CN102095777B - Method for detecting surface differential membrane protein of mesenchyme stem cells of placenta source - Google Patents
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Abstract
The invention relates to a method for detecting differential membrane protein of mesenchyme stem cells, and aims at providing a method for detecting surface differential membrane protein of mesenchyme stem cells of a placenta source. The method comprises the steps of: separating and culturing mesenchyme stem cells, extracting membrane protein from mesenchyme stem cells, obtaining membrane proteingel spectrum by bidirectional fluorescence difference gel electrophoresis, and carrying out mass spectrum analysis and verification. The method utilizes 2D-DIGE (Difference Gel Electrophoresis) for detecting special differential surface markers on the surface of the membrane of the stem cells for the first time, and can compare and analyze protein in different samples of the same gel quantitatively; an interior marker is added in each gel, and DIA and BVA software can automatically calibrate the expression quantity of protein according to the interior marker in each protein point, thus the error between gel of different batches is reduced to the maximum extent, the real change degree of protein is reflected, and the false positive rate and the false negative rate are reduced.
Description
Invention field
The present invention relates to a kind of detection method of stem cell difference memebrane protein, particularly mescenchymal stem cell (Mesenchymal stem cells, MSCSs) detection method of surface differences memebrane protein in placenta and umbilical cord source.
Background technology
(stem cells SC) is the multipotential cell that a class has the of self-replication capacity to stem cell, and under certain condition, it can be divided into multiple functioning cell.Mescenchymal stem cell (MSCSs) is the important member of stem cell family, derive from marrow the earliest, because it has powerful multiplication capacity and multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation to make it have potential applicability in clinical practice widely at aspects such as organizational project, cell therapy and gene therapies.In recent years, along with people learn the further investigation of characteristic and function to mesenchymal stem cell biological, successfully separation and identify MSCSs from tissues such as marrow, peripheral blood, muscle, fat, bleeding of the umbilicus, umbilical cord and placenta.Wherein, the easily separated cultivation of placenta source MSCSs, propagation can be divided into the clone in 3 germinal layer sources rapidly, and the ability of oriented injury migration; Placenta is discarded object clinically simultaneously, and it is restricted hardly to draw materials, and does not relate to ethics problem; Therefore be good experiment material and cell therapy carrier.And the mescenchymal stem cell in umbilical cord source, studies show that so far, it not only can become the desirable substitute of mesenchymal stem cells MSCs, and has bigger application potential, it expresses the peculiar molecular marker of multiple embryonic stem cell, have that differentiation potential is big, multiplication capacity is strong, immunogenicity is low, draw materials conveniently, the restriction of amoral ethics problem, be easy to feature such as preparation of industrialization, therefore also have the tool potential applicability in clinical practice.Therefore, the present invention selects these two kinds of cells as detected object.
The MSCS of derived from bone marrow can express surface markers such as CD29, CD44, CD73, CD90, CD105, CD106, CD166, stroma cell antigen l (Stro.1), stem cell antigen 1 (Sca.1), CXC class chemokine receptors 4 (CXCR-4) and CXCR.6, but does not express the hematopoietic cell surface marker: CD1 lb, CD14, CD31, CD33, CD34, CD133 and CD45.The MSCS in placenta source expresses CD29, CD44 and CD105, does not express CD34, CD45, CD19.The MSCS in umbilical cord source expresses CD13, CD29, CD44, CD105, does not express CD34, CD11a, CD14, CD31, CD45.But placenta and umbilical cord MSCS surface marker, different documents are reported for work and are not quite similar.In other words, marrow, placenta and umbilical cord MSCS do not have clear and definite cell surface marker.Umbilical cord is as the tubular structure that connects fetus and placenta, why not together, the difference of its isolated MSCS and placenta MSCS surface expression has, also there is not data to indicate at present, but this species diversity has significance in Developmental Biology, this patent is exactly to adopt a kind of new technological means, discloses isolated MSCS surface protein differential expression in placenta and the umbilical cord.
Summary of the invention
The problem to be solved in the present invention is, overcome deficiency of the prior art, a kind of detection method of mescenchymal stem cell surface differences memebrane protein is provided, and this method can be used for detecting the special memebrane protein sign on the mescenchymal stem cell surface of placenta, two kinds of separate sources of umbilical cord.
