KR102196352B1 - A biomarker for diagnosis of drowning and uses thereof - Google Patents

Abstract

The present invention provides a method of measuring the expression level of RAGE from a sample isolated from a drowning body as a subject in order to provide information for determining the cause of death as drowning. In addition, it provides a diagnostic kit for determining drowning, including an agent for measuring the expression level of RAGE.

Description

A biomarker for diagnosis of drowning and a method for determining drowning using the same {A biomarker for diagnosis of drowning and uses thereof}

The present invention relates to a RAGE gene, which is a biomarker for determining the cause of death as drowning, and more particularly, to a method for determining drowning using the gene and a diagnostic kit using the same.

Drowning is one of the main causes of accidents. Worldwide, it is known that between 150,000 and 800,000 people die of drowning every year. According to the WHO, drowning drowns in water, causing respiratory damage, and death through a variety of mechanisms including asphyxiation, electrolyte/osmotic disturbance, and hypothermia. Therefore, judging a cause of death as drowning is one of the most difficult forensic purposes. Moreover, it is a difficult problem to determine whether the cause of death was drowning or if the body was abandoned in water after death (postmortem submersion). In general, the judgment of drowning is made using a combination of autopsy, histological findings, toxicological analysis, diatom test, and forensic investigation. However, autopsy and diatom testing are not conclusive evidence. In addition, it is an important and difficult problem to distinguish between drowning in fresh water and drowning in seawater.

Judging the postmortem interval (PMI) for drowning is also important in criminal investigations. However, it is not easy to judge this, and bacteria and accumulated degree days (ADD) have also been used (Myburgh J et al, Forensic Sci Int 2013;229(1-3):165.e1-6). However, there is no known specific biomarker that can predict the elapsed time after death in drowning.

In general, drowning causes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which is a major cause of death. Therefore, it has been reported that the pulmonary surfactant and related cytokines may be markers for determining drowning (Miyazato T et al, Int J Legal Med. 2012 Jul;126(4):581-7) .

Surfactant-protein A (SP-A) is a surface-active protein present in alveolar type II cells and is known as an immunohistologic marker for determining drowning (Lee SY et al, J Forensic Sci. 2017;62: 1080-1088). AQP-5 (aquaporin 5) is also expressed in type I alveolar epithelial cells and is known as an immunohistologic marker for determining drowning (Hayashi T et al, Int J Leg Med. 2009;123:7-13).

However, previous studies have focused on seawater drowning, or have failed to distinguish between drowning and post-pitching or other suffocation.

A single biomarker that allows the distinction between drowning and post pitcher and freshwater drowning and seawater drowning, and predicting the elapsed time of drowning, has yet to be known. Therefore, there is a need to develop such a reliable biomarker.

Miyazato T et al, Int J Legal Med. 2012 Jul;126(4):581-7 Lee SY et al, J Forensic Sci. 2017;62:1080-1088 Hayashi T et al, Int J Leg Med. 2009;123:7-13

In order to solve the above problems, an object of the present invention is to provide a biomarker effective in determining drowning, and the biomarker according to the present invention can distinguish between drowning and post-pitching, freshwater drowning and seawater drowning. It is also possible to predict the elapsed time after death in drowning.

In addition, the present invention is to provide a kit and a determination method using the same.

In order to achieve the above object, the present invention (a) measuring the expression level of RAGE (SEQ ID NO: 1, GenBank Accession No. DQ104254.1) from a sample isolated from the drowned body, and (b) (a It provides a method of providing information for determining drowning as a cause of the subject, comprising the step of comparing the expression level of RAGE measured in step) with the expression level of RAGE of the normal control sample.

In order to achieve another object of the present invention, the present invention provides a diagnostic kit for determining drowning, including an agent for measuring the expression level of RAGE (SEQ ID NO: 1, GenBank Accession No. DQ104254.1).

In order to achieve another object of the present invention, the present invention is to (a) measure the expression level of RAGE from a sample isolated from the drowning body, and (b) drown the expression level of RAGE measured in step (a) It provides a method of providing information for estimating the postmortem interval (PMI) of a subject, comprising the step of comparing with the standard expression level of RAGE for each elapsed date.

