CN103468744A - VEGF165 gene modified hair follicle stem cells and preparation method thereof - Google Patents

Abstract

The present invention relates to VEGF165 gene-modified hair follicle stem cells and a preparation method thereof. The method comprises the following steps: 1) collecting hair follicles in good shape and in the anagen phase, cutting the hair follicles into three equal parts under a microscope, taking the middle part, and rinsing with PBS Put it into a 50mL culture flask and add DMEM/F12 supplement medium; 2) Purification of hair follicle stem cells; 3) Identification of hair follicle stem cells; 4) Packaging lentivirus; 5) Infection of hair follicle stem cells with lentivirus; 6) VEGF165 gene modification obtained Hair follicle stem cells. In the present invention, hair follicle stem cells are used as seed cells, which can rapidly expand in large quantities during in vitro culture, and the source is very rich, reducing immune rejection; at the same time, the hair follicle stem cells are modified by VEGF165 gene transfection, and new engineering with expansion and contraction functions can be formed. The vascular system will well solve the problem of easy necrosis and low survival rate of transplanted artificial skin.

Description

Hair follicle stem cells modified by VEGF165 gene and preparation method thereof

technical field

The invention relates to the field of skin tissue engineering, in particular to a method for gene modification of hair follicle stem cells and the hair follicle stem cells obtained by the method. the

technical field

As the largest organ of the human body, the skin has multiple functions such as sensation, temperature regulation, secretion and excretion, and prevention of water evaporation. The most important function is to serve as a barrier between the human body and the external environment to maintain the stability of the internal environment. important parts of. With the development of social economy, especially the prosperity and development of my country's manufacturing and handicraft industry as the "world factory", various traumas involving skin defects, especially burns, crushing injuries, and cutting injuries, are also increasing. Among them, large-area skin defects caused by trauma often lead to very serious physical disabilities and even death. Currently, the standard treatment for skin defects is autologous skin transplantation. Due to its characteristics of no immune rejection and high survival rate, it is widely used clinically and has achieved good curative effect. However, the disadvantages of autologous skin transplantation are also very obvious. Because it is an autologous material, it itself is a re-injury to the patient, which greatly increases the pain of the patient, and there is a risk of complications such as infection of the donor site and non-healing. What's more serious is that for the above-mentioned large-area skin defects, the lack of enough autologous skin for transplantation often leads to difficulties in wound repair, which seriously affects the treatment process and even leads to death. the

For this reason, scientists have been trying to find an exogenous skin substitute. As early as 1500 BC, skin xenografts were used to temporarily cover skin defect wounds. With the establishment and development of tissue engineering, skin tissue engineering has risen rapidly and become a research hotspot in the past ten years. Tissue engineering is a discipline that applies the principles and methods of engineering and life sciences to study biological substitutes for rebuilding, maintaining or improving tissue functions. At present, substantial progress has been made in foreign research on the application of skin tissue engineering to construct artificial skin. Products such as Apligraf, OrCel, Suprathel, Biobrane, OASIS, Integra, and Lyphoder have been approved by the U.S. Drug and Food Administration (FDA). It has been applied in the treatment of clinical skin defects. the

However, although the above several artificial skin products are currently available for clinical selection, they have indeed promoted the clinical treatment of patients with large-scale skin defects. However, according to the problems we have encountered in clinical application for many years, combined with literature reports, we believe that there are still many problems in artificial skin transplantation, including "hard injuries" that may lead to the final failure of treatment: ① Due to the above artificial skin products None of them has the ability to generate blood vessels, so the transplanted artificial skin has no vascular system to supply nutrients, so that the artificial skin is prone to necrosis and the transplantation fails. ②All kinds of allogeneic cells contained in artificial skin products are likely to cause immune rejection. Clinically, transplanted skin often appears necrosis, shedding, serious complications such as systemic immune response, and may spread diseases. ③At present, all artificial skin products can only restore part of the anatomical structure and physiological functions of normal skin, but cannot regenerate skin appendage structures with important functions, such as blood vessels, hair, sweat glands, etc. the

