本文關鍵詞： 妊娠期糖尿病 FoxO1 腫瘤壞死因子-α 滋養(yǎng)細胞　出處：《南京醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的： 研究妊娠期糖尿病(gestational diabetes mellitus,GDM)孕婦與正常孕婦胎盤和大網膜脂肪組織叉頭轉錄因子Fox01的表達情況以及腫瘤壞死因子-α(tumor necrosis factor-alpha,TNF-α)對滋養(yǎng)細胞Fox01的調控機制。 方法： 1.選取2013年1月至2013年4月在南京醫(yī)科大學第一附屬醫(yī)院產科門診常規(guī)產檢并入院行擇期剖宮產的GDM孕婦20例為GDM組,正常孕婦20例為對照組,檢測人體測量參數和生化指標。檢測兩組孕婦外周血空腹血糖(Fasting plasma glucose,FPG).胰島素(fasting serum insulin,FINS),計算出胰島素抵抗指數(HOMA-insulin resistance index,HOMA-IR)。免疫組化法對GDM及正常孕婦胎盤和脂肪組織Fox01蛋白進行定位,western blot法檢測兩組孕婦胎盤和脂肪組織Fox01和TNF-a蛋白的表達情況,熒光實時定量PCR測定兩組孕婦胎盤和脂肪組織FoxO1、TNF-amRNA的表達水平。比較兩組的檢測結果,并做相關性分析。 2.采用不同濃度的TNF-α(0,1,10,20,50和100ng/ml)分別作用于人滋養(yǎng)細胞株HTR-8/SVneo和BeWo細胞24小時,采用western blot檢測兩種細胞FoxO1蛋白水平的變化。Fox01siRNA和control siRNA寡聚核苷酸轉染滋養(yǎng)細胞株HTR-8/SVneo和BeWo,western blot和熒光實時定量PCR驗證干擾效果。將細胞分為3組,分別為NS siRNA組,NS siRNA+TNF-α組和FoxO1siRNA+TNF-α組,細胞干擾成功后,在后兩組中加20ng/ml TNF-α,24小時后分別收集細胞和細胞上清液,采用熒光實時定量PCR測定三組細胞白細胞介素-6(Inerleukin-6,IL-6)和白細胞介素一1p(Interleukin-1β,IL-1β)mRNA的含量,采用酶聯(lián)免疫吸附測定法(enzyme linked immunosorbent assay,ELISA)法檢測三組細胞上清液IL-6和IL-1p的濃度。 結果： 1.GDM組孕婦血清FPG、FINS及HOMA-IR顯著高于正常對照組。免疫組化結果顯示,兩組孕婦胎盤和脂肪組織均有Fox01蛋白表達,GDM組孕婦胎盤和脂肪組織FoxO1、TNF-α mRNA和蛋白表達水平較正常組升高,差異明顯。GDM組孕婦胎盤和脂肪組織FoxO1mRNA及蛋白表達水平與TNF-α呈正相關,胎盤Fox01蛋白與FIN、HOMA-IR呈明顯正相關。 2.隨著TNF-a濃度的增加,HTR-8/SVneo和BeWo細胞Fox01的蛋白表達量增加,在20ng/ml濃度時,Fox01蛋白表達量最高。與NS siRNA組相比,NS siRNA+TNF-α組細胞IL-6、IL-1βmRNA水平和細胞上清液IL-6、IL-1β濃度明顯升高。與NS siRNA+TNF-α組相比,FoxO1siRNA+TNF-α組細胞IL-6、IL-1βmRNA水平和細胞上清液IL-6、IL-1β濃度較低,差異有統(tǒng)計學意義。 結論： 1.GDM孕婦體內存在嚴重的胰島素抵抗,胎盤和大網膜脂肪組織FoxO1的過度表達可能參與了GDM胰島素抵抗的發(fā)生發(fā)展。 2.TNF-α參與滋養(yǎng)細胞Fox01表達的調控。在TNF-α作用下滋養(yǎng)細胞FoxO1表達升高,其可能通過促進炎癥因子IL-6、IL-1β的表達參與了GDM胰島素抵抗的發(fā)病過程。
[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- 偽 (TNF- 偽). Tumor necrosis factor-alpha. The regulatory mechanism of TNF- 偽 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- 偽 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- 偽 group and FoxO1siRNA TNF- 偽 group, after successful cell interference, 20 ng / ml TNF- 偽 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- 偽 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- 偽, 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- 偽 group, the level of IL-6 and IL-1 尾 mRNA and the supernatant IL-6 in NS siRNA TNF- 偽 group were higher than those in NS siRNA group. Compared with NS siRNA TNF- 偽 group, the IL-1 尾 concentration was significantly increased, and the cell IL-6 in FoxO1 siRNA TNF- 偽 group was significantly higher than that in NS siRNA TNF- 偽 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- 偽 participates in the regulation of Fox01 expression in trophoblastic cells. The expression of FoxO1 in trophoblastic cells is increased under the action of TNF- 偽, 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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.256

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