CN114622010B - Marker for identifying lethal hypothermia based on brown fat and forensic application - Google Patents

Marker for identifying lethal hypothermia based on brown fat and forensic application Download PDF

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CN114622010B
CN114622010B CN202210371404.6A CN202210371404A CN114622010B CN 114622010 B CN114622010 B CN 114622010B CN 202210371404 A CN202210371404 A CN 202210371404A CN 114622010 B CN114622010 B CN 114622010B
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death
freeze
hypothermia
adipose tissue
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赵锐
王宁
张淼
王林林
王鹏飞
张富源
郭相伸
曹志鹏
黄平
官大威
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China Medical University
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Abstract

The invention belongs to the field of forensics and biological medicines, and particularly relates to a marker for identifying lethal hypothermia based on brown fat and forensic application. According to the method for diagnosing freeze death based on the DIA proteomics technology, which is provided by the invention, the proteomics technology can detect various DEPs simultaneously, the DEPs can reveal complex and changeable chemical reactions involved in the freeze death process, can help people to understand the pathophysiological mechanism of freeze death more deeply, and is beneficial to better relieving hypothermia caused by cold exposure. The biological marker for rapidly diagnosing freeze death is closely related to freeze death, has the characteristics of relatively small influence of post-death change, small difference between parallel samples, convenient acquisition of required reagents, simple and rapid operation, high cost efficiency and the like, and is favorable for transforming fruits.

Description

Marker for identifying lethal hypothermia based on brown fat and forensic application
Technical Field
The invention belongs to the field of forensics and biological medicines, and particularly relates to a marker for identifying lethal hypothermia based on brown fat and forensic application.
Background
The human body is in Cold environment for a long time, and the heat dissipation capacity is far more than the heat generation capacity and the physiological limit of the human body temperature regulation due to the insufficient heat preservation of the human body, and the Death caused by the disturbance of the metabolism and the physiological functions of the substance is called as freeze Death (Death from Cold) or lethal hypothermia (Fatal Hypothermia). The occurrence of frostbite or freeze death is closely related to geographical environment, organism factors and the like. The lower the ambient temperature, the greater the wind speed and humidity, the higher the incidence of frostbite or freeze-death. One research report based on U.S. population surveys indicates that about 2000 americans die annually during 2006-2010 in association with extremely cold weather, with about 63% of the deaths being associated with exposure to cold environments. In recent years, with the popularization of outdoor entertainment activities, the occurrence rate of frostbite or freeze death has been increasing year by year in outdoor activities such as mountain climbing, skiing, hiking, camping, and the like. In addition, the occurrence of frostbite or freeze death is also closely related to factors such as age, hunger, fatigue, trauma, diseases, alcohol, drugs or individual cold tolerance differences.
In forensic practice, the incidence of death due to cold exposure is high in cold areas or in winter, spring and low temperature seasons. The frozen person can have abnormal desquamation phenomenon caused by central nervous system dysfunction due to hypothermia or bruise or contusion at the protruding part of the body due to falling and falling, and the phenomenon can sometimes generate a certain degree of interference on the diagnosis of frozen death and influence the judgment of the properties of the case. Currently, diagnosis of freeze death by traditional forensic identification often relies on gross changes in left and right ventricular blood color, bright red cadaveric plaques, acute gastric mucosal erosion, winieski plaques (Wischnevsky gastric lesions), and iliac psoas and pancreatic bleeding, but these changes lack specificity. In recent years, many research teams at home and abroad have made great progress in exploring specific diagnosis of freeze death. In 2002, G.Teresinski et al found that ketone bodies were present in high concentrations in blood, urine, and vitreous bodies of dead individuals in cases where low temperatures caused death. In 2009, jakubeniene, M, etc., found a significant increase in sodium content in skeletal muscle of the frozen. In 2012, oda, team T found that ATP binding proteins, amino acid metabolic proteins, urea cycle proteins, oxidative stress proteins, anti-apoptotic proteins, and negative apoptosis regulatory proteins were all significantly down-regulated in rat liver endoplasmic reticulum and mitochondria at low temperature. Our research team has investigated the possibility of pulmonary edema fluid as a freeze-death-specific diagnostic marker in 2019 using fourier-exchanged infrared spectroscopy in combination with chemometrics. In addition, there are research teams applying the second generation sequencing technique (Next Generation Sequencing, NGS) to find that the expression of 91 mRNA in ilium muscle tissue is dramatically increased under severe low temperature conditions during skeletal muscle thermogenesis, wherein mRNA of connective tissue growth factor CTGF, JUNB protooncogene, AP-1 transcription factor subunit, nuclear receptor subfamily members NR4A1, SDC4, etc. involved in muscle regeneration, tissue repair, lipid metabolism related proteins all appear to be significantly differentially expressed during low temperature injury. At present, the latest research shows that the application of ATR-FTIR spectrum analysis to the spectral change of lipid carbohydrate nucleic acid in plasma can accurately diagnose freeze death, but the related method has long time consumption, high cost and complex operation, and is not beneficial to popularization in primary forensic work. Therefore, in forensic practice, a diagnosis mode with accurate result, simple operation and high cost efficiency is found, which is more beneficial to basic forensic workers to improve the accuracy of freeze death diagnosis.
