CN103487585B - Western blotting detects the method for hair follicle stem cells VEGF165 protein expression - Google Patents
Western blotting detects the method for hair follicle stem cells VEGF165 protein expression Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5073—Stem cells
Abstract
The present invention relates to the method that Western blotting detects hair follicle stem cells VEGF165 protein expression, the method comprises the following steps: 1) hair follicle stem cells cracking is for subsequent use; 2) polyacrylamide gel electrophoresis comprises and a) prepares 8% separation gel; B) 5% spacer gel is prepared; C) 30% polyacrylamide 0.33ml, 10%SDS20 μ l, 10% Ammonium Persulfate 98.5 20 μ l, TEMED2 μ l, adds rear mixing, quick glue; The above-mentioned mixed liquor of 1.4ml injects layer glass sheet separation fast, inserts comb rapidly, carefully takes out comb after polymerization completely; D) electrophoresis; E) electrotransfer; F) protein on cellulose nitrate is detected; G) immunology detection.The present invention, owing to have employed above-mentioned technical scheme, achieves the detection of hair follicle stem cells VEGF165 protein expression after transfection.
Description
Technical field
The present invention relates to skin tissue engineering field, particularly relate to a kind of method that Western blotting detects hair follicle stem cells VEGF165 protein expression.
Technical field
Skin is as the maximum organ of human body, there is sensation, regulate body temperature, secretion and excretion, prevent the multiple effects such as moisture evaporation, wherein topmost function is that it is also immune important component part simultaneously as the barrier of human body and external environment to maintain the stable of environment.Along with socioeconomic development, particularly as the China's manufacturing industry of " world's factory " and the prosperity and development of handicraft, the various wound relating to defect of skin is particularly burnt, crush injury, incised injury etc. also get more and more.Wherein, the widespread skin defect that wound causes usually causes very serious physical disabilities, even dead.The standard treatments of current clinical treatment defect of skin is auto-skin grafting, and owing to having without immunological rejection, survival rate high, it applies very extensive clinically, and achieves good curative effect.But the shortcoming of auto-skin grafting also clearly, owing to being autologous drawing materials, itself being exactly the damage again to patient, greatly adding patient suffering, and have generation skin donor site to infect, the risk of the complication such as disunion.More seriously, for above-mentioned large skin defect, often enough can cause wound repair difficulty for the autologous skin transplanted owing to lacking, have a strong impact on treatment process, even caused death.
Just because of this, scientists attempts to find a kind of ectogenic Graftskin always.As far back as B.C. 1500, dermatoheteroplasty was just used to cover skin wound temporarily.And along with the foundation of organizational engineering and development, skin tissue engineering is risen rapidly, and become the focus studied nearly ten years.Organizational engineering is principle and the method for application project and life science, research biosubstitute, for the subject rebuilding, keep or improve function of organization.At present, the research that foreign applications skin tissue engineering builds artificial skin achieves substantial progress, and the products such as Apligraf, OrCel, Suprathel, Biobrane, OASIS, Integra, Lyphoder are successively ratified to go on the market by united states drug and food control office (FDA) and have been applied in the treatment of clinic skin defect.
But, although existing several artificial skin products above can supply selection of clinical at present, also really facilitate the clinical treatment to large skin defect patient.But, according to the problem run in our clinical application for many years process, in conjunction with bibliographical information, we think that current artificial skin is transplanted and also there is many problems, wherein even comprise and may cause treating " hard defects " that prove an abortion: 1. because above artificial skin product does not all have Angiogenesis ability, cause the artificial skin transplanted not have vascular system to supply nutrition, thus artificial skin is easily necrosed, make graft failure.2. contained in artificial skin product various variant cells easily cause immunological rejection, and often occur the cutaneous necrosis of transplanting clinically, come off, serious even occurs the severe complications such as systemic immune response, and likely spreads disease.3. all people's work skin products all can only recover part anatomical structure and the physiological function of normal skin at present, and cannot regenerate the cutaneous appendages structure with critical function, such as: blood vessel, hair, sweat gland etc.
Hair follicle stem cells is the stem cell that a class is present in raw coal bunker knuckle portion, has the features such as a point voltinism, self and in-vitro multiplication ability are strong.Hair follicle stem cells in vitro culture research shows high clonality, has very high regeneration potential.Because hair follicle stem cells derives from hair, can directly obtain from patient self, quantity is extremely abundant, and without any complication, to patient completely without wound, has now become the focus of skin tissue engineering research.(the TaylorG such as Taylor, Lehrer MS, Jensen PJ, et al.Involvement of follicular stem cells in forming not onlythe follicle but also the epidermis.Cell, 2000,102 (4): 451-461.) research finds that hair follicle stem cells can not only be differentiated to form hair follicle, but also take part in the forming process of epidermal tissue.The current research result that Stelios etc. deliver proves, containing a large amount of stem cells in hair follicle, is one of source of human stem cell the most easily obtained, and successfully hair follicle stem cells differentiation and development is generated new vascular system.Vascular endothelial growth factor 165 (VEGF165) is one of vascular endothelial growth factor 5 kinds of hypotypes, and its activity is the strongest, and distribution range is the widest, is the Main Subtype played a role in VEGF body.Domestic and international study hotspot is become in recent years around the revascularization gene therapy research centered by VEGF165.
