Compound for delaying senescence of human mesenchymal stem cells and application of same
A technology of compounds and stem cells, applied in the direction of effective ingredients of hydroxyl compounds, medical preparations containing active ingredients, drug combinations, etc., can solve the problems that the effect of quercetin has not been reported yet
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Embodiment 1
[0054] Embodiment 1, the screening of natural product library
[0055] This example involves the screening of a natural product library (133 compounds, purchased from Selleck, Cat. No. L1400) on human progeria mesenchymal stem cells (WS MSCs). The method used was the MTS method, and the reagents were purchased from Promega. The quercetin used in subsequent experiments was quercetin dihydrate, which was purchased from Selleck Company.
[0056] The specific method is as follows: Firstly, the fifth-generation WS MSCs were seeded in a 96-well plate, 3000 cells / well, cultured overnight, and replaced with MSC medium containing 1 μM of various compounds on the second day, cultured for 7 days, and the next day Change fluid. The negative control was the solvent dimethyl sulfoxide (DMSO, 1‰), and the positive control was Vitamin C (VC, 280 μM). After 7 days of drug treatment, MTS detection was carried out, the absorbance value (OD value) at 490 nm was measured, and the cell viability...
Embodiment 2
[0059] Embodiment 2, the effective concentration detection of preferred 10 small molecule compounds
[0060] After screening, 10 small molecules such as quercetin, rhein, luteolin, formononetin, honokiol, berberine, kaempferol, phylloxine, synephrine, licochalcone A, etc. were obtained , and studied the effective concentration at the level of WS MSCs cells. see results image 3 .
[0061] Detection method Cell counting. The fifth generation (p5) WS MSCs were seeded in a 6-well plate at 30,000 cells / well, cultured overnight, and replaced with medium containing 100nM, 250nM, 500nM and 1μM of various compounds on the next day, and cultured From about 20 days to the sixth passage, the medium was changed every other day (fresh MSC medium containing various compounds at different concentrations). The negative control was the solvent dimethyl sulfoxide (DMSO). After drug treatment to the sixth passage, cells were counted and analyzed after trypan blue staining. It was found tha...
Embodiment 3
[0081] Example 3. Drug-treated WS MSCs have better osteogenesis, cartilage differentiation ability and anti-aging phenotype.
[0082] Using quercetin 100nM and negative control DMSO (1‰) to treat the seventh generation of WS MSCs (drug treatment since the fifth generation), differentiate into osteoblasts, cartilage and adipocytes ( Figure 5 ). The results showed that the ability of WS MSCs to differentiate into osteoblasts and chondrocytes was enhanced after quercetin treatment.
[0083] Osteogenic Medium:
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[0086] Cartilage Medium:
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[0088] Fat Medium:
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[0090] At the same time, progeria mesenchymal stem cells treated with quercetin had a phenotype of delaying aging ( Image 6 ). A, Ki67 immunofluorescence staining of progeria mesenchymal stem cells treated with quercetin, the rate of Ki67 positive cells increased after quercetin treatment; B, Western blot protein quantification of senescence markers p16 and p21 proteins. ...
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