KR102312534B1 - A method for diagnosing delayed ischemia using a cerebrospinal fluid-derived mitochondrial marker - Google Patents

Abstract

The present invention provides a composition for diagnosing delayed ischemia comprising an agent capable of detecting von Willebrand factor (vWF)-positive mitochondria, a kit for diagnosing delayed ischemia comprising the composition, and diagnosis of delayed ischemia using the composition and kit It relates to a method of providing information for
According to the composition for diagnosing delayed ischemia, the kit for diagnosing delayed ischemia, and the method for providing information for diagnosing delayed ischemia of the present invention, by analyzing the characteristics of extracellular mitochondria in cerebrospinal fluid, vWF (von Willebrand factor)-positive mitochondria and GLAST ( By measuring the level of glutamate aspartate transporter)-positive mitochondria, it can be used to predict or diagnose the risk of developing delayed ischemia after subarachnoid hemorrhage.

Description

{A method for diagnosing delayed ischemia using a cerebrospinal fluid-derived mitochondrial marker}

The present invention provides a composition for diagnosing delayed ischemia comprising an agent capable of detecting von Willebrand factor (vWF)-positive mitochondria, a kit for diagnosing delayed ischemia comprising the composition, and diagnosis of delayed ischemia using the composition and kit It relates to a method of providing information for

Subarachnoid hemorrhage (SAH) due to rupture of a cerebral aneurysm is a life-threatening disease with a mortality rate of 45% within 30 days of onset. In addition, delayed cerebral ischemia (DCI) may occur within 30 to 40% of subarachnoid hemorrhage after subarachnoid hemorrhage, which is associated with a major cause of mortality. Patients with subarachnoid hemorrhage with delayed ischemia are more likely to have various complications such as pneumonia, cardiac arrhythmias, intestinal bleeding and urinary tract infection compared to patients without delayed ischemia, which leads to prolonged hospitalization and medical costs. increase, etc. The onset of this delayed ischemia is influenced by various clinical and radiological parameters. Well-known clinical risk factors for delayed ischemia are female sex, smoking, and low clinical grade at hospitalization. A high Hunt-Hess-grade (grade IV and V) or World Federation of Neurosurgical Societies (WFNS) grade increases the likelihood of developing delayed ischemia.

In relation to the onset of delayed ischemia, various genetic studies such as single nucleotide polymorphisms (SNP) or genome-wide association studies (GWAS) are being conducted. However, only 5 to 10% of the susceptibility genetic variants were observed, suggesting that there are still unknown various genetic variants beyond the DNA sequence scale.

Accordingly, the present inventors tried to develop an efficient and easy method for diagnosing the onset of delayed ischemia, predicting the onset risk, and predicting the prognosis after subarachnoid hemorrhage. The present invention was completed by developing a diagnostic composition capable of diagnosing or predicting

Korean Patent Publication No. 10-2019-0008216

An object of the present application is to provide a composition for diagnosing delayed ischemia, a kit for diagnosis, and a method for providing information for diagnosis, including an agent capable of detecting von Willebrand factor (vWF)-positive mitochondria.

The objects of the present invention are not limited to the above-mentioned objects, and other objects and advantages of the present invention not mentioned may be understood by the following description, and will be more clearly understood by the embodiments of the present invention. Moreover, it will be readily apparent that the objects and advantages of the present invention may be realized by the means and combinations thereof indicated in the claims.

A first aspect of the present application is to provide a composition for diagnosing delayed ischemia, comprising an agent capable of detecting von Willebrand factor (vWF)-positive mitochondria.

A second aspect of the present application is to provide a kit for diagnosing delayed ischemia, comprising the composition for diagnosing delayed ischemia.

A third aspect of the present application is to provide an informational method for diagnosing delayed ischemia, comprising measuring the level of von Willebrand factor (vWF)-positive mitochondria from a biological sample isolated from a subject.

A fourth aspect of the present application is an information providing method for diagnosing delayed ischemia, comprising measuring the level of extracellular mitochondria from a biological sample isolated from a subject, wherein the biological sample isolated from the subject is derived from cerebrospinal fluid It is to provide an information providing method for the diagnosis of delayed ischemia.

