阅读说明：本技术 尿液中低级别胶质瘤的蛋白标志物及其在早期诊断中的用途 (Protein marker of urine low-grade glioma and application of protein marker in early diagnosis ) 是由 季楠 张力伟 张扬 王燚 李春朝 于 2019-09-03 设计创作，主要内容包括：本申请涉及尿液中低级别胶质瘤的蛋白标志物及其在早期诊断中的用途。具体而言,本申请涉及选自以下的蛋白的鉴定试剂在制备用于低级别胶质瘤早期诊断的试剂中的用途：胞外基质蛋白4(Matrilin-4)、透明质酸酶1(Hyaluronidase-1)及其组合。这两种蛋白对于低级别胶质瘤(少枝,星形)的早期诊断具有较好的临床应用前景。(The present application relates to protein markers of low-grade glioma in urine and their use in early diagnosis. In particular, the present application relates to the use of an agent for identifying a protein selected from the group consisting of: extracellular matrix protein 4(Matrilin-4), Hyaluronidase 1(Hyaluronidase-1), and combinations thereof. The two proteins have good clinical application prospect for early diagnosis of low-grade glioma (oligodendron and asteroid).)

1. Use of an identifying agent for a protein selected from the group consisting of:

Extracellular matrix protein 4, hyaluronidase 1, or a combination thereof;

Preferably, the low grade glioma is selected from: astrocytoma, oligoblastoma, or a combination thereof.

2. The use of claim 1, wherein the diagnosis is an early diagnosis.

3. Use according to claim 2, wherein:

an increased expression level of extracellular matrix protein 4 compared to a healthy control, indicating that the subject has had a low grade glioma or that the subject has a statistically significant higher probability of having a low grade glioma than the healthy control; and/or the presence of a gas in the gas,

A decreased level of expression of hyaluronidase 1 as compared to a healthy control indicates that the subject has had a low grade glioma or that the subject has a statistically significant higher probability of having a low grade glioma than the healthy control.

4. The use according to any one of claims 1 to 3, wherein the identification agent is selected from the group consisting of: mass spectrometric identification of reagents, antibodies or antigen binding fragments thereof.

5. The use of claim 1, wherein the protein is from urine of a subject.

6. The use of claim 3, wherein the expression level is an expression level determined in the urine of the subject;

The subject is a human.

7. A device for diagnosing low-grade glioma comprising an identifying agent for a protein selected from the group consisting of: extracellular matrix protein 4, hyaluronidase 1, or a combination thereof;

The device is a kit or chip.

8. The device for diagnosing low-grade glioma according to claim 7, wherein: the identifying agent is selected from: mass spectrometric identification of reagents, antibodies or antigen binding fragments thereof.

9. the device for diagnosing low-grade glioma according to claim 7, wherein said diagnosis is an early diagnosis.

10. The device for diagnosing a low-grade glioma according to claim 7 wherein said low-grade glioma is selected from the group consisting of: astrocytoma, oligoblastoma, or a combination thereof.

Technical Field

The present application relates to the field of biotechnology, more specifically to protein markers of low-grade glioma (oligodendron, asteroid) in urine and their use in early diagnosis.

Background

The latest yearbook of the Ministry of health in 2013 shows that more than 30 ten thousand patients die of brain tumors every year in China. Glioma, the most common primary malignancy of the brain, accounts for 81% of all primary brain malignancies (us brain tumor registry report, CBTRUS, 2018) [1 ].

glioma has high malignancy degree and poor treatment effect. Median survival in glioblastoma patients is only 14.6 months after standard surgery, radiation and chemotherapy [2 ]. This not only causes great pain to the patient's body, but also causes a psychological heavy burden to the patient and his family members. In addition, the damage to brain function caused by glioma itself and the disability rate caused by surgical treatment are also high. Therefore, the disease brings great burden to national medical resources and social security systems.

According to the morphological division of tumor cells, brain gliomas are mainly classified according to their tumor cell morphology as follows: astrocytoma, oligoblastoma, ependymoma, mixed glioma (e.g., oligoastrocytoma-astrocytoma). Brain gliomas are classified into classes 1 (lowest malignancy, best prognosis) to 4 (highest malignancy, worst prognosis) according to the malignancy classification of the tumor cells (e.g., the grading system established by the world health organization WHO): low grade glioma (WHO grade 1-2); high grade glioma (WHO grade 3-4). According to the location of the tumor, brain glioma is divided into: supratentorial, infratentorial, pontocerebral gliomas.

the glioma is discovered in an early stage and is treated by an operation in time, the survival prognosis of a patient with the malignant glioma can be prolonged, and even a part of patients can be cured, so that the glioma has a vital function in early diagnosis. However, glioma is hidden and lacks of specific early symptoms, which leads to the discovery that the glioma is often in the later stage of the disease course, and the glioma is large, strong in invasiveness and poor in surgical treatment effect.

