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Rat dermal mesenchymal stem cell culture method

A technology of mesenchymal stem cells and dermis, applied in the field of isolation and culture of rat dermal mesenchymal stem cells, can solve problems such as data instability, and achieve the effect of avoiding pollution and better condition

Inactive Publication Date: 2014-06-25
JIANGSU UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since this method undergoes long-term trypsinization, the growth state of the obtained dermal cells is greatly affected, and the data obtained by using such cells in subsequent experimental research will be unstable.
[0005] It can be seen that there are disadvantages in the current culture method of dermal mesenchymal stem cells, and dermal mesenchymal stem cells play an important role in the development of skin tissue and tissue repair and regeneration, so the culture method of dermal mesenchymal stem cells with less pollution of epidermal cells and good state has been obtained It is very necessary to establish

Method used

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  • Rat dermal mesenchymal stem cell culture method

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Experimental program
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Embodiment 1

[0038] Embodiment 1, combined technical route figure 1 , the specific implementation steps of the present invention are described as follows:

[0039] (1) Six SD rat suckling mice born on the same day were purchased from the Experimental Animal Center of Jiangsu University and killed by neck dislocation, soaked in disinfectant alcohol for 5 minutes, then taken out from the ultra-clean workbench, and placed in a sterile 10cm large dish in PBS wash three times;

[0040](2) Cut off the back and neck skin of suckling mice soaked in disinfectant alcohol and washed with PBS, use ophthalmic tweezers to grab the head and back skin with a little force to tear off the back skin tissue, put the taken back skin into Soak in PBS containing penicillin and streptomycin for 5 minutes;

[0041] (3) Remove the subcutaneous fat, blood vessels and other subcutaneous tissue of the back skin tissue (the subcutaneous tissue has no obvious red blood vessels), and then cut the skin tissue into thin ...

Embodiment 2

[0045] Example 2, comparing the dermal mesenchymal stem cells obtained in the present invention with the cells obtained by the control method (direct skin tissue block attachment method and enzyme digestion method):

[0046] Using an inverted microscope to observe and compare the growth state of the P2 generation dermal mesenchymal stem cells obtained by the method of the present invention and the enzyme digestion method, the results show that the growth state of the cells obtained by the method of the present invention is obviously better than that of the enzyme digestion method, and can effectively avoid long-term enzyme digestion. Damage to cells caused by digestion ( image 3 );

[0047] Application of RT-PCR technology to the detection of the epidermal index CK19 in the P2 generation dermal mesenchymal stem cells obtained by the method of the present invention and the direct attachment method of skin tissue blocks (internal reference gene: β-actin), the results show that ...

Embodiment 3

[0051] Example 3, identification of surface markers and differentiation of dermal mesenchymal stem cells isolated and cultured in the present invention:

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Abstract

The invention discloses a rat dermal mesenchymal stem cell culture method. The method comprises the steps of digesting skin tissue and keeping the skin tissue for a whole night; separating epidermis and dermis of the skin tissue; sticking a dermis tissue block in a cell culture dish and adhering in an inverted incubator at 37 DEG C for half an hour, and cultivating the tissue block through low-sugar DMEM and 10% of FBS; implementing conventional pancreatin digestion subculture when the dish is full of cells cultivated from the dermis tissue block to obtain dermal mesenchymal stem cells. The method disclosed by the invention is a novel method combining a skin tissue block adherent method and an enzyme digestion method, not only effectively avoids epidermal keratinocyte pollution caused by the skin tissue block adherent method but also avoids influence of a long-time pancreatic function on a cell growth state in the enzyme digestion method. The culture method disclosed by the invention provides possibility for further research on effects of mesenchymal stem cells in the skin tissue on skin development, growth and damage repair.

Description

technical field [0001] The invention belongs to the field of in vitro culture of cells, and more specifically, the invention relates to a method for separating and culturing rat dermal mesenchymal stem cells. Background technique [0002] Dermal mesenchymal stem cells (dermal fibroblasts), as skin tissue cells, have very important biological functions, which are mainly reflected in the following aspects: ①As the stromal cells of the skin dermis, they play an important role in maintaining the balance and stability of the extracellular matrix; ②Affect the development and growth of the epidermis by secreting cytokines; ③Regulate the growth and regeneration of skin accessory structures such as hair follicles; ④It can be used as an important source of mesenchymal stem cells to transplant and repair other tissues and organs. Dermal mesenchymal stem cells cultured in vitro are of great significance to the study of the interaction between the dermis and epidermis during skin develop...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 张斌许文荣钱晖朱伟张徐王梅吴晓丹朱艳华史惠
Owner JIANGSU UNIV
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