CN101329300A - Construction and application of cardiac muscle cell protein difference expression atlas - Google Patents
Construction and application of cardiac muscle cell protein difference expression atlas Download PDFInfo
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Abstract
The invention provides a buildup method of a protein difference expression atlas when the committed differentiation of stem cells is induced by drugs, which detailedly establishes the protein difference expression atlas formed by that the stem cell is induced by the ICA and directionally differentiated to be a cardiac muscle cell; a model that the drug induces the stem cell to be directionally differentiated to be the cardiac muscle cell is established so as to prepare a two-dimensional electrophoresis protein sample; electrophoresis is gelled bidirectionally, images are collected and analyzed so as to confirm the difference protein. A comparison protein atlas is used for screening and identifying the difference expression protein when the ES cell is directionally differentiated to be the cardiac muscle cell by the inducing of the ICA; the difference expression protein is used as a drug effect target and applied to the preparation of a novel inducer which has high-efficiency and low-toxicity and is used for prompting the stem cell to be differentiated.
Description
Technical field
The invention belongs to pharmacology and medical technical field, relate to the structure and the application of the protein difference expression atlas of drug-induced stem cell directional differentiation, be specifically related to make up icariin induced dry-cell directed differentiation cardiac muscle cell's protein difference expression atlas, the evaluation icariin induces the characteristic of group and spontaneous differentiation group marking protein group to change.
Background technology
The external orientable cardiac muscle cell with typical structure and function that is divided into of embryonic stem cell (embryonic stem cell, ES cell), this differentiated system has become the important tool of expression of research cardiac gene and function.At present, it is still not fully aware of the ES cell directional to be divided in cardiac muscle cell's process numerous gene functions and signal transduction network regulation mechanism.Though have some researchs to carry out desk study, mainly concentrate on a kind of or a few known and protein to cardiac muscle cell's influence of last differentiation pathway eventually.And have numerous genes, transcription factor, protein and growth factor regulating and control cardiac muscle cell's differentiation in the ES cell directional atomization in different aspects and different time, if only utilize traditional gene, analysis of protein method, be difficult to system, explain the fundamental mechanism of whole heart development up hill and dale.Only still can not answer processing behind certain expression of gene time, expression, the protein translation and the situation of modifying or the like from dna sequence dna, therefore research protein expression and function become and become more and more important on integral level.
The proteomic techniques that development in recent years is got up is that all proteins with genome encoding is a research object, from the composition and the Changing Pattern thereof of cellular level and integral level research protein, thereby deeply is familiar with organic multiple physiology and pathologic process.Developing rapidly of its core technology dielectrophoresis, mass spectrophotometry and protein group information science can be finished the research that extremely complicated protein function and cell factor change.As be applied to the research of ES cells in vitro directed differentiation process, can further investigate cardiac muscle differentiation each phase characteristic gene transcript protein expression and functional mode, illustrate the early stage biological phenomena essence that takes place and break up of embryo heart, differentiation mechanism and the application of exploring stem cell is of great importance.
The previous work of present patent application person place research group has successfully made up the mouse embryo stem cell directed differentiation and has been cardiac muscle cell's medicine efficacy screening model, micromolecular compound drug effect in plurality of Chinese monomeric compound and the structure diversity storehouse is estimated, confirmed icariin (icariin, ICA) significantly improve the differentiation rate of ES cells in vitro directed differentiation after the intervention for the cardiac muscle cell, and differentiation phase is significantly shifted to an earlier date mutually, good dose-effect and time-effect relationship are arranged, and desk study the ICA expression that can promote cardiac muscle to grow dependent gene increase, and the time be sequencing on mutually.Be nowhere near but only study also, must study by genetic transcription and translate the process of protein from the angle of gene, could real essence and the rule that discloses biological phenomena.To ICA induce gene function in the ES cell directional differentiation phase signal transduction pathway the executor---the expression of functional protein does not conduct a research as yet yet.
Summary of the invention
An object of the present invention is to provide the construction method of drug-induced stem cell directional differentiation phase protein difference expression atlas, specifically made up the protein difference expression atlas of ICA induced dry-cell directed differentiation, realize by following steps for the cardiac muscle cell:
(1) set up drug-induced stem cell directional and be divided into cardiac muscle cell's model: the ES cell is cultivated through hanging drop and is formed embryoid body, adds medicine ICA and induces its directed differentiation to be the cardiac muscle cell.
