阅读说明：本技术 缺血性脑卒中的诊断标志物 (Diagnostic marker for cerebral ischemic stroke ) 是由 蔡学礼 谢慧蓉 舒小桓 孙景萍 黄慧芬 李威 兰卫明 吴骏 吴新校 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,具体涉及包括缺血性脑卒中的诊断标志物或其组合,同时本发明提供了检测中枢神经系统疾病的试剂盒、系统及其应用。优选地,所述标志物包括CACNA1E、PDCD1、USP14；所述中枢神经系统疾病是脑卒中,更优选地,所述中枢神经系统疾病是缺血性脑卒中。(The invention relates to the fields of biotechnology and biomedicine, in particular to a diagnostic marker for ischemic stroke or a combination thereof, and also provides a kit and a system for detecting central nervous system diseases and application thereof. Preferably, the markers include CACNA1E, PDCD1, USP 14; the central nervous system disease is stroke, more preferably, the central nervous system disease is ischemic stroke.)

1. A marker combination for the diagnosis of central nervous system disease, comprising any two or all of CACNA1E, PDCD1, USP 14.

2. The marker combination of claim 1 wherein the central nervous system disorder that the marker combination diagnoses includes stroke, epilepsy, alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, demyelinating disease, multiple sclerosis, schizophrenia, depression, and central nerve injury;

preferably, the stroke comprises ischemic stroke and hemorrhagic stroke.

3. The marker combination of claim 2, wherein the central nervous system disorder is ischemic stroke.

4. The marker combination of claim 1 wherein the diagnosis requires collection of a sample from the patient, the sample comprising tissue, blood 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 blood comprises: serum, plasma, whole blood;

preferably, the blood is whole blood.

5. The marker combination of claim 1 wherein CACNA1E is highly expressed in the patient, PDCD1 is lowly expressed in the patient and USP14 is lowly expressed in the patient.

6. A kit for detecting the mRNA expression level and/or protein expression level of at least one marker in the marker combination according to claim 1;

preferably, the kit comprises a reagent for detecting the expression quantity of mRNA;

preferably, the kit comprises a reagent for detecting the expression amount of the protein.

7. The kit of claim 6, further comprising at least one of an mRNA expression level auxiliary detection reagent, a protein expression level auxiliary detection reagent, an mRNA expression level auxiliary detection device, and a protein expression level auxiliary detection device.

8. A system for diagnosing a central nervous system disease, comprising an input device for inputting an expression level of at least any one of the marker combinations according to claim 1, and an output device for outputting a diagnosis result.

9. The system can also comprise a detection device for detecting the gene expression quantity, wherein the detection device comprises a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip and a chip signal reader.

10. The use of a reagent for detecting the expression level of at least one marker selected from the group consisting of CACNA1E, PDCD1 and USP14, the kit of claim 6 and the system of claim 8 for preparing a product for diagnosing whether a subject has a cns disease.

Technical Field

The invention relates to the fields of biotechnology and biomedicine, in particular to a diagnostic marker for ischemic stroke or a combination thereof.

Background

The cerebral apoplexy is a main clinical type of cerebrovascular disease, is a paroxysmal cerebral tissue blood circulation disorder disease, and is a group of diseases which are caused by arteriovenous stenosis, occlusion or rupture caused by various inducing factors to cause acute cerebral tissue blood circulation disorder and are clinically manifested as transient or permanent neurological impairment symptoms and physical signs. Stroke is one of three fatal diseases worldwide, and the high morbidity, high disability rate and high fatality rate of stroke cause heavy burden and pain to patients, families and society. The ischemic stroke is relatively common in clinic and is represented as clinical pathological processes of local cerebral tissue hypoxia, ischemia and neural necrosis, and the incidence rate of the ischemic stroke accounts for 60-80 percent of that of the cerebral stroke.

The clinical diagnosis classification terms for ischemic stroke are more, including cerebral infarction (or called cerebral infarction), cerebral thrombosis, cerebral embolism and the like. Patients with cerebral hemorrhage often show symptoms such as severe headache, frequent vomiting, hemiplegia, even coma and the like, and serious patients even die quickly. The most common cause of subarachnoid hemorrhage is rupture of intracranial aneurysm or rupture of cerebral vessel malformation, which is generally acute in onset and severe in headache, and is common to young and middle-aged people. The general symptoms of ischemic stroke are mild, and most patients have clear consciousness and are manifested as hemiplegia or weakness, dysphonia, limb numbness and the like.

