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CD29<+> human umbilical cord source mesenchymal stem cell and use thereof in preparation of drug for treating skeletal muscle atrophy in high-sugar and high-fat environments

A technology of mesenchymal stem cells and human umbilical cords, applied in bone/connective tissue cells, drug combinations, animal cells, etc., can solve problems of varying degrees, achieve the goal of improving hyperlipidemia, increased hyperglycemia, and improving symptoms of skeletal muscle atrophy Effect

Active Publication Date: 2018-01-09
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, alterations in skeletal muscle mass in type 2 diabetes occur less predictably and to varying degrees

Method used

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  • CD29&lt;+&gt; human umbilical cord source mesenchymal stem cell and use thereof in preparation of drug for treating skeletal muscle atrophy in high-sugar and high-fat environments
  • CD29&lt;+&gt; human umbilical cord source mesenchymal stem cell and use thereof in preparation of drug for treating skeletal muscle atrophy in high-sugar and high-fat environments
  • CD29&lt;+&gt; human umbilical cord source mesenchymal stem cell and use thereof in preparation of drug for treating skeletal muscle atrophy in high-sugar and high-fat environments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 CD29 + Isolation of mesenchymal stem cells derived from human umbilical cord

[0033] 1.1 Mix 2ul of anti-human CD29 antibody (R&D Systems, US) with 1mg of polylysine (Sigma) at 4°C, and spread it on a 90mm diameter petri dish (Shanghai Jingan Biotechnology Co., Ltd.), overnight at 4°C for standby .

[0034] 1.2 Aseptic operation, wash the umbilical cord with PBS (Gibco) until there is no blood stains and turn white.

[0035] 1.3 Cut the tissue into small sections with ophthalmological scissors, open it longitudinally, and cut the Walton glue into small pieces after removing the blood vessels and the outer skin of the umbilical cord.

[0036] 1.4 Put a small piece of umbilical cord tissue in a 50ml centrifuge tube and cut it into a slurry, add 0.1% collagenase IV (Gibco), digest at 37°C for 2 hours, add an appropriate amount of ɑ-MEM (Gibco) containing 10% fetal bovine serum to culture Mix the solution, centrifuge at 2000g for 10 minutes (L-800R Jiangdong Instruments...

Embodiment 2

[0040] Example 2 Surface marker inspection of human umbilical cord-derived mesenchymal stem cells

[0041] 2.1 Take the 5th generation 1×10 6 Cells / ml, 0.1ml / tube in the flow cytometry tube.

[0042] 2.2 Add directly labeled fluorescent antibodies CD29, CD34, CD45, HLA-DR, CD73, CD90, CD105 (Biolegend, US) and corresponding isotype control antibodies for immunolabeling reaction.

[0043] 2.3 After incubating at 4°C for 20-30 minutes, wash 1-2 times with PBS, and centrifuge to discard the supernatant.

[0044] 2.4 Add 0.5ml PBS buffer to suspend the cells into a single cell suspension.

[0045] 2.5 Use FACSCalibur Flow Cytometer Cellquset Software analysis software for each sample detection.

[0046] The test results are as Figure 4-9 As shown, the human umbilical cord-derived mesenchymal stem cells of the present invention express the following four mesenchymal stem cell membrane molecules: human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90, human...

Embodiment 3

[0047] Example 3 CD29 + Adipogenic differentiation of mesenchymal stem cells derived from human umbilical cord

[0048] 3.1 Select the P5 generation of umbilical cord-derived mesenchymal stem cells with good growth status, and when the cell fusion rate reaches 80%-90% fusion, trypsin digestion.

[0049] 3.2 Inoculate the cells in a six-well plate, about 2.5×10 per well 4 Cells, add 2ml / well of complete culture medium, put them in 37℃, 5% CO 2 Incubate in an incubator and a hypoxic incubator for 24 hours.

[0050] 3.3 After 24h, remove the old culture medium and add lipid-inducing liquid (α-MEM medium with 10% fetal bovine serum, 0.6mM IBMX, 12mg / L insulin, 10 -5 M dexamethasone and 250 μM indomethacin), induced for 21 days.

[0051] 3.4 Discard the culture medium in the culture plate, wash it twice with PBS, add 4% neutral formaldehyde, fix for 30 minutes, and remove the fixative.

[0052] 3.5 Add the freshly prepared and filtered Oil Red O staining solution, dye for 30 minutes, wash 3 ...

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Abstract

The invention discloses a CD29<+> human umbilical cord source mesenchymal stem cell and use thereof in the preparation of a drug for treating skeletal muscle atrophy in high-sugar and high-fat environments. The CD29<+> human umbilical cord source mesenchymal stem cell represents the following mesenchymal stem cell cytomembrane molecules: a human leukocyte differentiation antigen CD73, a human leukocyte differentiation antigen CD90, a human leukocyte differentiation antigen CD105 and a human leukocyte differentiation antigen CD29. According to the CD29<+> human umbilical cord source mesenchymalstem cell, the hyperlipidemia and hyperglycemia of a db<- / -> mouse can be obviously improved, muscle fiber cross sections of soleus and gastrocnemius muscle of the db<- / -> mouse can be increased, andthe contents and cell number of each myotube are increased, so that the symptoms of the skeletal muscle atrophy of the db<- / -> mouse are improved. According to the CD29<+> human umbilical cord sourcemesenchymal stem cell, the theoretical foundation and experiment basis are laid for the subsequent research and development of the drug for treating skeletal muscle atrophy in the high-sugar and high-fat environments, and the CD29<+> human umbilical cord source mesenchymal stem cell has wide application prospects.

Description

Technical field [0001] The present invention relates to the field of mesenchymal stem cells, specifically a CD29 + Human umbilical cord-derived mesenchymal stem cells and their use in preparing medicines for treating skeletal muscle atrophy in a high-sugar and high-fat environment. Background technique [0002] The quality and integrity of skeletal muscle are essential for the normal function of the musculoskeletal system and effective nutrient intake and storage. Skeletal muscle tissue is the body's largest metabolically active tissue, the main part of glucose metabolism and the fuel storage for other organs under pathological conditions. Muscle atrophy not only occurs with many common diseases, such as cancer, kidney / heart failure, sepsis, muscle genetic diseases and neurodegenerative diseases, etc., but also occurs in healthy individuals, such as in the fixation of leg fractures, during bed rest . The main determinants of adult muscle mass are exogenous essential amino acids...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P21/00A61P3/06A61P3/10
Inventor 唐静
Owner JILIN TUO HUA BIOTECH
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