CN113820488A

CN113820488A - Application of METs as molecular marker in evaluation of clinical prognosis of brain glioma patient

Info

Publication number: CN113820488A
Application number: CN202111161195.4A
Authority: CN
Inventors: 孟祥祺; 蔡金全; 蒋传路; 张严公
Original assignee: Harbin Medical University
Current assignee: Harbin Engineering University; Harbin Medical University
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-21

Abstract

The invention discloses the use of METs as molecular markers for assessing the clinical prognosis of patients with brain Glioma (GBM). According to the invention, according to the combination of clinical data and experimental results, the METs are found to be increased in the high-grade brain glioma, the METs can promote the proliferation, invasion and migration capacity of GBM, after being stimulated by the METs, the ubiquitination level of DDX5 in GBM cells is reduced, beta-catenin is increased into nuclei, and the expression level change of EMT related indexes and the malignant progression of the GBM cells can be regulated and controlled. The co-localized CD68 and citH3 in the biological sample are detected by technical methods such as immunofluorescence, immunoblotting experiment and the like, so that the level of METs can be indirectly detected, and the clinical prognosis of the GBM patient can be further evaluated. The invention provides a new technical means for guiding the individual diagnosis and treatment of the GBM patient, improving the clinical treatment effect of the patient and improving the overall life cycle of the GBM patient.

Description

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

Technical Field

The invention relates to a molecular marker related to diseases, in particular to application of METs as the molecular marker in evaluating clinical prognosis of patients with brain glioma. The invention belongs to the technical field of medicines.

Background

Brain glioma is a malignant tumor originated from intracranial neuroepithelial cells, is also the most common primary malignant tumor in the cranium, and accounts for 50% of malignant tumors in the central nervous system. Brain glioma has the characteristics of high invasion, malignant proliferation and the like, and the life cycle of a patient is often short. At present, the clinical treatment mode is that the operation treatment is combined with the synchronous radiotherapy and chemotherapy, but the 5-year survival rate of the patient with the brain glioma is still not more than 10 percent.

Glioblastoma (GBM) is the highest grade, worst prognosis, type of brain glioma. At present, GBM treatment is mainly surgical resection, and is combined with various treatment means such as synchronous radiotherapy and chemotherapy, immunotherapy, molecular targeted therapy and the like, but the treatment effect is poor. Clinical prognosis and molecular biology characteristics of brain gliomas of different grades have obvious differences. GBM has the progression of Epithelial-mesenchymal transition (EMT), the EMT progression indicates that the tumor is transformed to a subtype with higher malignant behavior, and the GBM has the characteristics of high expression of mesenchymal markers such as N-cadherin, Slug, beta-catenin and the like, and low expression of Epithelial markers such as E-cadherin, Claudin-1 and the like. EMT progression is involved in regulating biological activities such as invasion, migration, chemoradiotherapy tolerance and the like of GBM.

The EMT progression of GBM is closely related to the tumor microenvironment. The tumor microenvironment is composed of a variety of non-tumor cells, secreted factors, signaling molecules, and extracellular matrix components. The tumor microenvironment has unique myeloid cell subsets, such as tumor-related dendritic cells, tumor-related neutrophils and tumor-related microglia, and can promote malignant phenotypes of tumors, such as radiotherapy tolerance, chemotherapy tolerance, invasion migration and the like, by inhibiting the inhibitory action of immune cells in the tumor microenvironment on tumor cells. Microglia are inherent macrophages in the central nervous system, and can participate in biological processes of tumor, such as EMT (acute myocardial infarction), immunosuppression, angiogenesis, cell proliferation, migration, invasion and the like, through various modes of extracellular trap net release, cytokine and chemokine release and the like.

Extracellular trap nets (ETs) are DNA networks containing multiple protein components that lose the integrity of the intracellular membrane, extend chromatin, disagglomerate, disintegrate the nuclear membrane, and release them to the outside of the cell after stimulation. The common extracellular trapping net is derived from neutrophils, participates in immune reaction, and can enhance the infiltration capacity of tumor cells in diseases such as esophageal cancer, lung cancer, colon cancer and the like, promote the metastasis of tumors and regulate the adhesion of circulating tumor cells and endothelial cells. While the function of Microglia Extracellular Trap (METs) is the same as that of the microglia extracellular trap, the action mechanism of the microglioma is yet to be further researched.