Applicant has applied for a kind of method of VEGF165 genetic modification hair follicle stem cells for this reason, and the method is seed cell with hair follicle stem cells, can increase by rapid, high volume, originate very abundant, reduce immunological rejection when having in vitro culture; Utilize VEGF165 gene transfection to modify hair follicle stem cells simultaneously, the new engineered blood vessels system with enlargement and contraction function can be formed, well will solve transplanting artificial skin easily downright bad, the problem that survival rate is low.But how detecting hair follicle stem cells VEGF165 protein expression is a difficult problem.
Summary of the invention
In order to solve the above problems, the object of this invention is to provide a kind of method that Western blotting detects hair follicle stem cells VEGF165 protein expression.
In order to realize above-mentioned object, present invention employs following technical scheme:
Western blotting detects the method for hair follicle stem cells VEGF165 protein expression, and the method comprises the following steps:
1) hair follicle stem cells cracking
A) hair follicle stem cells is washed 3 times with the PBS of precooling;
B) the lysing buffer of precooling is added, 6 orifice plates: 80 μ l/well; 60mm double dish: 300 μ l/disk, hatches 20min for 4 DEG C;
C) with cell scraper, cell fragment is concentrated on side together with lysing buffer, hatch 40min and be transferred in the Eppendorf pipe of precooling for 4 DEG C; 12000g, 4 DEG C of centrifugal 5min;
D) supernatant is moved in another Eppendorf pipe, except part is used for surveying except protein concentration, all the other be directly used in experiment or-70 DEG C freezing for subsequent use;
2) polyacrylamide gel electrophoresis
A) 8% separation gel is prepared: 1.5mM Tris-HCl (pH8.8) 1.3ml, dddH
2o2.3ml, 30% polyacrylamide 1.3ml, 10%SDS50 μ l, 10% Ammonium Persulfate 98.5 50 μ l, TEMED3 μ l, adds rear mixing, quick glue; The above-mentioned mixed liquor of 3ml injects layer glass sheet separation fast, and immediately with a small amount of tri-distilled water sealing face, polymerized at room temperature 30min, sucks upper strata tri-distilled water;
B) 5% spacer gel is prepared: 1mM Tris-HCl (pH6.8) 0.25ml, ddH
2o1.4ml;
C) 30% polyacrylamide 0.33ml, 10%SDS20 μ l, 10% Ammonium Persulfate 98.5 20 μ l, TEMED2 μ l, adds rear mixing, quick glue; The above-mentioned mixed liquor of 1.4ml injects layer glass sheet separation fast, inserts comb rapidly, carefully takes out comb after polymerization completely;
D) electrophoresis: get Sample Dilution containing equal protein content to equal-volume, and 5 × sample buffer presses the mixing of 4:1 volume, 100 DEG C of water-bath 5min, take advantage of hot application of sample, every duct 30 μ l, after application of sample, gel is positioned over the electrophoresis tank that electrophoretic buffer is housed and carries out electrophoresis; Deposition condition: 95V × 15min in spacer gel, 165V × 50min in separation gel, when the bromophenol blue in sample migrates to the forward position of glue, terminates electrophoresis;
E) electrotransfer: after SDS-PAGE, polyacrylamide gel is unloaded, get and 6 of gel formed objects filter paper and a nitrocellulose membrane, in half-dried transfering buffering liquid, balance 15min, put 3 filter paper, gel, NC film, another 3 filter paper successively from top to bottom well, then be sandwiched in half-dried membrane-transferring device, film towards positive pole, glue to negative pole, according to membrane area size, with the constant current of 1.25mA/cm2, transferring film 90min;
F) detect the protein on cellulose nitrate: be immersed in by film in Ponceaux dyeing liquor and dye, about 3min red color visible protein colour band occurs, is then immersed in distilled water by film and decolours, shake several minutes gently, then changes destainer for several times, until background colour is very light;
G) immunology detection: 1, close: after transferring film, puts in TTBS by fine for nitre film, delays and shake 10min, then film 37 DEG C of slow yawing 2h of shaking table in confining liquid; 2, in conjunction with primary antibodie: after closing, fine for nitre film is moved into primary antibodie (1:1000), 4 DEG C are spent the night or room temperature yawing 3h; Reclaim primary antibodie, cellulose nitrate immerses in TTBS20ml, suddenly shakes 10min, continuous 3 times, remaining primary antibodie on cleaning film; 3, resist in conjunction with two: move in the horse anti-reactant liquor of anti-rabbit two (1:2000) by fine for nitre film, after room temperature yawing 2h, nitrocellulose membrane immerses in TTBS30ml, suddenly shakes 15min, continuous 3 times, and on cleaning film, remaining two resist; 4, the detection of immune complex: the development of GE ImageQuant LAS4000 chemiluminescence imaging analyser is taken pictures.
