CN104877966A - P2Y1/U2OS cell strain and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a P2Y1/U2OS cell strain and a preparation method and application thereof and belongs to the technical field of high throughput screening and detection of drugs. The P2Y1/U2OS cell strain is prepared by the following steps: firstly establishing a human P2Y1 receptor fluorescin eukaryotic expression vector P2Y1/pCMV6 and then transfecting a U2SO cell according to a Lipofectamine2000 operating manual; and screening the G418 (400 mu g/ml) in a pressurized manner for 2 weeks to obtain the P2Y1/U2OS cell strain. The P2Y1/U2OS cell strain disclosed by the invention and used for determining the biological activity of a P2Y1 receptor stimulant or an antagonist is easy to standardize and good in repeatability, has the characteristics of high specificity and sensitivity, low using cost and convenient operation for the P2Y1 receptor, and has a good application prospect in drug screening and efficacy evaluation.
Description
Technical field
The present invention relates to a kind of P2Y1/U2OS cell strain and preparation method thereof and application, belong to medicament high flux screening detection technique field.
Background technology
P2Y1 acceptor belongs to g protein coupled receptor, in the tissue such as the heart, brain, blood vessel, placenta, liver, lung of people, all have expression.P2Y1 acceptor and the coupling of Gq albumen phase, when this receptor is activated, can activate phospholipase Cβ (PLC β), protein kinase C (PKC), promotes intracellular calcium mobilisation.Research in recent years shows, P2Y1 acceptor plays an important role in hematoblastic activation, vasorelaxation and atherosclerosis.Such as: after ADP and P2Y1 receptors bind, Gq path can be triggered, cause cytoplasmic calcium ion levels to raise, thus make the gathering of thrombocyte generation Rapid reversible.In endotheliocyte, the mechanism that the activation of P2Y1 acceptor can be relied on by nitrogen protoxide causes coronary vasodilatation.Ultrapole L (LPA) can cause the formation of platelet activation and platelet-monocyte polymkeric substance thus form vascular plaque, and this effect can be blocked by P2Y1 receptor antagonist.P2Y1 acceptor can also the propagation of regulating cell, transfer, apoptosis and death process.In melanoma, nodus hemorrhoidalis intestinal tumor and brain tumor and other clone, P2Y1 receptor activation causes tumour cell quantity to reduce.In human prostata cancer PC-3 cell, P2Y1 receptor activation causes cell growth inhibition and death, and prompting P2Y1 acceptor promises to be the novel therapeutic target spot of prostate cancer.
At present, antiplatelet drug, as clopidogrel, prasugrel, has gone on the market for many years, has been widely used, determined curative effect, but also there is the problem such as clopidogrel Resistant, bleeding risk; In recent years, the antiplatelet compound being target spot with P2Y1 acceptor research day by day deeply, nucleotide derivative MRS2179 is the selective antagonist of P2Y1 acceptor, in vivo, external all have antithrombotic acitivity, be the instrument medicine of the most frequently used research P2Y1 acceptor, but there is the low defect of oral administration biaavailability.This research, by building the strain of fluorescent mark people P2Y1 albuminous cell, uses the means of high flux screening, ites is desirable to find some non-nucleotide classes, be convenient to oral P2Y1 receptor stimulant or antagonist.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of P2Y1/U2OS cell strain, specifically comprise the following steps:
(1) people P2Y1 acceptor fluorescent protein GFP carrier for expression of eukaryon P2Y1/pCMV6 is built;
(2) transfected with human osteosarcoma U2OS cell is carried out by Lipofectamine2000 process specifications, G418 pressurization screening 2 weeks, obtain the positive cell clone with antibiotics resistance, amplification cultivation is the U2OS clone of stably express people P2Y1, and ordering bright is P2Y1/U2OS cell strain.
Fluorescin of the present invention is GFP(green fluorescence protein) or any albumen with photoluminescent property, wherein GFP is green light albumen.
Another object of the present invention is to provide described P2Y1/U2OS cell strain for measuring the method for P2Y1 receptor stimulant or antagonist biological activity, comprise the steps: (GFP directly marks the C-terminal of P2Y1) under the P2Y1 acceptor gene condition that transfection GFP marks, induce in P2Y1/U2OS cell under receptor stimulant stimulates and produce fluorescence spot, analyze with high intension systematic observation and measure P2Y1/U2OS intracellular Fluorescence number of spots, detect the biological activity of P2Y1 receptor stimulant or inhibitor.
Receptor stimulant of the present invention is ATP, produce fluorescent spot after P2Y1 binding of receptor and ligand in cell to count out and to be proportionate with the activity of receptor stimulant, analyze with high intension systematic observation and measure P2Y1/U2OS intracellular Fluorescence number of spots, measure the biological activity of receptor stimulant.
