Application of miR-5189-5p as medicine target point to medicines for treating diabetes and nephropathy
A technology for diabetic nephropathy and drugs, which can be applied in the direction of drug combination, urinary system diseases, microbial measurement/testing, etc., and can solve problems such as lack of treatment methods
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Embodiment 1
[0025] Embodiment 1 MTT method detects the influence of each trigonelline on the proliferation of HMCs under high sugar
[0026] (1), inoculate cells:
[0027] Select the HMCs of passage 3-5, digest and treat the cells in the logarithmic growth phase with 0.25% trypsin, pipette with DMEM low-sugar culture medium containing 10% FBS, adjust to a suitable concentration of cell suspension, press 3×10 3 Cells per well were inoculated into a 96-well plate at a density of 200 μl per well, added in rows to reduce differences between groups, and the cell suspension was mixed by pipetting every 3 wells, and 6 replicate wells were set up for each group, and the outermost Wells are filled with sterile PBS.
[0028] (2), group intervention:
[0029] There are a normal group (Con group), a model group (HG group), a group with 6 kinds of concentrations of each traditional Chinese medicine, and a zero adjustment hole. Flat in 5% CO 2 , Incubate in a 37°C incubator for 6 hours, discard the...
Embodiment 2
[0045] Example 2 Real-time PCR detection of differential microRNA expression
[0046] (1), RNA extraction:
[0047] Use a small RNA kit specially designed for extraction of 200bp and below for full-type gold miRNAKit.
[0048] ① Discard the culture medium and wash once with PBS. every 10cm 2 Add 1ml of LB10 to the growing cultured cells, and place it horizontally for a while, so that the lysate is evenly distributed on the cell surface and the cells are lysed, and then use a pipette gun to blow the cells to make them fall off (for firmly adherent cultured cells, you can use a cell scraper to peel off the cells) .
[0049] ② Transfer the lysate containing cells to an RNase-free centrifuge tube, and pipette repeatedly until there is no significant precipitation in the lysate. Let stand at room temperature for 5 minutes.
[0050] ③ Add 0.2ml chloroform for every 1ml LB10 used, shake vigorously for 30 seconds, and incubate at room temperature for 3 minutes.
[0051] ④ Cent...
Embodiment 3
[0073] The mRNA and protein expression of embodiment 3 target gene HIF1AN
[0074] PCR results showed that, compared with the CON group, the relative mRNA expression of the target gene HIF1AN in the HG group was increased (P<0.01); compared with the HG group, the relative mRNA expression of the target gene HIF1AN in the HMCs cells of the TRL group was significantly decreased (P<0.05).
[0075] The results of Western Blot showed that, compared with the CON group, the relative protein expression of HIF1AN in the HMCs of the HG group was significantly increased (P<0.05); The relative expression of HIF1AN protein in the cells of the 100 μM group and the 200 μM group was significantly reduced (P<0.01), and the decrease in the 200 μM concentration group was more significant than that in the 100 μM concentration group.
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