Application of rps7 and srp14 genes in the treatment of renal insufficiency or renal injury
A technology of gene and gene suppression, applied in the field of application in the treatment of renal insufficiency or renal injury, can solve the problems such as apoptosis of renal tubular epithelial cells that have not yet been found
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Embodiment 1
[0031] SRP14 siRNA and RPS7 siRNA were used to knock down the expression of SRP14 or RPS7 in HK2 human renal tubular epithelial cells, respectively.
[0032] 1 Transfection of SRP14 siRNA
[0033] The concentration of SRP14 siRNA gene silencing was diluted to 5 nmol / ml according to the instructions (purchased from Thermo Fisher Company, #AM16708, AssayID#12804, RefSeq: NM_001309434.1).
[0034] 1) Dissolve 40pmol "SRP14 siRNA" in a 1.5ml EP tube containing 100μl Opti-DMEM-F12 serum-free and double-antibody-free medium, and mix gently.
[0035] 2) Dissolve 7.5 μl Lipo2000 in a 1.5ml EP tube containing 100 μl Opti-DMEM-F12 serum-free and double-antibody-free medium, mix well and place at room temperature for 5 minutes.
[0036] 3) Add the solution in 1) to the solution in 2), mix well and let stand for 15 minutes.
[0037] 4) When it is almost 13 minutes, remove the medium in the 6-well plate, wash the cells once with Opti-DMEM-F12 serum-free and double-antibody-free medium, a...
Embodiment 2
[0073] The cell treatment conditions of previous modeling were the same as in Example 1. The cell viability was measured by Japan Toren Cell Counting Kit-8 (CCK8, Cat. No. CK04) kit, the main component of CCK8 It can be reduced to water-soluble orange-yellow formazan by intracellular dehydrogenase, and the amount of formazan produced is proportional to the number of cells, which can indirectly measure the number of living cells.
[0074] see results figure 2 : In the normal negative control siRNA group (Silencer TM In Negative Control No.1siRNA (#AM4611, no significant similarity with mouse, rat or human gene sequence, that is, no interference, Thermo fisher, USA), the survival rate of cells after hypoxia and reoxygenation is significantly reduced (ctrl in the figure) , Knockdown of gene expression by SRP14 siRNA or RPS7 siRNA can significantly increase the survival rate of HK2 human renal tubular epithelial cells (IRI in the figure) after hypoxia-reoxygenation.
Embodiment 3
[0076] The cell treatment conditions of previous modeling were the same as in Example 1.
[0077] Detection of PI / Annexin V staining by flow cytometry
[0078] 1) Cell collection: Adherent HK2 cells were digested with trypsin and collected directly into a 5ml centrifuge tube. The number of cells per sample was (1-5)×10 6 / mL, centrifuge at 2500r / min for 5min, and discard the culture medium.
[0079] 2) Wash twice with pre-cooled PBS, and centrifuge at 2500r / min for 5min.
[0080] 3) Resuspend cells with 100ul of Binding Buffer, set blank control, single stain with PI, single stain with AnnexinV, double stain with experimental group, and incubate at room temperature in the dark for 10-15min.
[0081] 4) Flow cytometry analysis results
[0082] 5) Judgment of results: Apoptotic cells are resistant to all dyes used for cell viability identification, such as PI, but necrotic cells are not. The DNA of cells with damaged cell membranes can be stained with PI to produce red fluor...
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