CN100557447C - Utilize the method for proteomic techniques screening cardiac glycoside drug effect target position - Google Patents
Utilize the method for proteomic techniques screening cardiac glycoside drug effect target position Download PDFInfo
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- CN100557447C CN100557447C CNB2005101298396A CN200510129839A CN100557447C CN 100557447 C CN100557447 C CN 100557447C CN B2005101298396 A CNB2005101298396 A CN B2005101298396A CN 200510129839 A CN200510129839 A CN 200510129839A CN 100557447 C CN100557447 C CN 100557447C
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Abstract
Utilize the method for proteomic techniques screening cardiac glycoside drug effect target position, belong to medical technical field.Comprise: cardiac muscle cell's cultivation and cardiac glycoside medicine are to the foundation of myocardial cells effect model, preparation cardiac muscle cell two dimensional electrophoresis protein example, two dimensional gel electrophore-sis, image acquisition is analyzed, obtain the cardiac glycoside drug effect in cardiac muscle cell's differential expression protein, differential protein is behind in-gel digestion, and the utilization mass spectrum is measured differential protein peptide quality fingerprinting collection of illustrative plates; Enter sequence library, differential protein is differentiated and analyzed, and derive its dna sequence dna.Characteristic protein is carried out mass spectrophotometry,, filter out differential protein by comparing with mass spectral database; The biological function of checking differentially expressed protein.Drug effect target position with proteomic techniques screening cardiac glycoside medicine heart failure resistance can be directly used in the heart failure resistance medicine that instructs the new and effective low toxicity of research and development.
Description
(1) technical field
The present invention relates to utilize proteomic techniques screening cardiac glycoside drug effect in the method for cardiac muscle cell's target protein, screen the method for cardiac glycoside drug effect specifically, belong to medical technical field in cardiac muscle cell's target protein.
(2) background technology
Important disease of cardiovascular systems such as coronary heart disease, high blood pressure, chronic valvulopathy, congenital heart disease develop into and heart failure to a certain degree all can occur, become the topmost cause of death of above-mentioned disease.More back in heart failure is bad, the about 15-20% of total mortality ratio, and heavy patient's annual death rate can reach 50%.Therefore, the treatment heart failure is a global public health problem effectively.The cardiac glycoside medicine is the choice drug of treatment heart failure at present, but the effective range and the safe range of such drug therapy are less, therapeutic dose and dosis toxica have the 30%-50% overlaid approximately, the severe patient threat to life, in addition, also can cause central nervous system reaction, toxic and side effects such as gastrointestinal reaction have seriously limited the normal use of this medicine.This is unresolved always to contradiction for the effective therapeutic dose of this medicine and the toxic dose of generation, how to solve and is the focus of cardiovascular clinical and basic concern to contradiction from where setting about to solve this always.Studies confirm that the cardiac glycoside medicine all has positive inotropic action to the cardiac muscle cell of exsomatize papillary muscle and in vitro culture, therefore, its heart failure resistance effect is to realize by the direct effect to the cardiac muscle cell.Think that at present such medicine is by bringing into play positive inotropic action to the inhibition approach of myocardial cell membrane Na-KATP enzyme, but can not explain opposite effects such as the inhibition of the Na-KATP enzyme that shows at different drug concentrations or activation fully at this point, in recent years have and discover this medicine at the apoptosis involvement of cardiac muscle cell's level with influence cell signalling and intervention improves the heart function effect, and the inhibition of part mechanism and Na-KATP enzyme has nothing to do.This research is research object with cardiac muscle cell, and utilization comparison protein group learns a skill cardiac glycoside is studied at cardiac muscle cell's target site, resolves its target position associated protein, the drug target albumen that screening has potential value.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, utilize proteomic techniques screening digitaloid drugs to act on the method for cardiac muscle cell's target protein, be used for the research and development of the novel anti heart failure drugs of high-efficiency low-toxicity.
Technical scheme of the present invention is as follows:
(1) sets up the cardiac glycoside medicine to the myocardial cells effect model
When the cardiac muscle cell is cultured to 3 days, be divided into control group and drug effect group, add the variable concentrations medicine, be cardiac glycoside effect cardiac muscle cell's model.
