CN105462930B - Cell model for screening kappa opioid receptor agonist and screening method - Google Patents
Cell model for screening kappa opioid receptor agonist and screening method Download PDFInfo
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Abstract
The invention belongs to the field of cell biology and pharmacy, and relates to a cell model for screening OPRK opioid receptor agonist and a screening method. In particular, the invention relates to a cell which expresses both opioid receptors and PKA. The invention also relates to a method for screening opioid receptor agonists, a method for evaluating the agonistic activity or cytotoxicity of a compound or composition on opioid receptors, and the use of said cells in screening opioid receptor agonists. The cell model established by the invention is sensitive, efficient and reliable, and can be used for high-throughput screening and/or high-content screening of opioid receptors, particularly OPRK opioid receptor agonists.
Description
Technical Field
The invention belongs to the field of cell biology and pharmacy, and relates to a cell model for screening kappa opioid receptor (OPRK) agonists and a screening method.
Background
The drug screening model is an indispensable research platform for drug development. High content analysis or High Content Screening (HCS) is a cell-based detection object that relies on a high resolution cell imaging system, fully integrates the advantages of resources such as sample preparation technology, automation equipment, data management systems, detection reagents, bioinformatics, and the like, and achieves diversified, rapid, and large-scale Screening of candidate drugs at the cellular or molecular level. The HCS technology can simultaneously detect the influence of the candidate drug on various links of cell morphology, growth, differentiation, migration, apoptosis, metabolic pathways and signal transduction on the premise of keeping the integrity of cell structure and function, and acquire a large amount of related information in a single experiment to determine the biological activity and potential toxicity of the candidate drug.
Opioid receptors (OPRs), including kappa opioid receptor (OPRK), mu opioid receptor (OPRM), opioid receptor (OPRD), and opioid receptor-like receptor (ORL1), are currently the most important class of targets for potent analgesics, but are characterized by analgesia and dependence co-existence, thus severely impacting clinical utility.
In the last 30 years, the study of opioid receptors and opioids has become an active area in neuropharmacology, molecular pharmacology and the whole neurobiology research, and people hope to find novel powerful analgesics with strong analgesic effect and small dependence potential.
There is sufficient evidence that OPRK agonists have strong analgesic effects in both experimental animals and humans, have low self-dependent potential, and are able to combat mu opioid receptor agonist-induced addiction. Thus, the great attention of the vast pharmaceutical and pharmacological workers is drawn, and OPRK is considered to be an analgesic target with great research and development prospects.
OPRK genus G protein-coupled receptors (GPCRs), and Gi/oCoupled inhibition of Adenylyl Cyclase (AC) activity results in reduced cAMP production in nerve cells, thereby inhibiting neuronal activity.
OPRK comprises 3 binding sites (also reported as subtypes), meaning different sites of the same receptor to which a particular agonist has different binding capacity, designated kappa1、κ2And kappa3. As early asAttalii (Attalii B, Guarders C, Mazurquil H et al. event for multiple "kappa" binding sites byuse of opioid peptides in the guineae-pig-clinical laboratory. Neuroptides 1982; 3: 53-64) et al were used in 19823H-etorphine and unlabelled Dyn (1-27), compound DADLE and guinea pig spinal cord kappa receptor are subjected to competitive inhibition and saturable receptor binding tests, and the kappa receptor is found to have two binding sites with different characteristics, namely a DADLE sensitive type and a DADLE non-sensitive type. The authors call the former kappa1Subtype of kappa2The subtype is. Subsequently, Clark et al (Clark JA, Jin L, Price M et al. kappa. arbitrary receiver multiplicitu: evaluation for two U50488-sensory κ)1subtypes and a novelκ3subtype.j parmacoloexp ther.1989; 251: 461-76) in receptor binding assays of calf striatum3The subtype is.
Thus, since κ1、κ2And kappa3The subtypes belong to different sites of specific ligands on the same receptor, and the expressed full gene sequence of the kappa opioid receptor can achieve the same or similar technical effects.
cAMP is a second messenger of intracellular information transfer, and cAMP-dependent Protein Kinase A (PKA) is a serine/threonine protein kinase ubiquitous in eukaryotic cells, and plays an important role in information regulation and cell function regulation. The PKA holoenzyme is an R2C2 tetramer, comprising a regulatory (R) dimer and two identical catalytic (C) subunits. The catalytic subunit in the tetramer has no catalytic activity, the regulation subunit and the catalytic subunit are dissociated after PKA activation, and free catalytic subunits can freely move in cytoplasm and phosphorylate intracellular components.
In the cell model of the invention, the cell expresses a fusion protein of the catalytic subunit of human PKA and EGFP, namely PKAcat-EGFP. The activation of PKA is mainly influenced by intracellular cAMP concentration. In untreated cells, the cAMP concentration was low and PKAcat-EGFP exhibited high-brightness fluorescent particle aggregates in the cytoplasm. When the cAMP concentration is increased, PKAcat-EGFP is released from the aggregates and appears as a decrease in cytoplasmic fluorescent granules. The activation degree of OPRK can be detected by utilizing the redistribution phenomenon of PKAcat-EGFP.
Since OPRK is Gi/oCoupled to the receptor, activation inhibits cAMP formation, resulting in PKAcat-EGFP to form granules within the cytoplasm, consistent with untreated cell performance. Therefore, it is necessary to add AC agonist (such as sodium fluoride, forskolin, etc., preferably forskolin) to make the cAMP present in high concentration in the cell, so that PKAcat-EGFP is dispersed in the cytoplasm.
According to literature reports, most of the existing methods for screening the OPRK agonist are radioligand receptor binding experiments of cell membrane receptors, cAMP detection kits, dual-luciferase reporter gene analysis and the like, and have the following defects of large required cell amount, tedious process, long experimental period, radioactive determination, use of expensive acting substrates or auxiliary factors and more invested funds and manpower.