The influence of different GBM immune microenvironment on the GBM immune microenvironment is combined and the clinical prognosis of the patient is predicted, so that the individual treatment of the patient can be guided, and the treatment scheme is optimized to achieve the maximum benefit.

Disclosure of Invention

One of the objects of the present invention is to provide the use of METs as molecular markers for assessing the clinical prognosis of patients with brain glioma.

The invention also aims to provide application of the reagent for detecting METs in preparing a reagent or a kit for evaluating clinical prognosis of a patient with glioblastoma multiforme.

The invention also aims to provide a detection kit for evaluating the clinical prognosis of a patient with brain glioma.

In order to achieve the purpose, the invention adopts the following technical means:

the invention utilizes CD68 protein as a marker protein of microglia and the co-location with an extracellular trapping net specifically marked by citH3 to determine the level of the microglia extracellular trapping net (METs). According to the research result, clinical data and molecular biological experiments are combined, and the METs are found to exist in the brain glioma; as tumor grade increases, levels of METs increase; after METs are combined with brain glioma cell membrane protein, the ubiquitination level of intracellular DDX5 is reduced, the beta-catenin is promoted to enter the nucleus, the Epithelial-mesenchymal transition (EMT) of GBM is regulated, and the proliferation, invasion and migration of tumor cells are promoted.

On the basis of the research, the invention provides application of microglia extracellular trapping nets (METs) as molecular markers in preparing reagents or kits for evaluating clinical prognosis of glioblastoma multiforme patients.

Furthermore, the invention also provides application of the reagent for detecting METs in preparing a reagent or a kit for evaluating clinical prognosis of a patient with glioblastoma multiforme.

Preferably, the reagent for detecting METs consists of a reagent for detecting the microglia marker CD68 and a reagent for detecting the METs-specific marker citH 3.

Preferably, the reagent for detecting the microglia marker CD68 is a CD68 mouse monoclonal antibody, the reagent for detecting the METs specific marker citH3 is a citH3 rabbit monoclonal antibody, and the co-localized CD68 and citH3 in a biological sample are detected by immunofluorescence and immunoblotting, so that the level of METs is detected.

Furthermore, the invention also provides a detection kit for evaluating clinical prognosis of a patient with brain glioma, wherein the detection kit is used for detecting the level of METs by detecting co-localized CD68 and citH3 in a biological sample through immunofluorescence and immunoblotting, and the composition of the kit comprises: CD68 mouse monoclonal antibody, citH3 rabbit monoclonal antibody, goat anti-rabbit IgG antibody, goat anti-mouse IgG antibody, DAPI stain.

Preferably, the biological sample is a fresh pathological tissue of glioblastoma multiforme.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes the combination of METs and GBM cells to carry out co-immunoprecipitation combined with liquid phase mass spectrometry, and finds that the increase of METs release can promote the malignant progression of GBM. METs can be used as a marker for assessing brain glioma grading and prognosis and a molecular marker for GBM preoperative assessment to guide individualized diagnosis and treatment of patients, improve the clinical treatment effect of the patients, contribute to improving the overall life cycle of the GBM patients, and add tiles to the development of the precise medical industry in China.

Drawings

FIG. 1 is an application of using a METs kit to detect METs levels in different grades of brain gliomas, with citH3 and CD68 as characteristic markers. The level of METs in high grade brain gliomas is significantly increased compared to low grade gliomas;

FIG. 2 shows the binding of DNA fragments to the cell membrane protein CD109 in METs;

wherein: (A) harvesting DNA fragments in the METs with the length of 200-1000bp by an ultrasonic crusher; (B) silver staining results of binding of DNA fragments in METs and GBM cell membrane protein CD 109; (C) is the result of DNAPULL-Down that the DNA fragment in METs is combined with the GBM cell membrane protein CD 109; (D) the specific amino acid sequence of the combination of the cell membrane protein CD109 and the DNA fragment in the METs;