The present invention, owing to have employed above-mentioned technical scheme, achieves the detection of hair follicle stem cells VEGF165 protein expression after transfection.
Accompanying drawing explanation
Fig. 1, Fig. 2 are that primary hair follicle stem cells climbs out of from Hair Follicle Bulge portion, in nest like, epithelioid cell, arrange tight 40 ×.
Fig. 3 be P3 after IV Collagen Type VI screening purifying for hair follicle stem cells, in typical paving stone shape 100 ×.
Table 1 carries out Q-PCR detection for the P3 after sorting purifying for hair follicle stem cells, carries out the relative quantification of each gene expression by △ △ Ct method.
Fig. 4-Fig. 6 is the immunofluorescence dyeing of P3 for hair follicle stem cells, is respectively integrin β_1, integrin a6, keratin 15,100 ×.
Fig. 7-Fig. 9 is metal spraying display gelfoam three-dimensional rack scanning electron microscope, be respectively SEM50 ×, 200 ×, 400 ×.
Under Figure 10, Figure 11 are respectively inverted fluorescence microscope, P3 for the complete 72h of hair follicle stem cells slow-virus infection GFP figure and PH Figure 100 ×.
Figure 12 is the growth curve chart of the hair follicle stem cells after VEGF165 genetic modification.
Figure 13 is the RT-PCR result figure that the hair follicle after VEGF165 genetic modification does thin mRNA.
Figure 14 is the expression of results that the hair follicle after VEGF165 genetic modification does thin VEGF165 albumen.
Figure 15 ~ Figure 17 is respectively the HE dyeing of compound artificial skin, be respectively injection concentration 1 × 106,5 × 106,1 × 107/cm2, bar:200 ×.
Figure 18-19 is for being surface of a wound figure when rat back is transplanted.
Figure 20-23 is posttransplantation four the wound healing situations of 7d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
Figure 24-27 is posttransplantation four the wound healing situations of 14d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
Figure 28-31 is posttransplantation four the wound healing situations of 21d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
Figure 32 is the Wound healing rate of 7d, 14d, 21d tetra-group.
Figure 33-35 is to the HE dyeing that tissue of drawing materials does after 21d, observation vascularization situation.Be followed successively by VEGF165 group, empty plasmid group, acellular group.100×
Embodiment
The separation of embodiment 1 rat hair follicle stem cell, cultivation, qualification
1.1) select SD rat antenna in one week age portion skin, insert 0.25%Dispase enzyme 37 DEG C digestion 2h; With tweezers and disposable syringe syringe needle from hypodermis end pull-out hair follicle, it is intact and be in the hair follicle in growth period to collect form, under microscope, hair follicle is cut into three equal parts, get center section, put into 50mL culture flask after PBS rinsing, add DMEM/F12 supplementing culture medium, be placed in 37 DEG C, 5%CO
2in incubator, every 2d changes liquid once; Described DMEM/F12 supplementing culture medium composition is: 44mlDMEM/F12 nutrient solution, 5mlKSR serum substitute, 500 μ l mycillin mixed liquors, 500 μ l Glus, 500 μ l nonessential amino acid, 20ng/ml recombinant human epidermal growth factor, 10ng/ml recombination human basic fibroblast growth factor, 50 μ l hydroxyl ethanols, 10ng/ml hydrocortisone.Fig. 1, Fig. 2 are that primary hair follicle stem cells climbs out of from Hair Follicle Bulge portion, and in nest like, epithelioid cell, arrangement closely.
1.2) purifying of hair follicle stem cells: be coated in double dish by the IV Collagen Type VI of 100 μ g/ml according to the amount of 3ml/100mm dish, room temperature leaves standstill 1h; By the primary cell Trypsin Induced of 100mm double dish, after centrifugal collecting cell, blow and beat into single cell suspension and be inoculated in double dish, after 20min, by not adherent cell together with nutrient solution sucking-off; The complete medium culture of adherent cell, changes liquid in every 3 days; P2 for repurity once.If Fig. 3 is that IV Collagen Type VI screens the P3 after purifying for hair follicle stem cells, in typical paving stone shape.
1.3) qualification of hair follicle stem cells:
A, employing Q-PCR method: the P3 after sorting purifying carries out Q-PCR detection for hair follicle stem cells, carries out the relative quantification of each gene expression by △ △ Ct method.
Table 1 carries out Q-PCR detection for the P3 after sorting purifying for hair follicle stem cells, carries out the relative quantification of each gene expression by △ △ Ct method.
B, immunofluorescent staining method: by P3 for cell chulture to exponential phase, be seeded in after digestion on slide, after adhere-wall culture 2d, sop up nutrient solution, use PBST rinsing, add 4%PFA fixing after, more closed by 5%BSA room temperature.Add primary antibodie integrin β_1 (integrin-β 1) against murine polyclonal antibody (1:100), integrin a6(integrin-a6 respectively) polyclonal antibody (1:50) and keratin 15 (keratin-15) polyclonal antibody (1:100), incubated at room, after PBST washing, label two anti-lucifuge 30min again, add DAPI(1:2000) contaminate core 5min, lucifuge is dried, with mounting solution mounting.Fig. 4-Fig. 6 is the immunofluorescence dyeing of P3 for hair follicle stem cells, is respectively integrin β_1, integrin a6, keratin 15.