To date, there has been no report of a cell model based on high content analysis, high throughput screening of OPRK agonists.
Disclosure of Invention
The present inventors have established a cell model by creative efforts and extensive experiments and have surprisingly found that efficient screening for OPRK agonists or AC inhibitors can be achieved by a re-distribution of PKA within the cell and nuclear morphology analysis. The following invention is thus provided:
one aspect of the invention relates to a cell that expresses both opioid receptors and PKA.
The cell according to any one of the present invention, which satisfies any one or more of the following (1) to (5):
(1) the opioid receptors are kappa opioid receptors, mu opioid receptors, opioid receptors or opioid receptor-like receptors; in particular, the kappa opioid receptor is kappa1Type, kappa2Type or kappa3Kappa opioid receptors of type; the opioid receptors are of human origin; in particular, the opiatesThe amino acid sequence of the receptor is shown as SEQ ID NO: 2 is shown in the specification;
(2) the PKA is of human origin; specifically, the PKA is a PKA catalytic subunit; specifically, the PKA catalytic subunit is in single copy or multiple copies; specifically, the amino acid sequence of the PKA catalytic subunit is shown as SEQ ID NO: 1 is shown in the specification;
(3) opioid receptors and PKA are expressed non-fusionally; specifically, the opioid receptor and the PKA protein are constructed on different expression vectors;
(4) the PKA is linked to a detectable label; in particular, the detectable label is a reporter protein; more specifically, the reporter gene protein is an EGFP protein; specifically, the sequence of the EGFP protein is shown as SEQ ID NO: 3 is shown in the specification;
(5) the cells are mammalian cells cultured in vitro; chinese hamster ovary cells (e.g., Chinese hamster ovary cells CHO-K1) are preferred.
In the present invention, PKA and OPRK can refer to sequences of GenBank accession numbers NM002730 and AF498922, respectively, and encoded amino acid sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
In one embodiment of the invention, the amino acid sequence of the catalytic subunit of PKA is as follows (351 aa):
MGNAAAAKKGSEQESVKEFLAKAKEDFLKKWESPAQNTAHLDQFERIKTLGTGSFGRVMLVKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF(SEQ ID NO：1)
in one embodiment of the invention, the amino acid sequence of OPRK is as follows (380 aa):
MESPIQIFRGEPGPTCAPSACLPPNSSAWFPGWAEPDSNGSAGSEDAQLEPAHISPAIPVIITAVYSVVFVVGLVGNSLVMFVIIRYTKMKTATNIYIFNLALADALVTTTMPFQSTVYLMNSWPFGDVLCKIVISIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALDFRTPLKAKIINICIWLLSSSVGISAIVLGGTKVREDVDVIECSLQFPDDDYSWWDLFMKICVFIFAFVIPVLIIIVCYTLMILRLKSVRLLSGSREKDRNLRRITRLVLVVVAVFVVCWTPIHIFILVEALGSTSHSTAALSSYYFCIALGYTNSSLNPILYAFLDENFKRCFRDFCFPLKMRMERQSTSRVRNTVQDPAYLRDIDGMNKPV(SEQ ID NO：2)
in one embodiment of the invention, the amino acid sequence of EGFP is as follows (239 aa):
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO：3)
the cells of the invention may be used to screen for opioid receptor agonists or to evaluate the agonist activity of a test sample, e.g., a compound or composition, at an opioid receptor, i.e., as a cellular model for screening for opioid receptor agonists or evaluating the agonist activity of a compound or composition at an opioid receptor. Wherein, in particular, the opioid receptor is a kappa opioid receptor (OPRK). The reliability of this cell model is demonstrated in example 5 of the present invention.
In the cell model, the redistribution degree of a receptor-mediated cell signaling downstream protein molecule can reflect the activation degree of OPRK, and the nuclear morphology analysis can reflect cytotoxicity. The model is used for screening opioid receptor agonists, and the activity of activating OPRK of the compound is evaluated by detecting the redistribution degree of downstream protein molecules and the cytotoxicity is detected by nuclear morphology analysis.
The cell can be prepared by the following steps:
1) constructing a eukaryotic expression vector containing a human kappa opioid receptor gene;
2) transfecting a vector into a Chinese hamster ovary cell stably expressing a PKA protein (e.g., a PKAcat-EGFP fusion protein);
3) cell lines stably expressing the human kappa opioid receptor and PKA proteins (e.g., PKAcat-EGFP fusion protein) were screened.
The cells of the invention can also be made by co-transfecting empty chinese hamster ovary cells with a vector containing a human kappa opioid receptor gene, e.g., (OPRK gene), and a vector containing a PKA gene, e.g., PKAcat-EGFP gene.
PKAcat represents the catalytic subunit of PKA protein.
The invention also relates to a cell/cell strain (Chinese hamster ovary cell strain, CHO-PKAcat-EGFP/OPRK), the preservation number is CGMCC No.9002, the preservation date is 2014, 3 and 26 days, and the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms.
The cell strain is Chinese hamster ovary cells CHO-K1 capable of stably expressing OPRK and PKAcat-EGFP fusion proteins.
Another aspect of the invention relates to a method for screening for opioid receptor agonists or adenylate cyclase inhibitors, or for evaluating/determining the agonistic activity at the opioid receptor or the inhibitory activity at the adenylate cyclase or the cytotoxicity of a test sample, comprising the step of using a cell according to any one of the invention.
The method according to any one of the invention, comprising the steps of:
after or simultaneously adding a sample to be detected, adding an adenylate cyclase agonist, and detecting the redistribution degree of the PKA; preferably, cells in the same culture conditions but with addition of only adenylate cyclase agonist are used as control cells; in particular, the adenylate cyclase agonist is sodium fluoride or forskolin. Specifically, the final concentration of an adenylate cyclase agonist, such as forskolin, is 1-100. mu. mol/L, 1-50. mu. mol/L, 1-20. mu. mol/L, 1-15. mu. mol/L, 1-10. mu. mol/L, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. mu. mol/L.
