CN107703219A - The method that evaluation GFP genes transfection based on CILLC MS influences on hPMSCs metabolism group - Google Patents

The method that evaluation GFP genes transfection based on CILLC MS influences on hPMSCs metabolism group Download PDF

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CN107703219A
CN107703219A CN201710628078.1A CN201710628078A CN107703219A CN 107703219 A CN107703219 A CN 107703219A CN 201710628078 A CN201710628078 A CN 201710628078A CN 107703219 A CN107703219 A CN 107703219A
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hpmscs
metabolin
gfp
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mark
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CN107703219B (en
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曹红翠
李兰娟
俞炯
陈德莹
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Zhejiang University ZJU
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Abstract

The present invention relates to the extraction of placenta derived mesenchymal stem cell metabolin and detection technique, it is desirable to provide a kind of method that evaluation GFP genes transfection based on CILLC MS influences on hPMSCs metabolism group.The intracellular metabolin that the present invention passes through the hPMSCs before and after extracting, detecting the transfection of GFP genes, and it is quantified, the front and rear otherness metabolin of transfection and otherness metabolic pathway are analysed in depth, the biological safety of mark and tracer mescenchymal stem cell is used for from metabolism group angle estimator green fluorescent protein.The pioneering relatively hPMSCs of the present invention and the analysis process of GFP+hPMSCs metabolism group differences, two kinds of sample preparation methods are extracted with reference to stem cell metabolin is used for, endocellular metabolism thing is fully discharged.It is used for stem cell intracellular metabolism analyte detection with reference to CIL LC MS, realizes the accurate quantitative analysis of intracellular metabolin, and greatly increase the identifying species of metabolite.Present invention greatly enhances the sensitivity of result, reproducibility and reliability, avoids the upper caused malalignment of operation and contingency.

Description

What the evaluation GFP genes transfection based on CILLC-MS influenceed on hPMSCs metabolism group Method
Invention field
The present invention relates to the extraction of placenta derived mesenchymal stem cell metabolin and detection technique, more particularly to based on same position The evaluation Transfection of Enhanced Green Fluorescent of element mark liquid chromatograph mass spectrography (CIL LC-MS) is to placenta derived mesenchymal The method that stem cell metabolism group influences.
Background technology
Important member of the mescenchymal stem cell (MSCs) as stem cell line, be it is a kind of into fiber-like, have height can Plasticity and self-renewal capacity and the multipotential stem cell that can break up to a variety of thesocytes.Placenta is clinical waste, its placenta Derived mesenchymal stem cell (human placental mesenchymal stem cells, hPMSCs) have abundance, Separation method is easy, multiplication capacity is strong, low immunogenicity, the advantages that being limited without ethics, progressively turn into stem cell transplantation The advantage seed cell for the treatment of.
In regenerative medicine, green fluorescent protein because its phototoxicity is weak, compatibility well be commonly used for mark and tracking cells, To assess transplanted cells distribution in vivo and survival condition.But applied to it is preclinical, it is necessary to fully study green fluorescence egg In vain the transfection of (green fluorescent protein, GFP) gene whether genomics to mescenchymal stem cell, transcript profile , protein science, metabolism group are had an impact to meet the needs of different.Previously research has proven to the transfection of GFP genes to placenta The survival of the mescenchymal stem cell in source and differentiation potential do not make significant difference.But whether metabolism group is had an impact, due to The reasons such as mesenchymal stem cells metabolin extraction efficiency is low, detection sensitivity is low not yet confirm so far.
Study the horizontal change of different group cell metabolisms, it is necessary to comprising metabolic pathway as much as possible, thus whether Success extracts metabolin and detects that substantial amounts of metabolite is crucial.At present, many methods can apply to mammality patch The sample preparation of parietal cell metabonomic analysis.Such as 9/1v/v methanol/chloroform (MeOH/CHCl3) method or 1/1v/v Methanol/water (MeOH/H2O) method.According to the matching peak (peak pairs) detected, the latter apparently higher than the former, Wei Entu Prompting has common factor but specificity is significantly.But in the extraction of cell metabolite before and after being transfected for GFP genes, exclusive use is deposited Extraction efficiency is not good enough the problem of.