Above-mentioned cardiac muscle cell cultivates, and can select the arbitrary prior art in this area for use.The invention provides following method:
Get newborn 1-2 days rat, 10, cut open chest, it is dirty to core, and cleans up with D-Hanks liquid, adds 0.125% pancreatin and 0.02% divinyl tetraacethyl disodium, and 37 ℃ of water-baths 5 minutes are blown and beaten 1 minute, and supernatant is abandoned in natural sedimentation; Add 0.125% pancreatin, place 37 ℃ of water-baths 10 minutes, blow and beat 1～2 minute again, supernatant is drawn in natural sedimentation, filters the back with 400 order metal screens and moves into the 10ml centrifuge tube, adds equal-volume DMEM nutrient solution in the centrifuge tube.Repeat to digest 5 times to cardiac muscular tissue's piece be digested to substantially unicellular till.Then add DMEM nutrient solution (containing 10-15% volume hyclone), breaing up cell mass is single cell suspension.The adjustment cell number is 1 * 105/ml, and single cell suspension is inoculated in the culture flask.And culture vessel placed 37 ℃, 5%C O
2Cultivate in the incubator after 90 minutes, adopt the adherent isolation technics of differential, the cell suspension in the sucking-off culture hole is collected in another culture plate, splashes into 5-bromodeoxyuridine, and making its final concentration is 0.1mmol/L, and continues to cultivate.During cellular incubation to 3 day, be divided into control group and drug effect group, the drug effect group adds digitaloid drugs, acts on 24 hours.
(2) preparation cardiac muscle cell two dimensional electrophoresis protein example
Above-mentioned model sample cell grows to exponential phase, discards nutrient solution, and cell is scraped collecting cell to centrifuge tube, phosphate buffer flushing cell 3 times, blot the residual phosphoric acid damping fluid, add lysis buffer, multigelation cell 3-5 time, left standstill on ice 20 minutes, centrifugal 1 hour of 4 ℃ of 40000g get supernatant, survey protein concentration, packing ,-80 ℃ of storages are standby.
Preferably, it is the 8mol/L urea that above-mentioned lysis buffer is formed, 65mmol/L dithiothreitol (DTT), 4%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, the 40mmol/L trishydroxymethylaminomethane.
(3) two dimensional gel electrophore-sis
Each 100ug of albumen that gets " 80 ℃ of storages are standby " of two groups of steps 2 respectively mixes with swelling solution, add IPG and hold in the glue groove, IPG immobilized ph gradient strip glue faces down and puts into groove, on cover one deck mineral oil, add a cover and be placed on the IPGphor horizontal strip electrophoresis instrument battery lead plate, carry out isoelectric focusing.Isoelectric focusing finishes back adhesive tape balance 20 minutes in equilibrium liquid, and adhesive tape is moved on the SDS-PAGE separation gel, and the agarose sealing places electrophoresis tank with glass plate, with the current constant mode electrophoresis, migrates to apart from gel base 1 centimeters to the bromophenol blue forward position and to stop electrophoresis.Get gel.
It is 8mol/L urea that above-mentioned swelling solution is formed, 2%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 18mmol/L dithiothreitol (DTT), 0.5%IPG damping fluid.
(4) image acquisition analysis
After above-mentioned gained gel is dyed, use the gel scanner transmission scan, to obtain image analyzes with PDQUEST software, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, coupling between different gels, the standard molecular weight of mark 2DMARKER and isoelectric point are determined the relative molecular weight and the isoelectric point of protein site in the glue in the normalization of amount and the utilization.The dielectrophoresis collection of illustrative plates of cardiac muscle cell protein is with reference to glue under the normal condition, and the dielectrophoresis collection of illustrative plates of cardiac muscle cells protein carries out proportioning with it under the cardiac glycoside drug effect, obtains the cardiac glycoside drug effect in cardiac muscle cell's differential expression protein.
Prior art is adopted in the dyeing of gel.
(5) differential protein by mass spectrum, is measured differential protein behind in-gel digestion.Compare with mass spectral database, filter out differential protein.
Behind the clear and definite differential protein, its biological function can be determined.Also can do Western blot further verifies.
The invention has the beneficial effects as follows: with the drug effect target position or the relevant target position of proteomic techniques screening cardiac glycoside medicine heart failure resistance, through functional verification, can be directly used in the heart failure resistance medicine that instructs research and development or be transformed into new and effective low toxicity, produce huge economic and social benefit.
