阅读说明：本技术 24-羟基胆固醇在制备诊断或早期诊断精神***症的产品中的用途 (Use of 24-hydroxycholesterol in the manufacture of a product for the diagnosis or early diagnosis of schizophrenia ) 是由 李人 孙作厘 王传跃 于 2019-09-05 设计创作，主要内容包括：本发明提供了24-羟基胆固醇或检测24-羟基胆固醇的试剂在制备用于诊断精神分裂症的试剂或试剂盒中的用途,以及在制备用于精神分裂症一级亲属或精神分裂症临床超高危者早期诊断精神分裂症的试剂或试剂盒中的用途。24-羟基胆固醇可以作为诊断精神分裂症的生物标志物,对精神分裂症诊断有较高的灵敏度和特异性。同时,通过对高危或超高危患者的外周血24-羟基胆固醇浓度的分析,对精神分裂症进行早期诊断,从而指导早期干预、早期预防,进而改善预后。(The invention provides an application of 24-hydroxycholesterol or a reagent for detecting 24-hydroxycholesterol in preparing a reagent or a kit for diagnosing schizophrenia and an application in preparing a reagent or a kit for early diagnosing schizophrenia in first-class relatives of schizophrenia or clinical super-high risk people of schizophrenia. The 24-hydroxycholesterol can be used as a biomarker for diagnosing schizophrenia, and has higher sensitivity and specificity for schizophrenia diagnosis. Meanwhile, the schizophrenia is diagnosed at an early stage by analyzing the concentration of 24-hydroxycholesterol in peripheral blood of high-risk or ultra-high-risk patients, so that early intervention and early prevention are guided, and the prognosis is improved.)

Use of 24-hydroxycholesterol or a reagent for detecting 24-hydroxycholesterol in the manufacture of a reagent or kit for the diagnosis or early diagnosis of schizophrenia.

2.24-hydroxycholesterol or the use of a reagent for detecting 24-hydroxycholesterol in the preparation of a reagent or a kit for early diagnosis of schizophrenia in the first-class relatives of schizophrenia or in the clinical ultra-high risk of schizophrenia.

3. The use according to claim 1 or 2, wherein the kit is selected from a biochemical diagnostic kit, an immunodiagnostic kit or a molecular diagnostic kit; preferably, the kit is selected from one or more of Western blot kit, enzyme-linked immunosorbent assay (ELISA) kit, Radioimmunoassay (RIA) kit, radioimmunodiffusion kit, two-dimensional biphasic immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, Fluorescence Activated Cell Sorting (FACS) kit, aptamer chip kit, microarray kit and protein chip kit.

4. The use according to any one of claims 1-3, wherein the kit comprises a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

5. The use according to claim 1, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 24-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of the 24-hydroxycholesterol measured in the step 1 with a standard concentration; and

and step 3: determining the risk of the subject for schizophrenia;

preferably, in step 2, the standard concentration is the concentration of 24-hydroxycholesterol in peripheral blood of the normal population.

6. The use according to claim 2, wherein the early diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 24-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of the 24-hydroxycholesterol measured in the step 1 with a standard concentration; and

and step 3: determining the risk of the subject for schizophrenia;

preferably, in step 2, the standard concentration is the concentration of 24-hydroxycholesterol in peripheral blood of the normal population.

7. A kit for diagnosing or early diagnosing schizophrenia, the kit comprising 24-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

8. A kit for diagnosing or early diagnosing schizophrenia, the kit comprising a reagent for detecting 24-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

9. The use according to claim 1 or 2 or the kit according to claim 8, wherein the reagent for detecting 24-hydroxycholesterol is selected from one or more than two of 24-hydroxycholesterol ligand or an antibody to 24-hydroxycholesterol ligand, an antibody to 24-hydroxycholesterol, a chromogenic reagent, a luminescent marker, a dye, a fluorescent marker, a standard, a derivatizing reagent, an internal standard and an external standard; preferably, the standard is selected from serum albumin; more preferably, the alkaline hydrolysis reagent is selected from one or more of butyl hydroxy toluene, potassium hydroxide, ethylene diamine tetraacetic acid and ethanol; further preferably, the derivatization reagent is one or more than two selected from N' -diisopropyl carbodiimide, nicotinamide and 4-dimethylaminopyridine; further preferably, said internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol; further preferably, the external standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.

10. The use according to any one of claims 1 to 6 or the kit according to claim 8 or 9, wherein the kit further comprises a reagent for the detection of 27-hydroxycholesterol.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to application of a brain cholesterol metabolite 24-hydroxycholesterol (24OHC) in schizophrenia diagnosis and early diagnosis, and application in preparation of a corresponding reagent.

