KR101889594B1 - A composition for diagnosing vascular endothelial cell dysfunction and use thereof - Google Patents
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Abstract
The present invention relates to a composition for diagnosing dysfunction of vascular endothelial cells, a kit, a method for providing information for diagnosing dysfunction of vascular endothelial cells, and a method for screening a substance for improving function of vascular endothelial cells. The expression levels of Rab5C, clathrin, caveolin-1, EEA1, and KCa3.1 channels of the present invention were measured and compared, So that the vascular disease can be diagnosed early.
Description
The present invention relates to a composition for diagnosing dysfunction of vascular endothelial cells, a kit, a method for providing information for diagnosing dysfunction of vascular endothelial cells, and a method for screening a dysfunctional or improving inducer for vascular endothelial cells.
The function of the cell is controlled very sensitively by the ion channel. Ion channels control the flow of ions through the cell membrane to determine the makjeonap and has a significant effect on cell function, such as to adjust the concentration of Ca 2 + in the excitable cells through it. Ion channels are classified into Na + channel, Ca 2 + channel, and K + channel depending on the kind of ions passing through. There are several kinds of K + channels activated by intracellular Ca 2 + , and there are KCa1.1 channel, KCa2.3 channel, and KCa3.1 channel in vascular endothelial cells. These three channels play a different role in vascular endothelial cells, and the KCa3.1 channel regulates vascular contractility by inducing vascular endothelial cell hyperpolarization (Tharp DL, Bowles DK, 2009, Cardiovasc Hematol Agents Med Chem. 7, 1- 11), plays an important role in new blood vessel formation. In the cells other than vascular endothelial cells, these channels play an important role in regulating cell function. In addition, KCa3.1 channel is distributed in red blood cells, fibroblasts, proliferating vascular smooth muscle cells, and immune cells (T cells and B cells) and contributes to controlling the function of these cells.
Vascular endothelial cells play an important role in the regulation of vascular diameter and contractility. Vascular endothelial cells secrete NO or induce endothelium-dependent hyperpolarization to relax vascular smooth muscle and regulate vasoconstriction. When KCa3.1 channel is activated by intracellular calcium endothelial cell hyperpolarization is, vascular endothelial cell hyperpolarization promotes NO generation to induce intracellular Ca 2 + influx. Therefore, KCa3.1 channel regulates vascular smooth muscle contractility by inducing vascular endothelial cell hyperpolarization as well as NO secretion. In the case of vascular diseases such as hypertension, when NO relaxation is reduced, relaxation by hyperpolarity complements this and inhibits increase of vascular smooth muscle contractility.
Vascular endothelial dysfunction is a major cause of vascular disease and KCa3.1 channel, which plays an important role in vascular endothelial function, causes vascular diseases such as hypertension. For example, hypertension occurred in KCa3.1 channel deficient mice (KCa3.1 channel knockout mice) and decreased expression of KCa3.1 channel caused diseases such as pregnancy hypertension.
Because vascular endothelial cells are in direct contact with plasma, vascular endothelial dysfunction is caused by plasma vascular disease inducing substances. Such vascular disease-causing substances include oxidized low density lipoprotein (oxLDL), hyperglycemia, and ROS such as superoxide generated by these substances. For example, in the case of gestational hypertension and arteriosclerosis, oxidative low density lipoprotein is caused by blood, and diabetic vascular disease is caused by hyperglycemia and vascular endothelial dysfunction. As a result, the blood vessels are contracted and blood vessel diameter is decreased, and blood flow resistance is obstructed by increasing the resistance of the blood vessels. These vascular disease-inducing substances reduce vascular endothelial cell KCa3.1 channel expression and induce vascular endothelial dysfunction (Choi S et al, 2013a, Free Radic Biol Med, 57, 10-21)
The vascular disease is caused by structural abnormalities of vascular endothelial dysfunction, that is, the decrease of vascular diameter. Therefore, vascular endothelial dysfunction precedes the structural change of blood vessels. Therefore, the diagnosis of vascular endothelial dysfunction can be used for the early diagnosis of vascular disease (diagnosis of the onset of the disease before the structural change of the blood vessels), diagnosis of the progress and prognosis, and diagnosis of the therapeutic effect. Currently, several methods have been tried for early diagnosis in pregnancy-induced hypertension, including risk factors (age, etc.), uterine arterial blood flow measurement using Doppler, and sFlt-1 and endoglin measurements in plasma. However, there is no way to accurately diagnose the possibility of early onset of pregnancy. In the case of arteriosclerosis and diabetes, blood flow and blood vessel diameter are measured by angiography, CT angiography, Dopper Sono, etc. to diagnose the progress of the vascular disease. However, these methods have a problem that diagnosis can be made only after the disease progresses and the structural change (blood vessel change) of the blood vessel comes. There is no way to accurately predict the progression of vascular complications in patients with diabetes even though 50-80% of patients die from cardiovascular disease in most cases with diabetic complications.
However, since vascular endothelial cells can not be used for diagnostic purposes, it is impossible to diagnose vascular endothelial function using vascular endothelial cells of a patient. On the other hand, since vascular endothelial cell function is caused by vascular disease-causing substances in the blood, normal vascular endothelial cells cultured in vitro or using erythrocytes from patients exposed to the vascular disease-inducing substance can be administered to the patient's serum or After exposure to plasma, the vascular endothelial cells can be used to diagnose dysfunction.
Under these circumstances, the inventors of the present invention have made efforts to find a method for diagnosing vascular endothelial dysfunction in order to more effectively manage and treat vascular disease patients. As a result, it has been found that vascular endothelial cells treated with blood plasma or serum of a patient and red blood cells , The expression level of Rab5C, clathrin, caveolin-1, EEA1 (Early Endosomal Antigen 1) and KCa3.1 channel was measured to confirm the diagnosis of vascular endothelial dysfunction, Thereby completing the invention.