(2) preparation two dimensional electrophoresis protein example: the noble cells of collecting above-mentioned different differentiation phase phases, add the lysis buffer mixing, multigelation is 3 times in the liquid nitrogen, add 50 μ g/ml RNase and 200 μ g/mlDNase, placed 15 minutes at 4 ℃, 16100 leave the heart, abandon supernatant, use the pre-cold acetone of 1% dithiothreitol (DTT) (DTT) to wash twice again, drying at room temperature 1h, protein concentration is surveyed in hydrating fluid dissolving back.Preferential above-mentioned lysis buffer is formed and is contained 10% trichloroacetic acid acetone cold soln and 1%DTT.Hydrating fluid is formed and is contained 8mol/L urea, 4%CHAPS and 0.001% bromophenol blue.
(3) two dimensional gel electrophore-sis: each the 100 μ g of protein that get step (2) respectively mix with swelling solution, and adding IPG holds and carries out isoelectric focusing in the glue groove.Finish back adhesive tape balance 20 minutes in equilibrium liquid, adhesive tape is moved on the SDS-PAGE separation gel, get gel with the current constant mode electrophoresis.Above-mentioned swelling solution is formed and is contained 8mol/L urea, 4%CHAPS, 65mmol/L DTT, 0.2%Bio-Lyte and 0.001% bromophenol blue.
(4) image acquisition analysis: after above-mentioned gained gel was dyed, the image that scanning obtains was analyzed with PDQUEST software.The spontaneous dielectrophoresis collection of illustrative plates that is divided into cardiac muscle cell protein of ES cell is that ICA induces the dielectrophoresis collection of illustrative plates of group to carry out proportioning with it with reference to glue, obtains the differential expression protein that ICA induces differentiation.Above-mentioned gel-colored employing argentation.
(5) same step (3) obtains gel, adopts then and examines the dyeing of Ma Shi light blue method, and differential protein is identified and filtered out to differential protein by mass spectrum, behind in-gel digestion.
(6) behind the clear and definite differential protein, its biological function can be determined, also can make Western blot and further verify.
Another object of the present invention provides the application of protein difference expression atlas in preparation high-efficiency low-toxicity type promotion stem cell differentiating inducer of drug-induced stem cell directional differentiation, main utilization comparison protein collection of illustrative plates Screening and Identification goes out ICA and induces the ES cell directional to be divided into cardiac muscle cell's differentially expressed protein, the differentially expressed protein that shows in this collection of illustrative plates can be used as the drug effect target position, instructs the research and development of the novel promotion stem cell of preparation high-efficiency low-toxicity differentiating inducer.
Protein to above-mentioned differential expression carries out mass spectrum evaluation and analysis; filter out 49 differentially expressed protein points altogether; mainly can be divided into cyclin; protein anabolism albumen, energetic supersession albumen, autoprotection albumen; cytoskeletal protein; glycometabolism albumen, transcription factor, 7 classes such as signal transducer.Disclosed the action target spot that ICA induces the cardiac muscle cell to break up, induced the mechanism of action of cardiac muscle differentiation that experimental basis is provided, can be used as the application that the drug effect target position directly instructs the novel short cell-differentiation inducers of research and development simultaneously for illustrating ICA.
The invention has the beneficial effects as follows:
(1) the present invention has successfully made up the protein difference expression atlas that medicine ICA inducing mouse ES cell directional is divided into the cardiac muscle cell.
(2) protein to above-mentioned differential expression carries out mass spectrum evaluation and analysis; filter out 49 differentially expressed protein points altogether; mainly can be divided into cyclin; protein anabolism albumen, energetic supersession albumen, autoprotection albumen; cytoskeletal protein; glycometabolism albumen, transcription factor, 7 classes such as signal transducer.Disclosed the action target spot that ICA induces the cardiac muscle cell to break up, induced the mechanism of action of cardiac muscle differentiation that experimental basis is provided, can be used as the application that the drug effect target position directly instructs the novel short cell-differentiation inducers of research and development simultaneously for illustrating ICA.
Description of drawings
Fig. 1 is the variation of cellular morphology in the ES cell directional process of differentiation.
Fig. 2 is that different differentiation phase phases (differentiation d 5+3, d 5+7) are induced the cardiac muscle cell group that differentiates by the spontaneous differentiation of ES cell with by ICA, and the protein expression collection of illustrative plates diversity ratio that the process dielectrophoresis obtains.
Fig. 3 expresses for WESTERN BLOT method checking ICA induces the ES cell directional to be divided into cardiac muscle cell's differential protein.
The instantiation mode
The present invention is further described in conjunction with the accompanying drawings and embodiments.Should be understood that following preferred specific embodiments only illustrates, but not limit the scope of the invention by any way.
