Method and medium for amplifying bone-marrow mesenchymal stem cells and application
A bone marrow mesenchymal and stem cell technology, applied in the field of bioengineering, can solve the problems of limiting the research and clinical application of BMSCs, deterioration of biological activity and function, and decreased proliferation ability, achieving good application prospects and promoting the formation and proliferation of blood vessels. Ability Increase Effect
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Embodiment 1
[0060] 1. Isolation of rat BMSCs
[0061] 1) Male SD rats (purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine) were killed with ether, soaked in 75% alcohol for 5 minutes; a surgical towel was placed in the ultra-clean table, and the skin of the rat was cut in a sterile tray.
[0062] 2) Use sterile scissors and other surgical instruments to separate the rat femur, and use a syringe to absorb α-MEM culture solution (purchased from GIBCO) and repeatedly rinse the bone marrow cavity until the color turns white (about 4-5 times).
[0063] 3) Filter the washing solution with a 400-mesh sieve, centrifuge, discard the supernatant, and collect the cells.
[0064] 4) Add 5mL of rat lymphocyte separation medium with a density of 1.077g / mL (purchased from Tianjin Haoyang Company) into a centrifuge tube, resuspend the bone marrow cell pellet with 5mLα-MEM culture medium, and then lightly Lightly added to the rat lymphocyte separation me...
Embodiment 2
[0080] 1. Functional identification of BMSCs after combined treatment with bFGF+VEGF factors
[0081] 1) Take the BMSCs obtained from the bFGF treatment group, the bFGF+VEGF treatment group and the control group respectively in Example 1, digest and collect the cells by blowing and centrifuging.
[0082] 2) After resuspending with PBS, divide into 4 equal portions and add to 4 1.5mLEP tubes respectively.
[0083] 3) Centrifuge and remove the supernatant.
[0084] 4) Add antibodies to each tube, and incubate at 4 degrees for 30 minutes. Add 2 μL of PE-CD90 (purchased from Biolegend), PE-CD45 (purchased from Biolegend), and FITC-CD105 (purchased from Millipore) respectively, the system is 100 μL), and the other is the control group.
[0085] 5) After washing with PBS, the surface molecules of BMSCs were detected by flow cytometry (Becton Dickinson, USA). The expression results of the cell surface molecules are shown in Table 1.
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