According to the composition for diagnosing delayed ischemia, the kit for diagnosing delayed ischemia, and the method for providing information for diagnosing delayed ischemia of the present invention, by analyzing the characteristics of extracellular mitochondria in cerebrospinal fluid, vWF (von Willebrand factor)-positive mitochondria and GLAST ( By measuring the level of glutamate aspartate transporter)-positive mitochondria, it can be used to predict or diagnose the risk of developing delayed ischemia after subarachnoid hemorrhage.

1 is a view showing the results of observation of extracellular mitochondria present in the cerebrospinal fluid of a patient with subarachnoid hemorrhage with a transmission electron microscope. Black arrows in the figure indicate extracellular mitochondria.
2 is a view confirming the mitochondrial particle level in the extracellular cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia by flow cytometry.
3 is a diagram comparing the membrane potential level of extracellular mitochondria present in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia.
4 is a diagram comparing the membrane potential level of extracellular mitochondria by date in patients with delayed ischemia.
5 is a view showing the results of confirming the presence of vWF-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia using FACS.
6 is a diagram comparing the presence level of vWF-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia.
7 is a view showing the results of confirming the presence of GLAST-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia using FACS.
8 is a diagram comparing the presence level of GLAST-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia.
9 is a view showing the results of confirming the presence of CD45-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia using FACS.
10 is a view showing the results of confirming the presence of CD41/61-positive mitochondria in the cerebrospinal fluid of patients with delayed ischemia and patients with non-delayed ischemia using FACS.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

A first aspect of the present application provides a composition for diagnosing delayed ischemia, comprising an agent capable of detecting von Willebrand factor (vWF)-positive mitochondria.

As used throughout this specification, the term "delayed cerebral ischemia (DIC)" refers to the occurrence of a focal neurological disorder (eg, hemiparesis, aphasia, apraxia, hemianopia or neglect), or the Glasgow Coma Scale (total score or one of its individual components), the onset of delayed ischemia may be due to a variety of clinical and radiological variables. As used herein, the term "delayed ischemia" refers to "DIC", " It may be named interchangeably with "delayed cerebral ischemia".

In one embodiment of the present application, the composition may be a composition for diagnosis of delayed cerebral ischemia.

As used throughout this specification, the term "von Willebrand factor (vWF)" is a blood glycoprotein associated with hemostasis, and is called von Willebrand factor, von Willebrand factor, etc. When vWF is lacking or there is an abnormality, von Willebrand factor disease, and various diseases such as thrombotic thrombocytopenic purpura and Heyde's syndrome.

In one embodiment of the present application, the vWF-positive mitochondria may exist extracellularly, specifically, may exist in the cerebrospinal fluid.

In one embodiment of the present application, the vWF-positive mitochondria may be derived from endothelial cells.

In one embodiment of the present application, the composition includes an agent capable of confirming the origin of extracellular mitochondria present in the cerebrospinal fluid, and specifically, it may be a composition that can confirm from which cells the extracellular mitochondria are derived, However, the present invention is not limited thereto.

In one embodiment of the present application, the composition may include an agent capable of detecting endothelial cell-derived mitochondria.

In one embodiment of the present application, the composition may further include an agent capable of detecting glutamate aspartate transporter (GLAST)-positive mitochondria, but is not limited thereto.

As used throughout this specification, the term "glutamate aspartate transporter (GLAST)" is also called Excitatory amino acid transporter 1 (EAAT1), and is generally known to exist in the plasma membrane or inner mitochondrial membrane. . The GLAST serves to transport glutamate and aspartate, and is usually distributed in astrocytes.

In one embodiment of the present application, the GLAST-positive mitochondria may be derived from astrocytes.

In one embodiment of the present application, detecting the vWF-positive mitochondria and / or GLAST-positive mitochondria may be for measuring and comparing the presence, concentration, number, etc. of the mitochondria.

In one embodiment of the present application, the mitochondria may represent one or more of extracellular mitochondria, mitochondrial particles, extracellular cerebrospinal fluid mitochondria, and extracellular mitochondrial particles.

In one embodiment of the present application, the composition is an agent capable of detecting the expression level of DNA, RNA, mRNA or protein of vWF and/or GLAST in order to detect the vWF-positive mitochondria and/or GLAST-positive mitochondria may include.

According to one embodiment of the present application, the method for measuring the DNA, RNA and / or mRNA level is a polymerase reaction (PCR), a reverse transcriptase polymerization reaction (RT-PCR), a competitive reverse transcriptase polymerase reaction (competitive RT-PCR) ), real time quantitative RT-PCR, quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip method (DNA chip technology), but as long as it can measure the DNA, RNA and/or mRNA levels, it is not limited thereto.

