CN113373214B - The use of CD30 in the diagnosis of cranial nerve-related diseases - Google Patents

Abstract

The inventors screened for differentially expressed proteins in the peripheral venous blood serum of young men who faced long-term high mental and psychological stress before and after performing tasks, and screened out proteins that may be associated with cognitive decline. They also collected patient cases for further verification, proving that the concentration of CD30 in the serum of the subjects can be used to diagnose diseases related to cognitive function.

Description

Application of CD30 in the diagnosis of cranial nerve-related diseases

Technical field

The present invention relates to the fields of biotechnology and biomedicine, and specifically relates to the use of CD30 in diagnosing cranial nerve-related diseases.

Background technique

Dementia is a brain disease caused by abnormal degeneration of the brain. Statistics show that most of the patients are elderly. The clinical manifestations are: due to the decline of brain function, patients are affected in aspects such as memory, calculation, learning, understanding, and even language, judgment, and sense of direction. This disease not only causes pain to the patients themselves, but also has an extremely negative impact on their families and even society as a whole. As the number of elderly people increases, the number of patients with Alzheimer's disease is also increasing. Alzheimer's disease (AD) is a common form of degenerative dementia. There are currently more than 25 million AD patients in the world. Due to the characteristics of its onset age group, it has attracted much attention from countries in the aging stage. Therefore, the research on AD is of great significance.

Depressive disorder is one of the most common mental disorders in the population. According to the World Health Organization (WHO) survey, the incidence of depression worldwide is approximately 3.1%. Depression is a mood disorder or affective disorder caused by a variety of factors. The main symptoms are depression, slow thinking, decreased volitional activity, cognitive function impairment, loss of interest and ability in activities, loss of self-esteem, inappropriate guilt, death and suicidal thoughts, reduced concentration, sleep disorders and loss of appetite. , loss of sexual desire, and may be accompanied by various physical symptoms. In Western developed countries, the lifetime incidence of depression is between 6% and 8%. As the population gradually ages, the incidence of depression among people over 60 years old will be as high as 20% to 50%. The pathogenesis of depression is very complex, and the occurrence of depression may be related to the neurological imbalance of monoamine transmitters in the brain.

Contents of the invention

The inventors screened the differentially expressed proteins in the peripheral venous blood serum of young men who have been facing great mental and psychological stress for a long time before and after performing major tasks, screened out proteins that may be related to cognitive function, and collected patient cases. Further verification was conducted to prove that the concentration of CD30 in the subjects' serum can be used to diagnose cranial nerve-related diseases.

Applications for diagnosing diseases

On the one hand, the present invention provides the use of a reagent for detecting CD30 expression in preparing a product for diagnosing whether a subject suffers from cranial nerve-related diseases.

Preferably, the cranial nerve-related diseases include mental illness and/or dementia.

Preferably, the mental disorder includes anxiety and/or depression.

Preferably, the mental illness is depression or anxiety.

Preferably, the dementia includes Alzheimer's disease, vascular dementia, frontotemporal dementia, dementia with Lewy bodies, and Parkinson's disease.

Preferably, the dementia is Alzheimer's disease and/or vascular dementia.

Preferably, the CD30 is highly expressed in the patient.

Preferably, the high expression means that the CD30 expression level is greater than the CD30 expression level in the healthy control population, and is at least 1.1 times higher than the control expression level, such as at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times , 3.4 times, 3.5 times or more.

Preferably, the reagent for detecting the expression level of CD30 includes a reagent for detecting the expression level of CD30 protein and/or the expression level of CD30 mRNA.

Preferably, the reagents for detecting the expression amount of CD30 protein include reagents used in the following methods: Western Blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence separation technology and protein chip method.

Preferably, the reagents for detecting CD30 protein expression include reagents used by protein chip methods.

Preferably, the chip used in the protein chip method is the AAH-BLG-1 chip of Raybiotech Company.

Preferably, the reagent for detecting CD30 protein expression may also include a CD30 antibody or a fragment thereof, and the CD30 antibody or fragment thereof can specifically bind to the CD30 protein.

Preferably, the reagent for detecting CD30 protein expression also includes a secondary antibody, which can bind to the aforementioned CD30 antibody or fragment thereof and display a detectable signal.

