目的 建立基于囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator, CFTR)高通量筛选组胺H2受体(histocompatibility-2)调节剂的模型。方法 应用RT-PCR、Western blot检测H2受体在Fischer大鼠甲状腺滤泡上皮(FRT)细胞中表达情况。采用脂质体转染获取同时表达CFTR和黄色荧光蛋白双突变体YFP-H148Q/I152L的FRT细胞。荧光淬灭动力学实验检测细胞模型功能,判断该模型是否可以筛选H2受体的调节剂。结果 RT-PCR、Western blot结果显示,FRT内源性表达H2受体。采用倒置荧光显微镜观察,证实成功构建同时表达CFTR和YFP-H148Q/I152L的细胞模型。荧光淬灭动力学实验证明该模型具有cAMP激活氯离子通道的特性,可用于H2受体调节剂的筛选,且荧光斜率(slope)值与H2受体调节剂的浓度呈剂量依赖关系。结论 成功构建基于CFTR高通量筛选H2受体调节剂的细胞模型。
Abstract
OBJECTIVE To establish a high-throughput screening model of histamine H2 receptor regulators based on cystic fibrosis transmembrane conduction regulator (CFTR). METHODS RT-PCR and Western blot were used to detect the expression of H2 receptor in thyroid follicular epithelial (FRT) cells of Fischer rats. FRT cells expressing CFTR and YFP-H148Q/I152L were transfected with liposomes. Fluorescence quenching kinetics test was used to detect the function of the cell model to determine whether the model could screen regulators of H2 receptor. RESULTS RT-PCR and Western blot showed that FRT endogenous expressed H2 receptor. Fluorescence microscopy confirmed that CFTR and YFP-H148Q/I152L cell models were successfully constructed. The fluorescence quenching kinetics experiments showed that the model had the characteristics of cAMP activated chloride channels, and could be used to screen H2 receptor modulators. The slope of fluorescence was dose-dependent with the concentration of H2 receptor modulators. CONCLUSION The high-throughput screening cell model of H2 receptor modulators based on CFTR is successfully constructed.
关键词
囊性纤维化跨膜传导调节因子 /
H2受体 /
cAMP激活氯离子通道 /
大鼠甲状腺滤泡上皮细胞 /
高通量筛选细胞模型
{{custom_keyword}} /
Key words
CFTR /
H2 receptor /
cAMP activated chloride channel /
FRT cell /
high throughput screening cell model
{{custom_keyword}} /
中图分类号：
R965
R966
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] MORIGUCHI T, TAKAI J. Histamine and histidine decarboxylase: Immunomodulatory functions and regulatory mechanisms. Genes Cells, 2020, 25(7):443-449.
[2] PENG S Y, ZHUANG Q X, HE Y C, et al. Histamine excites neurons of the inferior vestibular nucleus in rats by activation of H1 and H2 receptors. Neurosci Lett, 2013, 541:87-92.
[3] TAHERPOUR A A, CHEGENI M, KHODAEI M M, et al. A first-principle DFT study of solvent effects on metiamide tautomers and imaginary interactions with H2-receptors. J Iran Chem Soc, 2017, 14(8):1-20.
[4] SHIM Y K, KIM N. The Effect of H2 Receptor Antagonist in Acid Inhibition and Its Clinical Efficacy. Korean J Gastroenterol, 2017, 70(1):4-12.
[5] ENNIS M, TILIGADA K. Histamine receptors and COVID-19. Inflamm Res, 2021, 70(1):67-75.
[6] YAMAMOTO T, ISONO A, MISHINA Y, et al. Gastroduodenal mucosal injury in patients taking low-dose aspirin and the role of gastric mucoprotective drugs: possible effect of rebamipide. J Clin Biochem Nutr, 2010, 47(1):27-31.
[7] LIU P, MCMENAMIN U C, JOHNSTON B T, et al. Use of proton pump inhibitors and histamine-2 receptor antagonists and risk of gastric cancer in two population-based studies. Br J Cancer, 2020, 123(2):307-315.
[8] YEO J H, LEE J. An Iodide-Yellow fluorescent protein-Gap Junction-intercellular communication assay. J Vis Exp, 2019, (144):e58966.
[9] GONG L L, FANG L H, PENG J H, et al. Development of high throughput screening method for Rho kinase inhibitor. Chin Pharm J(中国药学杂志), 2010, 45(8):580-584.
[10] JORDAKIEVA G, KUNDI M, UNTERSMAYR E, et al. Country-wide medical records infer increased allergy risk of gastric acid inhibition. Nat Commun, 2019, 10(1):3298.
[11] UNTERSMAYR E. Acid suppression therapy and allergic reactions. Allergo J Int, 2015, 24(8):303-311.
[12] WU L, ZHANG W, QIU X, et al. Identification of alkaloids from corydalisyanhusuo W. T. Wang as dopamine D1 receptor antagonists by using CRE-luciferase reporter gene assay. Molecules, 2018, 23(10):2585.
[13] BURFORD N T, WATSON J, ALT A. Standard curves are necessary to determine pharmacological properties for ligands in functional assays using competition binding technologies. Assay Drug Dev Technol, 2017, 15(7):320-329.
[14] OULD A Y, HEBERT-CHATELAIN E. Mitochondrial cAMP-PKA signaling: What do we really know. Biochim Biophys Acta Bioenerg, 2018, 1859(9):868-877.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金项目资助(81601234);吉林省教育厅基金项目资助(JJKH20170418KJ);吉林省卫生与健康技术创新项目资助(2018J113);吉林省大学生创新创业训练计划项目资助(202013706024);吉林省中医药管理局项目资助(2021092)
{{custom_fund}}
PDF(1330 KB)