FIG. 3 is a detailed mechanism of the promotion of EMT progression after binding of METs to CD 109;

wherein: (A) silver staining results for CD109 co-immunoprecipitated with DDX 5; (B) is an amino acid fragment of the amino acid sequence of DDX5 that binds to CD 109; (C) shows the amino acid fragment of DDX5 as the result of mass spectrum after the co-immunoprecipitation of CD109 and the cytoplasmic protein of glioma cells; (D) binding of CD109 and DDX5 was shown for co-immunoprecipitation; (E) for co-immunoprecipitation showing simultaneous binding of CD109 and DDX5 to USP34 and the level of DDX5 protein ubiquitination, the deubiquitination of DDX5 was verified; (F) to inhibit USP34 and CD109, the level of deubiquitination of DDX5 was elevated and inversely correlated with USP34 expression level; (G) exogenous addition of METs results in elevated levels of β -catenin; (H) the beta-catenin level is reduced after the CD109 is knocked down;

FIG. 4 is a graph of the effect of METs on tumor malignant progression in GBM;

wherein: (A) to verify the effect of different concentrations of METs on brain glioma invasion; (B) exogenous addition of METs can obviously promote the invasion capacity of brain glioma; (C) exogenous addition of METs can obviously promote the migration capability of brain glioma.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are, unless otherwise specified, conventional methods: the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 increase in METs levels around glioblastoma

1. Preparation of detection reagent

(1) Preparing a fluorescent antibody;

adding 10mg of bovine serum albumin dry powder to a 1.5mL EP tube, dissolving in 1mL of phosphate buffered saline (PBS, pH 7.4), and adding 1 μ g of CD68 mouse monoclonal antibody and 2 μ g of citH3 rabbit monoclonal antibody to the EP tube, mixing well with a pipette, and storing temporarily in a-20 ℃ refrigerator;

(2) preparing a fluorescent secondary antibody;

adding 10mg of bovine serum albumin dry powder into a 1.5mL light-proof EP tube, dissolving in 1mL PBS, taking 2 mu g of goat anti-rabbit IgG (H + L) Alexa Fluor 488 and 2 mu g of goat anti-mouse IgG (H + L) Alexa Fluor 594 under the light-proof condition, adding into the light-proof EP tube, fully mixing by using a pipettor, and temporarily storing in a refrigerator at-20 ℃ in a light-proof manner;

(3) preparing a DAPI stain;

add 10mg bovine serum albumin dry powder to 1.5mL light-resistant EP tube, dissolve in 1mL PBS, and add 5 μ g DAPI staining agent to light-resistant EP tube, pipette mix well, store in refrigerator at-20 ℃.

2. Experimental methods

2.1 fluorescent staining of tissue sections

(1) Making paraffin sections with the thickness of 3 mu m from the embedded brain glioma specimens of different grades;

(2) placing the paraffin section in a baking oven at 60 ℃ and baking for 2-8 h;

(3) the slices are immersed in a xylene solution for dewaxing for 2 times, each time for 30 min;

(4) the sections were immersed in ethanol rehydration at the following concentration gradient: 100% ethanol for 10min, 95% ethanol for 10min, 85% ethanol for 10min, 75% ethanol for 10min, 50% ethanol for 10min, distilled water for 10min, PBST solution for 10 min;

(5) antigen retrieval: placing the slices in 0.1mol/L citric acid repairing solution with pH of 6.0, maintaining the slices at 95-100 deg.C for 30min to complete antigen repairing, and naturally cooling to room temperature;

(6) and (3) sealing: washing the slices with PBST for 3 times, each for 3min, adding 5% BSA dropwise to block 37 deg.C, and maintaining for 30 min;

(7) primary antibody incubation: after dropwise adding the fluorescent antibody, putting the section into a wet box, incubating overnight at 4 ℃, and cleaning the section for 3 times and 3min each time by using a PBST solution after the overnight incubation;

(8) and (3) secondary antibody incubation: dripping corresponding fluorescent secondary antibody, placing the section in a wet box, incubating for 1h at room temperature in a dark place, washing and cutting for 3 times with PBST solution, each time for 3 min;

(9) and (3) cell nucleus staining: dripping DAPI staining solution, placing the slices in a wet box in a dark place at room temperature for 20min, and washing and cutting with PBST solution for 3 times, each time for 3 min;

(10) sealing: the slides were mounted using mounting medium and observed immediately under a fluorescent microscope.

2.2 tissue HE staining

(5) hematoxylin staining of cell nucleus: immersing the slices in hematoxylin staining solution for 3-8min, and washing with tap water for 5 min;

(6) eosin staining of cytoplasm: immersing the slices in eosin dye solution for dyeing for 1-3 min;

(7) dewatering and sealing: placing the slices in 95% ethanol for 5min, 100% ethanol for 5min, xylene solution for 5min, and xylene solution for 5min, dehydrating, and sealing with neutral gum.