Background

Schizophrenia is a common mental disease and is a major public health problem affecting the development of economic society. Schizophrenia has unknown etiology, poor prognosis and high disability rate, is one of the most serious and complex neuropsychiatric diseases, and has the clinical symptoms of hallucinations, delusions, neurophysiological dysfunction, neurocognitive dysfunction and the like. In recent years, development of science and technology such as neuroimaging, genomics, proteomics, immunohistochemistry and the like provides important tools for understanding the cause of schizophrenia at the gene and cell level, but the pathophysiological mechanism of schizophrenia is still unclear.

At present, most mental diseases, especially schizophrenia, lack specific pathological diagnosis indexes, clinical diagnosis can be judged by clinical experience only by depending on symptom description and duration and excluding other diseases, and diagnosis delay or misdiagnosis is very unfavorable for disease prognosis. Therefore, the search for a biomarker with high stability, specificity and sensitivity has become an important direction for the basic research and clinical research of schizophrenia.

Many experimental studies have long focused on neuronal and brain gray matter studies, however, some lines of research evidence suggest that some neurodevelopmental diseases may also occur in white matter, especially oligodendrocytes and their constituent myelin sheaths. The formation of myelin structures begins after birth and is completed in early adulthood, where the onset of schizophrenia happens to be high. In addition, extensive experimental studies on oligodendrocyte abnormalities in terms of cell number, space, morphology and myelination provide direct evidence for the presence of oligodendrocyte/myelination in schizophrenic patients. Cholesterol is the major constituent of myelin, where approximately 70% of the brain is stored. Dysmyelination in schizophrenic patients can lead to increased cholesterol, thereby affecting cholesterol metabolism. These suggest that schizophrenia patients may have cholesterol metabolic disorder in brain, and provide new direction for schizophrenia biomarkers.

Disclosure of Invention

The technical problem to be solved by the invention is that no blood biomarker is consistently recognized in the diagnosis and early diagnosis of schizophrenia at present, namely, a diagnosis method for objectively judging schizophrenia and an early diagnosis method are lacked in the prior art.

In order to solve the above technical problems, there is a need to screen biomarkers related to schizophrenia, thereby helping to reveal the underlying pathophysiological mechanism and developing objective and early diagnosis methods. Although some researchers believe that abnormal changes of total cholesterol in peripheral blood in schizophrenic patients and after treatment can be used as a biomarker, the conclusion of different researchers is not consistent, so that the biomarker is difficult to be used as an objective biomarker for diagnosis and early diagnosis of the schizophrenic disease.

The inventor finds that the main metabolite of the brain cholesterol is 24-hydroxycholesterol (24-hydroxycholesterol), and more than 90% of 24OHC in peripheral blood comes from the brain, which provides convenient conditions for detecting central lesion in peripheral blood. Therefore, 24OHC, a brain cholesterol metabolite in peripheral blood, has the potential to be a schizophrenia-associated biomarker.

The inventor further researches to find that the concentration of 24OHC in the peripheral blood plasma of the schizophrenic patients is obviously increased compared with the healthy population, and the clinical symptoms of the schizophrenic patients are obviously related to the level of hydroxycholesterol. Meanwhile, 24OHC concentration detection of first-class relatives and clinical super-high risk groups of schizophrenia patients finds that the plasma 24OHC concentration of the first-class relatives and the clinical super-high risk groups is obviously increased compared with that of healthy groups. Therefore, the blood plasma 24OHC level can be used as a biomarker for diagnosing schizophrenic patients and also can be used as a biomarker for early diagnosis and screening of high-risk or ultra-high-risk groups.

Based on the research findings, the invention provides the following technical scheme:

in one aspect, the invention provides the use of 24-hydroxycholesterol or a reagent for detecting 24-hydroxycholesterol in the manufacture of a reagent or kit for the diagnosis or early diagnosis of schizophrenia.

In another aspect, the invention provides the use of 24-hydroxycholesterol or a reagent for detecting 24-hydroxycholesterol in the preparation of a reagent or kit for early diagnosis of schizophrenia in first-class relatives of schizophrenia or in clinical super-high risk patients of schizophrenia.

Preferably, the above use, wherein said kit is selected from the group consisting of a biochemical diagnostic kit, an immunodiagnostic kit or a molecular diagnostic kit; preferably, the kit is selected from one or more of Western blot kit, enzyme-linked immunosorbent assay (ELISA) kit, Radioimmunoassay (RIA) kit, radioimmunodiffusion kit, two-dimensional biphasic immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, Fluorescence Activated Cell Sorting (FACS) kit, aptamer chip kit, microarray kit and protein chip kit.

