CN106967788A - A kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening - Google Patents
A kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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- C12Q2304/00—Chemical means of detecting microorganisms
- C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
Abstract
A kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening, the invention belongs to biomedicine field, is related to a kind of method for detecting cell phosphatase activity, comprises the following steps：A, the cell to culture carry out genetic engineering modification, import nonsecreting type luciferase report gene；B, addition pharmaceutical intervention C, suction are abandoned nutrient solution and washed, and add cell pyrolysis liquid cell lysis, obtain cell pyrolysis liquid；The steps such as D, detection E, calculating.The technical scheme that the present invention is provided with reference to luciferase report gene means by reaching the purpose that quantization cell vigor quantifies, so as to indicate the information of two dimensions of Cellular alkaline phosphatase vigor and cell viability in pharmaceutical intervention, and ultimately form influence of the parametrization index reflection different pharmaceutical to individual cells alkaline phosphatase activity.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of method for detecting cell phosphatase activity, especially relates to
And a kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening.
Background technology
Osteoporosis is osteoporosis, is one group of osteopathy caused by many reasons, and bone tissue has normal calcification, calcium salt
It is in normal rates with matrix, the metabolic bone disease with the characteristics of the reduction of unit volume inner bone tissues amount becomes.The generation and absorption of bone
It is a dynamic equilibrium, the tune for two process dynamics of bone information that the ostosis and osteoclast mediated by Gegenbaur's cell is connected to
Control.When bone information speed exceedes bon e formation, it may appear that bone amount is reduced, and occurs osteoporosis.The activity of alkaline phosphatase is often
As the mark of Bone m etabolism, because function and the osteogenetic process of alkaline phosphatase are closely related, so alkaline phosphatase activities
Belong to the bon e formation metabolic index of characteristic.
Substrate can be catalyzed in alkaline environment using alkaline phosphatase and produces colour developing or fluorescence reaction, so as to quantify
Detect the activity of different sample alkaline phosphatases.Prior art is mainly by improving chromogenic substrate so as to improve alkaline phosphatase
The sensitivity of viability examination.Such as Detection wavelength of the publication No. for CN1322255 alkaline phosphatase enzyme assay method 450nm or so
The method for being measured and proposing abatement hemoglobin interference.And for example publication No. is CN103645185A method, by enzymatic
Metallization and nanogold induction deposition of silver are combined, and improve the sensitivity of detection of alkaline phosphatase.And publication No. is
CN104109176A patent application, then by fluorescence probe be applied to alkaline phosphatase activities fluoroscopic examination, its accuracy and
Sensitivity all improves a lot.
But in drug study in such as medicament screening experiment, various concentrations drug solvent or auxiliary agent cell growth
In the case of being intervened, often lead to cell after pharmaceutical intervention and occur inactivation, apoptosis or the death of part, cause each sample
This cell quantity changes, and then the alkaline phosphatase activities change of each sample can not accurately show medicine in unit cell
The influence of alkaline phosphatase activities.Regular growth vigor testing methods are chromogenic reaction（Such as mtt assay or CCK8 methods）, but be due to
The viability examination of alkaline phosphatase often relies on chromogenic reaction and demarcated, and these conventional methods are not suitable for cell neutral and alkali
Phosphatase activity detection, the accuracy of meeting interference experiment.And prior art is detected to alkaline phosphatase activities, medicine is not considered
Or the influence of the toxicity rating cell of drug solvent in itself, these methods are not suitable for drug screening still.
The content of the invention
Problem to be solved：For problem above, the present invention, which is provided, can detect Cellular alkaline phosphatase vigor simultaneously
And the detection method of quantization cell vigor, is prevented effectively from drug study various concentrations drug solvent or auxiliary agent and cell is produced
Influence, increase the reliability of drug test, and this method cost is low, efficiency high.
