CN103468744A - VEGF165 gene modified hair follicle stem cells and preparation method thereof - Google Patents
VEGF165 gene modified hair follicle stem cells and preparation method thereof Download PDFInfo
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Abstract
The invention relates to VEGF165 gene modified hair follicle stem cells and a preparation method thereof. The preparation method comprises following step: 1) hair follicle stem cells, which possess excellent cellular morphology and are in growing period, are collected, and are equally cut into three parts; the part in the middle is taken, is rinsed with PBS, and is delivered into a 50mL culture bottle; and then DMEM/F12 supplemental medium is added into the culture bottle; 2) the collected hair follicle stem cells are purified; 3) the hair follicle stem cells are identified; 4) lentivirus are packaged; 5) the hair follicle stem cells are infected by lentivirus; and 6) the hair follicle stem cells are modified by obtained VEGF165 gene. According to the preparation method, the VEGF165 gene modified hair follicle stem cells are taken as seed cells, rapid and large-scaled amplification of the hair follicle stem cells can be realized by in vitro culture, resources of the hair follicle stem cells are quite abundant, and the hair follicle stem cells are capable of reducing immunological rejection. In addition, the hair follicle stem cells are transfected and modified by VEGF165 gene, so that novel engineering blood vessel systems with expansion and contraction functions can be formed, and problems of easily caused artificial skin necrosis in transplanting and low survival rate are solved perfectly.
Description
Technical field
The present invention relates to skin tissue engineering and learn field, relate in particular to a kind of method of hair follicle stem cells genetic modification and the hair follicle stem cells that the method obtains.
Technical field
Skin is as the organ of human body maximum, there is sensation, regulate body temperature, secretion and excretion, prevent the multiple effects such as moisture evaporation, wherein topmost function be as the barrier of human body and external environment to maintain the stable of interior environment, it is also immune important component part simultaneously.Along with socioeconomic development, particularly as the China's manufacturing industry of " world's factory " and the prosperity and development of handicraft, particularly burn, crush injury, incision etc. are also more and more for the various wounds that relate to skin injury.Wherein, the skin large defect that wound causes usually causes very serious physical disabilities, even dead.The standard treatments for the treatment of clinically skin injury at present is auto-skin grafting, owing to having without immunological rejection, and the surviving rate high, it applies very extensive clinically, and has obtained good curative effect.But the shortcoming of auto-skin grafting is also very obvious, owing to being autologous drawing materials, itself be exactly the damage again to the patient, greatly increased the patient suffering, and had the skin donor site of generation to infect, the risk of the complication such as disunion.More seriously, for above-mentioned large skin defect, often owing to lacking enough, can cause the wound repair difficulty for the autologous skin of transplanting, have a strong impact on the treatment process, even cause death.
Just because of this, scientists attempts to find a kind of ectogenic Graftskin always.As far back as B.C. 1500, dermatoheteroplasty just was used to cover the skin injury surface of a wound temporarily.And, along with foundation and the development of organizational engineering, skin tissue engineering is learned and risen rapidly, and become the focus of research nearly ten years.Organizational engineering is principle and the method for application project and life science, and the research biosubstitute, with the subject for reconstruction, maintenance or raising function of organization.At present, the foreign applications skin tissue engineering is learned the research that builds artificial skin and has been obtained substantial progress, and the products such as Apligraf, OrCel, Suprathel, Biobrane, OASIS, Integra, Lyphoder are successively gone on the market and have been applied in the treatment of clinical skin injury by united states drug and food control office (FDA) approval.
Yet, although existing above several artificial skin products can supply selection of clinical at present, also really promoted the clinical treatment to the 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 transplanting also exists many problems, wherein even comprise " hard defects " that may cause treatment to prove an abortion: 1. because above artificial skin product does not all have the vasculogenesis ability, cause the artificial skin of transplanting not have vascular system to supply with nutrition, thereby artificial skin is easily necrosed, make graft failure.2. in the artificial skin product, contained various variant cells easily cause immunological rejection, clinically the normal cutaneous necrosis that occurs transplanting, come off, serious the severe complication such as systemic immune response even occurs, and likely spread disease.3. current all people's work skin products all can only recover part anatomical structure and the physiological function of normal skin, and the cutaneous appendages structure that can't regenerate and there is critical function, such as: blood vessel, hair, sweat gland etc.
