KR20120004075A - Composition for diagnosing ischemia compriging sweet-taste receptor genes - Google Patents

Abstract

PURPOSE: A composition containing T1R2, T1R3 or alpha-gustducin gene is provided to ensure diagnosis accuracy and to early treat ischemia. CONSTITUTION: A composition for diagnosing ischemic diseases contains an agent for measuring mRNA or protein level of T1R2, T1R3, or alpha-gustducin gene. T1R2 and T1R3 has a base sequence of sequence number 1 and 2, respectively. A method or providing information for diagnosing ischemic diseases comprises: a step of measuring T1R2, T1R3, or alpha-gustducin gene and protein expression level from a biological sample of a patient; and a step of comparing expression and protein level with a control group.

Description

Composition for diagnosing ischemic disease containing sweet receptor genes {Composition for diagnosing ischemia compriging sweet-taste receptor genes}

The present invention relates to the use of one or more genes selected from the sweet receptors T1R2, T1R3 and their related α-guestucine as a diagnostic marker for ischemic disease, and to a composition for diagnosing ischemic disease containing the genes, a method for diagnosing ischemic disease, and the like. will be.

Recently, biomarker discovery has exploded with the completion of the genome project. Many biomarkers have been found that provide clues that are crucial for various cancers, cardiovascular and senile diseases. Recently, a new concept of epigenetic biomarkers has been discovered, and the application range of biomarkers has been widened.

Bio (gene) markers refer to markers in genes associated with a disease, which capture the clues of the disease through the presence or absence of these markers. By using the biomarker, a disease can be diagnosed accurately by a simpler method, and many researchers are devoted to the study of the biomarker. T1R2 and T1R3 related to the present invention are subunits that are specifically expressed in taste receptor cells and form a dimeric sweet taste receptor complex (T1R2 / T1R3 heterodimer), which are sweetened by the receptor TAS1R consisting of the T1R2 and T1R3. It is known that the detection of is mediated.

Both subunits belong to the so-called "receptor coupled to G-protein" or GPCR, in particular C-grade GPCR. T1R2 and T1R3 have recently been reported as glucose sensors, but there is no known association with ischemic disease.

In particular, diabetes-related changes in peripheral nerves that are deeply associated with cerebral ischemia among ischemic diseases include complex and diverse causes. The main pathogenic mechanism is the structural and functional impairment of the neuroepithelial microvessels originating in changes in nerve fibers caused by hypoxia or ischemia. The brain ischemia is fatal, even with a short time accompanied by paraplegia and dizziness blindness speech impairment.

Therefore, experiments for the study of brain ischemia have been conducted using several animals, and many studies have been conducted to elucidate the mechanism of ischemia using these models and to understand the effects of each drug. Difficulties exist in identifying and treating cases. In particular, there are various pathophysiological mechanisms involved in ischemic brain injury, so researches to discover new genes related to ischemic disease are very important.

Accordingly, the present inventors confirmed that up-regulation of taste-specific G-protein α-gutducin with taste receptors T1R2 and T1R3 in ischemic hippocampus was found in reactive astrocytes, and ischemic disease and T1R2, T1R3 or α-gutducin genes. The present invention was completed by examining the expression relevance and specificity of.

The present invention relates to the use of a T1R2, T1R3 or α-gutducin gene as an ischemic disease diagnostic marker based on the association between ischemic disease development, and an object of the present invention is an ischemia containing a T1R2, T1R3 or α-gutducin gene. It provides a composition for diagnosing a disease and a kit for diagnosing ischemic disease containing the same.

Another object of the present invention is to provide a method for providing information for diagnosing ischemic disease by measuring the expression level of a T1R2, T1R3 or α-Guustucine gene.

Still another object of the present invention is to provide a method for screening a substance for treating ischemic disease and a method for maintaining glucose homeostasis in the brain using a T1R2, T1R3, or α-guustucine gene.

In order to solve the above problems, the present invention provides a composition for diagnosing ischemic disease, comprising an agent for measuring the level of one or more gene mRNA or protein thereof selected from T1R2, T1R3 and α-guestucine.

In one embodiment of the present invention, the T1R2 has a nucleotide sequence of SEQ ID NO: 1, the T1R3 has a nucleotide sequence of SEQ ID NO: 2, the α-Guestucine may have a nucleotide sequence of SEQ ID NO: 3. .

In one embodiment of the invention, the agent for measuring the level of the gene mRNA may comprise a primer that specifically binds to one or more genes selected from T1R2, T1R3 and α-guestucine.

In one embodiment of the present invention, the agent for measuring the level of the protein may comprise an antibody that specifically binds to at least one protein selected from T1R2, T1R3 and α-guestucine.

In one embodiment of the present invention, the level of one or more gene mRNA or protein thereof selected from T1R2, T1R3 and α-guestucine may be to measure the level in the astrocytes.

In one embodiment of the present invention, the ischemic disease is wound healing, ischemic brain disease, ischemic heart disease, peripheral vascular disease, renal artery disease, gastrointestinal lesions, burns, skin grafts, vascular grafts, organ therapy, bone repair disease, liver Disease, uterine disease, retinal angiogenic disease, bone regeneration disease, cartilage repair disease, and smooth muscle cell disease.

In one embodiment of the present invention, the ischemic disease may be ischemic brain disease.

In one embodiment of the present invention, the gene is T1R2 and α- gustducin; T1R3 and α-gutducin; Or it may be measured by a combination of T1R2, T1R3, α-guestucine.

In addition, the present invention provides a kit for diagnosing ischemic disease comprising the composition according to the present invention.

In one embodiment of the present invention, the kit may be an RT-PCR kit, a DNA chip kit or a protein chip kit.

In addition, the present invention comprises the steps of measuring the level of expression of one or more genes selected from T1R2, T1R3 and α-guestucine or a protein encoded by a biological sample isolated from a patient; And comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene of the normal control sample.

In one embodiment of the present invention, the expression level of the gene is to measure the mRNA level of the gene, the protein level can be measured using an antibody that specifically binds to the protein.

In one embodiment of the present invention, the level of one or more gene mRNA or protein thereof selected from T1R2, T1R3 and α-guestucine may be to measure the level in the astrocytes.

In one embodiment of the present invention, the gene is T1R2 and α- gustducin; T1R3 and α-gutducin; Or it may be measured using a combination of T1R2, T1R3, α-guestucine.

In addition, the present invention,

(a) expressing at least one gene selected from among T1R2, T1R3 and α-guestucine in astrocytes in the presence of a candidate substance; And

(b) When the expression of one or more genes selected from among T1R2, T1R3 and α-guestucine is suppressed in astrocytes without candidates, the candidate substance is expressed as a substance for treating ischemic disease. It provides a method for screening a substance for treating ischemic disease, comprising the step of selecting.

In one embodiment of the present invention, the gene is T1R2 and α- gustducin; T1R3 and α-gutducin; Or it may be expressed by a combination of T1R2, T1R3, α-guestucine.

The present invention further provides a method of maintaining glucose homeostasis of the brain by reducing or increasing the expression of one or more genes selected from T1R2, T1R3 and α-guestucine in astrocytes.