Technical scheme
A kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening, including it is following
Step：
A, the cell to culture carry out genetic engineering modification, import nonsecreting type luciferase report gene；
Freezen protective is standby after B, transfectional cell cell or moves into container；
C, addition pharmaceutical intervention
D, suction are abandoned nutrient solution and washed, and add cell pyrolysis liquid cell lysis, obtain cell pyrolysis liquid；
E, detection：Take part cell pyrolysis liquid to add the solution containing luciferase luminous substrate and obtain sample, detection sample is obtained
To luminance value RLU；It is another to take part cell lysate or directly into the sample after luminous detection, add alkaline phosphatase colour developing
Substrate solution, with plain lysis liquid correction detection product absorbance；The standard curve calculated by standard solution of catabolite,
Calibration sample catabolite yield, is A；Above-mentioned detecting step is with plain lysis liquid level reference sample；
F, the Cellular alkaline phosphatase activity Q values corrected according to obtained RLU and A ratios, the final luciferin of calculating, formula is Q
=A/RLU。
Contain p-nitrophenyl phosphate (p-nitrophenyl phosphate, p- in common alkaline phosphatase chromogenic substrate
NPP), its catabolite is paranitrophenol (p-nitrophenol, p-NP).
Further, described non-secreting luciferase report gene is Renilla luciferase reporter gene, firefly
Luciferase report gene, bacteriofluorescein enzyme gene；Described step A processing cell is Gegenbaur's cell system, primary skeletonization is thin
Born of the same parents.
Further, described non-secreting luciferase report gene is Renilla luciferase reporter gene；Described
Gegenbaur's cell is Gegenbaur's cell system MC3T3-E1 or the cells of ROS 17/2.8.
Further, in described step A, luciferase report gene carrier is bacterial plasmid, and described carrier is opened
Mover is CMV, SV40.
Further, in described step B, provided with being further cultured for amplifying cells step after transfection.
Further, the container described in step B is sample-adding plate.
Further, the luminous substrate described in step E is coelenterazine, beetle fluorescein or reduced form flavine monokaryon glycosides
Acid；
Wherein, described is coelenterazine because of Renilla luciferase reporter gene correspondence luminous substrate（Coelenterazine）；
Described Fluc reporter gene correspondence luminous substrate is beetle fluorescein（Luciferin）；The bacterium fluorescent
The corresponding luminous substrate of plain enzyme gene is flavin mononucleotide reduced（FMNH2）
Gene reported above is unique corresponding relation in nonsecreting type luciferase with luminous substrate.
Described alkaline phosphatase Chromogenic Substrate Solution is to contain p-nitrophenyl phosphate (p-nitrophenyl
Phosphate, p-NPP) solution；Described catabolite is paranitrophenol (p-nitrophenol, P-NP)；Described E
Middle use cell pyrolysis liquid is NP40（NPE）, Triton (Qula lead to) X-100, Tween (tween) -20,
Digitonin（Digitonin）Or Sapponin（Saponin）Deng aqueous surfactant solution.
Further, described cell pyrolysis liquid composition is：1% triton x-100,1mM ethylenediamine tetra-acetic acids,
5mM magnesium chlorides, 5% glycerine, 25mM Tris-HCl pH of buffer 7.4, solvent is ultra-pure water；
Described Renilla luciferase luminous substrate solution composition is：Described Renilla luciferase luminous substrate solution composition
For：Coelenterazine 2.5uM, the M of sodium chloride 1.1, potassium dihydrogen phosphate 0.22M, pH 5.0, solvent is ultra-pure water；
Described Fluc luminous substrate solution composition is：25 mM glycylglycines, 15 mM potassium dihydrogen phosphates (pH
8.0), 4 mM ethylene glycol diethyl ethers ethylenediamine tetraacetic acid (EDTA) (EGTA), 2 mM atriphos (ATP), the thio threoses of 1 mM bis-
Alcohol, 15 mM magnesium sulfate, 0.1 mM coacetylases, 75 uM beetle fluoresceins.Solvent is ultra-pure water.