Hair follicle stem cells is the stem cell that a class is present in hair follicle external root sheath knuckle section, has the characteristics such as a minute voltinism, self and in-vitro multiplication ability are strong.Hair follicle stem cells in vitro culture research has shown high clonality, has very high regeneration potential.Because hair follicle stem cells derives from hair, can directly from patient self, obtain, quantity is extremely abundant, and, without any complication, the patient, fully without wound, has now been become to the focus that skin tissue engineering is learned research.(the Taylor G such as Taylor, Lehrer MS, Jensen PJ, et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell, 2000,102 (4): 451-461.) research finds that hair follicle stem cells not only can be differentiated to form hair follicle, but also has participated in the forming process of face tissue.The current research result that Stelios etc. deliver proves, contains a large amount of stem cells in hair follicle, is one of source of human stem cell the most easily obtained, and successfully the hair follicle stem cells differentiation and development is generated to new vascular system.Vascular endothelial growth factor 165 (VEGF165) is one of 5 kinds of hypotypes of vascular endothelial growth factor, and its activity is the strongest, and distribution range is the widest, is the Main Subtype played a role in the VEGF body.Become domestic and international study hotspot around the research of the revascularization gene therapy centered by VEGF165 in recent years.
Summary of the invention
The problems that may affect clinical application effect that exist in order to solve current artificial skin product, an object of the present invention is to provide the method for VEGF165 genetic modification hair follicle stem cells, another object of the present invention is to provide the VEGF165 genetic modification hair follicle stem cells that above-mentioned method obtains.The present invention be take hair follicle stem cells as seed cell, while having vitro culture, can increase by rapid, high volume, originates very abundant, reduces immunological rejection; Utilize the VEGF165 gene transfection to modify hair follicle stem cells simultaneously, can form the new engineering vascular system with enlargement and contraction function, will well solve easily necrosis of artificial skin, the problem that surviving rate is low of transplanting.
In order to realize first above-mentioned purpose, the present invention has adopted following technical scheme:
The method of VEGF165 genetic modification hair follicle stem cells, the method comprises the following steps:
1) select SD rat antenna section skin in one week age, insert 37 ℃ of digestion 2h of 0.25%Dispase enzyme; Pull out hair follicle with tweezers and disposable syringe syringe needle from the subcutis end, collect the intact and hair follicle in vegetative period of form, under microscope, hair follicle is cut into to three equal parts, get middle portion, put into the 50mL culturing bottle after the PBS rinsing, add the DMEM/F12 supplemental medium, be placed in 37 ℃, 5%C0
2in incubator, every 2d changes liquid once; Described DMEM/F12 supplemental medium composition is: 44mlDMEM/F12 nutrient solution, 5mlKSR serum substitute, 500 μ l mycillin mixed solutions, 500 μ l L-glutaminate, 500 μ l non-essential 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;
2) purifying of hair follicle stem cells: the amount by the IV Collagen Type VI of 100 μ g/ml according to 3ml/100mm dish is coated in culture dish, the standing 1h of room temperature; By the primary cell tryptic digestion of 100mm culture dish, after centrifugal collecting cell, blow and beat into single cell suspension and be inoculated in culture dish, after 20min, by not adherent cell together with the nutrient solution sucking-off; The complete culture medium culturing of adherent cell, change liquid in every 3 days; P2 for repurity once;
3) evaluation of hair follicle stem cells:
A, employing Q-PCR method: the P3 after the sorting purifying carries out the Q-PCR detection for hair follicle stem cells, the relative quantification that carries out each genetic expression by △ △ Ct method;
B, immunofluorescent staining method: P3 is arrived to logarithmic phase for cell cultures, be seeded in after digestion on slide, after adherent culture 2d, sop up nutrient solution, use the PBST rinsing, after adding 4%PFA fixing, then seal by the 5%BSA room temperature.Add respectively the anti-mouse polyclonal antibody of primary antibodie integrin beta 1, integrin a6 polyclonal antibody and keratin 15 polyclonal antibody, incubated at room, after the PBST washing, the two anti-lucifuge 30min that label again, add DAPI and dye core 5min, and lucifuge is dried, with mounting solution mounting, observe;
