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Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells

A technology of hair follicle stem cells and artificial skin, which is applied in the field of artificial skin and its preparation, can solve the problems of easy necrosis and low survival rate of transplanted artificial skin, achieve the effect of restoring and regenerating anatomical structure and physiological function, rich in sources, and easy to obtain

Active Publication Date: 2013-12-25
HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
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Problems solved by technology

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 transfection of VEGF165 gene, 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

Method used

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  • Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells
  • Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells
  • Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 Isolation, cultivation and identification of rat hair follicle stem cells

[0046] 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, and place at 37°C, 5%C0 2 In the incubator, the medium was changed every 2 days; the DMEM / F12 supplement medium composition was: 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 growth factor, 10ng / ml recombinant human basic fibroblast growth factor,...

Embodiment 2

[0053] Example 2 Preparation of Gelatin Sponge Three-dimensional Tissue Scaffold

[0054] 2.1) Preparation:

[0055] 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;

[0056] 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.

[0057] C. Freeze at -80°C for 2 hours

[0058] 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.

[0059] 2.2) Observation: Take a small amount of stent samples, observe and record with a scanning ele...

Embodiment 3

[0060] Example 3 Preparation of hair follicle stem cells modified with vascular endothelial growth factor 165 gene (VEGF165)

[0061] 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 0 to 500 μl as solution B, prepare another 500 μl of CaCl 2 Add solution A, then add solution B to solution A, make air bubbles, and place at room temperature for 2 minutes; add dropwise to the cell culture dish, shake horizontally for several times; after incubation for 10-12 hours, replac...

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Abstract

The invention relates to a preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells. The preparation method comprises following step: 1) hair follicle stem cells, which possess excellent cellular morphology and are in growing period, are collected; 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; 6) gelatin sponge three-dimensional tissue scaffolds are prepared; 7) the gelatin sponge three-dimensional tissue scaffolds which are preserved at a temperature of 4 DEG C are taken, and the modified hair follicle stem cells are inoculated in the gelatin sponge three-dimensional tissue scaffolds or on the surfaces of the gelatin sponge three-dimensional tissue scaffolds by injection so as to obtain the artificial skin. According to the preparation method, the VEGF165 gene modified hair follicle stem cells are taken as the 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 [0001] The invention relates to the field of skin tissue engineering, in particular to an artificial skin and a preparation method thereof. Background technique [0002] 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 def...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61L27/38A61L27/60
Inventor 全仁夫黄忠名郑宣许世超
Owner HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
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