Hair follicle stem cells are a type of stem cells that exist in the bulge of the outer root sheath of hair follicles, and have the characteristics of undifferentiation, self-renewal and strong proliferation in vitro. Hair follicle stem cells in in vitro culture studies exhibit high clonogenicity and high regenerative potential. Since hair follicle stem cells are derived from hair and can be obtained directly from the patient, the number is extremely abundant without any complications, and they are completely non-invasive to patients, so they have become the focus of skin tissue engineering research. Taylor et al. (Taylor G, 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.) found that hair follicle stem cells not only It can differentiate to form hair follicles, and also participates in the formation of epidermal tissue. The latest research results published by Stelios et al. prove that hair follicles contain a large number of stem cells, which are one of the most easily obtained sources of stem cells, and have successfully differentiated and developed hair follicle stem cells to generate new vascular systems. Vascular endothelial growth factor 165 (VEGF165) is one of the five subtypes of vascular endothelial growth factor. It has the strongest activity and the widest distribution range, and is the main subtype of VEGF in vivo. In recent years, research on angiogenesis gene therapy centered on VEGF165 has become a research hotspot at home and abroad. the

Contents of the invention

In order to solve many problems existing in artificial skin products that may affect the effect of clinical application, one object of the present invention is to provide a method for modifying hair follicle stem cells with VEGF165 gene, and another object of the present invention is to provide hair follicle stem cells modified with VEGF165 gene obtained by the above method . In the present invention, hair follicle stem cells are used as seed cells, which can rapidly expand in large quantities during in vitro culture, and the source is very rich, reducing immune rejection; at the same time, the hair follicle stem cells are modified by VEGF165 gene transfection, and new engineering with expansion and contraction functions can be formed. The vascular system will well solve the problem of easy necrosis and low survival rate of transplanted artificial skin. the

In order to achieve the above-mentioned first purpose, the present invention adopts the following technical solutions:

VEGF165 gene modification method for hair follicle stem cells, the method comprises the following steps:

1) Select the skin of the tentacles of one-week-old SD rats, put 0.25% Dispase enzyme in it to digest at 37°C for 2 hours; pull out the hair follicles from the end of the subcutaneous tissue with tweezers and disposable syringe needles, collect the hair follicles with good shape and in the anagen period, and examine them under a microscope Cut the hair follicle into three equal parts, take the middle part, rinse with PBS, put it into a 50mL culture bottle, add DMEM/F12 supplementary medium, place it in a 37°C, 5% CO ₂ incubator, and change the medium every 2 days; The above DMEM/F12 supplemented medium components are: 44ml DMEM/F12 culture medium, 5ml KSR serum substitute, 500μl penicillin-streptomycin mixed solution, 500μl L-glutamine, 500μl non-essential amino acids, 20ng/ml recombinant human epidermal cell growth Factor, 10ng/ml recombinant human basic fibroblast growth factor, 50μl hydroxyethanol, 10ng/ml hydrocortisone;

2) Purification of hair follicle stem cells: Coat 100μg/ml type IV collagen in a petri dish at an amount of 3ml/100mm dish, and let stand at room temperature for 1h; trypsinize the primary cells in a 100mm petri dish, and collect the cells by centrifugation After that, pipette into a single cell suspension and inoculate it in a culture dish. After 20 minutes, suck out the unattached cells together with the culture medium; culture the adherent cells with complete medium, and change the medium every 3 days; P2 generation is purified again once;

3) Identification of hair follicle stem cells:

a. Using Q-PCR method: after sorting and purifying the P3 hair follicle stem cells, conduct Q-PCR detection, and use the △△Ct method to perform relative quantification of the expression of each gene;

b. Cell immunofluorescence staining method: culture P3 generation cells to the logarithmic growth phase, inoculate them on glass slides after digestion, and after 2 days of adherent culture, suck off the culture medium, rinse with PBST, add 4% PFA to fix, and then use 5% BSA at room temperature to block. Add the primary anti-integrin β1 anti-mouse polyclonal antibody, integrin a6 polyclonal antibody and keratin 15 polyclonal antibody, incubate at room temperature, wash with PBST, add the labeled secondary antibody for 30 minutes, and add DAPI to stain the nucleus for 5 minutes. Light-dried, sealed with mounting solution, observed;