Beneficial effect of the present invention:
(1) the present invention P2Y1/U2OS cell strain detects P2Y1 receptor stimulant, antagonist, the easy stdn of this detection method described, reproducible, has that P2Y1 receptor-specific, cost are low, accurately and easily feature, has a good application prospect;
(2) the invention provides fluorescent mark people P2Y1 receptor protein cell strain P2Y1/U2OS, comparing domestic screening method such as homogeneous phase time discrimination fluorescence technology (HTRF) has simple to operate, visual in image, omit and add Buffer, primary antibodie, two tedious steps resisted, reduce testing cost.Owing to the present invention is to provide the reaction after direct-detection acceptor and part effect, so have more highly sensitive and specificity, greatly reduce false-positive generation, and can screen for mixture of Chinese herbal medicines.
Accompanying drawing explanation
Fig. 1 GFP marks P2Y1 schematic diagram;
Fig. 2 recombinant eukaryon expression vector P2Y1/pCMV6 schematic diagram;
The expression result of Fig. 3 high intension systematic observation analysis of cells surface P2Y1;
The systematic observation of Fig. 4 high intension is analyzed to stimulate in rear cell and is formed fluorescence spot;
Fig. 5 P2Y1/U2OS cell is active reaction result after ATP activates.
Embodiment
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
embodiment 1
The structure of GFP fluorescent mark people P2Y1 acceptor carrier for expression of eukaryon P2Y1/pCMV6, the foundation of P2Y1/U2OS cell strain, and the change of P2Y1/U2OS intracellular Fluorescence spot average number detects P2Y1 receptor stimulant.
One, the structure (as shown in Figure 2) of GFP fluorescent mark people P2Y1 acceptor (as shown in Figure 1) carrier for expression of eukaryon P2Y1/pCMV6:
(1) people P2Y1 cDNA(is purchased from OriGene company) amplification: with people P2Y1 cDNA for template, adopt the mode of PCR to increase total length people P2Y1 cDNA coding region, total length 1119bp;
Its upstream primer: 5 '-gaggcgatcgccATGACCGAGGTGCTGTGGCCGGC-3 ';
Downstream primer: 5 '-gcgacgcgtCAGGCTTGTATCTCCATTCTGCTTG-3 ' (primer is synthesized by Shuo Yang bio tech ltd).
(2) recovery, purified pcr product, be connected on pMD18-T Vector, obtains recombinant plasmid P2Y1/pMD18-T, utilize restriction enzyme
sgfi and
mlui carries out enzyme to plasmid and cuts.
(3) restriction enzyme is used
sgfi and
mlui digested plasmid G-D (pCMV6-AC-GFP-DR5).
(4) reclaim digestion products, connect 8 hours at 16 DEG C, obtain recombinant plasmid P2Y1/pCMV6, go intracellular toxin plasmid to extract test kit specification sheets in a large number according to OMEGA and extract P2Y1/pCMV6 plasmid.
The clone PCR reaction system of gene order is 50 μ L, is made up of 5 μ L 10 × Ex-Taq buffer, 1 μ L Ex-Taq archaeal dna polymerase 5 U/ μ L, 3 μ L 2.5 mM dNTP, 1 μ L upstream primer 10 pmol/ μ L, 1 μ L downstream primer 10 pmol/ μ L, 1 μ L plasmid (BC113493) 100 ng/ μ L and 38 μ L sterilized waters; PCR reaction conditions: 95 DEG C of denaturation 5 min, 95 DEG C of sex change 1 min, 65 DEG C of annealing 0.5 min, 72 DEG C extend 1 min, totally 30 circulations, and 72 DEG C extend 10 min; Containing Mg in 10 × Ex-Taq buffer used
2+.
It is 10 μ L that recovery PCR primer is connected to pMD18-T Vector reaction system, is made up of, reaction conditions: connect 4 hours at 16 DEG C 5 μ L recovery PCR primer, 4.5 μ L SolutionI, 0.5 μ L pMD18-T Vector.
Double digestion plasmid P2Y1-1R/pMD18-T reaction system is 50 μ L, by 2 μ L
sgfi(5 U/ μ L), 2 μ L
mlui(5 U/ μ L), 5 μ L10 × Tango Buffer, 25 μ LP2Y1/pMD18-T and 16 μ L sterilized waters composition; Reaction conditions: 37 DEG C of incubations 2 hours.
Double digestion plasmid G-D (pCMV6-AC-GFP-DR5) reaction system is 50 μ L, by 2 μ L
sgfi(5 U/ μ L), 2 μ L
mlui(5 U/ μ L), 5 μ L10 × Tango Buffer, 10 μ LG-D and 31 μ L sterilized waters composition; Reaction conditions: 37 DEG C of incubations 2 hours.
Construction recombination plasmid P2Y1/pCMV6 is with the restriction enzyme digestion product in step (2) and (3), under the condition of 16 DEG C, connect 8 hours; Reaction system is 11 μ L, by 5 μ L SolutionI, plasmid pCMV6 fragment in step (4) after restriction enzyme digestion with P2Y1 gene order fragment is respectively 1 μ L and 5 μ L form; Reaction conditions: connect 8 hours at 16 DEG C.The plasmid obtained in step (4) can transform further
e. colidH5 α carries out preservation amplification.
