Application of traditional Chinese medicine molecular sophocarpine in preparation of medicine for treating glioblastoma
A technology for glioblastoma and sophocarpine, applied in the fields of application, antineoplastic drugs, drug combinations, etc., can solve the problems of no effective treatment methods, and achieve good results, easy operation, and high success rate
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Embodiment 1
[0032] Embodiment 1: Detect whether sophocarpine has an effect on the proliferation of glioblastoma, and detect the effect of sophocarpine on the proliferation of glioblastoma by CCK8 experiment, PH3 immunofluorescence experiment, and cell clone formation experiment:
[0033] (1) CCK8 experiment: Inoculate the suspension of C6 and U251 cells in a 96-well plate (100 μL / well, 3000 cells / well). The culture plate was pre-incubated for 24 h in an incubator (37°C, 5% CO 2 ); replace the culture solution with structural formulas containing different concentrations (0, 0.5, 1, 2, 4mM) such as figure 1 (A) Complete culture solution of the indicated sophocarpine, 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 1-2 h. The absorbance at 450 nm was measured with a microplate reader.
[0034] Such as figure 1 (B) and (C) it can be seen that the higher the concentration of sophocarpine, the w...
Embodiment 2
[0040] Example 2: Detect whether sophocarpine affects the migration of glioblastoma, and detect the migration effect of sophocarpine on glioblastoma by scratch test and Transwell test:
[0041] (1) Scratch test: use a 20 μL 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 C6 and U251 cells under the condition were added with medium containing 2% serum, placed in a 37°C, 5% CO2 incubator, and cultivated; at 0h and 24h, samples were taken, photographed, and the imageJ software was used to analyze the healing of scratches.
[0042] figure 2 (A), (B), (E), (F) shows that the higher the concentration of sophocarpine, the weaker the migration ability of cells;
[0043] (2) Transwell experiment: Take 200 μL of C6 and U251 cell suspension (containing 104 cells, no serum) and add it to the Transwell chamber, add 500 μL serum-containing medium to the lower chamber of the 24-well plate...
Embodiment 3
[0046] Example 3: Detecting whether sophocarpine affects the cell cycle and apoptosis of glioblastoma, and detecting the effect of sophocarpine on the cell cycle and apoptosis of glioblastoma by flow cytometry, Western Blot and PI staining The role of:
[0047] (1) Western Blot combined with flow cytometry to detect the effect of sophocarpine on cell cycle arrest:
[0048] After the U251 cells grew to a suitable density in a 6cm dish, they were cultured for 24 hours in the culture medium of the control and 2mM sophocarpine, and the cells were collected by adding an appropriate amount of RIPA lysate containing protease inhibitors, and the protein was quantified by the BCA method. Loading Buffer sample preparation, SDS-PAGE gel running, PVDF membrane transfer under 20% methanol, 5% skimmed milk for blocking at room temperature for 1 hour, incubate antibody cyclin D1 overnight at 4°C, and incubate the corresponding species source-coupled HRP secondary antibody the next day, Afte...
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