The content of the invention
The problem to be solved in the present invention is to overcome deficiency of the prior art, there is provided a kind of evaluation based on CILLC-MS The method that the transfection of GFP genes influences on hPMSCs metabolism group, this method belong to new mescenchymal stem cell intracellular metabolin Extraction and detection method.
To solve technical problem, the present invention is realized by following technical scheme：
A kind of method that evaluation GFP genes transfection based on CILLC-MS influences on hPMSCs metabolism group is provided, including Following steps：
(1) Human plactnta derived mesenchymal stem cell being separately cultured and transfects
HPMSCs's is separately cultured：Clinical discarded fresh human placenta tissue is taken under aseptic condition, blood is fully washed with PBS After stain, the film for abandoning placenta tissue surface is cut.Remaining placenta tissue is shredded, digested with 0.2% 4 Collagenase Type, 37 DEG C of digestion 1h, filtering, rinsed with phosphate buffer (PBS), collect digestive juice and flushing liquor；It is special with MSCs after washing cell precipitation Culture medium is resuspended, and is placed in 37 DEG C, 5%CO2Saturated humidity incubator carries out cellar culture；
GFP transfects hPMSCs：When the merging rate up to 50% of hPMSCs of cellar culture, commercialization recombinant slow virus is added Suspension is transfected；Continue to cultivate 24h after transfection, then change old culture medium with new culture medium and continue to cultivate 3-4d, then add The puromycin for entering 10 μ g/mL is screened：The successful hPMSCs of untransfected is dead, Successful transfection GFP hPMSCs survivals, from And obtain expressing GFP placenta MSCs；
(2) extraction of metabolin
After the placenta MSCs for expressing GFP is passaged into 3-5 generations, removes old culture medium and clean cell surface, be quenched；Respectively with The methanol/water solvent extraction of the methanol of 9/1 volume ratio/chloroform solvent and 1/1 volume ratio, scrape cell, row ultrasonic wave added It is broken, the metabolin of centrifuging and taking supernatant, it is lyophilized rear standby to be concentrated and dried instrument；
(3) isotope marks
The metabolin after freezing is taken out, after being dissolved in water, adds acetonitrile and sodium carbonate/bicarbonate；Then use12C- pellet sulphonyl Chlorine marks light chain, uses13C-- dansyl Cls mark heavy chain, the reaction of water-bath line flag；Sodium oxide molybdena and formic acid neutralization reaction are hydrogenated with, from Quantified after the heart；
(4) liquid chromatogram-ultraviolet wide spectrum quantifies
Gradient elution is carried out based on liquid chromatogram-ultraviolet wide spectrum GC-MS, the temperature of sample manager is set, enters sample prescription Formula, column temperature, composition, the ratio of solvent and the steepness of gradient for optimizing mobile phase, make to reach optimal between each chromatographic peak Separation；
(5) liquid phase chromatogram-mass spectrometry combination method is identified
Each 6 parts of hPMSCs after experiment hPMSCs used and GFP marks, by each decile of the sample finally obtained, wherein Part mark12C- dansyl Cls, another part mark13C- dansyl Cls；According to LC-UV quantitative results, by same extraction Method, different type cell12C- dansyls chloride mark light chain with13The heavy chain 1 of C- dansyls chloride mark:In 1 mixing Machine；
Before detecting sample, blank, detection sample, Quality Control sample continuous sample introduction, the repeated of LC-MS and stably is investigated Property；4 purpose sample detection primary blanks, detection sample, Quality Control sample are often detected, with this alternately；
(6) data processing
The initial data obtained with R-based in-house softwares to detection and analysis pre-processes, and obtains comprising reservation The three-dimensional matrice table of time, mass-to-charge ratio and peak intensity；
By three-dimensional matrice table imported into the softwares of SIMCA-P 15.0 carry out multivariate data analysis, by principal component analysis, Offset minimum binary differentiates, volcano figure carries out metabolite data analysis；Mark database and mankind's generation are marked based on dansyl chloride A group database, metabolin database identification metabolin are thanked, it is poor based on MetaboAnalyst databases and KEGG database analysises Different in nature metabolic pathway.
