CN110609136B

CN110609136B - Application of ZYX (Zollinger X) porphyrins in diagnosis and treatment of malignant glioblastoma

Info

Publication number: CN110609136B
Application number: CN201910883467.8A
Authority: CN
Inventors: 文显梅; 姚月良; 罗韬; 罗春花; 王岩; 卞修武
Original assignee: First Affiliated Hospital of Army Medical University
Current assignee: First Affiliated Hospital of Army Medical University
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2022-08-09
Anticipated expiration: 2039-09-18
Also published as: CN110609136A

Abstract

The invention belongs to the technical field of biological medicines, and relates to a brain glioma-related tumor molecular marker and application thereof. The invention discloses an application of ZYX (Zollinger-associated protein) as a diagnosis marker of malignant glioblastoma multiforme, wherein the expression level of the ZYX-associated protein is positively correlated with the invasion intensity of glioma. The expression level of the ZYX porphyrins is positively correlated with the malignancy grade of glioma. The invention confirms the dynamic relation between the expression quantity of the ZYX adiponectin and the occurrence and development of glioma, and provides important guiding significance for the treatment, diagnosis, curative effect prediction, prognosis judgment and the like of glioma.

Description

Application of ZYX (Zollinger X) porphyrins in diagnosis and treatment of malignant glioblastoma

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a brain glioma-related tumor molecular marker and application thereof.

Background

Glioblastoma, also known as glioma, is the most common tumor of the nervous system, and among them, glioblastoma, which is a high grade glioma (WHO grade IV), has the characteristics of rapid progress, high fatality rate, low cure rate, and the like. In addition, high-grade glioma is difficult to realize complete excision in an operation due to the fact that the glioma has no boundary with brain tissue because of tumor invasive growth, and the glioma is difficult to solve in the current medical field.

Classical histopathology has strong subjectivity and cannot objectively, systematically and accurately reflect the genetic background and biological characteristics of tumor tissues. In 2008, the TCGA research team discovered 3 signaling pathways that are most frequently altered in glioblastoma genomic DNA, which provides a solid foundation for glioma pathogenesis and targeted therapy.

The current molecular markers for auxiliary diagnosis of glioma comprise IDH, MGMT, EGFR, P53, PTEN and the like, but no research and report on the application of ZYX adiponectin in diagnosis or treatment of malignant glioblastoma multiforme is available at present.

ZYX adiponectin (Zyxin), a cytoskeleton-associated phosphoprotein with a molecular weight of 69KD, is widely distributed in various cells of the body, especially in endothelial and smooth muscle cells. In the cytoplasm of normal cells, ZYX adiponectin is mainly located in adhesion spots, actin stress fibers and adhesion bands, participates in actin skeleton assembly and force transmission, mediates cell-to-cell and cell-to-matrix interaction, and finally influences cell migration, proliferation and death. In addition, ZYX porphyrins can enter the nucleus to regulate the expression of a plurality of important genes. In recent years, more and more researches show that when various malignant tumors occur in a human body, ZYX adiponectin plays various regulation roles in the occurrence and development of the tumors and can reflect the invasiveness degree of the glioma, so that ZYX adiponectin is expected to become a new potential tumor treatment target and a new prognosis index.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of ZYX adiponectin as a diagnostic marker for malignant glioblastoma and an application of ZYX adiponectin developed as a drug and/or a diagnostic kit for treating glioma.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the application of the ZYX porphyrins as diagnosis markers of malignant glioblastoma multiforme, wherein the expression level of the ZYX porphyrins is positively correlated with the invasion of glioma.

Further, the expression level of the ZYX porphyrins is positively correlated with the malignancy grade of glioma.

The invention verifies that ZYX in brain glioma increases with the malignant grade of glioma by 84 glioma pathological experiments, and the expression of ZYX increases.

Further, the amino acid sequence of the ZYX porphyrins is shown as Seq ID No. 1; the nucleotide sequence of ZYX porphyrins is shown in Seq ID No. 2.

The application of the ZYX zebra synalbumin in preparing an auxiliary diagnosis, curative effect prediction or prognosis judgment kit for malignant glioblastoma.

