Application of ADDA and/or DMTA in preparing medicine for improving expression level of gamma-globin
A technology of expression quantity and globin, which is applied in the direction of drug combination, pharmaceutical formula, medical preparations containing active ingredients, etc.
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[0021] 1. Materials and methods
[0022] 1) compound
[0023] ADDA (adenosine-2', 3'-dialdehyde), DMTA (5'-deoxy-5'(methyl-thio)-adenosine) were purchased from Sigma, USA. ADDA was dissolved in deionized water to make a 10mM stock solution; DMTA was dissolved in DMSO (dimethyl sulfoxide) to make a 0.5M stock solution.
[0024] 2) Cell culture
[0025] Human erythroid K562 cells were cultured at 37°C in RPMI1640 medium, 10% calf serum, 10 5 U / L penicillin, 100mg / L streptomycin, 5% CO 2 . K562 cells were treated with ADDA at a final concentration of 50 μM or 0.1 mM DMTA for 72 hours, and the cells were collected.
[0026] human bone marrow CD34 + Cells were separated with CD34 immunomagnetic beads (Miltenyi Biotec, USA), and 50 μM ADDA or 100 μM DMTA was added, and physiological saline or DMSO (volume ratio 2. Specifically, the cytokines SCF (100 ng / ml), IL-3 (10 ng / ml) and Flt-3 (10 ng / ml) were added to the culture medium for 1 week, and then only the cytokine EPO (5 U / m...
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