(4) description of drawings
Fig. 1 is the process flow diagram of the inventive method, 1 normal myocardium cell wherein, cardiac muscle cell behind 2 DEs, 3 dielectrophoresis, 4 dielectrophoresis, 5 silver medals dye, examine and dye or fluorescent dye, 6 silver medals dye, examine and dye or fluorescent dye, 7 protein spots (control group constitutive protein matter picture group spectrum), 8 protein spots (cardiac glycoside intervention group constitutive protein matter picture group spectrum), 9 quantitative computer analysis electrophoresis patterns, 10 differential protein spots, 11 tandem mass spectrum identification of protein, 12 protein information source sequence databases, 13 differential proteins are identified, 14 clear and definite cardiac glycoside effect rat myocardial cell associated protein.
Fig. 2 is the dielectrophoresis collection of illustrative plates of embodiment 1 normal myocardium cell.
Fig. 3 is the dielectrophoresis collection of illustrative plates of cardiac muscle cell behind embodiment 1 DE.
Fig. 4 is embodiment 1 differential protein spot 1158 ribonucleoprotein peptide dactylograms.
Fig. 5 is embodiment 1 differential protein spot 1090 actin peptide dactylograms.
Fig. 6 is embodiment 1 differential protein spot 1030 myosin peptide dactylograms.
Fig. 7 is the dielectrophoresis collection of illustrative plates of cardiac muscle cell after the embodiment 2 strophanthidin effects.
Fig. 8 is embodiment 2 differential protein spots 1026 calmodulin peptide dactylograms.
Fig. 9 is embodiment 2 differential protein spots 973 heat shock protein peptide dactylograms.
(5) embodiment
Embodiment 1:
(1) sets up the cardiac glycoside medicine to the myocardial cells effect model.
Get newborn 1-2 days Wistar rats, 10, cut open chest, it is dirty to core, and cleans up with D-Hanks liquid, adds 0.125% pancreatin and 0.02% divinyl tetraacethyl disodium, and 37 ℃ of water-baths 5 minutes are blown and beaten 1 minute, and supernatant is abandoned in natural sedimentation; Add 0.125% pancreatin, place 37 ℃ of water-baths 10 minutes, blow and beat 1～2 minute again, supernatant is drawn in natural sedimentation, filters the back with 400 order metal screens and moves into the 10ml centrifuge tube, adds equal-volume DMEM nutrient solution in the centrifuge tube.Repeat to digest 5 times to cardiac muscular tissue's piece be digested to substantially unicellular till; Then add the DMEM nutrient solution, wherein contain 15% hyclone, breaing up cell mass is single cell suspension; The adjustment cell number is 1 * 105/ml, and single cell suspension is inoculated in the culture flask; And culture vessel placed 37 ℃, 5%C O
2Cultivate in the incubator after 90 minutes, adopt the adherent isolation technics of differential, the cell suspension in the sucking-off culture hole is collected in another culture plate, splashes into 5-bromodeoxyuridine, and making its final concentration is 0.1mmol/L, and continues to cultivate.During cellular incubation to 3 day, be divided into control group and drug effect group, the drug effect group changes original fluid into serum-free medium, adds digoxin, and the medicine final concentration is 1ng/ml, acts on 24 hours.
(2) cardiac muscle cell's two dimensional electrophoresis protein example preparation
Above-mentioned model sample cell grows to exponential phase, discards nutrient solution, and cell is scraped collecting cell to centrifuge tube, phosphate buffer (PBS) flushing cell 3 times, the cell branch is filled to the 1.5ml centrifuge tube, blots the residual phosphoric acid damping fluid, add lysis buffer, multigelation cell 3-5 time left standstill 20 minutes on ice, 4 ℃, 40000g, centrifugal 60 minutes, remove supernatant, the mensuration protein concentration is 1.2ug/ul, total protein 1.12mg, divide to be filled to the 1.5ml centrifuge tube, be stored in-80 ℃ standby.It is the 8mol/L urea that described lysis buffer is formed, 65mmol/L dithiothreitol (DTT), 4%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, the 40mmol/L trishydroxymethylaminomethane.
(3) two dimensional gel electrophore-sis
Each 100ug of albumen that gets " 80 ℃ of storages are standby " of two groups of steps 2 respectively mixes with swelling solution, add IPG and hold in the glue groove, IPG immobilized ph gradient strip glue faces down and puts into groove, on cover one deck mineral oil, add a cover and be placed on the IPGphor horizontal strip electrophoresis instrument battery lead plate, carry out isoelectric focusing.Isoelectric focusing finishes back adhesive tape balance 20 minutes in equilibrium liquid, and adhesive tape is moved on the SDS-PAGE separation gel, and the agarose sealing places electrophoresis tank with glass plate, with the current constant mode electrophoresis, migrates to apart from gel base 1 centimeters to the bromophenol blue forward position and to stop electrophoresis.Get gel.