According to one embodiment of the present application, the method for measuring the level of the protein is Western blotting (Western blotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmuno-diffusion (radical immunodiffusion), Ouchterlony immunodiffusion, rocket immunoeletrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence ), immunochromatography, FACS analysis (fluorescenceactivated cell sorter analysis), flow cytometry or protein chip technology, but is not limited thereto as long as the level of the protein can be measured. .

According to one embodiment of the present application, "an agent capable of measuring the expression level" refers to a molecule that can be used to determine the expression level of a specific gene or protein encoded by the gene, specifically the gene or the It may include an agent capable of detecting and/or amplifying a protein, but is not limited thereto.

As used throughout this specification, the term "agent capable of detecting a specific gene or protein" refers to an agent capable of specifically binding to and recognizing the specific gene or protein, or detecting and amplifying, " An agent capable of amplifying a specific gene or protein" refers to an agent capable of increasing the number by repeating the replication of the specific gene or protein, for example, a polynucleotide containing the gene can be specifically amplified. It may mean a primer or a probe capable of specifically binding, but is not limited thereto.

According to one embodiment of the present application, the agent capable of measuring the expression level is a primer pair, a probe or an antisense nucleotide that specifically binds to the vWF and/or GLAST gene, or a protein encoded by the genes. It may be a specific antibody or an antibody to which a fluorescent material is bound, but is not limited thereto.

The primers used for measuring the expression level of the gene are prepared under suitable conditions (for example, 4 different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. -Can be a single-stranded oligonucleotide that can serve as a starting point for directing DNA synthesis, and the appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the polynucleotide of the region for measuring the expression level of the gene, but should be sufficiently complementary to hybridize.

According to one embodiment of the present application, as a result of analyzing the cell origin of extracellular mitochondria of cerebrospinal fluid derived from patients with delayed ischemia and patients with non-delayed ischemia using the antibody-conjugated fluorescent substance, patients with delayed ischemia were non - As it was confirmed that more vWF-positive mitochondria and less GLAST-positive mitochondria were detected than in patients with delayed ischemia, extracellular mitochondrial cells present in cerebrospinal fluid obtained from an individual suspected of developing delayed ischemia By analyzing the origin, it can be seen that delayed ischemia can be diagnosed.

According to one embodiment of the present application, vWF-positive mitochondria and/or GLAST-positive mitochondria may be included in the marker composition for diagnosis of delayed ischemia.

As used throughout this specification, the term “marker” refers to a biomarker, and it is possible to detect a change in a living organism and objectively measure the normal or pathological state of the organism, the degree of response to a drug, and the like.

According to one embodiment of the present application, the composition for diagnosis of delayed ischemia of the present application may be for diagnosing the onset of delayed ischemia, predicting the risk of onset of delayed ischemia, or predicting the prognosis of delayed ischemia, but is limited thereto. no.

As used throughout this specification, the term “diagnosis” refers to determining whether delayed ischemia has already occurred in a specific individual.

The term "prediction" used throughout the present specification refers to whether a specific individual is likely to develop delayed ischemia, and if there is a possibility of developing delayed ischemia, it is determined whether the likelihood of developing delayed ischemia is relatively high compared to a large number of unspecified individuals. means to do

According to one embodiment of the present application, the delayed ischemia may occur after subarachnoid hemorrhage, but is not limited thereto.

According to an embodiment of the present application, the extracellular mitochondria of the cerebrospinal fluid in patients with delayed ischemia were found to have more vWF-positive mitochondria and less GLAST-positive mitochondria compared to patients without delayed ischemia. As shown, it can be seen that by confirming the detection level of vWF-positive mitochondria and/or GLAST-positive mitochondria, the onset of delayed ischemia, the possibility of onset of delayed ischemia, and the like can be diagnosed.

In one embodiment of the present application, the composition may include an agent capable of detecting mitochondria, specifically, the mitochondria may be extracellular mitochondria, but is not limited thereto.

In one embodiment of the present application, the composition may include an agent capable of measuring the membrane potential of mitochondria, and specifically, the mitochondria may be extracellular mitochondria, but is not limited thereto.

In one embodiment of the present application, the agent capable of measuring the membrane potential of the mitochondria may include a JC-1 dye, but is not limited thereto.