The preparation of embodiment 2 gelfoam three-dimensional tissue support
2.1) gelfoam three-dimensional tissue support preparation:
A. get content be the Gelatin of 5% in the distilled water 10ml of 25 DEG C, add 0.05%6-sodium chondroitin sulfate (C6S) and 0.2% hyaluroni (HA) respectively;
B. use magnetic stirrer after 60 minutes under room temperature, crosslinking chemical 0.5%1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt acid salt solution (EDC) and 0.25%N-N-Hydroxysuccinimide solution (N-Hydroxysuccinimide) are instilled after mixing 5 minutes in solution, solution is injected the mould of 12 control-bureau's orifice plates, level is rocked evenly again.
C. at-80 DEG C freezing 2 hours
D. then go up freeze dryer, freeze-drying obtains the Gel-C6S-HA support that thickness is the porous spongy of 2mm for 24 hours.
2.2) observe: take a morsel support sample, scanning electron microscopic observation record, Fig. 7-Fig. 9 is metal spraying display gelfoam three-dimensional rack scanning electron microscope, be respectively SEM50 ×, 200 ×, 400 ×.
Embodiment 3 VEGF-165 gene (VEGF165) modifies the preparation of hair follicle stem cells
3.1 packaging slow viruss: the 293T cell in growth period of taking the logarithm, 24h is inoculated in 100mm double dish in advance, treats that next day, cell grew to 50%-70%; Virus packaging adopts calcium robin to carry out: be replaced by by 293T cell culture medium before transfection not containing dual anti-nutrient culture media, comprises the high sugar of 10%FBS+DMEM; Then, object plasmid pLenti-IRES-VEGF165-EGFP10ug and the 3 kind of each 5ug of packaging plasmid VSVG, RSV-REV, RRE adds in 50ulHBS liquid and mixes gently, then supplements ddH
2o to 500 μ l, as B liquid, separately prepares 500 μ lCaCl
2a liquid, then adds B liquid in A liquid, gets bubble, and room temperature places 2min; Dropwise add in Tissue Culture Dish, Cross Water yawing shakes repeatedly; Wait to hatch and cultivate 10-12h, be replaced by nutrient solution for dual anti-containing the high sugar+1% of 10%FBS+DMEM; After 48h cell occur melting be associated with strong green fluorescence express time, collect culture supernatant, after 0.45 μm of aperture membrane filtration, with ultracentrifuge 4 DEG C, the centrifugal 3h of 55000rpm/min, after removing supernatant, then add 100 μ l nutrient culture media piping and druming and be distributed into 2 pipes, preserve virus liquid for-80 DEG C.
3.2 slow-virus infection hair follicle stem cells: the hair follicle stem cells getting cultivation, 1 day in advance according to 1 × 10
5the hair follicle stem cells in P3 generation good for growth conditions digests by the concentration in/hole, be seeded in 24 orifice plates of Yu Shop glue after the piping and druming of centrifugal rear use 50 μ l virus stock solution used and the mixing of 50 μ l supplementing culture mediums, at 37 DEG C, 5%CO
2incubator leaves standstill 30min, then adds 400 μ l supplementing culture mediums continuation cultivations, changes liquid, observes green fluorescence after 48h, 72h under fluorescence inverted microscope, obtain VEGF165 genetic modification hair follicle stem cells after 24h.Under Figure 10, Figure 11 are respectively inverted fluorescence microscope, P3 is for GFP figure and the PH figure of the complete 72h of hair follicle stem cells slow-virus infection.
3.3 growth curves measure: by having infected the P3 of 72h for hair follicle stem cells, with 1 × 10 after digestion
5the cell concentration of/ml is seeded on 24 orifice plates, respectively at 1,2,3,4,5,6,7d carries out cell counting count board counting, arranges 6 multiple holes every day,
Take the mean.Then cell growth curve is drawn.As the growth curve chart that Figure 12 is the hair follicle stem cells after VEGF165 genetic modification.
3.4 reverse transcriptional PCRs (RT-PCR) detect the expression turning then VEGF165mRNA in hair follicle stem cells.
(1) total serum IgE is extracted: be placed on ice by six well culture plates being vaccinated with hair follicle stem cells, suck nutrient solution; Every hole adds the Trizol reagent 1ml of precooling, repeatedly blows and beats cracking and moves into fully afterwards in EP pipe; Add 0.2ml chloroform, after concuss mixing, place 2-3min; 12000g, 4 DEG C of centrifugal 15min; Moved into by supernatant in another EP pipe, about 0.5ml, adds equal-volume isopropyl alcohol, puts upside down mixing, and leave standstill 10min, centrifugal (12000g, 4 DEG C) 10min, abandons supernatant; With 75% alcohol 1ml washing precipitation of 0.5ml DEPC process, centrifugal (7500g, 4 DEG C) 5min; Incline alcohol, is inverted EP pipe, is dried to alcohol and waves
Send out;-70 DEG C, the water adding appropriate DEPC process is freezing for subsequent use.