The present invention has the effect of providing a composition and method for diagnosing ischemic disease, in particular cerebral ischemic disease, by confirming upregulation of T1R2, T1R3 and α-guthducin genes in astrocytes. In addition, the present invention may provide a method for screening a therapeutic agent for ischemic disease by screening for substances that inhibit upregulated expression of T1R2, T1R3 and α-guestucine genes in astrocytes. Therefore, the present invention can be usefully used for the study of the diagnosis and treatment of ischemic diseases by providing various uses according to novel T1R2, T1R3 and α-guestucine gene markers related to ischemic diseases.

1 is a photograph showing the results of immunoblotting of T1R2, T1R3, and α-guestucine after ischemia.
Figure 2 is a graph of the density analysis (Densitometric analysis) results of T1R2, T1R3 and α- gustdusin immunoblotting.
Figure 3 is a confocal micrograph of the immunoreactive changes of T1R2 (AF) and T1R3 (GL) in the hippocampus using immunohistochemical staining after ischemia.
Figure 4 is a photograph showing the phenotype of T1R2, T1R3 immunoreactive cells in the CA1 region of the hippocampus, 7 days after reperfusion.
Figure 5 is a photograph showing the transient profile and phenotype of α-gutducin immunoreactive cells in hippocampus after ischemia.

Definitions of terms used in the present invention are as follows.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether the ischemic disease. Among them, it is particularly useful for developing ischemic brain disease.

"Diagnostic markers, diagnostic markers, or diagnostic markers (diagnosis markers) is a substance that can diagnose the ischemic disease cells from normal cells, the polypeptide or nucleic acid showing an increase in the cells with ischemic disease compared to normal cells (Eg, mRNA, etc.), lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.), etc. For purposes of the present invention, ischemic disease diagnostic markers include T1R2, T1R3, or α. Gusdusin, genes that increase expression in astrocytes, preferably the T1R2 gene may be composed of the nucleotide sequence represented by SEQ ID NO: 1, the T1R3 may be composed of the nucleotide sequence represented by SEQ ID NO: 2 The α-guestucine may be composed of the nucleotide sequence represented by SEQ ID NO: 3.

The expression "upregulation" indicates that, compared to normal tissue cells, the expression of specific genes into mRNA or expression by proteins is significantly increased by intracellular transcription or translation in activated cells. it means.

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

A “fragment” of a tissue sample refers to one portion or piece of tissue sample, eg, a thin slice of tissue or cells cut from a tissue sample. If the present invention is understood to include a method in which the same fragment of a tissue sample is analyzed at both morphological and molecular levels or for both protein and nucleic acid, multiple sections of tissue samples are taken and subjected to the analysis according to the invention. It is understood that it can be done.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. One or both strands of a double stranded nucleic acid molecule and any fragment or portion of an intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

An “primer” is an oligonucleotide sequence that hybridizes to complementary RNA or DNA target polynucleotides and functions as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example by the action of nucleotidyltransferases that occur in polymerase chain reactions. Means.

A “protein” is also to include fragments, analogs and derivatives of a protein that possess essentially the same biological activity or function as the reference protein.

"Antibody" is used in its broadest sense and specifically refers to intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies and Antibody fragments that exhibit the desired biological activity.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates the detection of a reagent conjugated to, fused, conjugated or fused to a reagent, eg, a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

Techniques and procedures described or referenced herein are generally well understood by those of ordinary skill in the art, and are routine methods such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to protocols and / or parameters as specified by the manufacturer unless otherwise noted.

Before describing the methods and assays of the present invention, it should be understood that the present invention is not limited to the particular methods, protocols, cell lines, animal species or genus, constructs and reagents described herein, which may of course vary.

Hereinafter, the present invention will be described in detail.

The inventors have found that the expression of T1R2, T1R3 and α-Gurducin is upregulated in astrocytes in the presence of cerebral ischemia. In other words, the relationship between ischemic disease and sweet receptors T1R2 and T1R3, taste-specific G-protein α- gustducin expression changes were first identified.

Therefore, the present invention relates to the use of one or more genes selected from T1R2, T1R3 and α-guestucine as a diagnostic marker for ischemic disease, and diagnoses the onset of ischemic disease by confirming whether the gene is up-expressed in astrocytes. can do.

Disease to be diagnosed in the present invention is a disease associated with ischemia.

"Ischemia" refers to the improper entry or lack of blood into the body part, caused by contraction, blockage, or blockage of the blood supplying blood vessel. The ischemia causes tissue hypoxia. "Hypoxia" means insufficient oxygen levels in the blood or tissue, which may be the result of a lack of blood supply caused by, for example, vascular occlusion. This may occur due to thrombosis, embolism by any external circulatory substance, or obstruction of the vascular artery or vein by vascular disease such as atherosclerosis.

The ischemic disease can occur when the target tissue demand for oxygen is increased relative to the supply. Decrease in blood flow may begin suddenly and last briefly (acute ischemia) or may be started slowly and last long or appear frequently (chronic ischemia). Acute ischemia is often associated with local, irreversible tissue necrosis (infarction), while chronic ischemia is usually associated with transient hypoxia damage.

That is, the present invention thus relates to a method applied to the treatment of a medical wound associated with any ischemic disease, whether acute, transient or chronic. Ischemic diseases include hypoxia or ischemia, reduced tissue vascular or hypoglycemia, and acute ischemic events include those associated with surgery, organ transplantation, infarction (eg, brain, internal organs, myocardium, lungs, etc.), trauma, insults or injuries. It may include. Chronic events associated with ischemia can include hypertension, diabetes, obstructive arterial disease, chronic venous insufficiency, Raynaud's disease, cirrhosis, congenital heart disease, systemic sclerosis, and the like. Other diseases or conditions associated with ischemia include, but are not limited to wound healing, gastrointestinal lesions, autoimmune diseases and neurodegenerative diseases. Examples of special conditions include wound healing, ischemic attack, ischemic heart disease, peripheral vascular disease, renal artery disease, gastrointestinal lesions, burns, skin grafts, vascular grafts, organ regeneration (e.g., in vitro and in vivo), bone Repair disease, liver disease, uterine disease, retinal angiogenic disease, bone regeneration disease, cartilage regeneration disease, and smooth muscle cell disease.

In particular, the present invention is useful for cerebral ischemia, wherein ischemic brain disease is a diabetic nerve of peripheral nerves caused by structural and functional damage of the vascular mesothelial vessels originating in changes in nerve fibers caused by hypoxia or ischemia. Related to related changes. In addition, strict control of glucose is important for avoiding the ischemic brain disease progression. The present invention also relates to the maintenance of glucose homeostasis in the brain.

Diagnostic Marker  And diagnostic compositions

The present invention relates to a use as a diagnostic marker for at least one ischemic disease selected from T1R2, T1R3 and α-guestucine in one aspect.