In one embodiment of the present application, the composition includes an agent capable of detecting a mitochondrial state abnormality, and the state abnormality refers to any state that is not normal, specifically structural abnormalities and/or functional abnormalities. it could mean

A second aspect of the present application provides a kit for diagnosing delayed ischemia, comprising the composition for diagnosing delayed ischemia. The contents overlapping with the first aspect are equally applied to the kit for diagnosis of delayed ischemia of the second aspect.

According to one embodiment of the present application, the kit may be a PCR kit, RT-PCR kit, qRT-PCR kit, DNA chip kit, Western blot kit, ELISA kit, FACS kit or immunofluorescence assay kit, but is limited thereto no.

According to one embodiment of the present application, the kit may include polynucleotides, cDNAs, mRNAs, polypeptides, etc. of the present application, as well as other component compositions, solutions or devices suitable for the analysis method.

A third aspect of the present application provides an informational method for diagnosing delayed ischemia, comprising measuring the level of von Willebrand factor (vWF)-positive mitochondria from a biological sample isolated from a subject. The contents overlapping with the first aspect and the second aspect are equally applied to the information providing method for the delayed ischemia diagnosis of the third aspect.

As used throughout this specification, the term "individual" refers to any organism that develops or is likely to develop delayed ischemia, and specific examples include mice, monkeys, cattle, pigs, mini-pigs, livestock, humans, etc. It may include mammals, farmed fish, and the like, but is not limited thereto.

As used throughout this specification, the term "sample" refers to a material derived from an individual that has or is likely to develop delayed ischemia, and specifically, tissues, cells, whole blood, serum, plasma, saliva, sputum, urine, etc. may include, and specifically may be cerebrospinal fluid, but is not limited thereto.

In one embodiment of the present application, the sample may include mitochondria derived from an individual, and specifically may include extracellular mitochondria.

In one embodiment of the present application, a genetic sample may be obtained from these samples, and the genetic sample may include a nucleic acid, for example, DNA, mRNA, or cDNA synthesized from mRNA, from which the extracellular mitochondrial As long as the presence of cell-derived, specifically vWF-positive mitochondria and/or GLAST-positive mitochondria can be confirmed, the type is not limited thereto.

According to one embodiment of the present application, the subject may be a human, but is not limited thereto.

In one embodiment of the present application, the method comprises: measuring the level of vWF-positive mitochondria from a biological sample isolated from a control; and comparing the level of vWF-positive mitochondria of the subject and the control group.

In one embodiment of the present application, the method further comprises the step of diagnosing the subject as having developed delayed ischemia or predicting a high level of the risk of developing vWF-positive mitochondria in the subject when the level of vWF-positive mitochondria is higher than that of the control group. may include.

In one embodiment of the present application, the level of mitochondria may mean the amount, number, concentration, etc. of the mitochondria, specifically, the level of vWF-positive mitochondria of the subject is higher than the control, from the subject. It may mean that the number, concentration, or amount of vWF-positive mitochondria contained in the sample is higher than that of the control group.

In one embodiment of the present application, the method may further comprise measuring the level of glutamate aspartate transporter (GLAST)-positive mitochondria.

In one embodiment of the present application, the method comprises the steps of: measuring the level of GLAST-positive mitochondria from a biological sample isolated from a control; and comparing the level of GLAST-positive mitochondria of the subject and the control group.

In one embodiment of the present application, when the level of GLAST-positive mitochondria of the subject is lower than that of the control, diagnosing the subject as having developed delayed ischemia or predicting the risk of developing a high level of additional may include.

As used throughout this specification, the term “control group” may refer to a general individual or a non-patient group that has not developed delayed ischemia.

According to one embodiment of the present application, in order to perform risk prediction or diagnosis of the individual, the method further comprises analyzing non-genetic information, wherein the non-genetic information includes gender, age, hypertension, diabetes, It may be one or more selected from the group consisting of hyperlipidemia and smoking.

According to one embodiment of the present application, the method may further comprise the step of measuring the membrane potential of mitochondria from a biological sample isolated from an individual, and measuring the membrane potential of the mitochondria is normal mitochondria and abnormal mitochondria. may be used to distinguish between

According to one embodiment of the present application, measuring the membrane potential may be characterized in that the mitochondria are stained with a JC-1 dye or a JC-1 derivative, wherein the JC-1 is 5,5',6,6 '-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl- It may have the chemical formula of imidacarbocyanine iodide).