The biomarker according to the present invention can distinguish between drowning and post pitching, freshwater drowning and seawater drowning, and can predict the elapsed time after death in drowning. Specific information related to drowning can be determined by measuring the expression level of the biomarker according to the present invention in the sample isolated from the carcass.

1 is an RT-PCR result confirming the expression levels of RAGE, AQP5, SP-A, IL-6, and IL-1β genes. The expression level was standardized on the basis of GAPDH and expressed as a ratio. *P <0.05, **P <0.01 and ***P <0.005
(Con: control group, Hy: hypoxia group, FP: freshwater postmortem-submersion group), SP: seawater postmortem-submersion group, FD: freshwater drowning group , SD: seawater drowning.
2 is a RAGE protein western blotting analysis results. (a) is the result of Western blotting after electrophoresis, and (b) is the result of RAGE expression. *P <0.05 and **P <0.01
Figure 3 shows the results of H&E staining (a, b, c, d) and IHC staining (e, f, g, h) of RAGE. Control group (a, e), hypoxia group (b, f), post-seawater pitcher (c, g) and seawater drowning (d, h).
Figure 4 shows the results of H&E staining (a, b, c, d) and IHC staining (e, f, g, h) of RAGE. Control group (a, e), hypoxia group (b, f), freshwater post-pitcher (c, g) and freshwater drowning (d, h).
Figure 5 (a) is a result of RT-PCR confirming the RAGE gene expression level. The expression level was standardized on the basis of GAPDH and expressed as a ratio. *p <0.05, **p <0.01 and ***p <0.005. (b) is an RT-PCR trend line. y = -5.743ln(x) + 11.537.
6 is a result of Western blotting of RAGE protein. In (a), the intensity of the sRAGE band gradually fades over time. (b) is a Western blotting trend line. y = -11138ln(x) + 25061
7 is a result of lung tissue IHC staining of groups (AG) and control groups (H) from day 1 to day 7 after drowning.

In order to achieve the above object, the present invention (a) measuring the expression level of RAGE (SEQ ID NO: 1, GenBank Accession No. DQ104254.1) from a sample isolated from the drowned body, and (b) (a) It provides a method of providing information for determining drowning as a cause of a subject, comprising the step of comparing the expression level of RAGE measured in the step with the expression level of RAGE of a normal control sample.

RAGE (receptor for advanced glycation end products, SEQ ID NO: 1, GenBank Accession No. DQ104254.1) is known to be involved in the inflammatory response as a multi-ligand binding receptor. It is known to be expressed at low levels in cells, but is expressed in a large amount in the lungs. RAGE exists in two types, sRAGE (secretory RAGE) and mRAGE (membrane RAGE), and each has a molecular weight of 29kDa and 45kDa. sRAGE is a secreted form and is not well expressed in the transmembrane domain, so it acts as a decoy receptor. mRAGE is overexpressed in patients with airway epithelium and chronic obstructive pulmonary disease (COPD). RAGE is known to be associated with chronic inflammatory diseases, arteriosclerosis, cardiovascular disease, and lung disease.

"Drowning" according to the present invention means death in which the direct cause of death is drowning. It is a different concept from “postmortem submersion”, which falls into the water after death due to other causes.

The "subject" according to the present invention is an object for determining the cause of death, and may be a mammal. Mammals can be human, mouse, rat, cow, goat, pig, horse, sheep, dog, cat. The sample isolated from the subject is a tissue separated from the subject's body, and may be liver, lung, kidney, spleen, stomach, small intestine, or large intestine, preferably lung tissue.

The method for measuring the expression level according to the present invention may be a known method for measuring the expression level of mRNA or protein. Examples include RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, Western Blotting, ELISA, enzyme-linked immunosorbent assay. , Radioimmunoassay (RIA), radioimmunodiffusion method, okteroni immune diffusion method, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation, complement fixation method, flow cytometry (FACS) and protein chip It may be one or more methods selected from the group consisting of.