The method according to any of the invention, wherein:
adding the adenylate cyclase agonist after adding the sample to be detected; specifically, the adenylate cyclase agonist is added 1-60 minutes, 5-60 minutes, 10-50 minutes, 15-45 minutes, 20-40 minutes, 20-35 minutes (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 minutes), 25-35 minutes, or 30 minutes after the addition of the sample to be tested; and/or
The extent to which redistribution of PKA occurs is detected after 1-20 minutes, 1-15 minutes, 1-10 minutes, 3-15 minutes, 3-10 minutes or 5 minutes after addition of the adenylate cyclase agonist.
The method according to any one of the invention, comprising the steps of:
1) inoculating and culturing a cell according to any one of the present invention;
2) discarding the culture medium, and washing the cells at least 1 time with a serum-free culture medium;
3) adding a sample to be tested, and culturing in a serum-free culture medium;
4) adding forskolin, and continuing culturing in a serum-free culture medium;
5) detecting the redistribution degree of the PKA.
The method according to any one of the present invention satisfies any one or more of the following (1) to (4):
(1) the inoculation density in the step 1) is 0.6 multiplied by 104－2.0×104Culturing for 12-48 hours per mL; preferably, the incubation time is 24 hours;
(2) the culture conditions in step 1), 3) or 4) were 37 ℃ and 5% CO295% humidity;
(3) the serum-free culture medium in the step 2), 3) or 4) is F12;
(4) before detecting the redistribution degree of the PKA protein in the step 5), the method comprises the following pretreatment steps: the cells were fixed with a 12% formaldehyde solution diluted with PBS, left at room temperature for 20 minutes in the dark, discarded, added with PBS containing the nuclear dye hoechst33342, and left at room temperature for 30 minutes in the dark.
Without being limited by theory, the sample to be tested (kappa opioid receptor agonist) acts on OPRK firstly and mediates the inhibition of AC at the downstream of a signal path, and forskolin directly activates AC to reduce PKA-EGFP particles, so that forskolin is added after the sample to be tested is added, and at least the compound and the forskolin are added simultaneously to detect and analyze the redistribution degree of PKA and evaluate the agonistic activity of the compound on opioid receptors.
A method of assessing the cytotoxicity of an OPRK opioid receptor agonist or a compound or composition according to any one of the invention, comprising the steps of:
cells were treated with the compound for 24 hours, 30 minutes before the end, treated with hoechst33342 for 30 minutes, examined and analyzed for nuclear morphology, and the cytotoxicity of the compound was evaluated.
Hoechst33342 is a DNA dye which can emit indigo fluorescence at 461nm under the excitation of ultraviolet light at about 350 nm. Specifically for labeling DNA under fluorescent microscopic observation. Since the DNA can be labeled, such staining can also be used to identify the location of the nucleus and mitochondria containing the DNA.
According to the method of any one of the invention, by detecting whether the redistribution of PKAcat-EGFP with respect to control cells (cells of the same culture conditions but with addition of only adenylate cyclase agonist); if so, it is an opioid receptor agonist or an adenylate cyclase inhibitor. Specifically, a Cell image can be acquired by using In Cell Analyzer2000, and whether PKAcat-EGFP redistribution occurs or not can be determined; optionally, the extent of redistribution of PKAcat-EGFP is analyzed using a multi-target analysis module.
The invention also relates to cells obtained by the treatment according to any one of the methods described above and/or control cells.
Yet another aspect of the invention relates to a cell model or a kit comprising a cell according to any of the invention.
Yet another aspect of the invention relates to the use of the cell of any one of the above in screening for opioid receptor agonists or adenylate cyclase inhibitors, or in evaluating the opioid receptor agonistic activity or adenylate cyclase inhibitory activity or cytotoxicity of a test sample; specifically, the screening is high content screening; in particular, the opioid receptor is a kappa opioid receptor; in particular, the kappa opioid receptor is kappa1Type, kappa2Type or kappa3And (4) molding.
The method for screening opioid receptor agonists according to any one of the present invention, wherein the incubation time after addition of the OPRK opioid receptor agonist U50488 is 5-60 minutes; preferably, 20-35 minutes; more preferably, it is 30 minutes.
The method of screening for an OPRK opioid receptor agonist according to any one of the present invention, which satisfies one or more of the following (1) to (5):
(1) the inoculation density in step 1) is 6X 10 according to a 96-well plate4－20×104Culturing for 12-48 hours per mL; preferably 24 hours;
(2) the culture conditions in step 1), 3) or 4) were 37 ℃ and 5% CO295% humidity;
(3) the serum-free culture medium in the step 2), 3) or 4) is F12;
(4) the culture time after the compound or the composition to be detected is added in the step 3) is 15-45 minutes; preferably 30 minutes;
(5) before analyzing the redistribution degree of the PKA protein in the step 5), the method comprises the following pretreatment steps: the cells were fixed with a 12% formaldehyde solution diluted with PBS, left at room temperature for 20 minutes in the dark, discarded, added with PBS containing the nuclear dye hoechst33342, and left at room temperature for 30 minutes in the dark.
The above-described methods for screening for OPRK opioid receptor agonists are also useful as methods for evaluating the agonist activity of a compound or composition at an opioid receptor.
The screening method according to any one of the present invention, which is high content screening.
The High Content Screening (HCS) HCS is an automatic platform applying High-throughput fluorescence/bright field microscopic imaging and quantitative image analysis, integrates open reagents and a fluorescent label technology, simultaneously detects the influence of a screened sample on each link of cell morphology, growth, differentiation, migration, apoptosis, metabolic pathway and signal transduction on the premise of keeping the integrity of cell structures and functions, acquires a large amount of related information in a single experiment, and determines the biological activity and potential toxicity of the screened sample.
The invention also relates to cells/cell models obtained by treatment according to the screening method described in any one of the above.
