期71例的正常早期妊娠者作為對(duì)照組。2.免疫組織化學(xué)法及免疫熒光技術(shù)檢測(cè)子宮蛻膜組織中SGK1的表達(dá)。3.TRIZOL法抽提子宮蛻膜組織的總RNA,逆轉(zhuǎn)錄后Real time PCR測(cè)定兩組子宮蛻膜組織中SGK1 mRNA相對(duì)表達(dá)量。4.用含蛋白酶抑制劑的RIPA蛋白質(zhì)裂解液提取人子宮蛻膜組織蛋白質(zhì),采用BCA法進(jìn)行組織蛋白定量,Western blot技術(shù)進(jìn)行檢測(cè)人子宮蛻膜組織中SGK1蛋白質(zhì)相對(duì)表達(dá)量。結(jié)果:1.早期自然流產(chǎn)組和正常妊娠組在年齡、妊娠周數(shù)、初潮年齡、月經(jīng)周期、行經(jīng)天數(shù)等方面無(wú)顯著差異。早期自然流產(chǎn)患者血清雌二醇(E2)的水平低于正常妊娠者,差異有統(tǒng)計(jì)學(xué)意義(p0.001)。2.免疫組織化學(xué)法及免疫熒光發(fā)現(xiàn)SGK1在早期自然流產(chǎn)患者和正常妊娠者的子宮蛻膜組織中均有表達(dá)。3.早期自然流產(chǎn)患者蛻膜組織中的SGK1的mRNA水平較早期正常妊娠者蛻膜組織中的表達(dá)下降,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。4.早期自然流產(chǎn)患者子宮蛻膜組織中SGK1總的蛋白質(zhì)表達(dá)低于正常妊娠者,差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。并且,早期自然流產(chǎn)患者子宮蛻膜組織中SGK1磷酸化的蛋白表達(dá)低于正常妊娠者,差異具有統(tǒng)計(jì)學(xué)意義(P0.001),這與SGK1總蛋白在兩組之間的表達(dá)趨勢(shì)相一致。5.早期自然流產(chǎn)患者子宮蛻膜組織中SGK1的磷酸化蛋白與總蛋白的比值低于正常妊娠組,差異具有統(tǒng)計(jì)學(xué)意義(p0.001)。結(jié)論:1.SGK1在早期妊娠子宮蛻膜組織中表達(dá)。2.早期自然流產(chǎn)患者蛻膜組織中的SGK1的mRNA水平、總蛋白以及活化的蛋白表達(dá)均下降。第二部分SGK1的下調(diào)與早期自然流產(chǎn)發(fā)生相關(guān)機(jī)理研究目的:通過(guò)建立體外子宮蛻膜細(xì)胞,以蛻膜細(xì)胞中SGK1蛋白活性的變化為著眼點(diǎn),進(jìn)一步探究早期自然流產(chǎn)的發(fā)生機(jī)理,為病理妊娠發(fā)病機(jī)制及防治的研究提供新的思路和途徑。材料及方法:1.培養(yǎng)體外子宮蛻膜細(xì)胞,用免疫熒光法鑒定其純度。2.MTT比色試驗(yàn)檢測(cè)蛻膜細(xì)胞增殖活性,Annexin V-FITC/PI檢測(cè)蛻膜細(xì)胞凋亡。3.靶向性SGK1 siRNA干擾研究SGK1對(duì)蛻膜細(xì)胞凋亡的影響。4.脂多糖處理蛻膜細(xì)胞建立自然流產(chǎn)的體外細(xì)胞模型。5.酶聯(lián)免疫吸附測(cè)定蛻膜細(xì)胞上清中的細(xì)胞因子的含量。結(jié)果:1.靶向性SGK1 siRNA轉(zhuǎn)染24小時(shí)后的子宮蛻膜細(xì)胞的凋亡高于對(duì)照組蛻膜細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。2.經(jīng)雌二醇刺激蛻膜細(xì)胞中SGK1的表達(dá)高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。3.SGK1經(jīng)雌二醇活化后使脂多糖所引起的蛻膜細(xì)胞下降的增殖活性有一定程度的恢復(fù)。SGK1的活化使脂多糖所引起的蛻膜細(xì)胞凋亡的升高有一定程度的恢復(fù),差異有統(tǒng)計(jì)學(xué)意義(P0.01);并且使脂多糖所引起的蛻膜細(xì)胞抗凋亡基因BCL-2和XIAP的降低有一定程度的恢復(fù),差異有統(tǒng)計(jì)學(xué)意義(P0.01)4.SGK1經(jīng)雌二醇的刺激后活化增加IL-4和IL-5細(xì)胞因子的分泌,差異有統(tǒng)計(jì)學(xué)意義(P0.001和P0.01);SGK1活化后下調(diào)IFN-γ細(xì)胞因子的分泌,差異有統(tǒng)計(jì)學(xué)意義(P0.001);SGK1的活化上調(diào)細(xì)胞因子IL-4/IFN-γ的比值,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。5.SGK1經(jīng)雌二醇活化后可以使脂多糖處理所導(dǎo)致的增加的磷酸化的NF-κB蛋白下降以及活化的NF-κB蛋白下調(diào)(P0.01)。結(jié)論:1.蛻膜細(xì)胞中的SGK1經(jīng)雌二醇活化后促進(jìn)細(xì)胞增殖,降低脂多糖引起的蛻膜細(xì)胞的凋亡。2.SGK1經(jīng)雌二醇活化后抑制蛻膜細(xì)胞NF-κB的活性,從而減少蛻膜細(xì)胞Th1型細(xì)胞因子的分泌,增加Th2型細(xì)胞因子的分泌,糾正脂多糖引起的免疫耐受的偏移,有利于正常妊娠的維持。
[Abstract]:The expression of the expression of SGK1 in Decidua Tissue in early abortion: by measuring the expression of serum and glucocorticoid regulated protein kinase 1 (SGK1) in the early decidual tissue of pregnancy, the difference of SGK1 expression in early spontaneous abortion and normal pregnancy decidua was compared, and SGK1 was investigated in the maternal fetal interface microenvironment during early pregnancy. Biological significance, materials and methods: 1. select 88 cases of unexplained spontaneous abortion in early pregnancy as experimental study group, 71 normal early pregnant women at the same time as control group.2. immunofluorescence and immunofluorescence technique to detect the expression of SGK1 in Decidua Tissue by.3.TRIZOL method to extract the total RNA of the uterus Decidua Tissue, retrotranscriptase The relative expression of SGK1 mRNA in two groups of uterine decidua tissues was measured by Real time PCR. The protein of human decidua tissue was extracted from the RIPA protein lysate of protease inhibitor, the tissue protein was quantified by BCA method. Western blot technique was used to detect the relative expression of SGK1 protein in human decidua tissue. Results: 1. early self There was no significant difference in age, number of gestational weeks, age of menarche, menstrual cycle, and the number of days. The levels of serum estradiol (E2) in patients with early spontaneous abortion were lower than those of normal pregnancy. The difference was statistically significant (p0.001).2. immunofluorescence and immunofluorescence found that SGK1 was in early spontaneous abortion patients. The mRNA level of SGK1 in Decidua Tissue of patients with early spontaneous abortion of.3. and