Two dimensional gel electrophore-sis is the mainstream technology in the proteome research, but the repeatability of this technology and susceptibility are not good, and lack the accurate quantification to protein spots, the two-way fluorescence difference that occurs in recent years shows gel technique (two-dimensional difference gel electrophoresis, 2D-DIGE) overcome the above-mentioned shortcoming of traditional two dimensional gel electrophore-sis technology, 2D-DIGE in traditional double on the basis of gel electrophoresis technology, method in conjunction with the multi-fluorescence analysis, separate a plurality of by the fluorescently-labeled sample of difference at same glue, and introduced for the first time interior target concept, software is calibrated its expression according to the interior mark of each protein site automatically, guarantee that it is real that detected albumen abundance changes, greatly improved result's accuracy, reliability and repeatability, when having avoided using different gel in operational contingency and malalignment.
Therefore we we analyze the memebrane protein of separate sources by the gel electrophoresis of two-way fluorescence difference, study placenta and umbilical cord MSCS surface differences albumen, can prepare monoclonal antibody on the one hand on this basis, carry out the evaluation of MSCS, on the other hand, disclose both relations on Developmental Biology.
Be the technical solution problem, the present invention realizes by the following technical solutions:
The detection method of the mescenchymal stem cell surface differences memebrane protein in a kind of placenta source is provided, may further comprise the steps:
(1) separation of mescenchymal stem cell and cultivation
Be placenta source as mescenchymal stem cell, then: get fresh human placenta tissue and shred, collagenase digesting is collected the cell suspension that digestion obtains, and uses the Ficoll parting liquid to carry out density gradient centrifugation, collects the tunica albuginea layer, washes twice, collecting cell, conventional cultivation;
Be umbilical cord source as mescenchymal stem cell, then: get fresh umbilical cord tissue, peel off blood vessel, shred, collagenase digesting is collected the cell suspension that digestion obtains, and is centrifugal, collecting cell, the conventional cultivation;
All mescenchymal stem cells all are cultured to exponential phase, are used for transplanting;
(2) extraction of mescenchymal stem cell memebrane protein
The multigelation method makes clasmatosis, obtains containing the component of memebrane protein then by gradient centrifugation;
(3) two-way fluorescence difference gel electrophoresis obtains the memebrane protein gel pattern
By two-way fluorescence difference gel electrophoresis (2D-DIGE), obtain the fluorescein-labeled different types of memebrane protein gel pattern of Cy2, Cy3 and Cy5, adopt DeCyder 2D image analysis software to analyze, identify two group difference expressed proteins particles;
(4) mass spectrophotometry and checking
Choose the differential protein particle, the laggard capable mass spectrophotometry of enzymolysis in the glue, and to the internet database inquiry, identify differential protein, and seek the special surface sign of separate sources stem cell, adopt the Western-Blot checking then.
Beneficial effect of the present invention is:
The present invention is used for 2D-DIGE first to the detection of stem cell membrane surface specific differences surface marker, can be on same clotting glue to different samples in protein carry out the quantitative comparison analysis, in every clotting glue, added interior mark, DIA and BVA software can automatically be calibrated its expression according to the interior mark of each protein spots, reduced the error between different batches glue and the glue to the full extent, react the true change degree of protein, reduced false positive rate and false negative rate.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in detail.
(1) separation of mescenchymal stem cell cell is cultivated
The separation of placenta MSCSs is cultivated:
After under the aseptic condition placenta fully being washed the stain of dehematizing with the PBS of preheating, shred, be cut into the as far as possible little piece of organizing, place 0.1% 4 Collagen Type VI enzyme to digest, 37 ℃ of digestion, 30min, filter, with the PBS flushing, collect digestive juice and washing fluid, centrifugal 10 min of 1200 rpm, the collecting cell precipitation, resuspended with 5mlPBS, adding to 5ml contains on the Ficoll-PaqueTM PLUS human lymphocyte parting liquid (its relative density is 1.077g/L), carry out gradient centrifugation (20 ℃, 2000rpm, 25min).After centrifugal, tunica albuginea layer in the suction pipe, PBS washed twice, collecting cell.The MSCS special culture media is resuspended, is inoculated in 6 well culture plates, places 37 ℃, 5%CO
2In the saturated humidity incubator, the row routine is cultivated and is gone down to posterity.