Preferably, the use as described above, wherein the kit comprises a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

Preferably, the above use, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 24-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of the 24-hydroxycholesterol measured in the step 1 with a standard concentration; and

and step 3: determining the risk of the subject for schizophrenia;

preferably, in step 2, the standard concentration is the concentration of 24-hydroxycholesterol in peripheral blood of the normal population.

Preferably, the above use, wherein the early diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 24-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of the 24-hydroxycholesterol measured in the step 1 with a standard concentration; and

and step 3: determining the risk of the subject for schizophrenia;

preferably, in step 2, the standard concentration is the concentration of 24-hydroxycholesterol in peripheral blood of the normal population.

In another aspect, the present invention provides a kit for diagnosing or early diagnosing schizophrenia, the kit comprising 24-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

In another aspect, the present invention provides a kit for diagnosing or early diagnosing schizophrenia, the kit comprising a reagent for detecting 24-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

Preferably, the above-mentioned use or the above-mentioned kit, wherein the reagent for detecting 24-hydroxycholesterol is one or more selected from the group consisting of 24-hydroxycholesterol ligand or an antibody to 24-hydroxycholesterol ligand, an antibody to 24-hydroxycholesterol, a chromogenic reagent, a luminescent marker, a dye, a fluorescent marker, a standard, a derivatizing reagent, an internal standard and an external standard; preferably, the standard is selected from serum albumin; more preferably, the alkaline hydrolysis reagent is selected from one or more of butyl hydroxy toluene, potassium hydroxide, ethylene diamine tetraacetic acid and ethanol; further preferably, the derivatization reagent is one or more than two selected from N' -diisopropyl carbodiimide, nicotinamide and 4-dimethylaminopyridine; further preferably, said internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol; further preferably, the external standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.

Preferably, the use or the kit, wherein the kit further comprises a reagent for detecting 27-hydroxycholesterol.

The beneficial effects of the invention include:

the invention provides a corresponding diagnosis and early diagnosis reagent by detecting the level of 24-hydroxycholesterol as the basis of diagnosis and early diagnosis of schizophrenia, compared with the traditional diagnosis method which can be judged by clinical experience only after depending on symptom description and duration and eliminating other diseases, the invention has higher sensitivity and specificity for diagnosis of schizophrenia, can also predict schizophrenia in the early stage of onset and carry out early intervention, achieves the aims of early prevention and early treatment, and thus obviously improves prognosis.

On the other hand, the 24-hydroxycholesterol biomarker belongs to a peripheral blood index and can be conveniently obtained, and meanwhile, diagnosis and prediction can be carried out only by measuring one index of the peripheral blood 24-hydroxycholesterol level, so that the method is convenient to popularize and use in the future. Meanwhile, the method for diagnosing and early diagnosing by using the 24-hydroxycholesterol biomarker can be combined with other detection modes to obtain more reliable results.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings which are needed to be used are briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1: healthy controls and schizophrenic patients plasma 24OHC concentrations.

FIG. 2-1: correlation of positive symptom score of schizophrenic patients with plasma 24OHC concentration.

FIG. 2-2: correlation of negative symptom score with plasma 24OHC concentration in schizophrenic patients.

FIGS. 2 to 3: correlation of general psychotic symptom scores with plasma 24OHC concentrations in schizophrenic patients.

FIGS. 2 to 4: correlation of schizophrenia patient positive and negative symptom scale scores (PANSS scores) with plasma 24OHC concentrations.

FIG. 3: 24OHC as a ROC curve for diagnostic markers of schizophrenia.

FIG. 4: healthy controls, first degree relatives, clinical super high risk groups and schizophrenic patients plasma 27OHC concentrations.

FIG. 5-1: correlation of positive symptom score of schizophrenic patients with plasma 27OHC concentration.

FIG. 5-2: correlation of negative symptom score with plasma 27OHC concentration in schizophrenic patients.

FIGS. 5 to 3: correlation of general psychotic symptom scores with plasma 27OHC concentrations in schizophrenic patients.

FIGS. 5 to 4: correlation of schizophrenia patient positive and negative symptom scale scores (PANSS scores) with plasma 27OHC concentrations.

FIG. 6: 27OHC as ROC curve for diagnostic index of schizophrenia.

FIG. 7-1: plasma 24OHC concentrations in healthy controls and first degree relatives.

FIG. 7-2: plasma 24OHC concentrations in healthy controls and in clinical super-high risk groups.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.