The normal control sample according to the present invention is a sample of a corpse that has not been drowned or permeated after death, and is preferably a sample isolated from a corpse that has died due to an accident, not a corpse that has died due to a specific disease.

If the subject's cause of death is drowning, the level of RAGE expression is high. Compared to the case of the post pitcher, the expression level is higher in the case of drowning (Figs. 1 and 2). The same results were also shown in the immunohistochemistry test (FIGS. 3 and 4). Therefore, if it is confirmed that the RAGE expression level of the subject is high by comparing the expression level of RAGE measured from the sample isolated from the drowning body as a subject using these characteristics, the death of the subject is judged as drowning. can do.

In one specific embodiment, the present invention is a method for providing information for determining seawater drowning as a cause of death of a subject, comparing the expression level of RAGE in the subject measured in step (a) with the expression level of RAGE in a freshwater drowning sample. It may further include steps. Referring to Figs. 1(a) and 2(b), it can be seen that the RAGE expression rate of the seawater drowning body is higher than that of the freshwater drowning body. Therefore, when using a method further comprising the step of comparing the RAGE expression level of the subject with the RAGE expression level of the freshwater drowning sample, if it is confirmed that the RAGE expression level of the subject is higher than the RAGE expression level of the freshwater drowning sample, the subject's The cause of death can be judged as drowning in seawater. The freshwater is ordinary broth water with a low salt content, such as groundwater and river water, and seawater means seawater.

In another aspect of the present invention, the present invention provides a diagnostic kit for determining drowning, including an agent for measuring the expression level of RAGE (SEQ ID NO: 1, GenBank Accession No. DQ104254.1).

The agent for measuring the expression level of RAGE according to the present invention is a substance capable of measuring the expression level of RAGE nucleotide or protein.

An agent capable of measuring the expression level of RAGE nucleotides may be probes, primers, nucleotide sequences, or combinations thereof that specifically bind to RAGE nucleotides. Since the nucleotide sequence of the RAGE gene is known, a person skilled in the art can design a primer or probe that specifically binds to these gene sequences based on the above sequence according to a conventional method in the art.

An agent capable of measuring the expression level of the RAGE protein may be an antibody, antigen-binding fragment, polypeptide, protein, or a combination thereof that specifically binds to the RAGE protein or a fragment thereof.

In another aspect of the present invention, the present invention comprises the steps of (a) measuring the expression level of RAGE from a sample isolated from the drowning body, and (b) measuring the expression level of RAGE measured in step (a) by day after drowning It provides a method of providing information for estimating the postmortem interval (PMI) of a subject, comprising the step of comparing the RAGE standard expression level.

The standard expression level of RAGE for each day after drowning according to the present invention is the standard expression level of RAGE for each post-mortem date measured from samples isolated from control drowned bodies with different drowning days, specifically the first day of drowning and the second day of drowning. , It is the standard expression level of RAGE for each post-mortem day measured from each control drowned body such as drowning 3rd day, drowning 4th day, and drowning 5th day.

In drowning, RAGE has the highest expression level in drowned bodies on the day of drowning (day 1 of drowning), and in the case of drowning bodies with elapsed time, the expression level gradually decreases with elapsed time (FIGS. 5 and 6).

In one specific embodiment, in the method of providing information for estimating the postmortem interval (PMI) of the drowned body, the RAGE standard for each postmortem period measured from samples separated from control drowned bodies identified as different days after drowning. By calculating the expression level, a trend line can be made with the x-axis as the post-mortem date and the y-axis as the standard RAGE expression level for each elapsed date. At this time, by substituting the RAGE expression level of the subject from the trend line, the elapsed date of drowning of the subject can be estimated.

As one specific embodiment, in the method of providing information for estimating the elapsed date of drowning of the subject, the amount of RAGE expression may be the amount of expression of sRAGE. Referring to FIG. 6(a), the amount of expression of RAGE gradually decreases as time elapses after drowning. More specifically, the amount of expression of mRAGE is not significantly reduced, but the amount of expression of sRAGE is greatly reduced. Therefore, when comparing the expression level of sRAGE in a sample isolated from the drowning body as a subject and the standard expression level of sRAGE by day after drowning, it is possible to estimate the day after drowning of the subject.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[RAGE as a biomarker for judgment of drowning]

Materials and grouping

1-1. Experimental animals

The experimental animals were Sprague-Dawley rats of 5 weeks old purchased from DBL (Korea). All mice were approved by Soonchunhyang University Animal Experimental Ethics Committee (IACUC approval SCH16-0005).