Further, the active absorbance detection condition 400-415nm detections absorbance of described detection of alkaline phosphatase,
Described testing conditions are 405nm；Described detection luciferase absorbance detection condition is addition luciferase luminous substrate
Solution, is detected after thoroughly mixing in 10 minutes, and detection time is 10 seconds；It is preferred that condition hole-specifically to be added under the conditions of 21 DEG C,
Flow velocity 200ul/s, rotation concussion 5s, radius of turn 6mm, frequency 250rpmm, is spaced 5s, then detects luminosity to the hole
10s。
Further, this method can apply to osteosporosis resistant medicament screening experiment.
Further, described alkaline phosphatase Chromogenic Substrate Solution, which is constituted, is, 10mM p-NPP, 10 mM DEA,
0.5 mM magnesium chlorides（MgCl2）, pH 10.0,
Further, described ALP detections buffer solution composition is：10 mM DEA, 0.5 mM MgCl2, pH 10.0 detects
It is preceding that appropriate p-NPP is dissolved in detection buffer solution, the detection substrate solution that concentration is 10mM is configured to, is fully dissolved and mixed
It is even, place on ice.
Further, described catabolite pNP standard solution preparation methods are：It is dilute using ALP detection buffer solutions
Release pNP be 20 μM, 40 μM, 80 μM, 120 μM, 160 μM, 200 μM of paranitrophenol storing liquids be 0.5mM pNP, on demand to examine
Survey buffer solution doubling dilution.
Described nonsecreting type luciferase report gene is plasmid, described expression Renilla luciferase or firefly firefly
The plasmid sequence design of light element enzyme or bacteriofluorescein enzyme is known, and plasmid design can be found in websites such as Addgene simultaneously
Related plasmids are obtained, the commercial company such as Promega and Origene can provide the plasmid of these reporter genes.
The cell after obtained transfection after step A can be standby with freezen protective or moved into container.
Beneficial effect
The technical scheme that this patent is provided with reference to luciferase report gene means by reaching the mesh that quantization cell vigor quantifies
, so as to indicate the information of two dimensions of Cellular alkaline phosphatase vigor and cell viability in pharmaceutical intervention, and ultimately form ginseng
Influence of the numberization index reflection different pharmaceutical to individual cells alkaline phosphatase activity.
Because the cell quantity that luciferin luminescence method is detected has the broad range of linearity, corrected alkaline phosphatase
Activity has more comparativity, can preferably reflect influence of the medicine to alkaline phosphatase activities in itself, successfully solve
The problem of drug solvent or auxiliary agent interference alkaline phosphatase activities are detected in high-flux medicaments sifting, has filled up technical sky
In vain.
This method need not use intracellular dehydrogenase detection reagent, and cost is low and accuracy rate is high, efficient and convenient.
This efficiently detection method is more applicable for the high flux detection of mass efficient composition in drug screening, and can be with
Correct due to the influence that the cytotoxicity of solvent is brought to determination of alkaline phosphatase activity.
When luciferase in cell by the gene continuous expression that is transferred in advance when, the activity of luciferase in the time and
The related linear relationship of cell quantity.The sensitivity of luciferase detection is high, and substrate cost is low, and reaction speed is fast, can be complete
It is automatically performed in automatic ELIASA, it is simple to operate.Alkaline phosphatase activities detection is using fluorescence developing reaction, luciferase activity
By chemiluminescence determination, therefore the chromogenic reaction for not influenceing alkaline phosphatase activity to detect, transports while two kinds of detections
With influence that can be with integrated survey medicine to alkaline phosphatase activity and cell viability.
In large-scale medicine screening, alkaline phosphatase activity detection is often measured using cell pyrolysis liquid, and this is just
Selection to luciferase proposes limitation.In order to detect integrated application, it is necessary to from nonsecreting type fluorescent with alkaline phosphatase
Plain enzyme such as Renilla luciferase and Fluc etc., prevent from losing signal when abandoning nutrient solution.Although needing inspection simultaneously
Survey alkaline phosphatase activities, but this method can both be divided into two-step method detection, adapt to laboratory without multi-function microplate reader or
Section office are examined, compatibility is good；It can be continuously measured again using full-automatic ELIASA in single orifice plate, without changing hole
Plate, it is more convenient.