The term “JC-1” or “JC-1 dye” as used throughout this specification is a membrane-permeable dye, which is generally used to study apoptosis by monitoring the state, health, etc. of mitochondria. . The JC-1 dye exhibits a potential-dependent accumulation in mitochondria, which is indicated by a shift in fluorescence emission from green (~529 nm) to red (~ 590 nm), specifically, mitochondrial depolarization shows a decrease in the red/green fluorescence ratio. . This potential-sensitive color change is due to the concentration-dependent formation of J-aggregates exhibiting red fluorescence.

According to one embodiment of the present application, measuring the membrane potential is to measure the red/green ratio expressed or stained by JC-1 after reacting or staining the mitochondria with JC-1 dye or JC-1 derivative. can

In one embodiment of the present application, the method comprises the steps of: measuring a mitochondrial membrane potential from a biological sample isolated from a control; and comparing the level of the mitochondrial membrane potential of the individual and the control group, specifically measuring the red/green ratio expressed or stained by JC-1.

In one embodiment of the present application, when the level of the mitochondrial membrane potential of the subject is lower than that of the control, specifically, when the red/green ratio of JC-1 is lower than that of the control, the subject is considered to have developed delayed ischemia. It may further comprise the step of diagnosing or predicting the risk of developing a high level.

In one embodiment of the present application, the sample may be obtained from an individual 1 to 5 days after the occurrence of subarachnoid hemorrhage, specifically 1 day, 3 days, 5 days, and more specifically 5 days later may be obtained.

A fourth aspect of the present application provides an information providing method for diagnosing delayed ischemia, comprising measuring the level of extracellular mitochondria from a biological sample isolated from a subject. The contents overlapping with the first to third aspects are equally applied to the information providing method for the diagnosis of delayed ischemia of the fourth aspect.

In one embodiment of the present application, the biological sample isolated from the subject may be derived from cerebrospinal fluid, but is not limited thereto.

In one embodiment of the present application, the method comprises: measuring the level of extracellular mitochondria from a biological sample isolated from a control; and comparing the level of extracellular mitochondria of the subject and the control group.

In one embodiment of the present application, the method further comprises the step of diagnosing the subject as having developed delayed ischemia or predicting a high level of risk of developing the subject when the level of extracellular mitochondria of the subject is higher than that of the control group. may include.

In one embodiment of the present application, the level of mitochondria may mean the amount, number, concentration, etc. of the mitochondria, and specifically, that the level of the extracellular mitochondria is higher than that of the control means that the sample derived from the subject It may mean that the number of included extracellular mitochondria is higher than that of the control group, the concentration is higher, or the amount is higher.

[Example]

Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Electron Microscopy of Mitochondria

The cerebrospinal fluid sample was centrifuged at 4000 rpm for 10 minutes, and the pellet was examined using an electron microscope. Specifically, first, the pellet was fixed overnight at 4°C using cacodylate buffer (0.1 M sodium cacodylate, 2mM MgCl _{2 ) containing 2% glutaraldehyde.} After washing cacodyl salt 3 times at 4°C, the sample was post-fixed at 4°C for 1 hour using 2% osmium tetroxide. The samples were washed with deionized water and dehydrated for 20 minutes at each concentration using gradient ethanol (50% to 100%). The samples were then incubated with progressively concentrated propylene oxide dissolved in ethanol and impregnated with increasing concentrations of Eponate 812 resin. The samples were incubated overnight in an oven at 65° C. and cut using an Ultra microtome. The cross section was observed with a field emission transmission electron microscope (JEM-2100F, JEOL).

Example 2: Measurement of the potential of the mitochondrial membrane

Mitochondrial membrane potential is used as an important parameter of mitochondrial function. To measure the extracellular mitochondrial membrane potential in cerebrospinal fluid, the MitoProbe JC-1 assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer's instructions. The JC-1 monomer emits green fluorescence. When the monomer enters the mitochondria of healthy cells, JC-1 forms a complex called J-aggregates and emits high red fluorescence. The red/green fluorescence ratio of JC-1 is mainly used as a parameter to monitor the mitochondrial membrane potential. Based on this, the ratio of red/green fluorescence intensity was used to represent the mitochondrial membrane potential herein.

