CN116837086A - Use of markers in heat sensitive diagnosis or prediction - Google Patents
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Abstract
The invention discloses application of a marker in heat-sensitive diagnosis or prediction, in particular relates to a BCHE biomarker, and discovers that the abundance of the BCHE in serum of a heat-stressed subject is reduced for the first time, and whether the subject is heat-sensitive can be diagnosed by detecting the expression level of the BCHE, so that the convenient diagnosis of the subject is realized, and the method has higher practical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of a biomarker in heat-sensitive diagnosis or prediction.
Background
Heat stress refers to the sum of non-specific physiological responses of the body at high ambient temperatures to any demands placed on the body by the hot environment. The physiological reaction of human body under heat stress is destroyed in the temperature stress environment, and the human body generates a series of complex physiological and psychological changes. The physical function of the human or animal body is affected by heat stress.
Butyrylcholinesterase (BChE), also known as serum cholinesterase and pseudo cholinesterase, is widely distributed in the human central system and peripheral tissues including brain, skin, plasma, skeletal muscle, and the like; wherein brain BChE is highly expressed in amygdala, hippocampus and pituitary. Early BChE gene-silenced humans were not otherwise sensitive to individual anesthetics at the time of surgery, and early researchers thought that BChE physiological effects were not important and less studied. However, as Broomfield et al 1991 demonstrated that BChE has neuroprotective function against neurotoxic agents, from this point BChE has received extensive attention from researchers.
Disclosure of Invention
In view of the state of the art described above, the present invention aims to provide a biomarker for diagnosing or prognosticating heat sensitivity.
In a first aspect, the invention provides the use of an agent for detecting a BCHE biomarker in a sample in the manufacture of a product for diagnosing or prognosticating whether a subject is heat stress sensitive.
The terms "biomarker" or "biomarker" as used in the present invention are mutually generic and refer to a gene or gene product that is a target for modulating one or more phenotypes of interest (e.g., a phenotype of interest in a myeloid cell). In this case, the term "biomarker" is synonymous with "target". However, in some embodiments, the term also encompasses measurable entities that have been determined to be indicative of targets of a target outcome, e.g., one or more diagnostic, prognostic, and/or therapeutic outcome (e.g., for modulating an inflammatory phenotype, cancer status, etc.). In other embodiments, the term also encompasses compositions that modulate a gene or gene product, including anti-gene product antibodies and antigen-binding fragments thereof. Thus, biomarkers can include, but are not limited to, nucleic acids (e.g., genomic nucleic acids and/or transcribed nucleic acids), proteins, and antibodies (and antigen binding fragments thereof).
In some embodiments, a biomarker may be or include a marker of a particular disease state or stage, or a marker of the likelihood that a particular disease, disorder, or condition is likely to develop. In some embodiments, the biomarker may be a marker that includes a particular disease or treatment outcome or likelihood thereof.
Further, the sample comprises one of a tissue sample, primary or cultured cells or cell lines, cell supernatant, cell lysate, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, or a combination thereof.
Further, the test sample is serum.
Further, the subject includes a human or non-human mammal.
Further, the subject is a human.
The term "sample" as used herein refers to any sample obtained from an individual suffering from a disease to be treated by the methods of the present invention, including tissue samples (such as tissue slices and tissue needle biopsies); a cell sample (e.g., a cytological smear (such as a Pap or blood smear) or a cell sample obtained by microdissection); bone marrow samples (whole, whole cells, or a sub-population of cells therein); or a cell, fragment or organelle (such as obtained by lysing the cell and separating its components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle punch biopsy), nipple aspirate, vaginal fluid, saliva, swabs (such as oral swabs), or any material containing biomolecules derived from a first biological sample.
The term "subject" as used herein refers to an animal, vertebrate, non-human mammal or human, especially a person administering an agent, e.g., for experimental, diagnostic and/or therapeutic purposes or obtaining a sample or performing a procedure. In some embodiments, the subject is a mammal, such as a human, a non-human primate, a rodent (e.g., a mouse or a rat), a domestic animal (e.g., a cow, sheep, cat, dog, and horse), or other animal (e.g., a camel and a camel). In some embodiments, the subject is a human. The term "subject" is interchangeable with "patient".
Further, the reagent includes a primer or probe that specifically detects the expression level of the BCHE gene, or a reagent that specifically binds to a protein encoded by the BCHE gene.
