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A method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells

A technology derived from stem cells and adipose tissue, applied in the field of myogenic differentiation of stem cells, can solve the problems that the role of myoblast differentiation has not been reported, and achieve the goal of improving myogenic differentiation, increasing MyoD expression, and promoting myogenic differentiation Effect

Active Publication Date: 2021-08-03
NORTHERN JIANGSU PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no application of hydrogen in the field of stem cells, and its effect on ADSCs-induced myoblast differentiation has not been reported yet.

Method used

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  • A method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells
  • A method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells
  • A method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A eight-week large male New Zealand White Rabbit (Jiangsu University Animal Experiment Center) with a weight of 2.0kg (Jiangsu University Animal Experiment Center), raising and feeding rabbits under single cage feeding conditions. Control the temperature of the house (25 ° C), humidity (40-60%) and illumination (12h, bright and dark cycle). Observe the animal for a week before surgery to confirm that they are healthy.

[0045] In order to obtain ADSC, the animals were euthanized into the anesthesia chamber by passively inhaled the isoflurane until an excess occurred. The death of the rabbit is evaluated by monitoring the heart-breathing stop. Cut the hair from the abdomen area, cut a slit to expose the peritoneum, and remove the groin fat. The adipose tissue is then placed in a cellular chamber. The adipose tissue was washed 3 times with phosphate buffered saline (PBS) to remove red blood cells. The collected adipose tissue was cut into a small piece and transferred to a 20...

Embodiment 2

[0046] Example 2 Evaluation of Separation of Cells

[0047] There is no specific antigen in the MSC. Therefore, there is a need to simultaneously analyze a variety of surface antigen and cell morphology to determine the characteristics of mesenchymal cells. The ADSC surface marker is checked by flow cytometry. The third generation adhesive cells were treated with 0.25% trypsin (GIBCO, US) and washed twice with PBS. Cells were incubated with rabbit anti-CD31, anti-CD44, anti-CD45 and anti-CD90 antibodies (US Invitrogen and US GIBCO) were incubated at 4 ° C overnight. Unbound antibodies were removed by washing with PBS. After washing, cells were incubated with Cy3 labeled anti-goat / anti-rabbit secondary antibody incubation for 45 minutes at room temperature, and then resuspended in PBS for FACS analysis. Analysis of at least 1 × 10 per sample with flow cytometry (BD FACSVERSE, USA) 6 Cells.

[0048] In this study, the cells separated from adipose tissue have the following characte...

Embodiment 3

[0051] Third generation cells are digested with mixtures of trypsin and EDTA, 4 Single-cell suspensions of cells / mL then inoculated into a 6-well plate. The cells were divided into 4 groups. The first group is control group; the second group of inducers were treated with induced agent; the third group of inducers H 2 Treatment; 4th group of inducers 5-AZA + H 2 handle. The second group and the fourth group were induced with 10 μmol / l 5-Aza (Sigma, US) for 24 h, and was rinsed with D-HANKS equilibrium salt solution; then the medium was replaced with a high glucose DMEM containing 15% FBS. Group 1 and Group 2 are at 37 ° C and 5% Co in a conventional incubator. 2 Incubate together, while the third group and 4th group were in a hydrogen-containing incubator at 37 ° C, 25% h 2 Incubate; the oxygen concentration is 21%, the humidity is maintained in more than 90%, and the concentration is 5% concentration in the culture environment. 2 N-Ni NP 9% 2 . The medium was replaced with fre...

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Abstract

The invention discloses a method for inducing myogenic differentiation of adipose stem cells (ADSCs), the method comprising placing the adipose stem cells (ADSCs) in a 2 In a culture environment, induce 3-9 days at a temperature of 35-40°C. The present invention found that after 9 days of induction, the cells expressed troponin, myosin, MyoD and MHC, and at the same time, the expression of sarcomere α-actin and the differentiation rate of myoblasts were significantly higher than those of the control group. The present invention uses hydrogen to effectively promote the myogenic differentiation of ADSCs for the first time. Compared with 5-azacytidine (5-Aza) administration, hydrogen exposure is a simpler, non-toxic and cheap differentiation method. At the same time, Hydrogen also increased the effect of 5‑Aza on ADSC myoblast differentiation. It can be seen that hydrogen is the recommended inducer to realize the differentiation of MSCs into muscle fibers, and has broad application prospects.

Description

Technical field [0001] The present invention belongs to the field of stem cell active differentiation, and is specifically involved in a method of inducing a mesenchymal stem cells induced by a fat source. Background technique [0002] Wound, nerve injury, tumor resection or denatured muscle disease (such as shoulder sleeve after the fat infiltration after shoulder sleeve) is a serious clinical problem, apparently affects people's daily life and work, but the solution is very few. Stem cell transplantation is a promising technology developed in the 1990s to provide hope for the treatment of skeletal muscle disease. [0003] In recent years, the study of human MSC has been widely concerned, most of which is a bone marrow source of mesenchymal stem cell (BMSC) and fat-sized mesenchymal stem cells (ADSC). As shown in vitro, vivo evidence, these pluripotent cells can differentiate into mature fat cells, cartilage cells, osteoblasts, cardiomyocytes, hepatocytes, neurons and endothelia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2500/02C12N2500/40C12N2506/1384
Inventor 费文勇曹世超刘明生庞而凯
Owner NORTHERN JIANGSU PEOPLES HOSPITAL
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