CN107290552B - The biomarker of high coagulation and its application - Google Patents
The biomarker of high coagulation and its application Download PDFInfo
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- CN107290552B CN107290552B CN201710623514.6A CN201710623514A CN107290552B CN 107290552 B CN107290552 B CN 107290552B CN 201710623514 A CN201710623514 A CN 201710623514A CN 107290552 B CN107290552 B CN 107290552B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Abstract
The invention discloses the biomarker of high coagulation and its applications.The present invention uses three kinds of different coagulant objects (etamsylate, tranexamic acid and aminocaproic acid), the coagulation of rat is intervened respectively, successfully obtain the high coagulation model of rat acute, then the variation of the rat urine protein group of different coagulant object intervention groups is analyzed, under the high coagulation of system research for the first time, the variation characteristic of urine protein group, and search out high coagulation correlation urinary biomarkers object.The body that the present invention tentatively discloses high coagulation intervenes range and changed factor, and these can both prompt the variation characteristic of relevant physiological pathomechanism, signal pathway, it can also be used as important biomarker simultaneously, applied to the detection and/or diagnosis of the variation of high coagulation related pathologies, or to provide auxiliary information in this respect.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the biomarker of high coagulation and its application.
Background technique
When body is stimulated by physiology or pathological factor, a series of variation can be generated, wherein the variation that can be monitored is claimed
For biomarker.The most fundamental characteristic of biomarker is variation.After body is stimulated, the equilibrium state of internal script
It is broken, cell, tissue and organ can generate some abnormal metabolites, and discharge it in vitro, to restore original flat
Weighing apparatus maintains homeostasis.Compared with blood, urine since by the adjusting of body homeostatic mechanism, a large amount of variations can not accumulated,
The variation that body can preferably be reacted is a kind of source of good biomarker.Meanwhile urine have it is a large amount of, noninvasive,
It the advantages such as can continuously collect, the research of metabolites all kinds of in urine is increasingly valued by people.
Although urine has many advantages in terms of biomarker research, since protein group has apparent individual
Difference, and a variety of Physiological factors, such as age, gender, movement, diet can make urine protein group generate variation, this is just
So that the urine protein group for directly researching the mankind is relatively difficult.And animal model is used to carry out the research of urine protein group, sieve
Select possible candidate disease biomarker, so that it may by strict control experiment condition, by factors pair such as individual difference and environment
The influence of urine protein group is preferably minimized, and is at present more to obtain more believable experimental result in a relatively short period of time
A kind of method of popular searching urinary biomarkers object.
High blood coagulation state (hypercoagulable states) refers to that blood is in the state for being very easy to condensation, leads to
Often it is considered as prethrombotic state, eventually results in the formation of arteriovenous thrombus.Thrombotic diseases are often cardiovascular disease
Severe complication.The associated biomarkers of hypercoagulative state are studied, and visible report is mainly some blood biomarkers at present
Relevant report, and have no related urinary biomarkers object research report.Meanwhile clinically for the diagnosis side of hypercoagulative state
Method, in terms of focusing primarily upon blood testing, such as coagulation factor detection, d-dimer detection, clotting time detection.But
Summary of the invention
The present invention uses three kinds of different coagulant objects (etamsylate, tranexamic acid and aminocaproic acid), respectively to big
The coagulation of mouse is intervened, and the high coagulation model of rat acute is successfully obtained.Then completely the same analysis plan is used
Slightly, the variation of the rat urine protein group of different coagulant object intervention groups is analyzed, the high blood coagulation shape of system research for the first time
Under state, the variation characteristic of urine protein group, and search out high coagulation correlation urinary biomarkers object.
Present invention firstly provides the new applications of the system of detection X content.