In the present invention, the sample to be tested may be a compound or a mixture, including but not limited to: compositions, extracts, and the like.
In the present invention, the opioid receptor is a kappa opioid receptor (OPRK), a mu opioid receptor (OPRM), an opioid receptor (OPRD), or an opioid receptor-like receptor (OPRL 1); in particular, the kappa opioid receptor is selected from kappa1Type, kappa2Type and kappa3Any 1, 2 or 3 of the forms.
Advantageous effects of the invention
The cell model established by the invention is sensitive, efficient and reliable, and can be used for high-throughput screening and/or high-content screening of OPRK opioid receptor agonists. Compared with a radioligand receptor binding experiment, a cAMP detection kit, dual-luciferase reporter gene analysis and the like, the screening method established by the invention has the advantages of high sensitivity, few operation steps, no need of using isotopes, substrates or auxiliary factors, capability of initially judging cytotoxicity while detecting activity, intuitive result, simple and convenient operation steps and high speed.
Drawings
FIG. 1: u50488 induces redistribution of PKAcat-EGFP in cells expressing human OPRK. Wherein, FIG. 1A is a cell image of the redistribution of PKAcat-EGFP; FIG. 1B shows the degree of redistribution of PKAcat-EGFP as a particle formation index.
FIG. 2: u50488 induces a dose-effect relationship in which PKAcat-EGFP is redistributed. Wherein the final concentration of U50488 in FIGS. 2A-2J is 5.1 × 10-11、1.5/10-10、4.6×10-10、1.4×10-9、4.1×10-9、1.2×10-8、3.7×10-8、1.1×10-7、3.3×10-7、1.0×10-6Cell image of mol/L; FIG. 2K is a "S" type curve fitted by GraphPad Prism5 software Nonlinear regression (curve fit). n is 3, EC50It was 3.563. + -. 0.902 nM.
FIG. 3: u50488 has cytotoxic effect. n is 3.
FIG. 4: and (3) carrying out reliability analysis on a drug screening system for inducing redistribution of PKAcat-EGFP by U50488. n is 6, and the average Z' value is 0.596.
FIG. 5: effect of U50488 treated cell time on PKAcat-EGFP redistribution.
FIG. 6: effect of CHO-PKAcat-EGFP/OPRK cell seeding Density on PKAcat-EGFP redistribution.
FIG. 7: the OPRK agonist Dihydroetorphine (DHE) induces a dose-effect relationship in which PKAcat-EGFP is redistributed. (n is 3, EC)50The concentration was 5.030. + -. 0.421 nM. )
FIG. 8: the cytotoxic effects of DHE. n is 3.
Description of biological Material preservation
The present invention relates to the following biomaterials:
chinese hamster ovary cells (CHO-PKAcat-EGFP/OPRK) are preserved in China general microbiological culture Collection center (CGMCC) at 26 months and 3 months in 2014, the preservation number is CGMCC No.9002, the preservation address is No. 3 of the Beijing West Lu No. 1 of the morning district, the institute of microbiology, 100101.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Main drugs and reagents: plasmid miniprep kit was purchased from Omega Bio-tek; restriction enzymes were purchased from Thermo Scientific, and T4 ligase was purchased from TaKaRa; liposome Lipofectamine2000, hoechst33342, FBS, hygromycin B from Invitrogen; f12 was purchased from Hyclone; g418 was purchased from Merck; u50488 was purchased from Sigma; the receptor agonist used was synthesized by a chemical synthesis chamber; other reagents are domestic analytical pure reagents.
The main apparatus is as follows: in Cell Analyzer1000 or In Cell Analyzer2000GE healthcare science.
Cell line: CHO-PKAcat-EGFP Chinese hamster ovary cell line Thermo Scientific.
Example 1: establishment of cell lines stably expressing human OPRK and PKAcat-EGFP
1. Vector construction
Constructing pcDNA3.1/Hygro (+) -OPRK eukaryotic expression vector containing human OPRK full-length cDNA.
The purchased plasmids pcDNA3.1(+) -hKOR (purchased from Missouri University of Science & Technology) and pcDNA3.1/Hygro (+) plasmids were restriction-digested with Hind III, Xba I, the cleavage products were separated by electrophoresis on 1% agarose gel, and DNA fragment bands (hKOR fragment, SEQ ID NO: 4) were excised under a UV transilluminator. The agarose gel DNA recovery kit recovers DNA fragments. The recovered target fragment and vector fragment were ligated in a molar ratio (1: 1). The ligation products were transformed into DH5 alpha fresh competent cells and screened on LB plates (with ampicillin). Several single clones were inoculated separately in 5mL liquid LB medium (containing ampicillin) for scale-up culture. And taking the bacterial liquid and sending the bacterial liquid to a company for sequencing and comparison. And selecting the bacterial positive clone with the completely correct sequence alignment result for subsequent experimental operation.
