CN102095778A - Method for detecting surface difference membrane protein of mesenchymal stem cell of cord blood source - Google Patents

Abstract

The invention relates a method for detecting a difference membrane protein of a stem cell, and aims to provide a method for detecting the surface difference membrane protein of the mesenchymal stem cell of a cord blood source. The method comprises the following steps of: separating and cultivating the mesenchymal stem cell, extracting the membrane protein of the mesenchymal stem cell, obtaining a membrane protein gel graph by a two-directional fluorescence difference gel electrophoresis (2D-DIGE) method, and analyzing and checking the mass spectrum. In the method, the 2D-DIGE method is first used for detecting a special difference surface marker of the membrane surface of the stem cell so as to perform quantitative comparison and analysis on proteins in different samples on the same gel; an interior label is added into each gel; and DIA and BVA software can automatically calibrate an expression quantity of each protein point according to the interior label of the protein point, so the error between gel and gel in different batches is reduced to the maximum extent, the real change degree of the protein is reflected, and the false positive rate and the false negative rate are lowered.

Description

The detection method of the mescenchymal stem cell surface differences memebrane protein in bleeding of the umbilicus source

Invention field

The present invention relates to a kind of detection method of stem cell difference memebrane protein, particularly the mescenchymal stem cell in bleeding of the umbilicus source (Mesenchymal stem cells, MSCSs) and the detection method of the mescenchymal stem cell surface differences memebrane protein in placenta 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 of 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 people's bleeding of the umbilicus MSCSs has extremely consistent biological characteristics, and may be more original, and the proliferation and differentiation ability is stronger, easily preserves, and immunoreactivity is low.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 a discarded object clinically simultaneously, and it is restricted hardly to draw materials, and does not relate to ethics problem; Therefore also be good experiment material and cell therapy carrier.Therefore, the present invention selects these two kinds of cells as detected object.

It is generally acknowledged that flow cytometer detects, bleeding of the umbilicus MSCS expresses CD 29, CD 44, CD54, CD58, CD90, immunophenotypes such as C D 95, C D166, does not express immunophenotypes such as CD14, CD34, CD40, CD45, CD50, CD68, CD80, CD8 6, CD117 and CD152.Placenta MSCS expresses CD29, CD44 and CD105, does not express CD34, CD45, CD19.But bleeding of the umbilicus and placenta MSCS surface marker, different documents are reported for work and are not quite similar.Bleeding of the umbilicus and placenta MSCS do not have special cell surface marker.Bleeding of the umbilicus is that fetus is given birth to, ligature of the cord and remain in blood in placenta and the umbilical cord from having no progeny.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 on Developmental Biology, this patent is exactly to adopt a kind of new technological means, discloses isolated MSCS surface protein differential expression in bleeding of the umbilicus and the placenta.

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 bleeding of the umbilicus and two kinds of separate sources of placenta.

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 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, on same glue, separate a plurality of by the fluorescently-labeled sample of difference, and introduced for the first time interior target notion, 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 bleeding of the umbilicus and placenta 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 bleeding of the umbilicus source is provided, may further comprise the steps:

(1) separation of mescenchymal stem cell and cultivation

Bleeding of the umbilicus derived mesenchymal stem cell: get fresh bleeding of the umbilicus, use the Ficoll parting liquid to carry out density gradient centrifugation, collect the tunica albuginea layer, wash twice, collecting cell, the conventional cultivation;

Placenta derived mesenchymal stem cell: get the 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, 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, discern 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, identify differential protein to the Mascot internet database inquiry of MareixScience company, 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.

At first statement is used fresh human placenta tissue and fresh bleeding of the umbilicus though relate among the present invention, at the fresh umbilical cord tissue and the fresh bleeding of the umbilicus that have just broken away from human body, its concrete acquisition process itself does not belong to content of the present invention.The present invention to the detailed description of its preparation method just in order more clearly to introduce the set-up procedure of biomaterial, but this description and do not mean that the correlation technique means also belong to the claimed content of the present invention.

(1) separation and Culture of mescenchymal stem cell cell

The separation and Culture of bleeding of the umbilicus MSCSs:

Get under the aseptic condition about normal mature caesarean birth pregnant woman's Cord blood 50ml, dilute with phosphate buffer (PBS) by 1:1, bleeding of the umbilicus after the dilution is contained in the centrifuge tube of Ficoll-PaqueTM PLUS human lymphocyte parting liquid (its relative density is 1.077g/L) along tube wall slow the adding, carry out (20 ℃ of gradient centrifugations, 2000rpm, 25min).After centrifugal, tunica albuginea layer in the suction pipe, the PBS washed twice obtains Cord Blood Mononuclear Cell.The MSCS special culture media is resuspended, is inoculated in 6 well culture plates, places 37 ℃, 5%CO2 saturated humidity incubator, and the row routine is cultivated and gone down to posterity.

The separation and Culture of placenta MSCSs:

After under the aseptic condition placenta fully being washed the stain of dehematizing with the PBS of preheating, shred, be cut into as far as possible little piece of tissue, place 0.1% 4 Collagen Type VI enzyme to digest, 37 ℃ of digestion, 30min, filter,, collect digestive juice and washing fluid with the PBS flushing, 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.

(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 promptly 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 static 10 min of isopyknic 2 * sample-loading buffer (7mol/L urea, 2mmol/L thiocarbamide, 4%CHAPS, 65mmol/L DTT), sample in the preparation on the every glue on ice.

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 together with Cy2, Cy3, three kinds of fluorescence labeling sample mix of Cy5 in the glue 1～2.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, on filter paper, blot, 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 be used for 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% acetate).Shake and dye spend the night (about 13h), discard and examine dye liquor,, add 10% ethanol decolorization liquid 250ml again with distilled water washing 3 times, decolour on the shaking table to background clear till, image is preserved in scanning.

(5) mass spectrum sample preparation

1, on preparation glue, seeks the corresponding proteins point according to the analysis result of DeCyder 2D image analysis software, 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, the Promega trypsin enzyme liquid after the adding 5ml dilution in the Eppendorf pipe that blob of viscose is housed allows 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, positive ion mode is measured down, 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 the source document that the Data ExploreTM software processes AB MALDI-TOF-MS of company mass spectrum produces earlier.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: 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, an anti-(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 bleeding of the umbilicus source may further comprise the steps:

(1) separation of mescenchymal stem cell and cultivation

Get fresh bleeding of the umbilicus, use the Ficoll parting liquid to carry out density gradient centrifugation; Collect the tunica albuginea layer, wash twice, collecting cell, the conventional cultivation;

Get the fresh human placenta tissue, shred, collagenase digesting is collected the cell suspension that digestion obtains; Use the Ficoll parting liquid to carry out density gradient centrifugation, collect the tunica albuginea layer, wash twice, 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, obtain the fluorescein-labeled different types of memebrane protein gel pattern of Cy2, Cy3 and Cy5, adopt DeCyder 2D image analysis software to analyze, identification group difference expressed proteins particle;

(4) mass spectrophotometry and checking

Choose the differential protein particle, the laggard capable mass spectrophotometry of enzymolysis in the glue, and, identify differential protein to the Mascot internet database inquiry of MareixScience company, and seek the special surface sign of separate sources stem cell, adopt the Western-Blot checking then.