1-2. Seawater and freshwater harvesting

Seawater was collected from Janggo Port (37°03'N, 126°56'E), and freshwater was collected from Sapgyoho (36°87'N, 126°84'E). The collected water was immediately put in a sterile bottle and stored in a refrigerator. The salinity (NaCl) composition and COD (chemical oxygen demand) of seawater were 32.0mg/L and 1.6mg/L, respectively, and the BOD (biochemical oxygen demand) of freshwater was 4.4mg/L.

1-3. Grouping

For this experiment, mice were randomly divided into 4 groups. Hypoxic group (10 animals), drowning group (total 20, freshwater 10, seawater 10), post-pitch group (10, freshwater 5, seawater 5), control group (5). The drowning environment for the drowning group and the post-permeable group was created by filling a sterilized plastic tank (0.09 × 0.19 × 0.12m) with 11L of fresh water or seawater. The drowned group was anesthetized with Alfaxan (1.5 mL/kg, iv), and dive for 1 minute and breathing for 1 minute were repeated to reproduce the actual drowning situation in a tank filled with water, and then immersed in water for 2 hours. The post-permeable group was anesthetized with Alfaxan (1.5mL/kg, iv) and immersed in water after cervical dislocation. It was left for 2 hours each in fresh water and sea water. The hypoxia group was anesthetized with Alfaxan (1.5 mL/kg, iv) and placed in a place filled with 5% O ₂ and 95% N ₂ . The hypoxia group was exposed to the hypoxic environment for 8 minutes until the animals died, and then exposed to the air environment for 2 minutes. After autopsy, each lung tissue was fixed in 10% formalin for immunohistochemical staining, frozen in liquid nitrogen and stored at -80°C for western blotting and RNA extraction.

1-4. RNA extraction, cDNA synthesis

After homogenizing the frozen tissue sample with a mortar containing liquid nitrogen, RNA was extracted according to the tissue protocol of the manufacturer of the RNA isolation kit (MACHEREY-NAGEL, Germany). The extracted RNA was quantified with Biodrop μLite (Biodrop, UK), and cDNA was synthesized according to the instructions of Revertra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo, Japan).

Confirmation of RAGE expression

2-1. RT-PCR analysis

In order to find a new biomarker for determining drowning, the present inventors screened SP-A and AQP5, which are already known as biomarkers of drowning, and RAGE and cytokines IL-1β, IL-6, which are known as lung inflammation-related substances. I did.

All samples were amplified by real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA) device. PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, UK) and primers (Table 1). The PCR reaction solution contained 10 μL of the master mix, 1 μL of each primer (0.5 pmoL/μL), and 1 μL of a DNA sample, for a total of 20 μL. PCR conditions are after heating at 95°C for 10 minutes (initial denaturation), 30 seconds at 95°C (denaturation), 30 seconds at each primer binding temperature (binding), 30 seconds at 72°C (synthesis) for 40 cycles, Final extension was made at 72°C for 30 seconds. The Ct value was automatically calculated using the instrument software (StepOne Software v2.3). The expression level of the mRNA gene to be identified was standardized on the basis of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) as a control and analyzed using the ΔΔCt method.

As a result, SP-A, known as a biomarker of drowning, was highly expressed in hypoxic group and freshwater postpermeable group, showing different results from that known as a biomarker of drowning, and IL-6 was highly expressed only in freshwater drowning. RAGE and AQP5 were highly expressed in both freshwater and seawater drowning groups (Fig. 1).

2-2. Western blotting analysis

Western blotting was performed for RAGE highly expressed in both the freshwater and seawater drowning groups.