It is an object of the present invention to provide a composition for diagnosing dysfunction of vascular endothelial cells.
Another object of the present invention is to provide a kit for diagnosing dysfunction of vascular endothelial cells, which comprises the above composition.
It is still another object of the present invention to provide a method for providing information for diagnosing vascular endothelial dysfunction.
It is another object of the present invention to provide a method for screening a substance for improving function of vascular endothelial cells.
It is another object of the present invention to provide a method for screening for a dysfunctional inducer of vascular endothelial cells.
In order to achieve the above object, one aspect of the present invention provides a composition for diagnosing dysfunction of vascular endothelial cells.
In one aspect, the composition for diagnosing dysfunction of vascular endothelial cells comprises a group consisting of KCa3.1 channels and a group consisting of caveolin-1, Rab5C, clathrin, and EEA1 (Early Endosome Antigen 1) A composition for diagnosing dysfunction of vascular endothelial cells, comprising an agent for measuring the level of mRNA of one or more proteins or genes thereof selected from the group consisting of:
Although vascular endothelial dysfunction induces vascular disease, there is no way to directly measure vascular endothelial dysfunction in the human body. Therefore, by using the composition for diagnosing vascular endothelial dysfunction of the present invention, it is possible to diagnose vascular diseases early, diagnose progress, prognosis and therapeutic effect through diagnosis of vascular endothelial dysfunction. For example, in the case of pregnancy hypertension, it is possible to diagnose the development of vascular endothelial dysfunction and the possibility of the development of gestational hypertension by using the composition of the onset prior to the occurrence of hypertension, thereby developing new preventive and therapeutic methods. In the case of arteriosclerosis and diabetic vascular diseases, the diagnosis of vascular endothelial dysfunction using the composition of the present invention can be used to diagnose the progress of vascular diseases even before symptoms of vascular diseases appear, and to diagnose the progress, prognosis and therapeutic effect Do.
As used herein, the term " caveolin-1 " is a major component of the caveolae plasma membrane that can be found in most cell types, including the binding of integrins in the Ras-ERK pathway In the early stages and cell cycle progression. The inventors of the present invention firstly clarified the relationship between KCa3.1 channel and caveolin-1 in vascular endothelial dysfunction. The caveolin-1 sequence information of the present invention can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, the caveolin-1 of the present invention is NCBI GenBank Accession NO. But are not limited to, NM_001172897.1, NM_001243064.1, NM_031556.3, or NM_001135818.1.
As used herein, the term " Rab5C " is a GTPase (GTPase) that regulates the membrane traffic by regulating the fusion between early endosomes and the plasma membrane Protein refers to. The inventors of the present invention firstly clarified the relationship with Rab5C in the vascular endothelial dysfunction caused by a vascular disease-causing substance. The Rab5C sequence information of the present invention can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, Rab5C of the present invention may be, but is not limited to, NCBI GenBank Accession NO.CR541901.1, AB232595.1, NM_001105840.2 or NM_001246383.1.
As used herein, the term " clathrin " refers to a protein that exhibits a triskelion form with three branches consisting of three heavy chains and three light chains, one of the proteins that coat vesicles. The three branches interact to form a polyhedral lattice that surrounds the vesicles. In the endothelial dysfunction, the present inventors first clarified the relationship with clathrin. The clathrin sequence information of the present invention can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, the clathrin of the present invention can be obtained from NCBI GenBank Accession NO. But are not limited to, NM_001288653.1, NM_001003908.1, NM_019299.1, or XM_001136053.4.
As used herein, the term " EEA1 (Early Endosome Antigen 1) " is located in the early endosomes and plays an important role in endosomal trafficking. The present inventors first clarified the relevance of EEA1 in vascular endothelial dysfunction. The EEA1 sequence information of the present invention can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, EEA1 of the present invention can be used in combination with NCBI GenBank Accession NO. But are not limited to, NM_003566.3, NM_001001932.3, NM_001108086.1, or XM_522610.5.
In one embodiment of the present invention, the level of protein expression of caveolin-1, clathrin, Rab5C, and EEA1 was determined by treating vascular endothelial cells with vascular disease-causing substances (plasma or serum of pregnant hypertension, oxLDL, LPC, As a result, it was confirmed that the expression level of caveolin-1, clathrin, Rab5C, EEA1 protein was increased while the expression level of KCa3.1 channel protein was decreased. These results suggest that vascular endothelial dysfunction induced by vascular disease can be diagnosed by measuring the expression level of KCa3.1 channel or caveolin-1, Rab5C, clathrin, and EEA1.
In another embodiment of the present invention, the level of expression of Rab5C and EEA1 protein was determined according to the degree of plasma or serum dilution in pregnant hypertensive patients. As a result, it was confirmed that the expression level of Rab5C and EEA1 protein was increased, The levels of Rab5C and EEA1 protein were increased by the gestational hypertensive factor alone. In such a small amount, the expression level of the marker can be measured to diagnose vascular endothelial dysfunction, so that the inventive possibility of vascular disease can be predicted or early diagnosed.
In the present invention, the term " individual " means all animals including humans, and may be, but not limited to, humans.
As used herein, the term " vascular endothelial cells " plays a role in maintaining vascular homeostasis such as vasodilation and contraction, proliferation and migration of vascular smooth muscle, thrombogenesis and lysis.
As used herein, the term " dysfunction of vascular endothelial cells " means that vascular endothelial cells do not function and vascular homeostasis is broken in relation to vasodilatation and contraction, proliferation and migration of vascular smooth muscle, thrombus formation and dissolution, Thereby causing a vascular disease. Vascular endothelial dysfunction causes vascular wall damage and increased vasoconstriction, leading to vascular disease. In the present invention, the vascular disease may be, but not limited to, hypertension, arteriosclerosis, diabetic vascular disease, or a combination thereof. Specifically, the hypertension may be, but is not limited to, gestational hypertension.
