WO2017061809A1 - Composition for vascular endothelial cell dysfunction diagnosis, and use thereof - Google Patents

Abstract

The present invention relates to a composition, kit, and method for vascular endothelial cell dysfunction diagnosis and a method for screening a substance which induces the dysfunction or improved function of vascular endothelial cells. The method provided according to the present invention allows, when used, for the diagnosis of vascular endothelial cell dysfunction by using the expression levels of marker proteins, such as Rab5C, clathrin, caveolin-1, and early endosome antigen 1 (EEA1), and thus can be widely used for early diagnosis of various vascular diseases triggered by vascular endothelial cell dysfunction.

Description

A composition for diagnosing vascular endothelial dysfunction and its use

The present invention relates to a composition for diagnosing vascular endothelial dysfunction and its use. More specifically, the present invention relates to a composition for diagnosing vascular endothelial dysfunction, a kit, a method for diagnosing vascular endothelial dysfunction, and vascular endothelial dysfunction. The present invention relates to a method for screening an induction substance for improvement.

The function of cells is very sensitively regulated by ion channels. 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 ⁺ channels, Ca ^{2 +} channels, and K ⁺ channels according to the type of ions to pass through. In K ⁺ channels that are activated by intracellular Ca ^{2 +} There are several types of endothelial cells has KCa1.1 channel, KCa2.3 channel, KCa3.1 channel. These three channels play different roles in vascular endothelial cells. KCa3.1 channels induce vascular endothelial hyperpolarization to regulate vasoconstriction (Tharp DL, Bowles DK, 2009, Cardiovasc Hematol Agents Med Chem. 7, 1- 11) plays an important role in new angiogenesis. In cells other than vascular endothelial cells, these channels play an important role in cell function regulation. KCa3.1 channels are also distributed in erythrocytes, fibroblasts, proliferating vascular smooth muscle cells, and immune cells (T cells and B cells) and contribute to the regulation of their function.

The vascular endothelial cells play a very important role in the regulation of vascular aperture and contractility. Vascular endothelial cells secrete NO or induce endothelial cell-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 NO secretion as well as vascular endothelial cell hyperpolarization. In addition, in vascular diseases such as hypertension, when vascular relaxation due to NO decreases, relaxation due to hyperpolarization compensates for this and inhibits increase in vascular smooth muscle contractility.

If the function of these vascular endothelial cells is not normally performed, various vascular diseases may occur due to this. For example, KCa3.1 channels, which play an important role in the function of vascular endothelial cells, are known to cause vascular diseases such as hypertension if they are not functioning properly. In fact, KCa3.1 channel deficient mice (KCa3.1 channel knockout mice) Hypertension may occur and decreased expression of KCa3.1 channels is known to cause diseases such as gestational hypertension.

Because vascular endothelial cells are in direct contact with plasma, vascular endothelial dysfunction is caused by vascular disease-causing substances in plasma. Such vascular disease causing agents include oxidized low density-lipoprotein (oxLDL), hyperglycemia, and ROS such as superoxide generated by these substances. For example, vascular endothelial dysfunction occurs due to oxidized low density lipid protein in gestational hypertension and atherosclerosis, and hyperglycemia in diabetic vascular disease. As a result, an increase in vascular contractility and a decrease in vascular caliber are caused, and blood flow is disturbed by an increase in vascular resistance, and gas and nutrient supply to tissues are not performed smoothly, causing vascular diseases. These vascular disease-causing agents are known to cause vascular endothelial dysfunction by reducing the expression of vascular endothelial KCa3.1 channels (Choi S et al, 2013a, Free Radic Biol Med, 57, 10-21).

Vascular disease occurs when structural changes in blood vessels, ie, vascular diameter reduction, are caused by vascular endothelial dysfunction, and thus vascular endothelial dysfunction occurs before structural changes in blood vessels. Therefore, the diagnosis of vascular endothelial dysfunction can be used for early diagnosis of vascular disease (diagnosis of the disease before structural changes in blood vessels), progress and prognosis, and diagnosis of treatment effect.

Currently, in gestational hypertension, various methods such as measuring risk factors (age, etc.), uterine arterial blood flow using Doppler, and measuring gestational hypertension (sFlt-1, endoglin) in plasma are used for early diagnosis. No method has been developed to accurately diagnose the possibility of the disease. Accordingly, in the case of arteriosclerosis and diabetes, the progress of vascular disease is diagnosed by measuring blood flow and vascular diameter by angiography, CT angiography, Dopper Sono, etc. There is a problem that diagnosis is possible only after a structural change of the blood vessel (vascular change) is induced.

In addition, most cases of vascular disease complications occur in diabetes mellitus, and even though 50-80% of patients die from cardiovascular disease, a method for accurately predicting the progression of vascular disease complications in diabetics has not been developed yet.

The inventors of the present invention have made efforts to develop a method for more effectively diagnosing vascular endothelial dysfunction in order to more effectively manage and treat patients with vascular diseases. As a result, the inventors of vascular endothelial cells or subjects treated with plasma or serum of subjects We can confirm that vascular endothelial dysfunction can be diagnosed by measuring the expression levels of Rab5C, clathrin, caveolin-1, Early Endosome Antigen 1 (EAE1) and KCa3.1 channels in erythrocytes. The present invention was completed.

One object of the present invention is to provide a composition for diagnosing malfunction of vascular endothelial cells.

Another object of the present invention, to provide a kit for diagnosing dysfunction of vascular endothelial cells comprising the composition.

Still another object of the present invention is to provide a method for diagnosing vascular endothelial dysfunction.

Still another object of the present invention is to provide a method for screening a substance for improving the function of vascular endothelial cells.

Still another object of the present invention is to provide a method for screening a substance for inducing dysfunction of vascular endothelial cells.

Using the method provided by the present invention, the function of vascular endothelial cells using the expression levels of marker proteins such as Rab5C, clathrin, caveolin-1, and early endosome antigen 1 (EEA1). Since it is possible to diagnose whether there is a failure, it may be widely used for early diagnosis of various vascular diseases caused by dysfunction of vascular endothelial cells.

Figure 1 shows the results of comparing the expression level of KCa3.1 channel protein in umbilical vascular endothelial cells of normal mothers and gestational hypertension patients. ** means P <0.01 compared to control (normal maternal tissue or vascular endothelial cells) (NP: normal mother, PE: gestational hypertension, EC: vascular endothelial cells).

Figure 1a is a micrograph and graph comparing the expression level of KCa3.1 channel protein by immunovascular staining in umbilical vein (left) and umbilical artery (right) of normal mother and gestational hypertension.

