妊娠期糖尿病孕妇胎盘和大网膜脂肪组织Fox01的表达及意义

发布时间：2018-02-04 10:44

本文关键词： 妊娠期糖尿病 FoxO1 肿瘤坏死因子-α 滋养细胞　出处：《南京医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究妊娠期糖尿病(gestational diabetes mellitus,GDM)孕妇与正常孕妇胎盘和大网膜脂肪组织叉头转录因子Fox01的表达情况以及肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)对滋养细胞Fox01的调控机制。方法： 1.选取2013年1月至2013年4月在南京医科大学第一附属医院产科门诊常规产检并入院行择期剖宫产的GDM孕妇20例为GDM组,正常孕妇20例为对照组,检测人体测量参数和生化指标。检测两组孕妇外周血空腹血糖(Fasting plasma glucose,FPG).胰岛素(fasting serum insulin,FINS),计算出胰岛素抵抗指数(HOMA-insulin resistance index,HOMA-IR)。免疫组化法对GDM及正常孕妇胎盘和脂肪组织Fox01蛋白进行定位,western blot法检测两组孕妇胎盘和脂肪组织Fox01和TNF-a蛋白的表达情况,荧光实时定量PCR测定两组孕妇胎盘和脂肪组织FoxO1、TNF-amRNA的表达水平。比较两组的检测结果,并做相关性分析。 2.采用不同浓度的TNF-α(0,1,10,20,50和100ng/ml)分别作用于人滋养细胞株HTR-8/SVneo和BeWo细胞24小时,采用western blot检测两种细胞FoxO1蛋白水平的变化。Fox01siRNA和control siRNA寡聚核苷酸转染滋养细胞株HTR-8/SVneo和BeWo,western blot和荧光实时定量PCR验证干扰效果。将细胞分为3组,分别为NS siRNA组,NS siRNA+TNF-α组和FoxO1siRNA+TNF-α组,细胞干扰成功后,在后两组中加20ng/ml TNF-α,24小时后分别收集细胞和细胞上清液,采用荧光实时定量PCR测定三组细胞白细胞介素-6(Inerleukin-6,IL-6)和白细胞介素一1p(Interleukin-1β,IL-1β)mRNA的含量,采用酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)法检测三组细胞上清液IL-6和IL-1p的浓度。结果： 1.GDM组孕妇血清FPG、FINS及HOMA-IR显著高于正常对照组。免疫组化结果显示,两组孕妇胎盘和脂肪组织均有Fox01蛋白表达,GDM组孕妇胎盘和脂肪组织FoxO1、TNF-α mRNA和蛋白表达水平较正常组升高,差异明显。GDM组孕妇胎盘和脂肪组织FoxO1mRNA及蛋白表达水平与TNF-α呈正相关,胎盘Fox01蛋白与FIN、HOMA-IR呈明显正相关。 2.随着TNF-a浓度的增加,HTR-8/SVneo和BeWo细胞Fox01的蛋白表达量增加,在20ng/ml浓度时,Fox01蛋白表达量最高。与NS siRNA组相比,NS siRNA+TNF-α组细胞IL-6、IL-1βmRNA水平和细胞上清液IL-6、IL-1β浓度明显升高。与NS siRNA+TNF-α组相比,FoxO1siRNA+TNF-α组细胞IL-6、IL-1βmRNA水平和细胞上清液IL-6、IL-1β浓度较低,差异有统计学意义。结论： 1.GDM孕妇体内存在严重的胰岛素抵抗,胎盘和大网膜脂肪组织FoxO1的过度表达可能参与了GDM胰岛素抵抗的发生发展。 2.TNF-α参与滋养细胞Fox01表达的调控。在TNF-α作用下滋养细胞FoxO1表达升高,其可能通过促进炎症因子IL-6、IL-1β的表达参与了GDM胰岛素抵抗的发病过程。
[Abstract]:Objective: To study gestational diabetes mellitus in gestational diabetes mellitus. Expression of forkhead transcription factor Fox01 in placenta and omentum adipose tissue of pregnant women and normal pregnant women and tumor necrosis factor- 伪 (TNF- 伪). Tumor necrosis factor-alpha. The regulatory mechanism of TNF- 伪 on trophoblastic Fox01. Methods: 1. From January 2013 to April 2013, 20 pregnant women with GDM were selected as GDM group, who underwent routine birth examination and elective cesarean section in obstetrical outpatient department of the first affiliated Hospital of Nanjing Medical University from January 2013 to April 2013. 20 normal pregnant women were used as control group. The parameters of anthropometry and biochemical indexes were measured. Fasting plasma glucose in peripheral blood of pregnant women in both groups were measured. Serum casting (FINS). The insulin resistance index (HOMA-insulin resistance index) was calculated. The localization of Fox01 protein in placenta and adipose tissue of GDM and normal pregnant women was performed by immunohistochemistry. The expression of Fox01 and TNF-a protein in placenta and adipose tissue of pregnant women was detected by western blot method. The expression of TNF-a mRNA in placenta and adipose tissue of pregnant women was measured by real-time fluorescence quantitative PCR. The results of the two groups were compared and the correlation was analyzed. 