(2) the reverse transcription system of hair follicle stem cells RNA:
(3) PCR (PCR)
A) PCR primer VEGF165 protein primer design, Action in contrast.As Table1.
Table 1. primer sequence information
（Table1.Oligonucleotide sequences for PCR amplifycatio）
B) PCR reaction conditions, as Table2.
Table 2.PCR process
（Table2.The process of PCR）
C) PCR reaction system
(4) agarose electrophoresis: get amplified production 6 μ l loading, after 1.5% agarose gel electrophoresis, photograph under transmit ultraviolet light analyser, and with laser light density image scanner scanning.If Figure 13 is the RT-PCR result figure that hair follicle after VEGF165 genetic modification does thin mRNA.
3.5Western blotting method detects the expression of hair follicle stem cells VEGF165 albumen
(1) hair follicle stem cells cracking
A) hair follicle stem cells is washed 3 times with the PBS of precooling.
B) the lysing buffer(lysis buffer adding precooling is the damping fluid often used in biological experiment.Object is cell lysis), 6 orifice plates: 80 μ l/well; 60mm double dish: 300 μ l/disk, hatches 20min for 4 DEG C.
C) with cell scraper, cell fragment is concentrated on side together with lysing buffer, hatch 40min and be transferred in the centrifuge tube of precooling for 4 DEG C.Centrifugal (12000g, 4 DEG C) 5min.
D) supernatant is moved in another centrifuge tube, except part is used for surveying except protein concentration, all the other be directly used in experiment or-70 DEG C freezing for subsequent use.
(2) polyacrylamide gel electrophoresis
A) 8% separation gel is prepared: 1.5mM Tris-HCl (pH8.8) 1.3ml, dddH
2o2.3ml, 30% polyacrylamide 1.3ml, 10%SDS50 μ l, 10% Ammonium Persulfate 98.5 50 μ l, TEMED3 μ l(adds rear mixing, quick glue).The above-mentioned mixed liquor of 3ml injects layer glass sheet separation fast, and immediately with a small amount of tri-distilled water sealing face, polymerized at room temperature 30min, sucks upper strata tri-distilled water.
B) 5% spacer gel is prepared: 1mM Tris-HCl (pH6.8) 0.25ml, ddH2O1.4ml.
C) 30% polyacrylamide 0.33ml, 10%SDS20 μ l, 10% Ammonium Persulfate 98.5 20 μ l, TEMED2 μ l(adds rear mixing, quick glue).The above-mentioned mixed liquor of 1.4ml injects layer glass sheet separation fast, inserts comb rapidly, carefully takes out comb after polymerization completely.
D) electrophoresis: get Sample Dilution containing equal protein content to equal-volume, press the mixing of 4:1 volume with 5 × sample buffer, 100 DEG C of water-bath 5min, take advantage of hot application of sample, every duct 30 μ l, after application of sample, gel is positioned over the electrophoresis tank that electrophoretic buffer is housed and carries out electrophoresis.Deposition condition: 95V × 15min in spacer gel, 165V × 50min in separation gel, when the bromophenol blue in sample migrates to the forward position of glue, terminates electrophoresis.
E) electrotransfer: after SDS-PAGE, polyacrylamide gel is unloaded, get and 6 of gel formed objects filter paper and a nitrocellulose membrane (NC), in half-dried transfering buffering liquid, balance 15min, put 3 filter paper, gel, NC film, another 3 filter paper successively from top to bottom well, then be sandwiched in half-dried membrane-transferring device, film towards positive pole, glue to negative pole, according to membrane area size, with the constant current of 1.25mA/cm2, transferring film 90min.
F) detect the protein on NC film: be immersed in by film in Ponceaux dyeing liquor and dye, about 3min red color visible protein colour band occurs, is then immersed in distilled water by film and decolours, shake several minutes gently, then changes destainer for several times, until background colour is very light.
G) immunology detection: 1, close: after transferring film, puts in TTBS by fine for nitre film, delays and shake 10min, then film 37 DEG C of slow yawing 2h of shaking table in confining liquid.2, in conjunction with primary antibodie: after closing, fine for nitre film is moved into primary antibodie (1:1000), 4 DEG C are spent the night or room temperature yawing 3h.Reclaim primary antibodie, the fine film of nitre immerses in TTBS20ml, suddenly shakes 10min, continuous 3 times, remaining primary antibodie on cleaning film.3, resist in conjunction with two: move in the horse anti-reactant liquor of anti-rabbit two (1:2000) by fine for nitre film, after room temperature yawing 2h, the fine film of nitre immerses in TTBS30ml, suddenly shakes 15min, continuous 3 times, and on cleaning film, remaining two resist.4, the detection of immune complex: the development of GE ImageQuant LAS4000 chemiluminescence imaging analyser is taken pictures.Figure 14 is the expression of results that the hair follicle after VEGF165 genetic modification does thin VEGF165 albumen.