FIG. 1B is a photograph and a graph comparing or measuring the expression level of KCa3.1 channel protein by Western blot in umbilical vein tissue of normal mother and gestational hypertension patients.

Figure 1c is a photograph and a graph comparing or measuring the expression level of KCa3.1 channel protein in cultured umbilical vein endothelial cells by Western blot.

Figure 1d is a graph showing the results of measuring the KCa3.1 current in vascular endothelial cells cultured in the umbilical vein of normal mother and gestational hypertension patients.

Figure 2 shows the results of analyzing the effect of plasma or serum on the expression level of KCa3.1 channel protein of gestational hypertension patients (NP: normal mother, PE: gestational hypertension patients).

Figure 2a is a photograph and a graph showing the effect of gestational plasma or serum on the expression level of vascular endothelial KCa3.1 channel and the effect of antioxidant on it.

Figure 2b is a photograph showing the result of comparing the protein expression level of NOX2 (NADPH oxidase 2) in the plasma or serum of normal mothers and gestational hypertension patients.

FIG. 2C is a photograph and graph showing the effect of anti-LOX1 antibody on increasing protein expression level of NOX2 and decreasing protein expression level of KCa3.1 channel by gestational hypertension patients plasma or serum (LOX1: lectin-lik ox-LDL receptor-1, tempol and tiron: antioxidants). In this case, ** means P <0.01 compared to the control group (normal maternal plasma or serum-treated vascular endothelial cells), and # is a group treated with plasma or serum of gestational hypertension patients without LOX1 antibody. P <0.05 compared to

Figure 3a is a photograph and graph showing the results of analyzing the effect of oxidized low density lipid protein (oxidized low density-lipoprotein, oxLDL) on vascular endothelial cell KCa3.1 channel protein expression level. In this case, * and ** mean P <0.05 and P <0.01, respectively, compared to the control group (vascular endothelial cells not treated with vascular disease causing substances).

Figure 3b is a photograph and graph showing the results of analyzing the effect of hyperglycemia, a vascular disease causing substance, on the expression level of KCa3.1 channel protein of vascular endothelial cells. In this case, * and ** mean P <0.05 and P <0.01, respectively, compared to the control group (vascular endothelial cells not treated with vascular disease causing substances).

Figure 4 is a graph and photograph showing the results of analyzing the effect of ROS on the expression level of vascular endothelial KCa3.1 channel protein. 4 shows the effect of superoxide donor xantine and xantine oxidase mixture (Xantine and Xantine oxidase mixture, X / X0), and antioxidant (tempol, tiron) on the expression level of KCa3.1 channel protein . At this time, ** is P <0.01 compared to the control (vascular endothelial cells not treated with X / XO or antioxidant), and # and ## are blood vessels treated with X / XO (100 uM / 100 mU / ml). Compared with endothelial cells, P <0.05 and P <0.01, respectively.

5 shows the results confirming that caveolin-1 is involved in the degradation of KCa3.1 channel protein in vascular endothelial cells (NP: normal mother, PE: gestational hypertension patients).

5a is a photograph and a graph showing the effect of LPC, a vascular disease causing agent, on the protein expression level of vascular endothelial caveolin-1 and the effects of antioxidants on it.

Figure 5b is a photograph showing the effect of plasma or serum of gestational hypertension patients on the expression level of vascular endothelial caveolin-1 protein.

FIG. 5C is a photograph showing that the reduction of vascular endothelial KCa3.1 channel expression level by plasma or serum of gestational hypertension patients is induced by Cabolin-1. In this case, ** means P <0.01 compared to the control.

Figure 6 shows the results of analyzing the expression levels of clathrin, EEA1 (Early Endosome Antigen 1) according to the treatment of vascular disease causing agent.

Figure 6a is a photograph showing the results of observing the protein expression level of KCa3.1 channel, clathrin in the cell membrane of vascular endothelial cells treated with gestational hypertension patients plasma or serum.

Figure 6b is a photograph showing the results of observing the protein expression level of KCa3.1 channel, clathrin in the cell membrane of vascular endothelial cells treated with LPC.

Figure 6c is a photograph and a graph showing the result of comparing the expression level of EEA1 and KCa3.1 channel protein according to the concentration-specific treatment of X / X0.

Figure 7a is a photograph showing the results of comparing the expression level of Rab5C protein in vascular endothelial cells exposed to 10%, 25%, 100% concentration of gestational hypertension patients plasma or serum.

Figure 7b is a photograph showing the result of comparing the expression level of KCa3.1 channel protein in vascular endothelial cells exposed by diluting the plasma or serum of gestational hypertension patients to 10%, 25%, 100% concentration.

Figure 7c is a photograph showing the result of comparing the expression level of EEA1 protein in vascular endothelial cells exposed by diluting the plasma or serum of gestational hypertension patients to 10%, 25%, 100% concentration.

8 is a photograph and a graph showing the results of measuring the expression level of KCa3.1 channel and Rab5C protein using erythrocytes isolated from blood collected from normal mothers and gestational hypertension mothers (NP: normal mother, PE: gestational hypertension). patient).

9 is a photograph and graph showing the results of analyzing the effect of TBHP treatment on the expression level of KCa3.1 channel protein in vascular endothelial cells.

In order to achieve the above object, one embodiment of the present invention provides a composition for diagnosing dysfunction of vascular endothelial cells.

In one embodiment, the vascular endothelial dysfunction composition is composed of a group consisting of KCa3.1 channel and caveolin-1, Rab5C, clathrin, and Early Endosome Antigen 1 (EEA1). It may be a composition for diagnosing dysfunction of vascular endothelial cells, including an agent for measuring the level of mRNA of at least one protein or a gene thereof selected from the group.

Vascular endothelial dysfunction causes vascular disease, but there is no direct way to measure vascular endothelial dysfunction. Therefore, by using the composition for diagnosing vascular endothelial dysfunction of the present invention, vascular endothelial dysfunction can be diagnosed early, the course, the prognosis, and the treatment effect. For example, in the case of gestational hypertension, using the composition of the present disease prior to the development of hypertension, the diagnosis of vascular endothelial dysfunction and the possibility of the development of gestational hypertension can be diagnosed, and thus new prophylaxis and treatment can be developed. In the case of arteriosclerosis and diabetic-related vascular diseases, the diagnosis of vascular endothelial dysfunction using the composition of the present invention enables the diagnosis of vascular disease progression even before the symptoms of vascular disease appear, and thus the progress, prognosis, and treatment effect can be diagnosed Do.