Cell pyrolysis liquid, cell lysis effect is thorough, but the protein active extracted can be kept.Both alkaline phosphorus can be supported
Sour enzyme detection can support various luciferases to detect again.Two-step method, which adds, takes 25ul to do luciferase hair after 50ul cell pyrolysis liquids
Light is detected, separately takes 25ul to do alkaline phosphatase color developing detection.Full-automatic ELIASA method first detects firefly after adding 25ul cell pyrolysis liquids
Light element enzyme lights, after be directly added into alkaline phosphatase Chromogenic Substrate Solution and detected.
Luciferase luminous substrate solution, has the broad range of linearity under different cell quantities.Illumination effect is stable,
And compatible follow-up alkaline phosphatase detection.
Alkaline phosphatase Chromogenic Substrate Solution, thoroughly inactivation luciferase, color stability are changed using pH.
Brief description of the drawings
Fig. 1 is luciferase testing result in embodiment 1.
Fig. 2 be embodiment 1 in do not correct alkaline phosphatase testing result.
Fig. 3 is that the alkaline phosphatase of embodiment 1 corrects testing result.
Fig. 4 be embodiment 2 in vehicles cells result is manufactured separately.
Fig. 5 is the linear regression graph in embodiment 3 by the correction cell quantity drafting of luciferase luminescence method.
Fig. 6 be embodiment 3 in pass through lactic dehydrogenase detection method（CCK8 reagents）Correct linear time that cell quantity is drawn
Gui Tu.
Fig. 7 is that each sample alkaline phosphatase that embodiment 3 is calculated after correction cell viability according to luciferase activity is lived
Property Q1.
Fig. 8 is Ldh Activity in embodiment 3（CCK8 methods）Calculate each sample alkalescence after correction cell viability
Phosphatase activity Q2.
Embodiment
Embodiment 1：Screen effective anti-osteoporosis composition in a compound（It is introduced directly into luciferase plasmids）
First, 96 well plate methods prepare the skeletonization vehicles cells of expression Fluc
1. taking 96 well culture plates, transfection proxima luce (prox. luc) adds 4*10 into every hole4Individual MC3T3-E1 Gegenbaur's cells are cultivated, and 37
℃、5% CO2Overnight incubation.
2. it is prepared by transfection liquid：Following two liquid are prepared in polystyrene tube (to transfect the amount used in 1 hole cell, with this
Analogize).
A liquid：Luciferase over-express vector plasmid is diluted with not serum-containing media（Promega companies provide）Add
Measure as 0.2ug/50ul serum free mediums.Note：PGL4.74 [hRluc/TK] plasmid vector encodes sea pansy fluorescent plain gene, and
And high efficient expression is carried out by SV40 promoters, the plasmid is widely used for mammalian cell screening application.
B liquid：With not serum-containing media dilution lipofectamine Fugene HD（Promega companies provide）, use
Measure as 0.6ul/50ul serum free mediums.
It is gently mixed in A liquid, B liquid, room temperature and puts 15 minutes.
3. transfection：Original culture medium is replaced with AB compounds, shaken up, 6-24h in 37 DEG C of incubators is put, serum-free transfection is absorbed
Liquid, change normal incubation medium continues to cultivate.
2nd, dosing culture：
A kind of known Chinese medicine compound prescription, therefrom extracts 6 kinds of active ingredients, respectively the first and second the third fourths penta oneself, because polarity is different, most
A certain amount of organic solvent must be added in whole cell culture fluid.
First each active ingredient drying sterling is dissolved separately in after solvent, then is diluted with culture medium, as containing each
The nutrient solution of medicine uses for further cell experiment.
Such as：Take a certain amount of first composition to be first dissolved in anhydrous DMSO, after fully being dissolved through ultrasonic vibration, diluted with nutrient solution
For that can be used for cell experiment after 1%.Following organic solvent content is volume ratio.
| Medicine group | First | Second | Third | Fourth | Penta | Oneself |
| Containing solvent | 1% DMSO | 0.1%DMSO | 0.1%DMF | 1% methanol | 0.1%DMSO | 1% DMSO |
Described DMSO is dimethyl sulfoxide (DMSO).DMF is dimethylformamide.