The present invention provides the system of detection X content answering in the product of preparation diagnosis or the high coagulation of auxiliary diagnosis
With；
The present invention also provides the systems of detection X content in preparation diagnosis or auxiliary diagnosis thrombotic diseases and/or painstaking effort
Application in the product of pipe disease；
The present invention also provides application of the system of detection X content in diagnosis or the high coagulation of auxiliary diagnosis；
The present invention also provides the systems of detection X content in diagnosis or auxiliary diagnosis thrombotic diseases and/or cardiovascular disease
Application in disease；
The X be following X1) X2) or X3):
It X1) is Golgi apparatus protein I, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3, ribalgilase 4, chloride ion
Channel protein 1 intracellular, biglycan, Rab GDP dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde
Reductase member 2, Histidine Triad nucleotide binding protein 1, myosin light chain 6, L- xyloketose reductase and blood red egg
White β subunit；
It X2) is cytosol aminopeptidase, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanine-nucleotide-binding protein G (I)/G
(S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanylic acid combine
Protein G (I)/G (S)/G (T) β -2 subunit, angiotensin converting enzyme2 homologue, superoxide dismutase [Cu-Zn], starch
Sample albumen 2, calbindin, fumarylacetoacetase, myosin light chain 6, frizzled protein 2, Apoliprotein M, flesh are red
Albumen, ephrins B1, selenium Binding Protein 1 and calmodulin (CaM)；
It X3) is cell adhesion molecule 3, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin, thioredoxin (Trx), peptide
Base cis-trans propyl isomerism enzyme A, ABHD14B albumen, frizzled protein 4, α-enolase, anthrax toxin acceptor 1, frizzled protein 2,
Attract element, Neuroplastin, CD166 antigen, glutathione S-transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinoline promise
Oxidation of ketones reductase, Rho GDP- dissociate inhibitor 1, peroxiredoxin -6, polyphosphoinositide phosphatase 1, glucose -6-
Phosphoric acid isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative growth factor, chloride ion channel protein intracellular
4, Histidine Triad nucleotide binding protein 1, connection adhesion molecule C, this calcium element -1, Glutamate-cysteine ligase are urged
Change subunit, N (G), N (G)-Dimethylarginine dimethylaminohydro-lase 1, alpha-N-Acetylgalactosaminidase, fumaryl second
Ethyl acetoacetic acid enzyme, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan
4。
In above-mentioned application, the system of the detection X content can detect the X content institute for utilization method in the prior art
The reagent and/or instrument needed, reagent and/or instrument needed for X content as described in using mass spectrum or the detection of its relevant technologies, or it is sharp
Reagent and/or instrument needed for detecting the X content with immuning hybridization technology, or the X content institute is detected using elisa technique
The reagent and/or instrument needed, or reagent and/or instrument needed for detecting the X content using protein chip or test paper；The X
Content is X content in urine.
Present invention discover that after coagulant object (etamsylate, tranexamic acid and aminocaproic acid) is intervened, rat coagulation function
It changes, urine protein group is caused to change, illustrate that urine can reflect the variation of blood.Urine is internal as accumulation
The final place of metabolin, is not regulated and controled by homeostatic mechanism, can reflect a large amount of variation, and a large amount of variation means largely latent
In biomarker.
Etamsylate according to the present invention, Chinese nickname: etamcylate, dicynone, dicynon, Aglumin,
Chemical name is 2,5- dihydroxybenzenesulfonate salt, molecular formula: C10H17NO5S, molecular weight 263.31, molecular structure
Schematic diagram is as shown in Figure 1.The present invention is intervened using coagulation of the etamsylate to rat, successfully obtains rat acute height
Coagulation model, the proteome analysis of the high coagulation animal model of rat based on etamsylate building, obtains 20
A significant difference albumen, wherein have 13 for people's class homologous protein, i.e., above-mentioned X1) described in 13 albumen, and as
The biomarker of high coagulation caused by etamsylate induces.High coagulation caused by etamsylate induces is referred to due to first
High blood coagulation state caused by grade coagulation function enhances promotes the aggregation, adherency and work of blood platelet that is, by promoting vessel retraction
The release of sex factor, caused hypercoagulative state, alternatively referred to as prethrombotic state.