The vascular disease is a degree of vascular endothelial dysfunction, and it is known that the vascular disease progresses. The vascular endothelial dysfunction is caused by vascular disease-causing substances, which cause various pathways such as ROS Resulting in vascular endothelial dysfunction. Vascular endothelial dysfunction develops and vascular disease develops. The vascular disease-inducing substance affects the expression level of KCa3.1 channel in vascular endothelial cells.
In the present invention, the vascular disease-inducing substance is selected from the group consisting of oxidized low-density lipoprotein, lysophosphatidylcholine (LPC), sFlt-1, endoglin, hyperglycemia, superoxide anion donor superoxide anion donor, and the like.
The term " diagnosing " as used herein means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm whether or not vascular endothelial dysfunction is caused by the vascular endothelial cell dysfunction.
In addition, in another embodiment, the composition for diagnosing dysfunction of vascular endothelial cells of the present invention may further be a composition comprising an agent for measuring the level of mRNA of KCa3.1 channel protein or its gene.
The term " KCa3.1 channel " (subfamily N, member 4) in the present invention means a voltage-independent potassium channel in the form of heterotetramer activated by intracellular calcium . endothelial cells KCa3.1 channel to induce hyperpolarization to relax the blood vessels while the hyperpolarization of intracellular Ca 2 + increase the inlet thereby promoting the NO produced by eNOS relax the blood vessels. Thus, endothelial cells KCa3. The decrease in 1-channel expression induces vascular endothelial dysfunction, and the expression level of KCa3.1 channel protein or gene can be measured to confirm vascular endothelial dysfunction.
In one embodiment of the present invention, the expression level of KCa3.1 channel protein in the umbilical vein and the aortic tissue of the normal mother and the pregnant hypertensive patient was analyzed and the expression level of KCa3.1 channel protein was decreased in the pregnant hypertensive patient Respectively.
In another embodiment of the present invention, the effects of plasma or serum, oxLDL, LPC, and hyperglycemia in pregnant hypertensive patients on KCa3.1 channel protein expression levels in vascular endothelial cells of normal pregnant and gestational hypertensive patients were analyzed. The levels of KCa3.1 channel protein were decreased by plasma or serum, oxLDL, LPC and hyperglycemia in patients with hypertension. The results suggest that oxLDL and hyperglycemia, which are plasma or serum substances, are abnormally reduced in KCa3.1 channel expression in vascular endothelial cells, resulting in vascular endothelial dysfunction in pregnancy hypertension and diabetic vascular complication.
In another embodiment of the present invention, the effect of oxidative stress on the KCa3.1 channel protein expression level in vascular endothelial cells of normal pregnant and gestational hypertensive patients was analyzed by using oxidant treatment to determine KCa3.1 channel protein expression And it was confirmed to be recovered by antioxidant. This suggests that oxidative stress causes vascular endothelial dysfunction and that oxidative stress level can be measured by measuring the expression level of KCa3.1 channel protein.
In addition, KCa3.1 channel protein expression was measured in normal vascular endothelial cells (HUVECs) exposed to plasma or serum in patients with various stages of GHD, and the expression level of KCa3.1 channel protein And a decrease in the expression level can be confirmed even when diluted to about 1/10. Therefore, when the level of expression of the marker is measured, the possibility of developing a vascular disease can be predicted or diagnosed early.
In the present invention, the marker is a substance capable of distinguishing a normal group and an individual having a vascular endothelial dysfunction, wherein the marker is an increase or decrease in an individual having a vascular endothelial dysfunction of the present invention Polypeptides, proteins or nucleic acids, genes, lipids, glycolipids, glycoproteins or sugars, and the like. In particular, the present invention is not limited to a protein having a vascular endothelial dysfunction or a protein having a vascular disease induced by vascular endothelial dysfunction.
As used herein, the term " mRNA level measurement " refers to the measurement of the amount of mRNA by determining the expression level of genes for the diagnosis of vascular endothelial dysfunction in isolated cells to diagnose vascular endothelial dysfunction. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.
Specifically, primers or probes that specifically amplify a specific region of these genes based on the above sequence are known to those skilled in the art since nucleic acid information of the genes is known in GeneBank and the like. You can design.
The agent for measuring the mRNA level of the gene may comprise a primer pair, a probe or an antisense nucleotide that specifically binds to the gene.
Specifically, in order to diagnose dysfunction of vascular endothelial cells, the agent for measuring the mRNA level may be a primer pair specifically binding to a gene of a protein selected from Rab5C, clathrin, caveolin-1 and EEA1, a probe or an antisense oligo Nucleotides < / RTI >
As used herein, the term " primer pair " is a primer pair that contains all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, but specifically provides analysis results with specificity and sensitivity. The nucleic acid sequence of the primer is inconsistent with the non-target sequence present in the sample and can be given high specificity when it is a primer that amplifies only the target gene sequence containing a complementary primer binding site and does not induce nonspecific amplification .
As used herein, the term " probe " refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding do. The probe molecule may be a peptide nucleic acid (PNA), a peptide nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA, or a DNA. Specifically, it is PNA. In addition, the probe may be, for example, an enzyme, a protein, an antibody, a microorganism, an animal or animal cell or an organ, a nerve cell, DNA, and RNA DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
As used herein, the term " antisense oligonucleotide " refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which binds to a complementary sequence in the mRNA and inhibits translation of the mRNA into a protein . An antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to and capable of binding to the mRNAs of the genes. This can interfere with translation of the gene mRNA, translocation into the cytoplasm, maturation, or essential activity for all other overall biological functions. The length of the antisense oligonucleotide may be 6 to 100 bases, specifically 8 to 60 bases, more specifically 10 to 40 bases. The antisense oligonucleotides may be synthesized in vitro by conventional methods and administered in vivo or may be used to synthesize antisense oligonucleotides in vivo. One example for the synthesis of antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is a multi-cloning site (MCS) in the opposite direction. It is preferred that the antisense RNA is such that translation stop codon is present in the sequence so that it is not translated into the peptide sequence.