RIPA solution (500mM Tris-HCL, 10% NP-40, 10% sodium deoxycholate, 1.5M NaCl, 100mM EDTA, 100mM PMSF, 100mM Na ₃ VO ₄ , 1µg/1µl aprotinin, 1µg/µl leupeptin and distilled water) Was used to dissolve the lung tissue. The dissolved lung tissue was centrifuged at 14,000 rpm for 20 minutes at room temperature to obtain a supernatant and then diluted to the same concentration. The prepared sample was electrophoresed on SDS-PAGE, and the gel after electrophoresis was transferred to a nitrocellulose membrane (Immobilon-P, Merck KGaA, Germany). The membrane was immersed in 5% skim milk for 10 minutes to block non-specific binding. This blocking-treated membrane was reacted with RAGE antibody (1:5,000, abcam, Cambridge, MA, USA). After washing with phosphate buffered saline, a secondary antibody (1:10,000, Abcam, Cambridge, MA, USA) was added to the membrane and reacted at 4 °C for 2 hours, and then chemiluminescence (ECL) detection (Intron Biotechnology, Korea ) The band was observed using an instrument, and GAPDH was used as a reference gene to measure the protein expression level of each biomarker.

As a result of gel electrophoresis, RAGE was identified as two bands. RAGE expression was significantly higher in the drowning group than in the other groups (P <0.05), and among them, the seawater drowned group was expressed higher than that of the freshwater drowned group (FIG. 2 ).

Histopathology analysis

Paraffin blocks were made with the upper lobes of both lungs fixed in 10% formalin, and sections cut into 4 μm were prepared. After the slide tissue was treated with xylene and subjected to a hydration process, the tissue sections were used for hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining.

For IHC staining, the slides are immersed in 0.1M sodium citrate buffer and treated in a microwave for 10 minutes, and then the slides are treated with a protein blocking solution added with 2% normal goat serum for 30 minutes to block background staining. I did. Tissue sections were treated with PBS containing 1% of RAGE primary antibody (1:800, Abcam, Cambridge, MA, USA) and bovine serum albumin (BSA). After dropping the primary antibody in a container with high humidity and reacting overnight at 4°C, after washing with PBS, the immune reaction was performed by the horseradish peroxidase conjugated secondary antibody and HRP/DAB Detection IHC Kit (Abcam, Cambridge, MA, USA). 3,3'-diaminobenzidine (DAB) solution was dropped according to the instructions of to make the color appear. After washing the slides with tap water, control staining was performed using Mayer's hematoxylin (Sigma-Aldrich, USA).

H&E staining revealed pulmonary edema, pulmonary congestion and bleeding. This was clearly observed in the drowning group and the hypoxic group. As a result of IHC staining, RAGE expression was remarkably observed in epithelial cells associated with inflammatory cells. In the drowning group, RAGE particle-like staining was well observed in type I alveolar cells. Specifically, the strong and dense particle-shaped RAGE was most pronounced in the drowning group, but there was no significant difference in the freshwater drowning and seawater drowning groups (FIGS. 3 and 4).

[Change of RAGE expression level according to postmortem interval (PMI)]

Materials and grouping

4-1. Experimental animals

Experimental animals were 7-week-old Sprague-Dawley rats purchased from DBL (Korea). All animal experiments were approved by Soonchunhyang University Animal Experimental Ethics Committee (IACUC approval number: SCH180006).

4-2. Grouping

Seawater drowning samples were taken from the Asan embankment (36 ° 5409 "N 126 ° 54034"E). Water was collected in a sterilized bottle and stored in a refrigerator (4°C) until use in the experiment. Leave at room temperature one day before use. In this study, Sprague-Dawley rats were randomly assigned to 2 controls (negative control (CON), immersion control group (ICON), 5 animals in each group) and 7 drowning groups (5 per group) from 1 to 7 days. Marie), divided into 9 different groups. For the seawater drowning simulation model, mice were anesthetized with Alfaxan (1.5mL/kg, iv) and drowned in a water tank (0.09 x 0.19 x 0.12 m) filled with 11 L of seawater at a temperature of 15 ± 5 °C. After drowning, the mice were left in sea water for 1 to 7 days. The negative control group was anesthetized with Alfaxan (1.5mL/kg, iv) and sacrificed by cervical dislocation, and was not immersed in water (not in the drowning group and not in the post pitcher group). The immersion control group (ICON) was anesthetized in the same manner, euthanized by cervical dislocation, and then immersed in seawater for 1 day after death. Therefore, the immersion control (ICON) is a group in which the negative control is immersed for 1 day. The immersion control (ICON) study was conducted for 8 weeks along with the other 8 group studies.