The term "agent" as used herein refers to any small protein or other compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.
The term "primer" as used herein refers to an initial nucleic acid fragment, typically an RNA oligonucleotide, DNA oligonucleotide or chimeric sequence that is complementary to all or part of the primer binding site of a target nucleic acid molecule. The primer strand may comprise natural, synthetic or modified nucleotides. The lower limit of primer length is the minimum length required to form a stable duplex under the conditions of the nucleic acid amplification reaction.
The term "probe" as used herein refers to a synthetic or biological nucleic acid that contains a specific nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. One probe may be connected to a plurality of tag portions. Preferred moieties are identifying labels, such as fluorophores. The labeled probe may also comprise a plurality of different nucleic acid sequences, each labeled with one or more marker moieties. Each flag portion may be the same or different. It may be beneficial to label different probes (e.g., nucleic acid sequences) each having a different tag moiety. This can be achieved by having a single distinguishable portion on each probe. For example, probe A is attached to the X moiety and probe B is attached to the Y moiety. Alternatively, probe A is attached to the X and Y moieties and probe B is attached to the Z and W moieties. Alternatively, probe A is attached to the X and Y moieties and probe B is attached to the Y and Z moieties. All of the probes "A" and "B" described above will be distinguishable and uniquely labeled.
Further, the product comprises a reagent capable of detecting a BCHE biomarker, a kit, a chip, a nucleic acid membrane strip or a test paper comprising the reagent.
Further, the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the BCHE gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the BCHE protein.
Further, the kit includes reagents for detecting the expression level of BCHE gene or protein by western blotting, ELISA, radioimmunoassay, oxter lony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip assay.
The term "kit" as used in the present invention refers to a collection of compounds, means or reagents that may or may not be packaged together. The components of the kit may be contained in separate vials (i.e., the kit as a separate part) or provided in a single vial. Furthermore, it should be understood that the kit of the invention is preferably used for carrying out the above mentioned methods or uses. Preferably, it is envisaged that all components are provided in a ready-to-use manner for carrying out the above method or use. Furthermore, the kit preferably comprises instructions for carrying out the method or use. These instructions may be provided by a user manual in paper or electronic form. Furthermore, the manual may include instructions for administration and/or instructions for dosage using the kits of the invention.
Further, the chips include a gene chip including an oligonucleotide probe for BCHE gene for detecting a BCHE gene transcription level and a protein chip including a specific antibody or ligand of BCHE protein; the test paper comprises a gene test paper and a protein test paper.
The invention also provides a product for predicting or diagnosing whether a subject is susceptible to heat stress, the product comprising reagents for detecting the expression level of a BCHE gene or protein in a sample.
The term "diagnosis" as used herein refers to a method by which one of skill in the art can estimate and/or determine whether a patient has a given disease or disorder, and is an aid diagnosis. In the context of the present invention, "diagnosis" includes the use of the detection results for the heat stress sensitive biomarkers of the invention, optionally together with other clinical features, to achieve diagnosis (i.e., yes or no) of heat stress sensitivity from samples and assays obtained from the patient. Such a diagnosis is a "determination", which is not meant to imply that the diagnosis is 100% accurate. Thus, a measured level of biomarker on one side of the predetermined diagnostic threshold is indicative of a greater likelihood of disease occurrence in the subject than on the other side of the predetermined diagnostic threshold relative to the measured level.
Further, the subject includes a human or non-human mammal.
Further, the subject is a human.
Further, the sample comprises one of a tissue sample, primary or cultured cells or cell lines, cell supernatant, cell lysate, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, or a combination thereof.
Further, the test sample is serum.
Further, the product comprises a reagent capable of detecting a BCHE biomarker, a kit, a chip, a nucleic acid membrane strip or a test paper comprising the reagent.
Further, the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the BCHE gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the BCHE protein.
Further, the kit includes reagents for detecting the expression level of BCHE gene or protein by western blotting, ELISA, radioimmunoassay, oxter lony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip assay.
Further, the chips include a gene chip including an oligonucleotide probe for detecting a BCHE gene transcription level against the BCHE gene and a protein chip including a specific antibody or ligand of the BCHE protein.
The term "antibody" as used herein refers to a peptide or polypeptide derived from a substantially immunoglobulin based on a coding or immunoglobulin gene or fragment thereof that mimics or immunoglobulin genes, and is capable of specifically binding an antigen or epitope.
