KR20190031072A - Method for diagnosing vascular dementia using lipocalin-2 - Google Patents

Abstract

The present invention relates to a method for diagnosing vascular dementia using lipocalin-2. More particularly, the present invention relates to a kit and a composition for diagnosing vascular dementia comprising a preparation for measuring lipocalin-2 expression level, and a method for qualitative or quantitative analysis of lipocalin-2 to provide information necessary for diagnosis of vascular dementia. The level of lipocalin-2 is significantly increased in patients with vascular dementia compared to a normal group and is particularly easy to measure by using a body fluid sample such as plasma, thereby being useful as a biomarker for the diagnosis of vascular dementia.

Description

The present invention relates to a method for diagnosing vascular dementia using lipocalin-

The present invention relates to a diagnostic method for vascular dementia using lipocalin-2, and more particularly, to a diagnostic composition for diagnosing vascular dementia comprising an agent for measuring lipocalin-2 expression level, a diagnostic kit, and a diagnostic kit for diagnosing vascular dementia Lt; RTI ID = 0.0 > lipocalin-2 < / RTI > to provide information.

Various diseases of the nervous system have been reported. Especially, due to the aging of the population, about 1 out of every 10 elderly people aged 65 or older are suffering from dementia. Alzheimer's disease, cerebrovascular disease, depressive disorder, metabolic disease, thyroid disease, vitamin deficiency, infectious brain disease, and the like are the major causes of dementia, Disease, and normal pressure hydrocephalus. However, many of these causes are complex, and the occurrence of these causative diseases does not necessarily mean the occurrence of dementia, so the specific factors contributing to the causative diseases are not yet known Even so, researchers have a lot of controversy and need research.

Among the various dementia types, vascular dementia is important to prevent and diagnose cognitive dysfunction by controlling vascular risk factors. However, there are many difficulties in diagnosing vascular dementia in clinical practice, and effective therapies have not been developed so far. The reason is that vascular dementia includes a wide variety of diseases rather than a single disease. Often, it is easy to think that vascular dementia is caused simply by cerebrovascular disease, but the presence of cerebrovascular disease does not necessarily lead to vascular dementia. In fact, many factors affect the onset of dementia in order for vascular dementia to develop. These factors include vascular risk factors, changes in the cerebral cortex, cerebral white matter changes, individual characteristics and genetic factors.

On the other hand, some successive relationships have been reported between mild cognitive impairment (MIC), ischemic lesion of blood vessels, stroke, Alzheimer's dementia and vascular dementia, but this posterior relationship does not necessarily indicate the occurrence of posterior disease, There is a lot of controversy and research needs are being raised. For example, the association between preceding cognitive impairment and dementia has been reported, but the probability of a person diagnosed with amyotrophic mild cognitive impairment to develop actual vascular dementia is about 10 to 15% / year, and the predictive validity (Blossom CM Stephan et al., Beyond mild cognitive impairment: vascular cognitive impairment, no dementia (VCIND), Alzheimers Res Ther 2007: 4). For example, cerebral ischemic changes in the onset of vascular dementia are reported as one of the causes, but in fact, 30% of cases develop into vascular dementia within 6-12 months after a transient ischemic attack (Abhijeet Jagtap et al , Biomarkers in vascular dementia: A recent update, Biomarkers and Genomic Medicine (2015) 7, 43e56). In addition, the development of vascular dementia after stroke is only about 10-30% (Pendlebury ST et al., Dementia in patients hospitalized with stroke: rates, time course, and clinico-pathologic factors, Int J Stroke. 2012 Oct; 7 (7): 570-81)

Currently, dementia is largely classified as Alzheimer ' s Dementia, vascular dementia and other dementia. Accurate classification and differential detection of dementia have a great impact on treatment strategies. In addition, treatment of dementia that can be cured also causes structural changes in the brain, making it impossible to treat the disease. The recovery of treatable dementia may vary depending on the severity of damage, so if you have an accurate diagnosis and appropriate treatment, Can be obtained. It is very important to identify the specific type of disease early and apply the appropriate formulation in a timely manner. Therefore, there is an urgent need to determine the type of dementia based on biomarker analysis. In addition, it is desirable that such a biomarker is easily measured in order to facilitate accurate diagnosis early. In particular, in case of invasion of brain tissue or nerve tissue in brain diseases, there are considerable risks and difficulties such as causing secondary complications Should be considered.

