Application of cucurbitacin D in preparing glioma cell activity inhibitor
A technology for glioma cells and activity inhibitor, which is applied in the application field of cucurbitacin D in the preparation of glioma cell activity inhibitor, can solve the problem that there is no anti-cancer effect of malignant glioma and the like
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Embodiment 1
[0022] Example 1: Effects of cucurbitacin D on the proliferation and toxicity of glioma cell U-87MG
[0023] Glioma cell U-87MG (purchased from ATCC) was prepared with DMEM medium containing 10% fetal bovine serum, and added to a 96-well plate at 100 μl per well (5000 cells / well). The culture plate was pre-incubated for 24 hours in an incubator (37°C, 5% CO 2 ). DMEM medium containing 10% fetal bovine serum containing final concentrations of 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, and 1 μM cucurbitacin D was added to the wells of the culture plate, 200 μl per well. The plates were incubated in the incubator for 48 hours. Discard the medium and replace with DMEM medium containing 10% CCK8. Incubate the plate for 1 hour in the incubator. The absorbance (OD value) at 450 nm was measured with a microplate reader. The culture wells containing no glioma cells U-87MG and cucurbitacin D and containing CCK8 were used as blank control, and the culture wells containing no cucurbitacin D b...
Embodiment 2
[0030] Example 2: Inhibition of Cucurbitacin D on Proliferation of Different Types of Glioma Cells
[0031] 100 μl of glioma cells U-87MG (5000 / well) were prepared in a 96-well plate with DMEM medium containing 10% fetal bovine serum. The culture plate was pre-incubated for 24 hours in an incubator (37°C, 5% CO 2 ). DMEM medium containing 10% fetal bovine serum containing 0.5 μM cucurbitacin D was added to the culture plate, 200 μl per well, and the DMEM medium containing 10% fetal bovine serum containing an equal amount of PBS instead of cucurbitacin D was added as a control. The culture plate was incubated in the incubator for an appropriate period of time (0-6 days), and the cells in the well plate with and without drug addition were counted at a fixed time every day and the growth curve was drawn.
[0032] Under the same conditions, the glioma cell lines A172 and U-251 MG were used instead of U-87 MG for experiments, and the results were as follows: figure 2 As shown, ...
Embodiment 3
[0033] Example 3: Analysis of the effect of cucurbitacin D on cell proliferation by BrdU labeling method
[0034] 1. Cell sample preparation:
[0035] The U-87 MG cells with logarithmic growth were taken, and when the confluence reached 70%, the final concentrations of 0.25 μM and 0.5 μM cucurbitacin D were added to continue to culture for 48 h.
[0036] 2. BrdU mark:
[0037] A. After 48 hours, add BrdU (5-bromodeoxyuridine) at a final concentration of 90 g / L, and incubate at 37° C. for 2 hours.
[0038] B. After incubating the medium for 2 hours, discard the medium and wash the cells twice with PBS, 5 minutes each time.
[0039] 3. Cell immobilization:
[0040] Add 200 μl of cell fixation solution (4% paraformaldehyde) to each well and incubate at room temperature for 30 minutes, discard the fixation solution. Wash with PBS 3 times, 5 minutes each time.
[0041] 4. HCl fixation:
[0042] Add enough 2M HCl aqueous solution to cover the cells in each well of the culture ...
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