Thermogenesis is an important homeostatic mechanism necessary for mammalian survival and normal physiological function. The central nervous system has a body temperature regulating effect, in which brown adipose tissue (Brown Adipose Tissue, BAT) is involved in non-shivering thermogenesis. Skeletal muscle is in turn involved in shivering thermogenesis with a decrease in body temperature, and maintains body temperature in conjunction with non-shivering thermogenesis in which brown adipose tissue is involved. Studies have shown that when humans and rodents are exposed to cold environments, the sympathetic nervous system can be activated by cold stimulation, causing sympathetic nerves to release norepinephrine and activate β -adrenergic receptors (β -AR), increasing mitochondrial content, metabolic rate, and expression of thermogenic proteins and genes, thereby activating BAT thermogenic activity and improving cold tolerance. Therefore, on the basis of the early theory, the expression of differential proteins (Differentially Expressed Proteins, DEPs) in brown fat of the frozen mice is detected by utilizing a frozen mice model and adopting technical means such as proteomics, the biological markers with the largest differential amount and stable expression are screened, and the biological markers are further verified in a human frozen case sample, so that a new method is provided for forensic diagnosis of frozen mice.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a biomarker for diagnosing freeze death based on brown fat and a method for rapidly diagnosing freeze death. The invention proves the feasibility of diagnosing the freeze death by using brown fat, and can rapidly and accurately diagnose the freeze death by using the screened biological markers.
In order to achieve the above purpose, the present invention provides the following technical solutions.
The present invention provides a product for identifying lethal hypothermia, said product comprising a method of detection and calculation of biomarkers of pathological changes in brown adipose tissue, characterized in that said brown adipose tissue biomarkers comprise the diameter and/or number of fused lipid droplets in brown adipose tissue.
The invention also provides a product for identifying lethal hypothermia, the product comprising a detection reagent for brown adipose tissue biomarkers, characterized in that the brown adipose tissue biomarkers comprise the expression levels of RAB1B and/or GMFB in brown adipose tissue.
The invention also provides a product for identifying lethal hypothermia, said product comprising a method of detection and calculation of brown adipose tissue biomarkers, characterized in that said brown adipose tissue biomarkers comprise a combination of fusion lipid droplet diameter and/or number in brown fat and increased expression levels of RAB1B and GMFB.
Further, the product is a diameter and/or number of fused lipid droplets detected in brown adipose tissue by H & E staining, sudan III staining or transmission electron microscopy.
Still further, the diameter and/or number of intracellular fusion lipid droplets increases significantly in the lethal hypothermia group.
Further, the product is a detection of the expression level of RAB1B and/or GMFB in brown adipose tissue by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, northern blotting, chip, high throughput sequencing platform, immunohistochemical staining, western blotting, or enzyme-linked immunosorbent assay.
Still further, the expression level of RAB1B and/or GMFB is highly expressed in the lethal hypothermia group.