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements. The ischemic disease diagnostic marker of the present invention shows the same result in repeated experiments with genes whose expression is always increased by direct or indirect factors with the onset of ischemic disease, and the difference in expression level is very large when compared with the control group. These markers are highly reliable with little chance of falling. Therefore, the result of diagnosis based on the result obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The genes of the present invention are characterized in that they are upregulated and expressed in astrocytic cells of patients with ischemic disease. Induction of sweet signal molecules (T1R2, T1R3) in reactive astrocytic cells in ischemic hippocampus is that their expression is upregulated to maintain energy supplying neurons in response to altered extracellular levels due to ischemic injury. Therefore, it is possible to confirm the onset of ischemic disease through confirming upregulation of astrocytic cells of T1R2, T1R3 and α-Guestucine.

In particular, in using the markers, T1R2 and α-guestucine; T1R3 and α-gutducin; Or it is preferable to measure by the combination of T1R2, T1R3, and (alpha)-gustducin. After all, the present invention means that ischemic damage affects the expression of G-protein (α-gutducin) -coupled sweet receptors (T1R2, T1R3) in reactive astrocytes.

In the same aspect, the present invention relates to a composition for diagnosing ischemic disease, comprising an agent for measuring the level of one or more gene mRNAs or proteins thereof selected from T1R2, T1R3 and α-Guestucine.

Gene expression levels in biological samples, preferably astrocytes, can be confirmed by identifying the amount of mRNA or protein.

mRNA expression level measurement is a process of confirming the presence and expression level of mRNA of the marker genes in a biological sample in order to diagnose ischemic disease. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and RNase protection assay (RPA). , Northern blotting, DNA chips, etc., but are not limited to these.

Accordingly, in one specific aspect of the present invention, there is provided a diagnostic marker composition for ischemic disease comprising a primer sequence specific for at least one gene selected from T1R2, T1R3, and α-guestucine.

A primer is a nucleic acid sequence having a short free 3-terminal hydroxyl group, which refers to a short nucleic acid sequence capable of forming base pairs with complementary templates and serving as a starting point for template strand copying.

Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.

Primers of the invention can be chemically synthesized using other well known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.). Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

In addition, protein expression level measurement is a process of confirming the presence and expression level of a protein expressed from an ischemic disease marker gene in a biological sample in order to diagnose ischemic disease, preferably, specifically binding to the protein of the gene The amount of protein can be confirmed using an antibody. Analytical methods for this purpose include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited to these. . In one embodiment of the present invention, tissue immunostaining was used.

Accordingly, in another aspect, the present invention provides a diagnostic marker composition for ischemic disease comprising an antibody specific for at least one protein selected from T1R2, T1R3 and α-guestucine.

An antibody means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since marker proteins have been identified as described above, the production of antibodies using them can be readily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the ischemic disease marker protein antigen described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog. Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The following protocol related to the detection of specific markers in a sample is shown for illustration.

Protocols for investigating or testing for the presence of T1R2, T1R3, α-Gurducin protein in mammalian tissue or cell samples.

In one embodiment of the invention, the expression of the proteins in the sample was investigated using immunoblot analysis, immunohistochemistry and staining protocols. Immunohistochemical staining of tissue sections has proven to be a reliable method of assessing or detecting the presence of proteins in a sample. Immunohistochemistry (“IHC”) techniques utilize antibodies to probes and generally visualize cellular antigens in situ by color or fluorescent methods.

For sample preparation, tissue or cell samples from mammals (generally human patients) can be used. Samples can be obtained by a variety of procedures known in the art, including but not limited to surgical excision, aspiration or biopsy. The tissue may be fresh or frozen.

Tissue samples can be fixed (ie preserved) by conventional methods. Those skilled in the art will appreciate that the fixative may be selected depending on the purpose for which the sample is histologically stained or otherwise analyzed. One skilled in the art will also understand that fixation time is determined by the size of the tissue sample and the fixative used.

The method further includes a protocol for examining mRNA expression in tissue or cell samples. Methods of assessing mRNA in cells are known and include, for example, hybridization assays using complementary DNA probes and various nucleic acid amplification assays (eg, RT-PCR using complementary primers, etc.). Mammalian tissue or cell samples can be conveniently analyzed for mRNA using Northern blot, dot blot or PCR analysis. For example, RT-PCR analysis, quantitative PCR analysis is known in the art.

Methods for detecting mRNA in a biological sample include generating cDNA from the sample by reverse transcription using at least one primer, amplifying the resulting cDNA using polynucleotides as sense and antisense primers to amplify the cDNA, and amplifying the resulting cDNA. Detecting the presence of cDNA. In addition, the method may include one or more steps to enable determining the level of mRNA in a biological sample.

The method includes a protocol for examining or detecting mRNA in a tissue or cell sample by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control samples are reverse transcribed and labeled to generate cDNA probes. The probe is then hybridized to an array of nucleic acids immobilized on a solid support. The array is prepared to know the sequence and position of each member of the array. For example, genes with the ability to be expressed in certain disease states are arranged on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe is derived expresses the gene. Differential gene expression analysis of disease tissue can provide useful information.

T1R2, T1R3, and α-guestucine expression can be measured by the above protocol to diagnose ischemic disease. As mentioned above, the genes include T1R2 and α-Guestucine; T1R3 and α-gutducin; Or it is preferable to measure by the combination of T1R2, T1R3, and (alpha)-gustducin.

Diagnostic Kit

In a similar aspect, the present invention relates to a kit for diagnosing ischemic disease in humans. The kit is used to determine the presence of ischemic disease by analyzing the expression of one or more genes of T1R2, T1R3, α-guestucine, which can be performed in two ways: genetic analysis and immunity. Assay (immunoassay).

To this end, the kit comprises a primer or probe that specifically binds to a T1R2, T1R3, α-guthducin gene sequence; Or an antibody that specifically binds to T1R2, T1R3, α-gutducin protein.

That is, if the kit for diagnosing human ischemic disease of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermal stability DNA polymerases obtained from Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. In addition, when the kit for diagnosing human ischemia disease of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate of a label.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

In another aspect of the present invention, there is provided an ischemic disease diagnostic kit comprising the composition for diagnosing ischemic disease according to the present invention.

Preferably, the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary to perform reverse transcriptase. The reverse transcription polymerase kit contains each primer pair specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific for the nucleic acid sequence of the control gene. Other reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -May include DEPC-water, sterile water, and the like.

In another aspect, it may be a diagnostic kit, preferably comprising the necessary elements necessary to carry out the DNA chip. The DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent probe. The substrate may also comprise cDNA or oligonucleotide corresponding to the control gene or fragment thereof.

Also preferably, it may be a diagnostic kit characterized by including the necessary elements necessary to perform the ELISA. ELISA kits include antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit can also include antibodies specific for the control protein. Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.

Diagnosis method

In one aspect, the present invention provides a method for diagnosing ischemic disease or a method for providing information for diagnosing ischemic disease, including measuring expression levels of T1R2, T1R3, α-guestucine, based on such a finding.

In one embodiment, the method,

Measuring the expression level of at least one gene selected from T1R2, T1R3 and α-guestucine or the level of protein encoded by a biological sample isolated from a patient; And comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene of the normal control sample. In particular, it is characterized by measuring the expression level of the genes in the astrocyte (astrocyte). Other details are as described above.