In step (4) of the present invention, 4 DEG C of the temperature of sample manager is set；Input mode：Partial Loop enter Sample, 2 μ l；Column temperature：45℃；Mobile phase A：Water/formic acid (99.9:0.1, v/v), Mobile phase B：Acetonitrile/formic acid (99.9:0.1,v/ v)；Flow velocity：500uL/min；It is 85%A and 15%B when gradient elution program starts, after the concentration ratio keeps 1min, 2%A and 98%B are faded in 0.01min, after maintaining concentration ratio 1min, 85%A and 15%B are down in 0.5min, this is dense Degree ratio maintains 3.5min.
Inventive principle describes：
Evaluated the present invention relates to one kind based on the method for isotope marks liquid chromatograph mass spectrography (CIL LC-MS) Green fluorescent protein (GFP) gene transfects and expressed what Human plactnta derived mesenchymal stem cell (hPMSCs) metabolism group was influenceed Method.The present invention successfully extracts, detected the front and rear hPMSCs of GFP genes transfection intracellular metabolism by using new technological means Thing, and it is quantified, the front and rear otherness metabolin of transfection and otherness metabolic pathway are analysed in depth, from metabolism group angle Degree assesses green fluorescent protein and is used to mark the biological safety with tracer mescenchymal stem cell, and one kind is provided for cell therapy Study the new method of intracellular metabolism group.
Variant in view of the dissolubility of different in the mixed solvent metabolites, inventor uses 9/1MeOH/CHCl3 (method one) or 1/1MeOH/H2O (method two) is Extraction solvent, and according to the matching peak detected, method two is apparently higher than method One, Wei Entu prompting have common factor but specificity is significantly.Therefore the present invention combines method one and method two before being used for the transfection of GFP genes The extraction of cell metabolite afterwards, compare a kind of alone method or not good enough using extraction efficiency is improved for ACN or MeOH/ACN The problem of, and on the basis of high speed centrifugation smudge cells plus Ultrasound-assisted is used, make cell fully broken, intracellular metabolin fills Divide release.
LC-MS-MS (LC-MS) is to study the common method that intracellular is metabolized, but the repeatability of the technology It is bad with sensitiveness, and lack to the precisely quantitative of metabolite.The chromatography-mass spectroscopy of the chemical isotopes mark occurred in recent years Combination (CIL LC-MS) overcomes the disadvantages mentioned above of traditional LC-MS-MS, and CIL LC-MS are in traditional base On plinth, with reference to13C/12C-DNS labelling techniques, storehouse is identified from traditional HMDB, EML metabolin, to dansyl standard The increase of library databases, so as to expand the identifying species of metabolite and realize precisely quantitative.This method makes detection Sensitivity improves 1000 times, based on amine and phenols, can be introduced same with separating polar and ionic metabolin after mark Position element mark internal standard, drastically increases the accuracy of result, reproducibility and reliability, avoids not parallel caused by operation above Property and contingency.
The present invention proposes the generation for placenta derived mesenchymal stem cell before and after the transfection of GFP genes with reference to CIL LC-MS Group analysis process learned, the especially intracellular in the sample preparation of early stage and later stage metabolism analyte detection are thanked, on this basis on the one hand The metabolism of mescenchymal stem cell intracellular can be analyzed, understands the biological activity that has occurred and that of cell, on the other hand quantitatively can be with to it Compare hPMSCs and GFP+The difference of hPMSCs metabolism group, the transfection of GFP genes is assessed to placenta derived mesenchymal stem cell generation Thank to group influence learned.