Tranexamic acid according to the present invention, chemical name are 4- (aminomethyl) cyclohexanecarboxylic acid, also known as bright for tranexamic acid, biography
Acid, tranxamic acid, molecular formula C8H15NO2, molecular weight 157.21, schematic arrangement is as shown in Figure 1；Tranexamic acid
It can suppress with lysine binding sites (LBS) strong adsorption at the affine position of fibrin on fibrinolysin and plasminogen
Fibrinolysin, plasminogen are in conjunction with fibrin, so that the fibrin decomposition caused by fibrinolysin is consumingly inhibited, to reach
To high blood coagulation state.The present invention is intervened using coagulation of the tranexamic acid to rat, successfully obtains rat acute Gao Ning
Blood state model, the proteome analysis of the high coagulation animal model of rat based on tranexamic acid building, obtains 28
Significant difference albumen, wherein have 18 for people's class homologous protein, i.e., above-mentioned X2) described in 18 albumen, and as ammonia
The biomarker of high coagulation caused by first naphthenic acid induces.High coagulation caused by tranexamic acid induces refers to passing through inhibition
Hypercoagulative state caused by plasmin activity.
Aminocaproic acid according to the present invention, Chinese nickname: 6-aminocaprolc acid, lpsilon, Aminocaproic Acid, anti-blood fibre are molten
Acid, molecular formula C6H13NO2, molecular weight 131.17, schematic arrangement is as shown in Figure 1；Aminocaproic acid can inhibit fibre
Plasminogen activator, thus prevent plasminogen from activating as fibrinolysin, thus inhibit fibrinous dissolution,
Generate high coagulation.The present invention is intervened using coagulation of the aminocaproic acid to rat, successfully obtains rat acute height
Coagulation model, the proteome analysis of the high coagulation animal model of rat based on aminocaproic acid building, obtains 65
A significant difference albumen, wherein have 34 for people's class homologous protein, i.e., above-mentioned X3) described in 34 albumen, and as
The biomarker of high coagulation caused by aminocaproic acid induces.High coagulation caused by aminocaproic acid induces refers to passing through suppression
High coagulation caused by the activity factor of plasminogen processed.
It in practical applications can be according to the biomarker of the high coagulation of above-mentioned acquisition according in following (1)-(3)
Any method judges whether subject is in high coagulation:
(1) if the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribose core
The content of sour enzyme 4 is above at least one times of Healthy People, and chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is below at least one times of Healthy People, then
Subject is in or candidate is in high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate induction institute
Cause high coagulation.
If the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribonucleic acid
The content of enzyme 4 is not above at least one times of Healthy People or chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is not below at least one times of Healthy People,
Then subject is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate
High coagulation caused by inducing.
(2) if the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanylic acid combine
Protein G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanine
The content of nucleotide binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is above health
At least one times of people, and superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase,
Myosin light chain 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is below at least one times of Healthy People, then subject is in or candidate is in high coagulation；And this high blood coagulation
The Forming Mechanism of state is relevant to high coagulation caused by tranexamic acid induces.
If the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanine-nucleotide-binding protein
G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanosine
The content of acid binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is not above Healthy People
At least one times or superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase, flesh
Immunoglobulin light chains 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is not below at least one times of Healthy People, then subject is not at or candidate is not at high coagulation；And it is this
The Forming Mechanism of high coagulation is relevant to high coagulation caused by tranexamic acid induces.
(3) if the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also
The content of albumen (Trx) is above at least one times of Healthy People, and peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is below at least one times of Healthy People, then subject
It is in or candidate in high coagulation；And the Forming Mechanism of this high coagulation is relevant to Gao Ning caused by aminocaproic acid induces
Blood state.
If the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also egg
The content of white (Trx) is not above at least one times of Healthy People or peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is not below at least one times of Healthy People, then tested
Person is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to aminocaproic acid induction institute
Cause high coagulation.
It is a further object to provide the new applications using above-mentioned X as high coagulation marker.
The present invention provides preparing diagnosis or the high coagulation of auxiliary diagnosis using above-mentioned X as high coagulation marker
Product in application；
The present invention also provides preparing diagnosis or auxiliary diagnosis thrombotic disease using above-mentioned X as high coagulation marker
Application in the product of disease and/or cardiovascular disease.
