阅读说明：本技术 分子标志物在缺血性脑卒中诊治中的应用 (Application of molecular marker in diagnosis and treatment of ischemic stroke ) 是由 蔡学礼 兰卫明 孙景萍 舒小桓 谢慧蓉 詹艳丽 吴新校 吴骏 于 2021-07-30 设计创作，主要内容包括：本发明涉及生物技术、生物医药领域,具体涉及分子标志物在缺血性脑卒中诊治中的应用。本发明提供的标志物或标志物组合在临床诊断中有重要的意义；所述标志物包括ZNF22、RPS27、ZNF416,标志物的组合在诊断时AUC值会显著提高。(The invention relates to the fields of biotechnology and biomedicine, in particular to application of a molecular marker in diagnosis and treatment of ischemic stroke. The marker or the marker combination provided by the invention has important significance in clinical diagnosis; the markers comprise ZNF22, RPS27 and ZNF416, and the AUC value of the combination of the markers is obviously improved in diagnosis.)

1. A marker combination for diagnosing stroke, the markers comprising two or three of RPS27, ZNF22, ZNF 416.

2. The marker combination of claim 1 wherein RPS27 is underexpressed in a patient, wherein ZNF22 is underexpressed in a patient, and wherein ZNF416 is underexpressed in a patient.

3. The marker combination for diagnosing stroke as claimed in claim 1, wherein the stroke is ischemic stroke.

4. Use of a reagent for detecting the expression level of the marker combination of claim 1 or any one of the markers of claim 1 in the preparation of a product for diagnosing stroke in a subject.

5. The use of claim 4, wherein the agent for detecting the expression level comprises an agent for detecting the expression level of mRNA and/or the expression level of protein.

6. The use of claim 5, wherein the reagent for detecting the expression level of mRNA comprises a reagent used in the following method: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method.

7. The use according to claim 5, wherein the reagent for detecting the expression level of the protein comprises reagents used in the following methods: western blotting, enzyme linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytofluorimetry, and protein chip assay.

8. The use of any one of claims 4 to 7, wherein the diagnosis of the subject is determined by the detection of a sample taken from the patient, said sample comprising: blood, nasal epithelial cells, tissue, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid;

preferably, the sample is blood.

9. A kit for diagnosing whether a subject has suffered a stroke, the kit comprising reagents for detecting the expression level of the marker combination of claim 1 or any one of the markers of the marker combination of claim 1;

preferably, the kit further comprises an instrument used for detecting the expression amount.

10. Use of the kit of claim 9 for the manufacture of a product for diagnosing stroke in a subject.

Technical Field

The invention relates to the fields of biotechnology and biomedicine, in particular to application of a molecular marker in diagnosis and treatment of ischemic stroke.

Background

Ischemic stroke is also called cerebral infarction, and is called stroke or stroke in traditional Chinese medicine. The disease is local brain tissue regional blood supply disorder caused by various reasons, leads to cerebral tissue ischemia hypoxic lesion necrosis, and further produces clinically corresponding nerve function deficiency expression. Cerebral infarction is divided into main types such as cerebral thrombosis, cerebral embolism, lacunar infarction and the like according to different pathogenesis. Among them, cerebral thrombosis is the most common type of cerebral infarction, accounting for about 60% of all cerebral infarctions, and thus the so-called 'cerebral infarction' actually refers to cerebral thrombosis.

Because the etiology and basis of cerebral thrombosis are mainly atherosclerosis, the factor for generating atherosclerosis is the most common etiological factor for cerebral infarction. Recent research results conducted on a global scale have shown that: 90% of the risk of cerebral infarction can be attributed to 10 simple risk factors, which in turn are hypertension, smoking, excessive waist-hip ratio, improper diet, lack of physical exercise, diabetes, excessive alcohol consumption, excessive mental stress and depression, underlying heart disease, and hyperlipidemia, most of which are controllable.

The disease is better in middle-aged and old people over 50-60 years old, and male is slightly more than female. It often incorporates risk factors such as arteriosclerosis, hypertension, hyperlipidemia or diabetes or corresponding systemic non-specific symptoms. The precursor symptoms of cerebral infarction are not specific, and some patients may have transient ischemic attack such as dizziness, temporary limb numbness, weakness and the like. These symptoms are often overlooked by the patient and family members due to their short duration and mild degree. The onset of cerebral infarction is acute, and the cerebral infarction mostly occurs in rest or sleep, and the clinical symptoms of the cerebral infarction reach the peak hours or 1 to 2 days after the onset of the cerebral infarction.