Markers for distinguishing these demented symptoms have been reported in the art, but there is a lot of controversy about the marker that is practically useful. For example, homocysteine is known to be elevated in the serum of Alzheimer's patients, while plasma homocysteine levels are also elevated in patients with vascular dementia, which is reported to be related to hippocampal and cerebral cortical atrophy in vascular dementia patients (Abhijeet Jagtap et al., Biomarkers in vascular dementia: A recent update, Biomarkers and Genomic Medicine (2015) 7, 43e56) and the role of homocysteine is controversial. Thus, the development of biomarkers for diagnosing specific types of dementia differentially is required.

Accordingly, the present inventors confirmed the efficacy of lipocalin-2 as a biomarker for the diagnosis of vascular dementia in lipocalin-2 in a vascular dementia patient through remarkable elevation of levels in body fluids (especially, plasma) Respectively.

It is therefore an object of the present invention to provide a composition for the diagnosis of vascular dementia comprising an agent for measuring lipocalin-2 expression level and a kit for the diagnosis of vascular dementia comprising the same.

It is another object of the present invention to provide a method for qualitatively or quantitatively analyzing lipocalin-2 expression level from a sample collected from a subject to provide information necessary for diagnosis of vascular dementia.

In order to achieve the above object, the present invention provides a composition for diagnosing vascular dementia comprising an agent for measuring lipocalin-2 expression level and a kit for the diagnosis of vascular dementia comprising the same.

In order to achieve the other object of the present invention, the present invention provides a method for qualitatively or quantitatively analyzing lipocalin-2 expression level from a sample collected from a subject to provide information necessary for diagnosis of vascular dementia.

Hereinafter, the present invention will be described in detail.

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of an object, or therametrics (e.g., monitoring the status of an object to provide information about the therapeutic efficacy). Preferably, the diagnosis in the present invention may be to determine the level of expression of the lipocalin-2 protein to identify the presence or absence of vascular dementia.

The term 'vascular dementia (VaD)' in the present invention refers to dementia accompanied by a decline in cognitive function to an extent that can not be practiced in daily life and social life due to damage of cerebral blood vessels. Degenerative brain diseases (for example, Alzheimer's ) And dementia caused by the degenerative brain disease, and it is also distinguished from simple cognitive defect symptoms that may precede dementia such as mild cognitive impairment (MCI).

The degenerative brain disease refers to a disorder in which neurons in a part of the brain are gradually degenerated and disorder occurs in neurotransmission. Examples of degenerative neurological diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, multiple sclerosis, and the like.

 This mild cognitive impairment (MCI) is clinically characterized by minor impairment of recent memory without dementia, or significant impairment of other cognitive functions to an extent that exceeds what is expected of the age or educational background. Hard cognitive impairment is memory complaint; Abnormal daily activities; Abnormal general cognitive function; Abnormal memory compared to age; And the absence of dementia.

The expression " expression " in the present invention means that a protein or a nucleic acid is produced in a cell.

The term 'protein' as used herein is used interchangeably with 'polypeptide' or 'peptide', for example, a polymer of amino acid residues as commonly found in proteins in nature.

The term " polynucleotide " or " nucleic acid " as used herein refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' is RNA that transfers genetic information (gene-specific nucleotide sequence) to a ribosome in which amino acid sequence is specified from a specific gene during protein synthesis.

The present invention provides a composition for the diagnosis of vascular dementia comprising an agent for measuring lipocalin-2 expression level and a kit for the diagnosis of vascular dementia comprising the same.

The expression " measurement of lipocalin-2 expression level " is meant to measure the level of expression of lipocalin-2 protein or lipocalin-2 coding gene.

In the present invention, the lipocalin-2 protein is not particularly limited as long as it is known to be derived from human. Specific examples of the lipocalin-2 protein include GenBank accession number: NP_005555.1 (SEQ ID NO: 1). Preferably, the lipocalin-2 protein of the present invention may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, more preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The lipocalin-2 protein is known to be encoded by a polynucleotide sequence such as GenBank accession number: NM_005564 (mRNA, SEQ ID NO: 2).

The agent for measuring the expression level of the lipocalin-2 protein is not particularly limited as long as it is known in the art to be capable of measuring the expression level of the protein, but is preferably a lipocalin- Antibody or an aptamer.

