本文關鍵詞： KiSS-1 AMPK SP1 轉位　出處：《福建醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的:1.研究轉錄因子SP1對下丘腦GT1-7神經(jīng)細胞中KiSS-1基因啟動子活性的影響。2.探討AMPK對下丘腦GT1-7神經(jīng)細胞中轉錄因子SP1轉位的影響。 方法:1.建立KiSS-1基因啟動子報告基因體系,用Promega熒光素酶雙報告基因檢測系統(tǒng)檢測SP1SiRNA及SP1抑制劑Mithramycin A對GT1-7細胞KiSS-1基因啟動子活性的影響。2.用AMPK激動劑AICAR、AMPK抑制劑Compound C和AMPKα1-SiRNA干預GT1-7細胞后,分別提取細胞漿蛋白、核蛋白和總蛋白,用Western Blot方法檢測轉錄因子SP1蛋白表達的變化。3.用AICAR、Compound C孵育細胞,用免疫熒光法來進一步證實Western Blot的結果。 結果:1.用SP1SiRNA以及不同濃度的Mithramycin A作用于下丘腦GT1-7細胞后, KiSS-1基因的啟動子活性被明顯抑制。2. AICAR可以降低GT1-7細胞核蛋白中SP1的水平而相應的增加漿蛋白中SP1的水平,相反的, Compound C和AMPKα1-SiRNA都能夠增加GT1-7細胞核蛋白中SP1的水平而相應的降低漿蛋白中SP1的表達水平。無論是AICAR,還是Compound C或AMPKα1-SiRNA都沒有使GT1-7細胞總蛋白中SP1的含量發(fā)生顯著性的改變。3.免疫熒光的結果提示AICAR能抑制SP1向細胞核的轉位,而Compound C則能增加SP1向細胞核的轉位,與我們Western Blot的結果相符。 結論: AMPK對KiSS-1基因表達的抑制作用可能是通過抑制轉錄因子SP1的轉位而減弱了SP1對KiSS-1基因啟動子的激活作用而造成的。
[Abstract]:Objective:. 1. To study the effect of transcription factor SP1 on the activity of KiSS-1 gene promoter in hypothalamic GT1-7 neurons. 2. To investigate the effect of AMPK on transcription factor SP in hypothalamic GT1-7 neurons. 1 the effect of translocation. Methods: 1. To establish the reporter gene system of KiSS-1 gene promoter. Detection of SP1SiRNA and SP1 inhibitor Mithramycin by Promega luciferase double reporter Gene Detection system. Effect of A on the activity of KiSS-1 gene promoter in GT1-7 cells. 2. AMPK agonist AICAR was used. AMPK inhibitor Compound C and AMPK 偽 1-SiRNA were used to extract cytoplasmic protein, nuclear protein and total protein from GT1-7 cells. The expression of transcription factor SP1 protein was detected by Western Blot. The cells were incubated with AICAR-Compound C. The results of Western Blot were confirmed by immunofluorescence method. Results 1. The hypothalamic GT1-7 cells were treated with SP1SiRNA and different concentrations of Mithramycin A. The promoter activity of KiSS-1 gene was significantly inhibited. 2. AICAR could decrease the level of SP1 in GT1-7 nuclear protein and increase the level of SP1 in plasma protein. The opposite. Compound. Both C and AMPK 偽 1-SiRNA could increase the level of SP1 in the nuclear protein of GT1-7 and decrease the expression of SP1 in plasma protein, regardless of AICAR. Or Compound? C or AMPK 偽 1-SiRNA did not significantly change the content of SP1 in the total protein of GT1-7 cells. 3. The results of immunofluorescence suggested that AICAR could inhibit the nuclear orientation of SP1. The transposition. Compound C increased the translocation of SP1 to the nucleus, which was consistent with our results of Western Blot. Conclusion:. The inhibitory effect of AMPK on the expression of KiSS-1 gene may be due to the inhibition of translocation of transcription factor SP1 which weakens the activation of KiSS-1 gene promoter by SP1.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

【共引文獻】

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本文編號：1475897