Compared with prior art, the beneficial effects of the present invention are：
1st, the present invention creates the analysis process for comparing hPMSCs and GFP+hPMSCs metabolism group differences first, by by 9/ 1MeOH/CHCl3And 1/1MeOH/H2Two kinds of different sample preparation methods of O, which combine, is used for the extraction of stem cell metabolin, at a high speed On the basis of centrifugal breaking cell plus Ultrasound-assisted is used, makes cell fully broken, intracellular metabolin fully discharges.
2nd, the present invention combines CIL LC-MS and is used for stem cell intracellular metabolism analyte detection, Isotopic Internal Standard is introduced, from traditional HMDB, EML metabolin identify storehouse, to the increase of dansyl standard library databases, realize intracellular metabolin Accurate quantitative analysis, and greatly increase the identifying species of metabolite.
3rd, present invention greatly enhances the sensitivity of result, reproducibility and reliability, avoid caused by operation above not Collimation and contingency.
Embodiment
Illustrate first：The placenta tissue utilized in the present invention is clinical waste, is directed to where puerpera produces and cures Treat mechanism.Present disclosure is not directly related to take out or cut off placenta tissue based on the utilization to fresh human placenta tissue Operation.
With reference to specific embodiment, technical scheme is described in detail.
Compare hPMSCs and GFP+Metabolism group difference between hPMSCs
(1) mescenchymal stem cell being separately cultured and transfects
HPMSCs's is separately cultured：
After the discarded fresh human placenta tissue of clinic fully is washed into bloodstain with PBS under aseptic condition, cut and abandon placenta tissue The film on surface.Remaining placenta tissue is shredded to unqualified shape, is placed in 0.2% 4 Collagenase Type and digests, 37 DEG C of digestion 1h, mistake Filter, rinsed with phosphate buffer (PBS), collect digestive juice and flushing liquor, PBS repeated washings cell 2 times.With the special trainings of MSCs Support base to be resuspended, be inoculated according to cell concentration in T25 blake bottles, be placed in 37 DEG C, 5%CO2In saturated humidity incubator, carry out conventional Culture and passage.
HPMSCs transfection：
It is 100 addition recombinant slow virus by infection multiplicity (MOI) when the hPMSCs of cellar culture merges rate up to 50% (Lentivirus) suspension is transfected, and is changed old culture medium with new culture medium after culture 24h and is continued to cultivate, after usual 48h Visible fluorescence, 10 μ g/ml puromycins are screened after 3-4d, and the successful hPMSCs of untransfected is dead, Successful transfection GFP's HPMSCs is survived, so as to obtain expressing GFP placenta MSCs (GFP+hPMSCs)。
Recombinant slow virus suspension (slow virus carrier) is the gene to be grown up based on human immune deficiency I type viruses Therapy vector, compound is integrated before slow virus nucleoprotein to have and bite nuclear properties, viral genome is transported to nucleus, so that Slow virus can infect and be replicated in non-mitotic cell.The recombinant slow virus suspension of the present invention is commodity purchased in market, is purchased from Shanghai Ji Kai Gene Tech. Company Limited, official's network address：Www.genechem.com.cn, infection multiplicity 100.)
(2) extraction of metabolin
Use 9/1MeOH/CHCl3Extraction
1、GFP+HPMSCs cellar cultures are passaged to 3-5 generations in T175 blake bottles.
2nd, old culture medium is removed, with the ultra-pure water 20ml/ bottle Rapid Cleaning cell surfaces of 37 DEG C of preheatings.
3rd, liquid nitrogen (LN is used in 5s2) 15ml/ bottles are quenched.
4th, the 9/1MeOH/CHCl of every bottle of addition 3ml precooling3Extraction.
5th, cell scrapes cell, is transferred to 15ml centrifuge tubes, and row Ultrasound-assisted makes cell fully broken.
6th, packing 4 DEG C of centrifugation 10min of 16100g, takes supernatant, is concentrated and dried instrument and freezes supernatant, deposit to 1.5ml centrifuge tubes It is standby in -40 DEG C.
Use 1/1MeOH/H2O extractions
1、GFP+HPMSCs cellar cultures are passaged to 3-5 generations in T175 blake bottles.
