通用型NF-κB荧光素酶报告基因细胞系的构建
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摘要
建立一种通用型荧光素酶报告基因检测系统。以NF-κB信号通路为基础,用NF-κB-RE-Luc2P慢病毒感染HEK293细胞获得单克隆细胞系,并使用TNF-α进行了刺激验证;为证明该细胞系具备通用和改造能力进一步感染了FGF家族受体FGFRⅡ,筛选获得单克隆然后用FGF类药物进行刺激验证。结果表明,在TNF-α刺激实验中,响应值与TNF-α浓度呈现剂量依赖效应,且信噪比可达100倍以上,表明细胞系建立成功。FGF类药物刺激验证实验中,响应值与药物浓度可拟合成剂量依赖的"S"型曲线,表明该细胞系可用于FGF类药物活性检测。获得了NF-κB-RE-Luc2P HEK293和以此细胞系为基础改造得到的细胞系FGFRⅡ/NF-κB-RE-Luc2P HEK293,由于NF-κB是细胞内众多信号通路的交汇点,因此针对NF-κB信号通路,该细胞系具备通用性。
This work is aimed to establish luciferase reporter gene system with commonality. Lentivirus containing NF-κB-RE-Luc2 P was transduced into HEK293 and monoclonal HEK293 cell line expressing luciferase reporter gene under control of NF-κB response element was established and verified with TNF-α stimulation. To demonstrate the cell line capacity of commonality and modification,FGF family receptor FGFR Ⅱ was further transduced into this cell line and the screened FGFR Ⅱ/NF-κB-RE-Luc2 P HEK293 single clones was further validated by FGF analogue stimulation. Dose-response effect and more than 100 fold of signal to noise ratio were observed in this cell line in TNF-αstimulation,indicating the cell line was generated successfully. In response to FGF analogue stimulation,dose-response effect was fitted as the"S" curve,implying that the cell line can be applied to the activity detection of FGF-type medicine. The results showed that NF-κB-RE-Luc2 P HEK293 and modified one FGFR Ⅱ/NF-κB-RE-Luc2 P HEK293 was established. that the cell line has commonality regarding NF-κB signaling.
This work is aimed to establish luciferase reporter gene system with commonality. Lentivirus containing NF-κB-RE-Luc2 P was transduced into HEK293 and monoclonal HEK293 cell line expressing luciferase reporter gene under control of NF-κB response element was established and verified with TNF-α stimulation. To demonstrate the cell line capacity of commonality and modification,FGF family receptor FGFR Ⅱ was further transduced into this cell line and the screened FGFR Ⅱ/NF-κB-RE-Luc2 P HEK293 single clones was further validated by FGF analogue stimulation. Dose-response effect and more than 100 fold of signal to noise ratio were observed in this cell line in TNF-αstimulation,indicating the cell line was generated successfully. In response to FGF analogue stimulation,dose-response effect was fitted as the"S" curve,implying that the cell line can be applied to the activity detection of FGF-type medicine. The results showed that NF-κB-RE-Luc2 P HEK293 and modified one FGFR Ⅱ/NF-κB-RE-Luc2 P HEK293 was established. that the cell line has commonality regarding NF-κB signaling.
引文
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[6]王珺婷,钟鸣,刘洁. IL-33的生物学功能以及对肿瘤作用的研究进展[J].生物学杂志, 2018, 35(3):76-79.
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[11]Kawaguchi M, Yamamoto K, Kanemaru A, et al. Inhibition of nuclear factor-κB signaling suppresses Spint1-deletion-induced tumor susceptibility in the ApcMin/+model[J]. Oncotarget,2016, 7(42):68614.
[12]Ukaji T, Lin Y, Okada S, et al. Inhibition of MMP-2-mediated cellular invasion by NF-κB inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells:A model for early phase of metastasis[J]. Biochemical and Biophysical Research Communications, 2017, 485(1):76-81.