4) Packaging lentivirus: Take 293T cells in the logarithmic growth phase and inoculate them in a 100mm culture dish 24 hours in advance, and wait until the cells grow to 50%-70% the next day; the virus packaging is carried out by calcium transfer method: before transfection, the Replace the 293T cell culture medium with a medium without double antibodies, including 10% FBS+DMEM high glucose; then, add 5ug of the target plasmid pLenti-IRES-VEGF165-EGFP10ug and the three packaging plasmids VSVG, RSV-REV, and RRE to 50ulHBS Gently mix in the solution, then add ddH ₂ O to 500 μl as solution B, and prepare 500 μl CaCl ₂ A solution, then add B solution to A solution, make bubbles, and stand at room temperature for 2 minutes; add dropwise to the cell culture dish, cross Shake horizontally several times; after incubation for 10-12 hours, replace the culture medium with 10% FBS + DMEM high glucose + 1% double antibody; after 48 hours, when the cells appear confluent and express strong green fluorescence, collect the culture supernatant and use After filtering with a 0.45 μm pore size filter membrane, centrifuge at 4°C and 55,000 rpm/min for 3 hours in an ultracentrifuge, remove the supernatant, then add 100 μl of medium by pipetting and aliquot into 2 tubes, and store the virus solution at -80°C;

5) Infection of hair follicle stem cells with lentivirus: take the cultured hair follicle stem cells, digest the P3 generation hair follicle stem cells in good growth state at a concentration of 1×10 ⁵ /well 1 day in advance, centrifuge and mix with 50μl virus stock solution and 50μl supplementary medium After uniform pipetting, inoculate on the pre-coated 24-well plate, let it stand in a 37°C, 5% CO2 incubator for 30 minutes, then add 400 μl supplementary medium to continue the cultivation, change the medium after 24 hours, and under the fluorescent inverted microscope after 48 hours and 72 hours Observe the green fluorescence and obtain VEGF165 gene-modified hair follicle stem cells;

6) Obtained VEGF165 gene modified hair follicle stem cells. The proliferation ability of the cells, the expression of VEGF165mRNA by RT-PCR and the expression of VEGF165 protein by Western-Blot were detected respectively. the

In order to realize the above-mentioned second purpose, the present invention adopts the following technical solutions:

VEGF165 gene modifies hair follicle stem cells, and the hair follicle stem cells are obtained by the above-mentioned method. the

Due to the adoption of the above-mentioned technical scheme, the present invention uses hair follicle stem cells as seed cells and uses VEGF165 gene transfection to obtain hair follicle stem cells. The hair follicle stem cells can be planted on a three-dimensional gelatin sponge tissue scaffold to form a new composite with a new vascular system. Artificial skin model. This model has the following unique advantages:

①As stem cells, hair follicle stem cells have the characteristics of rapid and massive expansion during in vitro culture, long-term subculture in vitro, strong cell functions, etc., and have high regeneration and differentiation capabilities, which will greatly shorten the time for in vitro culture and expansion, and improve clinical outcomes. treatment efficiency;

②Because it can form a new engineered vascular system with expansion and contraction functions, it will well solve the problem of easy necrosis and low survival rate of transplanted artificial skin;

③The seed cells used in this study—hair follicle stem cells are taken from autologous hair, which are very abundant and easy to obtain, and will not cause any damage or pain to patients;

④Since the seed cells are taken from autologous tissue, problems such as immune rejection and disease transmission will be solved;

⑤ Compared with the current artificial skin products, this new type of artificial skin will restore and regenerate the anatomical structure and physiological functions of normal skin tissue to a greater extent. the

