稳定表达TRPA1通道的HEK-293T细胞模型的建立
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摘要
目的建立稳定表达TRPA1的HEK-293T细胞模型,并鉴定模型构建是否成功。方法构建TRPA1真核表达质粒,采用脂质体转染法将其转入HEK-293T细胞中,经G418筛选稳定表达株,采用RT-PCR、免疫组化技术,检测HEK-293T细胞TRPA1基因的转录和蛋白的表达。结果经酶切、测序证明,TRPA1基因的真核重组表达质粒已成功构建;PCR、免疫组化检测结果表明,将此重组质粒转入HEK-293T细胞可稳定表达TRPA1基因。结论成功构建了能稳定表达TRPA1通道的HEK-293T细胞株,为体外研究TRPA1生理病理功能和筛选相关TRPA1通道调节剂奠定了基础。
Aim To establish a HEK-293 T cell line model stably expressing TRPA1 channel,and to verify the successful establishment of the model.Methods The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293 T cells by liposome transfection method,the stable expression strain was screened by G418,and the transcription and protein expression of TRPA1 gene in HEK-293 T cells were detected by RT-PCR and immunohistochemical techniques. Results After restriction enzyme digestion and sequencing,it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293 T cells with TRPA1 gene stable expression.Conclusions A HEK-293 T cell line with stable expression of TRPA1 channel is successfully constructed,which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TRPA1 channel regulators.
Aim To establish a HEK-293 T cell line model stably expressing TRPA1 channel,and to verify the successful establishment of the model.Methods The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293 T cells by liposome transfection method,the stable expression strain was screened by G418,and the transcription and protein expression of TRPA1 gene in HEK-293 T cells were detected by RT-PCR and immunohistochemical techniques. Results After restriction enzyme digestion and sequencing,it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293 T cells with TRPA1 gene stable expression.Conclusions A HEK-293 T cell line with stable expression of TRPA1 channel is successfully constructed,which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TRPA1 channel regulators.
引文
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[11]Benemei S,Fusi C,Trevisan G,et al.The TRPA1 channel in migraine mechanism and treatment[J].Br J Pharmacol,2014,171(10):2552-67.
[12]Bautista D M,Jordt S E,Nikai T,et al.TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents[J].Cell,2006,124(6):1269-82.
[13]Leishman E,Kunkler P E,Manchanda M,et al.Environmental toxin acrolein alters levels of endogenous lipids,including TRP agonists:a potential mechanism for headache driven by TRPA1 activation[J].Neurobiol Pain,2017,1:28-36.
[14]Lv J,Cao L,Zhang R,et al.A curcumin derivative J147 ameliorates diabetic peripheral neuropathy in streptozotocin(STZ)-induced DPN rat models through negative regulation AMPK on TRPA1[J].Acta Cir Bras,2018,33(6):533-41.
[15]Maurisse R,De Semir D,Emamekhoo H,et al.Comparative transfection of DNA into primary and transformed mammalian cells from different lineages[J].BMC Biotechnol,2010,10(1):9.
[2]Story G M,Peier A M,Reeve A J,et al.ANKTM1,a TRP-like channel expressed in nociceptive neurons,is activated by cold temperatures[J].Cell,2003,112(6):819-29.
[3]Sotomayor M,Corey D P,Schulten K.In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats[J].Structure,2005,13(4):669-82.
[4]Kono T,Kaneko A,Omiya Y,et al.Epithelial transient receptor potential ankyrin 1(TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine[J].Am J Physiol Gastrointest Liver Physiol,2013,304(4):G428-36.
[5]Yang W,You H,Ping M,et al.Role of transient receptor potential ion channels and evoked levels of neuropeptides in a formaldehydeinduced model of asthma in Balb/c mice[J].PLo S One,2013,8(5):e62827.
[6]Earley S.TRPA1 channels in the vasculature[J].Br J of Pharmacol,2012,167(1):13-22.
[7]王胜兰,戴毅,刘晓丽,等.TRP通道与偏头痛[J].中国药理学通报,2013,29(10):1352-4.[7]Wang S L,Dai Y,Liu X L,et al.TRP channels and migraine[J].Chin Pharmacol Bull,2013,29(10):1352-4.
[8]Boiko N,Medrano G,Montano E,et al.TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to vinca alkaloids[J].PLo S One,2017,12(10):e0186888.
[9]Chianese G,Sirignano C,Shokoohinia Y,et al.TRPA1 modulating C14polyacetylenes from the iranian endemic plant echinophora platyloba[J].Molecules,2018,23(7):E1750.
[10]Preti D,Saponaro G,Szallasi A.Transient receptor potential ankyrin 1(TRPA1)antagonists[J].Pharm Pat Anal,2015,4(2):75-94.
[11]Benemei S,Fusi C,Trevisan G,et al.The TRPA1 channel in migraine mechanism and treatment[J].Br J Pharmacol,2014,171(10):2552-67.
[12]Bautista D M,Jordt S E,Nikai T,et al.TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents[J].Cell,2006,124(6):1269-82.
[13]Leishman E,Kunkler P E,Manchanda M,et al.Environmental toxin acrolein alters levels of endogenous lipids,including TRP agonists:a potential mechanism for headache driven by TRPA1 activation[J].Neurobiol Pain,2017,1:28-36.
[14]Lv J,Cao L,Zhang R,et al.A curcumin derivative J147 ameliorates diabetic peripheral neuropathy in streptozotocin(STZ)-induced DPN rat models through negative regulation AMPK on TRPA1[J].Acta Cir Bras,2018,33(6):533-41.
[15]Maurisse R,De Semir D,Emamekhoo H,et al.Comparative transfection of DNA into primary and transformed mammalian cells from different lineages[J].BMC Biotechnol,2010,10(1):9.
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