4) packing slow virus: the 293T cell in the vegetative period of taking the logarithm, in advance 24h is inoculated in the 100mm culture dish, treats that cell grew to 50%-70% and got final product next day; Virus packing employing calcium turns method and carries out: before transfection, the 293T cell culture medium is replaced by containing two anti-substratum, comprises that 10%FBS+DMEM is high sugared; Then, purpose plasmid pLenti-IRES-VEGF165-EGFP10ug and 3 kinds of packaging plasmid VSVG, RSV-REV, each 5ug of RRE add in 50ulHBS liquid and mix gently, then supplement ddH
2o to 500 μ l, as B liquid, separately prepares 500 μ lCaCl
2a liquid, then add B liquid in A liquid, gets bubble, and room temperature is placed 2min; Dropwise add in Tissue Culture Dish, the Cross Water yawing shakes repeatedly; Wait to hatch and cultivate 10-12h, be replaced by nutrient solution for anti-containing the high sugar of 10%FBS+DMEM+1% pair; After 48h, cell occurs melting while being associated with strong green fluorescence expression, collects culture supernatant, with after 0.45 μ m aperture membrane filtration, with 4 ℃ of ultracentrifuges, the centrifugal 3h of 55000rpm/min, after removing supernatant, then add 100 μ l substratum piping and druming and be distributed into 2 pipes, preserve virus liquid for-80 ℃;
5) slow virus infection hair follicle stem cells: get the hair follicle stem cells of cultivation, 1 day in advance according to 1 * 10
5be seeded in 24 orifice plates of Yu Shop glue after the piping and druming that the hair follicle stem cells digestion in P3 generation that the concentration in/hole is good by growth conditions, centrifugal rear use 50 μ l virus stock solution useds and 50 μ l supplemental mediums mix, at 37 ℃, the standing 30min of 5%CO2 incubator, adding 400 μ l supplemental mediums continues to cultivate again, change liquid after 24h, observe green fluorescence under the fluorescence inverted microscope after 48h, 72h, obtain VEGF165 genetic modification hair follicle stem cells;
6) the VEGF165 genetic modification hair follicle stem cells obtained.Detect respectively the multiplication capacity of this cell, expression and the Western-Blot detection VEGF165 protein expression situation that RT-PCR detects VEGF165mRNA.
In order to realize second above-mentioned purpose, the present invention has adopted following technical scheme:
VEGF165 genetic modification hair follicle stem cells, this hair follicle stem cells adopts above-mentioned method to obtain.
The present invention is owing to having adopted above-mentioned technical scheme, take hair follicle stem cells as seed cell, utilize the VEGF165 gene transfection to make hair follicle stem cells, this hair follicle stem cells can be planted in three-dimensional isinglass spongy tissue support, form into the NEW TYPE OF COMPOSITE artificial skin model with new vascular system.This model has advantages of following uniqueness:
1. as stem cell, when hair follicle stem cells has vitro culture, can increase by rapid, high volume, long-term subculture in vitro separately is cultivated, the characteristics such as cell function is vigorous, and have very high regeneration and differentiation capability, this will greatly shorten the cultured and amplified in vitro time, improve clinical treatment efficiency;
2. owing to can forming the new engineering vascular system with enlargement and contraction function, will well solve easily necrosis of artificial skin, the problem that surviving rate is low of transplanting;
3. in this research, seed cell-hair follicle stem cells used is taken from autologous hair, originates very abundant, also is very easy to obtain, and can cause any damage and misery to the patient;
4. because seed cell is taken from autologous tissue, immunological rejection and the problem such as spread disease will be readily solved;
5. with respect to current artificial skin product, this new-type artificial skin will recover and anatomical structure and the physiological function of the normal skin tissue of regenerating greatly.
The accompanying drawing explanation
Fig. 1, Fig. 2 are that primary hair follicle stem cells climbs out of from Hair Follicle Bulge section, are nest like, epithelioid cell, arrange tight 40 *.
Fig. 3 be P3 after IV Collagen Type VI screening purifying for hair follicle stem cells, be typical paving stone shape 100 *.
Table 1 carries out the Q-PCR detection for hair follicle stem cells, the relative quantification that carries out each genetic expression by △ △ Ct method for the P3 after the sorting purifying.
Fig. 4-Fig. 6 is the immunofluorescence dyeing of P3 for hair follicle stem cells, is respectively integrin beta 1, integrin a6, keratin 15,100 *.
Fig. 7-Fig. 9 is that metal spraying shows gelfoam three-dimensional rack scanning electron microscope, be respectively SEM50 *, 200 *, 400 *.