3. Results

CD68 as a microglia marker and citH3 as an extracellular trap specific marker co-localized to define release of METs. We found that CD 68-positive microglia and citH3 were elevated in peritumoral expression levels in high grade brain gliomas, and the results are shown in figure 1.

These results suggest that the expression level of co-localization of CD68 and citH3 correlates with brain glioma staging and prognosis. Thus, METs detection can be applied to assess the clinical prognosis of patients with brain glioma.

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

1. Experimental methods

1.1DNA extraction and preparation

(1) Adding 500nM phorbol ester (PMA) into LN229 and HG7 cell culture medium, placing into a cell incubator, standing for 4h, discarding the culture medium, adding 2mL precooled PBS solution, washing, centrifuging for 10min at 1000g, and collecting the supernatant;

(2) the extracted MET-DNA was disrupted by ultrasonication using a sonicator (power: 50w, ultrasonication: 10s, interval: 30s, 15 cycles in total) to obtain a DNA fragment of about 200-1000bp in length.

1.2DNAPull-down experiment

(1) 5. mu.g of biotin-labeled DNA and 500. mu.g of GBM cell membrane protein were mixed in advance and placed on ice;

(2) taking 100 mu L of Streptavidin-agarose G beads, washing with 0.5mL of precooled PBS solution, and centrifuging at 5000G for 30 seconds;

(3) adding the mixture of DNA and protein to beads, and resuspending the beads;

(4) incubating for 1h at 4 ℃;

(5)5000g, centrifuging for 30 seconds, removing supernatant, and collecting precipitate;

(6) washing beads with ice-cold PBS solution for 3 times, centrifuging at 5000g for 1min, removing supernatant as much as possible, and collecting precipitate;

(7) adding 10 μ L of non-denaturing lysis solution, and measuring the protein concentration by using BCA (bicinchoninic acid) method;

(8) adding 30 μ L protein loading buffer solution, resuspending the precipitate, boiling in boiling water for 10min, and taking out.

1.3 silver staining:

(1) electrophoresis: the experiment adopts discontinuous system protein SDS-PAGE, the concentration is 5% concentrated glue and 8-12% concentration separation glue; loading 40-60 μ g protein per well, starting voltage of 100v, adjusting to 120v after reaching separation gel, and performing electrophoresis for 90 min;

(2) after the PAGE electrophoresis is finished, transferring the PAGE protein gel into a glass plate or an enamel plate, and fixing for 30min by using a fixing solution;

(3) transferring the PAGE gel into a sensitizing solution to enable the dye solution to submerge the PAGE gel, and acting for 30min at room temperature;

(4) removing the sensitizing solution, rinsing with deionized water for 3 times, each time for 10 min;

(5) transferring the PAGE gel into a silver staining solution, enabling the staining solution to submerge the PAGE gel, and shaking for 40min at room temperature;

(6) transferring the PAGE glue into a developing solution, enabling the developing solution to submerge the PAGE glue, and shaking for 10min at room temperature;

(7) and (4) selecting proper time to terminate, discarding the color development liquid, and adding the termination liquid. And finally, signal collection and storage can be carried out.

1.4 Mass spectrometric detection

Extracting GBM membrane protein, mixing biotinylated METs sample and no biotin-linked agarose beads with the membrane protein, respectively, obtaining a sample through DNAPull-down experiment, and placing the sample in a 1.5mL EP tube. And sending the data to a third party detection mechanism for detection.

2 Co-immunoprecipitation

(1) Washing the cells twice with pre-cooled PBS (phosphate buffered saline), and finally blotting the PBS;

(2) add a precooled RIPABuffer (1 mL/10)⁷Individual cells, 10cm culture dish or 150cm²Culture flask, 0.5mL/10⁶Individual cells, 6cm petri dish or 75cm²Culture flasks);

(3) scraping the cells from a culture dish or a culture bottle, transferring the cell suspension into a 1.5mL EP tube, and slowly shaking for 15min at 4 ℃;

(4) centrifuging at 4 ℃ and 14000g for 15min, and immediately transferring the supernatant into a new centrifuge tube;

(5) preparing ProteinA agarose beads, washing the beads for 2 times by PBS, and preparing 50% solution by PBS;