As used herein, the term " protein level measurement " is a process for confirming the presence and expression level of marker proteins for the diagnosis of vascular endothelial dysfunction in isolated cells for diagnosis of vascular endothelial dysfunction. The expression level of the protein can be confirmed using an antibody that specifically binds to the marker protein. Specifically, the protein expression level itself is measured without using the antibody.
As the protein level measurement or comparative assay, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Flock Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronin immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography- Mass Spectrometry, LC-MS, LC-MS / MS, Western blot, and enzyme linked immunosorbent assay (ELISA).
Specifically, the agent for measuring the protein level for the diagnosis of dysfunction of vascular endothelial cells may include an antibody that specifically binds to a protein selected from KCa3.1 channel, Rab5C, clathrin, caveolin-1 and EEA1.
The term " antibody " as used herein refers to 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 one or more proteins selected from the above KCa3.1 channels, Rab5C, clathrin, caveolin-1 and EEA1, and includes polyclonal antibodies, monoclonal antibodies and recombinant antibodies . The production of the antibody can be easily carried out using techniques well known in the art.
The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.
Another aspect of the present invention provides a kit for diagnosing dysfunction of vascular endothelial cells, comprising the composition for diagnosing dysfunction of vascular endothelial cells. Specifically, the kit may be a reverse transcription polymerase chain reaction kit (RT-PCR), a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, .
The kit for diagnosing dysfunction of vascular endothelial cells may further comprise one or more other component compositions, solutions or devices suitable for the assay method.
Specifically, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription-polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each of the above genes, and has a length of about 7 bp to 50 bp, more specifically about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcriptase reaction kits include enzymes such as test tubes or other appropriate containers, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC- Water (DEPC-water), sterile water, and the like.
Specifically, the diagnostic kit may be a diagnostic kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.
In particular, the diagnostic kit may be a diagnostic kit characterized by comprising essential elements necessary for performing ELISA. ELISA kits include antibodies specific for the KCa 3.1 channel, Rab5C, clathrin, caveolin-1 and EEA1 proteins. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.
In particular, the diagnostic kit may be a rapid kit, which includes essential elements necessary for performing a rapid test to know the analysis result within 5 minutes. Rapid kits contain antibodies specific for proteins. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The rapid kit may also include antibodies specific for the control protein. Other rapid kits include reagents capable of detecting the bound antibody, such as a nitrocellulose membrane with a specific antibody and a secondary antibody immobilized thereon, a membrane bound to the bead conjugated with the antibody, Materials, and the like.
In particular, the diagnostic kit may be a multiple reaction monitoring (MRM) kit, which is an MS / MS mode, which includes essential elements necessary for performing mass analysis. While SIM (Selected Ion Monitoring) is a method of using ions generated by collision with the source part of the mass spectrometer, MRM once again selects a specific ion among the broken ions to connect another consecutively connected MS source And then collide with another once more, and then use ions obtained from the collision. More specifically, there is a problem in the SIM that disturbance may occur when the selected quantitative ions are ions that are also detected in plasma. On the other hand, in the case of using MRM, when ions having the same mass are further broken, the molecular structure is different and differentiated. Therefore, when the ion is used as a quantitative ion, disturbing peaks in the background are removed, Can be obtained. Thus, by using the MRM mode during mass spectrometry, desired materials can be simultaneously analyzed at better analytical sensitivity. Through the above-described MRM (Multiple Reaction Monitoring) analysis methods, it is possible to compare the protein expression level in the normal control group and the protein expression level in the vascular endothelial dysfunctional individuals in the vascular disease patients caused by vascular endothelial dysfunction The vascular endothelial cell dysfunction can be diagnosed by determining whether the expression level of the marker gene for the vascular endothelial cell dysfunction is increased or decreased.
Another aspect of the present invention provides a method for providing information for diagnosing dysfunction of vascular endothelial cells.
Specifically, the method of the present invention comprises the steps of: (a) measuring a level of at least one protein selected from the group consisting of Rab5C, clathrin, caveolin-1 and EEA1 or a gene thereof in a biological sample; And (b) comparing step (i) with step (a) with a normal control sample, to provide information for diagnosing dysfunction of vascular endothelial cells.
More particularly, the method of the present invention further comprises the step of (ii) measuring the level of expression of the KCa3.1 channel protein or a gene thereof and measuring (i) / (ii) and comparing the ratio calculated by the equation (ii) with a normal control sample, to provide information for diagnosing dysfunction of vascular endothelial cells.
Since the Rab5C, clathrin, caveolin-1, EEA1 and KCa3.1 channels are characterized in that the gene expression level or the expression level of the protein is changed in an individual having vascular endothelial dysfunction, Diagnosis of dysfunction and early diagnosis of vascular disease and progress status diagnosis will be possible.
As used herein, the term " biological sample " refers to a sample collected from the subject to diagnose vascular endothelial dysfunction of suspected individuals with impaired vascular endothelial function. But are not limited to, whole blood, plasma, serum, fractions thereof or cells contained therein, red blood cells, and the like.
Specifically, the expression level or the expression level of the gene of (i) Rab5C, clathrin, caveolin-1 protein in the biological sample of the suspected endothelial dysfunctional individual is lower than the expression level of the protein of the normal control sample or the expression level of the gene Or (ii) the expression level of the KCa3.1 channel protein or the expression level of the gene is lower than the protein expression level or the gene expression level of the normal control sample, it can be judged that the vascular endothelial dysfunction is caused.
More specifically, when the ratio of (i) / (ii) is higher than that of the normal control sample, it can be judged that the vascular endothelial cell function is impaired.