It is cardiac muscle cell's protein difference expression atlas that embodiment 1 makes up ICA induced dry-cell directed differentiation
(1) set up drug-induced stem cell directional and be divided into cardiac muscle cell's model: the ES cell is cultivated through hanging drop and is formed embryoid body, adds the medicine icariin and induces its directed differentiation to be the cardiac muscle cell.
(2) preparation two dimensional electrophoresis protein example: collect the noble cells of above-mentioned different differentiation phase phases, after the phosphate buffer flushing, blot residual liquid, add the lysis buffer mixing, multigelation is 3 times in the liquid nitrogen, adds 50 μ g/ml RNase and 200 μ g/ml DNase, placed 15 minutes at 4 ℃, 16100 leave the heart, abandon supernatant, use the pre-cold acetone of 1% dithiothreitol (DTT) (DTT) to wash again twice, drying at room temperature 1h, protein concentration, packing ,-80 ℃ of preservations are surveyed in hydrating fluid dissolving back.Preferential above-mentioned lysis buffer is formed and is contained 10% trichloroacetic acid acetone cold soln and 1%DTT.Hydrating fluid is formed and is contained 8mol/L urea, 4%CHAPS and 0.001% bromophenol blue.
(3) two dimensional gel electrophore-sis: each the 100 μ g of protein that get the step poly-(2) respectively mix 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 horizontal strip electrophoresis instrument battery lead plate, carry out isoelectric focusing.Finish back adhesive tape balance 20 minutes in equilibrium liquid, adhesive tape is moved on the SDS-PAGE separation gel, the agarose sealing places electrophoresis tank with glass plate, with the current constant mode electrophoresis, migrates to apart from 1cm place, gel base to the bromjophenol blue forward position and to stop electrophoresis, must gel.Above-mentioned swelling solution is formed and is contained 8mol/L urea, 4%CHAPS, 65mmol/L DTT, 0.2%Bio-Lyte and 0.001% bromophenol blue.
(4) image acquisition analysis: after above-mentioned gained gel was dyed, the image that scanning obtains was analyzed with PDQUEST software.Image analysis process comprises the shearing of collection of illustrative plates, the detection of protein spots, and the coupling between different gels, target standard molecular weight 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 spontaneous dielectrophoresis collection of illustrative plates that is divided into cardiac muscle cell protein of ES cell is that icariin induces the dielectrophoresis collection of illustrative plates of group to carry out proportioning with it with reference to glue, obtains the differential expression protein that icariin is induced differentiation.Above-mentioned gel-colored employing argentation.
(5) same step (3) obtains gel, adopts then and examines the dyeing of Ma Shi light blue method, and differential protein by mass spectrum, is identified differential protein behind in-gel digestion, compare with mass spectral database, filters out differential protein.
(6) behind the clear and definite differential protein, its biological function can be determined, also can make Western blot and further verify.
Embodiment 2 ICA inducing mouse ES cells in vitro directed differentiation are cultivated for the cardiac muscle cell
1.ICA inducing the differentiation of ES cell cultivates
(1) hanging drop is three days
With undifferentiated ES cell, make single cell suspension with ES cell differentiation nutrient culture media after, counting is diluted to 2.0 * 10 with the high sugared DMEM nutrient solution that contains 20% hyclone
4Individual/ml, the cell liquid (each hanging drop comprises 600 ES cells) that dropping 30 μ l/ drip on the double dish interior surface carries out hanging drop differentiation cultivation and treated that (embryonic body EB) formed embryoid body in 3 days.
(2) suspended two days
Double dish is spread bottom set portion with 1% agar solution in advance, the EB that forms is changed in the double dish that contains high sugared DMEM, 20% hyclone nutrient culture media suspended 2 days.
(3) adhere-wall culture
Embryoid body is transferred in advance on 24 orifice plates of handling with gelatin, is put into incubator and hatched 4-5 hour, treat that embryoid body is adherent after, every hole adds the 1ml differential medium and contains medicine 1 μ l ICA, the ICA final concentration is 10
-7Mol/L with the negative contrast of solubilizer DMSO, cultivates and collected sample in 3 days or 7 days.