(6) every 1mL of total protein was added with 100. mu.L of ProteinA agarose beads (50%), shaken at 4 ℃ for 10min to remove non-specific contaminating proteins and reduce background;

(7) centrifuging at 14000g for 15min at 4 ℃, transferring the supernatant into a new centrifuge tube, and removing Protein A beads;

(8) bca (bicinchoninic acid) method protein standard curve, protein concentration determination, total protein dilution at least 1: more than 10 times to reduce the effect of detergents in the cell lysate;

(9) diluting total protein to about 1 μ g/μ L with PBS to reduce the concentration of detergent in the lysate;

(10) adding a volume of primary antibody to 500 μ L total protein;

(11) the antigen antibody mixture was shaken slowly at 4 ℃ overnight or at room temperature for 2h, and kinase or phosphatase activity assays were recommended with 2h incubation at room temperature;

(12) adding 100 mu LProteinA agarose beads to capture the antigen-antibody complex, and slowly shaking the antigen-antibody mixture at 4 ℃ overnight or at room temperature for 1 h;

(13) centrifuging at 14000rpm for 5s, collecting agarose bead-antigen-antibody complex, washing with precooled RIPAbuffer 3 times 800 μ L/time;

(14) resuspending the agarose bead-antigen-antibody complex, and gently mixing;

(15) the sample was run at 100 ℃ for 5min, centrifuged with free antigen, antibody, beads, and the supernatant was electrophoresed to collect the remaining agarose beads and frozen at-20 ℃.

3 protein detection- -Westernblot

(1) Electrophoresis: the experiment was performed by polyacrylamide gel electrophoresis using 5% concentrated gel (H)₂O, 30% Acrylamide,1.0M Tris-HCl, 10% SDS, 10% ammonium persulfate, TEMED) and 8-12% strength separation gel (H)₂O, 30% Acrylamide,1.5M Tris-HCl, 10% SDS, 10% ammonium persulfate, TEMED); loading 40-60 μ g total protein per well, starting voltage of 100v, and adjusting to 120v after reaching separation gel;

(2) film transfer: the membrane transfer is constant current membrane transfer, the current is 200mA, and the time is selected for 60-120min according to the molecular weight of the target protein;

(3) and (3) sealing: placing the protein membrane into a TBST (Tris Buffered Saline with Tween-20) solution, and rinsing for 1-2min to wash off the membrane transferring solution on the membrane; adding 5% skimmed milk powder sealing solution, sealing at room temperature 50rpm for 60 min;

(4) incubating the primary antibody: cutting a PVDF (polyvinylidene fluoride) membrane according to a protein labeling indicator, respectively putting the PVDF membrane into a primary antibody solution, incubating the primary antibody at 4 ℃ overnight, then putting the PVDF membrane into a TBST washing solution, and washing for 3 times by shaking for 15min each time;

(5) incubation of secondary antibody: horseradish peroxidase (HRP) -labeled secondary antibodies were diluted proportionally with reference to secondary antibody concentration. Adding the PVDF membrane into the diluted secondary antibody, and shaking and incubating for 1h at room temperature; adding TBST washing solution, shaking and washing for 3 times, each time for 15 min;

(6) color development: luminescent liquid (liquid a, liquid B) 1: 1 configuration (care to keep out of the light), pipette the appropriate amount of the luminescent solution to cover the PVDF membrane, expose on an ECL luminometer and collect the image.

2. Results

LC-MS/MS results show that DNA in METs binds to CD109 protein on GBM cell membrane and promotes enhancement of binding of membrane proteins CD109 and DDX 5. After being stimulated by DNA in METs, CD109 is taken as a bridging molecule to promote the combination of the deubiquitinating protein UPS34 and DDX5, the deubiquitination of DDX5 is increased, the protein level of DDX5 is maintained stable, and the deubiquitination is not easy to degrade. The stably expressed DDX5 can be combined with beta-catenin in cytoplasm to promote the beta-catenin to enter nucleus, thereby promoting the expression level change of EMT related protein and improving the invasion and migration capacity of GBM cells, and the results are shown in figures 2 and 3.

Example 3METs can promote malignant progression of GBM

1. Method of producing a composite material

1.1 by applying 5. mu.g ml to GBM cell line LN229 and primary HG7^-1The cells were treated with METs for 12h and the change after treatment was observed using a Transwell chamber and cell scratch test. LN229 and HG7 cells were stimulated with 500nM PMA for 4h, the medium was discarded, 2mL of chilled PBS was added and the supernatant was removed for future use by centrifugation at 1000g for 10 min.