The present invention also provides using above-mentioned X as high coagulation marker in diagnosis or the high coagulation of auxiliary diagnosis
Application；
The present invention also provides using above-mentioned X as high coagulation marker in diagnosis or auxiliary diagnosis thrombotic diseases
And/or the application in cardiovascular disease.
It is a still further object of the present invention to provide diagnosis or the products of the high coagulation of auxiliary diagnosis.
The product of diagnosis provided by the invention or the high coagulation of auxiliary diagnosis is the system of above-mentioned detection X content.
The product includes the diagnostic card for recording following content:
(1) if the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribose core
The content of sour enzyme 4 is above at least one times of Healthy People, and chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is below at least one times of Healthy People, then
Subject is in or candidate is in high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate induction institute
Cause high coagulation.
If the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribonucleic acid
The content of enzyme 4 is not above at least one times of Healthy People or chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is not below at least one times of Healthy People,
Then subject is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate
High coagulation caused by inducing.
(2) if the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanylic acid combine
Protein G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanine
The content of nucleotide binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is above health
At least one times of people, and superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase,
Myosin light chain 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is below at least one times of Healthy People, then subject is in or candidate is in high coagulation；And this high blood coagulation
The Forming Mechanism of state is relevant to high coagulation caused by tranexamic acid induces.
If the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanine-nucleotide-binding protein
G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanosine
The content of acid binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is not above Healthy People
At least one times or superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase, flesh
Immunoglobulin light chains 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is not below at least one times of Healthy People, then subject is not at or candidate is not at high coagulation；And it is this
The Forming Mechanism of high coagulation is relevant to high coagulation caused by tranexamic acid induces.
(3) if the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also
The content of albumen (Trx) is above at least one times of Healthy People, and peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is below at least one times of Healthy People, then subject
It is in or candidate in high coagulation；And the Forming Mechanism of this high coagulation is relevant to Gao Ning caused by aminocaproic acid induces
Blood state.
If the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also egg
The content of white (Trx) is not above at least one times of Healthy People or peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is not below at least one times of Healthy People, then tested
Person is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to aminocaproic acid induction institute
Cause high coagulation.
Product of the present invention concretely kit.
Application of the said goods in preparation diagnosis or auxiliary diagnosis thrombotic diseases and/or the product of cardiovascular disease
It belongs to the scope of protection of the present invention.
Application of the said goods in the product of preparation detection or the assessment high coagulation of subject also belongs to of the invention
Protection scope.
Application of the said goods in diagnosis or auxiliary diagnosis thrombotic diseases and/or cardiovascular disease also belongs to the present invention
Protection scope.
Application of the said goods in the product for detecting or assessing the high coagulation of subject also belongs to protection of the invention
Range.
Final object of the present invention is to provide a kind of method detected or assess the high coagulation of subject.
The method of detection or the assessment high coagulation of subject provided by the invention is above-mentioned X content in detection subject.
In the above method, above-mentioned X content is above-mentioned X content in detection subject's urine in the detection subject.
In the above method, the method for assessing the high coagulation of subject is interior referring to documented by the diagnostic card in the said goods
Hold.
In the above method, the subject is mammal；Mammal is people or non-human mammal.
Test result of the invention shows: after three kinds of different clot promoting drugs are intervened respectively, 105 significant changes are obtained
Differential protein has 65 can find reciprocity albumen, i.e. human homology's albumen in the mankind in this 105 differential proteins, at this
In 65 people's class homologous proteins, 35 are mankind core urine protein group memberships, are illustrated under high blood coagulation state, mankind's core
Urine protein group may change.It has also been found that human homology's albumen of differential protein is distributed in each different tissues
In, but focus mostly in urinary system, digestive system and respiratory system, illustrate the change of coagulation function, may will affect these is
The function of system, also illustrating that the body of high coagulation intervenes range is that comparison is extensive, and the high coagulation of body is as a kind of aobvious
The pathophysiological change of work can have an impact a plurality of metabolic pathway of body.The present invention tentatively discloses the machine of high coagulation
The pre- range of soma and changed factor, and these can both prompt the variation characteristic of relevant physiological pathomechanism, signal pathway, together
When, it can also be used as important biomarker, applied to the detection and/or diagnosis of the variation of high coagulation related pathologies, or
Person is to provide auxiliary information in this respect.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of etamsylate, tranexamic acid and aminocaproic acid.A is the structural schematic diagram of etamsylate；
B is the structural schematic diagram of tranexamic acid；C is the structural schematic diagram of aminocaproic acid.
