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Pharmaceutical composition for treating glioblastoma and prognostic diagnostic reagent

A technology of glioblastoma and diagnostic reagents, applied in the field of biomedicine, can solve the problems that PDGFRA inhibitors cannot achieve anti-tumor goals, PDGFRA inhibitors cannot effectively anti-tumor, etc.

Active Publication Date: 2022-02-25
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem that the above-mentioned single PDGFRA inhibitor cannot achieve the purpose of anti-tumor, the present invention found in the research that simultaneously inhibiting EPHA2 and PDGFRA has a synergistic inhibitory effect on GBM cells in vivo and in vitro, thus proposing to jointly inhibit PDGFRA and EPHA2 as a method for treating GBM Solution to solve the problem of ineffective anti-tumor using a single PDGFRA inhibitor

Method used

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  • Pharmaceutical composition for treating glioblastoma and prognostic diagnostic reagent
  • Pharmaceutical composition for treating glioblastoma and prognostic diagnostic reagent
  • Pharmaceutical composition for treating glioblastoma and prognostic diagnostic reagent

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] By western blotting and immunofluorescence imaging experiments, the results are as follows figure 1 It can be concluded that PDGFA can activate EPHA2 independently of PDGFRA in GBM cells.

[0045] 1) Western blotting (Western blot) figure 1 A-E, G, K

[0046]① Extraction of total cell protein: Aspirate the medium, add 5ml of PBS and gently shake to wash the medium, then aspirate and discard the PBS. After adding 1ml of PBS, use a cell scraper to scrape off the cells, and transfer them to a 1.5ml centrifuge tube with a pipette gun. Centrifuge at 800g for 3min at 4°C, discard the supernatant and leave the cell pellet. Add about 10 times volume of RIPA lysate containing PMSF to the cell pellet, and lyse on ice for 30 min. After the lysis was completed, the cell lysate was placed in a centrifuge at 4°C and centrifuged at 25000g for 15min. Aliquot and transfer the centrifuged supernatant to a new 1.5ml centrifuge tube, which is the total cell protein.

[0047] ②Protein...

Embodiment 2

[0066] Through immunohistochemical analysis experiments, the results were as follows figure 2 The clinical significance of EPHA2 and PDGFRA in GBM was drawn.

[0067] 1) Immunohistochemical analysis figure 2 A

[0068] ①Paraffin section: put the paraffin specimen into -20℃ refrigerator for pre-cooling, then slice, spread and air-dry.

[0069] ② Dewaxing: Put the paraffin sections in an oven at 60°C for 30 minutes and then perform dewaxing steps: Xylene I 15 minutes, Xylene II 15 minutes, 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol 5min, rinse with tap water for 5min, and in PBS for 5min.

[0070] ③Antigen retrieval: Rinse the slices with PBS for 15 minutes. After configuring the repair solution corresponding to the antibody, add an appropriate amount of water to the pressure cooker and heat it to boiling. Put the slices into the pressure cooker for 2min 30s. Then immediately rinse the cooling pressure cooker with tap wa...

Embodiment 3

[0085] Through MTT experiments and mouse model experiments, the results are as follows image 3 It was concluded that the joint inhibition of PDGFRA and EPHA2 expression by IMA and ALW could inhibit GBM cells.

[0086] 1) MTT experiment image 3 A.C

[0087] ① Inoculate an appropriate amount of cells in a 96-well plate, 3000-5000 cells per well, and 100 μL of medium. After the cells adhered to the wall, they were treated with drugs and incubated in a 37°C incubator for 72 hours.

[0088] ② Take out the 96-well plate, discard the old medium, add 100 μL of medium containing MTT solution, continue to incubate at 37°C for 3 hours, then stop the culture, carefully aspirate and discard the medium in the well, add 100 μL DMSO to each well, shake until the crystals are fully dissolved .

[0089] ③Measure the OD value with a microplate reader at 570nm, and record the result.

[0090] 2) Antibody Array image 3 B

[0091] Using the antibody array kit from R&D Systems, scrape the ...

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Abstract

The invention provides a pharmaceutical composition for treating glioblastoma. The pharmaceutical composition comprises an expression inhibitor of hepatic ligand protein type A receptor 2 (EPHA2) and an expression inhibitor of platelet-derived growth factor receptor (PDGFRA). The invention also provides a diagnostic reagent for monitoring glioblastoma prognosis, which comprises a first composition for detecting the expression quantity of the EPHA2 gene and a second composition for detecting the expression quantity of the PDGFRA coding gene. The invention provides a scheme for treating GBM by jointly inhibiting PDGFRA and EPHA2 for the first time, and solves the problem that the anti-tumor effect cannot be achieved in clinical application by only using a single PDGFRA inhibitor.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a pharmaceutical composition for treating glioblastoma and a diagnostic reagent for prognosis. Background technique [0002] Glioma is the most common brain tumor. According to the WHO 2016 tumor pathological grading standard, central nervous system tumors are divided into four grades (grade I to grade IV). Although a small number of low-grade gliomas progress slowly and have a good prognosis, most of these tumors are malignant tumors with highly invasive growth and significantly shortened survival time. Take glioblastoma multiforme (grade IV) as an example, which is the most malignant form of glioma, and it is still difficult to cure despite the continuous progress of surgical, drug and other treatment measures. [0003] Platelet-derived growth factor receptor alpha (PDGFRA) and beta (PDGFRB) belong to the receptor tyrosine kinase (RTK) family and are receptors for platelet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/06C12Q1/6886G01N33/574A61P35/00
CPCA61K45/06C12Q1/6886G01N33/57407G01N33/57484A61P35/00C12Q2600/118C12Q2600/158G01N2333/715
Inventor 王岩卞修武盖曲倞吕胜青
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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