2. immunocytochemistry is identified the cardiac muscle cell
Immunofluorescence technique is identified the expression of myocardium specific marker thing troponin (Troponin T).The EB adhere-wall culture with the fixing 10min of pure cold acetone, is handled 30min with containing 10% sheep blood serum PBS after a couple of days again, adds an anti-Monoclonal mouse anti-cardiac troponin T (JLT-12) then, dilution in 1: 100,4 ℃ of overnight incubation.Gave a baby a bath on the third day after its birth time with PBS in second day, each 5min blots excess liquid, adds two anti-Rodamin-F (ab ') again
2Fragment goat anti-mouse IgG for troponin T, dilution in 1: 1000 is hatched 1.5h for 37 ℃.It is inferior to give a baby a bath on the third day after its birth with PBS again, and each 5min blots excess liquid, fluorescence microscope, Taking Pictures recording.Whether with what whether occur that sarcomere represents to be come by the ES cell differentiation is the cardiac muscle cell.
Referring to Fig. 1, the result shows that embryoid body is transferred in 24 orifice plates that are covered with gelatin and cultivated, and differentiation is continued in adherent back, is divided into the cardiac muscle cell by undifferentiated cell mass from cell mass.The similar spheroid of cardiac muscle cell group's shape, the center that has auto-rhythmicity to shrink.An embryoid body contains one or more center of compression, illustrates to have broken up at this moment to obtain the cardiac muscle cell.The adherent cardiac muscle cell who is divided into is carried out immunofluorescence dyeing, and visible cardiac muscle cell's sarcomere albumen troponin T presents red fluorescence, tangible sarcomere occurs, referring to Fig. 1 D.Fig. 1 is the variation of cellular morphology in the ES cell directional process of differentiation, and (A) ES cell is cloned (the arrow place is the ES cell clone) among the figure on the MEF feeder layer; (B) EB of suspension cultured; (C) grow and next spontaneous contraction pulsatile heart myocyte group by EB.(D) TnT (troponin T) immunofluorescence positive staining.Bar＝25μm(A，B，D)，10μm(C)。
Embodiment 3 two dimensional gel electrophore-sises separate the protein expression point that ICA induces ES cell directional cardiac muscle cell differentiation
Carry out isoelectric focusing electrophoresis earlier total protein is separated according to isoelectric point, and then carry out SDS-PAGE and further separate according to molecular size, the dyed electrophoretogram that obtains is the protein map of a Two dimensional Distribution.Be different differentiation periods of cardiac muscle cell and the ICA protein expression situation of change after inducing by control protein map analysis ES cell differentiation, thereby find and definite target protein.
(1) specimen preparation
A. cell sample
The sucking-off nutrient solution is scraped collecting cell (the spontaneous differentiation of adhere-wall culture 3 days and 7 days and induce through ICA the cardiac muscle cell who is divided into) with cell.Add D-Hanks solution, 1500 change, divided centrifugal 10 minutes, abandon supernatant.Repeat 3 times.Add 5 times of volume lysates (containing 10% trichloroacetic acid acetone cold soln and 1%DTT) digestion mixing.Multigelation is 3 times in the liquid nitrogen.Add 50 μ g/ml RNase and 200 μ g/ml DNase, placed 15 minutes at 4 ℃.16100 change, and 4 ℃ centrifugal 15 minutes.Collect supernatant, frozen after the packing in-80 ℃.
B. protein quantification: adopt the Bradford kit.
(2) first to isoelectric focusing
The aquation sample-loading buffer 400 μ l that taking-up is preserved from-20 ℃ of refrigerators (contain 8mol/L urea, 4%CHAPS, 0.001% bromophenol blue does not contain DTT, does not contain Bio-Lyte) tubule (1ml/ pipe), put the room temperature dissolving.Add 0.01g DTT in tubule, each 5 μ l of Bio-Lyte add 100 μ l samples, fully mixing.Take out the prefabricated adhesive tape of IPG (18cm pH3-10) of preserving from-20 ℃ of refrigerators, room temperature was placed 10 minutes.The edge of groove a to left side in focusing dish or the aquation dish and the right linear sample that adds.Applied sample amount is 100 μ g albumen/300 μ l.After all protein examples have all joined in focusing dish or the aquation dish, remove protective seam on the prefabricated IPG adhesive tape gently with tweezers.Distinguish the adhesive tape both positive and negative polarity, IPG adhesive tape glue is faced down places on focusing dish or the aquation dish sample solution gently, makes the positive pole of adhesive tape positive pole (indicating+) corresponding to the focusing dish.Guarantee that adhesive tape closely contacts with electrode.On every adhesive tape, cover 2-3ml mineral oil.To good positive and negative electrode, cover lid.It is the limiting current 50 μ A/ roots of every adhesive tape that the isoelectric focusing program is set, 20 ℃ of the temperature during isoelectric focusing.Carry out balance and second immediately to the SDS-PAGE electrophoresis with focusing on the adhesive tape that finishes.