2. Different concentrations of TNF- 伪 were used. 50 ng / ml and 100 ng / ml were exposed to human trophoblastic cell line HTR-8/SVneo and BeWo cells for 24 hours, respectively. Detection of FoxO1 protein levels in two kinds of cells by western blot .Fox01 siRNA and control. SiRNA oligodeoxynucleotides were transfected into trophoblast cell lines HTR-8/SVneo and BeWo. Western blot and real-time quantitative PCR were used to verify the interference effect. The cells were divided into three groups, NS siRNA group. NS siRNA TNF- 伪 group and FoxO1siRNA TNF- 伪 group, after successful cell interference, 20 ng / ml TNF- 伪 was added in the latter two groups. After 24 hours, the cell supernatants and cell supernatants were collected, and the levels of interleukin-6 and interleukin-6 in the three groups were measured by fluorescence real-time quantitative PCR. IL-6) and Interleukin-1 尾 -Interleukin-1 尾 -Interleukin-1 尾 -Interleukin-1 尾 -Interleukin-1 尾 -Interleukin-1 尾 -Interleukin-1 尾. Enzyme linked immunosorbent assay was detected by enzyme-linked immunosorbent assay (Elisa). The concentrations of IL-6 and IL-1p in supernatants of three groups were detected by Elisa. Results: 1. The expression of Fox01 protein in placenta and adipose tissue of pregnant women in GDM group was significantly higher than that in normal control group. The expression of TNF- 伪 mRNA and protein in placenta and adipose tissue of pregnant women in GDM group was higher than that in normal group. The expression of FoxO1mRNA and protein in placenta and adipose tissue of pregnant women was positively correlated with TNF- 伪, Fox01 protein and FIN in placenta. HOMA-IR was positively correlated. 2. With the increase of TNF-a concentration, the expression of Fox01 protein in HTR-8 / SVneo and BeWo cells increased, at the concentration of 20ng / ml. The expression of Fox01 protein was the highest. Compared with NS siRNA group, compared with NS siRNA TNF- 伪 group, the level of IL-6 and IL-1 尾 mRNA and the supernatant IL-6 in NS siRNA TNF- 伪 group were higher than those in NS siRNA group. Compared with NS siRNA TNF- 伪 group, the IL-1 尾 concentration was significantly increased, and the cell IL-6 in FoxO1 siRNA TNF- 伪 group was significantly higher than that in NS siRNA TNF- 伪 group. The levels of IL-1 尾 mRNA and IL-6 and IL-1 尾 in supernatant were significantly lower than those in control group. Conclusion: 1. There is severe insulin resistance in pregnant women with GDM. The overexpression of FoxO1 in placenta and omentum may be involved in the development of insulin resistance in GDM. 2. TNF- 伪 participates in the regulation of Fox01 expression in trophoblastic cells. The expression of FoxO1 in trophoblastic cells is increased under the action of TNF- 伪, which may be by promoting the inflammatory factor IL-6. The expression of IL-1 尾 is involved in the pathogenesis of GDM insulin resistance.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.256

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