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Drug Screening Model Construction Based on Inhibiting NF-κB Signal Pathway

Author: ZhangLiPeng
Tutor: LiuMingYao;LuoJian
School: East China Normal University
Course: Biomedical
Keywords: NF - kappa B Drug screening model TNFα IκBα Rattan acid
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 193
Quote: 0
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Abstract


Nuclear factor κB (nuclear factor ofκB, NF-κB) in the first 20 years mature plasma cells were found, with the immunoglobulin κ light chain intron enhancer sequence-specific binding and to promote transcription of a DNA gene binding complex, is widely found in eukaryotic cells. By regulating the expression of target genes, NF-κB transcription factor in a variety of cellular activities play an important role, such as cell proliferation, cell death, development, and innate and acquired immune response, especially in inflammation and tumor development process, is considered to be an ideal target for prevention and treatment of tumors and inflammation. Therefore, the establishment of inhibiting NF-κB signaling pathway drug screening model is essential. Biomedical and biological technology continues to evolve, so that high-throughput drug screening possible, and at the end of the last century developed the technology to random screening, a large number of samples to be screened, as an important technical means actively looking for drugs. High-throughput screening technologies to molecular and cellular level by means of experimental methods, the current diversified development trend, in the following three platforms, namely yeast platform to platform and animal cell platform. Luciferase reporter gene analysis in high-throughput screening of drugs play an important role. Studies show that, TNFα in vivo and in vitro activation of well NF-κB, can be used as NF-κB signaling pathway positive activator; IκBα with NF-κB binding, located in the cytoplasm, preventing NF-κB to the nucleus, the Executive its transcriptional activity, thus detects whether or not the degradation of IκBα indirectly reflects the activation status of NF-κB; vine acid (gambogic acid, GA) can inhibit the activity of NF-κB, can be used as positive inhibitor of NF-κB. In this paper, luciferase reporter gene technology and Western blot technique was to explore TNFα concentration and time on the NF-κB-luc reporter gene expression and NF-κB subunit inhibited the degradation of IκBα, and then construct the NF-κB signaling pathway inhibitor drugs screening model. In the luciferase reporter gene assay screening model, the first detection of the TNFα stimulated 293T cells optimal concentration: with different concentrations of TNFα (0,0.001,0.005,0.01,0.05,0.1 nM) stimulated 293T cells, and then select the most Jia TNF concentration to stimulate 293T cells with different time (0,3,6,12,18,24,36 h), the best time to detect TNF effect. Found that, 0.01nM TNFα for 24 h to stimulate NF-κB-luc reporter gene highly expressed in 293T cells, and its expression level was positively correlated with TNF alpha dose and treatment time. Garcinia acid verification showed that NF-κB luciferase reporter gene assay screening system stable and feasible. In the NF-κB subunit degradation inhibition screening model, the first detected Panc-28 cells stimulated TNFα best time: 0.01nM TNFα in a different time (0,5,15,30,60 min) Panc-28 cells stimulated and then select the better times with different concentrations of TNFα (0,0.01,0.05,0.1,0.5 nM) stimulated Panc-28, the optimal concentration detecting TNFα action. Found, 0.01 nM TNFα for 5 min that would allow Panc-28 cells was significantly IκBα degradation, the role of 20 ~ 30 min almost completely degraded, then IκBα levels began to increase. NF-κB inhibitors positive verification showed that Garcinia acid subunit of NF-κB inhibition of degradation screening system stable and feasible. Summary, luciferase reporter gene assay screening models and NF-κB inhibition subunit degradation screening model two screening model can be used NF-κB signaling pathway inhibitor drug screening.

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