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FTO gene-modified porcine adipose-derived stem cells as well as construction method and application thereof

A technology of genetic modification and construction method, applied in the direction of genetically modified cells, cell dissociation methods, cells modified by introducing foreign genetic material, etc., can solve the problem of tissue engineering skin that has not yet been reported, and improve the function of porcine adipose stem cells. , Reducing inflammatory response, the effect of strong differentiation ability

Active Publication Date: 2021-08-27
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The applicant's previous research (Wu et al., 2018, BBA-MOL CELL BIOL L) found that the FTO gene can significantly promote cell proliferation and differentiation, but the construction of tissue engineered skin has not been reported

Method used

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  • FTO gene-modified porcine adipose-derived stem cells as well as construction method and application thereof
  • FTO gene-modified porcine adipose-derived stem cells as well as construction method and application thereof
  • FTO gene-modified porcine adipose-derived stem cells as well as construction method and application thereof

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Experimental program
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Embodiment Construction

[0035] The present invention will be further elaborated below in conjunction with the accompanying drawings and embodiments.

[0036] (1) Isolation and identification of porcine adipose-derived stem cells

[0037] 1. Select 3-day-old piglets, anesthetize with 10 mg / kg body weight injection of Zyta 50 anesthesia in the neck, and disinfect the whole body with brogeramine and 75% alcohol after humane killing;

[0038] 2. Under the aseptic operation environment, collect the piglet neck, scapula, and back fat tissue, rinse with PBS (phosphate buffered saline solution) containing 5% double antibody (penicillin / streptomycin) three times, and transfer to a sterile petri dish , cut the tissue with ophthalmic scissors in the ultra-clean workbench;

[0039] 3. Transfer the shredded adipose tissue to a 50 mL sterile centrifuge tube, add an equal volume of 0.2% (w / v) type II collagenase for digestion for 1 h, and carry out the digestion reaction on a constant temperature shaker at 37°C, a...

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Abstract

The invention discloses FTO gene-modified porcine adipose-derived stem cells as well as a construction method and application thereof. The construction method comprises the following steps: 1) separating porcine adipose-derived stem cells (pADSCs); and 2) performing transient overexpression of the FTO gene by a liposome method, and constructing the FTO gene-modified porcine adipose-derived stem cells. The overexpression of FTO by pADSCs increases protein expression of cell cycle proteins CDK2 and CDK4, promotes transition from G1 phase to S phase of the cell cycle, and increases mRNA expression level of adipogenic differentiation key genes PPARgamma, FABP4 and C / EBP alpha. The stem cells are inoculated in a collagen sponge scaffold to construct a tissue engineering skin.

Description

technical field [0001] The invention transiently overexpresses FTO in porcine adipose stem cells, enhances its proliferation and differentiation ability, and combines collagen sponge scaffolds to prepare tissue engineering skin, which belongs to the application technology in the field of biology and modern agricultural technology. Background technique [0002] How to promote the rapid repair of skin wounds is an important direction of wound research. Using tissue engineering technology to construct skin substitutes is an important method to promote wound repair, and the key is to find ideal seed cells. Although embryonic stem cells and bone marrow mesenchymal stem cells have self-renewal and differentiation pluripotency, their widespread use is limited due to the small number of cells, difficulty in obtaining them, great damage to donors, and ethical issues. Adipose stem cells (ADSCs) are ideal seed cells for tissue engineering due to their wide source, abundant quantity, f...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/85C12N5/0775A61L27/60A61L27/38A61L27/24A61L27/54
CPCC07K14/47C12N15/85C12N5/0667A61L27/60A61L27/3804A61L27/24A61L27/54C12N2510/00C12N2509/00C12N2509/10A61L2430/40A61L2300/41
Inventor 刘有华王新霞
Owner ZHEJIANG UNIV
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