The separation of umbilical cord MSCSs is cultivated:
After under the aseptic condition umbilical cord fully being washed the stain of dehematizing with the PBS of preheating, peel off the navel artery and vein vascular, be cut into 1cm
3Square fritter places 0.1% 4 Collagen Type VI enzyme to digest, 37 ℃ of digestion, and 1h filters, and with the PBS flushing, collects digestive juice and washing fluid, 1200 rpm, centrifugal 10 min abandon supernatant, PBS washed cell precipitation.The MSCS special culture media is resuspended, is inoculated in 6 well culture plates, places 37 ℃, 5%CO
2In the saturated humidity incubator, the row routine is cultivated and is gone down to posterity.
(2)
The method of isolated cell memebrane protein:
1, cell is dissolved in buffer A (1mMkcl, 5mMNacl, the 3mM Mgcl2 that protease inhibitors is arranged after scraping cell on ice, 50mM Hepes, 1mM DTT, 0.5 μ g/ml Leupeptin, 20 μ M pmsf (PH=7.4)) in, multigelation is 2 times in room temperature and liquid nitrogen container.
2,5000 commentaries on classics, 4 degree are centrifugal, drive away nuclear and uncracked cell.
3, getting supernatant 12000 changes 4 degree and got precipitation in centrifugal 10 minutes and be dissolved in the buffer B (1mMkcl, 5mMNacl, 3mM Mgcl2,50mM Hepes, 1mM DTT, 0.5 μ g/ml Leupeptin, 20 μ M PMSF (PH=7.4)) that protease inhibitors is arranged.
4,12000 commentaries on classics, 4 degree were got to precipitate to be dissolved in the damping fluid of protease inhibitors C(0.5 μ g/ml Leupeptin are arranged in centrifugal 10 minutes, 20 μ M PMSF, 50mMTris-cl (PH=7.0)) extract the back in and survey protein concentration, the SDS-PAGE electrophoresis ,-20 degree are preserved standby after the packing.
(3) memebrane protein concentration determination
The protein extracting 2D Quant Kit quantification kit that adopts GE company to design at proteomics research specially.Its ultimate principle is with behind the protein precipitation, removes lysate, uses the molten protein precipitation of solution weight of copper ions again, and the copper ion with protein bound can not develop the color with the reaction of colour developing working fluid, and the depth of colour developing and the concentration of protein are inversely proportional to.
(4) 2D-DIGE
1, interior standard specimen product preparation: 2 samples (N1, N2) are respectively got 50 μ g, and volume 10 μ l join in the same eppendorf pipe, and the concussion mixing is centrifugal.The every pipe of 50 μ g is distributed into 2 pipes then.
2, the preparation of storage liquid: dyestuff is taken out from-20 ℃ refrigerator, gently revolve, room temperature leaves standstill 5min, inhales 5 μ l DMF to the dyestuff pipe, shakes centrifugally, is mixed with lnmol/ μ l storage liquid.
3, the preparation of working fluid: at first get 1.8 μ l DMF to the eppendorf pipe, draw then in 1.2 μ l storage liquid to the eppendorf pipe, shake the centrifugal working fluid that namely is made into 400 pmol/ μ l.Keep in Dark Place.
4, sample mark: the ratio of per 1 μ l working fluid and 50 μ g albumen is carried out fluorescence labeling.Lucifuge is carried out in the whole operation.Lucifuge is placed 30 min on ice, adds the reaction of l μ l lysine end mark in every tubulin
5, sample is prepared will be respectively to join in the same eppendorf pipe with the sample of Cy2, Cy3 and Cy5 mark, shakes mixing, and is of short duration centrifugal.The lucifuge operation.Add isopyknic 2 * sample-loading buffer (7mol/L urea, 2mmol/L thiocarbamide, 4%CHAPS, 65mmol/L DTT) static 10 min on ice on the every glue, sample in the preparation.