Description of drawings

Figure 1 and Figure 2 show the primary hair follicle stem cells crawling out from the hair follicle bulge, showing bird's nest-like, epithelial-like cells, arranged tightly 40×. the

Figure 3 is the P3 generation hair follicle stem cells after screening and purification of type IV collagen, showing a typical paving stone shape 100×. the

Table 1 shows the Q-PCR detection of the sorted and purified P3 hair follicle stem cells, and the relative quantification of the expression of each gene by the ΔΔCt method. the

Figure 4-Figure 6 shows the immunofluorescence staining of P3 generation hair follicle stem cells, respectively for integrin β1, integrin a6, and keratin 15,100×. the

Figures 7-9 are scanning electron microscopes showing the three-dimensional scaffold of gelatin sponge sprayed with gold, respectively SEM50×, 200×, and 400×. the

Figure 10 and Figure 11 are the GFP and pH images of the P3 hair follicle stem cells infected with lentivirus for 72 hours under an inverted fluorescence microscope, 100×. the

Fig. 12 is a growth curve of hair follicle stem cells after VEGF165 gene modification. the

Fig. 13 is a graph showing the RT-PCR results of the mRNA of the hair follicle stem after VEGF165 gene modification. the

Fig. 14 is the expression result of VEGF165 protein in hair follicle stem cells after VEGF165 gene modification. the

Figures 15 to 17 are the HE staining of the composite artificial skin, respectively, the injection concentrations are 1×106, 5×106, 1×107/cm2, bar: 200×. the

Figures 18-19 are pictures of wounds during back transplantation in rats. the

Figure 20-23 shows the healing of four wounds after 7 days of transplantation. The order is VEGF165 group, empty plasmid group, cell-free group and gauze group. the

Figure 24-27 shows the healing of four wounds after 14d transplantation. The order is VEGF165 group, empty plasmid group, cell-free group and gauze group. the

Figure 28-31 shows the healing of four wounds after 21d transplantation. The order is VEGF165 group, empty plasmid group, cell-free group and gauze group. the

Figure 32 is the wound healing rates of the four groups of 7d, 14d, and 21d. the

Figures 33-35 are the HE staining of the collected tissues after 21 days to observe the formation of blood vessels. The sequence is VEGF165 group, empty plasmid group and cell-free group. 100×

Detailed ways

The separation of embodiment 1 rat hair follicle stem cells, culture, identification

1.1) Select the vibrissae skin of one-week-old SD rats, put in 0.25% Dispase enzyme and digest at 37°C for 2 hours; use tweezers and disposable syringe needles to pull out the hair follicles from the end of the subcutaneous tissue, and collect the hair follicles with good shape and in the growth phase, and examine them under the microscope Cut the hair follicle into three equal parts, take the middle part, rinse with PBS, put it into a 50mL culture bottle, add DMEM/F12 supplementary medium, place it in a 37°C, 5% CO ₂ incubator, and change the medium every 2 days; The above DMEM/F12 supplemented medium components are: 44ml DMEM/F12 culture medium, 5ml KSR serum substitute, 500μl penicillin-streptomycin mixed solution, 500μl L-glutamine, 500μl non-essential amino acids, 20ng/ml recombinant human epidermal cell growth Factors, 10ng/ml recombinant human basic fibroblast growth factor, 50μl hydroxyethanol, 10ng/ml hydrocortisone. Figure 1 and Figure 2 show the primary hair follicle stem cells crawling out from the hair follicle bulge, showing bird's nest-like, epithelial-like cells, closely arranged.