Although the diagnosis of stroke is mostly clinical diagnosis, neuroimaging examination and bedside rapid blood glucose examination are still necessary. Within the first hours of the infarct, evidence on CT is minimal, even if large areas of the infarct are in the anterior circulation, including the disappearance of striae or islet cortex banding in the affected area, the disappearance of gray mass borders, and the high density shadow of the middle cerebral artery. In 6 to 12 hours of cerebral ischemia, medium to large infarcts begin to appear as CT-visible low density foci; small infarcts (e.g., lacunar infarcts) may only be found on MRI. Therefore, when ischemic stroke is suspected, diffusion weighted MRI (sensitive to early ischemic foci) can be examined immediately after the initial CT examination.

Stroke is a common acute cerebrovascular disease in the middle-aged and elderly people, and has a tendency to become younger. According to statistics, China died of nearly 300 million people with cerebrovascular diseases every year, which is 4 to 5 times higher than that of Europe and America, 3.5 times higher than that of Japan, and even higher than that of developing countries such as Thailand, India and the like; the incidence rate rises at a rate of 8.7% per year, the recurrence rate exceeds 30%, and the recurrence rate reaches 54% within 5 years; 75% of the survivors from stroke patients lose labor capacity to different degrees, and 40% are heavily disabled.

Disclosure of Invention

The inventor of the invention screens, verifies and provides a marker and a marker combination which can efficiently and accurately diagnose the central nervous system diseases by sequencing and analyzing collected patient samples and analyzing data of a data set. Also provides a kit and a system for detecting central nervous system diseases and application thereof.

In one aspect the invention provides a marker combination for the diagnosis of central nervous system disease comprising any two or three of CACNA1E, PDCD1, USP 14.

Preferably, the central nervous system disease comprises stroke, epilepsy, alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, demyelinating disease, multiple sclerosis, schizophrenia, depression and central nerve injury.

Preferably, the stroke comprises ischemic stroke and hemorrhagic stroke.

Preferably, the central nervous system disease is ischemic stroke.

Preferably, the subject is a mammal. Such as bovine, equine, ovine, porcine, canine, feline, rodent, primate.

Preferably, the subject is a human.

Preferably, the CACNA1E is highly expressed in the patient.

Preferably, the PDCD1 is low expressed in the patient.

Preferably, the USP14 is low expressed in patients.

In another aspect, the invention provides a kit for detecting the expression level of at least any one of the above marker combinations.

Preferably, the at least any one includes two or three.

Preferably, the at least any one marker comprises the following markers or marker combinations CACNA1E, PDCD1, USP14, CACNA1E + PDCD1, PDCD1+ USP14, CACNA1E + USP14, CACNA1E + PDCD1+ USP 14; wherein "+" represents a combination, and "_" represents the same meaning in other portions of the text.

Preferably, the expression amount includes an mRNA expression amount and/or a protein expression amount.

Preferably, the kit comprises a reagent for detecting the mRNA expression amount.

Preferably, the kit comprises a reagent for detecting the expression amount of the protein.

Preferably, the reagent for detecting the expression amount of mRNA comprises a reagent used in the following method: PCR-based quantitative detection method, Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO method, high throughput sequencing platform.

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

Preferably, the probe may be DNA, RNA, DNA-RNA chimeras, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

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 chips.

Preferably, the reagent for detecting the expression level of the protein comprises a specific antibody.

Preferably, the antibody comprises a monoclonal antibody, a polyclonal antibody.

Preferably, the antibody comprises an intact antibody molecule, any fragment of an antibody or an antibody with modifications, in particular, the antibody comprises a chimeric antibody, scFv, Fab, F (ab') 2, Fv, or the like. As long as the fragment retains the ability to bind to the protein. The preparation of antibodies for detecting protein levels is well known to those skilled in the art, and any method can be used in the present invention to prepare the antibodies.