Autopsy was performed immediately after the rat was removed from water on the designated date, and lung tissue samples were obtained. After careful examination with the naked eye, the lungs of each rat were removed and divided into three parts. Histological analysis of the lungs was performed. After autopsy, three parts of the lung were randomly designated A, B and C for the next analysis. Lungs of each animal were harvested and fixed in 10% formalin for histological and immunohistochemical analysis. Other lung tissues were immediately frozen using liquid nitrogen and stored at 80° C. for western blotting and RNA extraction.

4-3. RNA extraction and cDNA synthesis

The frozen lung tissue was ground with liquid nitrogen and homogenized, and then RNA was extracted using an RNA isolation kit (MACHEREY-NAGEL, Germany) according to the manufacturer's protocol. The concentration of extracted RNA was measured using Biodrop lite (Biodrop, UK). Revertra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo, Japan) cDNA was synthesized according to the manufacturer's instructions.

RAGE expression level check

5-1. RT-PCR analysis

qRT-PCR was performed in the StepOnePlus Real-Time PCR system (Applied Biosystems, USA) using SYBR Green PCR Master Mix (Applied Biosystems, UK), and the primers were the same as those used in <Example 2-1>. As for the PCR mixture, 10 μL of Master Mix, 1 μL of each primer (0.5 pmol/1 μL), and 1 μL of DNA sample (0.5 pmol/1 μL) were added to obtain a total volume of 20 μL. PCR conditions were heated for 10 minutes at 95°C (initial denaturation), 30 seconds at 95°C (denaturation), 30 seconds at each primer binding temperature (binding), and 30 seconds at 72°C (synthesis) for 40 cycles. Ct values were calculated using the instrument software (StepOne Software v2.3). Relative mRNA expression levels with other groups were analyzed using the ΔΔCt method using GAPDH as a reference gene.

As a result, there was no change in the expression level of RAGE mRNA in the negative control group (CON) and the submerged control group (ICON). In the drowning group, the drowned group on the first day showed a 12-fold higher RAGE expression rate compared to the negative control group. As the days after drowning increased, the rate of RAGE expression decreased, showing a two-fold or more decrease in the drowning group on the first day and the drowning group on the second day (Fig. 5(a)). The log graph showed almost the same trend line (Fig. 5(b))

5-2. Western blotting analysis

Lung tissue using RIPA solution (500mM Tris-HCL, 10% NP-40, 10% sodium deoxycholate, 1.5M NaCl, 100mM EDTA, 100mM PMSF, 100mM Na3VO4, 1μg/1μL aprotinin, 1μg/μL leupeptin and distilled water) Was dissolved. The dissolved lung tissue was centrifuged at 18,500 g for 20 minutes at room temperature to obtain a supernatant and then diluted to the same concentration. Protein concentration was measured based on a standard graph made by dilution of bovine serum albumin prepared in advance. The protein was boiled in the loading buffer at 100° C. for 5 minutes, and then the prepared sample was electrophoresed on SDS-PAGE, and the gel after electrophoresis was transferred to a nitrocellulose membrane (Immobilon-P, Merck KGaA, Germany). The membrane was blocked in phosphate buffered saline (PBS) mixed with 5% skim milk at room temperature for 2 hours, and then rabbit anti-RAGE primary antibody (1:5,000, Abcam, Cambridge, MA, USA) was added to 4°C. Reacted overnight at. After washing with PBS, the membrane was added with a goat anti-rabbit IgG secondary antibody (1:10,000, Abcam, Cambridge, MA, USA) and reacted at 4°C for 2 hours, and then chemiluminescence (ECL) detection (Intron Biotechnology, Korea) equipment is used to observe the band. GAPDH was used as a reference gene to measure the expression level.