2nd, old culture medium is removed, with the ultra-pure water 20ml/ bottle Rapid Cleaning cell surfaces of 37 DEG C of preheatings.
3rd, every bottle adds -20 DEG C of 1/1MeOH/H immediately2O 3ml carry out that extraction is quenched.
4th, cell scrapes cell, is transferred to 15ml centrifuge tubes, and row Ultrasound-assisted makes cell fully broken.
5th, packing 4 DEG C of centrifugation 10min of 16100g, takes the metabolin of supernatant, is concentrated and dried instrument and freezes to 1.5ml centrifuge tubes Supernatant, be stored in -40 DEG C it is standby.
(3) metabolin derivatization marks
1st, the metabolin after freezing is taken out, adds 50ul water to dissolve, after concussion mixes, 12000rpm centrifugations 15min.
2nd, 25ul ACN are added, add 15ul NA2CO3/NAHCO3(0.5mol/L, pH 9.5), after concussion mixes, centrifugation 10-15s, metabolin is set to be in alkaline environment.
3rd, 50ul is added12C- dansyl Cls：ACN=20mg/ml mark light chains connect, 50ul13C- dansyl Cls：ACN= After 20mg/ml marks heavy chain, concussion to mix, 10-15s is centrifuged.
4th, 60 DEG C of water-bath, 60min row dansyl reactions.
5th, adding 10ulNAOH (250mM) and consume unnecessary dansyl chloride, concussion mixes, 60 DEG C after centrifugation, water-bath 10min。
6th, 50ul (1%FA formic acid is added：10% (ACN:Water=1:1) 425mM) neutralize excess sodium hydroxide.
7th, concussion mixes, and after 14000rpm centrifugations 15min, takes 10ul12The carry out LC-UV of C- dansyls chloride mark is quantified Analysis.
(4) LC-UV is quantitative
It is as follows that liquid phase chromatogram condition is set in this experiment：
1. (photodiode array detector is carried, for 338nm using the Ultra Performance Liquid Chromatography instruments of Agilent 1290 Quantitative adsorption), and ACQUITY UPLC BEH C18 analytical columns (2.1 × 100mm, 1.7 μm, pore sizeWaters Co. chromatographic isolation) is carried out.
2. the temperature of sample manager：It is set in 4 DEG C
3. input mode：Partial Loop (a kind of input mode on mass spectrum machine menu), 2 μ l
4. column temperature：45℃
5. mobile phase A：Water/formic acid (99.9:0.1,v/v)
Mobile phase B：Acetonitrile/formic acid (99.9:0.1,v/v)
Flow velocity：500uL/min
The influence of column temperature, flow velocity to chromatographic peak in liquid phase chromatogram condition is little, should mainly optimize mobile phase composition, The ratio of solvent and the steepness of gradient, so that reaching optimal separation between each chromatographic peak, optimal conditions are as follows：
It is 85%A and 15%B when gradient elution program starts, after this concentration ratio keeps 1min, becomes in 0.01min To 2%A and 98%B, after maintaining concentration ratio 1min, 85%A and 15%B are down in 0.5min, this concentration ratio maintains 3.5min。
(5) LC-MS is identified
HPMSCs is all that biological specimen is repeated 3 times after experiment hPMSCs and GFP marks used, the sample that will be finally obtained Each decile, a portion mark12C, another part mark13C.According to LC-UV quantitative results, by same extracting process, Different type cell12C- dansyls chloride mark light chain with13The heavy chain 1 of C- dansyls chloride mark:The upper machine of 1 mixing.
It is as follows that liquid chromatography-mass spectrography condition is set in experiment：
1. using Agilent 1290UPLC systems, Waters ACQUITY UPLC BEH C are equipped with18Analytical column (2.1mm ×10cm,1.7μm particle size,Pore size), connect the electron spray ionisation flight time of Agilent 6230 Mass spectrograph (Model 6230, Agilent, Palo Alto, CA).
