A CD29 that improves skeletal muscle atrophy in a high-glucose and high-fat environment + Human umbilical cord-derived mesenchymal stem cell culture method

A technology of mesenchymal stem cells and human umbilical cords, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve problems of different degrees, such as increasing content and cell number, increasing hyperglycemia, and improving skeletal muscle atrophy The effect of symptoms

Active Publication Date: 2021-02-26
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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  • A CD29 that improves skeletal muscle atrophy in a high-glucose and high-fat environment <sup>+</sup> Human umbilical cord-derived mesenchymal stem cell culture method
  • A CD29 that improves skeletal muscle atrophy in a high-glucose and high-fat environment <sup>+</sup> Human umbilical cord-derived mesenchymal stem cell culture method
  • A CD29 that improves skeletal muscle atrophy in a high-glucose and high-fat environment <sup>+</sup> Human umbilical cord-derived mesenchymal stem cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 CD29 + Isolation of human umbilical cord-derived mesenchymal stem cells

[0037] 1.1 Mix 2ul anti-human CD29 antibody (R&D Systems, US) with 1mg poly-lysine (Sigma) at 4°C, spread it on a 90mm diameter petri dish (Shanghai Jingan Biotechnology Co., Ltd.), and set aside overnight at 4°C .

[0038] 1.2 Aseptic operation, wash the umbilical cord with PBS (Gibco) until there is no blood stain and turns white.

[0039] 1.3 Cut the tissue into small pieces with ophthalmic scissors, cut it longitudinally, and cut the Walton glue into small pieces after removing the blood vessels and umbilical cord skin.

[0040] 1.4 Put a small piece of umbilical cord tissue in a 50ml centrifuge tube, cut it into a muddy shape, add 0.1% collagenase IV (Gibco), digest at 37°C for 2 hours, add an appropriate amount of α-MEM (Gibco) containing 10% fetal bovine serum for culture Mix the solution, centrifuge at 2000g for 10 minutes (L-800R Jiangdong Instruments, China), discard the sup...

Embodiment 2

[0044] Example 2 Examination of surface markers of human umbilical cord-derived mesenchymal stem cells

[0045] 2.1 Take the 5th generation 1×10 6 Cells / ml, 0.1ml / tube in a flow detection tube.

[0046] 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.

[0047] 2.3 After incubating at 4°C for 20-30 minutes, wash with PBS 1-2 times, and centrifuge to discard the supernatant.

[0048] 2.4 Add 0.5ml PBS buffer to suspend the cells into a single cell suspension.

[0049] 2.5 Use FACSCalibur flow cytometer Cellquset Software analysis software to detect each sample.

[0050] Test results such 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 ...

Embodiment 3

[0051] Example 3 CD29 + Adipogenic differentiation of human umbilical cord-derived mesenchymal stem cells

[0052] 3.1 Select P5 generation of umbilical cord-derived mesenchymal stem cells in good growth state. When the cell fusion rate reaches 80%-90% fusion, trypsinize.

[0053] 3.2 Seed the cells in a six-well plate, about 2.5×10 per well 4 For each cell, add 2ml / well of complete culture medium, and put them in 37℃, 5% CO 2 Cultured in the incubator and hypoxic incubator for 24h.

[0054] 3.3 After 24 hours, remove the old culture medium, and add lipid induction solution (α-MEM medium supplemented with 10% fetal bovine serum, 0.6mM IBMX, 12mg / L insulin, 10 -5 M dexamethasone and 250 μM indomethacin), induced for 21 days.

[0055] 3.4 Discard the medium in the culture plate, rinse twice with PBS, add 4% neutral formaldehyde, fix for 30min, and absorb the fixative.

[0056] 3.5 Add the oil red O staining solution that is now filtered, stain for 30 minutes, wash with PBS ...

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Abstract

The invention discloses a CD29 for improving skeletal muscle atrophy under high-sugar and high-fat environment + The method for culturing human umbilical cord-derived mesenchymal stem cells, the steps are as follows: mix the anti-human CD29 antibody and polylysine, spread it on a culture dish; wash the umbilical cord with PBS solution until it turns white without blood stains; cut the tissue into small pieces Split longitudinally, remove blood vessels and umbilical cord skin, and cut Walton jelly into small pieces; cut small pieces of umbilical cord tissue into mud, add 0.25% collagenase IV, digest, add ɑ‑MEM with 10% fetal bovine serum for culture The solution was mixed evenly and centrifuged, and the lower part of the precipitated cells was added to the ɑ-MEM culture solution with 10% fetal bovine serum into the above-mentioned culture dish, and placed in 5% CO 2 Cultivate upside down in the incubator; discard the supernatant, add an appropriate amount of ɑ-MEM culture medium with 10% fetal bovine serum, change the medium every 3 days, and carry out subculture after the fusion rate reaches more than 85%. The invention lays a theoretical foundation and an experimental basis for subsequent research and development of drugs for treating skeletal muscle atrophy in high-sugar and high-fat environments, and has broad application prospects.

Description

technical field [0001] The invention relates to the field of mesenchymal stem cells, in particular to a CD29 that improves skeletal muscle atrophy in a high-sugar and high-fat environment + Method for culturing human umbilical cord-derived mesenchymal stem cells. Background technique [0002] Skeletal muscle mass and integrity are critical for proper function of the musculoskeletal system and efficient nutrient uptake and storage. Skeletal muscle tissue is the largest metabolically active tissue in the body, the major site of glucose metabolism and a fuel reservoir for other organs under pathological conditions. Muscle atrophy not only occurs with many common diseases, such as cancer, renal / heart failure, sepsis, muscle genetic diseases and neurodegenerative diseases, etc., but also occurs in healthy individuals, such as during the immobilization of a broken leg, during bed rest . The major determinants of adult muscle mass are exogenous essential amino acids (obtained th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 唐静
Owner JILIN TUO HUA BIOTECH
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