In other words, the expression of the T1R2, T1R3 and α-Guestucine gene is characterized as an indicator for ischemic disease. In other words, the method for diagnosing ischemic disease relates to investigating the expression of specific markers in astrocytes following ischemia, particularly cerebral ischemia, and the methods disclosed herein when assessing appropriate or effective therapies for treating patients with ischemic disease. It may provide a convenient, efficient and cost effective means of obtaining useful data and information.

For example, a biopsy can be performed to obtain a tissue or cell sample for a patient diagnosed with ischemia, and predict the effect of the therapeutic agent if the therapeutic agent is associated with, for example, the activity of T1R2, T1R3 or α-guustucine. The tissue or sample of the patient can be examined by various in vitro assays.

In a specific embodiment, the present invention comprises the steps of measuring mRNA from a biological sample of a suspected ischemic disease using a primer specific for one or more genes selected from T1R2, T1R3 and α-guestucine; And it provides a method for diagnosing ischemic disease comprising comparing the increase of the mRNA level with the mRNA level of the normal control sample.

Separation of mRNA from a biological sample can be carried out using a known process and mRNA levels can be measured by various methods.

As used herein, the term biological sample includes, but is not limited to, samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, in which gene expression levels of ischemic disease markers differ due to the development of ischemic diseases. Do not.

Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, and DNA chip.

Through the above detection methods, it is possible to compare the mRNA expression level in the normal control group and the mRNA expression level in the suspected ischemic disease patient, and to determine whether the expression level of the ischemic disease marker gene is increased by the mRNA. The diagnosis of ischemic disease can be diagnosed.

mRNA expression level measurement is preferably by using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for the gene used as an ischemic disease marker.

The reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band to determine the mRNA expression and extent of genes used as diagnostic markers for ischemic disease and to compare with the control group, ischemic disease occurrence It is easy to diagnose.

On the other hand, the DNA chip uses a DNA chip in which the nucleic acid corresponding to the ischemic disease marker gene or fragment thereof is attached to a glass-like substrate with high density, and isolates the mRNA from the sample, and the terminal or the inside is labeled with a fluorescent material. cDNA probes may be prepared, hybridized to DNA chips, and then read for the development of ischemic diseases.

In another specific embodiment, the present invention provides a method for determining protein levels by forming an antigen-antibody complex by contacting a biological sample of a suspected ischemic patient with an antibody specific for at least one protein selected from T1R2, T1R3, and α-guestucine. It provides a method for diagnosing ischemic disease, including comparing the increase in the amount of protein formation with the protein level of a normal control sample.

The separation of proteins from biological samples can be carried out using known processes and protein levels can be measured in a variety of ways.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto.

Through the above assay methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected patients with ischemic disease can be compared, and the significant increase in the expression level of the ischemic disease marker gene to the protein is increased. By determining whether or not, it is possible to diagnose whether the actual ischemic disease of the patient suspected of ischemic disease.

In the present invention, the term antigen-antibody complex means a combination of an ischemic disease marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label. .

Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives.

Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4-and the like. Radioisotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like.

Protein expression level measurement is preferably by using a Western blot using one or more antibodies against the ischemic disease marker. The whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody. By checking the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein produced by the expression of the gene can be confirmed to determine whether the ischemic disease occurs. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cells in which ischemic disease occurs. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

Also, preferably, immunohistostaining is performed using at least one antibody against the ischemic disease marker.

After collecting and fixing normal colon epithelial tissue and tissue suspected of ischemic disease, paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several um and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

In addition, preferably, at least one antibody against the ischemic disease marker is arranged at a predetermined position on the substrate to use a protein chip immobilized at a high density. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein, The presence of ischemic diseases can be checked.

Screening method

Meanwhile, in another aspect, the present invention relates to a method for screening a substance for treating ischemic disease, which suppresses expression of T1R2, T1R3, or α-guestucine, based on the above-described fact.

The method is an embodiment,

(a) expressing in the astrocytes at least one gene selected from T1R2, T1R3 and α-Gurducin in the presence of a candidate substance; And (b) a candidate substance when the expression of the genes is suppressed as compared with a case where one or more genes selected from T1R2, T1R3 and α-guestucine are expressed in astrocytes without candidates. It may include the step of selecting.

In the method of the present invention, first, cells containing T1R2, T1R3 or α-guestucine are cultured in the presence of a candidate. Such cells include cancer cells expressing a T1R2, T1R3 or α-gutducin gene or cells transformed to express an NLK gene.

Next, in the case where the expression of the T1R2, T1R3 or α-guestucine is suppressed, compared with the case where the cells transformed with the T1R2, T1R3 or α-Guestucine gene are expressed without the candidate, the gene is expressed. Candidates are selected for treatment of ischemic disease.

On the other hand, the present inventors confirmed that in the upregulated expression of T1R2, T1R3 or α-guestucine in ischemic disease tissues, their upregulation was found in reactive astrocytic cells, not any neurons in the ischemic hippocampus. In addition, the expression profiles of T1R2 and T1R3 were found to share overlapping expression patterns, and increased expression of the a-guustucine also showed spatiotemporal activity in ischemic hippocampus, consistent with the two sweet receptors. . That is, a-guustucine also occurred specifically among reactive astrocytes in ischemic hippocampus. This means that ischemic damage affects the expression of G-protein-coupled sweet receptors in reactive astrocytes, which is responsible for the maintenance of glucose homeostasis in the brain by the induction of sweet signaling proteins in reactive astrocytes manifested by cerebral ischemia. Suggests.

Thus, in another aspect, the present invention relates to a method of maintaining glucose homeostasis of the brain by reducing or increasing the expression of one or more genes selected from T1R2, T1R3 and α-guestucine in astrocytes.

In addition, the present invention extends to genetic approaches to up-regulation or down-regulation of expression of genes associated with expression of T1R2, T1R3 or α-guestucine. In general, it is more convenient to use genetic means to induce gene silencing such as pre- or post-transcriptional gene silencing. However, the general techniques used to upregulate expression are to increase the number of gene copies or antagonize inhibitors of gene expression.

As such, the present invention has been shown to increase the amount of expression of the T1R2, T1R3 or α- gustducin gene in ischemia, especially cerebral ischemia patients, and these results as a target gene for the development or screening for the treatment of ischemic diseases, The genes characterized above can be used.

