AMPK通过转录因子SP1影响下丘脑GT1-7神经元KiSS-1基因转录的机制研究

发布时间：2018-01-30 09:31

本文关键词： KiSS-1 AMPK SP1 转位　出处：《福建医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:1.研究转录因子SP1对下丘脑GT1-7神经细胞中KiSS-1基因启动子活性的影响。2.探讨AMPK对下丘脑GT1-7神经细胞中转录因子SP1转位的影响。方法:1.建立KiSS-1基因启动子报告基因体系,用Promega荧光素酶双报告基因检测系统检测SP1SiRNA及SP1抑制剂Mithramycin A对GT1-7细胞KiSS-1基因启动子活性的影响。2.用AMPK激动剂AICAR、AMPK抑制剂Compound C和AMPKα1-SiRNA干预GT1-7细胞后,分别提取细胞浆蛋白、核蛋白和总蛋白,用Western Blot方法检测转录因子SP1蛋白表达的变化。3.用AICAR、Compound C孵育细胞,用免疫荧光法来进一步证实Western Blot的结果。结果:1.用SP1SiRNA以及不同浓度的Mithramycin A作用于下丘脑GT1-7细胞后, KiSS-1基因的启动子活性被明显抑制。2. AICAR可以降低GT1-7细胞核蛋白中SP1的水平而相应的增加浆蛋白中SP1的水平,相反的, Compound C和AMPKα1-SiRNA都能够增加GT1-7细胞核蛋白中SP1的水平而相应的降低浆蛋白中SP1的表达水平。无论是AICAR,还是Compound C或AMPKα1-SiRNA都没有使GT1-7细胞总蛋白中SP1的含量发生显著性的改变。3.免疫荧光的结果提示AICAR能抑制SP1向细胞核的转位,而Compound C则能增加SP1向细胞核的转位,与我们Western Blot的结果相符。结论: AMPK对KiSS-1基因表达的抑制作用可能是通过抑制转录因子SP1的转位而减弱了SP1对KiSS-1基因启动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 伪 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 伪 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 伪 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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【共引文献】