1. the original nutrient solution of 96 orifice plates is abandoned in suction, it is separately added into the nutrient solution containing each medicine and continues to cultivate, every kind of medicine 4
Repeating hole.
2. a screening scheme hype 5 days, changes fresh pastille culture medium daily.
3. cell is washed with the PBS without calcium ions and magnesium ions.
4. suck PBS.
5. prepare cell pyrolysis liquid cell lysis：Cell pyrolysis liquid composition is 1% Qula X-100,1mM ethylenediamine tetra-acetic acids,
5mM magnesium chlorides, 5% glycerine, 25mM Tris-HCl pH of buffer 7.4, solvent is ultra-pure water.
Each hole adds 50ul cell pyrolysis liquids (full-automatic ELIASA rule adds 25ul) in 6.96 well culture plates, shakes at room temperature
Low speed is cracked 30 minutes in bed（(full-automatic ELIASA rule program, which is shaken, to be incubated 30 minutes）.
3rd, the comprehensive detection of Cellular alkaline phosphatase activity and luciferase activity：
Take 25ul cell lysates to add and contain coelenterazine（Coelenterazine）Luciferase luminous substrate solution, fluorescent
Plain enzyme luminous substrate solution formula is：Coelenterazine 2.5uM, the M of sodium chloride 1.1, potassium dihydrogen phosphate 0.22M, pH 5.0,
Solvent is ultra-pure water.It is kept in dark place when Coelenterazine is flat with 250uM ethanol solutions, the Chromogenic Substrate Solution of Fresh
It can be placed 6 hours on ice.It is now with the current.
Luciferase testing conditions are molten hole-specifically to add the luciferase luminous substrate containing coelenterazine under the conditions of 21 DEG C
Liquid, flow velocity 200ul/s, rotation concussion 5s, radius of turn 6mm, frequency 250rpmm, is spaced 5s, then detects luminosity to the hole
10s.(luciferase testing result is shown in Fig. 1)
It is another to take 25ul lysates or directly into the sample after luminous detection, add and contain p-nitrophenyl phosphate (p-
Nitrophenyl phosphate, p-NPP) alkaline phosphatase Chromogenic Substrate Solution (10mM p-NPP, 10 mM DEA,
0.5 mM MgCl2, pH 10.0), appropriate p-NPP is dissolved in detection buffer solution before detection（10 mM DEA, 0.5 mM
MgCl2, pH 10.0）In, the detection substrate solution that concentration is 10mM, fully dissolving and mixing are configured to, is put on ice with postponing
Put, it is now with the current.Each hole adds 100ul alkaline phosphatase Chromogenic Substrate Solutions in 96 well culture plates, and 410nm is examined after detection decomposition
Survey absorbance（Corrected with plain lysis liquid）.And it is molten with catabolite paranitrophenol (p-nitrophenol, P-NP) standard items
The standard curve that liquid is calculated, calibration sample P-NP yields, are A；
(the independent testing result of alkaline phosphatase is shown in that Fig. 2, * show that difference has statistical significance, P<0.05)
Numerical computations：Returned to zero with lysis buffer, measure sample dissociation thing luminance value RLU；Drawn according to p-NP standard curves
Sample dissociation liquid alkaline phosphatase decomposes the amount A that p-NPP produces p-NP.School is finally calculated by calculation formula Q=A/RLU
Each sample alkaline phosphatase activities Q after positive cell viability.The present embodiment need to provide specific data and curve map.