< Example >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

< Example  1>

temporary Whole brain  Ischemia Induction

All experimental animal courses have been approved by the Catholic Ethics Committee of the Catholic University of Korea (CUMC09U029), National Institutes of Health (NIH) Guide and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) Followed. Adult Sprague Dawley rats (250-300 g) were used for the experiment. Using a four-vessel occlusion (4-VO) and reperfusion method that blocks blood flow in the internal carotid artery region described in Pulsinelli and Brierley (Pulsinelli WA, Brierley JB (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat.Stroke 10: 267-272), which induced transient forebrain ischemia. That is, the vertebral artery was electrocauterized to stop circulation in the blood vessels. After 24 hours, the total carotid artery was occluded with a miniature aneurysm clip for 10 minutes. Only those animals showing complete flat electroencephalograms after vascular occlusion were classified as ischemia and used in the experiment. Body temperature was maintained at 37.5 ± 0.3 ° C. during ischemia and after ischemia with a heated lamp. After electrocoagulation of the vertebral arteries, rats that were caught around the carotid artery but not occluded were used as controls. No animals experienced cramps or died after sham-surgery or reperfusion. After 1, 3, 7, and 14 days post reperfusion, three mice at each time point were sacrificed for use in immunoblot analysis and seven were sacrificed for immunohistochemical analysis. Surgical animals were treated using the same method as the ischemia / reperfusion animal. At each time point following reperfusion, animals were deeply anesthetized with 16.9% urethane (10 ml / kg) and fixed by transcardial perfusion with fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Killing or by decapitation. Hippocampus was excised by rapid freezing in liquid nitrogen.

< Example  2>

Immunoblot  analysis( Immunoblot analysis )

For immunoblot analysis, coronal slices (400 μm thick) were cut at the septal hippocampus level with the McIlwain tissue sectioner (The Mickle Laboratory Engineering, Guildford, UK) and cut out the papillary papillae. Dorsal hippocampus and control rats 3, 7, 14 days after reperfusion, and the dermal papilla containing taste bumps were placed in ice-cold RIPA buffer (50 mM Tris buffer, pH 8.0; 150 mM NaCl; 1% NP-40; 0.5). % deoxycholate; and 0.1% SDS). Equal amounts of total protein (80 μg) from hippocampal tissue and dermal papilla were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto nitrocellulose membranes. Goat polyclonal antibody or α-Gurducin (Santa Cruz) against T1R2 (Santa Cruz Biotechonolgy, Inc., Santa Cruz, CA, USA; dilution 1: 100) or T1R3 (Santa Cruz Biotechonolgy, Inc .; dilution 1: 100) Blot immunostaining was performed using rabbit polyclonal antibodies against Biotechonolgy, Inc .; dilution 1: 200). The immunoreactive bands were developed with an improved chemiluminescence system (ECL kit, Amersham, Arlington Heights, IL, USA). Densitometric analysis was performed using the Eagle Eye TMII Still Video System (Stratagene, La Jolla, Calif., USA). Three animals were used for immunoblotting at each time point and each sample was tested for triplicate. After subtracting the peripheral density, the optical density ratio of the experimental rat to the surgical rat was measured. Statistical significance was analyzed by ANOVA according to Dunnetts t test; P <0.05 was considered significant.

As a result of the above analysis, as shown in FIG. 1, the immunoblot assay conducted with specific antibodies against T1R2, T1R3 or α-guestucine is consistent with the analysis in the papillary papilla (95 kDa (T1R2), 93 kDa). Immunoreactive species corresponding to proteins of (T1R3) or 38 kDa (α-Gurducin) were shown, respectively.

In addition, as shown in FIG. 2, in general, the expression level of each protein showed a similar transient pattern. The level of sweet signaling protein increased on day 3 after reperfusion, peaked on day 7, and then decreased. However, enhanced expression levels were maintained for at least 14 days.

< Example  3>

Immunohistochemical Analysis with Single, Double, and Triple Labels

Coronal freezing sections (25 μm thick) were used for T1R2, T1R3 and α-Guestucine immunohistochemical analysis. After 1 hour of blocking with 10% normal donkey serum, the sections were prepared for either T1R2 (Santa Cruz Biotechonolgy, Inc .; dilution 1: 100) or α-Guestucine (Santa Cruz Biotechonolgy, Inc .; dilution 1: 100). Incubated overnight at 4 ° C. with rabbit polyclonal antibody, or goat polyclonal antibody against T1R3 (Santa Cruz Biotechonolgy, Inc .; dilution 1: 100). Peroxidase-labeled donkey anti-goat. 0.05 with IgG (Jackson ImmunoResearch, West Grove, PA, USA; dilution 1: 100) or peroxidase-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch; dilution 1: 100), and 0.01% H ₂ O ₂ as substrate Primary antibody binding was visualized using% 3,3′-diaminobenzidine tetrahydrochloride. The specificity of the T1R2, T1R3 and α-Gustducin immunoreactivity in fragments without primary antibody or in non-specific rabbit or goat IgG was confirmed by lack of immunohistochemical staining. For double or triple-immunofluorescence histochemical analysis, sections were incubated overnight at 4 ° C with the following antibodies; Rabbit polyclonal antibody against T1R2, rabbit polyclonal antibody against α-gutducin, goat polyclonal antibody against T1R3, glial fibrillary acidic protein; Chemicon International Inc., Temecula, CA, USA; dilution 1: 500) Mouse monoclonal antibodies against, mouse monoclonal antibodies against OX-42 (Serotec, UK; dilution 1: 100) and mouse monoclonal antibodies against NeuN (anti-neuronal nuclear antigen; Chemicon International Inc .; dilution 1: 500).

The fragments were then divided into Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch; dilution 1: 1,000), Cy3-conjugated donkey anti-goat IgG (Jackson ImmunoResearch; dilution 1: 1,000), FITC-conjugated donkey anti -Mouse IgG (Jackson ImmunoResearch; dilution 1:50), FITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch; dilution 1:50), FITC-conjugated anti-rabbit antibody (Jackson ImmunoResearch; dilution 1:50) and Incubated with Cy5-conjugated anti-mouse antibody (Jackson ImmunoResearch; dilution 1: 500) mixture for 2 hours at room temperature, respectively. Control sections were also prepared as described above. Counterstaining of the cell nuclei was performed by incubating the sections with DAPI (4 ′, 6-diamidino-2′-phenyindole; Roche; dilution 1: 1000) for 10 minutes. Slides were observed with confocal microscopy (LSM 510 Meta, Carl Zeiss Co., Ltd., Germany).

<3-1> after ischemia T1R2 and Of T1R3  Temporal Profile and Phenotypic Analysis

Immunohistochemical analysis was performed to investigate the intracellular location and regional distribution of sweet receptors T1R2 and T1R3 in the hippocampus after ischemia.

As a result, all immunoreactivity specific for T1R2 or T1R3 were not seen when the primary antibody was omitted or replaced with nonspecific rabbit or goat IgG. In surgical rats, the immunoreactivity for T1R2 at all time points It was found in neurons of granule cells and pyramidal cell layers in hippocampus (FIGS. 2A and 2C). It is also evident in cerebral cortical neurons and hypothalamic neurons.

Furthermore, beginning one day after reperfusion, T1R2 immunoreactivity increased (data not shown) and increased preferentially in CA1 and CA3 regions and in the dentate hilar region by days 3 and 7 (FIG. 2B). ). At this time, the immunoreactivity of T1R2 was remarkable in small cells in the emissive layer and the directional layer of the CA1 region compared to the control group (C-E in FIG. 2). The distribution pattern and shape of these immunoreactive cells showed that they could be glial cells. On day 14, there was no change in the pattern of labeling, but the labeled cells showed a thickening aspect with strong immunoreactivity (FIG. 2F).

