Use of chrysophanic acid in inhibiting vascularization
An angiogenesis and rhein technology, applied in the field of compounds, can solve the problems of unclear anti-cancer mechanism and limited anti-cancer effect, and achieve the effects of less drug resistance, high curative effect and small dose
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Embodiment 1
[0018] Example 1: The inhibitory effect of rhein on the proliferation of human vascular endothelial cells.
[0019] Human umbilical vein endothelial cells (HUVEC, purchased from Cascade Biologics, Cot: C-003-5C) were cultured in Medium 200 (purchased from Cascade Biologics) containing 10% fetal bovine serum (FBS) (37°C, 5 %CO 2 , 95% humidity), the fourth-generation cells were inoculated in a 96-well culture plate at a density of 4000 / 250ul, and a control group, a drug group with various concentration gradients and a blank control were set, and 3 replicate holes were made in each group. When the cell growth density reaches 80%, add 5ul of rhein (dissolved in DMSO) with different concentration gradients respectively, so that the final concentrations are respectively 1000uM, 100uM, 10uM, 5uM and 1uM, add 5ulDMSO to the control group, and continue to cultivate (M200 medium, 2% FBS) 48 hours later, add 5mg / ml tetrazolium bromide (MTT) 20ul, continue to incubate at 37°C for 4 hour...
Embodiment 2
[0020] Example 2: Inhibitory effect of rhein on tube formation ability of human vascular endothelial cells.
[0021] Add BD Matrigel to each well of a 24-well culture plate TM Matrix original glue 200ul, make it polymerized into gel, the fourth generation human umbilical vein endothelial cell (HUVEC) suspension was inoculated into the 24-well plate coated with Matrigel glue at a density of 30000cell / 500ul, set the control group, each concentration gradient In the drug group, 10ul of rhein with a concentration gradient (respectively 1uM, 10uM, 100uM, 1000uM, dissolved in DMSO) was added to the drug group, and 10ul of sterile DMSO was added to the control group, and 3 replicate holes were made in each group. 37°C, 5% CO 2 , 95% humidity for 24 hours, OLYMPUS CK40-RFL optical microscope to observe the formation of vascular endothelial cell tubes, take 4 low-power fields from each well and count them, and take pictures with an OLYMPUS CK40 digital camera. The results are shown ...
Embodiment 3
[0022] Example 3: The inhibitory effect of rhein on the migration ability of human vascular endothelial cells.
[0023] 1. Preparation of chemokines
[0024] The NIH3T3 cells that grew well the next day after passage were used. Gently rinse with serum-free DMEM twice. Serum-free DMEM, 5% CO 2 , 37°C for 24-48 hours. Collect the cell supernatant. Centrifuge (12000g, 4°C, 10min). Filter the supernatant (0.22um filter membrane) and store in aliquots (-20°C).
[0025] 2. Matrigel invasion test
[0026] Take 25ul of diluted Matrigel (the original glue was diluted with DMEM at a ratio of 1:2) and add it to the chamber on the Transwell plate to cover the entire surface of the polyester film, and incubate at 37°C for 30min to make Matrigel polymerize into a gel. The fourth-generation human umbilical vein endothelial cell (HUVEC) suspension was inoculated into the upper chamber at a density of 30000 / 250ul, washed 3 times with PBS, digested and harvested from the culture flask. ...
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