NK1R与5-HT1AR双重过表达SK-MEL-2稳转细胞株的构建
Construction of NK1R and 5-HT1AR Dual-Overexpressing SK-MEL-2 Stable Cell Lines
DOI: 10.12677/hjbm.2026.164082, PDF,    科研立项经费支持
作者: 马裕添, 彭 赞, 尚 靖*, 岳芸芸*：中国药科大学中药学院，江苏 南京
关键词: NK1R；5-HT1AR；稳转细胞株；慢病毒载体；蛋白质二聚体；NK1R； 5-HT1AR； Stable Cell Line； Lentiviral Vector； Protein Dimer
摘要: 目的：构建神经激肽-1受体(NK1R)与5-羟色胺1A受体(5-HT1AR)双重过表达的SK-MEL-2稳转细胞株，为色素性疾病的研究提供体外模型。方法：采用聚合酶链式反应扩增目的基因，将两种目的基因分别插入空载体GL215 pcSLenti-EF1-Puro-CMV-MCS-HA-WPRE和H5297 pLenti-CMV-EGFP-3xFLAG-PGK-BSR-WPRE中，构建pcSLenti-EF1-Puro-CMV-HTR1A-linker-mcherry-HA-WPRE和pLenti-CMV-TACR1-linker-EGFP-3xFLAG-PGK-BSR-WPRE慢病毒载体。采用慢病毒感染SK-MEL-2细胞，并通过嘌呤霉素和杀稻瘟菌素筛选稳转株。利用荧光显微观察验证受体表达水平及膜定位特征。结果：成功获得NK1R、5-HT1AR单过表达及双重过表达SK-MEL-2稳转细胞株。荧光显微观察显示单过表达细胞红色或绿色荧光清晰，双重过表达细胞红色和绿色荧光均有表达且两荧光通道图像合并后呈黄色荧光，阳性率 ≥ 90%，且受体主要定位于细胞膜。结论：成功建立了NK1R与5-HT1AR的单过表达和双重过表达的SK-MEL-2稳转细胞模型，为色素性疾病机制研究和药物筛选提供了稳定可靠的体外平台。
Abstract: Objective: This paper aims to construct SK-MEL-2 stable cell lines with dual overexpression of neurokinin-1 receptor (NK1R) and 5-hydroxytryptamine 1A receptor (5-HT1AR), thereby providing a receptor-defined in vitro model for mechanistic studies of pigmentary disorders. Methods: The target genes were amplified by polymerase chain reaction (PCR) and inserted into the lentiviral vectors GL215 pcSLenti-EF1-Puro-CMV-MCS-HA-WPRE and H5297 pLenti-CMV-EGFP-3xFLAG-PGK-BSR-WPRE, respectively, to construct pcSLenti-EF1-Puro-CMV-HTR1A-linker-mCherry-HA-WPRE and pLenti-CMV-TACR1-linker-EGFP-3xFLAG-PGK-BSR-WPRE lentiviral packaging plasmids. Lentiviruses were packaged in 293T cells and used to infect SK-MEL-2 cells. Single-overexpressing and dual-overexpressing stable cell lines were obtained through puromycin and blasticidin selection. The expression level and membrane localization of the receptors were verified by fluorescence microscopy. Results: SK-MEL-2 stable cell lines with single overexpression of NK1R or 5-HT1AR, as well as dual overexpression of NK1R and 5-HT1AR, were successfully established. Fluorescence microscopy showed clear red or green fluorescence in single-overexpressing cells, while yellow fluorescence was observed in the merged images of dual-overexpressing cells. The positive rate was ≥90%, and the receptors were mainly localized on the cell membrane. Conclusion: SK-MEL-2 stable cell models with single and dual overexpression of NK1R and 5-HT1AR were successfully established. These models provide a stable and reliable in vitro platform for mechanistic studies of pigmentary disorders and drug screening.
文章引用：马裕添, 彭赞, 尚靖, 岳芸芸. NK1R与5-HT1AR双重过表达SK-MEL-2稳转细胞株的构建[J]. 生物医学, 2026, 16(4): 804-809. https://doi.org/10.12677/hjbm.2026.164082

参考文献

[1] D’Mello, S.A.N., Finlay, G.J., Baguley, B.C. and Askarian-Amiri, M.E. (2016) Signaling Pathways in Melanogenesis. International Journal of Molecular Sciences, 17, Article No. 1144.
https://doi.org/10.3390/ijms17071144
[2] Zhang, M.Y., Chen, T., Lu, X., Lan, X.B., Chen, Z.Q. and Lu, S.Y. (2024) G Protein-Coupled Receptors (GPCRs): Advances in Structures, Mechanisms and Drug Discovery. Signal Transduction and Targeted Therapy, 9, Article No. 88.
https://doi.org/10.1038/s41392-024-01803-6
[3] Milligan, G. (2009) G Protein‐Coupled Receptor Hetero‐Dimerization: Contribution to Pharmacology and Function. British Journal of Pharmacology, 158, 5-14.
https://doi.org/10.1111/j.1476-5381.2009.00169.x
[4] Steinhoff, M.S., von Mentzer, B., Geppetti, P., Pothoulakis, C. and Bunnett, N.W. (2014) Tachykinins and Their Receptors: Contributions to Physiological Control and the Mechanisms of Disease. Physiological Reviews, 94, 265-301.
https://doi.org/10.1152/physrev.00031.2013
[5] Harris, J.A., Faust, B., Gondin, A.B., Dämgen, M.A., Suomivuori, C., Veldhuis, N.A., et al. (2022) Selective G Protein Signaling Driven by Substance P-Neurokinin Receptor Dynamics. Nature Chemical Biology, 18, 109-115.
https://doi.org/10.1038/s41589-021-00890-8
[6] Albert, P.R. and Vahid-Ansari, F. (2019) The 5-HT1A Receptor: Signaling to Behavior. Biochimie, 161, 34-45.
https://doi.org/10.1016/j.biochi.2018.10.015
[7] Wu, H.L., Pang, S.L., Liu, Q.Z., Wang, Q., Cai, M.X. and Shang, J. (2014) 5-HT1A/1B Receptors as Targets for Optimizing Pigmentary Responses in C57BL/6 Mouse Skin to Stress. PLOS ONE, 9, e89663.
https://doi.org/10.1371/journal.pone.0089663
[8] Wu, H.L., Zhao, Y.C., Huang, Q.L., Cai, M.X., Pan, Q., Fu, M.S., et al. (2018) NK1R/5‐HT1AR Interaction Is Related to the Regulation of Melanogenesis. The FASEB Journal, 32, 3193-3214.
https://doi.org/10.1096/fj.201700564rr
[9] Ping, F.F., Shang, J., Zhou, J., Song, J. and Zhang, L. (2012) Activation of Neurokinin-1 Receptor by Substance P Inhibits Melanogenesis in B16-F10 Melanoma Cells. The International Journal of Biochemistry & Cell Biology, 44, 2342-2348.
https://doi.org/10.1016/j.biocel.2012.09.025
[10] Fogh, J. and Trempe, G.L. (1975) New Human Tumor Cell Lines. In: Fogh, J., Ed., Human Tumor Cells in Vitro, Springer, 115-159.
https://doi.org/10.1007/978-1-4757-1647-4_5
[11] Naldini, L., Blömer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., et al. (1996) In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science, 272, 263-267.
https://doi.org/10.1126/science.272.5259.263

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