To measure the mitochondrial membrane potential, first, a cerebrospinal fluid sample (100 μl) was mixed with JC-1 (1 μl of 1 μM in dimethylsulfoxide) in a 96-well flat-bottom polystyrene plate (SPL, Pocheon, Korea) 20 Incubated in the dark at 37°C for minutes. The fluorescence intensity of the green monomer (excitation/emission 485/516) and the red aggregate (excitation/emission 570/595) was measured using a GloMax explorer (Promega, WI, USA). The ratio was obtained by dividing the red fluorescence value by the green fluorescence value.

Example 3: Fluorescence-activated cell sorter analysis (FACS)

To analyze extracellular mitochondria in cerebrospinal fluid samples, flow cytometry was performed using a BD FACSCalibur (Becton-Dickinson, San Jose, CA, USA). First, a cerebrospinal fluid sample (100 μl) was stained with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) at room temperature for 30 minutes. In addition, the following fluorescence-conjugated antibodies were used to investigate the potential cellular origin of cerebrospinal fluid extracellular mitochondrial signaling: vWF-FITC (von Willebrand factor-fluorescein; 1:500, Abcam, Cambridge, MA, USA), GLAST -APC (glutamate aspartate transporter-allophycocyanin, 1:500, Miltenyl Biotec), CD45-FITC (1:500, Miltenyl Biotec SL, Madrid, Spain), and CD41/61-FITC (1:500, Miltenyi Biotec). The cerebrospinal fluid samples were immunostained with each antibody for 15 minutes at room temperature, and flow cytometry was performed using Flowjo version 10 software (FlowJo, Ashland, OR, USA). To minimize the effect of fluorescence spillover on the measured signal, an unstained single staining control was used to determine the appropriate gate, voltage, and fluorescence compensation for flow cytometry.

Example 4: Statistical Analysis

Continuous variables are expressed as mean and ± standard deviation (SD). Data were analyzed by Chi-square or Student's t-test. To evaluate the difference in JC-1 rates according to the presence of delayed ischemia, repeated measures of change analysis (ANOVA), Mauchly's spherical test and lower limit correction were performed. In addition, percentages and percentages of JC-1 according to specific types of mitochondria were compared using the Mann-Whitney U test. All statistical analyzes were performed using SPSS V.21 (SPSS, Chicago, IL, USA) and MedCalc software (Medcalc, Mariakerke, Belgium), and a P-value <0.05 was considered statistically significant.

Experimental Example 1: Confirmation of clinical characteristics of the cohort group

The clinical characteristics of the cohorts participating in the present study were not significantly different according to gender, hypertension, diabetes, hyperlipidemia, and smoking in delayed ischemia. In patients with subarachnoid hemorrhage with delayed ischemia, a Hunt and Hess (HH) grade of grade 3 or higher was observed more frequently than patients without delayed ischemia (66.7% vs. 42.9%), and clinical trials after 3 months were observed. The results were also poor (41.7% vs. 23.8%). However, the results between the two groups were not statistically significant. In addition, the aneurysm location (anterior cerebral circulation) and size were similar between the two groups.

Experimental Example 2: Confirmation of extracellular mitochondria and mitochondrial membrane potential of cerebrospinal fluid

As a result of observation with an electron microscope, it was confirmed that extracellular mitochondria released from the cerebrospinal fluid of patients with subarachnoid hemorrhage were present (FIG. 1). In addition, as a result of flow cytometry, it was confirmed that more extracellular mitochondrial particles were present in patients with delayed ischemia than patients with non-delayed ischemia ( FIG. 2 ). As a result of confirming the extracellular mitochondrial membrane potential at the JC-1 red/green ratio on days 1, 3, and 5 after the onset of subarachnoid hemorrhage, the JC-1 red/ It was confirmed that the green ratio was the highest.

Next, the Huynh-Feldt method showed an epsilon of 0.871 and a significant interaction (p < 0.001), and the ratio of JC-1 red/green ratios according to the day of measurement between patients with delayed ischemia and patients with non-delayed ischemia. shows a significant difference. In particular, patients with delayed ischemia tended to have a lower JC-1 red/green ratio than patients with non-delayed ischemia [at day 1, 3.35 samples red/green ratio (3.20-3.75) DCI vs. 3.70 (3.40-3.95) non-DCI, p=0.060; Day 3, 4.65 (4.45-5.00) DCI vs. 5.10 (4.65-5.30) non-DCI, p=0.087], the difference in red/green ratio between the two groups was most pronounced on day 5 [DCI, 3.05 (2.90-3.35) vs. non-DCI, 4.20 (4.10-4.50), p < 0.01] (Fig. 3).