The term “caveolin-1” of the present invention is a major component of the caveolae plasma membrane found in most cell types, and is an early step in integrin binding in the Ras-ERK pathway. It is associated with phase and cell cycle progression. The present inventors first identified the relationship between KCa3.1 channel and kaveolin-1 in vascular endothelial dysfunction.

The sequence information of the caveolin-1 can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, Cabolin-1 of the present invention is NCBI GenBank Acession NO. NM_001172897.1, NM_001243064.1, NM_031556.3 or NM_001135818.1, but is not limited thereto.

As used herein, the term “Rab5C” refers to a protein that plays a role in regulating membrane traffic by controlling fusion between early endosomes and plasma membranes as one of GTPases. . The present inventors first identified the relationship with Rab5C in vascular endothelial dysfunction caused by a vascular disease causing agent.

The Rab5C sequence information 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 Acession 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 having three branches composed of three heavy chains and three light chains. The three branches can interact to form a polyhedral lattice surrounding the vesicle. The present inventors first identified the relationship with clathrin in vascular endothelial dysfunction.

The sequence information of the clathrin can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, the clathrin of the present invention may be prepared by NCBI GenBank Acession NO. NM_001288653.1, NM_001003908.1, NM_019299.1 or XM_001136053.4, but is not limited thereto.

The term “Early Endosome Antigen 1” (EEA1) of the present invention is located in the early endosomes and plays an important role in endosomal trafficking. The present inventors first identified the relationship between EEA1 in vascular endothelial dysfunction.

The sequence information of EEA1 can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, EEA1 of the present invention is NCBI GenBank Acession NO. NM_003566.3, NM_001001932.3, NM_001108086.1 or XM_522610.5, but is not limited thereto.

In one embodiment of the present invention by treating the vascular endothelial cells (plasma or serum of gestational hypertension patients, oxLDL, LPC, hyperglycemia, etc.) in the vascular endothelial cells, protein expression levels of Cabolin-1, Clathrin, Rab5C and EEA1 As a result, it was confirmed that the expression level of KCa3.1 channel protein was decreased, whereas the protein expression levels of carveolin-1, clathrin, Rab5C and EEA1 were increased. This suggests that vascular endothelial dysfunction induced by vascular disease causing agents can be diagnosed by measuring the expression level of KCa3.1 channel, caveolin-1, clathrin, Rab5C or EEA1.

In another embodiment of the present invention, as a result of confirming the expression level of Rab5C and EEA1 protein according to the dilution of plasma or serum of gestational hypertension patients, it was confirmed that the expression level of Rab5C and EEA1 protein increased, about 1/10 It was confirmed that Rab5C and EEA1 protein expression levels were increased only by gestational hypertension inducing factor alone. In such a small amount, the expression level of the marker can be measured to diagnose vascular endothelial dysfunction, thereby predicting or early diagnosis of the possibility of vascular disease.

In the present invention, the subject means all animals including humans, and specifically, may be human, but is not limited thereto.

The term "vascular endothelial cell" of the present invention refers to one of the major cells that form the structure of the blood vessels, mainly serves to maintain vascular normality, such as expansion and contraction of blood vessels, proliferation and migration of vascular smooth muscle, thrombogenesis and lysis do.

The term “dysfunction of vascular endothelial cells” of the present invention means that vascular endothelial cells are not functioning, and thus vascular abnormalities are broken in connection with vasodilation and contraction, proliferation and migration of vascular smooth muscle, thrombogenesis and dissolution, and thereby vascular endothelial cells. Means the symptoms that cause the disease. The dysfunction of the vascular endothelial cells causes vascular wall damage and increased vascular contractility, through which vascular diseases occur.

In the present invention, the vascular disease caused by the dysfunction of the vascular endothelial cells is not particularly limited thereto. For example, the vascular disease may be hypertension, arteriosclerosis, diabetic vascular disease, or a combination thereof. Can be.

Since the vascular disease is caused by the dysfunction of vascular endothelial cells, the progress of vascular disease can be known according to the level of vascular endothelial dysfunction, and the vascular endothelial cells Dysfunction may also be caused. In addition, since dysfunction of the vascular endothelial cells can change the KCa3.1 channel expression level of the vascular endothelial cells, KCa3.1 channel expression level of the vascular endothelial cells may be affected by the substance causing the vascular disease. It may be.

In the present invention, as long as the vascular disease-causing substance can cause vascular endothelial dysfunction, cause the development of vascular disease, or change KCa3.1 channel expression level of vascular endothelial cells, Although not limited, it can be, for example, a substance that increases oxidative stress, and in another example, oxidized low-density lipoprotein, lysophosphatidylcholine (LPC), which can increase oxidative stress, Gestational hypertension (sFlt-1, endoglin), high blood sugar, superoxide anion donor (superoxide anion donor).

The term “diagnosis” of the present invention refers to a series of acts of identifying the presence or characteristic of a pathological condition.

For the purposes of the present invention, the diagnosis may be interpreted to mean the act of checking whether the vascular endothelial dysfunction is impaired, thereby predicting the onset of vascular disease due to the vascular endothelial dysfunction.

Meanwhile, the composition for diagnosing dysfunction of vascular endothelial cells of the present invention may further include an agent for measuring KCa3.1 channel protein or its mRNA level.

The term "KCa3.1 channel" (intermediate conductance calcium-activated potassium channel, subfamily N, member 4) refers to a voltage-independent potassium channel in the form of a heterotetramer that is activated by intracellular calcium. KCa3.1 channels of vascular endothelial cells induce hyperpolarization to relax blood vessels, while hyperpolarization increases intracellular Ca2 + influx and promotes NO production by eNOS, thereby relaxing blood vessels. When the expression of the channel is decreased, vascular endothelial dysfunction may be induced. By analyzing the expression level of the KCa3.1 channel protein or gene, it is possible to determine whether the vascular endothelial dysfunction is induced.

In one embodiment of the present invention, as a result of analyzing the expression level of KCa3.1 channel protein in the umbilical vein and umbilical artery tissue of normal maternal and gestational hypertension patients, the expression level of KCa3.1 channel protein decreases in gestational hypertension patients. Confirmed.

In another embodiment of the present invention, as a result of analyzing the effect of plasma or serum, oxLDL, LPC and hyperglycemia on vascular endothelial cells of normal maternal and gestational hypertension patients gestational hypertension patients, gestational It was confirmed that the expression level of KCa3.1 channel protein is reduced by plasma or serum, oxLDL, LPC and hyperglycemia of hypertensive patients. As a result, it was found that oxLDL and hyperglycemia, which are plasma or serum substances, abnormally reduce KCa3.1 channel expression of vascular endothelial cells in gestational hypertension and vascular complications of diabetes, thereby inducing vascular endothelial dysfunction.

