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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 yet

Active Publication Date: 2021-02-09
NORTHERN JIANGSU PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] An eight-week-old male New Zealand white rabbit (Jiangsu University Animal Experiment Center) with a body weight of 2.0 kg was used to raise and feed the rabbits under single-cage rearing conditions. The temperature (25° C.), humidity (40-60%) and light (12h, light-dark cycle) of the house were controlled. Animals were observed for one week prior to surgery to confirm that they were healthy and disease-free.

[0045] To obtain ADSCs, animals were euthanized in an anesthesia room by passive inhalation of isoflurane until overdose occurred. Rabbits were assessed for death by monitoring for cardio-respiratory arrest. Hair was clipped from the abdominal region, an incision was made to expose the peritoneum, and inguinal fat was removed. Then, place the adipose tissue in a laminar flow chamber. Adipose tissue was washed 3 times with phosphate-buffered saline (PBS) to remove red blood cells. The collected adipose tissue was cut into small pieces and transferred to a 20 mL...

Embodiment 2

[0046] Evaluation of Example 2 Isolated Cells

[0047] There are no specific antigens in MSCs. Therefore, simultaneous analysis of multiple surface antigens and cell morphology is required to characterize mesenchymal cells. ADSC surface markers were examined by flow cytometry. Passage 3 adherent cells were treated with 0.25% trypsin (Gibco, USA) and washed twice with PBS. Cells were incubated overnight at 4°C with rabbit anti-CD31, anti-CD44, anti-CD45 and anti-CD90 antibodies (Invitrogen, USA and Gibco, USA). Unbound antibody was removed by washing three times with PBS. After washing, cells were incubated with Cy3-labeled anti-goat / anti-rabbit secondary antibody for 45 min at room temperature in the dark, then resuspended in PBS for FACS analysis. Each sample was analyzed with a flow cytometer (B D FACSVerse, USA) at least 1 × 10 6 cells.

[0048] In this study, cells isolated from adipose tissue were characterized by (1) spindle-shaped adherent growth and colony format...

Embodiment 3

[0051] The third passage cells were digested with a mixture of trypsin and EDTA to make 10 4 cells / mL of single-cell suspension, and then seeded into 6-well plates. Cells were divided into 4 groups. Group 1 was the control group; Group 2 was treated with inducer 5-Aza; Group 3 was treated with inducer 5-Aza+H 2 treatment; group 4 with inducer H 2 deal with. Cells in groups 2 and 4 were induced with 10 μmol / L 5-Aza (Sigma, USA) for 24 hours, and washed with D-Hanks balanced salt solution; then, the medium was replaced with high glucose DMEM containing 15% FBS. Groups 1 and 2 were incubated in a conventional incubator at 37 °C and 5% CO 2 Incubate together while groups 3 and 4 at 37 °C in a hydrogen incubator, 25% H 2 Incubation; the oxygen concentration is 21%, the humidity is kept above 90%, and the culture environment contains a concentration of 5% CO 2 , and a concentration of 49% N 2 . The medium was replaced with fresh DMEM and FBS every 3 days until the test was s...

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Abstract

The invention discloses a method for inducing the myogenic differentiation of adipose-derived stem cells (ADSCs). The method comprises the following steps: placing the adipose-derived stem cells (ADSCs) in a culture environment containing an inducer H2, and performing induction for 3-9 days at 35-40 DEG C. After 9 days of induction, the cells express troponin, myosin, MyoD and MHC, and meanwhile,the expression of alpha-sarcomeric actin and a differentiation rate of myoblasts are obviously higher than those of a control group. According to the method, hydrogen is adopted for the first time toeffectively promote the myogenic differentiation of the ADSCs, compared with the application of 5-Azacytidine (5-Aza), the hydrogen exposure is a simpler, non-toxic and cheap differentiation method, and meanwhile, hydrogen can also increase the effect of 5-Aza on the differentiation of ADSC myoblasts. Therefore, hydrogen is a recommended inducer for realizing that MSCs are differentiated into muscle fibers, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of myogenic differentiation of stem cells, and in particular relates to a method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells. Background technique [0002] Loss of skeletal muscle function due to trauma, nerve injury, tumor resection, or degenerative muscle disease (e.g. fatty infiltration following a rotator cuff tear) is a serious clinical problem that clearly affects people's daily life and work, but solutions are elusive. few. Stem cell transplantation is a promising technique developed in the 1990s that offers hope for the treatment of skeletal muscle diseases. [0003] In recent years, studies on human MSCs, most of which are bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs), have received extensive attention. As shown by in vitro, ex vivo and in vivo evidence, these pluripotent cells can differentiate into mature...

Claims

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Application Information

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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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