(3) balance of adhesive tape
The adhesive tape glue that focusing is good faces up and is placed on the dried thick filter paper.With the thick filter paper of another part MilliQ water-soaked, squeeze and remove excessive moisture, directly place then on the adhesive tape, blot mineral oil and unnecessary sample on the adhesive tape gently.Adhesive tape is transferred in the swelling dish, and adhesive tape of each groove adds 5ml adhesive tape level pad (I) (containing 6mol/L urea, 2%SDS, 0.375mol/L Tris-HCl, 20% glycerine and 2%DTT) in the groove of adhesive tape is arranged.Sample aquation dish is placed on slowly rocked on the horizontal shaking table 15 minutes.After balance finishes for the first time, thoroughly outwell or sop up the adhesive tape level pad I in the sample aquation dish.And draw unnecessary equilibrium liquid with filter paper.Add adhesive tape level pad (II) (containing 6mol/L urea, 2%SDS, 0.375mol/LTris-HCl, 20% glycerine and 2.5% iodoacetamide) again, continue on horizontal shaking table, slowly to rock 15 minutes.
(4) second to the SDS-PAGE electrophoresis
Prepare two of 12.5% acrylamide gels.After treating that gel solidifies, remove the MilliQ water on separation gel surface, with the flushing of MilliQ water.Go excess liquid between the glass plate of SDS-PAGE polyacrylamide gel top with the filter paper suction.With second being placed on the desktop to gel of handling well, long glass plate is down, short glass plate up, the top of gel is facing to oneself.Agarose sealing liquid is carried out heating for dissolving.With 10 * electrophoretic buffer,, become 1 * electrophoretic buffer with 10 times of graduated cylinder dilutions.The rush bubble on damping fluid surface.After balance finishes for the second time, thoroughly outwell or sop up the adhesive tape level pad II in the sample aquation dish.The IPG adhesive tape is shifted out from sample aquation dish, and an end of clamping adhesive tape with tweezers makes the glue face soak the end fully in 1 * electrophoretic buffer.Then adhesive tape glue is faced up and be placed on the long glass plate of gel.The SDS-PAGE gel that is placed with adhesive tape is transferred on the encapsulating frame, and short glass plate one is facing to oneself.Above gel, add low melting-point agarose sealing liquid.Lightly adhesive tape is pushed away downwards with tweezers, make it to contact fully with polyacrylamide gel glue face.Placed 10 minutes, low melting-point agarose sealing liquid is thoroughly solidified.After low melting-point agarose sealing liquid solidifies fully.Gel is transferred in the electrophoresis tank.After electrophoresis tank adds electrophoretic buffer, energized, low current of using when initial or low-voltage treat that sample walks out the IPG adhesive tape fully, be condensed into a line after, strengthen electric current (or voltage) again, treat can stop electrophoresis when the bromophenol blue indicator reaches bottom margin.
(5) after electrophoresis finishes, pry open layer glass gently, take out gel, and corner cut is with marking.Adopt argentation to carry out gel-colored.
(6) image analysis: adopt PDQuest software to carry out labor, comparison protein spot difference.
The result shows, IPG adhesive tape with the long pH scope of 18cm 3-10 is separated as one, be separated as two with 12.5%SDS-PAGE, after the dyeing of SDS-PAGE gel argentation, the scanning picture is with the PDQuest software analysis, spontaneous differentiation d 5+3, d 5+7 cardiac muscle cell, ICA induce differentiation d 5+3, d 5+7 cardiac muscle cell group order, and protein expression is rise trend (referring to Fig. 2) on the whole successively.Protein expression is obvious differentiation phase phase, and along with differentiation is carried out protein expression quantity and increased progressively gradually, ICA of the same period induces the more spontaneous differentiation group of differentiation group to express obviously to be increased, and more approaching with the protein expression situation of mouse fetal rhythm tissue.Fig. 2 be different differentiation phase phases (differentiation d5+3 d5+7) induces the cardiac muscle cell group that differentiates by the spontaneous differentiation of ES cell with by ICA, the protein expression collection of illustrative plates diversity ratio that obtains through dielectrophoresis, the spontaneous differentiation of (A) ES cell is organized 5+3 days among the figure; (B) ICA induced group 5+3 days; (C) the spontaneous differentiation of ES cell is organized 5+3 days; (D) ICA induced group 5+7 days; (E) tire mouse cardiac muscular tissue.The protein spots of the numbering corresponding expression difference that marks among the figure.