The application of ZYX zebrina protein in preparing molecular targeted drugs for targeting malignant glioblastoma.

Further, the molecular targeting agent comprises a monoclonal antibody agent, an ADC, or a small molecule chemical agent.

Currently, in molecular targeted drugs for treating high-grade glioma, bevacizumab, a monoclonal antibody drug for vascular endothelial growth factor, is mainly applied. Bevacizumab is an artificially synthesized vascular endothelial growth factor monoclonal antibody drug, and can inhibit tumor growth, alleviate edema caused by tumor, and the like. However, bevacizumab is expensive and has been proved to have reduced sensitivity and efficacy for a long period of time, so that it is urgent to develop a drug which targets different targets and has longer lasting efficacy and lower price.

A medicament for targeted treatment of malignant brain glioblastoma that affects the progression of glioblastoma by inhibiting the activity of ZYX adiponectin.

Further, the medicament may be in any pharmaceutically acceptable dosage form, and may include any pharmaceutically acceptable carrier and/or adjuvant.

A glioma cell strain with a knockdown gene for ZYX adiponectin.

The glioma cell strain with the knocked-down ZYX zebra synaptoprotein gene is applied to auxiliary diagnosis, curative effect prediction or prognosis judgment of malignant glioblastoma.

The research team of the invention has proved that the life cycle of the glioma cells and the primary glioblastoma cells is prolonged after the ZYX protein is knocked down, so that the establishment of the glioma cell strain with the knocked-down ZYX adiponectin gene has important guiding significance in the aspects of subsequent clinical mechanism research and disease prediction and diagnosis.

The invention has the beneficial effects

The invention provides the application of the ZYX porphyrins as diagnosis markers of malignant glioblastoma multiforme and the application of the ZYX porphyrins in the development of drugs related to the treatment of glioma, confirms the dynamic relation between the expression quantity of the ZYX porphyrins and the occurrence and development of glioma, and provides important guiding significance for the treatment, diagnosis, curative effect prediction, prognosis judgment and the like of glioma.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.

FIG. 1 photograph of immunohistochemical detection of different grade glioma pathology sections.

FIG. 2Western blotting to detect ZYX expression patterns of glioma of different grades.

FIG. 3 Western blotting electrophoretogram of lentivirus-infected LN229 cells.

Fig. 4 survival plots of mice in the control and knockdown ZYX groups.

Fig. 5 immunohistochemical image of pathological sections after inoculation of brain glioma in control group and mice with knockdown of ZYX group, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1

Immunohistochemical staining

And 1, conventional paraffin section preparation: appropriate wax blocks were picked and sliced to give 6-8 μm slices.

And 2, dewaxing: baking the slices in an oven at 60 ℃ for half an hour, then immediately transferring the slices into xylene I, soaking for 15min, and then transferring the slices into xylene II, and soaking for 15 min.

And 3, hydration: dewaxing, sequentially passing through 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5min each time.

And 4, washing with distilled water for 3 times, and each time for 10 min.

Repair antigen: and (3) introducing 1000ml of the antigen repairing liquid into a pressure cooker, heating and boiling, putting the slices into a pressure cooker, exhausting the air for 2.5min, stopping the fire, and then carrying out waste heat for 1min, taking out the slices, putting the slices into the heat antigen repairing liquid, and naturally cooling the slices to room temperature.

Wash 3 times with PBS for 10min each.

And 7, blocking: the slices are soaked in blocking solution at 37 deg.C for 30 min.

Wash 3 times with PBS for 10min each.

9, applying primary antibody, diluting antibody according to ZYX (1:500), adding appropriate amount of antibody, and refrigerating at 4 deg.C overnight.

10, the next day, the slices were taken out and re-warmed at room temperature for 30 min.

Wash 3 times with PBS for 10min each. Universal secondary antibody was incubated for 1h at room temperature.

12, color development: DAB color development, under the mirror to maximum intensity, stop the reaction in tap water.

Staining nuclei: staining in hematoxylin for 20s, and then differentiating for 1sec with hydrochloric acid alcohol.