Table 1 marked member
| The glue number | CY2 | CY3 | CY5 |
| 1 | Each 25 μ g of two kinds of albumen of 50 μ g() | Placenta source stem cell membrane albumen 50 μ g | Umbilical cord source stem cell membrane albumen 50 μ g |
| 2 | Each 25 μ g of two kinds of albumen of 50 μ g() | Umbilical cord source stem cell membrane albumen 50 μ g | Placenta source stem cell membrane albumen 50 μ g |
6, isoelectric focusing
Respectively with Cy2, Cy3, three kinds of fluorescence labeling sample mix of Cy5 in the glue 1～2 together.Add an amount of hydrating fluid and (0.5%v/v) IPG buffer (pH 4-7) vibration mixing to make the cumulative volume of sample on each glue be 450 μ l, protein solution is drawn in the IPG adhesive tape groove, with IPG immobilized ph gradient strip (Ph4-7NL, 24cm) put into protein-contg adhesive tape groove, cover the about 2ml of one deck Immobiline Drystrip covering liquid, place on the IPGphor isoelectric focusing instrument, setting program, make aquation and focus on and all under 20 ℃, carry out, wherein in 30V low-voltage aquation 12h, pass through 100V 0.5h then, 500V 0.5h, 1000V 1h, 8000V 1h is stabilized at last and carries out isoelectric focusing 8h under the 8000V.
7, balance
Press from both sides out adhesive tape with tweezers, after the ultrapure water flushing, blot at filter paper, with the ultrapure water flushing, filter paper blots again, clamps adhesive tape with positive terminal (being acidic terminal) downwards with tweezers then, negative pole end (i.e. alkalescence end) upwards, put into balance test tube (tweezers folded be the alkalescence end, acidic terminal leaves bromjophenol blue and serves as a mark), with equilibrium liquid A (50mmolTriS-HCL, pH8.8,6mmol/L Urea, 30% glycerine, 1%SDS, 0.2%DTT, 0.1% bromophenol blue), equilibrium liquid B (50mmolTriS-HCL, pH8.8,6mmol/L Urea, 30% glycerine, 1%SDS, 3% iodoacetamide, 0.1% bromophenol blue) first back balance 15min.
8, second to vertical SDS-PAGE electrophoresis
The IPG adhesive tape is shifted out from level pad, 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 being faced up is placed on the long glass plate of gel, adhesive tape is pushed on the PAGE glue face gently, and glue face and adhesive tape will be combined closely, and the SDS-PAGE gel is transferred on the encapsulating frame again, adds 0.5% low melting-point agarose sealing liquid above gel.5W/ gel electrophoresis 30min then with the permanent power electrophoresis of 20W/ glue, arrives place, gel base until the bromjophenol blue index line and stops electrophoresis.
9, scanning analysis image
Typhoon multifunctional laser scanning instrument scanning SDS-PAGE gel, the image DeCyder after the scanning
TM2D6.5 analysis image is found out differential protein spot.
10, strengthening applied sample amount sets up preparation glue and uses coomassie brilliant blue staining
Each histone is mixed into 1000 μ g, carries out dielectrophoresis, use coomassie brilliant blue staining again, use ddH
2The O washed twice, each 15min pours Coomassie brilliant blue into and dyes blue dye liquor (0.25% Coomassie brilliant blue R-250 is dissolved in 50% methyl alcohol and 10% acetic acid).Shake and dye spend the night (about 13h), discard and examine dye liquor, with distilled water washing 3 times, add 10% ethanol decolorization liquid 250ml again, decolour on the shaking table clear to background till, image is preserved in scanning.
(5) mass spectrum sample preparation
1, seeks corresponding protein site according to the analysis result of DeCyder 2D image analysis software at preparation glue, cut protein spots with the Tip suction nozzle of the lml pipettor after the pruning from gel, in the Eppendorf pipe of packing into;
2, use the 50%ACN(acetonitrile)/100mM NH4HCO3(200ml, pH8.0) little blob of viscose is embathed 10min, 3 times repeatedly; Inhale at last and go washing lotion;
3, with Speed Vac blob of viscose is drained;
4, blob of viscose is immersed 10mM DTT/50mM NH4HCO3(pH8.0) in (usually 50ml), and incubation 1h(temperature is elevated to 65 ℃ gradually); Inhale afterwards and go immersion liquid;
5, blob of viscose is immersed 55mM iodoacetamide/50mM NH4HCO3(pH8.0) in (more slightly than 50ml), under the room temperature in the darkroom incubation 30min; Inhale afterwards and go immersion liquid;
6, blob of viscose is embathed 10min with 100ml 10mM NH4HCO3; After NH4HCO3 solution is removed in suction, embathe 10min with 100ml ACN again;
7, repeat the 6th operation that goes on foot one time;
8, supernatant is removed in suction, with Speed Vac blob of viscose is drained 5min;
9, add Promega trypsin enzyme liquid after the 5ml dilution in the Eppendorf pipe that blob of viscose is housed, allow enzyme liquid be absorbed by blob of viscose;
10, add the 10mM NH4HCO3 of capacity to cover the blob of viscose (about 35ml) of imbibition;
11, in 37 ℃ of incubation 3h or spend the night;
12, add isopyknic 60%ACN/about 40ml of 5% formic acid(), supersonic oscillations 10min, thus reach the effect of extracting.Centrifugal 2min collects and preserves supernatant.Add about 40ml 60%ACN/5% formic acid again to remaining blob of viscose, repeat operation just now, and preserve supernatant;
13, will collect the supernatant of preserving and drain about 1h;
14, use the ZipTipC18 desalination;
15, do maldi analysis before, with the sample dissolution drained in 50%ACN/0.1%TFA ,-20 ℃ of preservations.