The preparation of embodiment 4 compound artificial skin sample
1) preparation of sample: 4 DEG C of gelfoam supports preserved before getting, uses 75% ethanol disinfection, then uses PBS cleaning down; Be laid in 50mm double dish, be placed in 37 DEG C of CO
21h is hatched in incubator; After first being digested with 0.25%Trypsin-0.02%EDTA by the hair follicle stem cells after transfection 72h, obtained cell suspension, the centrifugal 5min of 500r/min, abandons supernatant, with 5ml DMEM/F12 liquid Eddy diffusion cell, by injection respectively with 1 × 10
6, 5 × 10
6, 1 × 10
7/ cm
2density cell is inoculated in gelfoam internal stent.The hair follicle stem cells preserved before getting again, equally with 1 × 10
6, 5 × 10
6, 1 × 10
7/ cm
2density hair follicle stem cells is inoculated in gelfoam rack surface, DMEM/F12 supplementing culture medium cultivate, be placed in the CO of 5%
2cultivate 2 weeks under 37 DEG C of liquid levels in incubator, after Fusion of Cells, change air-liquid interface culture 10d into.
2) detection of compound artificial skin sample: after cultivating 48h under liquid level, observation of cell growing state under inverted microscope.Every 24h observes once later.The compound artificial skin 4%PFA solution prepared is fixed, and carries out conventional dehydration, embedding, section, and HE dyes, light Microscopic observation.As Figure 15 ~ Figure 17 is respectively the HE dyeing of compound artificial skin, be respectively injection concentration 1 × 10
6, 5 × 10
6, 1 × 10
7/ cm
2, bar:200 ×.
The anthropomorphic rat skin defect model of embodiment 5 transplants artificial skin
1) preparation of transplantation experiments animal model and experiment grouping in body:
Healthy male SPF level Sprague-Dawley rat 18, body weight 200-220g.Preoperative 2d sloughs art district dorsal body setae.
Experiment grouping: every rat back center work 4 surface of a wound, be divided into 4 groups: A group (compound artificial skin of the hair follicle stem cells of injection of VEGF 165 genetic modification) at random, B group (injecting the compound artificial skin of the hair follicle stem cells of empty genetic modification), C group (not injecting the artificial skin of cell) and D group (surface of a wound covering gauze).A B group injection concentration be 1 × 10
7/ cm
2.As Figure 18-19.
2) preoperative rat weight, 1% yellow Jackets intraperitoneal injection of anesthesia, after fixing limbs, iodophor disinfection skin of back, the full thickness dermal wounds 4 pieces of 1.2cm × 1.2cm is done in design both sides, center, tail side, back, and methylene blue mark skin wipes out scope, Ge Shu district, every side 2, every block gap 1cm.Cut skin with sharp knife sheet along mark line, be deep to subcutaneous fascia superficialis layer, excision full thickness skin, forms the surface of a wound of exposed fascia.After abundant hemostasis, be divided into four groups at random afterwards, A, B, C group above-mentioned material is implanted Wound Defect respectively, and 5 one 0 wire discontinuous sewings are fixed on edge of wound skin, and D group petrolatum gauze and aseptic dressing cover, and packing is fixing.(sting for preventing rat oneself and damage affected part, pollute and destroy, design flexibility overcoat protects.
3) post-transplantation process: after laboratory animal anesthesia is revived, sends back between raising, and single cage is raised.Next day recovers normal diet, avoids art district gauze to soak, pollute as far as possible.Once outer dressing soaks, change with sterile gauze immediately.Every day is anti-infective with benzylpenicillin sodium for injection injection.
4) tissue specimen collection: respectively at postoperative 7d, 14d, 21d, gets 6 sacrifice of animal at every turn, takes pictures observe wound healing situation, as Figure 20-31 with common Sony camera W570.Calculate the Wound healing rate of the different group of different time.As Figure 32.
5) after 21d, HE dyeing is done to tissue of drawing materials, observe vascularization situation.As Figure 33-35.