In one embodiment of the present invention, the expression levels of KCa3.1 channel and Rab5C protein in red blood cells were examined, and it was confirmed that the expression level of KCa3.1 channel protein was decreased and the level of Rab5C protein was increased. It was found that the expression level of KCa3.1 channel protein and Rab5C protein can be measured in red blood cells to diagnose vascular endothelial dysfunction.
The expression level of the gene of the present invention can be used to measure or compare mRNA expression levels.
The measurement or comparison of the mRNA expression level may be performed using a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real time reverse transcription polymerase chain reaction, an RNase protection assay, a Northern blotting or a DNA chip, but the present invention is not limited thereto . Through these measurement methods, it is possible to confirm the mRNA expression level of the vascular disease patients induced by the amount of mRNA expression and vascular endothelial dysfunction in the normal control group, and to compare the degree of mRNA expression thereof to diagnose or predict the vascular disease.
The protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein. The antibody and the protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used.
As used herein, the term " antigen-antibody complex " refers to a conjugate of a corresponding protein antigen in a biological sample and an antibody recognizing it. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.
For the purpose of the present invention, the above protein expression level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronian immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, But are not limited to, liquid chromatography-mass spectrometry (LCMS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA).
Also, specifically, the method of the present invention comprises the steps of: (a) treating a biological sample with normal vascular endothelial cells; And (b) measuring the expression of at least one protein selected from the group consisting of Rab5C, clathrin, caveolin-1 and EEA1 or a gene thereof in the endothelial cells of step (a). (c) comparing the (i) measured in the step (b) with a normal control sample, to provide information for diagnosing dysfunction of vascular endothelial cells.
More specifically, the method of the present invention further comprises the steps of (ii) measuring the expression level of the KCa3.1 channel protein or a gene thereof, and (i) using the measured (i) and (ii) and comparing the ratio calculated by the equation (ii) with that of the normal control sample, to provide information for diagnosing dysfunction of vascular endothelial cells.
Another aspect of the present invention provides a method for screening a substance for improving function of vascular endothelial cells.
Specifically, there is provided a method for the treatment of a vascular endothelial cell, comprising the steps of: (a) treating a candidate substance which is expected to induce the function improvement of vascular endothelial cells in the separated cell; (b) measuring the expression level of at least one protein selected from the group consisting of Rab5C, clathrin, caveolin-1 and EEA1 or a gene thereof in a cell treated with the candidate substance; And (c) selecting the candidate substance as an inducer of vascular endothelial function improvement when the amount of (i) measured in the step (b) is decreased compared with a negative control not treated with the candidate substance. And may be a screening method of inducing substances for improving the function of vascular endothelial cells.
More specifically, the screening method of the inducer for improving vascular endothelial function of the present invention comprises the steps of (ii) adding the expression level of the KCa3.1 channel protein or a gene thereof to (i) and ii), when the ratio calculated by the formula (i) / (ii) is decreased as compared with the negative control not treated with the candidate substance, the candidate substance is selected as being a substance inducing the function improvement of vascular endothelial cells A method for screening a substance for improving function of vascular endothelial cells.
In one embodiment of the present invention, when the vascular endothelial growth factor TBHP was treated, the expression level of KCa3.1 channel protein was significantly increased. This is because the increase of KCa3.1 channel expression level is known to improve vascular endothelial function (Chadha PS et al, 2010, J Pharmacol Exp Ther, 335 (2), 284-293), the function of vascular endothelial cells Suggesting that it is possible to determine whether the candidate substance can induce improvement in vascular endothelial cell function by measuring the protein expression level of the KCa3.1 channel after treating the candidate substance expected to induce improvement will be.
In the present invention, the isolated cells may be cells separated from the endothelial dysfunction-induced cells or individuals, and specifically may be cells isolated from blood or blood vessels, more specifically, red blood cells or vascular endothelial cells, But is not limited thereto.
In addition, in the present invention, the term " individual " means an individual with or without induced endothelial dysfunction.
The term " test agent " as used herein includes any substance, molecule, element, compound, entity, or combination thereof. But are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or a combination of two or more substances.
Another embodiment of the present invention provides a method for screening a dysfunctional inducer of vascular endothelial cells.
Specifically, there is provided a method for treating a vascular endothelial cell, comprising the steps of: (a) treating a candidate cell which is expected to induce vascular endothelial dysfunction to separate cells; (b) measuring at least one protein selected from the group consisting of Rab5C, clathrin, caveolin-1 and EEA1 or a gene expression level thereof in cells treated with the candidate substance; And (c) selecting the candidate substance as a dysfunctional inducer of vascular endothelial cells when the (i) in the step (b) is increased as compared with the negative control group not treated with the candidate substance, And may be a screening method of an endothelial cell dysfunction inducer.
More specifically, the screening method for a vascular endothelial dysfunction inducer of the present invention comprises the steps of: (ii) measuring the KCa3.1 channel protein or its gene expression level and measuring (i) and (ii) ), When the ratio calculated by the formula (i) / (ii) is increased as compared with the negative control group in which the candidate substance is not treated, the candidate substance is selected as being a dysfunctional inducer of vascular endothelial cells And a method of screening for a vascular endothelial dysfunction inducer.
In the present invention, the separated cells mean cells isolated from normal or uninvolved endothelial dysfunction, and specifically cells isolated from blood or blood vessels, more specifically, red blood cells or vascular endothelial cells Cells, but are not limited thereto.
In one embodiment of the present invention, when LPC or X / XO, a vascular disease-causing substance, was treated on normal vascular endothelial cells, KCa3.1 channel protein decreased and caveolin-1, clathrin, and EEA1 protein levels were increased. Thus, a candidate substance expected to induce vascular endothelial dysfunction in normal vascular endothelial cells is treated, and the expression level of the protein is measured to determine whether the candidate substance can induce vascular endothelial dysfunction It can be judged.
