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Method for improving efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes

A technology of cardiomyocytes and brown fat, which is applied in the field of cell engineering, can solve the problems of low efficiency, achieve the effect of collecting a large amount of cells, rich sources, and improving efficiency

Inactive Publication Date: 2019-04-23
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current isolation and culture methods have low efficiency for the differentiation of brown adipose stem cells into cardiomyocytes, and it is urgent to find a way to improve their differentiation into cardiomyocytes

Method used

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  • Method for improving efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes
  • Method for improving efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes
  • Method for improving efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Differentiation of brown adipose stem cells to cardiomyocytes

[0019] 1. Take a newborn 1-day-old SD rat, open the chest, take the heart, put it in pre-cooled PBS liquid, cut it into pieces, and use 0.05% trypsin to digest it repeatedly until the tissue block is completely digested, and centrifuge at 1000rpm / 7 minutes to obtain cells The pellet was resuspended in high-glucose DMEM plus 15% fetal bovine serum (gibco) and seeded on a culture dish. After 1 hour, unattached cardiomyocytes were discarded to obtain primary cardiac fibroblasts.

[0020] 2. Using high glucose-DMEM plus 10% fetal bovine serum to culture myocardial fibroblasts to 85-90% confluence, digest them, inoculate them in 24 culture well plates, and cultivate them for 10 days. Discard the culture medium, and utilize PBS to wash 3 times, add the NH of 0.3% (volume / volume) Triton X-100 and 20mM 4 Treat with OH PBS at 37°C for 5 minutes, discard the digestion solution, wash twice with PBS, and obt...

Embodiment 2

[0023] Embodiment 2 conversion rate detection

[0024] 1. Use CCK-8 to detect the proliferation of BADSC for 1 / 2 / 3 / 4 / 5 / 7 days, wash the cultured cells with PBS 3 times, add 10% CCK-8 solution, and incubate at 37°C 1.5 hours, draw the supernatant, and use a microplate reader to detect the OD value under the condition of 450nm, such as figure 2 As shown in A, BADSCs seeded on derivatized matrix materials have a stronger proliferative ability. Using immunofluorescence to detect the expression of cell cycle-related checkpoint PH3, such as figure 2 As shown in B, the results also confirmed that the extracellular derived matrix promoted the cell cycle activation of BADSC, and the number of cells entering the S phase was significantly increased.

[0025] 2. To further detect the differentiation of BADSC to the myocardial lineage on the matrix material. The BADSC cells cultured for 3 days and 7 days were harvested, washed three times with PBS, fixed with 4% paraformaldehyde for 3...

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Abstract

The invention discloses a method for improving the efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes and belongs to the technical field of cell engineering. Cardiac stem cells from which brown adipose tissue comes are rich in source, the quantity of collected cells is large, and the cells are easily accepted by a patient; by means of the method, an extracellular matrix derived from cardiac fibroblasts is utilized for improving the differentiation of the brown adipose-derived stem cells into the cardiomyocytes, and the problem that a current isolation culture method is low in efficiency of differentiating brown adipose-derived stem cells into cardiomyocytes is solved.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a method for improving the differentiation efficiency of brown adipose stem cells into cardiomyocytes. Background technique [0002] Seeding cells is the basis of myocardial tissue engineering strategies and cell therapy strategies. The seed cells currently used for the repair of damaged myocardium include terminally differentiated cardiomyocytes, tissue stem cells from various sources and different developmental stages (such as adipose-derived mesenchymal stem cells (ADSCs), bone marrow mesenchymal stem cells, etc.), and totipotent Stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) etc. These cells can improve cardiac function to a certain extent, but there are also disadvantages such as limited source, low expansion efficiency, limited ability of myocardial differentiation, and certain tumorigenicity. At the same tim...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0656C12N5/0657C12N5/0667C12N2533/90C12N2509/00
Inventor 周瑾刘伟王常勇朱惠敏王春兰
Owner ACADEMY OF MILITARY MEDICAL SCI
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