Sequence table
<110> Hangzhou Xiaoshan Traditional Chinese Medical Hospital
<120> Western blotting detects the method for hair follicle stem cells VEGF165 protein expression
<160>4
<210> 1
<211> 20
<212> DNA
<213> Prof. Du Yucang
<400> 1
GTCCCTCACC CTCCCAAAAG 20
<210> 2
<211> 21
<212> DNA
<213> Prof. Du Yucang
<400> 2
GCTGCCTCAA CACCTCAACC C 21
<210> 3
<211> 20
<212> DNA
<213> Prof. Du Yucang
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CACCCACCCA CATACATACA 20
<210> 4
<211> 19
<212> DNA
<213> Prof. Du Yucang
<400> 4
CTCCCAACTC AAGTCCACA 19
Claims (1)
1. Western blotting detects the method for hair follicle stem cells VEGF165 protein expression, it is characterized in that the method comprises the following steps:
One, select SD rat antenna in one week age portion skin, insert 0.25% Dispase enzyme 37 DEG C digestion 2h; With tweezers and disposable syringe syringe needle from hypodermis end pull-out hair follicle, it is intact and be in the hair follicle in growth period to collect form, under microscope, hair follicle is cut into three equal parts, get center section, put into 50mL culture flask after PBS rinsing, add DMEM/F12 supplementing culture medium, be placed in 37 DEG C, 5%CO
2in incubator, every 2d changes liquid once; Described DMEM/F12 supplementing culture medium composition is: 44mlDMEM/F12 nutrient solution, 5mlKSR serum substitute, 500 μ l mycillin mixed liquors, 500 μ l Glus, 500 μ l nonessential amino acid, 20ng/ml recombinant human epidermal growth factor, 10ng/ml recombination human basic fibroblast growth factor, 50 μ l hydroxyl ethanols, 10ng/ml hydrocortisone;
Two, the purifying of hair follicle stem cells:
Be coated in double dish by the IV Collagen Type VI of 100 μ g/ml according to the amount of 3ml/100mm dish, room temperature leaves standstill 1h; By the primary cell Trypsin Induced of 100mm double dish, after centrifugal collecting cell, blow and beat into single cell suspension and be inoculated in double dish, after 20min, by not adherent cell together with nutrient solution sucking-off; The complete medium culture of adherent cell, changes liquid in every 3 days; P2 for repurity once;
Three, the qualification of hair follicle stem cells:
A, employing Q-PCR method: the P3 after sorting purifying carries out Q-PCR detection for hair follicle stem cells, carries out the relative quantification of each gene expression by △ △ Ct method;
B, immunofluorescent staining method: by P3 for cell chulture to exponential phase, be seeded in after digestion on slide, after adhere-wall culture 2d, sop up nutrient solution, use PBST rinsing, add 4%PFA fixing after, more closed by 5%BSA room temperature; Add primary antibodie integrin β_1 polyclonal antibody, integrin a6 polyclonal antibody and keratin 15 polyclonal antibody respectively, incubated at room, after PBST washing, label two anti-lucifuge 30min again, and add DAPI and contaminate core 5min, lucifuge is dried, with mounting solution mounting, observe;
Four, pack slow virus: the 293T cell in growth period of taking the logarithm, 24h is inoculated in 100mm double dish in advance, treats that next day, cell grew to 50%-70%; Virus packaging adopts calcium robin to carry out: be replaced by by 293T cell culture medium before transfection not containing dual anti-nutrient culture media, comprises the high sugar of 10%FBS+DMEM; Then, each 5 μ g of object plasmid pLenti-IRES-VEGF165-EGFP10 μ g and 3 kind of packaging plasmid VSVG, RSV-REV, RRE add in 50 μ lHBS liquid and mix gently, then supplement ddH
2o to 500 μ l, as B liquid, separately prepares 500 μ lCaCl
2a liquid, then adds B liquid in A liquid, gets bubble, and room temperature places 2min; Dropwise add in Tissue Culture Dish, Cross Water yawing shakes repeatedly; Wait to hatch and cultivate 10-12h, change nutrient solution for dual anti-containing the high sugar+1% of 10%FBS+DMEM; After 48h cell occur melting be associated with strong green fluorescence express time, collect culture supernatant, after 0.45 μm of aperture membrane filtration, with ultracentrifuge 4 DEG C, the centrifugal 3h of 55000rpm/min, after removing supernatant, then add 100 μ l nutrient culture media piping and druming and be distributed into 2 pipes, preserve virus liquid for-80 DEG C;
Five, slow-virus infection hair follicle stem cells: the hair follicle stem cells getting cultivation, 1 day in advance according to 1 × 10
5the concentration in/hole is seeded in 24 orifice plates overlaying glue, at 37 DEG C, 5%CO by after the digestion of the hair follicle stem cells in P3 generation good for growth conditions, centrifugal rear use 50 μ l virus stock solution used and 50 μ l supplementing culture mediums mixing piping and druming
2incubator leaves standstill 30min, then adds 400 μ l supplementing culture mediums continuation cultivations, changes liquid, observes green fluorescence after 48h, 72h under fluorescence inverted microscope, obtain VEGF165 genetic modification hair follicle stem cells after 24h;
Six, hair follicle stem cells cracking
A) hair follicle stem cells is washed 3 times with the PBS of precooling;
B) the lysing buffer of precooling is added, 6 orifice plates: 80 μ l/well; 60 mm double dish: 300 μ l/disk, hatch 20 min for 4 DEG C;