1.2 Using the Transwell technique to detect changes in cell migration and invasiveness in cell lines following the addition of METs

(1) Paving matrix glue: matrigel gel was mixed with serum-free cell culture medium at 4 ℃ in the following ratio 1: diluting at a ratio of 8, uniformly coating 100 μ L of the diluted solution on the surface of a polycarbonate membrane in an upper chamber, and standing at 37 ℃ for 0.5-1 h;

(2) cell culture: taking the cells to be detected in logarithmic growth phase, washing with PBS, suspending the cells with serum-free medium, and adjusting the cell density to 1-10 × 10⁵/mL；

(3) Inoculating cells: adding 500 μ L of culture medium containing 10% FBS into the lower chamber of 24-well plate, placing Transwell chamber into 24-well plate with tweezers, adding 200 μ L of cell suspension into the upper chamber, and culturing in incubator for 12-48 h;

(4) cell fixation: the chamber was removed, the medium removed, and the Matrigel and cells in the upper chamber were gently wiped with a cotton swab. Adding 600 μ L of 4% paraformaldehyde into a new 24-well plate, and fixing for 20-30 min;

(5) cell staining and counting: the fixative was discarded, stained with 0.2% crystal violet for 10min, washed 3 times with PBS, and the upper side of the chamber was wiped with a cotton swab to wipe off the dye that was non-specifically bound to the upper surface of the chamber. After proper air drying, cells were observed under a microscope and counted;

1.3 cell engraftment changes in cell lines after addition of METs were detected using the cell scratch technique.

(1) About 5X 10 of the additive is added into each hole of a six-hole plate⁵And (4) incubating the cells overnight to ensure adherent growth of the cells.

(2) And (4) making a blank scratch without cells in the orifice plate along the fixed direction by the gun head. Cells were washed 3 times with PBS, scraped cells were removed, and serum-free medium was added.

(3) Put in 5% CO at 37 DEG C₂Culturing in an incubator. The confluence of the cells in the scratch of the six-well plate is photographed at 0, 6, 12 and 24 h.

1.4 statistical analysis of results Using Image J software

2. Results

Similar to other epithelial tumors, GBM has the phenomenon of epithelial-mesenchymal transition, and the migration, invasion, metastasis and other capacities of tumor cells growing like stroma are obviously enhanced. EMT progresses and participates in malignant phenotypes of tumor such as chemotherapy and radiotherapy tolerance, apoptosis resistance, invasion and relapse and the like. Experiments show that the GBM cell migration and invasion capacity is increased after METs are added, and the METs can promote the malignant progression of brain glioma, and the results are shown in figure 4.

Claims

1. Application of Microglia Extracellular Trap (METs) as a molecular marker in preparing a reagent or a kit for evaluating clinical prognosis of a patient with glioblastoma multiforme.

2. Application of a reagent for detecting METs in preparing a reagent or a kit for evaluating clinical prognosis of a patient with glioblastoma multiforme.

3. Use according to claim 2, characterized in that the reagents for detecting METs consist of reagents for detecting the microglia marker CD68, reagents for detecting the METs-specific marker citH3 and a fluorescent dye capable of strongly binding to DNA.

4. The use according to claim 2, wherein the reagent for detecting the microglia marker CD68 is a CD68 mouse monoclonal antibody, the reagent for detecting the METs-specific marker citH3 is a citH3 rabbit monoclonal antibody, and co-localized CD68 and citH3 in the biological sample are detected by immunofluorescence and immunoblotting, thereby detecting the level of METs.

5. The use of claim 2, wherein the fluorescent dye is DAPI (4', 6-diamidino-2-phenylindole).

6. A detection kit for evaluating clinical prognosis of a patient with brain glioma is characterized in that the detection kit is used for detecting co-localized CD68 and citH3 in a biological sample by immunofluorescence and immunoblotting, and further detecting the level of METs, and the kit comprises the following components: CD68 mouse monoclonal antibody, citH3 rabbit monoclonal antibody, goat anti-rabbit IgG antibody, goat anti-mouse IgG antibody, DAPI stain.

7. The test kit of claim 5, wherein the biological sample is a fresh pathological tissue of glioblastoma multiforme.