It is 8mol/L urea that above-mentioned swelling solution is formed, 2%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 18mmol/L dithiothreitol (DTT), 0.5%IPG damping fluid.
(4) image acquisition analysis
Above-mentioned gel is dyeing earlier:
The operation manual that silver dyes by protein silver transfection reagent box carries out.The basic process of dyeing is followed successively by: with distilled water rinsing SDS-PA GE glue 5min; Fixing 60min in the immobile liquid that contains 40% ethanol, 10% acetate; Containing 30% ethanol, 0.2%Na
2S
2O
3, 6.8% sodium acetate and 0.125% glutaraldehyde sensitizing solution in sensitization 30min; Wash each 5min 3 times; Containing 0.25%AgNO
3Dye 20min with silver in the dyeing liquor of 0.0148% formaldehyde; Wash each 1min 2 times; Containing 2.5%Na
2CO
3With the protein clear spot to the gel that develops in the developer solution of 0.0148% formaldehyde; With 1.46%EDTANa
2H
2O stops 10min; Gel is stored in 4.6% glycerine standby.But result's repeated rows is examined and is dyed.
Examine and dye routinely the coomassie brilliant blue staining method and carry out.Its process is filter paper filtering dyeing liquor (50% methyl alcohol, 10% acetate, 0.25%CBB R350), washing glue 5min, and the 3h that dyes in the dyeing liquor, 5% acetate, the decolouring of 10% methanol solution is changed destainer repeatedly and is become clear until protein site on background.
After above-mentioned gel is dyed, use the gel scanner transmission scan, obtain digitized image (seeing Fig. 2 and 3 dielectrophoresis collection of illustrative plates), analyze with PDQUEST software, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, the coupling between different gels, the standard molecular weight of mark 2DMARKER and isoelectric point are determined the relative molecular weight and the isoelectric point of protein site in the glue in the normalization of amount and the utilization.The dielectrophoresis collection of illustrative plates of cardiac muscle cell protein is with reference to glue under the normal condition, the dielectrophoresis collection of illustrative plates of cardiac muscle cells protein carries out proportioning with it under the digoxin effect, obtain differential expression protein (Fig. 2,3 protein sites 1026,1173 that digitaloid drugs acts on the cardiac muscle cell, 1030,1199,1178,1127,1090,1181).
(5) the above-mentioned differential protein that screens is behind in-gel digestion, and utilization electron spray ion trap mass spectrometry (LTQ-MS) is measured differential protein (seeing Fig. 4,5,6).Differential protein through the visible abundance of Western blot apparently higher than control group (P＜0.05).
Embodiment 2:
(1) sets up digitaloid drugs to the myocardial cells effect model.
The cardiac muscle cell cultivates as described in the embodiment 1, and different is that the drug effect group changes digoxin into strophanthidin, and drug concentration is 1 * 10
-9Mol/L acts on 24 hours respectively, causes cardiac glycoside effect cardiac muscle cell model.
(2) cardiac muscle cell's two dimensional electrophoresis protein example preparation is with embodiment 1.
(3) two dimensional gel electrophore-sis is with embodiment 1
(4) image acquisition analysis is with embodiment 1
Gel is behind coomassie brilliant blue staining, use the gel scanner transmission scan, obtain digitized image, analyze with PDQUEST software, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, the coupling between different gels, the standard molecular weight of mark 2DMARKER and isoelectric point are determined the relative molecular weight and the isoelectric point of protein site in the glue in the normalization of amount and the utilization.The dielectrophoresis collection of illustrative plates of cardiac muscle cell protein is with reference to glue under the normal condition, the dielectrophoresis collection of illustrative plates of cardiac muscle cells protein carries out proportioning with it under the digitalis drug effect, obtain differential expression protein (Fig. 2,7 protein sites 1158,1173 that digitaloid drugs acts on the cardiac muscle cell, 1030,1199,973,1127,1026,1181).
(5) differential protein uses substance assistant laser desorpted ionized/flight time mass spectrum (MALDI-TOF-MS) behind in-gel digestion, measures differential protein peptide quality fingerprinting collection of illustrative plates.Clear and definite DE associated protein (seeing Fig. 8,9).Differential protein through the visible abundance of Western blot apparently higher than control group (P＜0.05).