hKOR fragment, 1143 bp:
ATGGAATCCC CGATTCAGAT CTTCCGCGGG GAGCCGGGCCCTACCTGCGC CCCGAGCGCCTGCCTGCCCC CCAACAGCAGCGCCTGGTTT CCCGGCTGGG CCGAGCCCGA CAGCAACGGCAGCGCCGGCTCGGAGGACGC GCAGCTGGAG CCCGCGCACATCTCCCCGGC CATCCCGGTC ATCATCACGGCGGTCTACTCCGTAGTGTTC GTCGTGGGCT TGGTGGGCAA CTCGCTGGTCATGTTCGTGA TCATCCGATACACAAAGATG AAGACAGCAACCAACATTTA CATATTTAACCTGGCTTTGG CAGATGCTTTAGTTACTACAACCATGCCCT TTCAGAGTAC GGTCTACTTGATGAATTCCT GGCCTTTTGG GGATGTGCTGTGCAAGATAGTAATTTCCAT TGATTACTAC AACATGTTCA CCAGCATCTTCACCTTGACC ATGATGAGCGTGGACCGCTA CATTGCCGTGTGCCACCCCG TGAAGGCTTT GGACTTCCGC ACACCCTTGAAGGCAAAGATCATCAATATC TGCATCTGGC TGCTGTCGTCATCTGTTGGC ATCTCTGCAA TAGTCCTTGGAGGCACCAAAGTCAGGGAAG ACGTCGATGT CATTGAGTGC TCCTTGCAGTTCCCAGATGA TGACTACTCCTGGTGGGACC TCTTCATGAAGATCTGCGTC TTCATCTTTG CCTTCGTGAT CCCTGTCCTCATCATCATCGTCTGCTACAC CCTGATGATC CTGCGTCTCAAGAGCGTCCG GCTCCTTTCT GGCTCCCGAGAGAAAGATCGCAACCTGCGT AGGATCACCA GACTGGTCCT GGTGGTGGTGGCAGTCTTCG TCGTCTGCTGGACTCCCATT CACATATTCATCCTGGTGGA GGCTCTGGGG AGCACCTCCC ACAGCACAGCTGCTCTCTCCAGCTATTACT TCTGCATCGC CTTAGGCTATACCAACAGTA GCCTGAATCC CATTCTCTACGCCTTTCTTGATGAAAACTT CAAGCGGTGT TTCCGGGACT TCTGCTTTCCACTGAAGATG AGGATGGAGCGGCAGAGCAC TAGCAGAGTCCGAAATACAG TTCAGGATCC TGCTTACCTG AGGGACATCGATGGGATGAATAAACCAGTA TGA(SEQ ID NO：4)
2. cell transfection
The pcDNA3.1/Hygro (+) -oprk eukaryotic expression vector was transfected by liposome method (Lipofectamine 2000 transfection reagent from Invitrogen, cat # 11668019) into a Chinese hamster ovary cell line (purchased from Thermo Scientific) that had been fused to express PKAcat-EGFP.
3. Monoclonal screening
Novel cell lines stably expressing human OPRK are obtained by resistance selection and a limiting dilution method. The specific operation steps are as follows:
48h after transfection, the cell culture medium was changed to F12 medium containing 200. mu.g/mL HygroB, 500. mu.g/mLG 418 and 10% fetal bovine serum, and the culture was continued for 10 days, with changes every 2 days. After 10 days, the polyclonal cells were digested, diluted and mixed at a density of 100 cells/20 mL, inoculated into a 96-well plate (0.2 mL/well), cultured for 3-4 days, observed under an inverted microscope, and wells containing only one clone were selected as a marker. After 7 days, cell clones were digested and a portion of the cells were plated into 96-well plates for PKA redistribution experiments.
Using F12 medium containing 10% fetal bovine serum, 500. mu.g/mL G418 and 50. mu.g/mL hygromycin B, the mixture was incubated at 37 ℃ with 5% CO2And culturing in an incubator with 95% humidity.
The chinese hamster ovary cells/cell lines (CHO-PKAcat-EGFP/OPRK) prepared in this example were collected in the common microorganism center of the committee for culture collection of chinese microorganisms (CGMCC) at 26 months 3 and 2014, with a collection number of cgmccno.9002, a collection address of north institute of china academy of sciences No. 1 north school 3 of the sunward district of beijing, and 100101.
Compared with the cells before transfection, the cell morphology obtained by establishment is not obviously changed, and the growth state is stable.
The cell line prepared in the embodiment can be used as a cell model for screening opioid receptor agonists or evaluating the agonist activity and cytotoxicity of compounds or compositions on the opioid receptors.
In the following examples, the cells/cell lines used were those constructed in the present example, unless otherwise specified.
Example 2: identification of Chinese hamster ovary cells (CHO-PKAcat-EGFP/OPRK)
The cell strain prepared in example 1 was treated with an agonist U50488 to activate OPRK, which in combination with forskolin induces the redistribution of PKAcat-EGFP. The receptor of the human kappa opioid receptor in the established cell strain is activated after being combined with a specific ligand U50488, the activity of AC is regulated, the concentration of cAMP is further influenced, and PKAcat-EGFP redistribution occurs after the cAMP and forskolin interact.
In order to quantitatively detect the redistribution degree of the PKAcat-EGFP induced by U50488, In this example, an In Cell Analyzer1000 or an In Cell Analyzer2000 was used to obtain a Cell image of the redistribution of PKAcat-EGFP, and the obtained Cell image was analyzed by a Multi-Target Analysis module (Multi Target Analysis) to express the redistribution degree of PKAcat-EGFP as a particle formation index. The particle formation index is equal to the Intensity-Background Intensity x TotalArea per well. The method comprises the following specific steps:
1) inoculating the cells in a black, bottom-permeable 96-well culture plate at 37 deg.C with 5% CO2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with 100. mu.L/well of serum-free F12 medium (hereinafter referred to as cell assay solution). Thereafter, 100. mu.L/well of cell assay solution was added.
3) Cell assay solution containing U50488 was added at 50. mu.L/well to give a final concentration of U50488 of 100nmol/L and incubated in a cell incubator at 37 ℃ for 30 minutes.
4) Cell assay solution containing forskolin was added at 50. mu.L/well to a final concentration of 10. mu. mol/L, and incubated in a cell incubator at 37 ℃ for 5 minutes.
5) Cells were fixed with 12% formaldehyde diluted with 1 × PBS, 100 μ L/well, and left at room temperature for 20 minutes in the dark.
6) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
7) Cell images were acquired using In Cell Analyzer1000, and the degree of redistribution of PKAcat-EGFP was analyzed using a Multi-target analysis module (Multi TargetAnalyzers).
The experiment process of the CHO-PKAcat-EGFP and the CHO-PKAcat-EGFP/OPRK cells is completely consistent, and forskolin is added.
If as shown in fig. 1A and 1B.