Figure 10, Figure 11 are respectively under inverted fluorescence microscope, P3 for the GFP figure of slow virus infection complete 72h and PH Figure 100 for hair follicle stem cells *.
The growth curve chart that Figure 12 is the hair follicle stem cells after the VEGF165 genetic modification.
The RT-PCR that Figure 13 is the dry thin mRNA of hair follicle after the VEGF165 genetic modification is figure as a result.
Figure 14 is the expression of results that the hair follicle after the VEGF165 genetic modification is done thin VEGF165 albumen.
Figure 15～Figure 17 is respectively the HE dyeing of compound artificial skin, is respectively injection concentration 1 * 106,5 * 106,1 * 107/cm2, bar:200 *.
Surface of a wound figure when transplant for doing rat back Figure 18-19.
Figure 20-23 are posttransplantation four the wound healing situations of 7d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
Figure 24-27 are posttransplantation four the wound healing situations of 14d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
Figure 28-31 are posttransplantation four the wound healing situations of 21d.Be followed successively by VEGF165 group, empty plasmid group, acellular group, gauze group.
The Wound healing rate that Figure 32 is 7d, 14d, tetra-groups of 21d.
The HE dyeing of Figure 33-35 for after 21d, the tissue of drawing materials being done, observe the vascularization situation.Be followed successively by VEGF165 group, empty plasmid group, acellular group.100×
Embodiment
Separation, the cultivation of embodiment 1 rat hair follicle stem cells, identify
1.1) select SD rat antenna section skin in one week age, insert 37 ℃ of digestion 2h of 0.25%Dispase enzyme; Pull out hair follicle with tweezers and disposable syringe syringe needle from the subcutis end, collect the intact and hair follicle in vegetative period of form, under microscope, hair follicle is cut into to three equal parts, get middle portion, put into the 50mL culturing bottle after the PBS rinsing, add the DMEM/F12 supplemental medium, be placed in 37 ℃, 5%CO
2in incubator, every 2d changes liquid once; Described DMEM/F12 supplemental medium composition is: 44mlDMEM/F12 nutrient solution, 5mlKSR serum substitute, 500 μ l mycillin mixed solutions, 500 μ l L-glutaminate, 500 μ l non-essential 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 section, are nest like, epithelioid cell, arrange closely.
1.2) purifying of hair follicle stem cells: the amount by the IV Collagen Type VI of 100 μ g/ml according to 3ml/100mm dish is coated in culture dish, the standing 1h of room temperature; By the primary cell tryptic digestion of 100mm culture dish, after centrifugal collecting cell, blow and beat into single cell suspension and be inoculated in culture dish, after 20min, by not adherent cell together with the nutrient solution sucking-off; The complete culture medium culturing of adherent cell, change liquid in every 3 days; P2 for repurity once.As Fig. 3 be P3 after IV Collagen Type VI screening purifying for hair follicle stem cells, be typical paving stone shape.
1.3) evaluation of hair follicle stem cells:
A, employing Q-PCR method: the P3 after the sorting purifying carries out the Q-PCR detection for hair follicle stem cells, the relative quantification that carries out each genetic expression by △ △ Ct method.As table 1
B, immunofluorescent staining method: P3 is arrived to logarithmic phase for cell cultures, be seeded in after digestion on slide, after adherent culture 2d, sop up nutrient solution, use the PBST rinsing, after adding 4%PFA fixing, then seal by the 5%BSA room temperature.Add respectively primary antibodie integrin beta 1(integrin-β 1) anti-mouse polyclonal antibody (1:100), integrin a6(integrin-a6) polyclonal antibody (1:50) and keratin 15 (keratin-15) polyclonal antibody (1:100), incubated at room, after the PBST washing, two anti-lucifuge 30min again label, add DAPI(1:2000) dye 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 beta 1, integrin a6, keratin 15.
The preparation of embodiment 2 gelfoam three-dimensional tissue supports
2.1) the support preparation of gelfoam three-dimensional tissue:
A. the gelatin that to get content be 5% is dissolved in the distilled water 10ml of 25 ℃, adds respectively 0.05%6-sodium chondroitin sulfate (C6S) and 0.2% hyaluroni (HA);
B. under room temperature, use magnetic stirrer after 60 minutes, after linking agent 0.5%1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt acid salt solution (EDC) and 0.25%N-N-Hydroxysuccinimide solution (N-Hydroxysuccinimide) are splashed in solution and mix 5 minutes, solution is injected to the mould of 12 control-bureau's orifice plates, level is rocked evenly again.