Screening and identifying early glioma diagnosis markers in body fluids by various omics methods is a conventional strategy for early glioma diagnosis research [3 ].

CN101298629A discloses the application of LRRC4 gene promoter region methylation detection in glioma diagnosis and a detection system thereof, and a brain tissue specific expression gene LRRC4 is a brain glioma specific methylation dissimilatory gene, thereby suggesting that LRRC4 gene promoter region methylation detection can be applied to early diagnosis of glioma.

CN101298629A, the prepared kit allows the methylation state of the plasma LRRC4 promoter region to be detected, and early diagnosis is made for patients with brain glioma.

CN103966337A discloses a long-chain non-coding RNAPRKAG2-AS1 derived from serum exosome for screening and early diagnosis of high-risk glioma population. The PRKAG2-AS1 expression level is reduced, and the specificity to early diagnosis of glioma is high.

CN107664696A discloses the application of serum CCKBR as a diagnostic marker of glioma, in particular to the early warning and early diagnosis of glioma.

CN107664696A establishes an enzyme linked immunosorbent assay kit based on the double antibody sandwich principle, and can rapidly and accurately detect CCKBR.

CN104076151A relates to a kit for early diagnosis of glioma, which comprises a mouse anti-human B7-H4 biotin labeled monoclonal antibody, detects a collected cerebrospinal fluid sample, and predicts the disease risk of glioma and early diagnoses patients with glioma by detecting the protein expression level of B7-H4 in the cerebrospinal fluid.

However, the above-described methods in the art require invasive procedures on the patient, such as drawing blood, and some even require puncturing to access the cerebrospinal fluid. In view of this, there remains a need in the art for early protein markers that are easy to manipulate (e.g., enable non-invasive collection of patient samples) and that better distinguish between normal human and glioma patients.

Disclosure of Invention

In view of the above-identified need in the art, there is provided according to some embodiments of the present disclosure use of an agent for identifying a protein for the manufacture of an agent for diagnosing a low-grade glioma, wherein the protein is selected from any one or a combination of: extracellular matrix protein 4(Matrilin-4), Hyaluronidase 1 (Hyaluronidase-1).

In a specific embodiment, the diagnosis refers to an early diagnosis of low grade glioma.

In the present application, the low grade glioma is selected from: astrocytoma, oligoblastoma, or a combination thereof.

In some embodiments, extracellular matrix protein 4 and hyaluronidase 1 can be used in combination or alone.

In particular embodiments, an increased expression level of extracellular matrix protein 4 and/or a decreased expression level of hyaluronidase 1 in the subject as compared to a healthy control indicates that the subject has a low-grade glioma. The term "suffering from" is to be understood in its broadest sense, including: has already suffered from; or at a set significance level, the probability of having the disease is statistically significantly higher than healthy controls.

In the context of the present application, a healthy control refers to an individual who does not have (is not clinically diagnosed with) a low-grade glioma.

identification reagents suitable for use in the present disclosure are mass spectrometry identification reagents or antibodies (or forms of antigen-binding fragments thereof).

In a specific embodiment, when mass spectrometric identification reagents are employed, data-independent acquisition methods and parallel reaction monitoring are used. The data-independent acquisition method divides the whole full scanning range of the mass spectrum into a plurality of windows, and selects, fragments and detects all ions in each window at high speed and circularly, so that all fragment information of all ions in a sample is obtained without omission or difference. The data-independent acquisition method works like blanket bombing, hitting all targets without omission. The parallel reaction monitoring is a target mass spectrum quantitative analysis technology based on a secondary mass spectrum signal, compared with the traditional selective reaction monitoring technology, the method does not need to design the parent ion/daughter ion pairing information of the target protein in advance, and saves the experimental design and the operation time; and the selectivity is higher, the sensitivity is better, the reproducibility is better, and the anti-interference capability in a complex background is stronger. Compared with immunization methods, the method is no longer limited by commercial antibodies, and overcomes the limitations of antibody specificity and titer based on immunization methods. The parallel reaction monitoring technology can perform qualitative and quantitative analysis on various proteins simultaneously. It should be understood that although a specific identification method is used in the specific example, the technical effect of the present application is not achieved depending on the specific identification method (e.g., mass spectrometry procedure, mass spectrometer model, parameters set in the mass spectrometry method, specific peptide sequence identified in the mass spectrometry, chromatography column model; antibody supplier, specific epitope targeted by antibody, antibody typing, immunological procedure and parameters), because the core of the technical solution of the present application is to find the relationship between the amount of extracellular matrix protein 4 and hyaluronidase 1 present in urine and the disease, and thus any means capable of determining the protein content is available.

