Method for culturing CD29<+> human umbilical cord mesenchymal stem cells for alleviating skeletal muscle atrophy in high glucose and lysophosphatidylcholine environment

A technology of mesenchymal stem cells and culture methods, applied in the field of mesenchymal stem cells, can solve problems of different degrees, achieve the effect of increasing content and cell number, increasing hyperglycemia, and improving symptoms of skeletal muscle atrophy

Active Publication Date: 2018-01-02
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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  • Method for culturing CD29&lt;+&gt; human umbilical cord mesenchymal stem cells for alleviating skeletal muscle atrophy in high glucose and lysophosphatidylcholine environment
  • Method for culturing CD29&lt;+&gt; human umbilical cord mesenchymal stem cells for alleviating skeletal muscle atrophy in high glucose and lysophosphatidylcholine environment
  • Method for culturing CD29&lt;+&gt; human umbilical cord mesenchymal stem cells for alleviating skeletal muscle atrophy in high glucose and lysophosphatidylcholine environment

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 ɑ-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 method for culturing CD29<+> human umbilical cord mesenchymal stem cells for alleviating skeletal muscle atrophy in a high glucose and lysophosphatidylcholine environment. The method comprises the following steps: uniformly mixing an anti-human CD29<+> antibody with polylysine, and spreading in a culture dish; washing an umbilical cord till the bloodstain has no bloodstain and is whitened by using a PBS (Poly Butylene Succinate) solution; cutting tissue into small fragments, splitting longitudinally, removing cortices of blood vessels and the umbilical cord, and cutting Wharton jelly into small pieces; cutting the small umbilical cord tissue pieces into mud, adding 0.25% collagenase IV, performing digestion, uniformly mixing an alpha-MEM (Minimum Eagle's Medium) culture liquid with 10% of fetal calf serum, centrifuging, putting a proper amount of the alpha-MEM culture liquid with 10% of fetal calf serum into the culture dish, and performing inverted culture inside a 5% CO2 culture tank; and removing supernate, adding a proper amount of the alpha-MEM culture liquid with 10% of fetal calf serum, replacing the liquid once every other three days, and performing continuous cell culture when the fusion rate is up to 85%. By adopting the method, a theoretical basis and an experiment basis for later study and research on medicines for treating skeletal muscleatrophy in high glucose and lysophosphatidylcholine environments are made, and the method has wide 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 Applications(China)
IPC IPC(8): C12N5/0775
Inventor 唐静
Owner JILIN TUO HUA BIOTECH
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