Further, the product contains a specific primer for amplifying RAB1B and/or GMFB genes, a probe hybridized with nucleotide sequences of RAB1B and/or GMFB genes or an antibody specifically binding to RAB1B and/or GMFB proteins.
Further, the specific primer sequences for amplifying RAB1B and/or GMFB genes in the products are as follows:
Gmfb：Forward 5’-AGAAACCCACAATGCTGCTATT-3'
Reverse5’-TACGAGACTCTGCCATCATCA-3’
Rab1b：Forward 5’-GAACCCCGAATATGACTACCTG-3’
Reverse5’-CGAATCTTGAAATCCACACCG-3’。
further, the brown adipose tissue biomarker in the product is used for preparing a detection reagent or a detection kit for identifying the lethal hypothermia or making a detection judgment industry standard.
Compared with the prior art, the invention has the beneficial effects.
(1) The invention provides a method for primarily identifying whether to undergo cold exposure or not based on the diameter and/or the number of the fused fat droplets in brown fat, and the operation method is simple and convenient.
(2) The invention provides a biological marker for rapidly diagnosing freeze death, wherein the expression change of the biological marker is closely related to freeze death, and the biological marker has the characteristics of relatively small influence of post-death change, small difference between parallel samples, convenient acquisition of required reagents, simple and rapid operation, high efficiency-cost ratio and the like, and is beneficial to fruit transformation.
(3) According to the method for diagnosing freeze death based on the DIA proteomics technology, which is provided by the invention, the proteomics technology can detect various DEPs simultaneously, the DEPs can reveal complex and changeable reactions involved in the freeze death process, can help people to know the pathophysiological mechanism of freeze death more deeply, and is beneficial to better curing hypothermia caused by cold exposure.
(4) The method for rapidly diagnosing freeze death based on the brown fat biological marker provided by the invention utilizes the proteomics technology to analyze a large number of DEPs, combines repeated verification of the molecular biology technology, can provide an accurate, rapid and economic identification means for forensic actual work, and is expected to provide more beneficial help for case detection in forensic practice.
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FIG. 1 results of morphological observation of brown adipose tissue of mice.
FIG. 2H & E staining results of brown adipose tissue of mice.
FIG. 3H & E statistics of brown adipose tissue of mice.
FIG. 4 results of Sudan III staining of brown adipose tissue of mice.
FIG. 5 results of brown adipose tissue electron microscopy of mice.
Figure 6 is based on DIA proteomic analysis.
FIG. 7 shows Wen diagram for up/down protein numbers expressed by different proteins (DEPs) between frozen groups (4 ℃ and-20 ℃) and control groups.
FIG. 8 uses GO functional analysis to analyze the relevant biological processes of Differentially Expressed Proteins (DEPs) in frozen groups (4℃and-20 ℃) mainly involving cellular processes, metabolic processes, biological regulation, etc.
FIG. 9 shows DEPs of pre-20 expressed in frozen groups (4℃and-20 ℃) using KEGG Top20 analysis.
FIG. 10 shows volcanic analysis of DEPs in frozen groups (4 ℃ C. And-20 ℃ C.).
FIG. 11 results of screening for biological markers of diagnostic interest for freeze-death in proteins co-expressed in the 4℃and-20℃freeze-death group.
FIG. 12 Western Blotting of 7 different proteins in brown adipose tissue of mice.
FIG. 13 Western Blotting statistics of 7 different proteins in brown adipose tissue of mice.
FIG. 14 results of qPCR detection of 7 different proteins in brown adipose tissue of mice.
FIG. 15 results of brown adipose immunohistochemical staining of mice.
FIG. 16 results of immunohistochemical staining of human brown adipose tissue.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will aid in the understanding of the present invention, but are merely illustrative of the invention and the invention is not limited thereto. The methods of operation in the examples are all conventional in the art.
Example 1 brown fat morphology identifies lethal hypothermia.