Preferably, the detectable signal may be emitted by a detectable label.

Preferably, the CD30 antibody or fragment thereof can also be combined with a detectable marker, and the detection of the detectable marker indicates the expression of the CD30 protein.

Preferably, the detectable label includes, but is not limited to, one or more of fluorescent dyes, fluorescent molecules, chemiluminescent labels, biotin, and radioactive isotopes.

Preferably, the fluorescent dye includes but is not limited to rhodamine, ROX dye, AlexaFluor dye, ATTO dye, DyLight dye, cyanine dye, FluoProbes dye, SulfoCy dye, Seta dye, IRIS dye, SeTau dye, SRfluor dye, Square dye.

Preferably, the fluorescent molecule includes FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LCRED460.

Preferably, the chemiluminescent label includes peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminal, luminol, isoluminol , acridinium ester, sulfonamide.

Preferably, the reagents for detecting CD30 mRNA expression include reagents used in the following methods: PCR-based detection methods, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO method, high-throughput sequencing platform method.

Preferably, the product also includes reagents for collecting and/or processing samples.

Preferably, the sample includes: serum, plasma, whole blood, urine, saliva, semen, milk, cerebrospinal fluid, tears, nasal epithelial cells, sputum, tissue, mucus, lymph, cytosol, ascites, pleural effusion , amniotic fluid, bladder wash fluid and bronchoalveolar lavage fluid.

Preferably, the sample is serum.

Preferably, the serum is isolated from peripheral venous blood.

product

In another aspect, the present invention provides a kit for diagnosing whether a subject suffers from a cranial nerve-related disease, the kit comprising the aforementioned reagent used for detecting the expression level of CD30 in the subject.

Preferably, the kit may also include any one or more of the following: auxiliary detection reagents for mRNA expression, auxiliary detection reagents for protein expression, auxiliary detection instruments for mRNA expression, and auxiliary detection instruments for protein expression.

Preferably, the auxiliary detection reagents for mRNA expression include but are not limited to: reaction reagents for visualizing the amplicons corresponding to the primers, for example, through agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, original method, etc. Reagents for visualizing amplicons such as bit hybridization and fluorescence detection methods, RNA extraction reagents, reverse transcription reagents, cDNA amplification reagents, standards used to prepare standard curves, positive controls, and negative controls.

Preferably, the auxiliary detection reagents for protein expression include but are not limited to: blocking solution, antibody diluent, washing buffer, color development stop solution, and standards for preparing standard curves.

On the other hand, the present invention provides a pharmaceutical composition for treating cranial nerve-related diseases. The pharmaceutical composition includes an inhibitor of CD30. The inhibitor can knock out the expression gene of CD30 or reduce the content of CD30.

Preferably, the inhibitor may be an antibody that specifically neutralizes CD30.

Preferably, the inhibitor can also be a reagent used by siRNA interference, CRISPR/cas9 method, homologous recombination, gene knockout, gene replacement, gene silencing, site-directed mutation, or chemical drug method.

Preferably, the pharmaceutical composition can be tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, Intravenous drip preparations, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, small pills, nasal preparations, pulmonary preparations, eye drops, etc., oral or parenteral preparations.

Preferably, the cranial nerve-related diseases include mental illness and/or dementia.

Preferably, the mental disorder is depressive anxiety syndrome.

Preferably, the dementia is Alzheimer's disease and/or vascular dementia.

method

On the other hand, the present invention provides a method for diagnosing whether a subject suffers from cranial nerve-related diseases. The method includes determining the subject's disease status through the detection results of CD30 expression in the subject.

Preferably, the method further includes the step of detecting the expression level of CD30 in the subject.

Preferably, the cranial nerve-related diseases include mental illness and/or dementia.

Preferably, the mental disorder is depressive anxiety syndrome.

Preferably, the dementia is Alzheimer's disease and/or vascular dementia.

Description of drawings

Figure 1 shows the results of analysis of the difference in CD30 density in serum of male youth before and after the task.

FIG. 2 is a graph showing the difference in CD30 density in the serum of the control group and the patients.

Figure 3 is the ROC curve chart for the diagnosis of anxiety and depression syndrome.

Figure 4 is the ROC curve chart for dementia diagnosis.