Preferably, the kit can also comprise an mRNA expression quantity auxiliary detection reagent, a protein expression quantity auxiliary detection reagent, an mRNA expression quantity auxiliary detection instrument and a protein expression quantity auxiliary detection instrument.

Preferably, the mRNA expression level auxiliary detection reagent includes, but is not limited to: a reaction reagent for visualizing the amplicon corresponding to the primer, for example, a reagent for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization, fluorescence detection method, or the like; RNA extraction reagent; a reverse transcription reagent; a cDNA amplification reagent; preparing a standard substance for a standard curve; a positive control; and (5) a negative control product.

Preferably, the protein expression amount auxiliary detection reagent includes but is not limited to: blocking solution, antibody diluent, washing buffer solution, chromogenic stop solution and standard substance for preparing a standard curve.

In another aspect, the present invention provides a system for diagnosing central nervous system diseases, comprising an input device for inputting the expression level of at least any one of the aforementioned combination of test markers, and an output device for outputting the diagnosis result.

Preferably, the system may further comprise a detection means for detecting the expression level of the combination of markers.

Preferably, the detection device comprises a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip and a chip signal reader.

Preferably, the chip comprises a probe for detecting the expression level of the marker.

Preferably, the chip further comprises an internal reference probe.

Preferably, the internal reference comprises GAPDH or β -Actin.

Preferably, the chip comprises a protein chip and/or a gene chip.

Preferably, the system further comprises an evaluation result transmission unit that can transmit the evaluation result of the subject to an information communication terminal device that can be referred to by the patient or medical staff.

In another aspect, the present invention provides a method for diagnosing a central nervous system disease, which comprises detecting the expression level of at least any one of the aforementioned marker combinations.

Preferably, the method further comprises the step of collecting the blood from the subject.

Preferably, the sample comprises: tissue, blood 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 blood comprises: serum, plasma, whole blood.

Preferably, the blood is whole blood.

Preferably, the method further comprises a step of judging the diseased state of the subject by the result of the detection of the expression amount after the detection.

In another aspect, the invention provides a reagent for detecting the expression level of at least any one of the CACNA1E, PDCD1 and USP14, a kit for detecting the expression level of the CACNA, and use of the system for detecting the expression level of the CACNA1E, the PDCD1 and the USP14 in preparation of a product for diagnosing whether a subject has a central nervous system disease.

Drawings

FIG. 1 is a box plot of the difference of the differential genes, A is USP14, B is PDCD1, and C is CACNA 1E.

FIG. 2 is a ROC curve chart of marker USP14 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 3 is a graph of ROC curve of the marker PDCD1 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 4 is a ROC plot of marker CACNA1E in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 5 is a ROC plot of marker combination PDCD1_ USP14 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 6 is a ROC plot of marker combination CACNA1E — USP14 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 7 is a ROC plot of marker combination CACNA1E _ PDCD1 in diagnosing ischemic stroke; the ordinate is sensitivity and the abscissa is specificity.

FIG. 8 is a ROC plot of marker combination CACNA1E _ PDCD1_ USP14 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-la b. githu. io/cufflinks/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 patient samples and databases are screened, the differential expression boxplot of the screened partial markers is shown in figure 1, and the expression changes in patients are shown in table 2.

TABLE 2 differentially expressed genes

	Preliminary studies	GSE22255
			CACNA1E	up	up
PDCD1	down	down
			USP14	down	down

The accuracy was further improved by using the data from the database to rank the markers shown in table 2, where the AUC values for each marker or combination of markers at diagnosis are shown in table 3, and the ROC curves are shown in fig. 2-8.

TABLE 3 AUC values for diagnosis

Marker/marker combination	AUC	Drawing (A)
			USP14	0.757	2
PDCD1	0.71	3
			CACNA1E	0.757	4
PDCD1_USP14	0.8175	5
			CACNA1E_USP14	0.7775	6
CACNA1E_PDCD1	0.8525	7
			CACNA1E_PDCD1_USP14	0.848	8

The AUC results in Table 3 show that each marker of CACNA1E, PDCD1 and USP14 can be used as a marker for diagnosing cerebral apoplexy independently, the AUC value after combination is further increased, higher diagnosis accuracy is obtained, any two markers have synergistic effect, and even if the AUC value of each independent marker is about more than 0.7, the combination is obviously improved.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.