The expression levels of Rab5C, clathrin, caveolin-1, EEA1, and KCa3.1 channels of the present invention were measured and compared, So that the vascular disease can be diagnosed early.
FIG. 1 is a graph showing the results of comparative measurement of expression levels of KCa3.1 channel protein in umbilical vascular endothelial cells of normal pregnant women and pregnant hypertensive patients. ** indicates P <0.01 compared to the control (normal maternal tissue or vascular endothelial cells) (NP: normal mother, PE: pregnant hypertensive patients, EC: vascular endothelial cells). A is a comparative measurement of the expression level of KCa3.1 channel protein in the umbilical vein (left) and the right aorta (right) in normal pregnant women and pregnant hypertensive patients by immunohistochemistry, and B and C are the measurements of maternal and gestational hypertension (B) or cultured umbilical vein endothelial cells (C) by Western blotting, and D represents the level of expression of KCa3.1 channel protein in blood vessels cultured in umbilical vein of normal maternal and gestational hypertensive patients This is a measurement of KCa3.1 current in endothelial cells.
Fig. 2 shows the effect of plasma or serum of pregnant hypertensive patients on the expression level of KCa3.1 channel protein (NP: normal mother, PE: pregnant hypertensive patient). A shows the effect of plasma or serum of the pregnant patient on the expression level of KCa3.1 channel in vascular endothelial cells and the effect of antioxidant on it and B shows the effect of NOX2 (NADPH oxidase 2) as compared to the control. C is a graph showing the effect of anti-LOX1 antibody (LOX1: lectin-like) on the increase of protein expression level of NOX2 by plasma or serum of pregnant hypertensive patients and the decrease of protein expression level of KCa3.1 channel ox-LDL receptor-1, tempol and tiron: antioxidants). ** indicates P <0.01 compared to the control group (normal maternal plasma or serum-treated vascular endothelial cells) and # indicates that plasma or serum of pregnant hypertensive patients was not treated with LOX1 antibody ≪ / RTI >
FIG. 3 is a graph showing the effect of oxidized low density lipoprotein (oxLDL) (A) and hyperglycemia (B) on vascular endothelial cell KCa3.1 channel protein expression level. On the other hand, * and ** mean P <0.05 and P <0.01, respectively, as compared to the control group (vascular endothelial cells not treated with vascular disease inducing substances).
Figure 4 shows the effect of ROS on the expression level of vascular endothelial KCa3.1 channel protein. (Xanthine and Xanthine oxidase mixture, X / X0) and an antioxidant (tempol, tiron) on the expression level of KCa3.1 channel protein. ** represents P < 0.01 compared to the control (X / XO or anti-oxidant untreated vascular endothelial cells), # and # are blood vessels treated with X / XO (100 uM / 100 mU / ml) P <0.05 and P <0.01, respectively, compared with endothelial cells.
FIG. 5 shows that caveolin-1 is involved in the degradation of KCa3.1 channel protein in vascular endothelial cells (NP: normal mother, PE: pregnant hypertensive patient). A shows the effect of LPC, a vascular disease-inducing substance, on the protein expression level of vascular endothelial cell caveolin-1 and the effect of antioxidant on the protein expression level, and B indicates that blood plasma or serum of pregnant hypertensive patients has vascular endothelial cell caveolin- And C shows the decrease in the level of expression of vascular endothelial KCa3.1 channel by plasma or serum of pregnant hypertensive patients with caveolin-1. On the other hand, ** indicates P < 0.01 as compared with the control group.
FIG. 6 is a graph showing the expression levels of clathrin and EEA1 (Early Endosomal Antigen 1) according to treatment of vascular disease-causing substances. A shows the level of protein expression of KCa3.1 channel and clathrin in plasma membranes of blood plasma or serum-treated vascular endothelial cells of pregnant hypertensive patients, B shows the KCa3.1 channel, clathrin, and C is a comparative measurement of the expression levels of EEA1 and KCa3.1 channel proteins according to X / X0 concentration treatment.
FIG. 7 is a graph comparing protein levels of Rab5C, EEA1 and KCa3.1 channels in vascular endothelial cells exposed by diluting plasma or serum of pregnant hypertensive patients with concentrations of 10%, 25% and 100%.
8 is a graph showing the expression levels of KCa3.1 channel and Rab5C protein using erythrocytes isolated from normal maternal and pregnant hypertensive mothers (NP: normal pregnant women, PE: pregnant hypertensive patients).
9 shows the effect of TBHP treatment on the expression level of KCa3.1 channel protein in vascular endothelial cells.
Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.
Example In vascular disease patients, vascular endothelial cells KCa3 . 1 channel Analysis of protein expression level
In order to investigate whether KCa3.1 channel expression is decreased in vascular endothelial cells, we compared the expression levels of KCa3.1 channel protein in vascular endothelial cells of normal pregnant and gestational hypertensive patients.
KCa3.1 channel protein expression levels in umbilical vein and aortic tissue were measured by histological analysis (Fig. 1A) and western blot (Fig. 1B), and vascular endothelial cells were measured in umbilical vein of normal mother and gestational hypertensive patients And the level of expression of KCa3.1 channel protein (C in Fig. 1) and the size of KCa3.1 current (D in Fig. 1) were measured. To determine the level of KCa3.1 channel protein expression, an antibody with the amino acid sequence of RQVRLKHRKLREQV (SEQ ID NO: 1) was used.
As a result, the level of protein expression of vascular endothelial KCa3.1 channel was significantly decreased and KCa3.1 current size was decreased in pregnant hypertensive patients compared with normal mothers.
Therefore, it was confirmed that the expression level of KCa3.1 channel in vascular endothelial cells is abnormally decreased when the pregnancy hypertension develops and induces vascular endothelial dysfunction.
