基于FRET技术研究AhR、HIF-1α与ARNT的相互作用

发布时间：2018-04-19 15:45

本文选题：芳香烃受体 + AhR核易位体蛋白　；参考：《内蒙古医科大学》2017年硕士论文

【摘要】：目的:本研究拟探讨在不同浓度的BaP处理下,采用CoCl2作为HIF-1α的激动剂,在A549细胞中基于FRET技术研究AhR、HIF-1α与ARNT的相互作用,以期对这两条重要的信号通路之间关系的研究提供一定的参考,为科学家们寻找阻断AhR和HIF-1信号通路的药物来预防和治疗癌症提供一定的实验依据。方法:(1)利用分子克隆基因重组技术获得目的质粒:从A549细胞中提取总RNA,采用RT-PCR法克隆目的基因AhR、ARNT片段,通过质粒载体的构建将两个目的基因AhR和ARNT分别重组到荧光表达载体质粒pAcGFP1-N1和pDsRed-Monomer-N1上,得到pAcGFP1-N1-AhR和pDsRed-Monomer-N1-ARNT两个重组质粒。(2)利用荧光共振能量转移实验技术研究AhR、HIF-1α与ARNT的相互作用:pAcGFP1-N1-AhR是目的基因Ah R与GFP融合表达,而pDsRed-Monomer-N1-ARNT是ARNT与pDsRed-Monomer-N1融合表达。检测AhR与ARNT是否发生了相互作用,就等同于检测pAcGFP1-N1和pDsRed-Monomer-N1是否发生了FRET。脂质体转染技术介导pAcGFP1-N1-AhR和pDsRed-Monomer-N1-ARNT两个重组质粒在A549细胞中的表达,用不同浓度的BaP(0μM、2μM、4μM、8μM,作用24h)和CoCl2(300μM,作用24h)处理A549细胞,运用高内涵细胞筛选系统的荧光成像模块,研究在A549细胞中用CoCl2激动HIF-1α进入核内时,根据荧光重组质粒的荧光强度的变化情况,判断AhR与HIF-1α是否竞争结合ARNT。结果:(1)成功构建pAcGFP1-N1-AhR和pDsRed-Monomer-N1-ARNT两个重组质粒,经酶切鉴定和DNA序列测定鉴定载体构建成功,转染细胞后pDsRed-Monomer-N1-ARNT和pAcGFP1-N1-AhR两个重组质粒在A549细胞中成功表达,转染率分别为15%和19%。(2)在细胞质中可观察到绿色荧光信号,在细胞核中可观察到红色荧光信号,表明这两种重组质粒在A549细胞中成功表达。此外,我们发现在细胞核中可检测到FRET荧光信号,表明AhR与ARNT在核内发生相互作用。(3)用不同浓度的BaP(0μM、2μM、4μM、8μM,作用24h)处理转染入A549细胞中的pAcGFP1-N1-AhR和pDsRed-Monomer-N1-ARNT两个重组质粒,FRET荧光信号强度与对照组相比明显增强且呈剂量依赖性,当BaP浓度为8μM时,FRET荧光信号强度约为对照组的2.3倍,且差异具有显著性(P0.05)。(4)加入300μM CoCl2和BaP 8μM处理24h后,FRET荧光信号强度与只加BaP 8μM,作用24h相比明显减弱,处理组的荧光信号强度为之前的0.6倍。结论:BaP能上调pAcGFP1-N1-AhR和pDsRed-Monomer-N1-ARNT两个重组质粒的FRET荧光信号并呈剂量依赖性;CoCl2处理后FRET荧光信号强度减弱说明HIF-1α竞争AhR结合ARNT蛋白,进一步说明HIF-1α信号通路的激活抑制AhR信号通路。
[Abstract]:Aim: to investigate the interaction between AhRHIF-1 伪 and ARNT in A549 cells by using CoCl2 as an agonist of HIF-1 伪 in A549 cells treated with different concentrations of BaP.In order to provide a certain reference for the study of the relationship between these two important signal pathways and provide some experimental basis for scientists to find drugs to block the AhR and HIF-1 signaling pathways to prevent and treat cancer.Methods the target plasmid was obtained by molecular cloning gene recombination technique: total RNAs were extracted from A549 cells, and the target gene AhRRARNT fragment was cloned by RT-PCR method.The two target genes, AhR and ARNT, were recombined into the fluorescent expression vector pAcGFP1-N1 and pDsRed-Monomer-N1 by construction of plasmid vector.Two recombinant plasmids, pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT, were obtained. The fluorescence resonance energy transfer technique was used to study the interaction between HIF-1 伪 and ARNT: pAcGFP1-N1-AhR is the fusion expression of the target gene Ah R and GFP, while pDsRed-Monomer-N1-ARNT is the fusion expression of ARNT and pDsRed-Monomer-N1.Detecting whether AhR interacts with ARNT is equivalent to detecting whether pAcGFP1-N1 and pDsRed-Monomer-N1 have fret.The expression of pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT in A549 cells was mediated by liposome transfection technique. A549 cells were treated with different concentrations of BaP(0 渭 MU 2 渭 MU 8 渭 M for 24 h) and CoCl2(300 渭 M for 24 h. The fluorescent imaging module of high intension cell screening system was used.In A549 cells, CoCl2 was used to stimulate HIF-1 伪 into the nucleus. According to the change of fluorescence intensity of the recombinant plasmid, the competitive binding of AhR and HIF-1 伪 was determined.Results two recombinant plasmids, pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT, were successfully constructed. The recombinant plasmids pDsRed-Monomer-N1-ARNT and pAcGFP1-N1-AhR were successfully expressed in A549 cells after transfection.The transfection efficiencies were 15% and 19%, respectively. Green fluorescence signals were observed in the cytoplasm and red fluorescence signals were observed in the nucleus, indicating that the two recombinant plasmids were successfully expressed in A549 cells.In addition, we found that FRET fluorescence signals could be detected in the nucleus.The results showed that the fluorescence signal intensity of pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT recombinant plasmids transfected into A549 cells was enhanced in a dose-dependent manner with different concentrations of BaP(0 渭 MU 2 渭 MU 4 渭 M + 8 渭 M and 24 h. The fluorescence signal intensity of pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT recombinant plasmids transfected into A549 cells was increased in a dose-dependent manner.When the concentration of BaP was 8 渭 M, the fluorescence signal intensity of fret was about 2.3 times of that of the control group, and the difference was significant (P 0.05). The intensity of fret fluorescence signal was significantly decreased after the addition of 300 渭 M CoCl2 and 8 渭 M BaP for 24 h, compared with that of BaP 8 渭 M alone for 24 h.The intensity of fluorescence signal in the treatment group was 0.6 times higher than that in the previous treatment group.Conclusion FRET fluorescence signal of pAcGFP1-N1-AhR and pDsRed-Monomer-N1-ARNT can be upregulated by% bap and the intensity of FRET fluorescence signal is decreased in a dose-dependent manner after treatment with CoCl2, which indicates that HIF-1 伪 competes with ARNT protein and further indicates that the activation of HIF-1 伪 signaling pathway inhibits AhR signaling pathway.
【学位授予单位】：内蒙古医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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