Blue return: flushing with running water for 10 min.

15, dehydration: passing through a cylinder in the order of 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, xylene I and xylene II.

And 16, sealing the piece, namely drying after dehydration, sealing the piece by using a neutral gum solution, and baking for 30min at 37 ℃.

Immunohistochemical scoring

Immunohistochemistry scoring was performed using Image-pro plus5.0 software. The specific operation is as follows:

1. adopt the drawing

The image acquisition is carried out under the same microscope and under the same brightness, magnification and software parameter setting. Each slice was randomly acquired 3 pictures adjusted to a clear field of view.

Software analysis

And 1) correcting the optical density of the picture, namely clicking a Files button to open the file, clicking a Calibration under a Measure button, clicking new in an Intensity window, and then selecting std option. And setting the content level to 230 in Options. The window is not closed.

And 2) color selection: selecting Count/size under Measure, clicking Select colors, selecting HSI mode, setting H to be 0-30, S to be 0-255 and I to be 0-230, then clicking file-save, saving the color selection setting, and directly loading the color selection setting file for each subsequent picture.

3) selecting measurement parameters: and clicking Measure on the Count/size interface, and selecting two parameters of IOD and Area under the select Measure.

And 4) measuring: the measurement area is circled with the irregular tool and the count button is clicked. The measured data is read in the view statistics window, and the IOD SUM value is used as the cumulative optical density value of the photo. Clicking the cover AOI(s) to select(s) under Edit, pressing the button again, displaying the measured data in the view statistics window, reading the Area SUM value as the Area value of the photo.

Calculating: the final relative optical density of each photograph is the IOD SUM divided by the Area SUM, and the average of the 3 relative optical densities of each specimen is the immunohistochemical score of that specimen.

Experimental results:

glioma pathology show: ZYX is expressed more highly in brain gliomas with higher grade. See figure 1.

Example 2

Western blot assay

1, glue preparation: cleaning and airing the glass plate, mounting the glass plate on a plate frame, preparing separation gel according to the molecular weight of protein, flattening the liquid surface by using absolute ethyl alcohol, standing at room temperature until the gel is solid, pouring out the absolute ethyl alcohol on the liquid surface, preparing concentrated gel, adding the concentrated gel, immediately inserting a comb, and standing at room temperature until the gel is solid.

SDS-PAGE electrophoresis

Adding the working liquid of the electrophoresis solution into an electrophoresis tank, and loading all the protein samples and the Marker in equal volume.

The electrophoresis parameters are set as: constant voltage electrophoresis, concentration gel voltage of 80V and separation gel voltage of 120V. And ending electrophoresis when the bromophenol blue reaches the bottom of the separation gel.

3, turning the film: a PVDF membrane with a proper size is taken, activated for 1min by methanol and then transferred into a membrane transfer buffer. The filter paper, the membrane, the glue and the filter paper are placed in sequence, bubbles are fully removed, and the electrophoresis tank is fully washed to remove residual SDS. Placing in ice water, and transferring into membrane at constant flow of 200mA for 90 min.

And 4, sealing: and taking the membrane out of the electric rotating tank, marking, placing in prepared sealing liquid, ensuring that enough sealing liquid can cover the surface of the membrane, and sealing by a table concentrator at room temperature for 2 hours.

5 primary antibody incubation and washing: after the blocking, the PVDF membrane was carefully removed with forceps, the excess was cut off, and the membrane was placed in the prepared primary antibody incubation solution overnight at 4 ℃. Washing, PBST washing 3 times, each time 10 min.

6 secondary antibody incubation and washing: appropriate HRP conjugated secondary antibodies were selected according to the source of the primary antibody, and the secondary antibodies were expressed as 1: at 5000 dilution, PVDF membrane was placed in it, allowed to cover completely, and shaken at room temperature for 1 h. Washing, membrane removal, PBST washing 3 times, each time 10 min.

7, developing: the PVDF membrane was fully coated with Western staining ECL HRP chemiluminescent developer and developed in a Bio-Rad gel imager.

Experimental results:

western blotting verified that the higher the expression of ZYX protein in gliomas with higher degrees of malignancy. See fig. 2.