C. under-80 ℃ freezing 2 hours
D. then go up Freeze Drying Equipment, freeze-drying obtains the Gel-C6S-HA support of the porous spongy that thickness is 2mm in 24 hours.
2.2) observe: the support sample that takes a morsel, scanning electron microscopic observation record, Fig. 7-Fig. 9 is that metal spraying shows gelfoam three-dimensional rack scanning electron microscope, be respectively SEM50 *, 200 *, 400 *.
The preparation that embodiment 3 VEGF-165 genes (VEGF165) are modified hair follicle stem cells
3.1 packing slow virus: the 293T cell in the vegetative period of taking the logarithm, in advance 24h is inoculated in the 100mm culture dish, treats that cell grew to 50%-70% and got final product next day; Virus packing employing calcium turns method and carries out: before transfection, the 293T cell culture medium is replaced by containing two anti-substratum, comprises that 10%FBS+DMEM is high sugared; Then, purpose plasmid pLenti-IRES-VEGF165-EGFP10ug and 3 kinds of packaging plasmid VSVG, RSV-REV, each 5ug of RRE add in 50ulHBS liquid and mix gently, then supplement ddH
2o to 500 μ l, as B liquid, separately prepares 500 μ lCaCl
2a liquid, then add B liquid in A liquid, gets bubble, and room temperature is placed 2min; Dropwise add in Tissue Culture Dish, the Cross Water yawing shakes repeatedly; Wait to hatch and cultivate 10-12h, be replaced by nutrient solution for anti-containing the high sugar of 10%FBS+DMEM+1% pair; After 48h, cell occurs melting while being associated with strong green fluorescence expression, collects culture supernatant, with after 0.45 μ m aperture membrane filtration, with 4 ℃ of ultracentrifuges, the centrifugal 3h of 55000rpm/min, after removing supernatant, then add 100 μ l substratum piping and druming and be distributed into 2 pipes, preserve virus liquid for-80 ℃.
3.2 slow virus infection hair follicle stem cells: get the hair follicle stem cells of cultivation, 1 day in advance according to 1 * 10
5be seeded in 24 orifice plates of Yu Shop glue after the piping and druming that the hair follicle stem cells digestion in P3 generation that the concentration in/hole is good by growth conditions, centrifugal rear use 50 μ l virus stock solution useds and 50 μ l supplemental mediums mix, at 37 ℃, 5%CO
2the standing 30min of incubator, then add 400 μ l supplemental mediums and continue to cultivate, change liquid after 24h, observe green fluorescence under the fluorescence inverted microscope after 48h, 72h, obtain VEGF165 genetic modification hair follicle stem cells.Figure 10, Figure 11 are respectively under inverted fluorescence microscope, and P3 is GFP figure and the PH figure with the complete 72h of slow virus infection for hair follicle stem cells.
3.3 growth curve is measured: will infect the P3 of 72h for hair follicle stem cells, after digestion with 1 * 10
5the cell concn of/ml is seeded on 24 orifice plates, respectively at 1,2,3,4,5,6,7d carries out the cell counting count board counting, and 6 multiple holes are set every day,
Take the mean.Then draw cell growth curve.As Figure 12 growth curve chart that is the hair follicle stem cells after the VEGF165 genetic modification.
3.4 reverse transcriptional PCR (RT-PCR) detects the expression turn VEGF165mRNA in hair follicle stem cells then.
(1) extract total RNA: six well culture plates that will inoculate hair follicle stem cells are placed on ice, suck nutrient solution; Every hole adds the Trizol reagent 1ml of precooling, repeatedly blows and beats in the fully rear EP of the immigration pipe of cracking; Add the 0.2ml chloroform, concuss mixes rear placement 2-3min; 12000g, 4 ℃ of centrifugal 15min; Supernatant liquor is moved in another EP pipe, and about 0.5ml, add the equal-volume Virahol, and put upside down and mix, standing 10min, centrifugal (12000g, 4 ℃) 10min, abandon supernatant; The 75% alcohol 1ml washing precipitation of processing with 0.5ml DEPC, centrifugal (7500g, 4 ℃) 5min; The alcohol that inclines, be inverted the EP pipe, is dried to the alcohol volatilization; Add water-70 that appropriate DEPC processes ℃ freezing standby.