(6) mass spectrophotometry
The point template for preparing is put into Applied Biosystems Voyager-DE STR 4307 MALDI-TOF-MS mass spectrometers to be analyzed, adopt reflective-mode, measure under the positive ion mode, the ion gun accelerating potential is 20000 V, the reflected voltage ratio is 1.12, N2 optical maser wavelength 337nm, pulse width is 3 ns, and ion postpones extracting 100 nsec, vacuum tightness 4x10-7Torr, acquisition quality scope m/z800-3000 dalton, the mass signal single sweep operation adds up 100 times, uses ACTH as external perimysium reference, the tryptose matter enzyme degradation peak (842.510 that autotomys, 2211.105) proofread and correct as internal standard, obtain peptide quality fingerprinting figure (PMF).
(7) data retrieval
MALDI-TOF-MS mass spectrogram spectrum unscrambling adopts the Mascot Distiller software of MatrixScience company to carry out, and calls Data ExploreTM software earlier and handles the source document that the AB MALDI-TOF-MS of company mass spectrum produces.Data base querying adopts the Mascot of MareixScience company, and its referral web site is http://www.matrixscience.com/cgi/search_form.pl FORMVER=2﹠amp; SEARCH=PMF.The database retrieval parameter is: the corresponding name of input and E-mail address in Your name and the E-Mail, database is NCBInr, species classification (Taxonomy) are human (Homo sapiens), enzyme is Trypsin, the not restriction enzyme site that allows is l, and fixing modify (Fixed modifications) is that iodoacetamide Carbamidomethyl (C) modifies, and variable modification is not selected, the fragments of peptides tolerance is lOOppm, and Mass values is MH
+, select Monoisotopic, select the temporary file of directly input Mascot Distiller generation or the tabulation (Peak Lists) of monoisotopic peak can carry out database retrieval.
(8) western-blot albumen checking
The preparation gel, separation gel is 10%, concentrated glue is 5%,
1, go up sample, every porin amount is 40 μ g;
2, electrophoresis, voltage are 150V, and the time is 90min;
3, change film, electric current is 400mA, and the time is 100min;
4, sealing 2h, confining liquid is the 1 * TBS solution that contains 5% skimmed milk power;
5, primary antibodie (RON 1:8000; Actin 1:800) 4 ℃ of refrigerator overnight;
6, the 1 * TBST that contains 0.05% tween washes film 4 times, 10min/ time;
7, two anti-(1:5000) room temperature reaction 2h;
8, the 1 * TBST that contains 0.05% tween washes film 5 times, 10min/ time;
9, ECL AB liquid colour developing, X-ray sheet exposure 30 seconds.
