[1]李玉蕾,龙 隆,温 泉,等.共表达人kappa阿片受体与PKAcat-EGFP的CHO稳定细胞株建立及功能鉴定[J].中国药理学通报,2018,(09):1321-1326.[doi:10.3969/j.issn.1001-1978.2018.09.025]
 LI Yu-lei,LONG Long,WEN Quan,et al.Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification[J].Chinese Pharmacological Bulletin,2018,(09):1321-1326.[doi:10.3969/j.issn.1001-1978.2018.09.025]
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共表达人kappa阿片受体与PKAcat-EGFP的CHO稳定细胞株建立及功能鉴定()
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《中国药理学通报》[ISSN:/CN:]

卷:
期数:
2018年09期
页码:
1321-1326
栏目:
实验方法学
出版日期:
2018-09-26

文章信息/Info

Title:
Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification
文章编号:
1001-1978(2018)09-1321-06
作者:
李玉蕾12龙 隆1温 泉12王莉莉1宫泽辉12苏瑞斌12
军事科学院军事医学研究院毒物药物研究所1.抗毒药物与毒理学国家重点实验室、 2.神经精神药理学北京市重点实验室,北京 100850
Author(s):
LI Yu-lei12 LONG Long1 WEN Quan12 WANG Li-li1 GONG Ze-hui12 SU Rui-bin12
1.State Key Lab of Toxicology and Medical Countermeasures, 2.Beijing Key Lab of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Academy of Military Sciences, Beijing 100850,China
关键词:
kappa阿片受体 蛋白激酶A 稳定转染 高内涵分析 信号转导 中国仓鼠卵巢细胞
Keywords:
kappa opioid receptor PKA stable transfection high-content analysis signal transduction Chinese hamster ovary cell(CHO cell)
分类号:
R322.65; R329.24; R345.57; R392.11; R394.2
DOI:
10.3969/j.issn.1001-1978.2018.09.025
文献标志码:
A
摘要:
目的 在中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞上建立人kappa阿片受体(human kappa opioid receptor, hKOR)及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记的蛋白激酶A催化亚基(catalytic domain of cAMP-dependent protein kinase A, PKAcat)融合蛋白(PKAcat-EGFP)稳定共表达的细胞模型,为体外高通量筛选作用于hKOR的药物及药物分子机制研究打下基础。方法 通过脂质体介导法将潮霉素B抗性的hKOR重组质粒[pcDNA3.1/Hygro(+)-hKOR]转染入已稳定表达PKAcat-EGFP的CHO细胞中,随后用含潮霉素B的选择性培养基培养细胞,有限稀释法挑取耐药单克隆,PKA重分布实验筛选阳性克隆,利用Z'因子对建立的细胞模型的可靠性进行评价,利用PKA重分布实验与LANCE cAMP 384 Kit检测受体功能。结果 PKA重分布实验与LANCE cAMP 384 Kit结果表明,CHO-PKAcat-EGFP/hKOR-13号克隆反应性良好,100 nmol·L-1的U-50488作用时的Z'平均值为0.596,证明了该细胞模型的可靠性,且经多次传代后的受体表达也能保持稳定。结论 成功建立了hKOR与PKAcat-EGFP融合蛋白稳定共表达的细胞模型CHO-PKAcat-EGFP/hKOR-13。
Abstract:
Aim To establish a cell model which stably co-express human kappa opioid receptor(hKOR)and enhanced green fluorescent protein(EGFP)labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat)fusion protein(PKAcat-EGFP)in Chinese hamster ovary(CHO)cells, laying the foundation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resistant hKOR recombinant plasmid [pcDNA3.1/Hygro(+)-hKOR] was transfected into CHO cells stably expressing PKAcat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The positive clones were selected by PKA redistribution assay. Z' factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1 U-50488 for 30 min, the average value of Z' factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.

参考文献/References:

[1] Ehrich J M, Messinger D I, Knakal C R, et al. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons[J]. J Neurosci,2015,35(37):12917-31.
[2] Schindler A G, Messinger D I, Smith J S, et al. Stress produces aversion and potentiates cocaine reward by releasing endogenous dynorphins in the ventral striatum to locally stimulate serotonin reuptake[J]. J Neurosci,2012,32(49):17582-96.
[3] Tejeda H A, Counotte D S, Oh E, et al. Prefrontal cortical kappa-opioid receptor modulation of local neurotransmission and conditioned place aversion[J]. Neuropsychopharmacology,2013,38(9):1770-9.
[4] Singh S, Carpenter A E, Genovesio A. Increasing the content of high-content screening: an overview[J]. J Biomol Screen,2014,19(5):640-50.
[5] 孙雪娇,刘 祎,李 城,等.抗肾纤维化药物高内涵筛选模型的建立及应用[J].中国药理学通报,2017,33(3):327-34.
[5] Sun X J, Liu Y, Li C, et al. Establishment and application of high content screening model for anti-renal fibrosis drugs[J].Chin Pharmacol Bull,2017,33(3):327-34.
[6] Zhang J H, Chung T D, Oldenburg K R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays[J]. J Biomol Screen,1999,4(2):67-73.
[7] Von Voigtlander P F, Lewis R A. U-50,488, a selective kappa opioid agonist: comparison to other reputed kappa agonists[J]. Prog Neuropsychopharmacol Biol Psychiatry,1982,6(4-6):467-70.
[8] Feliciello A, Gottesman M E, Avvedimento E V. The biological functions of A-kinase anchor proteins[J]. J Mol Biol,2001,308(2):99-114.
[9] Almholt K, Tullin S, Skyggebjerg O, et al. Changes in intracellular cAMP reported by a redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera[J]. Cell Signal,2004,16(8):907-20.
[10] Zaccolo M, De Giorgi F, Cho C Y, et al. A genetically encoded, fluorescent indicator for cyclic AMP in living cells[J]. Nat Cell Biol,2000,2(1):25-9.
[11] Kohno M, Fukushima N, Yoshida A, et al. Gi1 and GoA differentially determine kinetic efficacies of agonists for kappa-opioid receptor[J]. FEBS Lett,2000,473(1):101-5.
[12] Alasbahi R H, Melzig M F. Forskolin and derivatives as tools for studying the role of cAMP[J]. Pharmazie,2012,67(1):5-13.

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备注/Memo

备注/Memo:
收稿日期:2018-05-25,修回日期:2018-06-28
基金项目:国家科技部“重大新药创制”科技重大专项(No 2015ZX09501003)
作者简介:李玉蕾(1980-),女,硕士,实验师,研究方向:神经精神药理学,E-mail:bettylpaine@126.com;
苏瑞斌(1973-),男,博士,研究员,研究方向:神经精神药理学,通讯作者,E-mail:ruibinsu@126.com
更新日期/Last Update: 2018-08-26