Embodiment 4 mass spectrums identify that ICA induces the differential expression protein of ES cell directional cardiac muscle cell differentiation
1. mass spectrum is identified
Repeat the dielectrophoresis experiment, utilize coomassie brilliant blue staining, the glue behind the scanning analysis is placed under the bright lamplight, rifle head with silanization cuts protein spots, with enzymolysis protein in the trypsase glue, MOLDI-TOF-MS identifies, obtains the peptide quality fingerprinting collection of illustrative plates of test sample.
2. database retrieval
Protein database search (
Http:// mascot.proteomics.com.cn/search-form-PMF.html), isoelectric point and the molecular weight on glue finally determined protein according to Search Results and conjugated protein.
The result shows, with differential protein spot cutting enzymolysis among the embodiment 2, identifies by mass spectrum, obtains 49 differentially expressed proteins altogether, wherein raises 44,5 (table 1) of downward modulation.49 albumen of differential expression mainly can be divided into cyclin, protein anabolism albumen, energetic supersession albumen, autoprotection albumen, cytoskeletal protein, glycometabolism albumen, transcription factor, 7 classes such as signal transducer.The application of prompting bidirectional electrophoresis technique can disclose drug-induced ES cell differentiation on the protein integral level be differential expression situation in cardiac muscle cell's process, provides experimental basis for furtheing investigate myocardium each stage protein expression and functional mode and the drug-induced differentiation target spot of affirmation of breaking up.
Table 1 ICA induces the ES cell directional to be divided into differentially expressed protein in cardiac muscle cell's process
| The protein title | Numbering | Sequence number (NCBI) | Molecular weight (kDa) | Isoelectric point | Peptide sequence marking | Peptide sequence is beaten reliability |
| Cell cycle | ||||||
| The RAS autoploid A2 that is correlated with | F13 | 16923986 | 21768.1 | 5.83 | 216 | 100 |
| Cytoskeleton | ||||||
| Endoplasmic reticulum protein 29 precursor | E14 | 19526463 | 28805.1 | 5.9 | 338 | 100 |
| Keratin compound 1, acidity, gene 19 | E19 | 6680606 | 44514.7 | 5.28 | 374 | 100 |
| Capping protein (actin filament) flesh Z line α 1 | F1 | 33468887 | 32919.3 | 5.34 | 369 | 100 |
| Myosin light chain 2 isomeride MLC-2a | F17 | 627919 | 18252.9 | 4.48 | 193 | 100 |
| Vimentin | C24 | 2078001 | 51533.1 | 4.96 | 238 | 100 |
| Energetic supersession | ||||||
| Adenylate kinase 4 | D2 | 6753022 | 25046.1 | 7.02 | 364 | 100 |
| The ATP synzyme, the mitochondria F0 compound of commentaries on classics H+ effect, d subunit | D24 | 21313679 | 18737.6 | 5.52 | 259 | 100 |
| Electron transfer flavoprotein, the α polypeptide | E6 E8 E10 | 66911229 | 34987.5 | 8.62 | 177 218 190 | 100 100 100 |
| Isocitric dehydrogenase 3 (NAD+) α | E17 | 18250284 | 39613.1 | 6.27 | 417 | 100 |
| Protein synthesis/metabolism/processing/decomposition | ||||||
| Ubiquitin carboxyl terminal hydrolase-l 1 | D3 | 6755929 | 24821.5 | 5.33 | 180 | 100 |
| Ubiquitin-conjugating enzyme E2N | D16 E3 | 4507793 | 17127 | 6.13 | 207 413 | 100 100 |
| Proteasome Alpha 2 subunits | D6 | 6679497 | 25909.3 | 8.39 | 272 | 100 |
| Agavain, ATP dependence, protein protomer homolog | D21 | 8393156 | 29781.3 | 7.05 | 144 | 100 |
| Proteasome 26S | E4 | 6754724 | 36517.2 | 6.29 | 447 | 100 |
| Proteasome Alpha 6 subunits | E16 | 6755198 | 27354.8 | 6.34 | 253 | 100 |
| Fatty acid binding protein 5, the epidermis type | D18 E5 | 6754450 | 15127.4 | 6.14 | 96 106 | 99.997 100 |
| Other nucleotide metabolisms of RNA/ and transportation, nucleoprotein | ||||||
| Heterogeneous RNA nucleoprotein D-like | E22 | 7710036 | 33538.1 | 6.85 | 196 | 100 |
| 40S ribosomal protein S12 | F4 | 54039306 | 14515.6 | 6.82 | 110 | 100 |
| 5 ', 3 '-nucleotidase, endochylema | D17 | 7657031 | 23061.8 | 5.31 | 236 | 100 |
| The gene outcome of hypoxanthine phosphoribosyltransferase | E20 | 309315 | 24553.6 | 6.51 | 78 | 99.817 |
| Nudix (the chain primitive X of nucleoside diphosphate) motif 5 | F3 | 13435414 | 23996.1 | 5.34 | 74 | 99.552 |