After 36 male C57BL/6J mice of 1.6 weeks of age were adaptively bred at room temperature for 2 weeks by means of free diet, 12 hours of circadian rhythm, etc., 8-week-old mice (body weight 18.3-23.1 g) were randomly divided into a control group (Ctrl group) and an experimental group (frozen dead groups at 4 ℃ and-20 ℃) each of 12. Control mice were anesthetized with pentobarbital sodium and then allowed to die in a 4 ℃ climatic chamber (50% relative humidity) for 3.5 hours (taking the time average of 4 ℃ mice death in the experimental group), and the mice in the experimental group were placed in a 4 ℃ climatic chamber or-20 ℃ climatic chamber (50% relative humidity) until death.
After the molecular biology related samples are irrigated by normal saline, the brown adipose tissue of the scapular triangle of each group of mice is extracted and put into liquid nitrogen for freezing, and then the samples are quickly transferred into a deep freezing ice box at the temperature of minus 80 ℃ for preservation and detection.
The morphological related sample is not required to be irrigated, and is placed in 4% paraformaldehyde solution for fixing for 24 hours after the materials are obtained, and then related treatment is carried out according to the experimental requirements.
The experimental results are shown in FIG. 1, and compared with Ctrl group, the brown fat volume of mice in frozen groups (4 ℃ C. Group and-20 ℃ C.) is reduced, compact, and the veins are dilated and blood is stasis.
2. Tissue section preparation and H & E staining.
After the different groups of animal model samples were fixed in 4% paraformaldehyde solution for 24 hours, the running water was slowly rinsed for 12 hours.
(1) The brown fat of the mice in the control group is soaked in 80% ethanol solution for overnight at 4 ℃; soaking in 95% ethanol solution at room temperature for 2 hr; soaking 100% absolute ethyl alcohol I, II for 15 minutes at room temperature respectively for dehydration; the xylene is transparent for 30 seconds to 1 minute; soaking paraffin wax I, II and III (52-54 ℃) for 1 hour; paraffin (60-62 ℃) embedding.
(2) Brown fat of mice in the frozen dead group at 4 ℃ in the experimental group is soaked in 80% ethanol solution at 4 ℃ overnight; soaking in 90% ethanol solution at room temperature for 1 hr and 20 min; soaking in 95% ethanol solution at room temperature for 40 min; soaking 100% absolute ethyl alcohol I, II for 10 minutes at room temperature respectively for dehydration; xylene is transparent for 1 minute; soaking paraffin wax I, II and III (52-54 ℃) for 1 hour; paraffin (60-62 ℃) embedding.
(3) The brown fat of the mice frozen at-20 ℃ in the experimental group is soaked in 80% ethanol solution for overnight at 4 ℃; soaking in 95% ethanol solution at room temperature for 2 hr; soaking 100% absolute ethyl alcohol I, II for 30 minutes at room temperature respectively for dehydration; the xylene is transparent for 30 seconds to 1 minute; soaking paraffin wax I, II and III (52-54 ℃) for 1 hour; paraffin (60-62 ℃) embedding.
Each group of wax blocks was sliced to 5 μm for use. H & E staining was routinely performed according to its standard procedure.
H & E staining results indicated that the number of fusion lipid droplets was significantly increased, the volume was increased, the veins were distended, and blood was stasis in the frozen groups (4 ℃ and-20 ℃) compared to Ctrl groups (as shown in fig. 2). (bar=100 μm).
Under the 400-time visual field of H & E sections (figure 3), 3 different visual fields of brown fat of each group are randomly selected, the diameter average number of fat drops of a Ctrl group is taken as a reference standard, the diameter size of the fat drops and the number of the fat drops of frozen dead groups (4 ℃ group and-20 ℃ group) are counted, and the result shows that the diameter and the number of the fused fat drops of the frozen dead groups are obviously increased compared with those of the Ctrl group. (#: P < 0.01).
3. Sudan III staining (Sudan III Staining).
Different groups of mouse brown fat were fixed in 4% paraformaldehyde solution for 24 hours; precipitating sugar with 15%, 20%, 30% I, 30% II sucrose solution for 24 hr respectively; slowly freezing at-20deg.C for 30 min; after embedding with OCT in a frozen microtome, a frozen section of 20 μm was prepared for use (box temperature-35 ℃ C., cutter head temperature-35 ℃ C.). And (3) after the prepared frozen slices are dried at room temperature, fixing the frozen slices in a formaldehyde-calcium solution for 10 minutes, rinsing the frozen slices in distilled water for 1 minute, dip-dying the frozen slices in hematoxylin dye liquor for 2 minutes, rinsing the frozen slices in tap water for 1 minute, rinsing the frozen slices in distilled water for 1 minute, dip-dying the frozen slices in Sudan III dye liquor for 30 minutes, rinsing the frozen slices in 70% ethanol solution for 15 seconds to remove floating color, and sealing the frozen slices with glycerinum glue.