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 mothers and gestational hypertension patients, KCa3.1 channel protein expression through oxidant treatment It was confirmed that the decrease, and recovered by the antioxidant. Through this, oxidative stress caused vascular endothelial dysfunction, and it was found that oxidative stress level can be measured by measuring the expression level of KCa3.1 channel protein.

On the other hand, KCa3.1 channel protein expression level was measured in normal vascular endothelial cells (HUVECs) exposed to plasma or serum of gestational hypertension patients diluted in various stages. Since dilution to about 1/10 diminishes the expression level, it was confirmed that measuring the expression level of the KCa3.1 channel can predict the onset or early diagnosis of vascular disease.

The term "mRNA level measurement" of the present invention is a method of confirming the expression level of genes for diagnosing vascular endothelial dysfunction in isolated cells for diagnosing vascular endothelial dysfunction. Means the way. Specific methods for measuring the mRNA level is not particularly limited, but as an example, reverse transcriptase (RT-PCR), competitive reverse transcriptase (Competitive RT-PCR), real-time reverse transcriptase (Real- time RT-PCR), RNase protection assay (RPA), Northern blotting, DNA chips and the like can be used.

The agent used to measure the mRNA level is specifically a primer pair, a probe or an antisense oligonucleotide, and since the nucleic acid information of the genes is known to GeneBank, etc., those skilled in the art can specifically detect specific regions of these genes based on the sequence. Primer pairs, probes or antisense oligonucleotides can be designed.

Specifically, for diagnosing dysfunction of vascular endothelial cells, the agent for measuring the mRNA level is a primer pair, a probe specifically binding to a gene encoding a protein selected from Rab5C, clathrin, caveolin-1 and EEA1 Or antisense oligonucleotides.

The term “primer pair” of the present invention is not particularly limited as long as it can recognize and amplify a gene sequence encoding KCa3.1 channel, Rab5C, clathrin, caveolin-1 or EEA1 provided herein. As an example, it may be a primer pair of any combination consisting of forward and reverse primers capable of binding to each gene, and as another example, may be a primer pair capable of binding with specificity and sensitivity to each gene. have. For example, the primer consists of a sequence whose nucleic acid sequence is inconsistent with the non-target sequence present in the sample, thus amplifying only the target gene sequence containing the complementary primer binding site and not causing nonspecific amplification. It may be a primer pair having.

The term "probe" of the present invention refers to a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample through the binding. Component of the probe is not particularly limited as long as it can specifically bind to the target material, for example, peptide nucleic acid (PNA), locked nucleic acid (LNA), peptides, polypeptides, proteins, RNA, DNA and the like, and as other examples, DNA such as cDNA, genomic DNA, etc .; RNA such as genomic RNA, mRNA and the like; And proteins such as antibodies, antigens, enzymes, peptides, and the like.

As used herein, the term “antisense oligonucleotide” refers to a DNA or RNA or derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence in the mRNA and inhibits translation of the mRNA into a protein. Do it. Antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of said genes and capable of binding to said mRNA. This may inhibit the essential activity of the translation of the gene mRNA, translocation into the cytoplasm, maturation or any other overall biological function. 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 in a conventional manner to be administered in vivo or to allow the antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using 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 in the opposite direction of the multicloning site (MCS). The antisense RNA is preferably such that the translation stop codon is present in the sequence so that it is not translated into the peptide sequence.

The term "measurement of protein levels" of the present invention refers to the KCa3.1 channel, Rab5C, clathrin, caveolin-1, or EEA1, a marker protein for diagnosing vascular endothelial dysfunction in isolated cells for diagnosing vascular endothelial dysfunction. It means the process of checking the presence and the degree of expression.

The method for measuring the protein level is as long as it can measure the expression level of the marker protein KCa3.1 channel, Rab5C, clathrin, caveolin-1 or EEA1, which is a diagnostic marker protein for vascular endothelial dysfunction provided by the present invention. Examples include, but are not limited to, protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and Surface Enhanced Laser Desorption / Ionization Time of Flight (SELDI-TOF). Mass Spectrometry Analysis, Radioimmunoassay, Radioimmunoassay, Oukteroni Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complementary Assay, Two-Dimensional Electrophoresis, Liquid Chromatography-Mass Spectrometry, LC-MS), liquid chromatography-mass spectrometry / mass spectrometry, western blot, and enzyme linked immunosorbentassay (ELISA).

In the present invention, the method for measuring the protein level, KCa3.1 channel, Rab5C, clathrin, caveolin-1 or a marker protein for diagnosing vascular endothelial dysfunction, to diagnose vascular endothelial dysfunction It can be interpreted by using an antibody that specifically binds to EEA1.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site.

In the present invention, the antibody may be interpreted to mean an antibody that specifically binds to a KCa3.1 channel, Rab5C, clathrin, caveolin-1, or EEA1 protein, which is a marker protein for diagnosing vascular endothelial dysfunction. .

The antibody may include all polyclonal antibodies, monoclonal antibodies, recombinant antibodies and the like, and may include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. have. The functional fragment of the antibody molecule means a fragment having at least antigen binding function, and examples thereof include Fab, F (ab '), F (ab') 2, and Fv.

In another aspect, the present invention provides a kit for diagnosing dysfunction of vascular endothelial cells, comprising the composition for diagnosing dysfunction of vascular endothelial cells.

The kit for diagnosing dysfunction of vascular endothelial cells provided by the present invention is not particularly limited thereto. For example, a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, Protein chip kits, rapid kits, multiple reaction monitoring (MRM) kits, and the like.

The kit for diagnosing dysfunction of vascular endothelial cells may further include one or more other component compositions, solutions or devices suitable for analytical methods.

As an example, the 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. A primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, the length of about 7 bp to 50 bp, more specifically 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- It may include DEPC-water, sterile water, and the like.

As another example, the kit may be a diagnostic kit, which includes an essential element necessary to perform a 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, a preparation, 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.

As another example, the kit may be a diagnostic kit characterized by including essential elements necessary to perform an ELISA. ELISA kits include antibodies specific for the KCa3.1 channel, Rab5C, clathrin, caveolin-1 and EEA1 proteins. 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.

As another example, the kit may be a rapid kit, characterized in that it contains the essential elements necessary to perform a rapid test that can be analyzed within 5 minutes. Rapid kits include antibodies specific for a 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. Rapid kits may also include antibodies specific for the control protein. Other rapid kits include reagents that can detect bound antibodies, such as nitrocellulose membranes to which specific and secondary antibodies are immobilized, membranes bound to beads to which antibodies are bound, absorbent pads, and sample pads. Substances and the like.