| Uridine diphosphate guanosine triphosphate (ester) enzyme | F6 | 21281687 | 17373.7 | 5.74 | 304 | 100 |
| Carbohydate metabolism/transportation | ||||||
| Prediction: be similar to GAPD | D20 | 94387168 | 36280.3 | 7.62 | 113 | 100 |
| Aldose reductase (AR) | E24 | 1351911 | 35709.4 | 6.71 | 132 | 100 |
| Phosphogluconolactonase 2, the B chain | F23 | 6678674 | 36549 | 5.7 | 179 | 100 |
| The 6-phosphogluconic acid lactonase | D7 | 13384778 | 27237.4 | 5.55 | 201 | 100 |
| Phosphoglycerate phosphomutase 1 | D10 | 10179944 | 28918.9 | 6.19 | 184 | 100 |
| The glyceraldehyde-3-phosphate dehydrogenase analog | E7 | 55153885 | 35751.1 | 7.59 | 120 | 100 |
| PDHC | F2 | 12805431 | 34813.8 | 5.63 | 209 | 100 |
| Phosphotriose isomerase | D4 F10 | 54855 | 26678.8 | 6.9 | 269 280 | 100 100 |
| Stress reaction | ||||||
| Heat shock protein β-1 | D19 | 547679 | 22999.7 | 6.12 | 232 | 100 |
| (heat shock 27kDa albumen) (HSP27) | F5 F11 F15 | 397 442 69 | 100 100 98.549 | |||
| Heat shock protein HSP27 | D23 | 424145 | 21961.2 | 6.45 | 304 | 100 |
| Crystalline protein, α B | E13 | 6753530 | 20056.4 | 6.76 | 430 | 100 |
| Superoxide dismutase 1, solubility | E9 | 45597447 | 15932.8 | 6.02 | 427 | 100 |
| Superoxide dismutase 2 is in the mitochondria | F8 | 31980762 | 24587.5 | 8.8 | 261 | 100 |
| Peroxide oxidation reductase 4 | F9 | 7948999 | 31033.1 | 6.67 | 197 | 100 |
| Peroxide oxidation reductase 6 (anti-oxidant albumen 2) | F7 | 3219774 | 24855 | 5.71 | 626 | 100 |
| Transcribe | ||||||
| Basic transcription factor 3 isomeride B | D22 | 20070130 | 17688.2 | 6.85 | 263 | 100 |
| Protein kinase C q interaction protein PICOT | E23 | 6840949 | 37758.4 | 5.42 | 143 | 100 |
| Signal transduction | ||||||
| Voltage dependent anion channel 2 | E2 | 6755965 | 31712.6 | 7.44 | 107 | 100 |
| Rho GDP inhibiting factor (GDI) α that dissociates | D1 | 31982030 | 23392.8 | 5.12 | 345 | 100 |
| Phosphotidylethanolabinding binding protein | F19 | 1517864 | 20847.3 | 5.19 | 133 | 100 |
| Other | ||||||
| Haemoglobin (people δ 2/ mouse β 2) B chain | F12 | 18655687 | 15607.1 | 7.27 | 202 | 100 |
| 3-Mercaptopyruvate sulfurtransferase (MST) | F21 | 90110410 | 33002.5 | 6.11 | 18 | 100 |
| Carbonic anhydrase 2 | E15 | 33243954 | 29023.5 | 6.52 | 513 | 100 |
| Putative protein LOC68045 | E18 | 13386026 | 28134.8 | 6.4 | 346 | 100 |
| Cytokine induction protein 29 kDa | E12 | 13384730 | 23518.3 | 6.29 | 89 | 99.986 |
| Red blood cell esterase-1 | D8 | 20070420 | 28072.8 | 9 | 190 | 100 |
Embodiment 5 adopts WSTERN BLOT method to identify that ICA induces the differential expression protein of ES cell directional cardiac muscle cell differentiation
Collection is respectively organized sample and add lysis buffer (PH7.5 Tris-hydrochloric acid 20mmol/L on 4 ℃ of ice bath, NaCl 150mmol/L, EDTA 1mmol/L, Triton X-1001%, deoxysodium cholate 0.5%, PMSF1mmol/L, leupeptin 10 μ g/ml, aprotitin 30 μ g/ml), repeatedly blow and beat 30min repeatedly on ice, 4 ℃ of centrifugal 30min of 13000 * g obtain supernatant and are total protein, Lowry kit method (DC protein detection kit, Bio-Rad, CA, US) protein concentration is carried out quantitatively, frozen standby after the packing.SDS-PAGE, the protein immunization marking and film optical density analytical approach are operated routinely.The albumen applied sample amount is 80 μ g/ roads.Behind the SDS-PAGE albumen electricity is changeed film to nitrocellulose filter, 5% skim milk sealing 1h, one anti-ly is added in 5% skimmed milk that dissolves with 0.05% tween PBS incubated at room 2h or 4 ℃ of overnight incubation on the shaking table with 1: 1000 dilutability.Wash film 3 times with the PBS that contains 0.05% tween, anti-to remove non-specific binding one.Continue to hatch two anti-1-2h, two anti-dilutabilitys are 1: 5000.Wash film under the same terms three times, use the ECL reagent colour development, with the X-ray film exposure, full-automatic sheet-punching machine develops photographic film in the darkroom.Protein band carries out densitometric analysis on the film after scanning.In this experiment with GAPDH as house keeping protein.