The sudan III staining results indicated (fig. 4) that orange fusion-like lipid droplets appeared in both frozen groups (4 ℃ and-20 ℃) compared to Ctrl groups, consistent with H & E staining results. (bar=100 μm).
4. Transmission electron microscopy (Transmission electron microscope, TEM).
Different groups of mice were taken 1mm brown fat 3 Fixing the tissue in 2.5% glutaraldehyde fixing solution for 6 hr, and rinsing with buffer solution for 3 times; fixing 1% Russian acid for 1 hour, and rinsing 3 times by using buffer solution; respectively dehydrating with 30%, 50% and 70% ethanol for 20 min, respectively dehydrating with 80%, 90% and 100% acetone for 20 min; embedding for 1 hour by using acetone/embedding agent (1/1), and then embedding for 3 hours by using acetone/embedding agent (1/3); curing for 12 hours by using a temperature box at 35 ℃ and 45 ℃ respectively, and then curing for 24 hours by using a temperature box at 60 ℃; the ultrathin section machine is used for preparing 50nm sections for later use; after 3% uranium acetate-lead citrate is dyed for 15 minutes, JEM-1400Flash transmission electron microscope is used for shooting.
The electron microscope results showed (FIG. 5) that compared with Ctrl group, obvious mitochondrial damage (shown as ≡) and lipid droplet fusion (shown as ≡) appear in brown adipocyte interstitial of frozen dead group (4 ℃ group and-20 ℃ group)Shown). (bar=1 μm)
Example 2 screening for the presence of differentially expressed protein markers in brown fat based on DIA proteomics techniques.
Proteomic analysis:
after the DIA technical principle is applied, the steps of sample preparation, spectrogram database establishment, DIA data acquisition and the like are carried out, qualitative and quantitative analysis of protein is carried out, the quality control of the original mass spectrum data is carried out by adopting QuiC (Biognosys) software, and the database is built by adopting Pulsar software for the data obtained in the DDA acquisition mode, and the analysis of protein annotation, sample relation, protein difference and the like are carried out. The library identification standard is as follows: precursor Threshold 1.0%FDR,Protein Threshold 1.0%FDR. DEPs analysis was performed based on the quantitative results of brown intrafat protein in control and experimental mice. To meet the need for freeze-death method medical diagnostics, we screened DEPs co-expressed in freeze-death groups at 4℃and-20℃and based on this, were expressed in |log 2 FC|>1.6 as a standard to obtain 7 candidate protein indexes, namely GMFB, KDM1A, DDX6, RAB1B, SHMT1, CLPTM1 and LMF1. In combination with forensic practice, to avoid post-mortem changes and the effects of false negatives on outcome decisions, the protein indicators selected should meet the requirements of stable expression and relatively small molecular weight within the group. Based on the above screening criteria, all the 7 indexes meet the screening conditions and can be listed as candidate biological markers for diagnosing freeze death. The results of the relevant proteomic screening are shown in FIGS. 6-11.
Example 3. Validation of screening for the presence of differentially expressed proteins in brown fat based on DIA proteomics techniques.
1. Protein extraction:
according to INVENT Minute TM Total protein extraction kit for adipose tissue and cell (AT-022) the protein extraction was performed in the instruction procedure. The brief steps are as follows: taking 80mg of brown fat sample, wrapping with filter paper, lightly pressing the tissue, and removing part of lipid components; transferring the rest tissues into a 1.5mL centrifuge tube with the kit, adding 100mg of protein extraction grinding powder, adding 50 mu L of buffer solution A, and fully grinding the tissues to be uniform slurry; 200. Mu.L of buffer A was added again and the grinding continuedGrinding for 30 seconds; centrifuging at 2000rpm for 1 minute, and transferring the supernatant into a centrifugal column; after uncapping and incubating for 20 minutes in an environment of minus 20 ℃, rapidly transferring to a low-temperature centrifuge (4 ℃ and 2000 rpm), uncapping and centrifuging for 2 minutes, and discarding a centrifugal column; adding buffer solution B with the volume capacity of 1/10 of the protein solution into the filtered protein solution, fully vibrating and uniformly mixing, and storing in a deep freezing refrigerator at-80 ℃ for standby.