As another example, the kit may be a multiple reaction monitoring (MRM) kit, which is an MS / MS mode characterized by including the necessary elements necessary to perform mass spectrometry. Whereas Selected Ion Monitoring (SIM) is a method of using ions generated by collisions once in the source portion of the mass spectrometer, MRM selects a particular ion one more time from the broken ion to detect another continuously connected source of MS. It is a method of using the ions obtained from the collision after passing through it once more. More specifically, in SIM, there is a problem in that the selected ions may interfere with quantification when the selected ions are ions that are also detected in plasma. On the other hand, in case of using MRM, even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. Can be obtained. Thus, by using the MRM mode in mass spectrometry it is possible to simultaneously analyze the desired materials with better analytical sensitivity. Through the multiple reaction monitoring (MRM) analysis method, it is possible to compare protein expression level in normal control group and vascular endothelial dysfunction in patients with vascular disease caused by vascular endothelial dysfunction. In addition, it is possible to diagnose whether the vascular endothelial dysfunction is determined by determining whether the expression level of the vascular endothelial dysfunction diagnostic marker gene is significantly increased or decreased.

In another aspect, the present invention provides a method for diagnosing malfunction of vascular endothelial cells.

The Rab5C, clathrin, caveolin-1, EEA1 and KCa3.1 channels are characterized by a change in the expression level of the gene or the level of the protein in an individual with dysfunction of vascular endothelial cells. If the vascular endothelial dysfunction can be diagnosed, it can be diagnosed early on the development of vascular disease or the progress of the vascular disease.

As one example, the method for diagnosing vascular endothelial dysfunction of the present invention includes (a) Rab5C, clathrin, caveolin-1, EEA1, and the like in biological samples of individuals suspected of vascular endothelial dysfunction. Measuring protein or mRNA levels of a marker selected from the group consisting of a combination of; And (b) comparing the measured level (i) with the level measured from a normal control sample.

In this method, the protein or mRNA level measurement is the same as defined above.

The term "biological sample" of the present invention refers to a sample collected from the individual for diagnosing vascular endothelial dysfunction of a suspected vascular endothelial dysfunction, particularly as long as the vascular endothelial dysfunction can be confirmed. The present invention may be, but is not limited to, whole blood, plasma, serum, fractions thereof, or cells contained therein, and red blood cells.

In addition, the method may further comprise the step (a) measuring the protein or mRNA level of (ii) KCa3.1 channel.

In addition, the method measures the protein or mRNA level of (ii) KCa3.1 channel in step (a), and using the measured (i) and (ii) calculated by the formula of (i) / (ii) Comparing the calculated rate to the rate calculated from the normal control sample.

In the method, (i) Rab5C, clathrin, caveolin-1 or EEA1 protein or mRNA levels in biological samples of individuals suspected of vascular endothelial dysfunction are relatively higher than those measured from normal control samples. Levels, or (ii) if the protein or mRNA levels of the KCa3.1 channel are relatively lower than those measured from normal control samples, the subject may be determined to have vascular endothelial dysfunction. have.

In particular, when the ratio of (i) / (ii) calculated from the subject indicates a relatively higher level than that calculated from the normal control sample, it may be determined that dysfunction of vascular endothelial cells appeared in the subject.

In one embodiment of the present invention, as a result of confirming the expression level of KCa3.1 channel and Rab5C protein in red blood cells, it was confirmed that the expression level of KCa3.1 channel protein is decreased and the expression level of Rab5C protein is increased. Through this, the expression levels of KCa3.1 channel protein and Rab5C protein were measured in erythrocytes, indicating that vascular endothelial dysfunction could be diagnosed.

As another example, the method for diagnosing dysfunction of vascular endothelial cells of the present invention may comprise the steps of: (a) treating a biological sample of a subject suspected of dysfunction of vascular endothelial cells to normal vascular endothelial cells; (b) protein or mRNA levels of markers selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in vascular endothelial cells treated with the biological sample of step (a) Measuring; And (c) comparing said measured level (i) with a level measured from normal vascular endothelial cells to which said biological sample has not been treated.

In this method, the protein or mRNA level measurement is the same as defined above.

In addition, the method may further comprise the step of (ii) (ii) measuring the protein or mRNA level of the KCa3.1 channel.

In addition, the method measures the protein or mRNA level of (ii) KCa3.1 channel in step (b), and using the measured (i) and (ii) calculated by the formula of (i) / (ii) Comparing the calculated rate to a rate calculated from normal vascular endothelial cells to which the biological sample has not been treated.

In the above method, (i) the protein or mRNA level of Rab5C, clathrin, caveolin-1, or EEA1 measured in vascular endothelial cells treated with a biological sample of an individual suspected of vascular endothelial dysfunction is the biological The sample exhibited a relatively higher level than that measured from untreated normal vascular endothelial cells, or (ii) the protein or mRNA levels of the KCa3.1 channel were measured from normal vascular endothelial cells not treated with the biological sample. If the level is relatively low, it may be determined that dysfunction of vascular endothelial cells has occurred in the subject.

In particular, if the ratio of (i) / (ii) calculated from treated vascular endothelial cells of the subject is relatively higher than that derived from normal vascular endothelial cells without treatment It may be determined that dysfunction of vascular endothelial cells has appeared in the subject.

In another aspect, the present invention provides a method for screening a substance for inducing a function of vascular endothelial cells.

As described above, when vascular endothelial dysfunction occurs, the expression levels of the Rab5C, clathrin, caveolin-1, EEA1 and KCa3.1 channels change, and thus Rab5C, clathrin, caveolin-1 In the case where the changed expression levels of the EEA1 and KCa3.1 channels are restored to normal levels, it can be predicted that vascular endothelial dysfunction is resolved.

Therefore, a candidate substance predicted to induce improvement of vascular endothelial cells is treated to vascular endothelial cells in which dysfunction has occurred, and a failure marker of vascular endothelial cells expressed in the vascular endothelial cells to which the candidate substance has been treated. By measuring the expression level of the Rab5C, clathrin, caveolin-1, EEA1 and KCa3.1 channels, and then confirming whether the expression level of each marker measured is restored to normal levels, the candidate substance is endothelium It can be determined whether it is possible to induce improvement of the function of the cell.

