Method for differentiating epidermal stem cells to 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, which can solve problems such as lack of skin attachments

Inactive Publication Date: 2015-03-11
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

Method used

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  • Method for differentiating epidermal stem cells to sweat gland-like epithelial cells
  • Method for differentiating epidermal stem cells to sweat gland-like epithelial cells
  • Method for differentiating epidermal stem cells to 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 steps are as follows:

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

[0069] 2) Take the tendon: remove the skin, rinse 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 at 4°C for more than 48 hours, and shake it repeatedly during the period;

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

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

Embodiment 2

[0072] Example 2 Cultivation 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 Department of Urology, the First Affiliated Hospital of the Third Military Medical University of the Chinese People’s Liberation Army and the Medical Beauty Center of the Third Affiliated Hospital of the Third Military Medical University of the Chinese People’s Liberation Army, all patients are aware of And agree. The same below), after immersing and wiping in the clean bench with 75% alcohol for 5 minutes, rinse with PBS solution 3 times, each time for 3-5 minutes;

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

[0076] 3) Take out the skin pi...

Embodiment 3

[0085] Example 3 Cultivation of epidermal stem cells

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

[0087] The specimen was taken from a healthy foreskin.

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

[0089] 1) After soaking in alcohol for 5 minutes, place it on a clean bench and gently wipe the specimen three times with an alcohol cotton ball;

[0090] 2) Move the specimen to a petri dish, rinse the specimen 3 times with PBS balanced salt solution, and rinse every corner, not less than 5 minutes each time, until the specimen becomes whitish and swollen;

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

[0092] 4) Put the leather strips in a petri dish, add 0.25% separating enzyme to submerge the tissue (the ratio of separating enzyme to specimen is 5:1);

[0093] 5) Digested overn...

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PUM

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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 differentiation of epidermal stem cells into sweat gland-like epithelial cells. Background technique [0002] Severe skin defects, such as large-scale deep burns, can damage the entire layer of the skin and its appendages. It is difficult to regenerate the skin only by the wound itself, and sufficient skin substitutes are needed to repair it. Although the constructed epidermis double-layer tissue engineered skin has been used in clinics 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, ESC) are skin tissue-specific stem cells located in the basal layer of the epidermis. They play an important role in maintain...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/24A61L27/22A61L27/20A61L27/60A61L27/52C12N5/074C12N5/071
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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