Application of cholecystokinin receptor inhibitor-Devazepide in preparation of anti-fibrosis pharmaceutical preparations
A receptor inhibitor, cholecystokinin technology, which is used in the preparation of anti-myofibroblast proliferation and anti-fibrosis therapeutic drugs.
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Embodiment 1
[0028] Example 1 Devazepide inhibits the proliferation of myofibroblast WPMY-1.
[0029] Purchase prostate tissue-derived myofibroblast WPMY-1 (ATCC), culture WPMY-1 cells in DMEM medium containing 10% FBS and a cell incubator with 5% CO2 at 37 degrees, and when the cells are in good condition, follow the daily Hole 1*10 4 Cells were inoculated into 96-well cell culture plates at a density of 24 h, and the cells were treated with different concentrations of Devazepide ( figure 1 ), using the CCK-8 cell proliferation detection kit to detect cell proliferation activity 48h after drug treatment ( figure 2 ).
[0030] WPMY-1 cells in good condition were used to make cell slides, the cells were treated with Devazepide (25uM), and the expression of KI-67 related to WPMY-1 cell proliferation was detected by immunofluorescence ( image 3 ). At the same time, the RNA of WPMY-1 in myofibroblasts treated with Devazepide (25uM) for 24 hours was extracted, and the expressions of KI-67...
Embodiment 2
[0031] Example 2 Devazepide induced myofibroblast WPMY-1 cell cycle G2 / M arrest.
[0032] Cultivate WPMY-1 cells in vitro, and inoculate 6-well cell culture plates when the cell viability is good. After the cells adhere to the wall, treat WPMY-1 cells with 25uM Evazepide, harvest the cells at 24 and 48 hours after treatment, and fix with 70% ice ethanol 48h, after further processing, the cell cycle was detected by flow cytometry ( Figure 5 ). The expression of cell cycle-related proteins was detected by immunoblotting ( Image 6 ). In summary, it was confirmed that Devazepide caused WPMY-1 cell cycle G2 / M phase arrest, and up-regulated the expression of p-Chk1 and p-Chk2.
Embodiment 3
[0033] Example 3 Devazepide promotes apoptosis of myofibroblast WPMY-1.
[0034] WPMY-1 cells were cultured in vitro, and when the cells were in good condition, the cells were treated with Devazepide (25uM), and the apoptosis was detected by AnnexinV-FITC / PI double staining after treatment ( Figure 7 , Figure 8 ); extract the total protein of cells treated with Devazepide, detect the expression of apoptosis-related proteins, and up-regulate the expression of activated Caspase3 ( Figure 9 ). In summary, it is confirmed that Devazepide induces the apoptosis of myofibroblast WPMY-1.
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