(alkaline phosphatase correction testing result is shown in that Fig. 3, * show that difference has statistical significance, P<0.05)
It can be outputed from result above, if between individually detection of alkaline phosphatase vigor, A/D medicines, between B/D medicines, D/E medicines
Statistical significance is not embodied between thing, reason is probably that the larger methanol of D group reagents addition toxicity has shadow to cell viability
Caused by ringing.After influence of the methanol to cell viability is corrected, alkaline phosphorus can actually be promoted by preferably embodying D group reagents
Sour enzyme activity.And between A/B medicines, between B/E medicines, between B/F medicines, in individually detection alkaline phosphatase between C/D medicines
False positive is presented during enzyme activity, the generation of false positive is restrained effectively after being corrected by luciferase.
Embodiment 2：Screen effective anti-osteoporosis composition (independently prepared vehicles cells) in a compound
In addition to directly being transfected and being detected using 96 orifice plates, can also using individually progress cell preparation and storage, with
Utilized during standby screening.
First, the MC3T3-E1 Gegenbaur's cells of expression Fluc are prepared：
1. taking 6 well culture plates, transfection proxima luce (prox. luc) adds 4x105 MC3T3-E1 Gegenbaur's cell into every hole and cultivated, 37 DEG C,
5% CO2, which is cultivated to 40%-60%, to be converged.The transfection same day is changed with 2ml fresh cultures.
2. it is prepared by transfection liquid：Following two liquid are prepared in polystyrene tube (to transfect the amount used in 1 hole cell, with this
Analogize).
A liquid：Make concentration containing Fluc over-express vector plasmid pGL4.51 with the dilution of not serum-containing media
For 4ug/250ul.B liquid：Lipofectamine2000 liposome reagents are diluted with not serum-containing media, concentration is 10ul/
250ul.It is gently mixed in A liquid, B liquid, room temperature and puts 15 minutes.
Note:[luc2/CMV/Neo] plasmid that pGL4.51 provides for Promega companies.The plasmid contains firefly fluorescence
Plain enzyme gene, and started by CMV promoter, the plasmid contributes to efficient sieve also comprising a neomycin resistance gene
Select stable expression pGL4.51 MC3T3 light emitting tool cells
Transfection：A/B compounds are slowly added into culture medium, shaken up, 6-24h in 37 DEG C of incubators is put, serum-free transfection liquid is absorbed,
Normal incubation medium is changed to continue to cultivate.
3. original nutrient solution is abandoned in suction, add the nutrient solution containing 0.4mg/mlG410 and continue to cultivate.
4. every 2 days change fresh medium, until cell confluency degree about 90%-100%.
5. wash cell with 5 milliliters of PBSs without calcium ions and magnesium ions.
6. suck PBS.
7. with 1 milliliter of 0.25% Trypsin Induced dissociated cell (5 minutes, 37 °C), blood is then contained with 5ml
Clear complete culture solution terminates digestion.
8. disperseing cell by blowing and beating repeatedly, cell suspension is transferred to centrifuge tube.
9. centrifuged 5 minutes with 400g acceleration.
10. original nutrient solution is sucked, by cell with appropriate complete culture solution（G418 concentration is reduced to containing 0.2mg/ml dimensions
Hold dosage）It is resuspended.
11. count after cell, with 1 × 106Per 10cm2It is distributed into blake bottle.
12. the step for being repeated per 2-4 days is with amplifying cells (keeping about 80 % density growths)
13. take 4 parts of each 50000 cells to be cracked at random（The step 5-6 of method be the same as Example 1）And use light of firefly luciferin
Enzyme activity is verified.
Fluc luminous substrate solution composition is：25 mM glycylglycines, 15 mM potassium dihydrogen phosphates (pH
8.0), 4 mM ethylene glycol diethyl ethers ethylenediamine tetraacetic acid (EDTA) (EGTA), 2 mM atriphos (ATP), the thio threoses of 1 mM bis-
Alcohol, 15 mM magnesium sulfate, 0.1 mM coacetylases, 75 uM beetle fluoresceins.Solvent is ultra-pure water.It need to be eventually adding during configuration
Atriphos and beetle fluorescein, are placed on ice with postponing, now with the current.Fluc testing conditions are 21
The luciferase luminous substrate solution containing beetle fluorescein is hole-specifically added under the conditions of DEG C, 5s is shaken in flow velocity 200ul/s, rotation,
Radius of turn 6mm, frequency 250rpmm, are spaced 5s, then detect luminosity 10s to the hole.