The term " antibody " in the present invention means an immunoglobulin which specifically binds to an antigenic site. The lipocalin-2 antibody in the present invention is an antibody that specifically binds only to lipocalin-2 protein without reacting with other proteins other than lipocalin-2. In the present invention, the antibody specifically binding to the lipocalin-2 protein may preferably be an antibody that specifically binds to a protein (lipocalin-2) comprising the amino acid sequence represented by SEQ ID NO: 1. The antibody may be obtained by cloning a lipocalin-2 coding gene into an expression vector to obtain a protein encoded by the gene, and obtaining an antibody produced by injecting the resulting protein into an animal, such as by a conventional method in the art . The lipocalin-2 antibody may be produced through a lipocalin-2 full-length sequence protein, or may be produced using a polypeptide fragment containing an antigenic site of lipocalin-2 protein. The form of the antibody of the present invention is not particularly limited and includes a polyclonal antibody or a monoclonal antibody. Also included in the antibody of the present invention are a portion of the whole antibody as well as any immunoglobulin antibody that specifically binds to lipocalin-2, if it has antigen-antibody binding (response). F (ab '), F (ab '), < / RTI > which have antigen binding functions, as well as complete forms of antibodies with two full length light chains and two full length heavy chains, 2, and Fv. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies, chimeric antibodies, and recombinant antibodies, as long as they can specifically bind lipocalin-2 protein.

In the present invention, the term 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample , The presence of the target protein in the sample can be confirmed specifically. Aptamer is produced by determining the sequence of an oligonucleotide having a selective and high binding ability to a target protein (lipocalin-2 protein in the present invention) to be identified according to a general method of producing an aptamer and then synthesizing an oligonucleotide But not limited to, -SH, -COOH, -OH, or NH2 so that the 5 'terminal or the 3' terminal of the aptamer chip can be bound to the functional group of the aptamer chip.

In the present invention, measuring the expression level of the lipocalin-2 coding gene means measuring the expression level of transcripts derived from the lipocalin-2 gene. For example, when the lipocalin-2 mRNA expression level . ≪ / RTI >

Therefore, the agent for measuring the level of expression of the lipocalin-2 coding gene is not limited thereto, but may preferably mean an agent for detecting mRNA expressed from the gene. Therefore, the agent for detecting the lipocalin-2 coding gene is not particularly limited as long as it is a ligand that specifically binds or hybridizes to the mRNA expressed from the gene. For example, a primer (pair) .

The term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a template complementary to a nucleic acid sequence having a short free 3 'hydroxyl group and acting as a starting point for template strand replication . Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

The sequence of the primer does not need to have a sequence completely complementary to a part of the nucleotide sequence of the template. It is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, in the present invention, the primer for measuring the level of expression of lipocalin-2 mRNA does not need to have a perfectly complementary sequence to the lipocalin-2 gene sequence, and it is possible to use lipocalin-2 mRNA or lipocalin- It is sufficient that the specific region of the cDNA is amplified to have a length and complementarity to the purpose of measuring the amount of lipocalin-2 mRNA. The primer for the amplification reaction is a set (pair) which is complementarily bound to a template (or sense) at opposite ends of a specific region of the lipocalin-2 mRNA to be amplified and an opposite region (antisense), respectively . Primers can be readily designed by those skilled in the art with reference to the lipocalin-2 mRNA or cDNA sequence.

The term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA having a base pair length of several to several hundreds, which can specifically bind to a specific gene mRNA or cDNA (complementary DNA) And it is labeled so that the presence or expression level of mRNA or cDNA to be bound can be confirmed. For the purpose of the present invention, a probe complementary to lipocalin-2 mRNA can be used for the diagnosis of vascular dementia by measuring the expression level of lipocalin-2 mRNA by performing hybridization with a sample of a subject. The selection and hybridization conditions of the probes can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. The primers or probes can also be modified in various ways according to methods known in the art, so long as they do not interfere with hybridization with lipocalin-2 mRNA. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ) Or charged conjugates (eg, phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescence or enzymes.

In the diagnostic kit of the present invention, in order to measure the level of expression of lipocalin-2, an antibody that selectively recognizes lipocalin-2 protein as a marker, a primer that recognizes an aptamer or lipocalin-2 mRNA as a marker, One or more other component compositions, solutions or devices suitable for the method may be included.