normal pregnancy was lower than that in the Decidua Tissue of early normal pregnancy. The difference was statistically significant (P0.001) the total protein expression of SGK1 in the Decidua Tissue of early.4. patients with.4. was lower than that of normal pregnancy. The difference was statistically significant (P0.001). And the protein expression of SGK1 phosphorylation in the uterine decidua tissues of early spontaneous abortion patients was lower than those of normal pregnancy, and the difference was statistically significant (P0.001). This was in accordance with the expression trend of SGK1 total protein in the two groups, which was consistent with the phosphorylated eggs of SGK1 in the uterine decidua tissues of patients with early spontaneous abortion. The ratio of white to total protein was lower than that in normal pregnancy group, the difference was statistically significant (p0.001). Conclusion: the expression of SGK1, total protein and activated protein in decidua tissues of early pregnancy induced abortion patients with.2. decreased. The decrease of the second part of SGK1 and the occurrence of early spontaneous abortion in the decidua tissues of early spontaneous abortion patients. Objective: by establishing ecutic cells in vitro, the changes in the activity of SGK1 protein in decidua cells are the eye points, and the mechanism of early spontaneous abortion is further explored, and new ideas and ways are provided for the pathogenesis and prevention and treatment of pathological pregnancy. Materials and methods: 1. the culture of ecutic cells in vitro and immunization of the ecutic cells in vitro Fluorescence assay to identify the proliferation activity of decidual cells by.2.MTT colorimetric assay, Annexin V-FITC/PI detection of.3. targeting SGK1 siRNA interference in decidual cell apoptosis the effect of SGK1 on the apoptosis of decidua cells.4. LPS treated decidual cells to establish spontaneous abortion in vitro cell model.5. enzyme linked immunosorbent assay in decidua cell supernatant Results: the apoptosis of the uterus decidua cells after the transfection of 1. targeted SGK1 siRNA was higher than that of the control group decidua cells. The difference was statistically significant (P0.01), the expression of SGK1 in the decidual cells stimulated by estradiol was higher than that of the control group. The difference was statistically significant (P0.01).3.SGK1 was activated by estradiol to make lipopolysaccharide. The proliferation activity of decidual cells caused by decidua caused by the activation of.SGK1 caused a certain degree of recovery of the apoptosis of the decidua cells caused by lipopolysaccharide, and the difference was statistically significant (P0.01), and the reduction of the anti apoptotic gene BCL-2 and XIAP of the decidual cells caused by lipopolysaccharide recovered to a certain extent. There were statistically significant (P0.01) 4.SGK1 activation by estradiol to increase the secretion of IL-4 and IL-5 cytokines, the difference was statistically significant (P0.001 and P0.01), and the secretion of IFN- gamma cytokines after SGK1 activation was statistically significant (P0.001), and the ratio of activation up to IL-4/IFN- gamma of SGK1 was statistically significant (P). 0.01).5.SGK1 can increase the increased phosphorylation of NF- kappa B protein induced by lipopolysaccharide treatment and the activation of NF- kappa B protein down regulation (P0.01). Conclusion: SGK1 in the 1. decidual cells promotes cell proliferation after estradiol activation, and reduces the apoptosis of the decidual cells induced by lipopolysaccharide after the activation of estradiol activation. The activity of NF- kappa B in decidual cells reduces the secretion of Th1 - type cytokines in decidual cells, increases the secretion of Th2 - type cytokines, and corrects the migration of immune tolerance induced by lipopolysaccharide, which is beneficial to the maintenance of normal pregnancy.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.21

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