2.Western Blotting verification:
brown fatty proteins extracted by the above method were operated according to Western Blotting standard procedures, taking care during the experiment that the primary antibody used was incubated overnight at 4 ℃. The actual antibodies and corresponding concentrations used in the present invention are now described as follows:
。
western Blotting verification results show (FIG. 12), compared with the Ctrl group, only two differential expression proteins of RAB1B and GMFB have the same proteomic expression trend, and the expression of the two differential expression proteins is obviously up-regulated in the freezing groups (4 ℃ group and-20 ℃ group), so that the two indexes can be used as stable biological markers for diagnosing freezing.
Western Blotting statistics showed (FIG. 13) that the expression of both RAB1B and GMFB proteins was significantly elevated in the frozen groups (4℃and-20 ℃) compared to the Ctrl group, whereas the remaining five proteins (KDM 1A, CLPTM1, LMF1, DDX6, SHMT 1) were not statistically different or were not completely consistent with proteomics results. P < 0.05; # P < 0.01;:. Ns: no statistical significance).
qPCR detection.
Designing related primers for 7 candidate indexes screened by proteomics, wherein the sequence information of the primers is as follows:
。
placing 50-100mg brown adipose tissue into 1.5mL centrifuge tube, cutting tissue with sterile scissors, adding 1mL TRIzol, and homogenizingThe slurry was allowed to stand at room temperature for 5min. Adding 0.2mL of chloroform, oscillating for 15s, and standing for 2min; centrifuging at 4deg.C, 12000g×15min, and collecting supernatant. 0.5mL of isopropyl alcohol was added, and the liquid in the tube was gently mixed and allowed to stand at room temperature for 10min. Centrifuge at 4℃12000 g.times.10 min, discard supernatant. 1mL of 75% ethanol was added and the precipitate was gently washed 3 times. The supernatant was discarded at 4℃and 7500 g.times.5 min. Air drying, adding proper amount of DEPC H 2 O-dissolving (dissolving promotion at 65 ℃ for 10-15 min) RNA for standby. Taking 2 mu L of RNA solution, and measuring the concentration of RNA of a sample and the absorbance value (OD 260, OD 280) of the sample at 260nm and 280nm by using NanoDropTMONE, wherein the ratio of OD260/OD280 is 1.8-2.0, so that the purity of the sample to be measured is qualified. In use, the samples were diluted to 250 ng/. Mu.L with DEPC water depending on the RNA concentration of the samples. The qPCR was performed according to the instructions of TaKaRa kit (Cat#RR047A, cat #RR820A), including cDNA synthesis, PCR amplification, etc., and the results were shown in FIG. 14.
The results of qPCR of the brown adipose tissue of mice showed (fig. 14) that the expression of Rab1b and Gmfb mRNA was significantly elevated and statistically different in both the freeze-dead groups (4 ℃ and-20 ℃ groups) compared to Ctrl groups, with a trend consistent with Western Blotting, whereas mRNA of the remaining five proteins (Kdm 1a, clptm1, lmf1, ddx6, shmt 1) were not statistically different or consistent with protein trend. P < 0.05; # P < 0.01;:. Ns: no statistical significance).
4. And (5) verifying the brown adipose tissue immunohistochemical staining of the mice.
The preparation of tissue sections and H & E staining "according to the embedding conditions described in example 1"2 was performed according to the standard procedure for immunohistochemical staining, the invention relates to antibodies and corresponding concentrations as follows:
。
the color development time of the present invention has a direct effect on the result, and thus a special explanation is made: RAB1B color development time is 5 seconds; the GFMB development time was 2 seconds.