Example 2. Vascular endothelial cells caused by vascular disease-inducing substances KCa3 . 1 channel Analysis of protein expression level
In order to investigate whether the decrease of KCa3.1 channel expression in vascular endothelial cells is caused by vascular disease substances in blood plasma or serum, blood plasma or serum, oxLDL, LPC and hyperglycemia of normal pregnant and pregnant hypertensive patients were observed in vessels And the effect on the expression level of KCa3.1 channel in endothelial cells was compared and analyzed.
As a result of the above experiment, the expression level of NOX2 protein that produces ROS in vascular endothelial cells exposed to plasma or serum of pregnant hypertensive patients is increased and the level of KCa3.1 channel is increased compared with vascular endothelial cells exposed to normal maternal plasma or serum It was confirmed that the level of protein expression was remarkably decreased (Fig. 2B). A decrease in the level of KCa3.1 channel expression in exposed vascular endothelial cells of plasma of this pregnant hypertensive patient plasma or serum was inhibited by an antioxidant (Fig. 2A) or an antibody against LOX-1 (Fig. 2C). In other words, the plasma or serum of pregnant hypertensive patients increased the expression level of NOX2 protein through LOX-1, thereby increasing the production of ROS and decreasing the KCa3.1 channel protein expression level, and the plasma or serum major vascular disease The inducer could be oxLDL. Also, the expression level of KCa3.1 channel protein in vascular endothelial cells was decreased by oxLDL or hyperglycemia (Fig. 3).
Therefore, it was confirmed that oxLDL and hyperglycemia, plasma or serum substances, such as pregnancy hypertension and vascular complication of diabetes, abnormally decrease the expression level of KCa3.1 channel in vascular endothelial cells, leading to induction of vascular endothelial dysfunction.
Example 3. Oxidative Stress in Vascular Endothelial Cells KCa 3.1 Analysis of channel expression level
To investigate the effect of oxidative stress on the expression of KCa3.1 channel, superoxide anion donors, which are oxidants in vascular endothelial cells exposed to plasma or serum of normal maternal vascular endothelial cells and pregnant hypertensive patients, The effect on KCa3.1 channel protein was measured by Western blot. And the level of expression of? -Tubulin or? -Actin was measured as a control in each sample.
As a result of the above experiment, the level of KCa3.1 channel protein expression was decreased by oxidant (X / XO) treatment and the decrease of KCa3.1 channel protein expression level by oxidant was recovered by treatment with antioxidant (FIG. 4)
Therefore, in view of the above results, it was confirmed that the expression level of KCa3.1 channel protein in vascular endothelial cells was dependent on oxidative stress, especially superoxide anion. The expression level of KCa3.1 channel was measured, It was confirmed that the oxidative stress applied to the cells, especially the level of superoxide anion, can be determined.
These results suggest that the oxidative stress induced by the vascular disease induces a decrease in the expression level of KCa3.1 channel, resulting in a vascular endothelial dysfunction.
Example 4. Vascular Endothelial Cells Following Treatment of Vascular Diseases caveolin -1, clathrin, Rab -5C, EEA1 Analysis of protein expression level
The levels of caveolin-1, clathrin, Rab-5C, EEA1, and KCa3.1 channel proteins were measured using Western blot after treating vascular disease-causing substances in normal vascular endothelial cells (FIGS. 5 and 6). (SEQ ID NO: 2), PQLMLTAGPSVAVPPQAPFGYGYTAPPYGQPQPGFGYS (SEQ ID NO: 3), ASRGATRPNGPNT (SEQ ID NO: 4) and FCAECSAKNALTPSSKKPVR (SEQ ID NO: 5), respectively, in order to measure the levels of caveolin-1, clathrin, Rab5C and EEA1 protein expression Antibodies were used.
As a result, when the plasma or serum of LPC or gestational hypertensive patients was treated, the expression level of caveolin-1 protein was increased, and it was confirmed that caveolin-1 and KCa3.1 channel protein coexisted by immunoprecipitation (FIG. 5 C). The KCa3.1 channel protein was decreased by the vascular disease-inducing substance, and the clathrin and the intracellular EEA1 protein expression levels were increased (FIG. 6C, FIG. 7). On the other hand, KCa3.1 decreased and clathrin increased in the cell membrane due to vascular disease-causing substances (Fig. 6, A, B).
Therefore, it was found that the increase of caveolin-1, clathrin and EEA1 protein was caused by the vascular disease-causing substance in the light of the above results.
This suggests that vascular endothelial dysfunction induced by vascular disease-inducing substances can be diagnosed by measuring the expression level of KCa3.1 channel or caveolin-1, clathrin, and EEA1.
In addition, after treating candidate substances expected to induce vascular endothelial dysfunction in normal vascular endothelial cells, the level of protein expression of KCa3.1 channel or caveolin-1, clathrin, EEA1 was measured, Suggesting that the substance can determine whether it can induce vascular endothelial dysfunction.
Example 5. In vascular endothelial cells according to plasma or serum dilution of pregnant hypertensive patients KCa3 . 1 channel , Rab5C , EEA1 Analysis of protein expression level
To investigate the effect of plasma or serum dilution of human vascular endothelial cells treated with pregnancy hypertension on the expression levels of Rab5C and EEA1 protein, which cause the decrease of KCa3.1 channel protein and its expression level, plasma of pregnant hypertensive patients Or serum was mixed with a culture medium and human vascular endothelial cells cultured in a solution of plasma or serum at a concentration of 10, 25, 100% (without dilution) were exposed for 24 hours and then cultured in a Western blot to KCa3 The level of expression of Rab5C protein and the expression level of EEA1 protein were measured through the expression of 1 channel protein and small GTPase. In each sample, the expression level of β-actin was measured as a control.
