Application of 2,3-dihydrogen-3-hydroxymethyl-6-amino-[1,4]-benzoxazine in preparing medicaments for inducing embryonic stem cells to differentiate towards vascular endothelial cells
A technology of embryonic stem cells and benzoxazine, applied in the field of benzoxazine derivatives
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Embodiment 1
[0066] Example 1 Preparation of 2,3-dihydro-3-hydroxymethyl-6-amino-[1,4]-benzoxazine
[0067] Addition of 2,4-dinitrophenoxymethyloxirane (0.480 g, 2 mmol) and acetic acid (1 mL, 17.5 mmol) in EtOH / H at reflux temperature 2 Iron powder (0.670 g, 12 mmol) was added to the O (v / v=6:1, 100 mL) solution. The reaction mixture was continued to reflux for 120 minutes. Cool to room temperature to saturate Na 2 CO 3 The solution was adjusted to pH=8, filtered with celite, the ethanol and water in the filtrate were concentrated, and the obtained solid was washed with ethanol (10 mL×4). ethanol phase with anhydrous MgSO 4 Dry and filter to obtain a crude product; the above crude product is separated by silica gel column chromatography (ethanol as a developing solvent) to obtain 2,3-dihydro-3-hydroxymethyl-6-amino-[1,4]-benzoxane Azine (0.149 g, 42%).
Embodiment 2
[0068] Example 2 Detection of the influence of 2,3-dihydro-3-hydroxymethyl-6-amino-[1,4]-benzoxazine on the morphology of embryonic stem cells
[0069] Embryonic stem cells were divided into 2×10 4Cells / well were evenly seeded in a 24-well plate with DMEM containing leukemia inhibitory factor, L-glutamine, penicillin, β-mercaptoethanol, non-essential amino acids, nucleotides, and 15% fetal calf serum. (high glucose) for 24 hours. After the cells adhered to the wall, the waste liquid was discarded and washed with 1×PBS. Set normal group: cultivated under the culture condition of removing leukemia inhibitory factor; solvent control group: add DMSO and culture under the culture condition of removing leukemia inhibitory factor; experimental group: add concentration of 0.5μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM of 2,3-dihydro-3-hydroxymethyl-6-amino-[1,4]-benzoxazine were incubated. Culture conditions: 37°C, CO 2 Cultured in an incubator. The medium was changed regularly...
Embodiment 3
[0071] Example 3 Detection of Vascular Endothelial Cell Marker (Flk-1)
[0072] Embryonic stem cells were evenly seeded in 48-well plates, and DMEM (high glucose ) for 24 hours. After the cells adhered to the wall, the waste liquid was discarded and washed with 1×PBS. Set up normal group: cultured under the culture condition of removing leukemia inhibitory factor; solvent control group: added DMSO culture under the culture condition of removing leukemia inhibitory factor; experimental group: added concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM and 40 μM of 2,3-dihydro-3-hydroxymethyl-6-amino-[1,4]-benzoxazine were incubated. 37°C, CO 2 Culture in the incubator, replace the culture medium every day, discard the waste liquid after 10 days, wash with 0.1×PBS three times, fix with 4% paraformaldehyde for 15 minutes, after washing, block with goat serum for 20 minutes, add Flk-1 The primary antibody was incubated overnight at 4°C, washed three times with 0.1×PBS, add...
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