Example 3

Establishment of ZYX knockdown cell line

Lentiviral construction

(1) construction, sequencing, plasmid extraction and packaging of ZYX-shRNA interference lentivirus by Shanghai Kjeka Biotech.

(2) viruses were used with the GV112 vector, the sequence of elements: hU6-MCS-CMV4-Puro ^r 。

(3) ZYX-shRNA-sh RNA interference and control sequences are shown in Table 1

TABLE 1

Serial number	shRNA	Sequence(5′-3′)
			SEQ ID NO.3	shZYX#1	TCCACATGAAGTGTTACAA
SEQ ID NO.4	shZYX#2	GTTCCAAGTCCAGTACCAA
			SEQ ID NO.5	shCtrl	TTCTCCGAACGTGTCACGT

Lentiviral infection of LN229 cells

1. (1) LN229 cell viral infection: 5u shCtr, sh ZYX #1 and sh ZYX #2 viruses (the virus titer is more than 10^8PFU/ml) are respectively added when the cell fusion degree in the six-hole plate is about 80 percent.

(2) post-infection screening: after 48h of infection, puromycin (5. mu.g/mL) was added for screening, and the solution was changed and dosed every 2 days for 2 weeks to obtain stable cell lines. Different groups of proteins were collected and examined for ZYX expression according to the Western blot protocol above.

Experimental results:

the expression of the ZYX protein in the ZYX knockdown group of cells is obviously reduced, which indicates that the construction of the ZYX knockdown cell strain is successful. See fig. 3.

Example 4

In situ graft neoplasia

An animal: NOD/SCID mice, 4-6 weeks old, were used and cultured under standard sterile conditions.

(2) anesthesia: chloral hydrate 5% (8. mu.L/. mu.g), anesthesia was given by intraperitoneal injection.

(3) cells: LN229/Ctr, LN229/sh ZYX #1, GBM1/Ctr and GBM1/sh ZYX #1 cells with luciferase reporter gene virus were constructed and each mouse was inoculated with 2X 10 cells ⁵ And (4) cells.

(4) orthotopic transplantation tumor procedure: after the mice were fully anesthetized, a needle (pre-cut hair to fully expose) was inserted 0.5mm before the coronal suture, 2mm from the pre-halogenated point. Sterilizing the needle insertion position with 75% alcohol, slowly inserting the micro-syringe into the needle to a depth of about 1-2mm and having a breakthrough feeling, and slowly injecting the cell suspension; after the injection is finished, staying for 1-2min, and then slowly withdrawing; the needle insertion site is sterilized again.

(5) observing the state of the mice daily and weighing the body weight; observing the growth state of the transplanted tumor in a living animal imaging system after 1 month; mice were recorded for death time and dead specimens were processed in time.

Experimental results:

orthotopic transplantation neoplasia indicates that the survival curve of mice is significantly prolonged after knockdown of ZYX. FIG. 4 shows

Control group shows highly aggressive growth of malignant glioblastoma; after the ZYX is knocked down, the edges of the tumor and tissues beside the cancer are clearly visible. See fig. 5.

The present invention is not limited to the embodiments described above, which are only illustrative and not restrictive, and those skilled in the art can make many modifications without departing from the spirit and the scope of the appended claims.

<110> first subsidiary hospital of China civil liberation army, military and medical university

Application of ZYX (Zollin-X) porphyrins in diagnosis and treatment of malignant glioblastoma

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Claims

The application of the ZYX porphyrins in preparing the kit for detecting the malignant glioblastoma, which is characterized in that the expression level of the ZYX porphyrins is high or low and is positively correlated with the invasion of the malignant glioblastoma.
2. The use of claim 1, wherein the level of expression of ZYX adiponectin is positively correlated with the malignancy grade of glioma.
3. The use according to claim 1 wherein the amino acid sequence of ZYX adiponectin is as set forth in Seq ID No. 1.
The application of the ZYX zebra synprotein in preparing an auxiliary diagnosis, curative effect prediction or prognosis judgment kit for malignant glioblastoma.