The spatiotemporal distribution pattern of T1R3 immunoreactivity was also almost identical to that of T1R2. In the hippocampus of control animals, the underlying T1R3 immunoreactivity was predominantly present in neurons as well as in the dentate gyrus (Figure 2 G, I).

At 3 days (FIG. 2 J) and 7 days (FIG. 2 H, K) after reperfusion, T1R3 immunoreactivity was prominent in the emissive layer of the hippocampus proper and in the hilar region of the dentate gyrus. In T1R3 immunoreactive cells appeared to be glial. After 2 weeks of ischemia, T1R3 immunoreactivity was prominently located in the CA1 region and labeled cells showed a hypertrophic appearance (Fig. 2L).

In addition, double labeling was performed using cell type-specific markers, including GFAP, OX-42 and NeuN, to identify cell morphologies expressing sweet receptors in ischemic hippocampus.

As a result, most T1R2 immunoreactive cells were GFAP-immunoreactive stellate cells in the emissive layer of the CA1 region following ischemic injury (FIG. 3 AC), but not OX-42-immunoreactive microglia (FIG. 3 DF). In addition, some T1R2 immunoreactive cells in the pyramidal cell layer were conserved neurons showing NeuN immunoreactivity (FIG. 3 GI). Double labeling immunofluorescence of T1R3 and these markers showed that the T1R3 immunoreactivity was located in reactive astrocytic cells (Fig. 3 J-L) and conserved neurons but not microglia.

In addition, triple labeling of T1R2, T1R3 and GFAP showed that most T1R2 and T1R3 double-labeled cells were positive for GFAP in the CA1 region of ischemic hippocampus (FIG. 3 MP).

In the lactate (lactic acid) shuttle model, astrocytes transport and metabolize glucose to lactate, which can be used as an energy source around neurons. Cerebral ischemia causes major changes in oxygen and glucose supply, cellular metabolism, and reduced levels of glucose in ischemic tissues are known to play an important role in ischemic injury. In adaptive response to hypoxia, astrocytic cells enhance anaerobic glycolysis by increasing glycolytic enzyme activity to maintain intracellular energy and control brain energy metabolism in pathological conditions such as normal and ischemic damage. It plays a decisive role.

Thus, the induction of sweet signal molecules in reactive astrocytic cells in ischemic hippocampus suggests that their expression is upregulated to maintain energy supply to neurons in response to altered extracellular levels due to ischemic injury.

<3-2> Α- after ischemia Gustducin  Temporal Profile and Phenotypic Analysis of Immune Reactive Cells

Similarly to the above method, the immunoreactivity of α-guestucine was examined by immunohistochemical analysis.

As a result, the immunoreactivity of α-guestucine was observed in the surgical and ischemic hippocampus. In surgical animals, immunoreactivity was located in neurons of the granulocyte and pyramidal cell layers (Figures 4 A, C). On day 3 (FIG. 4D) and day 7 (FIG. 4B, E) after ischemic injury, -gutducin immunoreactivity was seen in myriad glia-like cells, in particular CA1 region and alveolar closure site It was remarkable in the emitting layer and the directing layer of. At 14 days after reperfusion, the labeled pattern of α-guestucine remained unchanged compared to the case at day 7. However, labeled cells showed a thickening appearance at a higher rate as shown in F of FIG. 4.

The distribution pattern and morphology of the α-gutducin-reactive cells suggest that they may be glial. This shows that α-gutducin-expressing cells in the ischemic CA1 region are reactive astrocytes, not OX-42-immunoreactive microglial cells (data not shown) (FIG. 4 GI), α-gutducin and GFAP or OX Confirmed by double labeling of -42.

In addition, some immunoreactive cells were conserved neurons showing NeuN reactivity (FIG. 4 JL). In addition, triple labeling of T1R3, α-gutducin and GFAP showed that most T1R3 and α-gutducin double label cells in the CA1 region of the ischemic hippocampus were positive for GFAP (FIG. 4 MP).

In conclusion, the present invention confirmed that the sweet receptors T1R2 and T1R3 were significantly upregulated in reactive astrocytic cells using transient whole brain ischemic rats. Taste-specific G-protein α-gutducin to which they are associated was selectively increased in reactive astrocytic cells within the same hippocampal region.

These results indicate that ischemic damage affects the expression of G-protein-coupled sweet receptors in reactive astrocytes, which suggests that sweet signaling protein induction of reactive astrocytes manifested by cerebral ischemia maintains glucose homeostasis in the brain. Implying involvement