Claims (1)
1. the detection method of the mescenchymal stem cell surface differences memebrane protein in placenta source is characterized in that, may further comprise the steps:
(1) separation of mescenchymal stem cell and cultivation
Get the fresh human placenta tissue and shred, collagenase digesting is collected the cell suspension that digestion obtains, and uses Ficoll-PaqueTM PLUS human lymphocyte parting liquid to carry out density gradient centrifugation, collects the tunica albuginea layer, washes twice, collecting cell, the conventional cultivation; Concrete steps are:
After under the aseptic condition placenta fully being washed the stain of dehematizing with the PBS of preheating, shred, be cut into the as far as possible little piece of organizing, place 0.1% 4 Collagen Type VI enzyme to digest, 37 ℃ of digestion, 30min, filter, with the PBS flushing, collect digestive juice and washing fluid, the centrifugal 10min of 1200rpm, collecting cell precipitation, resuspended with 5mlPBS, add to the 5ml relative density and be containing on the Ficoll-PaqueTM PLUS human lymphocyte parting liquid of 1.077g/L, carry out gradient centrifugation, centrifugal condition is 20 ℃, 2000rpm, 25min; After centrifugal, tunica albuginea layer in the suction pipe, PBS washed twice, collecting cell; The mescenchymal stem cell special culture media is resuspended, is inoculated in 6 well culture plates, places 37 ℃, 5%CO
2In the saturated humidity incubator, the row routine is cultivated and is gone down to posterity;
Get fresh umbilical cord tissue, peel off blood vessel, shred, collagenase digesting is collected the cell suspension that digestion obtains, and is centrifugal, collecting cell, the conventional cultivation; Concrete steps are:
After under the aseptic condition umbilical cord fully being washed the stain of dehematizing with the PBS of preheating, peel off the navel artery and vein vascular, be cut into 1cm
3Square fritter places 0.1% 4 Collagen Type VI enzyme to digest, 37 ℃ of digestion, and 1h filters, and with the PBS flushing, collects digestive juice and washing fluid, 1200rpm, centrifugal 10min abandons supernatant, PBS washed cell precipitation; The mescenchymal stem cell special culture media is resuspended, is inoculated in 6 well culture plates, places 37 ℃, 5%CO
2In the saturated humidity incubator, the row routine is cultivated and is gone down to posterity;
All mescenchymal stem cells all are cultured to exponential phase, are used for transplanting;
(2) extraction of mescenchymal stem cell memebrane protein
The multigelation method makes clasmatosis, obtains containing the component of memebrane protein then by gradient centrifugation;
(3) two-way fluorescence difference gel electrophoresis obtains the memebrane protein gel pattern
By two-way fluorescence difference gel electrophoresis, obtain the fluorescein-labeled different types of memebrane protein gel pattern of Cy2, Cy3 and Cy5, adopt the DeCyder2D image analysis software to analyze, identification group difference expressed proteins particle; Specifically may further comprise the steps:
1. interior standard specimen product preparation: two samples of N1, N2 are respectively got 50 μ g, and volume 10 μ l join in the same eppendorf pipe, and the concussion mixing is centrifugal; The every pipe of 50 μ g is distributed into 2 pipes then;
2. the preparation of storage liquid: fluorescein is taken out from-20 ℃ refrigerator, gently revolve, room temperature leaves standstill 5min, inhales 5 μ lDMF to the dyestuff pipe, shakes centrifugally, is mixed with lnmol/ μ l storage liquid;
3. the preparation of working fluid: at first get 1.8 μ l DMF to the eppendorf pipe, draw then in 1.2 μ l storage liquid to the eppendorf pipe, shake the centrifugal working fluid that namely is made into 400pmol/ μ l; Keep in Dark Place;
4. sample mark: the ratio of per 1 μ l working fluid and 50 μ g albumen is carried out fluorescence labeling; Lucifuge is carried out in the whole operation; Lucifuge is placed 30min on ice, adds the reaction of l μ l lysine end mark in every tubulin;
5. sample is prepared: will be respectively join in the same eppendorf pipe with the sample of Cy2, Cy3 and Cy5 mark, shake mixing, and of short duration centrifugal; The lucifuge operation; Add isopyknic 2 * sample-loading buffer static 10min on ice on the every glue, sample in the preparation, damping fluid: 7mol/L urea, 2mmol/L thiocarbamide, 4%CHAPS and 65mmol/L DTT;
Marked member is described below:
Intend adding the sample in the glue 1: with the sample of Cy2 mark, contain placenta source stem cell membrane albumen and umbilical cord source each 25 μ g of stem cell membrane albumen; With the sample of Cy3 mark, contain placenta source stem cell membrane albumen 50 μ g; With the sample of Cy5 mark, contain umbilical cord source stem cell membrane albumen 50 μ g;