1.2) Purification of hair follicle stem cells: Coat 100μg/ml type IV collagen in a petri dish at an amount of 3ml/100mm dish, and let stand at room temperature for 1h; trypsinize the primary cells in a 100mm petri dish, and collect the cells by centrifugation After that, pipette into a single cell suspension and inoculate it in a culture dish. After 20 minutes, suck out the unattached cells together with the culture medium; culture the adherent cells with complete medium, and change the medium every 3 days; P2 generation is purified again once. Figure 3 shows the P3 generation of hair follicle stem cells after screening and purification of type IV collagen, showing a typical paving stone shape. the

1.3) Identification of hair follicle stem cells:

a. Using the Q-PCR method: the sorted and purified P3 hair follicle stem cells were detected by Q-PCR, and the relative quantification of the expression of each gene was carried out by the △△Ct method. as table 1

b. Cell immunofluorescence staining method: culture P3 generation cells to the logarithmic growth phase, inoculate them on glass slides after digestion, and after 2 days of adherent culture, suck off the culture medium, rinse with PBST, add 4% PFA to fix, and then use 5% BSA at room temperature to block. Add primary anti-integrin-β1 (integrin-β1) anti-mouse polyclonal antibody (1:100), integrin a6 (integrin-a6) polyclonal antibody (1:50) and keratin-15 (keratin-15) polyclonal antibody Antibody (1:100), incubate at room temperature, wash with PBST, add labeled secondary antibody for 30 minutes in the dark, add DAPI (1:2000) to stain the nuclei for 5 minutes, dry in the dark, and seal with mounting solution. Figure 4-Figure 6 shows the immunofluorescent staining of P3 generation hair follicle stem cells, including integrin β1, integrin a6, and keratin 15, respectively. the

The preparation of embodiment 2 gelatin sponge three-dimensional tissue support

2.1) Preparation of gelatin sponge three-dimensional tissue scaffold:

A. Dissolve 5% gelatin in 10ml of distilled water at 25°C, add 0.05% 6-chondroitin sulfate sodium salt (C6S) and 0.2% hyaluronic acid sodium salt (HA) respectively;

B. After stirring with a magnetic stirrer at room temperature for 60 minutes, the cross-linking agent 0.5% 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution (EDC) and 0.25% N -N-Hydroxysuccinimide solution (N-Hydroxysuccinimide) was dropped into the solution and mixed for 5 minutes, and then the solution was injected into the mold of a 12-hole plate, and the solution was shaken evenly. the

C. Freeze at -80°C for 2 hours

D. Then put on the freeze dryer, and freeze dry for 24 hours to obtain a porous sponge-like Gel-C6S-HA scaffold with a thickness of 2 mm. the

2.2) Observation: Take a small amount of stent samples, observe and record them with a scanning electron microscope. Figures 7-9 are scanning electron microscopes showing three-dimensional stents of gelatin sponge sprayed with gold, respectively SEM50×, 200×, 400×. 

Example 3 Preparation of Hair Follicle Stem Cells Modified by Vascular Endothelial Growth Factor 165 Gene (VEGF165)

3.1 Packaging lentivirus: Take 293T cells in the logarithmic growth phase and inoculate them in a 100mm culture dish 24 hours in advance, and wait until the cells grow to 50%-70% the next day; the virus packaging is carried out by calcium transfer method: before transfection, 293T Replace the cell culture medium with a double antibody-free medium, including 10% FBS+DMEM high glucose; then, add 5ug of the target plasmid pLenti-IRES-VEGF165-EGFP10ug and the three packaging plasmids VSVG, RSV-REV, and RRE to 50ulHBS solution Gently mix in medium, then add ddH ₂ O to 500μl as B solution, and prepare 500μl CaCl ₂ A solution, then add B solution to A solution, make bubbles, and place at room temperature for 2min; add dropwise to the cell culture dish, cross level Shake several times; after incubation for 10-12 hours, replace the culture medium with 10% FBS + DMEM high glucose + 1% double antibody; after 48 hours, when the cells appear confluent and have strong green fluorescence expression, collect the culture supernatant and use 0.45 After filtering with a filter membrane with a pore size of μm, centrifuge at 4°C and 55,000rpm/min for 3h in an ultracentrifuge, remove the supernatant, then add 100μl of medium by pipetting and aliquot into 2 tubes, and store the virus solution at -80°C.

