PDGFR-beta gene expression inhibitor and application thereof
A β gene and inhibitor technology, applied in the field of PDGFR-β gene expression inhibitor, can solve the problem that GRPC molecules cannot be used alone, and achieve the effect of inhibiting the expression level
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Embodiment 1
[0062] Example 1 Preparation of mPG molecules and mPG-TAT molecules
[0063] 1. Preparation of mPG molecules
[0064] In this example, the porphyrin group was coupled to guanine and a porphyrin-guanine coupling compound was constructed. Porphyrin MPIX is a typical G-quadruplex-specific binding compound, which mainly binds to the terminal plane of the G-quadruplex interaction occurs, the structure of which is as figure 1 Shown is a derivative of the NMM molecule. Using a (PEG)2 linker would be as figure 2 The peptide nucleic acid guanine OGkk containing two D-lysines is ligated with MPIX to obtain image 3 The shown porphyrin-guanine coupling compound (MPIX-(PEG)2-G(PNA)- D- Lys- D- Lys), referred to as mPG. Wherein, the peptide nucleic acid guanine OGkk containing two D-lysines is composed of such as Figure 4 The shown guanine and two D-lysines at the N-terminal of guanine constitute the ESI mass spectrum identification diagram of the peptide nucleic acid guanine OGkk...
Embodiment 2
[0073] Example 2 mPG inhibits the expression level of PDGFR-β gene
[0074] In this example, mPG, mPG-TAT and TMPyP4, a ligand molecule with a classical G-quadruplex structure, were used to treat U2OS cells respectively, and the expression level of PDGFR-β gene was detected by qRT-PCR and Western blot experiments.
[0075] Wherein, the steps of U2OS cell culture include:
[0076] Cells were incubated in DMEM medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin and streptomycin at 37°C, 5% CO 2 conditions, U2OS cells were seeded in 6-well plates (2×10 5 cells / well) and cultured for 12 hours, then added mPG or mPG-TAT with a final concentration of 0, 2, 10 μM and incubated for 48 hours, harvested cells from each culture plate well;
[0077] qRT-PCR quantitative detection steps include:
[0078] Total RNA was extracted from the cells using an RNA extraction kit (Tiangen, China) according to the manufacturer’s instructions, and 1.0 μg of total RNA was reverse...
Embodiment 3
[0083] Example 3 Effects of mPG molecules on the growth of U2OS cells
[0084] In this example, the effects of mPG and mPG-TAT on the growth of U2OS cells were also detected.
[0085] Wherein, the steps of U2OS cell culture include:
[0086] Cells were incubated in DMEM medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin and streptomycin at 37°C, 5% CO 2 conditions, U2OS cells were seeded in 96-well plates (5×10 3 cells / well) and cultured for 12 hours, then added mPG or mPG-TAT with a final concentration of 10 μM for 24 hours or 48 hours after incubation, and set up a DMSO control experimental group at the same time, and then took cell microscope photos or MTT cell viability detection. .
[0087] The experimental steps of MTT cell viability detection include: add 10 μM mPG or mPG-TAT molecules to the cells for 24 hours or 48 hours, add 10 μL MTT solution to each well, so that the final concentration of MTT in each well is 0.5 mg / ml, lightly Mix gently a...
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