Application of gastrodin to preparation of medicine for protecting human umbilical vein endothelial cells
A technology of vascular endothelium and umbilical vein, applied in the field of cell biology and pharmacology, can solve the problems of unreported hypertension and achieve the effect of small side effects, good effect and low dosage
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Embodiment 1
[0016] The establishment of embodiment 1 cell damage model
[0017] Grouping: The experiment was divided into a blank group, a control group, and a different concentration of Hcy group; each group had 5 replicate wells.
[0018] Cell model establishment:
[0019] (1) Collect HUVEC cells in the logarithmic phase. After the cells are 90% full, aspirate the original medium, add 150 μL of Hcy solution with different concentrations in the experimental group, add 150 μL of medium in the control group and blank group, and the final volume of each well is 150 μL.
[0020] (2) Place in 5% CO2, 37°C cell culture incubator and incubate for 6, 12, 18, 24, 30 h, observe under an inverted microscope; add 20 μL of MTT solution to each well, and continue culturing in a 37°C constant temperature incubator for 4 h.
[0021] (3) Aspirate the supernatant, add 150 μL DMSO to each well, shake on a shaker in the dark for 10 minutes to fully dissolve the blue crystals.
[0022] (4) Measure the abso...
Embodiment 2
[0024] The influence of embodiment 2 gastrodin on cell activity
[0025] Drug Action Concentration Establishment:
[0026] (1) Collect HUVEC cells in the logarithmic phase. After the cells are 85% full, suck out the original medium, add 100 μL of 2 mmol / L Hcy solution and 100 μL of different drug concentration solutions in the experimental group, and add 200 μL of 0.5 mmol / L Hcy in the modeling group 150 μL of culture medium was added to the control group and the blank group.
[0027] (2) Incubated in 5% CO2, 37°C cell culture incubator for 24 hours, observed under an inverted microscope; 20 μL of MTT solution was added to each well, and cultured in a constant temperature incubator at 37°C for 4 hours.
[0028] (3) Aspirate the supernatant, add 150 μL DMSO to each well, shake on a shaker in the dark for 10 minutes to fully dissolve the blue crystals.
[0029] (4) Measure the absorbance of each well at a wavelength of 490 nm with a microplate reader.
[0030] Let the relativ...
Embodiment 3
[0031] The influence of embodiment 3 gastrodin on ROS
[0032] (1) Spread the cells on a 6-well plate. After the cells grow to about 70%, aspirate the original medium, add 1mL of 2mmol / L Hcy solution and 1mL of different drug concentration solutions in the experimental group, add 1mmol / L Hcy in the modeling group 2mL, the control group was added 2mL of culture medium, and the final volume of each well was 2mL.
[0033] (2) Incubated in 5% CO2, 37°C cell culture incubator for 24 hours, observed under an inverted microscope, sucked out the original medium, washed 3 times with pre-cooled PBS, added pre-cooled PBS containing 1 μmol / L DCFH-DA, 37 Incubate the cells at ℃ for 30 min.
[0034] (3) Aspirate the original PBS, wash with pre-cooled PBS 3 times, add 500 μL of paraformaldehyde, and place in a 37°C incubator for 15 minutes to fix.
[0035] (4) Pour off the paraformaldehyde, wash 3 times with pre-cooled PBS, and finally add 2 mL of pre-cooled PBS. Observe cells under a flu...
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