The results of the immunohistochemical staining of brown fat of mice show (as shown in FIG. 15) that RAB1B and GMFB are strongly positive expressed in brown fat of frozen groups (4 ℃ and-20 ℃ groups) compared with Ctrl groups, and the results are consistent with Western Blotting. (bar=100 μm).
5. And (5) verifying immune histochemical staining of brown adipose tissue of a human body.
The invention performs the verification of the brown adipose tissue immunochemical staining of the human body on the premise of agreeing with the ethical committee of the university of Chinese medical science. The operation steps are as follows:
(1) Extracting brown fat of a triangle area in a human neck, and fixing the brown fat in 4% paraformaldehyde solution for 24 hours;
(2) Flushing with running water for 12 hours;
(3) 60% ethanol solution, soaking for 12 hours at 4 ℃;
(4) 85% ethanol solution, soaking for 1 hour at room temperature;
(5) 95% ethanol solution I, II, each soaked for 2.5 hours at room temperature;
(6) 100% ethanol solutions I, II and III are respectively soaked for 2 hours at room temperature;
(7) Xylene I, II and III are respectively soaked for 50 minutes and transparent at room temperature;
(8) Paraffin wax I, II and III (52-54 ℃) are soaked for 1 hour respectively;
(9) Embedding paraffin (60-62 ℃);
(10) Preparing a 5 mu m thick slice for later use by a slicer;
(11) Immunohistochemical staining was performed according to conventional standard procedures.
The invention relates to antibodies and corresponding concentration specifications as follows:
。
the color development time of the present invention has a direct effect on the result, and thus a special explanation is made: RAB1B development time was 15 seconds and GFMB development time was 20 seconds.
The results of immunohistochemical staining of human brown adipose tissue showed that (fig. 16) compared with the human brown adipose sample of non-freezing case, both RAB1B and GMFB were expressed strongly positive in brown adipose of freezing case, proving that RAB1B and GMFB can be used as biological markers for diagnosing freezing. (bar=100 μm).
In summary, the biological markers referred to in the present invention are specific for diagnosis of freeze death. Therefore, in forensic practice, the specific biological markers related to the invention can be subjected to immunohistochemical staining by utilizing brown adipose tissue of a human body, so that forensic diagnosis can be accurately, quickly and conveniently made on freeze death.
The applicant states that the process of the present invention is illustrated by the following examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the following process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
SEQUENCE LISTING
<110> university of medical science in China
<120> a marker for identification of lethal hypothermia based on brown fat and forensic applications
<130> 2022-03-29
<160> 16
<170> PatentIn version 3.5
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Claims (4)

1. Use of an agent that detects a biomarker in brown adipose tissue, said biomarker being the diameter and number of fused lipid droplets in brown adipose tissue, and the expression levels of RAB1B and GMFB in brown adipose tissue, in the manufacture of a product for identifying lethal hypothermia.
2. The use according to claim 1, wherein the diameter and number of said fusogenic lipid droplets are both significantly increased in a lethal hypothermia group; the expression levels of RAB1B and GMFB are highly expressed in the lethal hypothermia group.
3. The use according to claim 1, wherein said product comprises specific primers for amplification of RAB1B and GMFB genes.
4. The use according to claim 3, wherein the specific primer sequences for amplifying the RAB1B and GMFB genes are shown in SEQ ID No.1-SEQ ID No. 4.
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CN109078188A (en) * 2018-08-21 2018-12-25 浙江大学 A kind of action target spot and anti-tumor drug of anti-tumor drug
WO2021225525A1 (en) * 2020-05-08 2021-11-11 Agency For Science, Technology And Research A biomarker for brown fat activity detection

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US20150147292A1 (en) * 2011-09-15 2015-05-28 Joslin Diabetes Center, Inc. Novel surface markers for adipose tissue

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Publication number Priority date Publication date Assignee Title
CN109078188A (en) * 2018-08-21 2018-12-25 浙江大学 A kind of action target spot and anti-tumor drug of anti-tumor drug
WO2021225525A1 (en) * 2020-05-08 2021-11-11 Agency For Science, Technology And Research A biomarker for brown fat activity detection

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