2. the setting of ion gun condition is as follows：
Nitrogen nebulizer gas (nitrogen atomizer air pressure):1.38Bar
dry gas flow:5L/min (flows of dry gases)
Dry temperature (drying temperature):325℃,
Capillary voltage (capillary voltage):4000V
End plate offset (end plate offset voltage):120V
Mass range (mass range):m/z up to 1700
Spectra rate (spectrum rate):1Hz.
Resolving power (resolution capability)：11000,FWHM at m/z 622.
Mass spectrum wave spectrum is obtained under positive ion mode.
Still it is that 85%A and 15%B, 15min linearly become when being 85%A and 15%B, 2min when 3. gradient elution program starts Turn to 55%A and 45%B, after 20min, fade to 35%A and 65%B, fade to 2%A and 98%B during 26min, this concentration ratio After keeping 3min, then 85%A, 15%B are returned in 0.1min.
4. before starting to detect sample, BLANK, AA, QC sample continuous sample introduction 3 times, investigate the repeated and steady of UPLC-MS It is qualitative.If instrument is reproducible, start testing goal sample.Often detect BLANK, AA, QC sample of 4 purpose sample detections This, with this alternately.
(6) identification of Data Analysis Services and otherness material
1st, what is obtained is tested and analyzed to UPLC-MS with R-based in-house software Is soMS (Guo and Li, 2009) Initial data is pre-processed, it is filtered make an uproar, peak identification, peak extraction, peak alignment and normalization, zero-fill program generations After missing values, corresponding centralization, scaling, conversion are carried out to data, obtained comprising retention time, mass-to-charge ratio and peak intensity Three-dimensional matrice table.
2nd, three-dimensional matrice table is imported into SIMCA-P 15.0 (Umetrics, Umea, Sweden) software and carries out multivariate data Analysis, the analysis metabolite data such as (PLS-DA), volcano figure is differentiated by principal component analysis (PCA), offset minimum binary.First, use PCA is analyzed the data of all samples including QC samples, to verify the reliability of UPLC-MS detections, is obtained total Body variation tendency, hPMSCs groups and GFP+Fail to separate well between hPMSCs groups.In order to further discriminate between hPMSCs and GFP+ HPMSCs group difference, pattern recognition analysis is carried out to two groups of data using PLS-DA, OPLS, Volcano plot, obtained The FC in the figure of volcano>1.5 or FC>2.0 and t examines p<0.05 material, i.e., change in GFP transfection group cells obvious Material, then these materials are analyzed and identified, only 2% metabolin changes, and does not also have in strata figure Significant difference.
3rd, this experiment detects 1151 peak pairs altogether, according to accurate mass-to-charge ratio to HMDB, EML database and Dansyl standard library are scanned for, and are selected according to retention time.Will likely material standard items and need The material to be identified carries out retention time and MS/MS fragment ion patterns are contrasted, so as to finally determine material 739, bag Include otherness material 11.Compose what storehouse was contrasted by the standard items retrieved metabolite profile picture library and established with this laboratory Method identifies the metabolin 5 of biological significance.
4th, based on MetaboAnalyst (www.metaboanalyst.ca) and KEGG databases to otherness metabolin, Otherness metabolic pathway is analyzed.It is related to nine metabolic pathways, due to otherness metabolin (taurine, uracil, r- Aminobutyric acid etc.) position that occupies is relatively inessential, do not cause significantly changing for metabolic pathway.Therefore CIL LC-MS are based on The transfection of GFP genes the metabolism group of placenta derived mesenchymal stem cell is not made significant difference, the mark of cell can be continued on for And tracer.

