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Application of METs as molecular marker in evaluation of clinical prognosis of glioma patient

A molecular marker and glioblastoma technology, applied in the field of medicine, can solve the problems of less than 10% 5-year survival rate of glioma patients, short survival period of patients, glioma invasion and other problems, and improve the overall Survival period, the effect of improving clinical treatment effect

Pending Publication Date: 2021-12-21
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glioma is characterized by high invasion and malignant proliferation, which often leads to short survival of patients
At present, the clinical treatment is surgical treatment combined with concurrent radiotherapy and chemotherapy, but the 5-year survival rate of patients with glioma is still less than 10%.
Microglial extracellular traps (Macrophage / microglia extracellular traps, METs) have the same function, but the mechanism of action on glioma needs further study

Method used

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  • Application of METs as molecular marker in evaluation of clinical prognosis of glioma patient
  • Application of METs as molecular marker in evaluation of clinical prognosis of glioma patient
  • Application of METs as molecular marker in evaluation of clinical prognosis of glioma patient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 The level of METs around the tumor of brain glioblastoma increased

[0030] 1. Preparation of detection reagents

[0031] (1) Preparation of fluorescent antibody;

[0032] Add 10 mg bovine serum albumin dry powder to a 1.5 mL EP tube, dissolve in 1 mL phosphate buffered saline (PBS, pH=7.4), and add 1 μg CD68 mouse monoclonal antibody and 2 μg citH3 rabbit monoclonal antibody to the EP tube , mixed thoroughly with the pipette, and temporarily stored in a -20°C refrigerator;

[0033] (2) Preparation of fluorescent secondary antibodies;

[0034] Add 10 mg bovine serum albumin dry powder to a 1.5 mL light-proof EP tube, dissolve it in 1 mL PBS, and take 2 μg goat anti-rabbit IgG (H+L) Alexa Fluor 488 and 2 μg goat anti-mouse IgG (H +L) Add Alexa Fluor 594 into a light-proof EP tube, mix thoroughly with a pipette, and temporarily store in a -20°C refrigerator away from light;

[0035] (3) Preparation of DAPI staining agent;

[0036] Add 10mg bovine serum albu...

Embodiment 2

[0060] DNA fragments in Example 2METs bind GBM cell membrane proteins and promote downstream signaling pathways

[0061] 1. Experimental method

[0062] 1.1 DNA extraction and preparation

[0063] (1) Add 500nM phorbol ester (PMA) into the culture medium of LN229 and HG7 cells, put it in the cell incubator and let it stand for 4h, discard the culture medium, add 2mL of pre-cooled PBS solution to wash, and centrifuge at 1000g for 10min to get the supernatant;

[0064] (2) Using a sonicator (power: 50w, sonication: 10s, interval: 30s, a total of 15 cycles) to crush the extracted MET-DNA to obtain DNA fragments with a length of about 200-1000bp.

[0065] 1.2 DNA pull-down experiment

[0066] (1) Mix 5 μg of biotin-labeled DNA and 500 μg of GBM cell membrane protein in advance, and place on ice;

[0067] (2) Take 100 μL of Streptavidin-agarose G beads, wash with 0.5 mL of pre-cooled PBS solution, and centrifuge at 5000 g for 30 seconds;

[0068] (3) Add the mixture of DNA and ...

Embodiment 3

[0109] Example 3 METs can promote the malignant progression of GBM

[0110] 1. Method

[0111] 1.1 By using 5 μg ml of GBM cell line LN229 and primary cell HG7 -1 The cells were treated with METs for 12 hours, and the changes after treatment were observed by Transwell chamber and cell scratch experiment. Stimulate LN229 and HG7 cells with 500nM PMA for 4h, discard the medium, add 2mL cold PBS to wash, centrifuge at 1000g for 10min, and take the supernatant for later use.

[0112] 1.2 Using Transwell technology to detect changes in cell migration and invasiveness in cell lines after adding METs

[0113] (1) Spread Matrigel: Dilute Matrigel gel with serum-free cell culture medium at a ratio of 1:8 at 4°C, take 100 μL and evenly spread it on the surface of the polycarbonate membrane in the upper chamber, and place it at 37°C for 0.5-1h;

[0114] (2) Cell culture: Take the cells to be tested in the logarithmic growth phase, wash with PBS, suspend the cells with serum-free mediu...

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Abstract

The invention discloses particularly discloses an application of METs as a molecular marker in evaluation of clinical prognosis of the patient suffering from brain glioma. According to clinical data in combination with experimental results, it is found that the level of METs in high-grade brain glioma is increased, METs can promote proliferation, invasion and migration ability of GBM, after METs stimulation, the ubiquitination level of DDX5 in GBM cells is reduced, beta-catenin nucleation is increased, and expression level changes of EMT related indexes and malignant progression of GBM cells can be regulated and controlled. Co-localized CD68 and citH3 in a biological sample are detected through technical methods such as immunofluorescence and immunoblotting experiments, and the level of METs can be indirectly detected so as to evaluate clinical prognosis of GBM patients. The invention provides a new technical means for guiding individualized diagnosis and treatment of the GBM patient, improving the clinical treatment effect of the patient and prolonging the overall lifetime of the GBM patient.

Description

technical field [0001] The invention relates to a disease-related molecular marker, in particular to the application of METs as a molecular marker in evaluating the clinical prognosis of glioma patients. The invention belongs to the technical field of medicine. Background technique [0002] Glioma is a malignant tumor originating from intracranial neuroepithelial cells, and it is also the most common primary malignant tumor in the brain, accounting for 50% of malignant tumors in the central nervous system. Glioma is characterized by high degree of invasion and malignant proliferation, which often leads to short survival time of patients. Currently, the clinical treatment is surgery combined with concurrent radiotherapy and chemotherapy, but the 5-year survival rate of glioma patients is still less than 10%. [0003] Glioblastoma (GBM) is the highest grade type of glioma with the worst prognosis. At present, GBM treatment is mainly based on surgical resection, combined wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/577
CPCG01N33/57484G01N33/57407G01N33/577G01N2800/52
Inventor 孟祥祺蔡金全蒋传路张严公
Owner HARBIN MEDICAL UNIVERSITY
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