As a result of gel electrophoresis, RAGE was identified as two bands: sRAGE of 29 kDa and mRAGE of 45 kDa. The expression level decreased from day 1 to day 7, but there was little change in mRAGE, whereas sRAGE showed a marked decrease trend (Fig. 6).

Histopathology analysis

The tissue fixed with 10% formalin was dehydrated by gradually increasing the concentration of ethanol. After the tissue was cleared with xylene, a paraffin block was made. The prepared paraffin block was cut to a thickness of about 4 μm for histological staining. After removing paraffin from the slide tissue section with xylene and undergoing a hydration process, the tissue section was used for hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining.

For IHC staining, after removing paraffin, the slides were immersed in 0.1M sodium citrate buffer and treated in a microwave for 10 minutes to perform antigen retrieval. The slides were treated with a protein blocking solution to which 2% normal goat serum was added for 30 minutes to block background staining. The primary antibody, rabbit polyclonal antibody (Abcam, Cambridge, MA, USA) was diluted 1:100 to treat tissue sections, placed in a container with humidity, and reacted overnight at 4°C. After washing with PBS, horseradish peroxidase (HRP)-conjugated secondary goat antibody was dropped and left for 2 hours for immune response. According to the manufacturer's instructions of the HRP/DAB Detection IHC Kit (Abcam, Cambridge, MA, USA), a 3,3'-diaminobenzidine (DAB) solution was dropped to make the color appear. After washing the slides with tap water, control staining was performed using Mayer's hematoxylin (Sigma-Aldrich, USA).

As a result of IHC staining, it was confirmed that RAGE expression decreased as the drowning period elapsed. Specifically, the lung tissues of the 1st, 2nd, and 3rd day groups showed distinct RAGE particle-like staining in type I alveolar cells, and a clear line shape along the alveolar edge. After the 4th and 5th days, the particle staining disappeared and only the lines remained, and on the 6th and 7th days, the line shape was also weakened (Fig. 7). However, as a result of H&E staining, all the drowned groups were similar without distinct histological changes, and congestion, hemorrhage, and edema were observed.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> A biomarker for diagnosis of drowning and uses thereof <130> 2019-0051 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 1429 <212> DNA <213> Homo sapiens <400> 1 aggaagcagg atggcagccg gaacagcagt tggagcctgg gtgctggtcc tcagtctgtg 60 gggtgagcca ctccctcaac cccactgacc ctccctgcag aaagcacttt aaccccacac 120 cccagtcgtc ctagaacttt tcccagaacc cgaggaagtg cctttcaagg tccctcaccc 180 accctgtcca aattttgtta gccctcattc ccttcctacc cctctaccat ggtgctatct 240 cccaggggca gtagtaggtg ctcaaaacat cacagcccgg attggcgagc cactggtgct 300 gaagtgtaag ggggccccca agaaaccacc ccagcggctg gaatggaaac tgaacacagg 360 ccggacagaa gcttggaagg tcctgtctcc ccagggagga ggcccctggg acagtgtggc 420 tcgtgtcctt cccaacggct ccctcttcct tccggctgtc gggatccagg atgaggggat 480 tttccggtgc caggcaatga acaggaatgg aaaggagacc aagtccaact accgagtccg 540 tgtctaccag attcctggga agccagaaat tgtagattct gcctctgaac tcacggctgg 600 tgttcccaat aaggtgggga catgtgtgtc agagggaagc taccctgcag ggactcttag 660 ctggcacttg gatgggaagc ccctggtgcc taatgagaag ggagtatctg tgaaggaaca 720 gaccaggaga caccctgaga cagggctctt cacactgcag tcggagctaa tggtgacccc 780 agcccgggga ggagatcccc gtcccacctt ctcctgtagc ttcagcccag gccttccccg 840 acaccgggcc ttgcgcacag cccccatcca gccccgtgtc tgggagcctg tgcctctgga 900 ggaggtccaa ttggtggtgg agccagaagg tggagcagta gctcctggtg gaaccgtaac 960 cctgacctgt gaagtccctg cccagccctc tcctcaaatc cactggatga aggatggtgt 1020 gcccttgccc cttcccccca gccctgtgct gatcctccct gagatagggc ctcaggacca 1080 gggaacctac agctgtgtgg ccacccattc cagccacggg ccccaggaaa gccgtgctgt 1140 cagcatcagc atcatcgaac caggcgagga ggggccaact gcaggctctg tgggaggatc 1200 agggctggga actctagccc tggccctggg gatcctggga ggcctgggga cagccgccct 1260 gctcattggg gtcatcttgt ggcaaaggcg gcaacgccga ggagaggaga ggaaggcccc 1320 agaaaaccag gaggaagagg aggagcgtgc agaactgaat cagtcggagg aacctgaggc 1380 aggcgagagt agtactggag ggccttgagg ggcccacaga cagatccca 1429 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 2 atgactctac ccacggcaag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 3 gatctcgctc ctggaagatg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for AQP5 <400> 4 tctgggtagg gcctattgtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for AQP5 <400> 5 cagctcgatg gtcttcttcc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for RAGE <400> 6 agcttcagtc tgggccttc 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for RAGE <400> 7 cagctgaatg ccctctgg 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SP-A <400> 8 ctaagtgctg ccctctgacc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for SP-A <400> 9 aggagccata catgccaaac 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for IL-6 <400> 10 cccttcagga acagctatga a 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for IL-6 <400> 11 acaacatcag tcccaagaag g 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for IL-1b <400> 12 cacctctcaa gcagagcaca g 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for IL-1b <400> 13 cacctctcaa gcagagcaca g 21