[13]Freitas RHCN, Fraga CAM. NF-κB-IKKβpathway as a target for drug development:realities, challenges and perspectives[J].Current Drug Targets, 2018, 19(16):1933-1942.
[14]王晓晨,吉爱国. NF-κB信号通路与炎症反应[J].生理科学进展, 2014, 45(1):68-71.
[15]Hayden MS, Ghosh S. Shared principles in NF-κB signaling[J].Cell, 2008, 132(3):344-362.
[16]Sun SC. The noncanonical NF-κB pathway[J]. Immunological Reviews, 2012, 246(1):125-140.
[17]Wang F, Yin J, Lu Z, et al. Limb ischemic preconditioning protects against contrast-induced nephropathy via renalase[J].EBioMedicine, 2016, 9(1):356-365.
[2]Oeckinghaus A, Ghosh S. The NF-κB family of transcription factors and its regulation[J]. Cold Spring Harbor Perspectives in Biology,2009, 1(4):1-15
[3]Niu J, Chang Z, Peng B, et al. Keratinocyte growth factor/fibroblast growth factor-7-regulated cell migration and invasion through activation of NF-κB transcription factors[J]. J Biol Chem, 2007,282(9):6001-6011.
[4]Di Prisco G, Iannaccone M, Ianniello F, et al. The neonicotinoid insecticide Clothianidin adversely affects immune signaling in a human cell line[J]. Sci Rep, 2017, 7(1):13446.
[5]包琦,赵凯,段子渊.猪Btnl5基因的克隆,表达分析及对NF-κB信号通路的调控作用[J].生物技术通报, 2017, 33(6):142-148.
[6]王珺婷,钟鸣,刘洁. IL-33的生物学功能以及对肿瘤作用的研究进展[J].生物学杂志, 2018, 35(3):76-79.
[7]Thompson AG, Thomas R. Induction of immune tolerance by dendritic cells:implications for preventative and therapeutic immunotherapy of autoimmune disease[J]. Immunology and Cell Biology, 2002, 80(6):509-519.
[8]印翠,张俊玲,施志仪,等.应用于miRNA靶标检测的双荧光素酶报告载体的构建及鉴定[J].生物技术通报, 2015, 31(12):180-185.
[9]Thorne N, Inglese J, Auld DS. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology[J]. Chemistry&Biology, 2010, 17(6):646-657.
[10]Baird AM, Godwin P, Heavey S, et al. 4:DHMEQ a novel therapeutic option for the treatment of inflammatory mediated cisplatin resistant non-small cell lung cancer[J]. Lung Cancer,2015, 87:S2.
[11]Kawaguchi M, Yamamoto K, Kanemaru A, et al. Inhibition of nuclear factor-κB signaling suppresses Spint1-deletion-induced tumor susceptibility in the ApcMin/+model[J]. Oncotarget,2016, 7(42):68614.
[12]Ukaji T, Lin Y, Okada S, et al. Inhibition of MMP-2-mediated cellular invasion by NF-κB inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells:A model for early phase of metastasis[J]. Biochemical and Biophysical Research Communications, 2017, 485(1):76-81.
[13]Freitas RHCN, Fraga CAM. NF-κB-IKKβpathway as a target for drug development:realities, challenges and perspectives[J].Current Drug Targets, 2018, 19(16):1933-1942.
[14]王晓晨,吉爱国. NF-κB信号通路与炎症反应[J].生理科学进展, 2014, 45(1):68-71.
[15]Hayden MS, Ghosh S. Shared principles in NF-κB signaling[J].Cell, 2008, 132(3):344-362.
[16]Sun SC. The noncanonical NF-κB pathway[J]. Immunological Reviews, 2012, 246(1):125-140.
[17]Wang F, Yin J, Lu Z, et al. Limb ischemic preconditioning protects against contrast-induced nephropathy via renalase[J].EBioMedicine, 2016, 9(1):356-365.