Specifically, the method for screening a function improving inducer of vascular endothelial cells of the present invention comprises the steps of: (a) treating candidate cells expected to be capable of inducing functional improvement of vascular endothelial cells to separated cells; (b) measuring the protein or mRNA levels of a marker selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in cells treated with the candidate substance; And (c) comparing the level of the marker measured in step (b) with the level of the marker measured in isolated cells not treated with the candidate substance.

In the present invention, the candidate material refers to a substance capable of regulating dysfunction of vascular endothelial cells, for example, to restore vascular endothelial cells in which dysfunction has occurred to a normal state, or It may be a substance that is expected to exhibit or induce an effect that can alleviate, ameliorate, or treat dysfunction, and as another example, may cause dysfunction from vascular endothelial cells or prevent dysfunction of vascular endothelial cells. It may be a substance that is expected to be inducible.

The candidate is not particularly limited as long as it is expected to have an effect of regulating dysfunction of vascular endothelial cells, but as an example, any substance, molecule, element, compound ), Entities, etc., and may be other examples, such as natural products, synthetic compounds, and the like, and as another example, proteins, polypeptides, small organic molecules, polysaccharides, and the like. (polysaccharide), polynucleotide and the like.

In the present invention, the isolated cell is not particularly limited as long as it can be used as a target for confirming whether or not it can induce improvement of vascular endothelial cell function of the candidate, but as an example, vascular endothelial cell function Cells isolated from the subject causing the failure, as another example, cells isolated from the blood or blood vessels of the subject causing vascular endothelial dysfunction, and as another example, vascular endothelial dysfunction Erythrocytes isolated from the blood of the induced individual or vascular endothelial cells isolated from the blood vessels of the individual.

In addition, the method may further comprise the step of (ii) (ii) measuring the protein or mRNA level of the KCa3.1 channel.

In addition, the method measures the protein or mRNA level of (ii) KCa3.1 channel in step (b), and using the measured (i) and (ii) calculated by the formula of (i) / (ii) Comparing the proportions calculated to the proportions calculated from cells in which the candidate material has not been treated.

In the method, (i) protein or mRNA levels of Rab5C, clathrin, caveolin-1 or EEA1 measured in cells treated with the candidate are relatively higher than those measured from cells not treated with the candidate. The candidate is a function of vascular endothelial cells if the protein or mRNA level of the KCa3.1 channel is at a lower level than that measured from cells not treated with the candidate. It can be determined that improvement can be induced.

In particular, when the ratio of (i) / (ii) calculated in the cells treated with the candidate material is relatively lower than that calculated from the cells in which the candidate material is not treated, the candidate material is vascular endothelial. It can be determined that the improvement of the function of the cell can be induced.

In one embodiment of the present invention, when treated with TBHP, a vascular endothelial function inducing substance, the expression level of KCa3.1 channel protein was confirmed to increase significantly in concentration. It is known that an increase in KCa3.1 channel expression levels can improve vascular endothelial function (Chadha PS et al, 2010, J Pharmacol Exp Ther, 335 (2), 284-293). After treating candidate substances that are expected to induce improvement, the protein expression levels of KCa3.1 channels are measured, suggesting that the candidates can determine whether they can induce improvement of vascular endothelial function. will be.

In another aspect, the present invention provides a method for screening a substance for inducing dysfunction of vascular endothelial cells.

As described above, when vascular endothelial dysfunction occurs, the expression levels of the Rab5C, clathrin, caveolin-1, EEA1 and KCa3.1 channels are changed, which may induce improvement of vascular endothelial cells. Candidates predicted to be treated are treated with vascular endothelial cells, and the candidate substances are treated with Rab5C, clathrin, caveolin-1, EEA1, and KCa3.1 channels, which are dysfunction markers of vascular endothelial cells. After measuring the expression level, it is possible to determine whether the candidate substance can induce dysfunction of vascular endothelial cells by checking whether the expression level of each measured marker is changed.

Specifically, the method of screening a substance for inducing dysfunction of vascular endothelial cells of the present invention comprises the steps of: (a) treating a candidate material that is expected to induce dysfunction of vascular endothelial cells to an isolated cell; (b) measuring the protein or mRNA levels of a marker selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in cells treated with the candidate substance; And (c) comparing the level of the marker measured in step (b) with the level of the marker measured in isolated cells not treated with the candidate substance.

In the present invention, the candidates and the isolated cells are the same as defined above.

In addition, the method may further comprise the step of (ii) (ii) measuring the protein or mRNA level of the KCa3.1 channel.

In addition, the method measures the protein or mRNA level of (ii) KCa3.1 channel in step (b), and using the measured (i) and (ii) calculated by the formula of (i) / (ii) Comparing the proportions calculated to the proportions calculated from cells in which the candidate material has not been treated.

In the method, (i) protein or mRNA levels of Rab5C, clathrin, caveolin-1 or EEA1 measured in cells treated with the candidate are relatively higher than those measured from cells not treated with the candidate. The candidate material functions as a vascular endothelial cell if the protein or mRNA level of the KCa3.1 channel is relatively lower than that measured from cells not treated with the candidate material. It can be determined that failure can be induced.

In particular, when the ratio of (i) / (ii) calculated in the cells treated with the candidate material is relatively higher than that calculated from the cells in which the candidate material is not treated, the candidate material is vascular endothelium. It can be determined that the cell can be inferior in function.

In one embodiment of the present invention, when treated with vascular disease-causing LPC or X / XO to normal vascular endothelial cells, KCa3.1 channel protein is reduced and the expression level of the caveolin-1, clathrin, EEA1 protein is increased It was. Through this process, after treating a candidate substance expected to induce vascular endothelial dysfunction in normal vascular endothelial cells, the expression level of the protein is measured to determine whether the candidate substance can induce vascular endothelial dysfunction. It can be seen that judging.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1: Endothelial Cells in Patients with Vascular Disease KCa3 . 1 channel  Protein expression level analysis

To determine whether KCa3.1 channel expression decreases in vascular endothelial cells of patients with vascular disease, the expression levels of KCa3.1 channel proteins in vascular endothelial cells of normal mothers and gestational hypertension patients were compared.

KCa3.1 channel protein expression levels in umbilical veins and umbilical artery tissues were measured by histological analysis (A in FIG. 1), Western blot (B in FIG. 1), and vascular endothelial cells in umbilical veins in normal maternal and gestational hypertension. The isolation culture was performed to measure the expression level of KCa3.1 channel protein (C in FIG. 1) and the magnitude of KCa3.1 current (D in FIG. 1). To measure KCa3.1 channel protein expression levels, antibodies having the amino acid sequence of RQVRLKHRKLREQV (SEQ ID NO: 1) were used.

