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
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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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