Methods of Differentiation of Epidermal Stem Cells into Sweat Gland-like Epithelial Cells

A technology of epidermal stem cells and epithelial cells, which is applied in the field of differentiation of epidermal stem cells into sweat gland-like epithelial cells, and can solve problems such as lack of skin attachments

Inactive Publication Date: 2016-03-16
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the epidermal double-layer tissue engineered skin currently constructed has been applied clinically and basically solved the problem of wound coverage, it has not achieved the complete reconstruction of the real skin tissue structure and function. The biggest problem is the lack of skin accessories, such as sebaceous glands, Appendages such as sweat glands

Method used

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  • Methods of Differentiation of Epidermal Stem Cells into Sweat Gland-like Epithelial Cells
  • Methods of Differentiation of Epidermal Stem Cells into Sweat Gland-like Epithelial Cells
  • Methods of Differentiation of Epidermal Stem Cells into Sweat Gland-like Epithelial Cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 : Preparation of Rat Tail Collagen

[0067] The specific operation steps are as follows:

[0068] 1) Thawing: freeze 3 rat tails of about 300g, and thaw them in 75% alcohol solution for 1 hour;

[0069] 2) Take the tendon: peel off the skin, wash it with D-Hanks solution 3 times, take out the tail tendon, cut it into mud, dissolve it in 250ml of sterile 1% glacial acetic acid, store it at 4°C for more than 48 hours, and shake it repeatedly;

[0070] 3) Centrifugation: centrifuge at 3000rpm for 1 hour, and take the supernatant;

[0071] 4) Storage: The resulting milky white translucent viscous liquid was stored at 4°C for later use, and the measured concentration was 20 mg / ml (wet weight).

Embodiment 2

[0072] Example 2 Culture of Human Fibroblasts

[0073] Specific steps are as follows:

[0074] 1) The specimen is the foreskin of healthy adult circumcision (healthy foreskin, provided by the Urology Department of the First Affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army and the Medical Cosmetology Center of the Third Affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army, and the patients were informed And agree. The same below), after bathing and wiping the purification workbench with 75% alcohol for 5 minutes, rinse it repeatedly with PBS solution for 3 times, each time for 3-5 minutes;

[0075] 2) Remove the subcutaneous tissue and dermal reticular layer as much as possible, cut the surface dermis into a size of 2cm×0.3cm in another clean petri dish, transfer it into a glass vial containing 10-20ml of 0.25% separase, cover it and place it Digest at 4°C for 18-20 hours;

...

Embodiment 3

[0085] Example 3 Culture of epidermal stem cells

[0086] 1. Primary isolation and culture of human epidermal stem cells

[0087] Specimens were taken from healthy foreskins.

[0088] After the specimen is removed, soak it in PBS balanced salt solution and store it in a refrigerator at 4°C. Follow the steps below:

[0089] 1) After soaking in alcohol for 5 minutes, put it on the ultra-clean workbench, and gently wipe the specimen three times with alcohol cotton ball;

[0090] 2) Move the specimen to a petri dish, rinse the specimen with PBS balanced salt solution repeatedly for 3 times, every corner should be rinsed, each time for no less than 5 minutes, until the specimen turns white and swells;

[0091] 3) Cut off the subcutaneous tissue with surgical scissors in a petri dish, and cut the specimen into leather strips with a size of 0.3cm×2cm;

[0092] 4) Put the leather strip in a petri dish, add 0.25% separase to immerse the tissue (the ratio of separase to specimen is...

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Abstract

The invention relates to a method for differentiating epidermal stem cells to sweat gland-like epithelial cells. The method comprises the following steps: preparation of rat tail collagen, culture of human fibroblast cells, culture of the epidermal stem cells, preparation and observation of composite chitosan tissue engineered skin and preparation and observation of composite chitosan tissue engineered gel. The method proves that the three-dimensional culture of the composite chitosan tissue engineered skin and gel and the induction and vertical shaking culture of a certain concentration of epidermal growth factors (EGF) are utilized in vitro to form a tube-like structure, the formed tube-like structure is similar to sweat gland secretion portion cells in vivo on morphology, histology and function and has the physiological structure and the physiological effect basis similar to those of normal sweat gland secretion portion cells.

Description

technical field [0001] The invention relates to a method for separating and culturing cells, in particular to a method for differentiating epidermal stem cells into sweat gland-like epithelial cells. Background technique [0002] Severe skin defects, such as large-area deep burns, damage the whole layer of skin and its appendages. It is difficult to achieve skin regeneration only by the wound itself, and sufficient skin substitutes are needed for repair. Although the epidermis double-layer tissue-engineered skin currently constructed has been applied clinically and basically solved the problem of wound coverage, it has not achieved the complete reconstruction of the real skin tissue structure and function. The biggest problem is the lack of skin accessories, such as sebaceous glands, Appendages such as sweat glands. Epidermal stem cells (Epidermal stem cells, ESCs) are skin tissue-specific stem cells, located in the basal layer of the epidermis, and play an important role i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074A61L27/38A61L27/24A61L27/22A61L27/20A61L27/60A61L27/52C12N5/071
Inventor 王元元伍津津杨亚东鲁元刚
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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