(2) the reverse transcription system of hair follicle stem cells RNA:
(3) polymerase chain reaction (PCR)
A) PCR primer VEGF165 albumen design of primers, 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
| cDNA | 5.0μl |
| Target mRNA | Each 1.0 μ l |
| Actin upstream and downstream primer | Each 1.0 |
| 2 * Tag enzyme | 12.5μl |
[0073]?
| Aseptic tri-distilled water | 5.5μl |
| ? | 25.0μl |
(4) agarose electrophoresis: get amplified production 6 μ l loadings, after 1.5% agarose gel electrophoresis, photograph under the transmit ultraviolet light analyser, and by laser light density image scanner scanning.As Figure 13 RT-PCR that is the dry thin mRNA of hair follicle after the VEGF165 genetic modification figure as a result.
3.5Western the blotting method detects the expression of hair follicle stem cells VEGF165 albumen
(1) hair follicle stem cells cracking
A) with the PBS scouring of wool capsule stem cell of precooling 3 times.
B) the lysing buffer(lysis buffer that adds precooling is the damping fluid often used in biological experiment.Purpose is lysing cell), 6 orifice plates: 80 μ l/well; The 60mm culture dish: 300 μ l/disk, hatch 20min for 4 ℃.
C) with cell scraper, cell debris is concentrated on to a side together with lysing buffer, hatch 40min and be transferred in the centrifuge tube of precooling for 4 ℃.Centrifugal (12000g, 4 ℃) 5min.
D) supernatant is moved in another centrifuge tube, except the part be used for surveying protein concentration, all the other be directly used in the experiment or-70 ℃ freezing standby.
(2) polyacrylamide gel electrophoresis
A) prepare 8% separation gel: 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, mix after TEMED3 μ l(adds, fast glue).The above-mentioned mixed solution of 3ml injects the layer glass sheet separation fast, and immediately with a small amount of tri-distilled water sealing face, polymerized at room temperature 30min, suck the upper strata tri-distilled water.B) prepare 5% spacer gel: 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, mix after TEMED2 μ l(adds, fast glue).1.4ml above-mentioned mixed solution injects the layer glass sheet separation fast, inserts rapidly comb, the careful comb that takes out after polymerization fully.
D) electrophoresis: the sample of getting containing equal protein content is diluted to equal-volume, and 5 * sample buffer mixes by the 4:1 volume,
100 ℃ of water-bath 5min, take advantage of hot application of sample, every duct 30 μ l, and after application of sample, gel is positioned over the electrophoresis chamber that electrophoretic buffer is housed and carries out electrophoresis.Deposition condition: 95V * 15min in spacer gel, 165V * 50min in separation gel, when the tetrabromophenol sulfonphthalein in sample migrates to the forward position of glue, finish electrophoresis.
E) electrotransfer: after SDS-PAGE, polyacrylamide gel is unloaded, get 6 filter paper and a nitrocellulose membrane (NC) with the gel formed objects, balance 15min in half-dried transfering buffering liquid, 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 is towards positive pole, and glue is to negative pole, according to the membrane area size, with the constant current of 1.25mA/cm2, transferring film 90min.
F) detect the protein on the NC film: film is immersed in the ponceau staining fluid and dyes, and about 3min red color visible protein colour band occurs, then film is immersed in distilled water and decolours, and shakes gently several minutes, then changes destainer for several times, until background colour is very light.G) immunology detection: 1, sealing: after transferring film, the fine film of nitre is put in TTBS to slow 10min, then the film 37 ℃ of slow yawing 2h of shaking table in confining liquid of shaking.2, in conjunction with primary antibodie: after sealing, the fine film of nitre is moved into to primary antibodie (1:1000), 4 ℃ 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, cleans remaining primary antibodie on film.3, anti-in conjunction with two: by the fine film immigration horse anti-reaction solution of anti-rabbit two of nitre (1:2000), after room temperature yawing 2h, nitre fibre film immerses in TTBS30ml, suddenly shakes 15min, continuous 3 times, cleans remnants two on film and resists.4, the detection of immunocomplex: GE ImageQuant LAS4000 chemoluminescence imaging analysis instrument develops and takes pictures.Figure 14 is the expression of results that the hair follicle after the VEGF165 genetic modification is done thin VEGF165 albumen.