14. examination criteria is：Cell is continued to multiply in the pharmaceutical culture mediums of G418 containing 0.2mg/ml of continuous 7 days, multiplication
Time is less than 72h；50000 cell Fluc vigor are not less than 400000LUM；Cell morphology keeps Gegenbaur's cell
Double angle shuttle-type and expression bon e formation reaction GAP-associated protein GAP such as collagen 1, osteopontin etc..It is now stable expression
PGL4.51 MC3T3 light emitting tool cells, can be frozen on demand.
15. being made using the cell before bone active drugs screening, the light emitting tool cell for freezing or cultivating is taken by 10000
Cell per well is inoculated in 96 orifice plates.Second and third part in later step be the same as Example 1.
The method of screening purifying cells can increase vehicles cells luciferase activity（Fig. 4）, but for the alkaline phosphorus of detection
Sour enzyme does not influence.Independently prepared vehicles cells can more easily detect medicine skeletonization efficiency at any time.
The Cellular alkaline phosphatase activity test method of the luciferin of embodiment 3 correction and the comparison of the past method
1. the comparison of preliminary cellular quantity detectability：Cell in embodiment 2 is pressed into varying number：0、800、4000、
20000th, 100000 it is inoculated in per hole in 96 orifice plates, passes through luciferase luminescence method and lactic dehydrogenase detection method respectively（CCK8
Reagent）Correct cell quantity.And draw linear regression graph, such as Fig. 5 respectively whereby.Luciferase luminescence method detection method is the same
Text, and CCK8 method lactic dehydrogenases are detected as general technology, it is known to those skilled in the art.It is summarized as follows：In 96 orifice plates
Middle inoculating cell (the μ l of nutrient solution 100/hole).10 μ l CCK-8 solution is added to every hole and is incubated in incubator 2 small
When.The absorbance at 450 nm is determined with ELIASA.
The range of linearity from Fig. 5, Fig. 6 visible luciferase luminescence method detection cell viability is broad, cell quantification have compared with
High sensitivity and reliability.But the range of linearity of conventional cell viability quantitative approach-Lactate dehydrogenase C CK8 determination methods compared with
Small, detectable cell quantity scope is narrower.
2. the comparison of alkaline phosphatase calibration result：Utilize alkaline phosphatase enzyme activity of the method in embodiment 2 to medicine to be measured
Power is detected, and show that sample dissociation liquid alkaline phosphatase decomposes the amount that p-NPP produces p-NP according to p-NP standard curves
A.According to Ldh Activity（CCK8 methods）Calculate each sample alkaline phosphatase activities Q2 after correction cell viability.Another root
Each sample alkaline phosphatase activities Q1 after correction cell viability is calculated according to luciferase activity.As a result Fig. 8 is seen.
As seen from Figure 8 because CCK8 detection sensitivities are relatively low, the range of linearity is narrower, and the Q2 qualities of data drawn are poor to be difficult to
The difference reflected between two kinds of medicines.
Claims (10)
1. a kind of application of Cellular alkaline phosphatase activity test method based on luciferin correction in drug screening, its feature
It is, comprises the following steps：
A, the cell to culture carry out genetic engineering modification, import nonsecreting type luciferase report gene；
B, addition pharmaceutical intervention
C, suction are abandoned nutrient solution and washed, and add cell pyrolysis liquid cell lysis, obtain cell pyrolysis liquid；
D, detection：Take part cell pyrolysis liquid to add the solution containing luciferase luminous substrate and obtain sample, detection sample is obtained
To luminance value RLU；It is another to take part cell lysate or directly into the sample after luminous detection, add alkaline phosphatase colour developing
Substrate solution, with plain lysis liquid correction detection product absorbance；The standard curve calculated by standard solution of catabolite,
Calibration sample catabolite yield, is A；Above-mentioned detecting step is with plain lysis liquid level reference sample；
E, the Cellular alkaline phosphatase activity Q values corrected according to obtained RLU and A ratios, the final luciferin of calculating, formula is Q
=A/RLU。
2. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that described non-secreting luciferase report gene is Renilla luciferase reporter gene, firefly
Noctiluca luciferase reporter gene, bacteriofluorescein enzyme gene；Described step A processing cell be Gegenbaur's cell system, it is primary into
Osteocyte.
3. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 2, it is special
Levy and be, described non-secreting luciferase report gene is Renilla luciferase reporter gene；Described Gegenbaur's cell is into
Osteocyte system MC3T3-E1 or the cells of ROS 17/2.8.
4. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that in described step A, luciferase report gene carrier is bacterium or virus particle, institute
The Vector promoter stated is CMV, SV40.
5. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that after described step A, can be provided with and be further cultured for amplifying cells after transfection and screen purifying
The step of.
6. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that the container described in step A is sample-adding plate.
7. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that the luminous substrate described in step D is coelenterazine, beetle fluorescein or reduced form flavine
Mononucleotide；
Described alkaline phosphatase Chromogenic Substrate Solution is the solution containing p-nitrophenyl phosphate；Described catabolite for pair
Nitrophenols；It is NPE, triton x-100, Tween-20, ocean ground that cell pyrolysis liquid is used in described E
Yellow soap glycosides or saponin aqueous surfactant solution.
8. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 or 7
Application in drug screening, it is characterised in that described cell pyrolysis liquid, which is constituted, is：1% triton x-100,1mM ethylenediamines
Tetraacethyl, 5mM magnesium chlorides, 5% glycerine, 25mM Tris-HCl pH of buffer 7.4, solvent is ultra-pure water；
Described Renilla luciferase luminous substrate solution composition is：Coelenterazine 2.5uM, the M of sodium chloride 1.1, biphosphate
Potassium 0.22M, pH 5.0, solvent is ultra-pure water；
Described Fluc luminous substrate solution composition is：25 mM glycylglycines, pH is 8.0 15 mM phosphorus
Acid dihydride potassium, 4 mM ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)s, 2 mM atriphos, 1 mM dithiothreitol dithios, 15 mM
Magnesium sulfate, 0.1 mM coacetylases, 75 uM beetle fluoresceins, solvent is ultra-pure water.
9. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in screening, it is characterised in that the active absorbance detection condition of described detection of alkaline phosphatase detects for 400-415nm
Absorbance, described testing conditions are 405nm；Described detection luciferase absorbance detection condition is sent out to add luciferase
Light substrate solution, is detected after thoroughly mixing in 10 minutes, and detection time is 10 seconds；Condition is hole-specifically to be added under the conditions of 21 DEG C,
Flow velocity 200ul/s, rotation concussion 5s, radius of turn 6mm, frequency 250rpmm, is spaced 5s, then detects luminosity to the hole
10s。
10. a kind of Cellular alkaline phosphatase activity test method corrected based on luciferin according to claim 1 is in medicine
Application in thing screening, it is characterised in that applied to osteosporosis resistant medicament screening experiment.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006000576A2 (en) * | 2004-06-24 | 2006-01-05 | Galapagos N.V. | Methods and compositions to promote bone homeostasis |
| CN104593415A (en) * | 2015-01-07 | 2015-05-06 | 中山大学 | High-throughput drug screening model using human pregnane X receptor (hPXR)-mediated dual-luciferase reporter gene technique |
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2017
- 2017-03-28 CN CN201710192378.XA patent/CN106967788A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006000576A2 (en) * | 2004-06-24 | 2006-01-05 | Galapagos N.V. | Methods and compositions to promote bone homeostasis |
| CN104593415A (en) * | 2015-01-07 | 2015-05-06 | 中山大学 | High-throughput drug screening model using human pregnane X receptor (hPXR)-mediated dual-luciferase reporter gene technique |
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| Title |
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| 赵玉岩等: ""雌激素对成骨细胞增殖及IGF-2表达的调控作用"", 《中国组织化学与细胞化学杂志》 * |
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