Claims (2)

1. the method that the evaluation GFP genes transfection based on CILLC-MS influences on hPMSCs metabolism group, it is characterised in that including Following steps：
(1) Human plactnta derived mesenchymal stem cell being separately cultured and transfects
HPMSCs's is separately cultured：Clinical discarded fresh human placenta tissue is taken under aseptic condition, bloodstain is fully washed with PBS Afterwards, the film for abandoning placenta tissue surface is cut；Remaining placenta tissue is shredded, digested with 0.2% 4 Collagenase Type, 37 DEG C of digestion 1h, filtering, is rinsed with phosphate buffer, collects digestive juice and flushing liquor；After washing cell precipitation, with MSCs special culture medias It is resuspended, is placed in 37 DEG C, 5%CO2Saturated humidity incubator carries out cellar culture；
GFP transfects hPMSCs：When the merging rate up to 50% of hPMSCs of cellar culture, add recombinant slow virus suspension and turned Dye；Continue to cultivate 24h after transfection, then change old culture medium with new culture medium and continue to cultivate 3-4d, add 10 μ g/mL's Puromycin is screened：The successful hPMSCs of untransfected is dead, Successful transfection GFP hPMSCs survivals, so as to be expressed GFP placenta MSCs；
(2) extraction of metabolin
After the placenta MSCs for expressing GFP is passaged into 3-5 generations, removes old culture medium and clean cell surface, be quenched；Respectively with 9/1 The methanol/water solvent extraction of the methanol of volume ratio/chloroform solvent and 1/1 volume ratio, scrapes cell, and row ultrasonic wave added is broken It is broken, the metabolin of centrifuging and taking supernatant, it is lyophilized rear standby to be concentrated and dried instrument；
(3) isotope marks
The metabolin after freezing is taken out, after being dissolved in water, adds acetonitrile and sodium carbonate/bicarbonate；Then use12C- dansyl Cl marks Remember light chain, use13C-- dansyl Cls mark heavy chain, the reaction of water-bath line flag；Sodium oxide molybdena and formic acid neutralization reaction are hydrogenated with, after centrifugation Quantified；
(4) liquid chromatogram-ultraviolet wide spectrum quantifies
Gradient elution is carried out based on liquid chromatogram-ultraviolet wide spectrum GC-MS, set the temperature of sample manager, input mode, Column temperature, composition, the ratio of solvent and the steepness of gradient for optimizing mobile phase, make to reach optimal point between each chromatographic peak From；
(5) liquid phase chromatogram-mass spectrometry combination method is identified
Each 6 parts of hPMSCs after experiment hPMSCs used and GFP marks, by each decile of the sample finally obtained, wherein one Minute mark is remembered12C- dansyl Cls, another part mark13C- dansyl Cls；According to LC-UV quantitative results, by same extraction side Method, different type cell12C- dansyls chloride mark light chain with13The heavy chain 1 of C- dansyls chloride mark:In 1 mixing Machine；
Before detecting sample, blank, sample, Quality Control sample continuous sample introduction are detected, investigate LC-MS repeatability and stability；Often 4 purpose sample detection primary blanks, detection sample, Quality Control sample are detected, with this alternately；
(6) data processing
The initial data obtained with R-based in-house softwares to detection and analysis pre-processes, when obtaining comprising retaining Between, the three-dimensional matrice table of mass-to-charge ratio and peak intensity；
Three-dimensional matrice table is imported into the softwares of SIMCA-P 15.0 and carries out multivariate data analysis, by principal component analysis, partially minimum Two multiply differentiation, volcano figure carries out metabolite data analysis；Mark database and mankind's metabolism group are marked based on dansyl chloride Database, metabolin database identification metabolin, based on MetaboAnalyst databases and KEGG database analysis otherness generations Thank to approach.
2. according to the method for claim 1, it is characterised in that in the step (4), set the temperature 4 of sample manager ℃；Input mode：Partial Loop sample introductions, 2 μ l；Column temperature：45℃；Mobile phase A：Water/formic acid is 99.9:0.1,v/v；Flowing Phase B：Acetonitrile/formic acid is 99.9:0.1,v/v；Flow velocity：500uL/min；It is 85%A and 15%B when gradient elution program starts, After the concentration ratio keeps 1min, 2%A and 98%B are faded in 0.01min, after maintaining concentration ratio 1min, in 0.5min 85%A and 15%B are down to, this concentration ratio maintains 3.5min.
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