3.2 Infection of hair follicle stem cells with lentivirus: take the cultured hair follicle stem cells, digest the hair follicle stem cells of P3 generation in good growth state at a concentration of 1×10 ⁵ /well 1 day in advance, centrifuge and mix with 50 μl virus stock solution and 50 μl supplementary medium After pipetting, inoculate the 24-well plate with pre-coated gel, and let it stand in the incubator at 37°C and 5% CO ₂ for 30 minutes, then add 400 μl of supplementary medium to continue the cultivation, change the medium after 24 hours, and under the fluorescent inverted microscope after 48 hours and 72 hours Observing the green fluorescence, the VEGF165 gene modified hair follicle stem cells were obtained. Figure 10 and Figure 11 are the GFP image and pH image of P3 hair follicle stem cells infected with lentivirus for 72 hours under an inverted fluorescence microscope, respectively.

3.3 Growth curve measurement: P3 generation hair follicle stem cells infected for 72 hours were digested and inoculated on a 24-well plate at a cell concentration of 1×10 ⁵ /ml for 1, 2, 3, 4, 5, 6, and 7 days respectively. For cell counting plate counting, 6 multiple wells were set up every day,

Take the average. Cell growth curves were then drawn. Figure 12 is a growth curve of hair follicle stem cells after VEGF165 gene modification. the

3.4 Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of VEGF165mRNA in the hair follicle stem cells after transformation. the

(1) Extraction of total RNA: Place the six-well culture plate inoculated with hair follicle stem cells on ice, absorb the culture medium; add 1ml of pre-cooled Trizol reagent to each well, repeatedly pipette and lyse and transfer to EP tube; add 0.2ml Chloroform, vigorously oscillate and mix well, then place for 2-3min; centrifuge at 12000g, 4°C for 15min; transfer the supernatant to another EP tube, about 0.5ml, add an equal volume of isopropanol, mix by inverting, let stand for 10min, and centrifuge (12000g, 4℃) for 10min, discard the supernatant; wash the precipitate with 0.5ml DEPC-treated 75% alcohol and 1ml, centrifuge (7500g, 4℃) for 5min; pour off the alcohol, invert the EP tube, and dry until the alcohol evaporates; DEPC-treated water was frozen at -70°C for later use. the

(2) Reverse transcription system of hair follicle stem cell RNA:

(3) Polymerase chain reaction (PCR)

a) PCR primers VEGF165 protein primer design, Action as a control. Such as Table1.

Table 1. Primer sequence information

(Table1. Oligonucleotide sequences for PCR amplifycatio)

b) PCR reaction conditions, such as Table2.

Table 2. PCR process

(Table2.The process of PCR)

c) PCR reaction system

cDNA 5.0μl target mRNA 1.0μl each Actin upstream and downstream primers 1.0μl each 2×Tag enzyme 12.5μl

[0073] sterile triple distilled water 5.5μl the 25.0μl

(4) Agarose electrophoresis: Take 6 μl of the amplified product and load it on a 1.5% agarose gel. After electrophoresis on a 1.5% agarose gel, take pictures under a transmission ultraviolet light analyzer, and scan with a laser densitometric image scanner. Figure 13 is a graph showing the RT-PCR results of the mRNA of the hair follicle stem after the VEGF165 gene modification. the

3.5 Western blotting method to detect the expression of VEGF165 protein in hair follicle stem cells

(1) Lysis of hair follicle stem cells

a) Wash the hair follicle stem cells 3 times with pre-cooled PBS. the

b) Add pre-cooled lysing buffer (lysis buffer is a buffer often used in biological experiments. The purpose is to lyse cells), 6-well plate: 80 μl/well; 60mm culture dish: 300 μl/disk, incubate at 4°C for 20 minutes. the

c) Concentrate the cell fragments together with the lysing buffer on one side with a cell scraper, incubate at 4°C for 40 min and transfer to a pre-cooled centrifuge tube. Centrifuge (12000g, 4°C) for 5min. the

d) Transfer the supernatant into another centrifuge tube, except for a part used to measure the protein concentration, the rest can be directly used for experiments or frozen at -70°C for future use. the

