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Sweat gland differential induction medium and applications thereof

A technology of differentiation induction and culture medium, applied in the field of cell biology, can solve the problems of unclear key regulatory genes, inability to develop widely, and inability to prepare sweat gland differentiation induction medium, etc., and achieve stable and reliable results.

Active Publication Date: 2015-05-06
许永安 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For the above sweat gland induction methods, on the one hand, it is necessary to wait to obtain normal SGs, and on the other hand, the key regulatory genes that induce MSCs to differentiate into SGCs are not clear, so it is impossible to prepare a finished sweat gland differentiation induction medium. The above induction methods are limited by the source of SGs. due to the limitations of the

Method used

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  • Sweat gland differential induction medium and applications thereof
  • Sweat gland differential induction medium and applications thereof
  • Sweat gland differential induction medium and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Isolation, culture and identification of umbilical cord mesenchymal stem cells

[0036] 1. Isolation and culture of umbilical cord mesenchymal stem cells

[0037] (1) After the fresh umbilical cord tissue was collected as medical waste with the informed consent of the patient, it was stored in a transfer box at 4°C in a sterile manner, and the umbilical cord with clip marks and congestion on both sides was excised within 6 hours part;

[0038] (2) Flush the periphery of the umbilical cord and the lumen of the umbilical vein with a phosphate solution (PBS buffer) containing penicillin (100 U / ml) and streptomycin (100 μg / ml);

[0039] (3) Then cut the umbilical cord tissue into 0.5-1.0mm 3 directly add 1g / L type II collagenase solution and incubate in a 37°C incubator for 16h;

[0040] (4) After washing the umbilical cord tissue with PBS solution, add 25g / L trypsin and continue to incubate in a 37°C incubator for 0.5h;

[0041] (5) adding a culture medium with a vo...

Embodiment 2

[0097] Isolation and identification of sweat gland cells

[0098] 1. Isolation and culture of normal sweat gland cells

[0099] (1) Place the skin in D-Hank’s balanced salt solution containing penicillin (100 U / ml) and streptomycin (100 μg / ml), rinse it 3 times, 5 minutes each time, to remove excess blood stains;

[0100] (2) Then remove the subcutaneous fat tissue, and continue to rinse repeatedly with D-Hank's balanced salt solution, each time for 5 minutes, to remove the residual fat tissue;

[0101] (3) Trim the skin into a microparticle skin with a diameter of about 0.5-0.8 mm by the method of making microparticle skin, add 3 ml of type II collagenase solution with a concentration of 2.5 mg / ml, and suspend the microparticle skin in the type II collagenase solution ;

[0102] (4) Then place the type II collagenase solution containing the skin particles in an incubator with a temperature of 37.5° C. and a humidity of more than 95% to promote the enzymatic digestion of the...

Embodiment 3

[0131] Preparation and Application of Medium for Inducing Sweat Gland Differentiation

[0132] 1. Preparation of Sweat Gland Differentiation Induction Medium

[0133] Group 1: Add the following components in the low-sugar DMEM medium: based on the volume of the low-sugar DMEM medium, the volume fraction of special grade fetal bovine serum is 10%, hemisuccinyl hydrocortisone 0.4 μg / ml, and rh-KGF is respectively 10ng / ml, 20ng / ml, 40ng / ml, 80ng / ml 100ng / ml, triiodothyronine 2nmol / ml, rh-EGF 10ng / ml, insulin-transferrin-sodium selenite solution 0.1ml / ml, penicillin 100U / ml, streptomycin 100μg / ml.

[0134] Wherein, the insulin-transferrin-sodium selenite solution comprises the following components: 0.01 g / L of insulin, 0.0055 g / L of human transferrin, and 0.000006 g / L of selenite.

[0135] Group 2: Add the following components in the low-sugar DMEM medium: based on the volume of the low-sugar DMEM medium, the volume fraction of special grade fetal bovine serum is 8%, hemisuccin...

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Abstract

The invention relates to the field of cytobiology, and particularly relates to a sweat gland differential induction medium and applications thereof. The sweat gland differential induction medium is prepared by adding the following components into a low-sugar DMEM medium: based on the volume of the low-sugar DMEM medium, 8-15% by volume of fetal calf serum, 0.3-0.5mu g / ml of semi-hydrocortisone succinate, 10-100ng / ml of rh-KGF, 1-3nmol / ml of triiodothyronine, 10-100ng / ml of rh-EGF, and 0.05-0.2ml / ml of an insulin-transferrin-sodium selenite solution. The sweat gland differential induction medium is constructed by taking a recombinant human keratinocyte growth factor as a key regulation factor, constructing the finished product of the sweat gland differential induction medium, inducing the differentiation of human umbilical cord mesenchymal stem cells to sweat gland sample cells. By using the medium disclosed by the invention, the result is stable and reliable, and the sweat gland differential induction medium can be widely used for related studies and clinical application of differential induction of various MSCs to the sweat gland cells.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a sweat gland differentiation induction medium and its application. Background technique [0002] At present, to induce the differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) and / or umbilical cord mesenchymal stem cells (hUC-MSCs) into sweat gland cells, co-culture with heat shock sweat gland cells or culture in heat shock sweat gland medium can be used. The supernatant method was used to obtain BM-MSCs and / or hUC-MSCs with sweat gland cell phenotype characteristics. However, obtaining sweat gland-like cells (SGCs) using the above methods generally requires obtaining normal sweat gland tissue (sweat glands, SGs) from a donor, and then adopting a corresponding induction method to obtain SGCs. For the above sweat gland induction methods, on the one hand, it is necessary to wait to obtain normal SGs, and on the other hand, the key regulatory genes that induce MSCs to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 张茂许永安
Owner 许永安
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