基于CFTR的胞浆内第二信使cAMP检测方法的建立*

doi:10.12047/j.cjap.6208.2022.015

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (1): 79-84.doi: 10.12047/j.cjap.6208.2022.015

基于CFTR的胞浆内第二信使cAMP检测方法的建立^*

吴明达^1,2, 刘雪莹³, 冯剑南³, 高雪伟³, 郝峰^1△, 高俊涛^4△

1.吉林医药学院检验学院, 吉林 132013;
2.延边大学医学院, 延吉 133002;
3.北华大学医学技术学院, 吉林 132013;
4.吉林医药学院基础医学院, 吉林 132013

收稿日期:2021-05-13 修回日期:2021-12-15 出版日期:2022-01-28 发布日期:2022-05-30
通讯作者: ^△Tel: 13689881986; E-mail: haof863@126.com,15948628662@163.com
基金资助:
* 国家自然科学基金(81601234);吉林省教育厅基金(JJKH20170418KJ);吉林省卫生与健康技术创新项目(2018J113);吉林省科技发展计划项目(20210101239JC);吉林省科技发展计划项目(20191102060YY);吉林省教育厅课题(JJKH20220462KJ)

Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm

WU Ming-da^1,2, LIU Xun-ying³, FENG Jian-nan³, GAO Xue-wei³, HAO Feng^1△, GAO Jun-tao^4△

1. College of Laboratory Medicine, Jilin Medical College, Jilin 132013;
2. School of Medicine, Yanbian University, Yanji 133002;
3. School of Medical Technology, Beihua University, Jilin 132013;
4. School of Basic Medicine, Jilin Medical College, Jilin 132013, China

Received:2021-05-13 Revised:2021-12-15 Online:2022-01-28 Published:2022-05-30

摘要/Abstract

摘要： 目的: 建立一种基于CFTR可敏感检测胞浆内第二信使cAMP的检测方法。方法: 构建CFTR和YFP-H148Q/I152L真核表达载体,应用脂质体转染法构建共表达CFTR和YFP-H148Q/I152L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;荧光淬灭动力学实验验证细胞模型的有效性;荧光淬灭动力学实验验证细胞模型可筛选CFTR调节剂;放射免疫法检测加入CFTR激活剂后细胞内的cAMP浓度。结果: 倒置荧光显微镜下观察到CFTR表达在细胞膜上,YFP-H148Q/I152L表达于胞浆中;成功构建共表达CFTR和YFP-H148Q/I152L的FRT细胞模型;荧光变化斜率值与CFTR调节剂浓度成剂量依赖关系,该模型可筛选CFTR调节剂;荧光变化斜率值可反映胞浆内cAMP浓度,该模型可敏感检测胞浆内cAMP浓度。结论: 此细胞模型可以高效敏感检测胞浆内第二信使cAMP浓度,为cAMP信号相关靶点的研究提供一种简便快捷的方法。

关键词: 囊性纤维化跨膜传导调节因子, 细胞模型, 第二信使, cAMP浓度

Abstract: Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.

Key words: cystic fibrosis transmembrane conductance regulator, cell model, second messenger, cAMP concentration

中图分类号:

Q2-33

吴明达, 刘雪莹, 冯剑南, 高雪伟, 郝峰, 高俊涛. 基于CFTR的胞浆内第二信使cAMP检测方法的建立^*[J]. 中国应用生理学杂志, 2022, 38(1): 79-84.

WU Ming-da, LIU Xun-ying, FENG Jian-nan, GAO Xue-wei, HAO Feng, GAO Jun-tao. Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm[J]. CJAP, 2022, 38(1): 79-84.

参考文献

[1] Wang JL, Gareri C, Rockman HA. G-protein-coupled receptors in heart disease[J]. Circ Res, 2018, 123(6): 716-735.
[2] Mao CQ, Zhuo X, Jiang YH. G protein-coupled receptors in cancer stem cells[J]. Curr Pharm Des, 2020, 26(17): 1952-1963.
[3] Heike B, Gunnar K. 3-iodothyronamine induces diverse signaling effects at different aminergic and non-aminergic G-protein coupled receptors[J]. Exp Clin Endocrinol Diabetes, 2020, 128(6-7): 395-400.
[4] Hopkins AL, Groom CR. The druggable genome[J]. Nat Rev Drug Discov, 2002, 1(9): 727-730.
[5] Akiyama H, Kamiguchi H. Second messenger networks for accurate growth cone guidance[J]. Dev Neurobiol, 2015, 75(4): 411-422.
[6] Yang L. Neuronal cAMP/PKA signaling and energy homeostasis[J]. Adv Exp Med Biol, 2018, 1090: 31-48.
[7] 孔祥权, 杨玉雯, 蒋静涵, 等. 尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道[J].中国应用生理学杂志, 2012, 28(1): 24-27.
[8] Peverelli E, Giardino E, Mangili F, et al. cAMP/PKA-induced filamin A (FLNA) phosphorylation inhibits SST2 signal transduction in GH-secreting pituitary tumor cells[J]. Cancer Lett, 2018, 435: 101-109.
[9] Line V, Hans B, Jesper MM. A cAMP biosensor-based high-throughput screening assay for identification of Gs-coupled GPCR ligands and phosphodiesterase inhibitors[J]. J Biomol Screen, 2015, 20(7): 849-857.
[10] Linsdell P. Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure[J]. Channels (Austin), 2018, 12(1): 284-290.
[11] Rodrigues DJ, McLoughlin D. Using reporter gene technologies to detect changes in cAMP as a result of GPCR activation[J]. Methods Mol Biol, 2009, 552: 373-382.
[12] 郑锴, 朱杭飞, 王长文, 等. YFP-H148Q/I152L在FRT细胞中的表达及其对碘离子敏感特性的研究[J]. 中国兽医杂志, 2015, 51(10): 11-13.
[13] Borovac J, Bosch M, Okamoto K. Regulation of actin dynamics during structural plasticity of dendritic spines: Signaling messengers and actin-binding proteins[J]. Mol Cell Neurosci, 2018, 91: 122-130.
[14] Kunapuli P, Ransom R, Murphy KL, et al. Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening[J]. Anal Biochem, 2003, 314(1): 16-29.
[15] 吴颖旭, 程杰坤, 李乐, 等. 杭白菊提取物对异丙肾上腺素诱导小鼠心肌肥厚的影响[J]. 中国应用生理学杂志, 2019, 35(5): 399-401.
[16] Wu LH, Zhang WY, Qiu X, et al. Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D₁ Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay[J]. Molecules, 2018, 23(10): 2585.
[17] Burford NT, Watson J, Alt A. Standard curves are necessary to determine pharmacological properties for ligands in functional assays using competition binding technologies[J]. Assay Drug Dev Technol, 2017, 15(7): 320-329.
[18] Ould AY, Hebert-Chatelain E. Mitochondrial cAMP-PKA signaling: What do we really know[J]. Biochim Biophys Acta Bioenerg, 2018, 1859(9): 868-877.
[19] Yangthara B, Mills A, Chatsudthipong V, Tradtrantip L, Verkman AS. Small-molecule vasopressin-2 receptor antagonist identified by a g-protein coupled receptor“pathway”screen[J]. Mol Pharmacol, 2007, 72(1): 86-94.

基于CFTR的胞浆内第二信使cAMP检测方法的建立^*

Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价