Next, the association between the JC-1 rate and delayed ischemia development was confirmed. Specifically, 12 of the cohorts were subarachnoid hemorrhage patients who experienced delayed ischemia, and the number of patients with different onset of delayed ischemia was as follows: Day 4, n=1 (8.3%); Day 5, n=1 (8.3%); Day 6, n=3 (25.0%); Day 7, n=5 (41.7%); Day 8, n=2 (16.7%). Two patients (Patients 2 and 4, red and brown lines in Fig. 4) with onset of delayed ischemia on days 4 and 5 showed a lower JC-1 red/green ratio than the other patients. In contrast, the two patients who started delayed ischemia on day 8 (patients 3 and 8 in Figure 4, blue lines and dark blue lines) had a higher JC- It was confirmed that the ratio of 1 was relatively high (FIG. 4).

Experimental Example 3: FACS analysis result

To confirm the potential cellular origin of the extracellular cerebrospinal fluid mitochondria, flow cytometry was performed on day 5 cerebrospinal fluid using a fluorescence-conjugated antibody. The antibody was used to identify the following cell origins: GLAST (astrocyte), vWF (endothelial cells), CD45 (microglia/microphage), and CD41/61 [platelet].

As a result, it was confirmed that patients with delayed ischemia had a higher proportion of vWF-positive mitochondrial particles than patients with non-delayed ischemia [40.10% (38.25%-44.90%) vs. 30.20% (25.70%-36.68%), p < 0.01] ( FIGS. 5 and 6 ). In contrast, patients with delayed ischemia had fewer GLAST-positive particles than patients with non-delayed ischemia [29.55% (20.65%-31.90%) DCI vs. 38.50% (29.90%-40.00%) non-DCI, p < 0.01] ( FIGS. 7 and 8 ). In addition, for CD45-positive and CD41/61-positive mitochondrial particles, no difference was found between patients with delayed and non-delayed ischemia [CD45: 12.50% (10.90%-14.55%) DCI vs. 10.70% (9.43%-13.98%) non-DCI, p=0.369; CD41/61: 9.95% (7.25%-12.05%) DCI vs. 12.40% (9.13%-14.63%) non-DCI, p=0.155] ( FIGS. 9 and 10 ).

Based on the above results, it can be seen that, in patients with delayed ischemia, the extracellular mitochondria of the cerebrospinal fluid are vWF-positive and GLAST-negative. It can be seen that it does not originate from , and based on this, it is possible to diagnose or predict patients with delayed ischemia in advance.

So far, the present invention has been focused on preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

As described above, although the present invention has been described with reference to limited embodiments and drawings, the present invention is not limited thereto, and the technical idea of the present invention and the following by those of ordinary skill in the art to which the present invention pertains. Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

Claims (11)

A composition for diagnosing delayed ischemia, comprising an agent capable of detecting vWF (von Willebrand factor)-positive mitochondria in extracellular mitochondria in cerebrospinal fluid. delete According to claim 1,
The vWF-positive mitochondria are derived from endothelial cells, a composition for diagnosis of delayed ischemia.
According to claim 1,
Wherein the delayed ischemia is onset after subarachnoid hemorrhage, delayed ischemia diagnostic composition.
A kit for diagnosing delayed ischemia, comprising the composition of any one of claims 1, 3 and 4.
A method for providing information for diagnosing delayed ischemia, comprising measuring the level of von Willebrand factor (vWF)-positive mitochondria in extracellular mitochondria in cerebrospinal fluid from a biological sample isolated from a subject.
7. The method of claim 6, wherein the method
measuring the level of vWF-positive mitochondria from the biological sample isolated from the control; and
comparing the level of vWF-positive mitochondria of the subject and the control.
Further comprising a, information providing method for delayed ischemia diagnosis.
8. The method of claim 7, wherein the method
If the level of vWF-positive mitochondria of the subject is higher than that of the control, diagnosing the subject as having developed delayed ischemia or estimating the risk of developing delayed ischemia at a high level, diagnosing delayed ischemia How to provide information for
9. The method of claim 8, wherein the method
GLAST (glutamate aspartate transporter)- information providing method for the diagnosis of delayed ischemia, further comprising the step of measuring the level of positive mitochondria.
delete delete

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