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Composition for diagnosing ischemia compriging sweet-taste          receptor genes <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 3060 <212> DNA <213> TIR2 DNA sequence <400> 1 gggcatcgtc taaggctgtt acttggctgg catgggaccc caggcgagga cactccattt 60 gctgtttctc ctgctgcatg ctctgcctaa gccagtcatg ctggtaggga actccgactt 120 tcacctggct ggggactacc tcctgggtgg cctctttacc ctccatgcca acgtgaagag 180 cgtctctcac ctcagctacc tgcaggtgcc caagtgcaat gagtacaaca tgaaggtctt 240 gggctacaac ctcatgcagg ccatgcgatt cgccgtggag gaaatcaaca actgtagctc 300 tctgctgccc ggcgtgctgc tcggctacga gatggtggat gtctgctacc tctccaacaa 360 tatccagcct gggctctact tcctgtcaca gatagatgac ttcctgccca tcctcaaaga 420 ctacagccag tacaggcccc aagtggtggc cgtcattggc ccagacaact ctgagtccgc 480 catcaccgtg tccaacattc tctcctactt cctcgtgcca caggtcacat atagcgccat 540 caccgacaag ctgcgagaca agcggcgctt ccctgccatg ctgcgcactg tgcccagcgc 600 cacccaccac atcgaggcca tggtgcaact gatggttcac ttccagtgga actggatcgt 660 ggtgctggtg agcgatgacg attatggccg agagaacagc cacctgctga gccagcgtct 720 gaccaacact ggcgatatct gcattgcctt ccaggaggtt ctgcctgtac cagaacccaa 780 ccaggccgtg aggcctgagg agcaggacca actggacaac atcctggaca agctgcggcg 840 gacctcggcg cgtgtggtgg tgatattctc gccagagctg agcctgcaca acttcttccg 900 cgaggtgctg cgctggaact tcacaggctt tgtgtggatt gcctctgagt cctgggccat 960 cgaccctgtt ctacacaacc tcacagagct gcgccacacg ggcactttcc tgggcgtcac 1020 catccagagg gtgtccatcc ctggcttcag ccagttccga gtgcgccacg acaagccaga 1080 gtatcccatg cctaacgaga ccagcctgag gactacctgt aaccaggact gtgacgcctg 1140 catgaacatc accgagtcct ttaacaacgt tctcatgctt tcgggggagc gtgtggtcta 1200 cagtgtgtac tcggccgtct acgcggtagc ccacaccctc cacagactcc tccactgcaa 1260 ccaggtccgc tgcaccaagc aaatcgtcta tccatggcag ctactcaggg agatctggca 1320 tgtcaacttc acgctcctgg gcaaccagct cttcttcgac gaacaagggg acatgccgat 1380 gctcctggac atcatccagt ggcaatgggg cctgagccag aaccccttcc aaagcatcgc 1440 ctcctactcc cccaccgaga cgaggctgac ctacattagc aatgtgtcct ggtacacccc 1500 caacaacacg gtccccatat ccatgtgttc taagagttgc cagcctgggc aaatgaaaaa 1560 acccataggc ctccacccgt gctgcttcga gtgtgtggac tgtccgccgg gcacctacct 1620 caaccgatca gtagatgagt ttaactgtct gtcctgcccg ggttccatgt ggtcttacaa 1680 gaacaacatc gcttgcttca agcggcggct ggccttcctg gagtggcacg aagtgcccac 1740 tatcgtggtg accatcctgg ccgccctggg cttcatcagt acgctggcca ttctgctcat 1800 cttctggaga catttccaga cgcccatggt gcgctcggcg ggcggcccca tgtgcttcct 1860 gatgctggtg cccctgctgc tggcgttcgg gatggtcccc gtgtatgtgg gcccccccac 1920 ggtcttctcc tgtttctgcc gccaggcttt cttcaccgtt tgcttctccg tctgcctctc 1980 ctgcatcacg gtgcgctcct tccagattgt gtgcgtcttc aagatggcca gacgcctgcc 2040 aagcgcctac ggtttctgga tgcgttacca cgggccctac gtctttgtgg ccttcatcac 2100 ggccgtcaag gtggccctgg tggcaggcaa catgctggcc accaccatca accccattgg 2160 ccggaccgac cccgatgacc ccaatatcat aatcctctcc tgccacccta actaccgcaa 2220 cgggctactc ttcaacacca gcatggactt gctgctgtcc gtgctgggtt tcagcttcgc 2280 gtacgtgggc aaggaactgc ccaccaacta caacgaagcc aagttcatca ccctcagcat 2340 gaccttctcc ttcacctcct ccatctccct ctgcacgttc atgtctgtcc acgatggcgt 2400 gctggtcacc atcatggatc tcctggtcac tgtgctcaac tttctggcca tcggcttggg 2460 gtactttggc cccaagtgtt acatgatcct tttctacccg gagcgcaaca cttcagctta 2520 tttcaatagc atgattcagg gctacacgat gaggaagagc tagtcccacc caccgcctca 2580 gcagcagagc ccccagccac gctcccggtg ttccttgttc ctcagcattc tttgcagtgt 2640 agctattttt tttacccaca tagctcttga aattacccat aatgcactcc cactgccccc 2700 tcctccaatc cccgaacccc gcccgagcgg ggggttcact ggctaggacc taccacccac 2760 ttatggaaga aaccaccaag gtgacctatg tggctccaag gatggcccac cactgccatc 2820 tggtggccat agagagcacc tgcggggctg tggcccatgc cttcctgctc atggctagtg 2880 tggctgtgag accaaccatg gtggctactg tgctctccag atgtctgtta atctgtgttt 2940 cattccggtt cctggggagc acagactggg actcctgtgt tctaatggtc agatgagcat 3000 catgggtcct ccattgttgc ttatgaataa atttcccttg ggtgaaaaaa aaaaaaaaaa 3060                                                                         3060 <210> 2 <211> 2559 <212> DNA <213> TIR3 DNA sequence <400> 2 atgctgggcc ctgctgtcct gggcctcagc ctctgggctc tcctgcaccc tgggacgggg 60 gccccattgt gcctgtcaca gcaacttagg atgaaggggg actacgtgct gggggggctg 120 ttccccctgg gcgaggccga ggaggctggc ctccgcagcc ggacacggcc cagcagccct 180 gtgtgcacca ggttctcctc aaacggcctg ctctgggcac tggccatgaa aatggccgtg 240 gaggagatca acaacaagtc ggatctgctg cccgggctgc gcctgggcta cgacctcttt 300 gatacgtgct cggagcctgt ggtggccatg aagcccagcc tcatgttcct ggccaaggca 360 ggcagccgcg acatcgccgc ctactgcaac tacacgcagt accagccccg tgtgctggct 420 gtcatcgggc cccactcgtc agagctcgcc atggtcaccg gcaagttctt cagcttcttc 480 ctcatgcccc aggtcagcta cggtgctagc atggagctgc tgagcgcccg ggagaccttc 540 ccctccttct tccgcaccgt gcccagcgac cgtgtgcagc tgacggccgc cgcggagctg 600 ctgcaggagt tcggctggaa ctgggtggcc gccctgggca gcgacgacga gtacggccgg 660 cagggcctga gcatcttctc ggccctggcc gcggcacgcg gcatctgcat cgcgcacgag 720 ggcctggtgc cgctgccccg tgccgatgac tcgcggctgg ggaaggtgca ggacgtcctg 780 caccaggtga accagagcag cgtgcaggtg gtgctgctgt tcgcctccgt gcacgccgcc 840 cacgccctct tcaactacag catcagcagc aggctctcgc ccaaggtgtg ggtggccagc 900 gaggcctggc tgacctctga cctggtcatg gggctgcccg gcatggccca gatgggcacg 960 gtgcttggct tcctccagag gggtgcccag ctgcacgagt tcccccagta cgtgaagacg 1020 cacctggccc tggccaccga cccggccttc tgctctgccc tgggcgagag ggagcagggt 1080 ctggaggagg acgtggtggg ccagcgctgc ccgcagtgtg actgcatcac gctgcagaac 1140 gtgagcgcag ggctaaatca ccaccagacg ttctctgtct acgcagctgt gtatagcgtg 1200 gcccaggccc tgcacaacac tcttcagtgc aacgcctcag gctgccccgc gcaggacccc 1260 gtgaagccct ggcagctcct ggagaacatg tacaacctga ccttccacgt gggcgggctg 1320 ccgctgcggt tcgacagcag cggaaacgtg gacatggagt acgacctgaa gctgtgggtg 1380 tggcagggct cagtgcccag gctccacgac gtgggcaggt tcaacggcag cctcaggaca 1440 gagcgcctga agatccgctg gcacacgtct gacaaccaga agcccgtgtc ccggtgctcg 1500 cggcagtgcc aggagggcca ggtgcgccgg gtcaaggggt tccactcctg ctgctacgac 1560 tgtgtggact gcgaggcggg cagctaccgg caaaacccag acgacatcgc ctgcaccttt 1620 tgtggccagg atgagtggtc cccggagcga agcacacgct gcttccgccg caggtctcgg 1680 ttcctggcat ggggcgagcc ggctgtgctg ctgctgctcc tgctgctgag cctggcgctg 1740 ggccttgtgc tggctgcttt ggggctgttc gttcaccatc gggacagccc actggttcag 1800 gcctcggggg ggcccctggc ctgctttggc ctggtgtgcc tgggcctggt ctgcctcagc 1860 gtcctcctgt tccctggcca gcccagccct gcccgatgcc tggcccagca gcccttgtcc 1920 cacctcccgc tcacgggctg cctgagcaca ctcttcctgc aggcggccga gatcttcgtg 1980 gagtcagaac tgcctctgag ctgggcagac cggctgagtg gctgcctgcg ggggccctgg 2040 gcctggctgg tggtgctgct ggccatgctg gtggaggtcg cactgtgcac ctggtacctg 2100 gtggccttcc cgccggaggt ggtgacggac tggcacatgc tgcccacgga ggcgctggtg 2160 cactgccgca cacgctcctg ggtcagcttc ggcctagcgc acgccaccaa tgccacgctg 2220 gcctttctct gcttcctggg cactttcctg gtgcggagcc agccgggccg ctacaaccgt 2280 gcccgtggcc tcacctttgc catgctggcc tacttcatca cctgggtctc ctttgtgccc 2340 ctcctggcca atgtgcaggt ggtcctcagg cccgccgtgc agatgggcgc cctcctgctc 2400 tgtgtcctgg gcatcctggc tgccttccac ctgcccaggt gttacctgct catgcggcag 2460 ccagggctca acacccccga gttcttcctg ggagggggcc ctggggatgc ccaaggccag 2520 aatgacggga acacaggaaa tcaggggaaa catgagtga 2559 <210> 3 <211> 1174 <212> DNA <213> Galpagust <400> 3 gctgcctgtt gtagcgagca ccgctcatat gtcctatatc taaactacag ctgtgctccg 60 tgtttgaaaa gtttgagcaa atcaactgcc cagccactaa catcaaaaga tgggaagtgg 120 aattagttca gagagcaagg aatcagccag aaggtccaaa gaactggaga agaagcttca 180 ggaggatgct gagcgggatg caagaactgt gaagttactg ctactaggag caggtgaatc 240 tgggaaaagc actattgtta aacaaatgaa gatcatccat aagaatggtt acagcaaaca 300 agaatgcatg gagtttaaag cagtgattta cagtaacaca ttgcagtcca tcctagctat 360 tgtgaaagcc atggctacac tggggattga ttatgtcaat cctaggagcc gagaggacca 420 agaacaactt cactcaatgg caaatacact agaagatggt gatatgactc cacagctggc 480 tgaaatcatt aaacgactgt ggggggatcc aggaatccaa gcctgctttg aaagggcatc 540 tgaataccag ctcaatgact ctgcagctta ctaccttaat gacttagaca gactcacagc 600 ccctgggtac gtgccaaatg agcaagatgt tctacattcc cgggtgaaaa ccactggtat 660 cattgaaact caattctcct ttaaagactt gaacttcaga atgtttgatg taggtggcca 720 gagatcagag agaaaaaaat ggatccactg ctttgaagga gtcacctgca ttatattttg 780 cgcagcgcta agtgcctatg acatggtgct tgtagaagat gaggaggtga acagaatgca 840 tgaaagtctt cacctgttta acagcatatg taatcataag tacttcgcaa ccacctccat 900 tgttctgttt cttaacaaga aagatctctt ccaggagaaa gtggctaagg tgcacctcag 960 catttgcttt ccagaatata ctggaccaaa cacatttgaa gatgcaggga actacatcaa 1020 gaaccagttt ctagacctga acttaaaaaa agaagataag gaaatctatt ctcacatgac 1080 ctgtgctact gacacacaaa atgtcaaatt tgtgtttgat gcagtgacag atataataat 1140 aaaagagaac ctcaaagact gtgggctctt ctga 1174

