血清和糖皮质激素调节蛋白激酶1在早期自然流产中的机制研究

发布时间：2018-07-22 13:33

【摘要】：第一部分SGK1在早期流产子宫蜕膜组织中的表达改变目的:通过测定妊娠早期蜕膜组织中血清和糖皮质激素调节蛋白激酶1(SGK1)的表达,比较早期自然流产和正常妊娠蜕膜组织中SGK1表达的差异,探讨SGK1在妊娠早期母胎界面微环境中的生物学意义。材料及方法:1.选择妊娠早期不明原因自然流产者88例作为实验研究组,同期71例的正常早期妊娠者作为对照组。2.免疫组织化学法及免疫荧光技术检测子宫蜕膜组织中SGK1的表达。3.TRIZOL法抽提子宫蜕膜组织的总RNA,逆转录后Real time PCR测定两组子宫蜕膜组织中SGK1 mRNA相对表达量。4.用含蛋白酶抑制剂的RIPA蛋白质裂解液提取人子宫蜕膜组织蛋白质,采用BCA法进行组织蛋白定量,Western blot技术进行检测人子宫蜕膜组织中SGK1蛋白质相对表达量。结果:1.早期自然流产组和正常妊娠组在年龄、妊娠周数、初潮年龄、月经周期、行经天数等方面无显著差异。早期自然流产患者血清雌二醇(E2)的水平低于正常妊娠者,差异有统计学意义(p0.001)。2.免疫组织化学法及免疫荧光发现SGK1在早期自然流产患者和正常妊娠者的子宫蜕膜组织中均有表达。3.早期自然流产患者蜕膜组织中的SGK1的mRNA水平较早期正常妊娠者蜕膜组织中的表达下降,差异有统计学意义(P0.001)。4.早期自然流产患者子宫蜕膜组织中SGK1总的蛋白质表达低于正常妊娠者,差异具有统计学意义(P0.001)。并且,早期自然流产患者子宫蜕膜组织中SGK1磷酸化的蛋白表达低于正常妊娠者,差异具有统计学意义(P0.001),这与SGK1总蛋白在两组之间的表达趋势相一致。5.早期自然流产患者子宫蜕膜组织中SGK1的磷酸化蛋白与总蛋白的比值低于正常妊娠组,差异具有统计学意义(p0.001)。结论:1.SGK1在早期妊娠子宫蜕膜组织中表达。2.早期自然流产患者蜕膜组织中的SGK1的mRNA水平、总蛋白以及活化的蛋白表达均下降。第二部分SGK1的下调与早期自然流产发生相关机理研究目的:通过建立体外子宫蜕膜细胞,以蜕膜细胞中SGK1蛋白活性的变化为着眼点,进一步探究早期自然流产的发生机理,为病理妊娠发病机制及防治的研究提供新的思路和途径。材料及方法:1.培养体外子宫蜕膜细胞,用免疫荧光法鉴定其纯度。2.MTT比色试验检测蜕膜细胞增殖活性,Annexin V-FITC/PI检测蜕膜细胞凋亡。3.靶向性SGK1 siRNA干扰研究SGK1对蜕膜细胞凋亡的影响。4.脂多糖处理蜕膜细胞建立自然流产的体外细胞模型。5.酶联免疫吸附测定蜕膜细胞上清中的细胞因子的含量。结果:1.靶向性SGK1 siRNA转染24小时后的子宫蜕膜细胞的凋亡高于对照组蜕膜细胞,差异有统计学意义(P0.01)。2.经雌二醇刺激蜕膜细胞中SGK1的表达高于对照组,差异有统计学意义(P0.01)。3.SGK1经雌二醇活化后使脂多糖所引起的蜕膜细胞下降的增殖活性有一定程度的恢复。SGK1的活化使脂多糖所引起的蜕膜细胞凋亡的升高有一定程度的恢复,差异有统计学意义(P0.01);并且使脂多糖所引起的蜕膜细胞抗凋亡基因BCL-2和XIAP的降低有一定程度的恢复,差异有统计学意义(P0.01)4.SGK1经雌二醇的刺激后活化增加IL-4和IL-5细胞因子的分泌,差异有统计学意义(P0.001和P0.01);SGK1活化后下调IFN-γ细胞因子的分泌,差异有统计学意义(P0.001);SGK1的活化上调细胞因子IL-4/IFN-γ的比值,差异有统计学意义(P0.01)。5.SGK1经雌二醇活化后可以使脂多糖处理所导致的增加的磷酸化的NF-κB蛋白下降以及活化的NF-κB蛋白下调(P0.01)。结论:1.蜕膜细胞中的SGK1经雌二醇活化后促进细胞增殖,降低脂多糖引起的蜕膜细胞的凋亡。2.SGK1经雌二醇活化后抑制蜕膜细胞NF-κB的活性,从而减少蜕膜细胞Th1型细胞因子的分泌,增加Th2型细胞因子的分泌,纠正脂多糖引起的免疫耐受的偏移,有利于正常妊娠的维持。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.21

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