As can be seen from the results, significant redistribution of PKAcat-EGFP in human OPRK expressing CHO-PKAcat-EGFP/OPRK cells occurred compared to CHO-PKAcat-EGFP cells not expressing human OPRK at a concentration of 100nmol/L of U50488 (FIG. 1, A-B).
In addition, since CHO-PKAcat-EGFP cells do not express human OPRK and therefore do not respond to U50488, but only to forskolin, AC is activated and ultimately appears as a reduction in particles.
Example 3: determination of half-effective concentration of U50488 induced PKAcat-EGFP redistribution
In [ 2 ]35S]EC of U50488 in GTP γ S binding experiments50＝3.1nM(Zhu J,Luo LY,LiJG.Activation of the cloned human kappa opioid receptor by agonists enhances[35S]GTPgammaS binding to membranes:determination of potencies and efficaciesof ligands[J].J Pharmacol Exp Ther,1997,282(2):676-84)。
The method comprises the following steps:
1) the cells prepared in example 1 were prepared to 1X 105one/mL cell suspension was plated in black, bottomless 96-well plates at 100. mu.L/well.
2) At 37 ℃ with 5% CO2And culturing in a 95% humidity incubator for 24 hours.
3) Cells were washed 1 time with cell assay solution at 100 μ L/well; the solution was discarded and 100. mu.L/well of cell assay solution was added.
4) Adding diluted U50488 to make the final concentration 5.1 × 10-11、1.5/10-10、4.6×10-10、1.4×10-9、4.1×10-9、1.2×10-8、3.7×10-8、1.1×10-7、3.3×10-7、1.0×10-6mol/L，37℃，5％CO2Incubate 30 minutes in a 95% humidity incubator.
5) Adding cell analysis solution containing forskolin to a final concentration of 10. mu. mol/L at 37 ℃ and 5% CO2Incubate 5 minutes in a cell incubator at 95% humidity.
6) Add cell fixative at 100. mu.L/well, mix well by shaking the plate gently, and leave it in the dark at room temperature for 20 minutes.
7) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
8) Cell images were acquired using In Cell Analyzer2000 and the degree of redistribution of PKAcat-EGFP was analyzed using a multi-target analysis module.
The results are shown in FIGS. 2A-2K.
As shown in FIGS. 2A-2J, the PKAcat-EGFP fusion protein began to redistribute at a concentration of 1nmol/L of U50488. The extent of redistribution of the PKAcat-EGFP fusion protein was maximized at a concentration of 500nmol/L of U50488. Fitting the "S" type curve (FIG. 2K) with GraphPad Prism5 software Nonlinear regression (curve fit) yields the half-effective concentration (EC 50488) for activating OPRK receptor in CHO-PKAcat-EGFP/OPRK cells to induce redistribution of the fusion protein PKAcat-EGFP (EC 50488 activation of OPRK receptor in CHO-PKAcat-EGFP/OPRK cells)50) The value was 3.563. + -. 0.902 nmol/L.
Example 4: u50488 assay for CHO cytotoxicity
The method comprises the following steps:
1) inoculating the cells in a black, bottom-permeable 96-well culture plate at 37 deg.C with 5% CO2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with 100. mu.L/well of serum-free F12 medium (hereinafter referred to as cell assay solution). Thereafter, 100. mu.L/well of cell assay solution was added.
3) Cell assay solution containing U50488 was added at 50. mu.L/well to give a final concentration of U50488 of 100nmol/L and incubated at 37 ℃ for 24 hours in a cell incubator.
4) Cell assay solution containing hoechst33342 was added 0.5 hour before the end of the administration at 50. mu.L/well to a final concentration of 100nmol/L, and incubated in a cell culture chamber at 37 ℃ for 30 minutes.
5) Cells were fixed with 12% formaldehyde diluted with 1 × PBS, 100 μ L/well, and left at room temperature for 20 minutes in the dark.
6) Discard solution, add 1 × PBS solution, 200 μ L/well.
7) Cell images were acquired using In Cell Analyzer1000 and U504884 was analyzed for CHO cytotoxic effects using a Multi-target analysis module (Multi TargetAnalysis).
U50488 had no toxic effect on CHO cells at a concentration of 100nmol/L of U50488 (FIG. 3).
Example 5: reliability evaluation of CHO-PKAcat-EGFP/OPRK cell model
This example uses the Z' factor to evaluate the reliability of the newly constructed cell model of example 1. The Z' factor is used as an important parameter for evaluating the reliability of an experimental system, and is widely applied to evaluating the stability and reliability of high-throughput screening and high-content analysis experimental systems. The formula for the Z' factor is as follows:
wherein σ is a standard deviation (standard deviation); μ is mean signal; c + is a positive control (positive control); c-is a negative control (negative control).
The value of the Z' factor is between 0 and 1. When the value of the Z' factor is 0, the experimental system is not established. When 0.5> Z' >0, the stability of the experimental system is poor and unreliable. When the 1 is more than Z' and is more than or equal to 0.5, the experimental system is good in stability and reliability. If the Z' factor has a value of 1, this is a standard deviation of 0 for the positive and negative controls, indicating an ideal experimental system (Ji-Hu Zhang, Thomas D.Y.Chung and Kevin R.Oldenburg.A Simple statistical parameter for Use in Evaluation and variation of High Throughput screening assays [ J ]. J Biomol Screen.1999.4: 67).
The steps for evaluating the reliability of the experimental system established by the invention are as follows:
1) making cells into 1 × 105Cell suspension/mL, inoculated into black, bottomless 96-well culture plate, 100. mu.L/well, at 37 deg.C and 5% CO2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with cell assay solution at 100 μ L/well; the solution was discarded and 100. mu.L/well of cell assay solution was added.
3) A dilution of U50488 was added to the cell assay to give a final concentration of 100nmol/L, and the cell assay without U50488 was added as a control, and the cells were incubated at 37 ℃ for 30 minutes in a cell incubator.