In a specific embodiment, the kit can be used in a variety of forms including Western blot, enzyme-linked immunospecific assay (ELISA), radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, immuno-staining, But are not limited to, diagnostic kits that include known essential components and ancillary elements necessary to perform a fixed assay, a fluorescence activated cell sorter (FACS), or a protein chip method.

In one embodiment, the kit comprises an antibody specific for the lipocalin-2 protein. The antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the target marker protein and little cross reactivity to other proteins. The kit may further comprise an antibody specific for a control protein (e. G., A normal donor cell specific marker protein). The kit may further comprise a reagent capable of detecting the bound antibody, for example, a labeled secondary antibody, chromophores, an enzyme (in conjugated form with the antibody) and a substrate or antibody capable of binding to the substrate Other materials, and the like. In addition, the kit of the present invention may include a washing solution or an eluting solution capable of removing surplus chromogenic substrate and unbound protein and retaining only the protein marker bound to the antibody.

The kit is not particularly limited as long as it is known in the art as an assay kit for providing a primer (primer pair) or a probe as a component. For example, a PCR (polymerase chain reaction) , Northern blotting, Southern blotting or kits for DNA microarray chips, and the like.

For example, the diagnostic kit may be a diagnostic kit characterized by comprising essential elements necessary for performing a polymerase reaction. The polymerase chain reaction kit contains the respective primer pairs specific for the marker gene (mRNA). A primer is a nucleotide having a sequence specific to the nucleotide sequence of each marker gene (mRNA), and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other polymerase enzyme reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), DNA polymerases (such as Taq polymerase) DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.

The present invention also provides a method for qualitatively or quantitatively analyzing the level of lipocalin-2 expression from a sample collected from a subject to provide information necessary for the diagnosis of vascular dementia.

Specifically, the method comprises: (a) measuring the level of lipocalin-2 expression from a sample of a subject; And

(b) comparing the lipocalin-2 expression level with that of a normal person, and determining that the subject having an increased level of lipocalin-2 expression compared to a normal person has a vascular dementia.

The term 'analysis' in the present invention may preferably mean 'measurement', and the qualitative analysis may be a measurement and confirmation of the presence or absence of a target substance, It may be meant to measure and confirm changes in the level of expression (level of expression) or amount. In the present invention, the analysis or measurement can be performed without limitation including both qualitative and quantitative methods, and preferably, a quantitative measurement can be performed.

Hereinafter, the above method will be described in accordance with each step.

The step (a) is a step of measuring the level of lipocalin-2 expression in the sample provided from the subject (latent patient).

The sample can be used without limitation as long as it is collected from a subject to be diagnosed as having vascular dementia. For example, the sample can be a cell or tissue obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, Urine, feces, and the like. Preferably, a biological fluid sample (fluid sample) may be used. For example, blood, plasma, serum, saliva, saliva, sputum, capsular fluid, amniotic fluid, ascites, cervix or vaginal discharge, urine and cerebrospinal fluid Lt; / RTI > The present invention is technically significant in that the diagnosis can be made through a body fluid sample (for example, plasma) other than a direct brain lesion tissue in which vascular dementia has been caused, thereby increasing the efficiency, promptness, convenience, and safety of diagnosis. In the present invention, the sample is most preferably blood, plasma, or serum.

The sample may be pretreated prior to use for detection or diagnosis. For example, homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like can be included.

The measurement of lipocalin-2 expression level in step (a) above means measuring the level of lipocalin-2 protein or lipocalin-2 coding gene expression.

If the protein expression level is determined by a protein expression assay method known in the art, the measurement method is not particularly limited. For example, Western blotting, enzyme-linked immunospecific assay (ELISA), radioimmunoassay, Immunoassay, immobilization assay, complement fixation assay, FACS (Fluorescence activated cell sorter), or protein chip method is used as the immunoassay method, Lt; / RTI >

If the gene expression level is determined by a gene expression assay method known in the art, the measurement method is not particularly limited. Preferably, the gene expression level is determined by PCR (polymerase chain reaction), RNase protection assay, northern blotting, southern blotting, and DNA chips.

In the step (b), the lipocalin-2 expression level of the test sample measured in the step (a) is compared with that of a normal person, and it is judged that the test subject having increased lipocalin-2 expression level as compared to a normal person has vascular dementia .