As a result, it was confirmed that the expression level of KCa3.1 channel protein was decreased but the level of Rab5C and EEA1 protein was increased in proportion to the degree of dilution in vascular endothelial cells exposed to plasma or serum of pregnant hypertensive patients (FIG. 7).
Thus, in view of the above results, the level of expression of Rab5C and EEA1 protein is increased by about 1/10 of the gestational hypertensive factor alone, compared with the case where the gestational hypertension is completely expressed, and the expression of vascular endothelial KCa3.1 channel protein Which is the reason for the decrease in the level of the.
In this way, the level of expression of KCa3.1 channel, Rab5C, EEA1 protein is measured at the stage before the disease is completely developed, in which the concentration of the factor that causes vascular endothelial dysfunction is low, to diagnose vascular endothelial dysfunction As a result, it was found that the possibility of developing a vascular disease can be predicted or early diagnosed by measuring the expression level of the marker.
Example 6. Red blood cells KCa3 . 1 channel and Rab5C's Analysis of protein expression level
In order to confirm that the endothelial cells and RBCs showed similar KCa3.1 channel and Rab5C expression patterns in vascular endothelial dysfunction, blood samples collected from normal mothers and pregnant hypertensive patients were analyzed by Western blot using KCa3.1 channel protein And the expression levels of Rab5C protein were measured. In each sample, the expression level of β-actin was measured as a control.
As a result, it was confirmed that the expression level of KCa3.1 channel protein was decreased and the expression level of Rab5C protein was increased similarly to the vascular endothelial cells in the red blood cells of pregnant hypertensive patients compared to the control group (red blood cells of normal mother) (Fig. 8).
Thus, in view of the above results, it was confirmed that the expression level of vascular endothelial KCa3.1 channel protein in the pregnant hypertensive patients can be determined by measuring KCa3.1 channel protein or Rab5C in erythrocytes.
Example 7. TBHP The endothelial cells KCa3 . 1 channel Analysis of protein expression level
The vascular endothelial cells were treated with TBHP, an inducer of vascular function improvement, and the expression level of KCa3.1 channel was measured using Western blot.
As a result, it was confirmed that KCa3.1 channel expression was significantly increased by TBHP treatment (Fig. 9).
This is because the increase of KCa3.1 channel expression level is known to improve vascular endothelial function (Chadha PS et al, 2010, J Pharmacol Exp Ther, 335 (2), 284-293), the function of vascular endothelial cells The candidate substance expected to induce the improvement can be treated to vascular endothelial cells and the protein expression level of KCa3.1 channel can be measured to judge whether the candidate substance can induce improvement of vascular endothelial cell function .
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
<110> Ewha University - Industry Collaboration Foundation <120> A composition for diagnosing vascular endothelial cell dysfunction and use thereof <130> KPA150137-KR <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> KCa3.1 antibody <400> 1 Arg Gln Val Arg Leu Lys His Arg Lys Leu Arg Glu Gln Val 1 5 10 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> caveolin-1 antibody <400> 2 Met Ala Asp Glu Leu Ser Glu Lys Gln Val Tyr Asp Ala His Thr Lys 1 5 10 15 Glu Ile Asp <210> 3 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> clathrin antibody <400> 3 Pro Gln Leu Met Leu Thr Ala Gly Pro Ser Val Ala Val Pro Pro Gln 1 5 10 15 Ala Pro Phe Gly Tyr Gly Tyr Thr Ala Pro Pro Tyr Gly Gln Pro Gln 20 25 30 Pro Gly Phe Gly Tyr Ser 35 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Rab-5C antibody <400> 4 Ala Ser Arg Gly Ala Thr Arg Pro Asn Gly Pro Asn Thr 1 5 10 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EEA1 antibody <400> 5 Phe Cys Ala Glu Cys Ser Ala Lys Asn Ala Leu Thr Pro Ser Ser Lys 1 5 10 15 Lys Pro Val Arg 20
Claims (21)
(b) comparing the ratio calculated by the equation below with (i) and (ii) measured in step (a) with a normal control sample, wherein the function of the vascular endothelial cell by oxidative stress A method of providing information for diagnosis of impotence;
Ratio = (i) / (ii).
(b) measuring the expression level of at least one protein selected from the group consisting of Rab5C, clathrin and EEA1 (Early Endosomal Antigen 1) or a gene thereof in the endothelial cells of step (a) ) Measuring the expression level of a KCa3.1 channel protein or a gene thereof; And
(c) comparing the ratio calculated by the equation below with (i) and (ii) measured in step (b) with a normal control sample, wherein the function of the endothelial cell by oxidative stress A method of providing information for diagnosis of impotence;
Ratio = (i) / (ii).
(b) measuring the level of expression of at least one protein selected from the group consisting of Rab5C, clathrin, and Early Endosomal Antigen 1 (EEA1) or a gene thereof in a cell treated with the candidate substance, (ii) measuring KCa3 Measuring the level of expression of a protein or a gene thereof; And
(c) when the ratio calculated by the following equation using (i) and (ii) measured in the step (b) is increased as compared with the negative control not treated with the candidate substance, A method for screening for a dysfunctional inducer of vascular endothelial cells by oxidative stress, comprising the step of selecting as a dysfunctional inducer of vascular endothelial cells by stress;
Ratio = (i) / (ii).
(ii) measuring the level of expression of one or more proteins selected from the group consisting of Rab5C, clathrin and EEA1 (Early Endosomal Antigen 1) or a gene thereof in cells treated with the candidate substance; and (ii) Measuring the expression level of the KCa3.1 channel protein or its gene; And
(c) when the ratio calculated by the following equation using (i) and (ii) measured in the step (b) is increased as compared with the negative control not treated with the candidate substance, A method for screening for a dysfunctional inducer of vascular endothelial cells by oxidative stress, comprising the step of selecting as a dysfunctional inducer of vascular endothelial cells by stress;
Ratio = (i) / (ii).
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