(2) Polyacrylamide gel electrophoresis

a) Preparation of 8% separating gel: 1.5mM Tris-HCl (pH8.8) 1.3ml, dddH ₂ O 2.3ml, 30% polyacrylamide 1.3ml, 10% SDS 50μl, 10% ammonium persulfate 50μl, TEMED 3μl (after adding Mix well, make glue quickly). 3ml of the above mixed solution was quickly injected into the gap between the two layers of glass plates, and then a small amount of triple-distilled water was used to seal the rubber surface, polymerized at room temperature for 30 minutes, and the upper layer of triple-distilled water was absorbed. b) Prepare 5% stacking gel: 0.25ml of 1mM Tris-HCl (pH6.8), 1.4ml of ddH2O.

c) 0.33ml of 30% polyacrylamide, 20μl of 10% SDS, 20μl of 10% ammonium persulfate, and 2μl of TEMED (mix well after adding to quickly make gel). Quickly inject 1.4ml of the above mixture into the gap between the two glass plates, insert the comb quickly, and take out the comb carefully after the polymerization is complete. the

d) Electrophoresis: Dilute the sample containing the same amount of protein to the same volume, and mix it with 5×sample buffer at a volume of 4:1,

Water bath at 100°C for 5 minutes, add samples while heating, 30 μl per well, after adding samples, place the gel in an electrophoresis tank filled with electrophoresis buffer for electrophoresis. Electrophoresis conditions: 95V×15min in the stacking gel, 165V×50min in the separating gel, when the bromophenol blue in the sample migrates to the front of the gel, the electrophoresis ends. the

e) Electrotransfer: After SDS-PAGE, unload the polyacrylamide gel, take 6 pieces of filter paper and a piece of nitrocellulose membrane (NC) of the same size as the gel, equilibrate in semi-dry transfer buffer for 15min, and sequentially transfer from Put 3 pieces of filter paper, gel, NC membrane, and another 3 pieces of filter paper from bottom to top, and then sandwich them in a semi-dry transfer membrane device, with the membrane facing the positive electrode and the glue facing the negative electrode. Flow, transfer membrane 90min. the

f) Detection of protein on NC membrane: Dip the membrane in ponceau staining solution for staining, and a red protein band appears in about 3 minutes, then immerse the membrane in distilled water for decolorization, shake gently for a few minutes, and then replace the decolorization solution Several times until the background color is very light. g) Immunological detection: 1. Blocking: After transferring the membrane, put the nitrocellulose membrane in TTBS, shake slowly for 10 minutes, and then shake the membrane slowly in the blocking solution for 2 hours at 37° C. on a shaker. 2. Conjugate the primary antibody: After blocking, transfer the nitrocellulose membrane into the primary antibody (1:1000), shake overnight at 4°C or at room temperature for 3 hours. To recover the primary antibody, immerse the nitrocellulose membrane in 20ml of TTBS, shake vigorously for 10min, 3 times in a row, and wash the residual primary antibody on the membrane. 3. Conjugated secondary antibody: transfer the nitrocellulose membrane into the horse anti-rabbit secondary antibody reaction solution (1:2000), shake it at room temperature for 2 hours, then immerse the nitrocellulose membrane in TTBS30ml, shake it rapidly for 15 minutes, continuously for 3 times, and wash the remaining residue on the membrane. Secondary Antibodies. 4. Detection of immune complexes: develop and take pictures with GE ImageQuant LAS4000 Chemiluminescence Imaging Analyzer. Fig. 14 is the expression result of VEGF165 protein in hair follicle stem cells after VEGF165 gene modification. the