Intend adding the sample in the glue 2: with the sample of Cy2 mark, contain umbilical cord source stem cell membrane albumen and placenta source each 25 μ g of stem cell membrane albumen; With the sample of Cy3 mark, contain umbilical cord source stem cell membrane albumen 50 μ g; With the sample of Cy5 mark, contain placenta source stem cell membrane albumen 50 μ g;
6. isoelectric focusing
To intend respectively adding in glue 1, the glue 2 three kinds of fluorescence labeling sample mix of Cy2, Cy3, Cy5 together; It is 450 μ l that the 0.5%v/v IPG buffer vibration mixing that adds an amount of hydrating fluid and pH4-7 makes the cumulative volume of sample on each glue, protein solution is drawn in the IPG adhesive tape groove, with pH4-7NL, the IPG immobilized ph gradient strip of 24cm is put into protein-contg adhesive tape groove, cover one deck Immobiline Drystrip covering liquid 2ml, place on the IPGphor isoelectric focusing instrument, setting program, make aquation and focus on and all under 20 ℃, carry out, wherein in 30V low-voltage aquation 12h, pass through 100V0.5h then, 500V0.5h, 1000V1h, 8000V1h is stabilized at last and carries out isoelectric focusing 8h under the 8000V;
7. balance
Press from both sides out adhesive tape with tweezers, after the ultrapure water flushing, blot at filter paper, with the ultrapure water flushing, filter paper blots again, clamping adhesive tape with tweezers then is that acidic terminal is downward with positive terminal, negative pole end i.e. alkalescence end is upwards put into the test tube of balance, and what tweezers were folded is the alkalescence end, acidic terminal leaves bromophenol blue and serves as a mark, with equilibrium liquid A and the back balance 15min of equilibrium liquid B elder generation; Equilibrium liquid A is 50mmol Tris-HCl, pH8.8,6mmol/L urea, 30% glycerine, 1%SDS, 0.2%DTT, 0.1% bromophenol blue, and equilibrium liquid B is 50mmol Tris-HCl, pH8.8,6mmol/L urea, 30% glycerine, 1%SDS, 3% iodoacetamide, 0.1% bromophenol blue;
8. second to vertical SDS-PAGE electrophoresis
The IPG adhesive tape is shifted out from level pad, and an end of clamping adhesive tape with tweezers makes the glue face be immersed in 1 * electrophoretic buffer fully; Then adhesive tape glue is faced up and be placed on the long glass plate of SDS-PAGE gel, gently adhesive tape is pushed on the SDS-PAGE gel glue face, SDS-PAGE gel glue face and adhesive tape will be combined closely, again the SDS-PAGE gel is transferred on the encapsulating frame, above the SDS-PAGE gel, added 0.5% low melting-point agarose sealing liquid; 5W/ gel electrophoresis 30min then with the permanent power electrophoresis of 20W/ glue, arrives place, gel base until the bromophenol blue index line and stops electrophoresis;
9. scanning analysis image
Typhoon multifunctional laser scanning instrument scanning SDS-PAGE gel, the image after the scanning is found out differential protein spot with DeCyder2D image analysis software analysis image;
10. strengthening applied sample amount sets up preparation glue and dyes blue dye liquor dyeing with Coomassie brilliant blue
Each histone is mixed into 1000 μ g, carries out dielectrophoresis, dye blue dye liquor dyeing with Coomassie brilliant blue again, use ddH
2The O washed twice, each 15min pours Coomassie brilliant blue into and dyes blue dye liquor, and it is that 0.25% Coomassie brilliant blue R-250 is dissolved in 50% methyl alcohol and 10% acetic acid that Coomassie brilliant blue dyes blue dye liquor; Shake to dye and spend the night, discard Coomassie brilliant blue and dye blue dye liquor, with distilled water washing 3 times, add 10% ethanol decolorization liquid 250ml again, decolour on the shaking table clear to background till, image is preserved in scanning;
(4) mass spectrophotometry and checking
Choose the differential protein particle, the laggard capable mass spectrophotometry of enzymolysis in the glue, and to the Mascot internet database inquiry of MatrixScience company, identify differential protein, and seek the special surface sign of separate sources stem cell, adopt the Western-Blot checking then.
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| Jeroen D.Langereis,et al..A 2D-DIGE Approach To Identify Proteins Involved in Inside-Out Control of Integrins.《Journal of Proteome Research》.2009,第8卷(第8期), |
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| 沙文琼 等.人胚胎滋养细胞和胎盘间充质干细胞的分离与纯化.《中国组织工程研究与临床康复》.2010,第14卷(第10期), |
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