Two, the foundation of the U2OS clone of stable transfection people P2Y1-1R:
U2OS cell containing 10% (volume fraction) calf serum DMEM substratum in, in 37 DEG C, 5% (volume fraction) CO
2saturated humidity under cultivate; Until cytogamy to the transfection carrying out recombinant expression vector P2Y1/pCMV6 during 70%-80% by Lipofectamine2000 process specifications; After transfection 48h, with trypsin digestion and cell, containing G418(400 μ g/ml) Selective agar medium in pressurize screening 2 weeks, obtain the positive cell clone with antibiotics resistance, amplification cultivation is the U2OS clone of stably express people P2Y1, called after P2Y1/U2OS cell strain.By the expression result of high intension systematic observation P2Y1.
Three, the change of P2Y1/U2OS intracellular Fluorescence spot average intensity detects P2Y1 agonist, antagonist:
The P2Y1/U2OS cell of above-mentioned acquisition is incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, 37 DEG C, 5% CO
2cultivate 24 hours under condition; Within second day, remove perfect medium, use PBS buffer solution for cleaning, add different concns sample to be tested ATP, and remove solution in hole after 20 minutes, fix 30 minutes by 4% paraformaldehyde room temperature, 3 times are washed with PBS damping fluid, add DAPI dyestuff lucifuge to dye 15 minutes, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, with high intension systematic observation analysis of fluorescence spot, and calculate the average blob number of each cell with Transfluor module analysis.
Four, the expression of P2Y1 on P2Y1/U2OS cytolemma:
P2Y1/U2OS cell is incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, and 37 DEG C, 5% CO
2cultivate 24 hours under condition; Within second day, remove perfect medium, use PBS buffer solution for cleaning, fix 30min by 4% paraformaldehyde room temperature, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, with high intension systematic observation fluorescence.
Five, P2Y1/U2OS cell expressing P2Y1 receptor activity assay:
P2Y1/U2OS cell is incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, and 37 DEG C, 5% CO
2cultivate 24 hours under condition; Within second day, remove perfect medium, use PBS buffer solution for cleaning, add sample to be tested ATP, room temperature places 20 minutes, removes solution in hole, fixes 30min by 4% paraformaldehyde room temperature, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, by high intension systematic observation change in fluorescence; Result shows to produce fluorescence spot (as shown in Figure 4) in cell, and the U2OS cell of untransfected P2Y1 gene does not have any change (as shown in Figure 3); Above experimental result confirms that the P2Y1 albumen of restructuring P2Y1/U2OS cell expressing has the physiologically active of natural P2Y1 protein similar, has specificity.
Six, the Determination of biological activity of P2Y1 receptor stimulant ATP:
Get the ATP of different concns, the P2Y1/U2OS cell in step 5 in method stimulating growth, the average number of the fluorescence spot after each sample stimulation P2Y1/U2OS cell is analyzed in high intension systematic observation; Take concentration of specimens as Transverse coordinate, with the number of each cell fluorescence spot for ordinate zou, make curve; Result represents, the number forming spot after the ATP stimulation P2Y1/U2OS cell of different concns exists dosage effect (as shown in Figure 5) dependency.
Claims (4)
1. a preparation method for P2Y1/U2OS cell strain, is characterized in that, specifically comprises the following steps:
(1) people P2Y1 acceptor fluorescent protein GFP carrier for expression of eukaryon P2Y1/pCMV6 is built;
(2) stable transfection human osteosarcoma U2OS cell is carried out by Lipofectamine2000 process specifications, G418 pressurization screening 2 weeks, obtain the positive cell clone with antibiotics resistance, amplification cultivation is the U2OS clone of stably express people P2Y1, and ordering bright is P2Y1/U2OS cell strain.
2. the preparation method of P2Y1/U2OS cell strain according to claim 1, is characterized in that: described fluorescin is GFP or any albumen with photoluminescent property.
3. P2Y1/U2OS cell strain according to claim 1 is for measuring P2Y1 receptor stimulant or antagonist biological activity, under it is characterized in that the P2Y1 acceptor gene condition comprising the steps: that transfection GFP marks, the fluorescence spot that in P2Y1/U2OS cell, generation can be detected is induced under receptor stimulant stimulates, analyze with high intension systematic observation and measure P2Y1/U2OS intracellular Fluorescence number of spots, detect the biological activity of P2Y1 receptor stimulant or inhibitor.
4. P2Y1/U2OS cell strain according to claim 4 is for measuring P2Y1 receptor stimulant or antagonist biological activity, it is characterized in that: described P2Y1 receptor stimulant is ATP or ADP.
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| CN111100841A (en) * | 2018-10-25 | 2020-05-05 | 中国医学科学院药物研究所 | Establishment and application of genetic engineering cell strain and high-throughput drug screening model of anti-obesity drug target UCP1 |
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Cited By (2)
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| CN111100841A (en) * | 2018-10-25 | 2020-05-05 | 中国医学科学院药物研究所 | Establishment and application of genetic engineering cell strain and high-throughput drug screening model of anti-obesity drug target UCP1 |
| CN111100841B (en) * | 2018-10-25 | 2022-07-19 | 中国医学科学院药物研究所 | Establishment and application of genetic engineering cell strain and high-throughput drug screening model of anti-obesity drug target UCP1 |
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