The lipocalin-2 expression level (including gene or protein level) of the test sample measured by the method of step (a) described above is compared with the lipocalin-2 expression level of a normal sample measured by the same method. At this time, the sample is preferably a sample obtained by the same kind or the same method between the subject and the normal person. It is judged that the level of expression of lipocalin-2 is increased compared to a healthy normal person and the subject is suffering from vascular dementia.

Lipocalin-2 not only shows a significantly increased level in vascular dementia patients compared to the normal group, but also is useful as a biomarker for the diagnosis of vascular dementia because it is easy to measure because it can be measured through a body fluid sample such as plasma Do.

Figure 1 shows the results of comparative evaluation of plasma lipocalin-2 (LCN2) levels in untreated two-tailed Student's t test (p <.0001) in normal control and VaD patients. The horizontal bar in each experimental group represents the average value of LCN2 level.
Figure 2 shows the receiver operating charateristic curve (AUC: area under curve) for plasma lipocalin-2 (LCN2) levels in vascular dementia (VaD) patients as biomarkers.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

&Lt; Example 1 >

Validation of plasma lipocalin-2 as a biomarker for the diagnosis of vascular dementia (VaD)

1) Experimental method

Patient: We recruited participants from patients who visited Kyungpook National University Hospital dementia clinic (Daegu, Korea). Information was obtained from all participants or guardians. This study was approved by the Delayed Ethics Committee. From January 2014 to May 2016, a total of 37 patients were enrolled in this study, consisting of 9 patients who were diagnosed with VaD and 28 healthy subjects at Kyungpook National University Hospital. Patients with VaD were selected according to the following criteria: (1) 40 years of age or older; (2) meeting the DSM-IV standard (Black & Grant, 2014) or / and the NINDS-AIREN standard (Roman et al., 1993); (3) evidence of significant cerebral vascular changes on the skull-MRI (cranial-MRI); (4) Mini-Mental State Examination score (MMSE) 10-26. At the time of diagnosis, blood screening tests were performed to exclude patients with cognitive impairment due to metabolic causes. Patients diagnosed with neurodegenerative disorders, space-occupying lesions, or lobar hemorrhage were also excluded from this study. Control subjects, 28 subjects, were healthy, intellectually active, and had no history of cerebrovascular, cardiovascular or neurological disorders. Cranial MRI and blood screening tests were also performed to exclude unknown causes.

Plasma samples: patients who fasted more than 8 hours the day before Blood collected from the large vena cava was placed in an EDTA tube, and a protease inhibitor cocktail (Roche Diagnostics) was added and gently mixed. The EDTA-treated blood was centrifuged at 3000 rpm for 10 minutes to separate plasma. The collected plasma samples were immediately frozen on dry ice and stored in a -80 ° C freezer.

Plasma lipocalin- 2 measurement: LCN2 levels were measured in human plasma samples using Quantikine kit (R & D systems) or Duo-set ELISA kit (R & D systems). The ELISA assay was performed in a 96-well plate using 100 쨉 l of plasma (1: 400) according to the manufacturer's protocol. The standard concentration of human recombinant LCN2 used is 39.06-2500 pg / ml. The level of recombinant LCN2 protein was normalized to the total protein content of plasma samples. All measurements were performed in duplicate.

Statistical analysis: Statistical analysis was performed by comparing the mean of the other groups using one-way ANOVA or two-way ANOVA with unpaired two-tailed Student's t- test, Tukey multiple comparison test. Data sets were analyzed using SPSS version 14.0K software (SPSS Inc., Chicago, IL). Statistical significance was established at p <0.05.

2) Experimental results

As shown in FIG. 1, the mean value of the normal control group was 33.376 ± 3.53 ng / ml, but the VaD patients had a value of 90.06 ± 612.71 ng / ml, and the plasma level of lipocalin Respectively.

The ROC curve was also used to evaluate the predictive significance of the level of LCN2 in the plasma of patients with VaD as biomarkers. As shown in FIG. 2, the area under the ROC curve (AUC, ie, accuracy) of the VaD patients was 0.952 (95% CI: 0.000 to 1.000, P = 0.00005, cutoff value: 49.32, sensitivity: 88.9%, specificity: ).