Further, the test paper comprises a gene test paper and a protein test paper.
The invention also provides a system for diagnosing or predicting whether a subject is sensitive to heat stress by using a biomarker BCHE, which comprises a result judging unit, wherein the obtained biomarker abundance is compared with an average value of the biomarker abundance detected in a heat stress sensitive individual sample.
Further, the system comprises a detection unit or a data processing unit.
Further, the detection unit is used for detecting individual information to obtain a detection result.
Further, the data processing unit is used for analyzing and processing the detection result of the detection unit.
The invention also provides a method of diagnosing or prognosticating whether a subject is susceptible to thermal stress, the method comprising the step of detecting the level of gene or protein expression of a biomarker as described hereinbefore in a sample from the subject.
The invention also provides a method of screening for a drug for therapeutic or prophylactic treatment of heat stress injury for non-therapeutic or non-diagnostic purposes, the method comprising the step of detecting the level of gene or protein expression of a biomarker as described hereinbefore in a sample from a subject.
The term "drug" as used herein refers to a mixture of one or more compounds or physiologically acceptable salts thereof with other chemical ingredients, such as physiologically acceptable carriers and excipients. The purpose of the drug is to facilitate administration of the compound to the organism. The medicaments herein also include pharmaceutically acceptable pharmaceutical carriers that do not cause significant irritation to the organism and do not abrogate the biological activity and properties of the administered compound, e.g.: diluents such as water and the like; fillers such as starch, sucrose, etc.; binders, such as cellulose derivatives, alginates, gelatin, polyvinylpyrrolidone; humectants, such as glycerol; disintegrants such as agar-agar, calcium carbonate and sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate and magnesium stearate, polyethylene glycol, and the like. In addition, other auxiliary materials such as flavoring agent and sweetener can be added into the above medicine.
Drawings
FIG. 1 is a ROC graph of BCHE before and after heat stress for a group 1 population;
FIG. 2 is a ROC graph of BCHE before and after heat stress for a group 2 population;
FIG. 3 is a graph of analysis of serum BCHE expression levels before and after heat stress in the total population;
FIG. 4 is a ROC graph of BCHE before and after heat stress in the general population;
FIG. 5 is a graph of ROC of control rats and heat stressed rats BCHE in group A rats;
FIG. 6 is a graph of ROC of control rats and heat stressed rats BCHE in group B rats;
FIG. 7 is a graph of analysis of serum BCHE expression levels in control rats and heat stressed rats in total rats;
FIG. 8 is a graph of ROC of control rats and heat stressed rats BCHE in total rats;
FIG. 9 is a graph showing ACHE standard curve detection and analysis of serum ACHE expression levels of control rats and heat stressed rats in total rats, wherein A is a graph showing ACHE standard curve detection and B is a graph showing analysis of serum ACHE expression levels of control rats and heat stressed rats in total rats.
Detailed Description
For a detailed description of the present invention, the following examples are provided for reference, it being understood and clearly recognized that the examples are intended for the purpose of illustration and are not intended to limit the scope of the present invention.
As used herein, the term "sensitivity" refers to the number (%) of truly positive patients relative to the total number of patients (100%). The individual is any selected subject. Sensitivity was calculated by the following formula: sensitivity = TP/(tp+fn) (TP = true positive; FN = false negative).
As used herein, the term "specificity" relates to the number (%) of true negative individuals relative to the total number of healthy subjects (100%). The specificity was calculated by the following formula: specificity = TN/(tn+fp) (TN = true negative; FP = false positive).
As used herein, the term "AUC" refers to the abbreviation of the area under the curve. In particular, it refers to the area under the subject's operating characteristics (ROC) curve. As used herein, the term "subject operating characteristic (ROC) curve" is a plot of true positive rate versus false positive rate indicating different possible tangent points for a diagnostic test. It shows a trade-off between sensitivity and specificity, depending on the chosen cut-off (any increase in sensitivity is accompanied by a decrease in specificity). The area under the ROC curve is a measure of the accuracy of the diagnostic test (the larger the area, the better, optimally 1, the ROC curve for the random test is located on the diagonal, with an area of 0.5).
