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A method for promoting differentiation and growth of epidermal stem cells

A technology of epidermal stem cells and neuron cells, applied in the field of stem cells, can solve the problem that epidermal stem cells are not induced into neuron cells, and achieve good proliferation effects and good application prospects

Active Publication Date: 2020-12-01
华夏源(上海)生命科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the problem that the existing epidermal stem cells are not induced to become neurons, the object of the present invention is to provide a neuron cell derived from epidermal stem cells, a preparation method and its application in the treatment of central nervous system injuries

Method used

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  • A method for promoting differentiation and growth of epidermal stem cells
  • A method for promoting differentiation and growth of epidermal stem cells
  • A method for promoting differentiation and growth of epidermal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of epidermal stem cells

[0025] Take the skin specimens, rinse the skin specimens 3 times with D-hank's solution containing gentamicin 800u / ml under aseptic conditions. Use ophthalmic scissors to cut the skin slices into 1cm*1cm size skin slices, put them into a culture bottle, add 0.25% Trypsin-0.02% EDTA dropwise, the volume ratio of the two is 1:6, and digest them at 4°C for 10h. Take out the skin slices, add a little fetal bovine serum dropwise to stop digestion, and rinse with D-hank's solution twice. The epidermis and dermis were separated, and the epidermis was gently uncovered with ophthalmic forceps; K-SFM was added, and the epidermis was carefully blown repeatedly for 15 minutes with a straw. Filter through a 200-mesh sieve, centrifuge at 1000 rpm for 5 minutes, discard the wound, and add K-SFM to make a cell suspension. The cells were inoculated on the pretreated type IV collagen culture plate, and cultured in a constant temperature i...

Embodiment 2

[0029] Example 2 Induced Differentiation of Epidermal Stem Cells to Neurons by Compounds

[0030] The compound of formula (1) was synthesized and prepared according to the method of CN101437785B.

[0031] The epidermal stem cells prepared in Example 1 of the 4th passage were digested with 0.25% trypsin-EDTA and inoculated in a 24-well plate coated with polylysine. After the cells adhered to the wall, the culture medium was replaced with the induction medium (experimental group ): DMEM / F12 + 1% penicillin + 20 μg / L bFGF + 20 μg / L compound of formula (1), change the medium in half the next day. Detection was carried out 7 days after induction. In addition, the epidermal stem cell group prepared in Example 1 at the 4th passage with the addition of DMEM / F12+1% penicillin+20 μg / L bFGF as the induction solution was used as control 1, and the epidermal stem cell group prepared in Example 1 at the 4th passage without the induction solution The epidermal stem cell group was used as c...

Embodiment 3

[0033] Example 3 Cell proliferation after induction

[0034] MTT method to detect the proliferation of cells before and after induction: the neuron cells with the highest expression of the marker protein in Example 2 were diluted to a cell concentration of about 5×10 6 L -1 , the cell suspension was inoculated in a 96-well plate, and 200 μL was added to each well. After the cells adhered to the wall, the culture medium in each well was sucked out. The experiment was divided into 3 groups: the experimental group corresponding to Example 2, control 1, and control 2, 6 holes per group. Blank group: Add freshly prepared DMEM medium containing 10% calf serum by volume to wells with cells, 200 μL / well. After culturing for 72 h, take out the 96-well plate, add 25 μL / well of MTT, incubate in the dark for 4 h, aspirate the liquid, dissolve 20% sodium lauryl sulfate in 50% dimethylformamide, and add 25 μL / well of MTT to each well of the 96-well plate Add 100 μL of the above mixture,...

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Abstract

The invention relates to a method for promoting differentiation and growth of epidermal stem cells. The method comprises the following steps: performing inducing on the epidermal stem cells by using an inducer containing a compound to obtain neuronal cells derived from the epidermal stem cells, and detecting the proliferation activity of the neuronal cells and the electrical characteristics of neurons. It is proved that the induced neuronal cells have a relatively good proliferation effect and corresponding neuron characteristics and have a relatively good application prospect.

Description

technical field [0001] The invention belongs to the field of stem cells, and in particular relates to a method for promoting the differentiation and growth of epidermal stem cells. Background technique [0002] The cells of the central nervous system of the brain include functional nerve cells and neural stem cells (Neural Stem Cells, NSCs) that maintain brain function. Functional nerve cells mainly include neurons, oligodendrocytes, and astrocytes. Neural stem cells are adult stem cells with self-proliferating ability and multi-lineage differentiation potential. Under physiological conditions, dead or apoptotic functional nerve cells are replaced by functional nerve cells differentiated from neural stem cells. Because of their self-proliferation ability and multi-directional differentiation potential, neural stem cells have brought a new dawn for the repair of nervous system damage and the treatment of cerebral ischemic diseases. A large number of experimental studies ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2501/115C12N2501/999C12N2506/1392
Inventor 刘欢李蒙
Owner 华夏源(上海)生命科技有限公司
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