Claims (1)
1. utilize the method for proteomic techniques screening cardiac glycoside drug effect target position, step is as follows:
(1) sets up the cardiac glycoside medicine to the myocardial cells effect model
When the cardiac muscle cell is cultured to 3 days, be divided into control group and drug effect group, the cardiac glycoside medicine to drug effect group adding variable concentrations is cardiac glycoside drug effect cardiac muscle cell's model;
(2) preparation cardiac muscle cell two dimensional electrophoresis protein example
Above-mentioned model sample cell grows to exponential phase, discards nutrient solution, and cell is scraped collecting cell to centrifuge tube, phosphate buffer flushing cell 3 times, blot the residual phosphoric acid damping fluid, add lysis buffer, multigelation cell 3-5 time, left standstill on ice 20 minutes, centrifugal 1 hour of 4 ℃ of 40000g get supernatant, survey protein concentration, packing ,-80 ℃ of storages are standby;
It is the 8mol/L urea that described lysis buffer is formed, 65mmol/L DTT, 4%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, the 40mmol/L trishydroxymethylaminomethane;
(3) two dimensional gel electrophore-sis
Each 100ug of albumen that gets " 80 ℃ of storages are standby " of two groups of steps (2) respectively mixes with swelling solution, add IPG and hold in the glue groove, IPG immobilized ph gradient strip glue faces down and puts into groove, on cover one deck mineral oil, add a cover and be placed on the IPGphor horizontal strip electrophoresis instrument battery lead plate, carry out isoelectric focusing; Isoelectric focusing finishes back IPG immobilized ph gradient strip balance 20 minutes in equilibrium liquid, the IPG immobilized ph gradient strip is moved on the SDS-PAGE separation gel agarose sealing, glass plate is placed electrophoresis tank, with the current constant mode electrophoresis, migrate to apart from gel base 1 centimeters to the bromophenol blue forward position and to stop electrophoresis, gel;
It is 8mol/L urea that described swelling solution is formed, 2%3-[(3-courage amido propyl)-diethylamine]-propane sulfonic acid, 18mmol/L dithiothreitol (DTT), 0.5%IPG damping fluid;
(4) image acquisition analysis
After above-mentioned gained gel is dyed, use the gel scanner transmission scan, to obtain image analyzes with PDQUEST software, image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, coupling between different gels, the standard molecular weight of mark 2DMARKER and isoelectric point are determined the relative molecular weight and the isoelectric point of protein site in the glue in the normalization of amount and the utilization; The dielectrophoresis collection of illustrative plates of cardiac muscle cell protein is with reference to glue under the normal condition, and the dielectrophoresis collection of illustrative plates of cardiac muscle cells protein carries out proportioning with it under the cardiac glycoside drug effect, obtains the cardiac glycoside drug effect in cardiac muscle cell's differential expression protein;
(5) differential expression protein by mass spectrum, is measured differential expression protein behind in-gel digestion; Compare with mass spectral database, filter out differential expression protein.
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| CN101871881B (en) * | 2009-04-22 | 2012-09-26 | 中国科学院电子学研究所 | Method for detecting protein content in solution |
| CN103233060B (en) * | 2013-04-26 | 2015-08-12 | 天津科技大学 | A kind of method analyzing protein expression in fungi fermentation process |
| CN103275993A (en) * | 2013-04-26 | 2013-09-04 | 温州医学院 | Method for obtaining spirulina platensis salt-tolerant functional gene by utilizing proteome technology |
| CN104087550B (en) * | 2014-07-10 | 2017-01-04 | 上海益诺思生物技术有限公司 | A kind of cultural method of rat myocardial cell |
| CN104502610B (en) * | 2014-12-25 | 2016-04-06 | 中山大学 | A kind of protein-bonded method of screening microbiotic |
| CN104483371A (en) * | 2014-12-30 | 2015-04-01 | 青岛市市立医院 | Method for screening drug action target position of tacrolimus in immunized T cell |
| CN104483376A (en) * | 2014-12-30 | 2015-04-01 | 青岛市市立医院 | Method for screening drug action target position of cyclosporine A in immunized T cell |
| CN105301082B (en) * | 2015-07-23 | 2018-01-23 | 海南师范大学 | Alpinetin and CDK1 and the differential protein detection method of serum effect |
| CN109828121A (en) * | 2019-03-18 | 2019-05-31 | 青岛农业大学 | A kind of proteomic analytical methods and its application |
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