The result shows, select several albumen ubiquitin carboxyl terminal hydrolase-l 1s (UCH-L1) of checking, myosin light chain 2 isomeride (MLC-2a), proteasome Alpha 2 subunits (PAMA2), crystalline protein α B (AB-CRY), protein expression level trend that detects through WESTERN BLOT method and dielectrophoresis be basically identical as a result.The result is referring to Fig. 3.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (2)
1. the construction method of a cardiac muscle cell protein difference expression atlas is characterized in that: realize by following steps:
(1) set up drug-induced stem cell directional and be divided into cardiac muscle cell's model: the ES cell is cultivated through hanging drop and is formed embryoid body, adds medicine ICA and induces its directed differentiation to be the cardiac muscle cell;
(2) preparation two dimensional electrophoresis protein example: the noble cells of collecting above-mentioned different differentiation phase phases, add the lysis buffer mixing, multigelation is 3 times in the liquid nitrogen, add 50 μ g/ml RNase and 200 μ g/mlDNase, placed 15 minutes at 4 ℃, 16100 leave the heart, abandon supernatant, wash twice with the pre-cold acetone of 1% dithiothreitol (DTT) again, drying at room temperature 1 hour, protein concentration is surveyed in hydrating fluid dissolving back, and above-mentioned lysis buffer is formed and is contained 10% trichloroacetic acid acetone cold soln and 1% dithiothreitol (DTT), hydrating fluid is formed and is contained 8mol/L urea, 4%CHAPS and 0.001% bromophenol blue;
(3) two dimensional gel electrophore-sis: each the 100 μ g of protein that get step (2) respectively mix with swelling solution, adding IPG holds and carries out isoelectric focusing in the glue groove, finish back adhesive tape balance 20 minutes in equilibrium liquid, adhesive tape is moved on the SDS-PAGE separation gel, get gel with the current constant mode electrophoresis, above-mentioned swelling solution is formed and is contained 8mol/L urea, 4%CHAPS, 65mmol/L DTT, 0.2%Bio-Lyte and 0.001% bromophenol blue;
(4) image acquisition analysis: after above-mentioned gained gel is dyed, the image that scanning obtains is analyzed with PDQUEST software, the spontaneous dielectrophoresis collection of illustrative plates that is divided into cardiac muscle cell protein of ES cell is with reference to glue, ICA induces the dielectrophoresis collection of illustrative plates of group to carry out proportioning with it, obtain the differential expression protein that ICA induces differentiation, above-mentioned gel-colored employing argentation;
(5) same step (3) obtains gel, adopts then and examines the dyeing of Ma Shi light blue method, and differential protein is identified and filtered out to differential protein by mass spectrum, behind in-gel digestion;
(6) behind the clear and definite differential protein, its biological function can be determined, or make Western blot and further verify.
2. the application of cardiac muscle cell protein difference expression atlas in preparation high-efficiency low-toxicity type promotion stem cell differentiating inducer that makes up according to claim 1 method, it is characterized in that: utilization comparison protein collection of illustrative plates Screening and Identification goes out ICA and induces the ES cell directional to be divided into cardiac muscle cell's differentially expressed protein, the utilization variance expressing protein is as the drug effect target position, promotes application in the stem cell differentiating inducer in preparation high-efficiency low-toxicity type.
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