The preparation of embodiment 4 compound artificial skin samples
1) preparation of sample: the gelfoam support of 4 ℃ of preservations before getting, use 75% ethanol disinfection, then use the PBS cleaning down; Be laid in the 50mm culture dish, be placed in 37 ℃ of CO
2hatch 1h in incubator; First by the hair follicle stem cells after transfection 72h with after 0.25%Trypsin-0.02%EDTA digestion, obtained cell suspension, the centrifugal 5min of 500r/min, abandon 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 to the gelfoam internal stent.The hair follicle stem cells of preserving before getting again, equally with 1 * 10
6, 5 * 10
6, 1 * 10
7/ cm
2density hair follicle stem cells is inoculated in to the gelfoam rack surface, the DMEM/F12 supplemental medium is cultivated, and is placed in 5% CO
2the interior 37 ℃ of submerged cultivation of incubator 2 weeks, change liquid-vapo(u)r interface into and cultivate 10d after cytogamy.
2) detection of compound artificial skin sample: after submerged cultivation 48h, observation of cell growing state under inverted microscope.Later every 24h observes once.The compound artificial skin prepared is fixed with 4%PFA solution, carries out conventional dehydration, embedding, section, and HE dyeing, observe under light microscopic.The HE that is respectively compound artificial skin as Figure 15～Figure 17 dyes, and is 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 is transplanted artificial skin
1) preparation of transplantation experiments animal model and experiment grouping in body:
18 of healthy male SPF level Sprague-Dawley rats, body weight 200-220g.Preoperative 2d sloughs art district dorsal body setae.
The experiment grouping: 4 surface of a wound are made in every rat back center, be divided at random 4 groups: A group (compound artificial skin of the hair follicle stem cells of injection of VEGF 165 genetic modifications), B group (the injection space base is because of the compound artificial skin of the hair follicle stem cells of 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 is weighed, and 1% vetanarcol intraperitoneal injection of anesthesia, after fixing limbs, the iodophor disinfection skin of back, 4 of the full thickness dermal wounds of 1.2cm * 1.2cm are done in both sides, design back tail side center, and methylene blue mark skin is wiped out scope, every side 2 ，Mei block gap, Ge Shu district 1cm.Cut skin with the sharp knife sheet along mark line, be deep to subcutaneous fascia superficialis layer, excision holostrome skin, the surface of a wound of formation exposed fascia.Fully after the hemostasis, be divided at random four groups afterwards, A, B, C group above-mentioned materials are implanted respectively to Wound Defect, 5 one 0 wire discontinuous sewings are fixed in edge of wound skin, and petrolatum gauze and aseptic dressing cover for the D group, and packing is fixing.(damage affected part for preventing that rat oneself stings, pollute and destroy, the protection of design flexibility overcoat.
3) post-transplantation is processed: after laboratory animal anesthesia is revived, send back between raising, single cage is raised.Recover normal diet next day, avoid art district gauze to soak, pollute as far as possible.Once outer dressing soaks, with sterile gauze, change immediately.Every day is anti-infective with the benzylpenicillin sodium for injection injection.
4) tissue sample collection: respectively at postoperative 7d, 14d, 21d gets 6 sacrifice of animal at every turn, takes pictures and observes the wound healing situation with the common camera W570 of Sony, as Figure 20-31.Calculate the Wound healing rate of the different groups of different time.As Figure 32.
5) after 21d, the tissue of drawing materials is done to HE dyeing, observe the vascularization situation.As Figure 33-35.