Claims (16)

A composition for diagnosing ischemic disease, comprising an agent for measuring the level of one or more gene mRNAs selected from T1R2, T1R3, and α-guestucine or a protein thereof. The method of claim 1,
The T1R2 has a nucleotide sequence of SEQ ID NO: 1, the T1R3 has a nucleotide sequence of SEQ ID NO: 2, the α- gustducin has a nucleotide sequence of SEQ ID NO: 3 composition for diagnosing ischemic disease. The method of claim 1,
The agent for measuring the level of the gene mRNA composition for diagnosing ischemic disease, characterized in that it comprises a primer that specifically binds to at least one gene selected from T1R2, T1R3 and α-guestucine. The method of claim 1,
The agent for measuring the level of the protein is a composition for diagnosing ischemic disease, characterized in that it comprises an antibody that specifically binds to at least one protein selected from T1R2, T1R3 and α-guestucine. The method of claim 1,
At least one gene mRNA selected from T1R2, T1R3 and α-guestucine or a protein thereof is a composition for diagnosing ischemic disease, characterized in that for measuring the level in the astrocytes (astrocyte). The method of claim 1,
The ischemic disease is wound healing, ischemic brain disease, ischemic heart disease, peripheral vascular disease, renal artery disease, gastrointestinal lesions, burns, skin grafts, vascular grafts, long-term treatment, bone repair disease, liver disease, uterine disease, retinal angiogenic disease , Ischemic disease, cartilage repair disease, and smooth muscle cell disease composition for diagnosing ischemic disease, characterized in that selected from the group consisting of. The method of claim 6,
The ischemic disease is ischemic brain disease, characterized in that the composition for diagnosing ischemic disease. The method of claim 1,
The genes include T1R2 and α-Guestucine; T1R3 and α-gutducin; Or T1R2, T1R3, α-guestucine is measured by a combination composition for diagnosing ischemic disease. Kit for diagnosing ischemic disease comprising a composition according to any one of claims 1 to 8. 10. The method of claim 9,
The kit is a diagnostic kit for ischemic disease, characterized in that the RT-PCR kit, DNA chip kit or protein chip kit. Measuring at least one gene expression level selected from T1R2, T1R3 and α-Guestucine or a protein encoded by the gene from a biological sample isolated from the patient; And
Comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene of the normal control sample. The method of claim 11,
The expression level of the gene is to measure the mRNA level of the gene, the protein level is a method of providing information for diagnosing ischemic disease, characterized in that by using an antibody that specifically binds to the protein. The method of claim 11,
The method of providing information for diagnosing ischemic disease, characterized in that the level of at least one gene mRNA or protein thereof selected from T1R2, T1R3, and α-guestucine is measured in astrocytes. The method of claim 11,
The genes include T1R2 and α-Guestucine; T1R3 and α-gutducin; Or T1R2, T1R3, α-guestucine is measured using a combination of the information providing method for diagnosing ischemic disease. (a) expressing at least one gene selected from among T1R2, T1R3 and α-guestucine in astrocytes in the presence of a candidate substance; And
(b) When the expression of one or more genes selected from among T1R2, T1R3 and α-guestucine is suppressed in astrocytes without candidates, the candidate substance is expressed as a substance for treating ischemic disease. Screening method for treating ischemic disease, comprising the step of selecting. 16. The method of claim 15,
The genes include T1R2 and α-Guestucine; T1R3 and α-gutducin; Or T1R2, T1R3, α-guestucine in combination with a method for screening a substance for treating ischemic disease.

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