As described above, the present invention relates to a method for diagnosing vascular dementia using lipocalin-2, and more particularly, to a composition for diagnosing vascular dementia including a preparation for measuring lipocalin-2 expression level, a diagnostic kit, And a method for qualitative or quantitative analysis of lipocalin-2 to provide information necessary for diagnosis of dementia.

The lipocalin-2 not only shows a significantly increased level in vascular dementia patients compared with the normal group but also is useful as a biomarker for the diagnosis of vascular dementia because it is easy to measure because it can be measured through a body fluid sample such as plasma It is highly likely to be used industrially in fields such as an in vitro diagnostic industry.

<110> Kyungpook National University Industry-Academic Cooperation Foundation          KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL <120> Method for diagnosing vascular dementia using lipocalin-2 <130> NP17-0059 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> human lipocalin-2 <400> 1 Met Pro Leu Gly Leu Leu Gly Leu Gly Leu Ala Leu   1 5 10 15 His Ala Gln Ala Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro              20 25 30 Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln          35 40 45 Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu      50 55 60 Asp Lys Asp Pro Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu  65 70 75 80 Asp Lys Ser Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys                  85 90 95 Asp Tyr Trp Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe             100 105 110 Thr Leu Gly Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val         115 120 125 Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys     130 135 140 Lys Val Ser Gln Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg 145 150 155 160 Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser                 165 170 175 Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Ile             180 185 190 Asp Gln Cys Ile Asp Gly         195 <210> 2 <211> 597 <212> DNA <213> Artificial Sequence <220> <223> human lipocalin-2 mRNA <400> 2 atgcccctag gtctcctgtg gctgggccta gccctgttgg gggctctgca tgcccaggcc 60 caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 120 aacttccagg acaaccaatt ccaggggaag tggtatgtgg taggcctggc agggaatgca 180 attctcagag aagacaaaga cccgcaaaag atgtatgcca ccatctatga gctgaaagaa 240 gacaagagct acaatgtcac ctccgtcctg tttaggaaaa agaagtgtga ctactggatc 300 aggacttttg ttccaggttg ccagcccggc gagttcacgc tgggcaacat taagagttac 360 cctggattaa cgagttacct cgtccgagtg gtgagcacca actacaacca gcatgctatg 420 gtgttcttca agaaagtttc tcaaaacagg gagtacttca agatcaccct ctacgggaga 480 accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 540 ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggctga 597

Claims (12)

A composition for the diagnosis of vascular dementia comprising an agent for measuring lipocalin-2 expression level.
2. The composition of claim 1, wherein said lipocalin-2 expression level measurement measures the level of expression of lipocalin-2 protein or lipocalin-2 coding gene.
The composition according to claim 1,
(a) an antibody or aptamer that specifically binds to lipocalin-2 protein; or
(b) a primer or a probe that specifically binds to lipocalin-2 mRNA.
4. The composition of claim 3, wherein the lipocalin-2 protein comprises the amino acid sequence of SEQ ID NO: 1.
4. The composition of claim 3, wherein the lipocalin-2 mRNA comprises the nucleotide sequence of SEQ ID NO: 2.
A kit for the diagnosis of vascular dementia comprising an agent for measuring lipocalin-2 expression level.
A method for qualitatively or quantitatively analyzing the level of lipocalin-2 expression from a sample collected from a subject to provide information necessary for diagnosis of vascular dementia.
8. The method of claim 7,
(a) measuring the level of lipocalin-2 expression from a sample of the subject; and
(b) comparing the level of lipocalin-2 expression to that of a normal person, and determining that the subject having increased levels of lipocalin-2 expression relative to a normal person is undergoing vascular dementia.
9. The method according to claim 7 or 8, wherein the sample is a body fluid sample selected from the group consisting of blood, plasma, serum, saliva, saliva, sputum, capsular fluid, amniotic fluid, ascites, cervix or vaginal discharge, urine and cerebrospinal fluid &Lt; / RTI &gt;
9. The method of claim 8, wherein said lipocalin-2 expression level measurement measures lipocalin-2 protein or lipocalin-2 coding gene expression level.
11. The method of claim 10, wherein the protein expression level is measured by Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Oucheroni immunodiffusion, rocket immunoelectrophoresis, immunoassay, immunoprecipitation assay, Wherein the protein is selected from the group consisting of protein chips.
11. The method according to claim 10, wherein the gene expression level measurement is by any one selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting and DNA chip .

Images