Sequence table
<110 > Hangzhou Xiaoshan Traditional Chinese Medical Hospital
<120>
hair follicle stem cells of VEGF165 genetic modification and preparation method thereof
<160>4
<210>?1
<211>?20
<212>?DNA
<213 > synthetic
<400>?1
GTCCCTCACC?CTCCCAAAAG?20
<210>?2
<211>?21
<212>?DNA
<213 > synthetic
<400>?2
GCTGCCTCAA?CACCTCAACC?C?21
<210>?3
<211>?20
<212>?DNA
<213 > synthetic
<400>?3
CACCCACCCA?CATACATACA?20
<210>?4
<211>?19
<212>?DNA
<213 > synthetic
<400>?4
CTCCCAACTC?AAGTCCACA?19
Claims (2)
1.VEGF165 the method for genetic modification hair follicle stem cells, is characterized in that the method comprises the following steps:
1) select SD rat antenna section skin in one week age, insert 37 ℃ of digestion 2h of 0.25%Dispase enzyme; Pull out hair follicle with tweezers and disposable syringe syringe needle from the subcutis end, collect the intact and hair follicle in vegetative period of form, under microscope, hair follicle is cut into to three equal parts, get middle portion, put into the 50mL culturing bottle after the PBS rinsing, add the DMEM/F12 supplemental medium, be placed in 37 ℃, 5%CO
2in incubator, every 2d changes liquid once; Described DMEM/F12 supplemental medium composition is: 44mlDMEM/F12 nutrient solution, 5mlKSR serum substitute, 500 μ l mycillin mixed solutions, 500 μ l L-glutaminate, 500 μ l non-essential 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;
2) purifying of hair follicle stem cells: the amount by the IV Collagen Type VI of 100 μ g/ml according to 3ml/100mm dish is coated in culture dish, the standing 1h of room temperature; By the primary cell tryptic digestion of 100mm culture dish, after centrifugal collecting cell, blow and beat into single cell suspension and be inoculated in culture dish, after 20min, by not adherent cell together with the nutrient solution sucking-off; The complete culture medium culturing of adherent cell, change liquid in every 3 days; P2 for repurity once;
3) evaluation of hair follicle stem cells:
A, employing Q-PCR method: the P3 after the sorting purifying carries out the Q-PCR detection for hair follicle stem cells;
B, immunofluorescent staining method: P3 is arrived to logarithmic phase for cell cultures, be seeded in after digestion on slide, after adherent culture 2d, sop up nutrient solution, use the PBST rinsing, after adding 4%PFA fixing, then seal by the 5%BSA room temperature; Add respectively the anti-mouse polyclonal antibody of primary antibodie integrin beta 1, integrin a6 polyclonal antibody and keratin 15 polyclonal antibody, incubated at room, after the PBST washing, the two anti-lucifuge 30min that label again, add DAPI and dye core 5min, and lucifuge is dried, with mounting solution mounting, observe;
4) packing slow virus: the 293T cell in the vegetative period of taking the logarithm, in advance 24h is inoculated in the 100mm culture dish, treats that cell grew to 50%-70% and got final product next day; Virus packing employing calcium turns method and carries out: before transfection, the 293T cell culture medium is replaced by containing two anti-substratum, comprises that 10%FBS+DMEM is high sugared; Then, purpose plasmid pLenti-IRES-VEGF165-EGFP10ug and 3 kinds of packaging plasmid VSVG, RSV-REV, each 5ug of RRE add in 50ulHBS liquid and mix gently, then supplement ddH
2o to 500 μ l, as B liquid, separately prepares 500 μ lCaCl
2a liquid, then add B liquid in A liquid, gets bubble, and room temperature is placed 2min; Dropwise add in Tissue Culture Dish, the Cross Water yawing shakes repeatedly; Wait to hatch and cultivate 10-12h, be replaced by nutrient solution for anti-containing the high sugar of 10%FBS+DMEM+1% pair; After 48h, cell occurs melting while being associated with strong green fluorescence expression, collects culture supernatant, with after 0.45 μ m aperture membrane filtration, with 4 ℃ of ultracentrifuges, the centrifugal 3h of 55000rpm/min, after removing supernatant, then add 100 μ l substratum piping and druming and be distributed into 2 pipes, preserve virus liquid for-80 ℃;
5) slow virus infection hair follicle stem cells: get the hair follicle stem cells of cultivation, 1 day in advance according to 1 * 10
5be seeded in 24 orifice plates of Yu Shop glue after the piping and druming that the hair follicle stem cells digestion in P3 generation that the concentration in/hole is good by growth conditions, centrifugal rear use 50 μ l virus stock solution useds and 50 μ l supplemental mediums mix, at 37 ℃, 5%CO
2the standing 30min of incubator, then add 400 μ l supplemental mediums and continue to cultivate, change liquid after 24h, observe green fluorescence under the fluorescence inverted microscope after 48h, 72h, obtain the hair follicle stem cells of VEGF165 genetic modification.
2. the hair follicle stem cells of the VEGF165 genetic modification that method according to claim 1 obtains.
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| CN105670987A (en) * | 2016-03-21 | 2016-06-15 | 杭州市萧山区中医院 | Inhibition method for induced differentiation of hair follicle stem cells into vascular endothelial cells |
| CN105754930A (en) * | 2016-03-21 | 2016-07-13 | 杭州市萧山区中医院 | Culture method for induced differentiation of hair follicle stem cells into vascular endothelial cells |
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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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