In one embodiment of the invention, 24-hydroxycholesterol is used as a biomarker in a reagent or kit for diagnosing schizophrenia or in a reagent or kit for early diagnosis of schizophrenia. By detecting the 24OHC concentration in the peripheral blood of a subject to be tested using the above-mentioned reagent or kit, schizophrenia can be diagnosed or schizophrenia can be diagnosed at an early stage. Specifically, peripheral blood of a subject to be tested can be extracted, plasma can be separated, and the 24OHC concentration can be measured, the blood extraction time can be carried out before treatment is received so as to diagnose the diseased state of the subject, and meanwhile, the blood extraction time can also be carried out along with the treatment process or after the treatment is finished so as to monitor the treatment effect.

Herein, the term "schizophrenia" is a psychiatric disorder characterized primarily clinically by hallucinations, delusions, neurophysiological and neurocognitive dysfunction.

The term "diagnosis" refers to a method of detecting and/or identifying the pathological state of a subject, identifying and/or determining the course of whether a subject has a given disease or disorder, estimating or determining the future clinical progression of a subject, following the onset of symptoms.

The term "early diagnosis" refers to a method of detecting and identifying a pathological condition in a subject prior to a specific or non-specific symptom.

Herein, the term "24-hydroxycholesterol" (24-hydroxycholesterol, 24OHC) is a major metabolite of brain cholesterol and has the following structural formula:

there are mainly 2 cholesterol clearance pathways in the brain: one is transport out of the brain through cerebrospinal fluid via apolipoprotein e (apoe); another more major pathway is the oxidative metabolic mode: approximately 6-7mg daily of cholesterol enters the blood circulation through the blood-brain barrier in the form of 24-hydroxycholesterol, catalyzed by cholesterol-24 hydroxylase (CYP46), a cytochrome oxidase P450, which is unique to neurons in the brain. 24-hydroxycholesterol is substantially identical to cholesterol distribution in the brain, and is mainly present in myelin sheaths.

In a specific embodiment of the present invention, the reagent for diagnosing schizophrenia, the reagent for early diagnosis of schizophrenia are prepared as a kit, which may be a biochemical diagnosis kit, an immunodiagnostic kit, a molecular diagnosis kit, or the like, preferably as the immunodiagnostic kit.

The diagnostic kit is prepared by adopting the principles or methods of immunology, microbiology, molecular biology and the like, and is used for diagnosing, detecting and epidemiological investigation of human diseases in vitro and the like.

The diagnostic kit may be of a type well known in the art, including, but not limited to, Western blot kit, enzyme linked immunosorbent assay (ELISA) kit, Radioimmunoassay (RIA) kit, radioimmunodiffusion kit, ouchterlony immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, Fluorescence Activated Cell Sorting (FACS) kit, aptamer chip kit, microarray kit, protein chip kit, and the like.

In the kit, the reagent for detecting 24OHC is included, and the reagent refers to a molecule capable of specifically determining the concentration of 24OHC, and includes but is not limited to nucleic acid molecules, proteins, compounds and the like which can specifically bind to 24 OHC.

In one embodiment of the present invention, a protein capable of specifically binding to 24OHC may be selected as the reagent for detecting 24OHC, and in another embodiment of the present invention, an antibody, which may be an antibody specifically binding to a marker protein, including but not limited to polyclonal antibody, monoclonal antibody, recombinant antibody and antigen binding fragment thereof, is preferred as the reagent for detecting 24OHC, as long as it retains an antigen binding function.

In the present invention, the reagent for detecting 24OHC may be linked with a detectable label molecule.

The present invention will be described in more detail with reference to specific examples, which, however, are for illustrative purposes only and do not limit the present invention.

The reagents and equipment sources used in the following examples are shown in Table 1 below, and reagents or equipment or procedures not described herein are routinely determined by one of ordinary skill in the art:

TABLE 1 reagents and apparatus used in the examples

Reagent/instrument	Model/specification	Manufacturer of the product
			Ammonium acetate	The content of 500g is more than or equal to 98 percent	Chemical reagent for national medicine group
Formic acid	50ml	Fluka
			Methyl tert-butyl ether	The content of 1L is more than or equal to 99.9 percent	Merck
N' -diisopropylcarbodiimide	The content of D12540725 g is more than or equal to 99 percent	Merck
			Nicotinamide	72340100 g content is more than or equal to 99 percent	Merck
4-dimethylaminopyridine	10770025 g content is more than or equal to 99 percent	Merck
			Methanol	The content of 348604L is more than or equal to 99.9 percent	Merck
High performance liquid chromatograph	Agilent G1312B	Agilent
			Mass spectrometer	AB Sciex Qtrap 6500	ABI of America