Application of plumbagin in preparing the medicine for preventing the blood vessel from regenerating
A technique for angiogenesis and plumbagin is applied in the application field of plumbagin in the preparation of angiogenesis-inhibiting drugs, and can solve the problems of no plumbagin inhibiting angiogenesis and the like.
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Embodiment 1
[0020] Example 1: plumbagin inhibits proliferation of endothelial cells.
[0021] Purpose and principle: MTS assay was used in endothelial cell proliferation assay. MTS assay is a colorimetric method to determine the number of cell proliferation. In living cells, NADPH dehydrogenase converts MTS tetrazolium salt compound into a colored substance soluble in tissue culture fluid. The number of living cells of these cell lines was highly correlated within a certain range. This model can be used to evaluate the effect of drugs on the proliferation ability of each batch of cells.
[0022] Method: Divide 1×10 5 cells / ml HMEC-1 (human microvascular endothelial cells) or HUVEC (human umbilical vein endothelial cells) cell suspension was inoculated in 96-well plates, 50 μl per well, and 50 μl of culture medium containing plumbagin with different concentrations were given at 5% CO 2 Incubate at 37°C for 48 to 72 hours, then add 20 μl of detection solution to each well, incubate for ...
Embodiment 2
[0024] Example 2: plumbagin inhibits endothelial cell migration
[0025] Purpose and principle: The cell migration test is also called in vitro scratch test. In this experiment, scratches were made on a single layer of endothelial cells, and the migration of cells at the edge of the scratches to the gap was observed and recorded under a microscope to evaluate the effect of drugs on the migration ability of endothelial cells.
[0026] Method: Place 4×10 4 Each / ml HUVEC cells were seeded in a 6-well plate, 2ml / well. Make a scratch in each well after the cells grow to more than 90%. Wash the dead cells floating on the upper layer at this time, and then add the medium containing 4ng / ml VEGF with different drug concentrations into the six-well plate, in 5% CO 2 Culture in an incubator at 37°C.
[0027] Results and evaluation: Observing under a microscope, after the scratch gaps in the VEGF control group were completely covered by cells, photographs were taken under the microsco...
Embodiment 3
[0028] Example 3: Experiment of plumbagin inhibiting endothelial cell migration in Boyden chamber
[0029] Purpose and principle: Boyden cell culture chamber was used in this experiment. Under the induction of growth factors, cells can pass through the polycarbonate filter membrane with a pore size of 8 μm. This experiment can detect the effect of drugs on the migration ability of endothelial cells under resistance conditions.
[0030] Method: Coat the 6-well plate with 0.1% gelatin, add 600 μl of VEGF containing 10ng / ml to the six-well plate outside the small chamber 165 endothelial cell culture medium. The concentration of 50 μl inserted into the small chamber is 4×10 5 cells / ml HUVEC cell suspension and 50 μl medium containing different concentrations of plumbagin, 37°C 5% CO 2 Incubate for four hours in the incubator.
[0031] Results and evaluation: Four hours later, the cell culture medium inside and outside the small chamber was aspirated, and the migrating cells in...
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