Application of linarin to preparing kidney tubular epithedial cell ischemia reperfusion injury protecting drugs/drug combinations
An ischemia-reperfusion and epithelial cell technology, which can be used in drug combinations, pharmaceutical formulations, and medical preparations containing active ingredients, etc. It can solve the problem of unclear role of ETS2 protein, and achieve the effect of broadening the scope of application.
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Embodiment 1
[0023] Example 1 HK2 cell ischemia-reperfusion model
[0024] Plant HK2 cells into a 35mm cell culture dish and culture in 2mL complete medium (89% DMEM + 10 fetal bovine serum + 1% penicillin streptomycin mixture), 5% carbon dioxide, and a humid environment to 70% confluence At the same time, the cells were divided into the model group and the control group. The cells in the model group were removed from the complete medium, washed twice with PBS, and then added 2 mL of sugar-free and serum-free DMEM medium (containing 1% penicillin-streptomycin mixed solution) into 1 %Oxygen, 5% carbon dioxide, and 94% nitrogen were cultured in a humid environment for 3h, 6h, 12h, 24h, 36h, and 48h respectively, while the cells in the control group were sucked out of the original medium, washed twice with PBS, and then added 2mL of complete medium , cultured in 5% carbon dioxide, normal oxygen, and humid environment for the same time. After the hypoxic culture, the model group was replaced w...
Embodiment 2
[0025] ETS2mRNA level detection in the model of embodiment 2
[0026] 2.1 mRNA extraction
[0027] Add 200 μL of chloroform to the collected trizol and cell lysate solution per milliliter of trizol, mix manually for 1 minute, place on ice for 3 minutes, then centrifuge at 12,000 rpm at 4°C, collect the supernatant, add an equal volume of isopropanol, and place at 4°C for 10 minutes After centrifugation, discard the supernatant and add 1mL of 75% ethanol, vortex, centrifuge again, absorb the supernatant, add appropriate amount of DEPC water to dissolve the liquid after drying, and finally use a spectrophotometer to detect the RNA concentration. The OD260 / OD280 ratios of the extracted RNAs were all between 1.8 and 2.0.
[0028] 2.2 Reverse transcription, real-time fluorescent quantitative PCR
[0029] TAKARA reverse transcription kit (RR037a) was used to perform reverse transcription according to its instructions, and the obtained cDNA was used as a template for real-time fluo...
Embodiment 3
[0031] Example 3 The Toxic Effect of Menghuaside on HK2 Cells
[0032] The same amount of HK2 cells was planted into each well of a 96-well plate, and when the cells reached 70% confluence, they were replaced with serum-free DMEMf12 medium, and 5 μM, 10 μM, 30 μM, 50 μM, 70 μM, and 100 μM mongoside were added to different wells respectively. Cultivate for 48h. After the cultivation, the liquid in the wells was aspirated, and 10 μL of CCK8 working solution and 100 μL of DMEM f12 medium were added to each well, and the absorbance at 560 nm was detected in a microplate reader after incubation at 37 °C for 4 h. The toxic effect of mongoside was judged by calculating the cell viability.
[0033] The result shows, as figure 2As shown, at the concentration > 50 μM, the activity of HK2 cells began to decline, but the maximum non-toxic concentration of succulentin to heart cells in previous studies was 30 μM (cultured for 48 h), considering the application of succulentin in human bo...
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