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Skin substitute for automatically capturing endothelial progenitor cells and promoting vascularization and construction method thereof

A technology of automatic capture of endothelial progenitor cells, applied in the field of new skin substitutes, can solve the problems of low survival rate, poor EPC adhesion, stem cell differentiation, etc., and achieve the effect of improving chemotaxis induction, improving safety, and accelerating vascularization

Inactive Publication Date: 2011-03-16
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

[0004] However, due to the immature method of isolating and culturing autologous EPC, the application of EPC is greatly limited.
Even if a sufficient amount of EPC can be cultured and amplified, it can be inoculated on the surface of the dermal scaffold for culture, and the EPC has poor adhesion and low survival rate, and it is easy to cause stem cell differentiation and lose angiogenesis ability after long-term culture (Hofmann NA, Reinisch A, Strunk D.Isolation and large scale expansion of adulthuman endothelial colony forming progenitor cells.J VisExp.2009;28(32):1524-29)
Therefore, there are various technical bottlenecks in promoting the vascularization of skin substitutes using the traditional methods of isolating, culturing, and transplanting EPCs.

Method used

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  • Skin substitute for automatically capturing endothelial progenitor cells and promoting vascularization and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1. Preparation of a new skin substitute that can automatically capture endothelial progenitor cells to promote vascularization

[0032] 1. Isolation and culture of human keratinocytes and fibroblasts:

[0033] The discarded foreskin tissue after circumcision was used to separate and culture keratinocytes and fibroblasts by enzymatic digestion. Separately prepared into a single cell suspension, according to 2-3 × 10 5 Cells per ml were seeded in culture flasks and placed in a 37°C incubator. Keratinocytes were cultured in serum-free medium (DK-SFM, Gibco, USA), and fibroblasts were cultured in complete DMEM medium (Gibco, USA). USA) for culture, and when the cells reach 70%-80% confluence, passage and expansion are carried out.

[0034] 2. Construction of vectors containing SDF-1α gene expression

[0035] Sequence of SDF-1α gene:

[0036] ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAG...

Embodiment 2

[0046] Embodiment 2. Animal transplantation test of skin substitute of the present invention

[0047] Male Balb / c-nu mice (nude mice, provided by Shanghai Xipro-Bichem Experimental Animal Co., Ltd.), weighing 18±2 grams, 40 were divided into four groups. Skin substitutes containing transfected SDF-1α gene fibroblasts, skin substitutes containing transfected ends plus pcDNA3.1-9HRE-CMV-SDF-1α fibroblasts, fibroblasts containing transfected empty vector Skin substitutes of cells and normal fibroblasts that have not been transfected with the gene.

[0048] After the nude mice were anesthetized with ketamine intraperitoneally, the skin and hair on the back were shaved, and the full-thickness skin on the ventral side of the spine and the deep fascia up to the muscle layer were excised. The active skin substitutes cultured in vitro for 2-3 weeks as described in Example 1 were transplanted on the wound surface. In order to prevent periwound skin shrinkage and epidermis crawling, wh...

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Abstract

The invention relates to the technical field of tissue engineering and medical wound healing. The conventional skin substitute has low vascularization speed and low survival rate, and cannot be clinically popularized and applied after being grafted at present. The invention constructs a novel skin substitute capable of automatically capturing endothelial progenitor cells in peripheral blood and accelerating vascularization. A stroma cell derived factor 9HRE-CMV gene regulated by a hypoxic response element is used to transfect fibroblasts; epidermal cells and transgenic fibroblasts are implanted on both sides of a dermal scaffold respectively to form the sandwich skin substitute; and the skin substitute duly and moderately expresses and secretees SDF-1 alpha after being grafted, and endothelial progenitor cells (EPC) in the mediated peripheral blood are mediated to a skin substitute grafting place so as to promote the vascularization of the skin substitute and improve the survival rate after grafting. The skin substitute can obviously improve the chemotactic induction to the EPC and accelerates the vascularization of the skin substitute. The average survival rate of the skin substitute is 95 percent, while the average survival rate of the conventional skin substitute not containing the transgenic fibroblasts is 72 percent.

Description

technical field [0001] The invention relates to the technical fields of tissue engineering and medical wound repair, in particular to a novel skin substitute capable of automatically capturing peripheral blood endothelial progenitor cells and promoting vascularization, which can significantly improve the transplant survival rate of the skin substitute. Background technique [0002] After more than 20 years of development of skin tissue engineering, the culture and transplantation of simple epidermal cells, the development and application of dermal scaffolds have been relatively mature, and have been successfully applied in clinical practice. On this basis, the researchers planted the epidermal cells cultured in vitro on the surface of the dermal scaffold to construct a sandwich skin substitute containing the epidermal cell layer. Figure 1 One-time repair of defective epidermis and dermis. However, when the sandwich-type skin substitute is transplanted on the escharectomy wo...

Claims

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Application Information

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IPC IPC(8): A61L27/60C12N15/12C12N15/63C12N5/10
Inventor 肖仕初纪世召夏照帆朱世辉王光毅李恒宇王俊杰
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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