Application of atractylone in preparation of Sirt3 gene activator
A technology of activator, atractylodes ketone, applied in the application field of atractylodes ketone in the preparation of Sirt3 gene activator
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Embodiment 1
[0025] Embodiment 1: atractylone is pair SIRT3 expression of influence
[0026] For the detection of atractylone SIRT3 The effect of expression was detected by Western Blot, immunofluorescence and RT-PCR. The specific operation is as follows:
[0027] 1) Western Blot detection of glioma treated with atractylone SIRT3 Changes in protein expression: Inoculate the cell suspension in a 6-well plate, add a culture solution containing 100 μM atractylone, and culture in an incubator for 24 h. The protein was extracted, electrophoresed by 10% SDS-PAGE, transferred to the membrane (320 mA, 70 min), blocked at room temperature for 1 h, and incubated overnight at 4 °C with the primary antibody. The next day, wash with TBST 3 times, 5 min each time. After incubation with the secondary antibody for 1 h at room temperature, the cells were washed 3 times with TBST, 15 min each time. development.
[0028] 2) Immunofluorescence detection of glioma cells treated with atractylone SIR...
Embodiment 2
[0031] Example 2: Effect of atractylone on the proliferation of glioma cells
[0032] In order to detect whether atractylone has an effect on the proliferation of glioma, we detected the effect of atractylone on the proliferation of glioma by CCK8 assay, clone formation assay, and PH3 immunofluorescence. The specific operation is as follows:
[0033] 1) Inoculate the cell suspension in a 96-well plate (100 μL / well, 3000 cells / well). The plate was pre-incubated for 24 h in an incubator (37°C, 5% CO 2 ). Change the culture medium to one containing different concentrations of atractylone, and add 10 μL of CCK8 solution to each well (be careful not to generate air bubbles in the well). Incubate the culture plate in the incubator for 1-4 h. The absorbance at 450 nm was measured with a microplate reader.
[0034] 2) Inoculate the cell suspension (500 cells / well) in a 6-well plate, add the culture solution containing 100 μM atractylone, and culture it in the incubator for 5 days...
Embodiment 3
[0037] Example 3: Effect of atractylone on migration of glioma cells
[0038] In order to detect whether atractylone has an effect on the migration of glioma, we tested the effect of atractylone on the migration of glioma by scratch test and transwell. The specific operation is as follows:
[0039] 1) Use a 1 mL pipette tip to evenly draw a horizontal line along the cover of the 6-well plate, and try to hang down to the horizontal line scratch on the back. The pipette tip should be vertical and cannot be tilted. Wash the cells 3 times with PBS, remove the scratched cells, and add serum-free medium. Put in 37℃, 5% CO 2 Incubator, culture. Take samples at 0, 24, and 48 hours and take pictures.
[0040] 2) Take 200 µL of cell suspension (containing 10 4 cells without serum) into the transwell chamber, and 500 µL of serum-containing medium in the lower chamber of the 24-well plate. Cultivate for 24 h. PFA fixed, stained with crystal violet, and photographed.
[0041] Such ...
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