Novel medical compound being capable of synergetically suppressing growth of tumors
A composition and drug technology, applied in the field of new pharmaceutical compositions capable of synergistically inhibiting tumor growth
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Embodiment 1
[0054] The synergistic inhibitory effect of LDM and TMZ on the proliferation of rat glioma C6 cells and human glioma U87 cells
[0055] The cytotoxicity of the two drugs was detected by the MTT method, and the operation was as follows:
[0056] 1). Take the cells in the logarithmic growth phase, digest and count them at 1×10 3 cells / well (C6 cells), 1.5×10 3 pcs / well (U87 cells) or 2×10 3 Cells were seeded in 96-well cell culture plates at a density of one per well (rCMEC cells), with at least 3 parallel wells in each group.
[0057] 2). After 24 hours, different concentrations of drugs were added for treatment.
[0058] 3). After 72 hours, add 5 mg / ml MTT solution prepared with PBS buffer, add 20 μl to each well, and incubate at 37° C. for 4 hours.
[0059] 4). Aspirate the supernatant, add 150 μl DMSO, and shake for 10 minutes to fully dissolve the crystals.
[0060] 5). Measure the absorbance (A) of each well at 570 nm with a microplate reader.
[0061] 6). The calcu...
Embodiment 2
[0064] The synergistic effect of the combined application of LDM and TMZ on the apoptosis of rat glioma C6 cells and human glioma U87 cells
[0065] Annexin V-FITC / PI (fluorescein isothiocyanate-labeled annexin V / propidium iodide) method was used to detect the effect of drug combination on cell apoptosis. The operation is as follows:
[0066] 1) Inoculate cells: 10×10 4 pcs / well (C6 cells), 15×10 4 pcs / well (U87 cells) or 20×10 4 Cells were seeded at a density of 1 / well (rCMEC cells) in 6-well cell culture plates.
[0067] 2) Cultivate in an incubator with 5% CO2 concentration at 37°C for 24 hours.
[0068] 3) Drug-treated cells, rat brain microvascular endothelial cells and rat glioma C6 cells were treated with LDM 0.1nmol / L and TMZ 300μmol / L or a combination of the two; human glioma U87 cells were treated with LDM 0.05 nmol / L and TMZ 210μmol / L or a combination of both.
[0069] 4) Continue culturing for 72 hours in an incubator with 5% CO2 concentration at 37°C.
[0...
Embodiment 3
[0077] The synergistic inhibitory effect of LDM and TMZ on the tubule formation ability of rat brain microvascular endothelial cells
[0078] Tube formation (Tube formation) assay was used to detect the effect of drug combination on angiogenesis. The operation is as follows:
[0079] 1) Pre-cool the pipette tip and the 96-well culture plate.
[0080] 2) Operate on an ice platform, add 50 μl of Matrigel gel, and place at 37° C. for 30 minutes to pre-solidify.
[0081] 3) Collect 2-3 generations of cells in good growth condition, digest with trypsin, centrifuge and count.
[0082] 4) Take 2×10 diluted 180 μl culture solution (containing 10% common fetal bovine serum) 4 cells / well.
[0083] 5) Treatment with different drug combinations (LDM: 0.1 nmol / L, TMZ: 200 μmol / L, and LDM 0.1 nmol / L+TMZ 200 μmol / L).
[0084] 6) Put it in the incubator for 16 hours until the lumen-like structure is formed.
[0085] 7) Take a picture under an inverted microscope at a magnification of ×...
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