Use of fingolimod or its salt in the treatment of cystic diseases
A technology for polycystic kidney disease and cyst, applied in the field of medicine, can solve the problems of the impact on the quality of life of patients, the lack of effective treatment methods for ADPKD, and the lack of drugs to delay the disease.
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Embodiment 1
[0082] Example 1: Effect of fingolimod on proliferation of human polycystic kidney cyst lining epithelial cell line WT9-12
[0083] Human polycystic kidney cyst lining epithelial cell line WT9-12 was cultured in DMEM / F12 medium with 10% fetal bovine serum, added with double antibodies, digested with trypsin and passaged. After about 70% of cell confluence, trypsinize, make cell suspension, count cells under microscope, and then inoculate in 96-well plate, 6×10 per well 3 cells. After synchronization, fingolimod treatment was given, and the concentration gradient of fingolimod was 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.175 μM, 1.588 μM, and 0.781 μM. The changes of cell proliferation after administration for 24, 48 and 72 hours were observed by MTT method. Aspirate the culture medium at each time point, add 10 μl of MTT (5 mg / ml aqueous solution) to each well, incubate at 37°C for 3 hours, suck out the culture medium, add 100 μl of DMSO to each well, shake at room temperature for ...
Embodiment 2
[0088] Example 2: Effects of fingolimod on apoptosis and cell cycle of human polycystic kidney cyst lining epithelial cells WT9-12
[0089] WT9-12 cells were treated with 2×10 5 Cells / well were seeded in a 6-well plate, and when 60-70% confluence was reached, the cells were treated with 1 μM, 2 μM, and 10 μM of fingolimod for 24 hours. Cells and supernatant were collected, trypsinized and centrifuged, washed with cold 1×PBS and then centrifuged at 1000 rpm / min for 10 minutes. Discard the supernatant, add 1×binding buffer, adjust the cell concentration to 1×10 6 cells / ml. Add 5 μl Annexin V and 1 μl PI (100 μg / ml) working solution to every 100 μl cell suspension, incubate at room temperature for 15 minutes in the dark, then add 400 μl 1× binding buffer, mix well and place on ice, then use flow cytometry as soon as possible Apoptosis was measured by cytometer, and the results are shown in Table 2 and figure 2 , fingolimod can significantly induce apoptosis in WT9-12 cells. ...
Embodiment 3
[0097] Example 3: Effect of fingolimod on HDACs in human polycystic kidney cyst lining epithelial cell line WT9-12
[0098] WT9-12 cells in 8×10 5 Cells / dish were seeded in a 10 cm culture dish, and when 60-70% confluence was reached, the cells were treated with 0 μM or 2.5 μM of fingolimod for 24 hours. Trypsinize and harvest the cells. Wash the cells with cold 1×PBS and centrifuge at 1000rpm / min for 10 minutes to remove the PBS. Add 1ml Trizol solution to dissolve the cells completely and extract RNA. Use HisScript 1 st The strand cDNA synthesis kit (Vazyme) reverse transcribes RNA into cDNA. The cDNA obtained by reverse transcription was used for Real time-PCR analysis. Such as Figure 4 As shown, fingolimod can inhibit the transcription of some HDACs.
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