As a result, the protein expression level of vascular endothelial KCa3.1 channel was significantly decreased and the magnitude of KCa3.1 current was decreased in gestational hypertension patients.

Therefore, it was confirmed that when gestational hypertension develops, vascular endothelial dysfunction is induced by abnormally decreasing the expression level of KCa3.1 channel of vascular endothelial cells.

Example  2: vascular endothelial cells caused by vascular disease causing substances KCa3 . 1 channel  Protein expression level analysis

To determine if the decrease in KCa3.1 channel expression in vascular endothelial cells of patients with vascular disease is caused by vascular disease causing agents in plasma or serum, plasma or serum, oxLDL, LPC and hyperglycemia of normal maternal and gestational hypertension patients The effect on the expression level of KCa3.1 channel in endothelial cells was analyzed.

As a result of the experiment, compared with vascular endothelial cells exposed to normal maternal plasma or serum, the expression level of NOX2 protein that produces ROS in plasma or vascular endothelial cells exposed to gestational hypertension increased and the expression of KCa3.1 channel was increased. Protein expression levels were found to be markedly reduced (FIG. 2B). Reduction of KCa3.1 channel expression levels in exposed vascular endothelial cells of gestational hypertension patients plasma or serum was inhibited by antioxidants (A in FIG. 2) or antibodies against LOX-1 (C in FIG. 2). In other words, the plasma or serum of gestational hypertension increased the expression level of NOX2 protein through LOX-1, increased ROS production and decreased KCa3.1 channel protein expression level, and major vascular disease of plasma or serum of gestational hypertension patients. It was found that the trigger was oxLDL. In addition, the expression level of KCa3.1 channel protein in vascular endothelial cells also decreased by oxLDL or hyperglycemia (FIG. 3).

Therefore, it was confirmed that oxLDL and hyperglycemia, which are plasma or serum substances, abnormally reduce KCa3.1 channel expression levels of vascular endothelial cells in gestational hypertension and vascular complications of diabetes mellitus, thereby inducing vascular endothelial dysfunction.

Example  3: oxidative stress in vascular endothelial cells KCa  3.1 Expression level analysis of channels

To investigate the effect of oxidative stress on KCa3.1 channel expression, superoxide anion donors, oxidizing agents, were observed in vascular endothelial cells of normal mothers and vascular endothelial cells exposed to plasma or serum of gestational hypertension. The effect on KCa3.1 channel protein was measured using Western blot. In each sample, the expression level of α-tubulin or β-actin was measured as a control.

As a result of the experiment, KCa3.1 channel protein expression level was decreased by oxidizing agent (X / XO) treatment, and KCa3.1 channel protein expression level was decreased by oxidizing agent (FIG. 4).

Therefore, in view of the above results, it was confirmed that the decrease in the expression level of KCa3.1 channel protein in vascular endothelial cells was due to the degree of oxidative stress, especially superoxide anion, and the expression level of KCa3.1 channel was measured. It was confirmed that the level of oxidative stress applied to the cells, in particular, superoxide anion.

Through this, it was found that the oxidative stress caused by the vascular disease-causing factor decreases the expression level of the KCa3.1 channel and causes dysfunction of vascular endothelial cells.

Example  4: vascular endothelial cells following treatment with vascular disease causing agent caveolin Analysis of Expression Levels of --1, clathrin, Rab-5C, and EEA1 Protein

After treating vascular endothelial cells to normal vascular endothelial cells, Western blot was used to measure the expression levels of caveolin-1, clathrin, Rab-5C, EEA1 and KCa3.1 channel proteins (FIGS. 5 and 6). To determine Caveolin-1, clathrin, Rab5C, and EEA1 protein expression levels, MADELSEKQVYDAHTKEID (SEQ ID NO: 2), PQLMLTAGPSVAVPPQAPFGYGYTAPPYGQPQPGFGYS (SEQ ID NO: 3), ASRGATRPNGPNT (SEQ ID NO: 4), FCASKKKNA having the sequence SEQ ID NO: 4) Antibodies were used.

As a result, when the plasma or serum treatment of LPC or gestational hypertension patients, the expression level of caveolin-1 protein was increased, it was confirmed that caveolin-1 and KCa3.1 channel protein together by immunoprecipitation method (Fig. 5) C). In addition, KCa3.1 channel protein was reduced by the vascular disease-causing agent, and clathrin and EEA1 protein expression levels were increased (FIG. 6C and 7). Meanwhile, KCa3.1 decreased and clathrin increased due to vascular disease-causing agents in cell membranes (A and B in Figure 6).

Therefore, in view of the above results, it was found that caveolin-1, clathrin, EEA1 protein is increased by the vascular disease causing agent.

This suggests that vascular endothelial dysfunction induced by vascular disease causing agent can be diagnosed by measuring the expression level of KCa3.1 channel or caveolin-1, clathrin, EEA1.

In addition, after treating the candidate substance expected to induce vascular endothelial dysfunction in normal vascular endothelial cells, and measuring the protein expression level of KCa3.1 channel or caveolin-1, clathrin, EEA1 candidate It suggests that the substance can determine whether it can induce vascular endothelial dysfunction.

Example  5: Analysis of KCa3.1 channel, Rab5C, EEA1 protein expression levels in vascular endothelial cells according to the plasma or serum dilution of gestational hypertension

To analyze the effect of plasma or serum dilution on the expression levels of KCa3.1 channel protein and Rab5C and EEA1 protein, which causes decreased expression levels of gestational hypertension treated with human vascular endothelial cells, Alternatively, the serum was mixed with the culture medium, and the human vascular endothelial cells incubated in a solution containing plasma or serum concentrations of 10, 25, 100% (without dilution) were exposed for 24 hours, followed by Western blot using KCa3. The expression level of Rab5C protein and the expression level of EEA1 protein were measured by the expression level of .1 channel protein, small GTPase. In each sample, the expression level of β-actin was measured as a control.

As a result, the expression level of KCa3.1 channel protein was decreased but Rab5C and EEA1 protein expression levels were increased in proportion to the degree of dilution in vascular endothelial cells exposed to plasma or serum of gestational hypertension patients (FIG. 7).

Therefore, in the light of the above results, the expression level of Rab5C and EEA1 protein was increased by only about 1/10 of gestational hypertension-inducing factor and vascular endothelial cell KCa3.1 channel protein expression compared to the case where gestational hypertension was fully expressed. It was confirmed that it causes a decrease in levels.

By measuring the expression level of KCa3.1 channel, Rab5C, and EEA1 protein in the stage before the onset of disease, the concentration of factors causing the vascular endothelial dysfunction is low. As can be seen, it can be seen that by measuring the expression level of the marker, it is possible to predict or diagnose the possibility of vascular disease.