Cerebral infarction is an acute disease, and is also a disease with high disability rate and high lethality rate. The treatment principle of the disease is as follows: strive for ultra-early treatment, intravenous thrombolysis treatment as far as possible within 4.5 hours of onset, and hospital conditioned within 6-8 hours of onset can carry out appropriate acute-phase intravascular intervention; the individual and integrated treatment scheme is determined, corresponding targeted treatment is adopted according to the risk factors, the disease degree and the like of the patient, and integrated treatment is realized by combining the efforts of multiple departments such as neurosurgery department, rehabilitation department, nursing department and the like, so that the treatment effect is improved to the maximum extent and the prognosis is improved.

Disclosure of Invention

The inventor screens markers capable of diagnosing ischemic stroke by performing bioinformatics analysis on collected patient samples and database information, and further combines the markers to obtain a more efficient diagnostic marker combination, wherein the markers or the marker combination have important significance in clinical application.

Marker combination

In one aspect, the present invention provides a marker combination for diagnosing stroke, wherein the markers comprise at least two of RPS27, ZNF22 and ZNF 416.

Preferably, the stroke comprises ischemic stroke and hemorrhagic stroke.

Preferably, the stroke is ischemic stroke.

Preferably, the RPS27 is low expressed in the patient.

Preferably, the ZNF22 is underexpressed in the patient.

Preferably, the ZNF416 is underexpressed in the patient.

Preferably, the high expression means that the expression level of the marker in the patient is greater than the expression level in the healthy control population by at least 1.1 fold, specifically at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, or 3.5 fold or more relative to the control expression level.

Preferably, the low expression means that the level of expression of the marker in the patient is less than the level of expression in a healthy control population, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the control expression.

Use for diagnosing diseases

In one aspect, the invention provides a use of an agent for detecting an expression level of the aforementioned marker combination or any one of the aforementioned marker combinations in the preparation of a product for diagnosing whether a subject has stroke.

Preferably, the reagent for detecting the expression amount comprises a reagent for detecting the expression amount of mRNA and/or the expression amount of protein.

Preferably, the detection comprises a quantitative or qualitative detection.

Preferably, the reagent for detecting the expression amount of mRNA comprises a reagent used in the following method: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method.

Preferably, the PCR-based detection method comprises at least any one of: a step of extracting RNA, a step of reverse transcribing mRNA into cDNA, and a step of measuring the content of cDNA.

Preferably, the method of measuring the content of cDNA includes, but is not limited to, PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.

Preferably, the reagent for detecting the mRNA expression level comprises a specific primer and/or a probe.

Preferably, the probes include hybridization-type probes and hydrolysis-type probes (Taqman probes).

Preferably, the two ends of the probe are connected with a quenching group and/or a fluorescent group.

Preferably, the fluorescent group includes, but is not limited to, FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red 460.

Preferably, the quenching group includes, but is not limited to DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, BHQ-3.

Preferably, the reagent for detecting the expression level of the protein comprises reagents used in the following methods: western blotting (Western Blot), enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytofluorimetry, and protein chip methods.

Preferably, the reagent for detecting the expression level of the protein comprises an antibody or a fragment thereof, wherein the antibody or the fragment thereof can specifically bind to the protein.

Preferably, the reagent for detecting the expression level of a protein further comprises a secondary antibody capable of binding to the aforementioned antibody or a fragment thereof and developing color.

Preferably, the visualization is embodied by a visualization reagent including, but not limited to, a fluorescent dye (including fluorescent molecules), a chemiluminescent label, luciferase, a metal ion, biotin, a radioisotope, a molecule that absorbs in the UV spectrum, a molecule that absorbs in the near infrared radiation, or a molecule that absorbs in the far infrared radiation.

Preferably, the fluorescent dye includes, but is not limited to, rhodamine, p-methylaminophenol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, amino-p-methylaminophenol, carboxy-p-methylaminophenol, chloro-p-methylaminophenol, methyl-p-methylaminophenol, sulfop-methylaminophenol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and sulforhodamine, cyanine, indocyanine, oxonol, thiacyanine, merocyanine, cyanine dye, oxadiazole derivative, pyridyloxazole, nitrobenzene oxadiazole, benzonitrobenzene, pyrene derivative, waterfall blue, oxazine derivative, nile red, nile blue, cresol purple, oxazine 170, azo derivative, proflavin, acridine orange, acridine yellow, arylmethine derivative, auramine, thioxanthene dye, Thioxanthene sulphonated dyes, alexas fluorescence (AlexaFluor), crystal violet, malachite green, tetrapyrrole derivatives, porphyrins, phthalocyanines, bilirubin, cy5.5, indocyanine green (ICG), DyLight750, or IRdye 800.