4) Cell assay solution containing forskolin was added at 50. mu.L/well to a final concentration of 10. mu. mol/L, and incubated in a cell incubator at 37 ℃ for 5 minutes.
5) Add cell fixative at 100. mu.L/well, mix well by shaking the plate gently, and leave it in the dark at room temperature for 20 minutes.
6) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
7) Cell images were acquired using In Cell Analyzer2000, the degree of redistribution of PKAcat-EGFP was analyzed using a multi-target analysis module, and the value of the Z' factor was calculated.
The screening system established by the invention has an average Z' value of 0.596 when acted by 100nmol/L U50488 (figure 4).
The results show that the cells of the invention are very reliable and feasible as cell models for screening opioid receptor agonists or evaluating the agonistic activity of compounds or compositions at opioid receptors.
Example 6: effect of U50488 time to treatment of cells on PKAcat-EGFP redistribution
The method comprises the following steps:
1) the cells prepared in example 1 were prepared to 1X 105Cell suspension of one/mLInoculating to black 96-well culture plate with transparent bottom, 100 μ L/well, and culturing at 37 deg.C with 5% CO2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with cell assay solution at 100 μ L/well; the solution was discarded and 100. mu.L/well of cell assay solution was added.
3) U50488 diluted with cell assay solution was added every 5 minutes at 50. mu.L/well to give a final concentration of 100nmol/L, and the cell assay solution containing no U50488 was added as a control, and the cells were incubated at 37 ℃ in a cell incubator for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, respectively, for agonist treatment.
4) Cell assay solution containing forskolin was added at 50. mu.L/well to a final concentration of 10. mu. mol/L, and incubated in a cell incubator at 37 ℃ for 5 minutes.
5) Add cell fixative at 100. mu.L/well, mix well by shaking the plate gently, and leave it in the dark at room temperature for 20 minutes.
6) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
7) Cell images were acquired using In Cell Analyzer2000 and the degree of redistribution of PKAcat-EGFP was analyzed using a multi-target analysis module.
After addition of U50488, PKAcat-EGFP rapidly redistributed. The extent of PKAcat-EGFP redistribution was higher compared to the control during 5 min to 60 min of agonist treatment cells (fig. 5).
Example 7: effect of cell seeding Density on PKAcat-EGFP redistribution
The method comprises the following steps:
1) the cells prepared in example 1 were seeded in black, transparent 96-well plates at 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000 cells/well, respectively, and 5% CO was added at 37 deg.C2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with cell assay solution at 100 μ L/well; the solution was discarded and 100. mu.L/well of cell assay solution was added.
3) Cell assay diluted U50488, 50. mu.L/well was added to a final concentration of 100nmol/L, and the cells were incubated at 37 ℃ for 30 minutes in a cell incubator with the addition of cell assay without U50488 as a control.
4) Cell assay solution containing forskolin was added at 50. mu.L/well to a final concentration of 10. mu. mol/L, and incubated in a cell incubator at 37 ℃ for 5 minutes.
5) Add cell fixative at 100. mu.L/well, mix well by shaking the plate gently, and leave it in the dark at room temperature for 20 minutes.
6) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
7) Cell images were acquired using In Cell Analyzer2000 and the degree of redistribution of PKAcat-EGFP was analyzed using a multi-target analysis module.
As shown in FIG. 6, the cell inoculation density was from 6000 to 20000/well, and 100nmol/L of U50488 factor treated cells for 30 minutes, the degree of inducing redistribution of PKAcat-EGFP was higher.
Example 8: determination of half-effective concentration of dihydroetorphine for redistribution of GFP-PKA in cell models
Dihydroetorphine structural formula (DHE) is an opioid receptor agonist and is effective in inhibiting AC. We evaluated the agonistic activity of DHE using the OPRK agonist screening model established in example 1. The method comprises the following steps:
1) CHO-PKAcat-EGFP/OPRK cells were plated at 1.0X 104One cell/well was inoculated in a black, clear-bottomed 96-well plate at 100. mu.L/well in 5% CO at 37 ℃2And culturing in a 95% humidity incubator for 24 hours.
2) Discard the medium and wash the cells 1 times with cell assay solution at 100. mu.L/well.
3) Adding cell analysis solution, 100 μ L/hole; DHE in DMSO was diluted by adding cell assay, 50. mu.L/well at 37 ℃ in 5% CO2Incubate 30 minutes in a 95% humidity incubator.
4) Cell assay solution containing forskolin was added at 50. mu.L/well to a final concentration of 10. mu. mol/L, and incubated in a cell incubator at 37 ℃ for 5 minutes.
5) A formaldehyde solution (12%) diluted with 1 XPBS solution was added to the wells at 100. mu.L/well, and the mixture was left to stand at room temperature in the dark for 20 minutes.
6) The solution was discarded, and a 1 XPBS solution containing hoechst33342 was added thereto at a concentration of 100. mu.L/well, and the mixture was left to stand at room temperature for 30 minutes in the absence of light.
7) Acquiring a Cell image by using In Cell Analyzer1000, analyzing the redistribution degree of PKAcat-EGFP by using a multi-target analysis module, fitting an S-shaped curve by using GraphPad Prism5 software Nonlinear regression (curve fit), and calculating EC50The value is obtained.
As a result, as shown in FIG. 7, DHE was significantly (n-3; EC) in the OPRK agonist screening cell model in which DHE activates human OPRK and induces GFP-PKA redistribution505.030 ± 0.421nM) promotes GFP-PKA redistribution to occur.
Example 9: determination of DHE cytotoxicity in CHO
The method comprises the following steps:
1) inoculating the cells in a black, bottom-permeable 96-well culture plate at 37 deg.C with 5% CO2And culturing in a 95% humidity incubator for 24 hours.
2) Cells were washed 1 time with 100. mu.L/well of serum-free F12 medium (hereinafter referred to as cell assay solution). Thereafter, 100. mu.L/well of cell assay solution was added.
