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Composite skin taking hair follicle stem cells as seed cell and construction method thereof

A technology of hair follicle stem cells and seed cells, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of insufficient skin source, unstable amplification factor, and low survival rate and other problems, to achieve strong proliferation, improve anti-infection ability, and promote proliferation

Inactive Publication Date: 2008-05-21
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the in vitro construction and transplantation of composite skin, researchers have found through long-term practice that the in vitro culture and expansion of epidermal cells have the following insurmountable defects: ①The culture conditions are harsh, the culture period is long, and the expansion factor is unstable
②Although in the early stage of culture, a part of epidermal cells still have the characteristics of epidermal stem cells, but after long-term culture and passage, epidermal cells gradually lose their proliferation potential and become terminal undifferentiated cells, which fall off and survive due to hyperkeratosis after transplantation low rate
③ For patients with extremely large area burns, the source of skin is seriously insufficient, and the source of epidermal cells is greatly limited
Therefore, the clinical application of composite skins containing autologous epidermal cells has been greatly limited.

Method used

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Embodiment 1

[0026] Embodiment 1 Construction of the composite skin of the present invention

[0027] Isolation and culture of hair follicle stem cells: take a small piece of scalp (2cm 2 ) was washed with PBS (containing penicillin 50U / ml, streptomycin 50μg / ml), carefully scraped off subcutaneous fat tissue, cut into 0.5cm×1.0cm size, placed in 5mg / ml Dispase II (Gibco company), digested at 4°C Overnight (14-16 hours), wash with serum-free DMEM, carefully pull out the hair, cut off the hair bulb, add 0.125% trypsin (Gibco company) to digest at 37°C for 10 minutes, pipette up and down, add DMEM containing 10% calf serum to stop digestion , filtered, centrifuged at 800 rpm for 8 min, washed twice in DMEM, diluted with complete DMEM culture medium and counted, according to 2.5×10 5 piece / cm 2 Cells were seeded in culture flasks at a density, placed in a 37°C incubator for culture with complete DMEM medium, and passaged and expanded when the cells reached 70% to 80% confluence.

[0028] Cl...

Embodiment 2

[0031] Embodiment 2 Construction of the composite skin of the present invention

[0032] When preparing composite skin, the hair follicle stem cells transfected by gene were divided into 1×10 5 piece / cm 2 Density inoculated on the surface of the acellular dermal substitute, cultured in complete DMEM medium at 37°C for 1 week, and changed the medium every 2 days to form a composite skin containing a single layer of hair follicle cells. All the other are with embodiment 1.

Embodiment 3

[0033] Embodiment 3 Construction of the composite skin of the present invention

[0034] When preparing composite skin, the hair follicle stem cells transfected by gene were divided into 1×10 5 piece / cm 2 Density seeding on the surface of the sponge-like collagen membrane dermal substitute, cultured in complete DMEM medium at 37°C for 2 weeks, changing the medium every 2 days, to form a composite skin containing stratified hair follicle stem cells. All the other are with embodiment 1.

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Abstract

The invention relates to the field of the medical wound surface restoration technology. The construction in vitro, which contains that composite skin of self epidermal cells can be used for transplantation, has become the hot point of the research on the field of the wound surface restoration; however, the culture of amplified epidermis cells in vitro has the defects that the culture conditions are stern; the epidermis source is seriously lacking, etc. The object of the invention is to supply composite skin which is constructed by taking hair follicle stem cells as seed cells, and a construction method thereof; the invention cultures the hair follicle stem cell in an isolating way, and the stem cell is inoculated on the surface of a dermis substitute through a plasmid vector transfection mutant Beta-catenin gene to form the composite skin after the culture cultivation in vitro. The invention can control the differentiation of the hair follicle stem cell and promote the multiplication of the hair follicle to multiple the stem cell in a short time; in this way, the stem cell can meet the requirement of application; moreover, the hair follicle stem cell has strong multiplication property which can promote the anti-infection capability after the transplantation of the composite skin.

Description

technical field [0001] The invention relates to the technical field of medical wound repair, in particular to a composite skin capable of repairing full-thickness skin defect wounds using hair follicle stem cells as seed cells and a construction method thereof. The hair follicle stem cells are transfected with a mutant β-catenin gene. Background technique [0002] In order to solve the problem of insufficient skin sources in patients with large-area deep burns, the construction of composite skins containing autologous epidermal cells in vitro for transplantation has become a research hotspot in the field of wound repair. The current conventional method for preparing composite skin is: take a small piece of skin specimen (2cm 2 ~4cm 2 ), isolated autologous epidermal cells, cultured and expanded in vitro, and then inoculated on the surface of dermal substitutes (such as acellular dermis, spongy collagen membrane, hyaluronic acid membrane, polylactic acid / polyglycolic acid me...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N5/10A61L27/60
Inventor 肖仕初夏照帆朱世辉杨晓妍田建广
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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