The preparation of embodiment 4 composite artificial skin samples

1) Sample preparation: take the gelatin sponge scaffold stored at 4°C, disinfect it with 75% ethanol, and then rinse it thoroughly with PBS; spread it on a 50mm petri dish, and incubate it in a CO ₂ incubator at 37°C for 1 hour; After 72 hours of transfection, the hair follicle stem cells were digested with 0.25% Trypsin-0.02% EDTA, the cell suspension was taken, centrifuged at 500r/min for 5min, the supernatant was discarded, and the cells were resuspended with 5ml DMEM/F12 solution, and injected at 1× Cells were seeded inside the gelatin sponge scaffold at a density of 10 ⁶ , 5×10 ⁶ , and 1×10 ⁷ /cm ² . Then take the hair follicle stem cells that were preserved before, and inoculate the hair follicle stem cells on the surface of the gelatin sponge scaffold at the same density of 1×10 ⁶ , 5×10 ⁶ , and 1×10 ⁷ /cm ² . % CO ₂ incubator for 2 weeks at 37°C under the liquid surface, and then changed to air-liquid interface culture for 10 days after cell fusion.

2) Detection of the composite artificial skin samples: After cultured under the liquid surface for 48 hours, the cell growth was observed under an inverted microscope. After that, observe once every 24 hours. The prepared composite artificial skin was fixed with 4% PFA solution, followed by routine dehydration, embedding, sectioning, HE staining, and observation under a light microscope. Figures 15 to 17 show the HE staining of the composite artificial skin, with injection concentrations of 1×10 ⁶ , 5×10 ⁶ , and 1×10 ⁷ /cm ² , bar: 200×.

Example 5 Humanoid Rat Skin Defect Model Transplanted with Artificial Skin

1) In vivo transplantation experimental animal model preparation and experimental grouping:

There were 18 healthy male SPF grade Sprague-Dawley rats, weighing 200-220g. The back hair of the operation area was removed 2 days before the operation. the

Experimental grouping: 4 wounds were made in the middle of the back of each rat, and they were randomly divided into 4 groups: Group A (composite artificial skin injected with VEGF165 gene-modified hair follicle stem cells), group B (composite artificial skin injected with empty gene-modified hair follicle stem cells). ), group C (artificial skin without cell injection) and group D (wound covering gauze). The injection concentration of groups A\B was 1×10 ⁷ /cm ² . As shown in Figure 18-19.

2) Rats were weighed before the operation, anesthetized by intraperitoneal injection of 1% pentobarbital sodium, and after the limbs were fixed, the skin on the back was disinfected with povidone iodine, and four full-thickness skin defect wounds of 1.2 cm × 1.2 cm were designed on both sides of the middle of the caudal side of the back , methylene blue marks the skin excision range, 2 operation areas on each side, each with an interval of 1cm. Cut the skin along the marked line with a sharp blade, go deep to the superficial subcutaneous fascia layer, and excise the whole thickness of the skin to form a wound that exposes the fascia. After adequate hemostasis, they were randomly divided into four groups. The above materials were implanted into the defect wounds in groups A, B, and C, and fixed on the wound edge skin with 5-0 silk sutures. Group D was covered with Vaseline gauze and sterile dressings. Package fixed. (In order to prevent rats from biting the affected area by themselves, causing pollution and damage, an elastic jacket is designed for protection. 

3) Treatment after transplantation: After the experimental animals wake up from anesthesia, they are sent back to the breeding room and raised in single cages. Return to normal diet the next day, and try to avoid soaking and polluting the gauze in the operation area. Once the outer dressing is soaked, change it with sterile gauze. Anti-infection was injected with penicillin sodium for injection every day. the

4) Collection of tissue samples: 7 days, 14 days, and 21 days after the operation, 6 animals were sacrificed each time, and a normal Sony camera W570 was used to take pictures to observe the wound healing, as shown in Figure 20-31. The wound healing rates of different groups at different times were calculated. Figure 32. the

5) After 21 days, HE staining was done on the collected tissues to observe the formation of blood vessels. As shown in Figure 33-35. the

sequence listing

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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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