the tag peptide is a peptide fragment capable of representing a certain protein, and is characterized by existence and specificity only in an amino acid sequence of the certain protein. In some embodiments, the identification agents of the present application are capable of identifying, or binding, or searching for, or monitoring, or targeting such tag peptides (sequences in extracellular matrix protein 4 and/or hyaluronidase 1). In other embodiments, the identification agents of the present application are capable of recognizing, or binding, or searching for, or monitoring, or targeting, epitopes of proteins (epitopes in extracellular matrix protein 4 and/or hyaluronidase 1, both linear and non-linear epitopes apply).

It should be understood that although the proteins are identified and quantified in the specific examples based on a particular sequence (e.g., SEQ ID No.1 and SEQ ID No.2), this does not mean that peptide fragments elsewhere in extracellular matrix protein 4 and hyaluronidase 1 cannot be used, as long as such fragments can distinguish different proteins from each other, and are suitable for use in the present application. The position or length of the fragments can be determined by the skilled person in accordance with conventional techniques in combination with the operational requirements of the identification method used, given the teaching of the present application.

In a specific embodiment, the expression level is selected from the protein level.

In a specific embodiment, the expression level is the level of protein expression in a urine sample from the subject.

In specific embodiments, the subject is a human.

in another aspect of the present application, there is provided a kit or chip for early diagnosis of low-grade glioma, comprising an identifying agent for a protein selected from the group consisting of: an agent for identifying extracellular matrix protein 4, an agent for identifying hyaluronidase 1, and a combination thereof.

In particular embodiments, the identification reagent is a mass spectrometry identification reagent.

In a specific embodiment, the diagnosis is an early diagnosis of low-grade glioma.

another aspect of the present application provides a method for diagnosing a low grade glioma in a subject comprising the steps of:

1) A urine sample is obtained from the subject,

2) Optionally, separating the protein from the urine sample,

3) Determining the expression level of a protein selected from the group consisting of: extracellular matrix protein 4, hyaluronidase 1, and combinations thereof.

in specific embodiments, the expression level is determined using mass spectrometry methods.

When mass spectrometry is used to determine the protein and its expression level, a digestion step may also be included after the step of obtaining a urine sample. In a specific embodiment, the proteins in the urine sample are digested with trypsin.

In specific embodiments, the mass spectrometry method is a data independent acquisition mode or a parallel reaction monitoring acquisition mode. Specifically, the data-independent detection method divides the whole full scanning range of the mass spectrum into a plurality of windows, and selects, fragments and detects all ions in each window at high speed and in a circulating manner, so that all fragment information of all ions in a sample is obtained without omission or difference. The parallel reaction monitoring technology collects all daughter ions of parent ions corresponding to the tag peptide segment through the tag peptide segment of the protein. Quantification is performed based on the signal intensity of the daughter ions.

the quantitative detection method based on the two proteins in urine can be used for establishing the baseline of the two proteins in a crowd by combining with a standard substance, and can be used for early diagnosis of low-grade glioma patients based on the content range of a normal control group.

drawings

Fig. 1A and 1B are PCA plots (fig. 1A) and OPLS-DA plots (fig. 1B) of the data independent acquisition mode for the protein group to distinguish low grade glioma from normal control group.

Fig. 2A and 2B show data independent detection of changes in the levels of extracellular matrix protein 4 (fig. 2A), hyaluronidase 1 (fig. 2B) proteins.

FIG. 3A is a graph of: the content of the extracellular matrix protein 4 tag peptide segment LEDLENQLANQK (SEQ ID No.1) is changed.

FIG. 3B is a diagram: the content of hyaluronidase 1 tag peptide ALYPSIYMPAVLEGTGK (SEQ ID No.2) was varied.

FIG. 4: the combination of the two proteins predicts the ROC curve for low-grade gliomas.

Detailed Description

The present application is further illustrated by the following examples, but is not to be construed as limited thereby, in conjunction with the following figures. The following provides specific materials and sources thereof used in embodiments of the present application. However, it should be understood that these are merely exemplary and are not intended to limit the present application, and that materials that are the same as or similar to the type, model, quality, nature, or function of the following reagents and instruments may be used in the practice of the present application. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.