Example 1
1. Experimental method
1. Heat stress population:
study object: the study involved 50 young healthy men, aged 17-20 years, had no habit of smoking and drinking, and had approximately the same daily performance, length of work, exercise intensity and dietary structure. During the experimental implementation, all the participants were randomly divided into two groups, numbered 1 and 2, 25 in each group, and exposed to a high temperature environment of 40 ℃ for 4 hours for 7 consecutive days.
Sample collection: before the experiment was carried out at 7: venous blood is taken 1 time by 00 am; venous blood is taken for the second time 7:00a day earlier after the end of heat stress, and is preserved for testing.
2. Heat stressed rats:
study object: male SD rats of 8 weeks of age were used, and were SPF-grade (Beijing Bei Fu) compliant for 20. After weighing and marking, the rats are randomly divided into two groups, namely a group A and a group B, wherein 5 rats in a control group and 5 rats in a heat stress group are arranged in each group; the rats in the control group are subjected to normal 12-hour illumination and 12-hour darkness, and are fed at room temperature of 22 ℃; the rats in the heat stress group are exposed to heat stress at 38 ℃ and an environmental temperature cabin with humidity of >70% for 2 hours (15:00-17:00) per day for 3 consecutive days, and the rats can eat and drink water freely during the exposure period.
Sample collection: blood is taken from the eyeballs at the early 7:00 stage on the 2 nd day after modeling, and serum is separated and stored for testing.
3. And (3) index detection: the expression level of BCHE in human and rat serum is detected by an enzyme-linked immunosorbent assay (ELISA). The detection kit is provided by cloud cloning company.
2. Experimental results
1) The ROC curve is drawn by taking 1 group of 25 young healthy men as a study object and BCHE as a detection variable, and the result is shown in figure 1, wherein the AUC value is 0.8909, the sensitivity is 1, the specificity is 0.8182, the optimal critical value point is 0.8182, and the BCHE is used as the detection variable for diagnosing heat stress sensitivity, so that the sensitivity and the sensitivity are higher, and the higher diagnosis efficiency is shown.
2) To verify the conclusion in the experimental result 1), 2 groups of 25 young healthy men are taken as study objects, BCHE is taken as a detection variable, and a ROC curve is drawn, and the result is shown in fig. 2, wherein AUC value is 0.8200, sensitivity is 0.8, specificity is 0.8, and optimal critical value point is 0.6, so that BCHE is proved to have higher specificity and sensitivity as a detection variable for diagnosing heat stress sensitivity, and higher diagnosis efficiency is shown.
3) Detecting the serum BCHE expression level before and after the heat stress of the population. ELISA test results were analyzed using Gragh Prism 9.0 software in two groups of 50 young healthy men, and P <0.05 was considered statistically different. As shown in fig. 3, the BCHE expression level was significantly reduced after the detection of heat stress in the human population as shown in fig. 3; the ROC curve is drawn by taking two groups of 50 young healthy men as study subjects and BCHE as a detection variable, and the result is shown in figure 4, wherein the AUC value is 0.8357, the sensitivity is 1, the specificity is 0.5714, the optimal critical value point is 0.5714, and the BCHE is used as the detection variable for diagnosing heat stress sensitivity, so that the sensitivity and the sensitivity are higher, and the higher diagnosis efficiency is shown.
4) The result is shown in fig. 5, which shows AUC value of 0.875, sensitivity of 0.75, specificity of 1, and optimal critical point of 0.75, and shows that BCHE is used as detection variable for diagnosing heat stress sensitivity with higher specificity and sensitivity, and higher diagnosis efficiency.
5) To verify the conclusion of experiment result 4), the ROC curve is drawn by taking the group B rats as a study object and BCHE as a detection variable, and the result is shown in fig. 6, wherein AUC value is 0.8984, sensitivity is 0.75, specificity is 0.875, and optimal critical point is 0.625, which proves that the use of BCHE as a detection variable for diagnosing heat stress sensitivity has higher specificity and sensitivity, and higher diagnosis efficiency is shown.
6) Control rats and heat stressed rats were tested for serum BCHE expression levels. ELISA detection results were analyzed using Gragh Prism 9.0 software with two groups of rats as subjects, and P <0.05 was considered statistically different. As shown in fig. 7, it can be seen from fig. 7 that the BCHE expression level in the serum of the heat-stressed rats was significantly reduced compared to the control rats; the ROC curve is drawn by taking two groups of rats as study objects and taking BCHE as a detection variable, and the result is shown in figure 8, wherein the AUC value is 0.8926, the sensitivity is 0.8125, the specificity is 0.9375, the optimal critical value point is 0.75, and the effect that the BCHE is taken as the detection variable for diagnosing heat stress sensitivity has higher specificity and sensitivity and higher diagnosis efficiency.