C) with cell scraper, cell fragment is concentrated on side together with lysing buffer, hatch 40min and be transferred in the Eppendorf pipe of precooling for 4 DEG C; 12000 g, 4 DEG C of centrifugal 5 min;
D) supernatant is moved in another Eppendorf pipe, except part is used for surveying except protein concentration, all the other be directly used in experiment or-70 DEG C freezing for subsequent use;
Seven, polyacrylamide gel electrophoresis
A) prepare 8% separation gel: the pH of 1.5 mM Tris-HCl 1.3 ml, Tris-HCl is 8.8, dddH2O 2.3 ml, 30% polyacrylamide 1.3 ml, 10%SDS 50 μ l, 10% Ammonium Persulfate 98.5 50 μ l, TEMED 3 μ l, adds rear mixing, quick glue; The above-mentioned mixed liquor of 3 ml injects layer glass sheet separation fast, and immediately with a small amount of tri-distilled water sealing face, polymerized at room temperature 30 min, sucks upper strata tri-distilled water;
B) 5% spacer gel is prepared: the pH of 1 mM Tris-HCl 0.25 ml, Tris-HCl is 6.8, ddH2O 1.4 ml;
C) 30% polyacrylamide 0.33 ml, 10%SDS 20 μ l, 10% Ammonium Persulfate 98.5 20 μ l, TEMED 2 μ l, adds rear mixing, quick glue; The above-mentioned mixed liquor of 1.4 ml injects layer glass sheet separation fast, inserts comb rapidly, carefully takes out comb after polymerization completely;
D) electrophoresis: get Sample Dilution containing equal protein content to equal-volume, press the mixing of 4:1 volume with 5 × sample buffer, 100 DEG C of water-bath 5 min, take advantage of hot application of sample, every duct 30 μ l, after application of sample, gel is positioned over the electrophoresis tank that electrophoretic buffer is housed and carries out electrophoresis; Deposition condition: 95 V × 15 min in spacer gel, 165 V × 50 min in separation gel, when the bromophenol blue in sample migrates to the forward position of glue, terminate electrophoresis;
E) electrotransfer: after SDS-PAGE, polyacrylamide gel is unloaded, get and 6 of gel formed objects filter paper and a nitrocellulose filter, in half-dried transfering buffering liquid, balance 15 min, put 3 filter paper, gel, NC film, another 3 filter paper successively from top to bottom well, be then sandwiched in half-dried membrane-transferring device, film is towards positive pole, glue to negative pole, according to membrane area size, with 1.25 mA/cm
2constant current, transferring film 90 min;
F) protein on nitrocellulose filter is detected: be immersed in by film in Ponceaux dyeing liquor and dye, about 3 min red color visible protein colour bands occur, are then immersed in distilled water by film and decolour, shake several minutes gently, then destainer is changed for several times, until background colour is very light;
G) immunology detection:
1) close: after transferring film, nitrocellulose filter is put in TTBS, delay and shake 10 min, then film 37 DEG C of shaking table slow yawing 2 h in confining liquid;
2) in conjunction with primary antibodie: after closing, nitrocellulose filter is moved into primary antibodie, 4 DEG C are spent the night or room temperature yawing 3 h; Reclaim primary antibodie, nitrocellulose filter immerses in TTBS 20 ml, suddenly shakes 10 min, continuous 3 times, remaining primary antibodie on cleaning film;
3) resist in conjunction with two: moved into by nitrocellulose filter in the anti-reactant liquor of horse anti-rabbit two, after room temperature yawing 2 h, nitrocellulose filter immerses in TTBS 30 ml, suddenly shakes 15 min, continuous 3 times, and on cleaning film, remaining two resist;
4) detection of immune complex: the development of GE ImageQuant LAS 4000 chemiluminescence imaging analyser is taken pictures.
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| CN101418042A (en) * | 2007-10-24 | 2009-04-29 | 苏州思坦维生物技术有限责任公司 | Separation and purification of recombinant human blood vessel endothelia cell growth factor, chemical labeling in vitro and use thereof |
| CN101914495A (en) * | 2010-07-22 | 2010-12-15 | 吉林大学 | Culture method for largely amplifying hair follicle stem cells in vitro |
| CN102830235A (en) * | 2012-08-28 | 2012-12-19 | 邹检平 | Luminescence detection kit and preparation method thereof |
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| CN101914495A (en) * | 2010-07-22 | 2010-12-15 | 吉林大学 | Culture method for largely amplifying hair follicle stem cells in vitro |
| CN102830235A (en) * | 2012-08-28 | 2012-12-19 | 邹检平 | Luminescence detection kit and preparation method thereof |
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| 张向荣.血管内皮细胞生长因子165基因转染人骨髓间充质干细胞构建组织工程皮肤的实验研究.《中国博士学位论文全文数据库(电子期刊) 医药卫生科技辑》.2010,(第3期),E080-1,正文第19-30页. * |
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Inventor after: Quan Renfu Inventor after: Huang Zhongming Inventor after: Zeng Linru Inventor after: Xu Jinwei Inventor after: Li Wei Inventor after: Xie Lijun Inventor after: Xu Canda Inventor before: Quan Renfu Inventor before: Huang Zhongming Inventor before: Zheng Xuan Inventor before: Xu Shichao |
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Free format text: CORRECT: INVENTOR; FROM: QUAN RENFU HUANG ZHONGMING ZHENG XUAN XU SHICHAO TO: QUAN RENFU HUANG ZHONGMING CENG LINRU XU JINWEI LI WEI XIE LIJUN XU CANDA |