Preferably, the chemiluminescent label includes, but is not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, isoluminol, acridinium esters, sulfonamides, and the like.

Preferably, the luciferase includes, but is not limited to, a pleiones magna (Gaussia) luciferase, a Renilla (Renilla) luciferase, a dinoflagellate luciferase, a firefly luciferase, a fungal luciferase, a bacterial luciferase, and a glowworm (vargula) luciferase.

Preferably, the antibody or fragment thereof is also capable of binding directly to a chromogenic agent, which is detected to indicate expression of the protein.

Preferably, the detection requires the collection of a sample from the patient.

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

Preferably, the sample is blood.

Product(s)

In another aspect, the present invention provides a kit for diagnosing whether a subject has cerebral stroke, wherein the kit comprises a reagent for detecting the aforementioned marker combination or any one of the aforementioned markers in the aforementioned marker combination.

Preferably, the kit further comprises the instrument for detecting the expression level of the subject.

Preferably, the kit further comprises reagents and/or instruments for detecting other disease markers.

In another aspect, the invention also provides application of the kit in preparing a product for diagnosing cerebral apoplexy.

Method

In another aspect, the present invention provides a method for diagnosing whether a subject has suffered from stroke, the method comprising detecting the expression level of the aforementioned marker combination or any one of the aforementioned markers in the aforementioned marker combination in the subject.

Drawings

FIG. 1 is a differential boxplot of differential genes, A is RPS27, B is ZNF22, and C is ZNF 416.

FIG. 2 is a ROC plot of marker RPS27 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 3 is a ROC curve graph of a marker ZNF22 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 4 is a ROC curve graph of a marker ZNF416 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 5 is a ROC graph of marker RPS27_ ZNF22 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 6 is a ROC curve graph of a marker ZNF22_ ZNF416 in the process of diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 7 is a ROC graph of the marker RPS27_ ZNF416 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 8 is a ROC graph of the marker RPS27_ ZNF22_ ZNF416 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 screening and validation of diagnostic Effect of marker combinations

Collecting blood samples of patients, wherein the information of the patients is shown in table 1, sequencing, and screening genes which are differentially expressed between healthy people and patients; differential expression analysis using software: ballgown (http:// cole-trap-lab. githu. io/cufflins/cuffdiffdiff/index. html). Screening is carried out by using differential expression screening standards to obtain 479 differential genes, wherein 346 genes with up-regulated expression are obtained, and 133 genes with down-regulated expression are obtained.

TABLE 1 sample information

Downloading a network database GSE22255 which is a blood genome expression profile of ischemic stroke, and carrying out the same difference analysis in the gene expression profile of the database; the markers with consistent changes detected in the patient samples and databases were screened, and the differential expression box plots of the partial markers obtained from the screening are shown in FIG. 1, the expression changes in the patients are shown in Table 2, and Ture indicates consistent results in the two analyses.

TABLE 2 differentially expressed genes

	Preliminary studies	GSE22255
				ZNF22	down	down	Ture
RPS27	down	down	Ture
				ZNF416	down	down	Ture

The markers shown in table 2 were ranked and combined using the data of the database to further screen for more accurate marker combinations, the AUC value of each marker or combination of markers at diagnosis is shown in table 3, the ROC curves are shown in fig. 2-8, and the relationship between the figures and the markers or marker combinations is shown in the last column.

TABLE 3 AUC values for diagnosis

Marker/marker combination	AUC	Drawings
			RPS27	0.703	2
ZNF22	0.733	3
			ZNF416	0.74	4
RPS27_ZNF22	0.765	5
			ZNF22_ZNF416	0.7775	6
RPS27_ZNF416	0.795	7
			RPS27_ZNF22_ZNF416	0.818	8

Note: combinations of markers for the symbols in the tables

According to the AUC result, each marker of RPS27, ZNF22 and ZNF416 can be independently used as a marker for diagnosing cerebral apoplexy, and the AUC value can be further increased after combination, so that higher diagnosis accuracy is obtained.