Detailed ways

The present invention will be further described below in conjunction with the examples. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled person familiar with the art may make use of the technical content disclosed above. Changes to equivalent embodiments of equivalent variations. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention fall within the protection scope of the present invention.

Example 1. Protein chip screening

For young men who faced great mental and psychological stress during a long-term mission, peripheral venous blood was collected before and after the mission, including 8 samples before the mission and 12 samples after the mission. Raybiotech's AAH-BLG-1 chip was used to screen target proteins from 507 factors. The readout is fluorescence intensity, which is normalized so that higher values indicate higher protein concentrations. The results showed that CD30 in serum increased (p=0.0331038). The specific results are shown in Figure 1.

Example 2: Clinical verification and differential diagnosis of patients with anxiety and depression

Obtain 5 ml of blood from the median cubital vein of all subjects, let it stand at room temperature for 30 minutes, centrifuge at 1000×g for 15 minutes, aliquot and freeze in a -70 degree refrigerator. A customized Raybiotech AAH-BLG-1 chip was used to specifically analyze CD30 levels in serum. The sources of patients and testing data are shown in Table 1 below.

Patients with depression and anxiety syndrome:

A total of 24 patients with depression and anxiety syndrome were treated at the First Medical Center of the People's Liberation Army General Hospital from June 2019 to August 2019 (the first 24 test data in the depression and anxiety column in Table 1). There were 19 patients with depression and anxiety syndrome hospitalized in the Fourth People's Hospital of Chengdu, Sichuan Province on October 23, 2020 (test data 25-43 in the depression and anxiety column in Table 1).

Schizophrenia patients:

On October 23, 2020, 20 patients with schizophrenia were hospitalized in the Fourth People's Hospital of Chengdu, Sichuan Province (test data 1-20 in the schizophrenia column in Table 1).

Dementia:

On October 23, 2020, 13 patients with Alzheimer's disease and 6 patients with vascular dementia were hospitalized in the Fourth People's Hospital of Chengdu, Sichuan Province (the data of patients with Alzheimer's disease and vascular dementia were combined into dementia, which is 19 data in the dementia column in Table 1).

Contrast:

The controls were from healthy volunteers who underwent physical examination at the First Medical Center of the People's Liberation Army General Hospital during the same period but had normal indicators. A total of 20 cases were collected.

Table 1. CD30 levels in patient serum

Result analysis:

Anxiety and depression syndrome: Both the anxiety and depression syndrome patients in Beijing and the patients in Sichuan showed elevated serum CD30 levels. The difference between the Beijing patients and the healthy controls was significant (p = 0.003259), and the difference between the Sichuan patients and the healthy controls was also significant (p = 0.00274). There was no statistical difference between the Beijing patients and the Sichuan patients. The difference analysis results of anxiety and depression syndrome are shown in the left figure of Figure 2, and the diagnostic ROC curve is shown in Figure 3.

Dementia: The difference between dementia patients and healthy controls was significant (p=0.000529). The difference analysis results of anxiety-depressive syndrome, schizophrenia, and dementia are shown in the right picture of Figure 2. The ROC curve for dementia diagnosis is shown in Figure 4.

Schizophrenia patients: There is no statistical difference between serum CD30 levels in schizophrenia patients and healthy controls.

Therefore, the above data show that CD30 has certain diagnostic significance. It can be used in the diagnosis of anxiety, depression and dementia, and it is suggested that the increase in its serum level is related to the decline of cognitive function.

Claims (6)

1. Use of an agent for detecting the amount of soluble CD30 expressed in serum for the preparation of a product for diagnosing whether a subject has alzheimer's disease, said soluble CD30 being highly expressed in serum of a patient.

2. The use according to claim 1, wherein the agent for expressing the amount of soluble CD30 in serum comprises an agent used in the following method: western blot, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescence assay techniques, and protein chip method.

3. The use according to claim 2, wherein the reagent for detecting the expression level of the soluble CD30 protein comprises a reagent used in a protein chip method.

4. The use of claim 1, wherein the reagents further comprise reagents for collecting and/or processing a sample.

5. The use of claim 4, wherein the sample is serum.

6. The use according to claim 5, wherein the serum is isolated from peripheral venous blood.