3) Cell assay solution containing DHE was added at 50. mu.L/well to a final DHE concentration of 100nmol/L and incubated in a cell culture chamber at 37 ℃ for 24 hours.
4) Cell assay solution containing hoechst33342 was added 0.5 hour before the end of the administration at 50. mu.L/well to a final concentration of 100nmol/L, and incubated in a cell culture chamber at 37 ℃ for 30 minutes.
5) Cells were fixed with 12% formaldehyde diluted with 1 × PBS, 100 μ L/well, and left at room temperature for 20 minutes in the dark.
6) Discard solution, add 1 × PBS solution, 200 μ L/well.
7) Cell images were acquired using In Cell Analyzer1000 and DHE cytotoxic effects on CHO were analyzed using a multi-target analysis module.
DHE had no toxic effect on CHO cells at a concentration of 100nmol/L DHE (FIG. 8).
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (23)
1. The cell has the preservation number of CGMCC No.9002 and the preservation date of 2014, 3 months and 26 days, and the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms.
2. A method of screening for kappa opioid receptor agonists or adenylate cyclase inhibitors or a method of assessing/determining the agonistic activity towards kappa opioid receptors or the inhibitory activity towards adenylate cyclase or the cytotoxicity of a test sample comprising the step of using the cell of claim 1.
3. The method of claim 2, comprising the steps of:
after or simultaneously with the addition of the sample to be tested, an adenylate cyclase agonist is added to detect the degree of redistribution of PKA.
4. The method according to claim 3, wherein cells in the same culture conditions but with addition of only adenylate cyclase agonist are used as control cells.
5. The method of claim 3, wherein the adenylate cyclase agonist is sodium fluoride or forskolin.
6. The method according to claim 3, wherein the adenylate cyclase agonist is added 1-60 minutes after the addition of the test sample.
7. The method according to claim 3, wherein the adenylate cyclase agonist is added 10-50 minutes apart after the test sample is added.
8. The method according to claim 3, wherein the adenylate cyclase agonist is added at an interval of 20-40 minutes after the addition of the sample to be tested.
9. The method of claim 3, wherein the extent to which redistribution of PKA occurs is detected after 1-20 minutes following addition of the adenylate cyclase agonist.
10. The method of claim 3, wherein the extent to which redistribution of PKA occurs is detected after 1-15 minutes following addition of the adenylate cyclase agonist.
11. The method of claim 3, wherein the extent to which redistribution of PKA occurs is detected after 1-10 minutes following addition of the adenylate cyclase agonist.
12. The method of claim 3, wherein the final concentration of the adenylate cyclase agonist is 1-100 μmol/L.
13. The method of claim 3, wherein the final concentration of the adenylate cyclase agonist is 1-50 μmol/L.
14. The method of claim 3, wherein the final concentration of the adenylate cyclase agonist is 1-20 μmol/L.
15. The method of claim 3, comprising the steps of:
1) inoculating and culturing the cell of claim 1;
2) discarding the culture medium, and washing the cells at least 1 time with a serum-free culture medium;
3) adding a sample to be tested, and culturing in a serum-free culture medium;
4) adding forskolin, and continuing culturing in a serum-free culture medium;
5) detecting the redistribution degree of the PKA.
16. The method of claim 15, wherein the seeding density in step 1) is 0.6 x 104－2.0×104One cell per mL, and the culture time is 12-48 hours.
17. The method according to claim 16, wherein the culturing time in step 1) is 24 hours.
18. The method according to claim 15, wherein the culture conditions in step 1), 3) or 4) are 37 ℃ and 5% CO295% humidity.
19. The method according to claim 15, wherein the serum-free medium in step 2), 3) or 4) is F12.
20. The method of claim 15, wherein the step 5) of detecting the degree of redistribution of the PKA protein comprises the following pre-treatment steps: the cells were fixed with a 12% formaldehyde solution diluted with PBS, left at room temperature for 20 minutes in the dark, discarded, added with PBS containing the nuclear dye hoechst33342, and left at room temperature for 30 minutes in the dark.
21. A cell model or a kit comprising the cell of claim 1.
22. Use of the cell of claim 1 for screening kappa opioid receptor agonists or adenylate cyclase inhibitors, or for evaluating the agonistic activity towards kappa opioid receptors or the inhibitory activity towards adenylate cyclase or the cytotoxicity of a test sample.
23. The use of claim 22, wherein the screening is high content screening.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102266324A (en) * | 2011-08-26 | 2011-12-07 | 中国人民解放军第四军医大学 | New application of kappa-opioid receptor agonist |
| WO2012118908A1 (en) * | 2011-03-01 | 2012-09-07 | Corning Incorporated | Assessment of opioid receptor ligands |
| CN102719404A (en) * | 2011-03-30 | 2012-10-10 | 中国人民解放军军事医学科学院毒物药物研究所 | Cell model and method for screening c-Fms kinase inhibitor |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102719404A (en) * | 2011-03-30 | 2012-10-10 | 中国人民解放军军事医学科学院毒物药物研究所 | Cell model and method for screening c-Fms kinase inhibitor |
| CN102266324A (en) * | 2011-08-26 | 2011-12-07 | 中国人民解放军第四军医大学 | New application of kappa-opioid receptor agonist |
Non-Patent Citations (3)
| Title |
|---|
| Changes in intracellular cAMP reportd by a Redistribution assay using a cAMP-dependentprotyein kinase-green fluorescent protein chimera;Almholt K,et al;《Cellular Signalling》;20040831;第16卷(第8期);摘要 * |
| G(i1) and G(oA) differentially determine kinetic efficacies of agonists for kappa-opioid receptor;Kohno M,et al;《Febs Letters》;20000504;第473卷(第1期);全文 * |
| 镁离子对术后病人自控镇痛的疗效观_省略_介导腺苷酸环化酶活性影响初步研究;吴宏亮;《中国博士学位论文全文数据库》;20061231(第12期);摘要，第4页、第26页 * |
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