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

A hair follicle stem cell and artificial skin technology, applied in the field of artificial skin and its preparation, can solve the problems of low survival rate, easy necrosis of transplanted artificial skin, etc., and achieve the effects of abundant sources, restoration and regeneration of anatomical structures and physiological functions, and easy to obtain effects.

Active Publication Date: 2015-05-13
HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Isolation, culture and identification of rat hair follicle stem cells

[0046] 1.1) Select one-week-old SD rat tentacle skin, put 0.25% Dispase enzyme into it to digest at 37°C for 2 hours; use forceps and a disposable syringe needle to pull out the hair follicles from the end of the subcutaneous tissue, collect the hair follicles that are in good shape and in the growing phase, and look under the microscope. Cut the hair follicle into three equal parts, take the middle part, rinse with PBS, put it into a 50mL culture flask, add DMEM / F12 supplemented medium, and place it at 37°C, 5% CO. 2 In the incubator, the medium was changed every 2 days; the DMEM / F12 supplemented medium was composed of: 44ml DMEM / F12 medium, 5ml KSR serum substitute, 500μl penicillin-streptomycin mixture, 500μl L-glutamine, 500μl non- Essential amino acids, 20ng / ml recombinant human epidermal growth factor, 10ng / ml recombinant human basic fibroblast growth factor, 50 μl hydroxyethanol, 10...

Embodiment 2

[0053] Example 2 Preparation of gelatin sponge three-dimensional tissue scaffolds

[0054] 2.1) Preparation:

[0055] A. Take 5% gelatin and dissolve it in 10ml of distilled water at 25°C, add 0.05% chondroitin 6-sulfate sodium salt (C6S) and 0.2% hyaluronate sodium salt (HA) respectively;

[0056] B. After stirring with a magnetic stirrer for 60 minutes at room temperature, the crosslinker 0.5% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (EDC) and 0.25% N -Hydroxysuccinimide solution (N-Hydroxysuccinimide) was dropped into the solution and mixed for 5 minutes, then the solution was poured into the mold of the 12-well plate, and shaken horizontally evenly.

[0057] C. Freeze at -80°C for 2 hours

[0058] D. Then put on a freeze dryer, freeze-dried 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 scanning electron microscope...

Embodiment 3

[0060] Example 3 Preparation of hair follicle stem cells modified by 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 The cell culture medium was replaced with a medium without dual antibodies, including 10% FBS+DMEM high glucose; then, 50 ug of the target plasmid pLenti-IRES-VEGF165-EGFP and 5 ug of each of the three packaging plasmids VSVG, RSV-REV, and RRE were added to 50 ul of HBS solution Mix gently in medium, then add ddH 2 O to 500μl as B solution, prepare another 500μl CaCl 2 Solution A, then add solution B to solution A, blow out bubbles, and leave at room temperature for 2 minutes; add dropwise to the cell culture dish, shake horizontally for several times; after incubation for 10...

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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 various functions such as feeling, regulating body temperature, secretion and excretion, and preventing water evaporation. important parts of. With the development of society and economy, especially the prosperity and development of my country's manufacturing and handicraft industries as "world factories", various traumas involving skin defects, especially burns, crush injuries, and cutting injuries, are also increasing. Among them, large-scale skin defects caused by trauma often lead to very serious physical disability and even death. At present, the standard treatment method for skin defects in clinical practice is autologous skin transplantation. Due to the characteristics of no immune rejection and high survival rate, it is widely use...

Claims

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Application Information

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Patent Type & Authority Patents(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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