7) The serum ACHE expression levels of rats in the control group and the heat-stressed rats are detected by taking the two groups of rats as study objects, and a standard curve is detected, and the result is shown in figure 9, ELISA detection results are analyzed by adopting Gragh Prism 9.0 software, wherein P <0.05 is regarded as statistical difference, the standard curve is detected to have the highest value of 100ng/mL and the lowest concentration of 0ng/mL, and the OD values of the healthy group and the heat-stressed group are close to the lowest OD value of the standard curve, so that the secretion of ACHE in serum is very low and is close to 0 and the difference is avoided.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. Use of a reagent for detecting a BCHE biomarker in a sample in the preparation of a product for diagnosing or predicting whether a subject is heat stress sensitive;
preferably, the sample comprises one of a tissue sample, primary or cultured cells or cell lines, cell supernatant, cell lysate, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, emulsion, whole blood, blood derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, or a combination thereof;
preferably, the test sample is serum;
preferably, the subject comprises a human or non-human mammal;
preferably, the subject is a human.
2. The use according to claim 1, wherein the reagent comprises a primer or probe for specifically detecting the expression level of the BCHE gene, or a reagent for specifically binding to a protein encoded by the BCHE gene.
3. The use according to claim 1, wherein the product comprises reagents capable of detecting BCHE biomarkers, a kit, a chip, a nucleic acid membrane strip or a test paper comprising said reagents;
preferably, the kit comprises a gene detection kit comprising a reagent or chip for detecting BCHE gene transcription level, a protein detection kit comprising a reagent or chip for detecting BCHE protein expression level;
preferably, the kit comprises reagents for detecting the level of BCHE gene or protein expression by western blotting, ELISA, radioimmunoassay, oxter lony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip assay.
4. The use according to claim 3, wherein the chip comprises a gene chip comprising oligonucleotide probes for BCHE genes for detecting the transcription level of BCHE genes and a protein chip comprising antibodies or ligands specific for BCHE proteins; the test paper comprises a gene test paper and a protein test paper.
5. A product for predicting or diagnosing whether a subject is heat stress sensitive, said product comprising an agent for detecting the expression level of a BCHE gene or protein in a sample;
preferably, the subject comprises a human or non-human mammal;
preferably, the subject is a human.
6. The product of claim 5, wherein the sample comprises one of a tissue sample, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood derived cells, urine, cerebral spinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, tissue culture fluid, tissue extracts, homogenized tissue, tumor tissue, cell extracts, or a combination thereof;
preferably, the test sample is serum.
7. The product of claim 5, wherein the product comprises reagents capable of detecting BCHE biomarkers, a kit, a chip, a nucleic acid membrane strip, or a test strip comprising the reagents;
preferably, the kit comprises a gene detection kit comprising a reagent or chip for detecting BCHE gene transcription level, a protein detection kit comprising a reagent or chip for detecting BCHE protein expression level;
preferably, the kit comprises reagents for detecting BCHE gene or protein expression levels by western blotting, ELISA, radioimmunoassay, oxter lony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip assay;
preferably, the chip comprises a gene chip comprising oligonucleotide probes for BCHE genes for detecting BCHE gene transcription levels and a protein chip comprising antibodies or ligands specific for BCHE proteins;
preferably, the test strip comprises a gene test strip and a protein test strip.
8. A system for diagnosing or predicting whether a subject is susceptible to heat stress using a biomarker BCHE, the system comprising a result determination unit that compares the resulting abundance of the biomarker to an average of the abundance of the biomarker detected in a sample of heat stress sensitive individuals;
preferably, the system comprises a detection unit or a data processing unit.
9. A method of diagnosing or prognosticating whether a subject is susceptible to thermal stress, comprising the step of detecting the level of gene or protein expression of a biomarker as claimed in claim 1 in a sample from the subject.
10. A method for screening for a drug for therapeutic or prophylactic treatment of heat stress injury for non-therapeutic non-diagnostic purposes, the method comprising the step of detecting the level of gene or protein expression of a biomarker as claimed in claim 1 in a sample from a subject.
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