Example  6: in red blood cells KCa3 . 1 channel Rab5C  Protein expression level analysis

To determine whether the expression of KCa3.1 channels, Rab5C and clathrin in vascular endothelial cells and erythrocytes is the same in patients with vascular endothelial dysfunction, KCa3. Expression levels of one channel, Rab5C and clathrin protein were measured. In each sample, the expression level of β-actin was measured as a control.

As a result, the expression level of KCa3.1 channel protein was decreased and the expression level of Rab5C and clathrin protein was increased in the erythrocytes of gestational hypertension patients compared with the control group (normal maternal red blood cells). 8).

Therefore, in view of the above results, it was confirmed that the expression level of vascular endothelial KCa3.1 channel protein in gestational hypertension patients can be determined by measuring KCa3.1 channel protein or Rab5C, clathrin, etc. in erythrocytes.

Example  7: TBHP  Endothelial Cells Following Treatment KCa3 . 1 channel  Expression level analysis of proteins

After vascular endothelial cells were treated with TBHP, a substance for improving blood vessel function, Western blot was used to measure the expression level of KCa3.1 channel.

As a result, it was confirmed that KCa3.1 channel expression significantly increased in concentration by TBHP treatment (FIG. 9).

It is known that an increase in KCa3.1 channel expression levels can improve vascular endothelial function (Chadha PS et al, 2010, J Pharmacol Exp Ther, 335 (2), 284-293). After treating candidate vascular endothelial cells that are expected to induce improvement, the protein expression level of KCa3.1 channel can be measured to determine whether the candidate substance can induce improvement of vascular endothelial function. Is to suggest.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (23)

(a) Rab5C, clathrin, caveolin-1, Early Endosome Antigen 1 (EEA1), and combinations thereof, in biological samples of individuals suspected of vascular endothelial dysfunction. Measuring protein or mRNA levels of a marker selected from the group consisting of; And (b) comparing the measured level (i) with a level measured from a normal control sample. (a) treating a normal vascular endothelial cell with a biological sample of a subject suspected of dysfunction of vascular endothelial cells; (b) protein or mRNA levels of markers selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in vascular endothelial cells treated with the biological sample of step (a) Measuring; And (c) comparing said measured level (i) with a level measured from normal vascular endothelial cells to which said biological sample has not been treated. The method according to claim 1 or 2, Wherein said biological sample is a sample selected from the group consisting of whole blood, plasma, serum, erythrocytes, and combinations thereof isolated from individuals suspected of dysfunction of vascular endothelial cells. The method according to claim 1 or 2, The mRNA level measurement of the marker is performed using a method selected from the group consisting of reverse transcriptase, competitive reverse transcriptase, real time reverse transcriptase, RNase protection assay, northern blotting and DNA chip assay. , Way. The method according to claim 1 or 2, The protein level measurement of the marker is performed using an antibody that specifically binds to the marker protein. The method according to claim 1 or 2, Protein level measurement of the markers can be performed by protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry Analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry, And LC-MS), LC-MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blot and ELISA (enzyme linked immunosorbentassay). The method according to claim 1 or 2, The dysfunction of the vascular endothelial cells will cause the development of atherosclerosis, hypertension or diabetes-related vascular disease. The method of claim 1, In the step (a) (ii) further comprising the step of measuring the protein or mRNA level of the KCa3.1 channel. The method of claim 1, In step (a), (ii) the protein or mRNA level of the KCa3.1 channel was measured, and the ratio calculated by the following formula using the measured (i) and (ii) was calculated from the normal control sample. Further comprising comparing with; Ratio = (i) / (ii). The method of claim 2, In step (b) (ii) further comprising the step of measuring the protein or mRNA level of the KCa3.1 channel. The method of claim 2, In step (b), (ii) the protein or mRNA level of the KCa3.1 channel was measured, and the ratio calculated by the following formula using the measured (i) and (ii) was determined in the following manner. Further comprising comparing with a ratio calculated from vascular endothelial cells; Ratio = (i) / (ii). Comprising an agent that measures the protein or mRNA levels of a marker selected from the group consisting of Rab5C, clathrin, caveolin-1, Early Endosome Antigen 1 (EEA1), and combinations thereof A composition for diagnosing malfunction of vascular endothelial cells. The method of claim 12, Wherein the composition further comprises an agent for measuring protein or mRNA levels of KCa3.1 channels. The method according to claim 12 or 13, The agent measuring the protein level is an antibody that specifically binds to the protein. The method according to claim 12 or 13, The agent measuring the mRNA level is a primer pair, probe or antisense oligonucleotide that specifically binds to cDNA derived from the mRNA. A kit for diagnosing malfunction of vascular endothelial cells, comprising the composition of claim 12 or 13. The method of claim 16, The kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, DNA chip kit, Enzyme-linked immunosorbent assay (ELISA) kit, protein chip kit, rapid kit or multiple reaction monitoring (MRM) kit, Kit. (a) treating the isolated cells with a candidate substance which is expected to induce an improvement in the function of vascular endothelial cells; (b) measuring the protein or mRNA levels of a marker selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in cells treated with the candidate substance; And (c) comparing the level of the marker measured in step (b) with the level of the marker measured in the isolated cells not treated with the candidate material, the method for screening a substance for improving the function of vascular endothelial cells. The method of claim 18, In step (b) (ii) further comprising the step of measuring the protein or mRNA level of the KCa3.1 channel. The method of claim 18, In step (b), (ii) the protein or mRNA level of the KCa3.1 channel was measured, and the ratio calculated by the following formula using the measured (i) and (ii) was determined. Further comprising comparing with a ratio calculated from the cells; Ratio = (i) / (ii). (a) treating the isolated cells with candidate substances that are expected to induce dysfunction of vascular endothelial cells; (b) measuring the protein or mRNA levels of a marker selected from the group consisting of (i) Rab5C, clathrin, caveolin-1, EEA1, and combinations thereof in cells treated with the candidate substance; And (c) comparing the level of the marker measured in step (b) with the level of the marker measured in the isolated cells not treated with the candidate substance, the method for screening the insufficiency inducing substance of vascular endothelial cells. The method of claim 21, In step (b) (ii) further comprising the step of measuring the protein or mRNA level of the KCa3.1 channel. The method of claim 21, In step (b), (ii) the protein or mRNA level of the KCa3.1 channel was measured, and the ratio calculated by the following formula using the measured (i) and (ii) was determined. Further comprising comparing with a ratio calculated from the cells; Ratio = (i) / (ii).

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