Claims (9)

As a method of determining drowning as the cause of the subject and determining the elapsed time of drowning,
(a) measuring the expression level of RAGE (SEQ ID NO: 1, GenBank Accession No. DQ104254.1) from the sample isolated from the drowning body as a subject;
(b) comparing the RAGE expression level measured in step (a) with the RAGE expression level of a normal control sample;
(c) determining the death of the subject as drowning when it is confirmed that the RAGE expression level of the subject is higher than the RAGE expression level of the normal control sample; And
(d) measuring the expression level of sRAGE from the RAGE of the subject to determine the elapsed time of drowning of the subject; including,
The normal control sample is a method of determining drowning as a cause of death of the subject, and determining the elapsed time of drowning, characterized in that the sample is a sample of a body that has not drowned. delete The method of claim 1,
In step (a), the expression level measurement method is RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, Western Blotting, ELISA, enzyme. Immunoassay (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion method, octeroni immune diffusion method, rocket immune electrophoresis, immunohistochemistry, immunoprecipitation, complement fixation assay , Flow cytometry (FACS) and a method for determining drowning as a cause of death of the subject, characterized in that at least one method selected from the group consisting of protein chips and the elapsed time of drowning. The method of claim 1,
To judge seawater drowning as a cause of the subject,
Comprising the step of comparing the expression level of RAGE of the subject measured in step (a) with the expression level of RAGE of the freshwater drowned sample, determining drowning as a cause of the subject and determining the elapsed time of drowning. Way. delete delete delete As a judgment method that can predict the elapsed time after death from a subject due to drowning,
(a) measuring the expression level of sRAGE from the sample isolated from the drowning body as a subject;
(b) comparing the expression level of sRAGE measured in step (a) with the standard expression level of sRAGE by day after drowning; And
(c) estimating the postmortem interval (PMI) of the subject; includes,
The standard expression level of sRAGE for each day after drowning is the standard expression level of sRAGE for each post-mortem day measured from samples separated from control drowned bodies with different days after drowning. How to determine the elapsed time of drowning. The method of claim 8,
The step of (c) estimating the postmortem interval (PMI) of the subject,
A trend line with the standard expression level of sRAGE for each post-mortem day measured from samples separated from the control drowning bodies identified as different drowning days, and the x-axis as the post-mortem date and the y-axis as the sRAGE standard expression level for each elapsed date. Making steps; And
Substituting the standard expression level of sRAGE of the subject on a trend line; determining drowning as a cause of death of the subject and determining the elapsed time of drowning.

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