Fig. 2 is the method for building up that etamsylate, tranexamic acid and aminocaproic acid cause the high coagulation animal model of rat
Schematic diagram.A is the schematic diagram for the method for building up that etamsylate causes the high coagulation animal model of rat；B is that tranexamic acid causes greatly
The schematic diagram of the method for building up of the high coagulation animal model of mouse；C is that aminocaproic acid causes the high coagulation animal model of rat
The schematic diagram of method for building up.
Fig. 3 is the influence of etamsylate, tranexamic acid and aminocaproic acid to rat APTT and CT.A is etamsylate to big
The influence of mouse APTT and CT；B is influence of the tranexamic acid to rat APTT and CT；C is aminocaproic acid to rat APTT and CT
It influences.
Fig. 4 is control group (physiological saline group) and the urine protein SDS-PAGE analysis of experimental group.A is physiological saline group
It is analyzed with the urine protein SDS-PAGE of etamsylate group；B is the urine protein of physiological saline group and tranexamic acid group
SDS-PAGE analysis；C is the urine protein SDS-PAGE analysis of physiological saline group and aminocaproic acid group.
Fig. 5 is the tissue expression analysis of high coagulation urine differential protein.A is that the high coagulation urine of etamsylate is poor
The tissue expression analysis of M-band；B is the tissue expression analysis of the high coagulation urine differential protein of tranexamic acid；C is amino
The tissue expression analysis of the high coagulation urine differential protein of caproic acid.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
SPF grade male Wistar rat in following embodiments, 180-200g tie up tonneau China experimental animal skill purchased from Beijing
Art Co., Ltd, adaptive feeding starts to test after a week under standard environment.
Activated partial thromboplastin time (Activated Partial Thromboplastin is used in following embodiments
Time, APTT) and the clotting time (Clotting Time, CT) assessment rat coagulation, to determine the high blood coagulation model of rat
Whether it is successfully established, specific assay method is as follows:
1, the measurement of activated partial thromboplastin time
After the blood sampling of rat angular vein, according to blood: lemon is added in the ratio of sodium citrate anticoagulant=9:1 (volume ratio)
Sour sodium anti-coagulants, upper and lower gentle inversion mix 5~6 times, centrifugation: 4 DEG C, 2500g, 15min, centrifugation gained supernatant is blood
Slurry.Take measurement of the 100 μ L blood plasma for partial thromboplastin time.
Measuring method follows the product description of kit: 37 DEG C of 100 μ L ellagic acid reagents of warm bath (being no more than 15min),
Then 100 μ L test plasmas are added, the 0.5mol/L of 37 DEG C of preheatings is added in 37 DEG C of warm bath 5min (no less than 5min) later
CaCl2100 μ L of solution, mixes and starts timing.Stop timing immediately when there is flocculent deposit, as activated partial blood coagulation is living
The enzyme time.
2, coagulation time test
Clotting time is measured using glass capillary method.After the blood sampling of rat angular vein, 25 μ L are not added into anti-coagulants immediately
Venous blood sucking glass capillary in, start timing.In timing course, capillary is overturned back and forth horizontal by ± 60 ° of angles
Glass tube stops timing, as clotting time until the blood in glass tube no longer moves back and forth.
It is as shown in table 1 that reagent used in the high coagulation animal model of rat is constructed in following embodiments, instrument and consumptive material
As shown in table 1.
Reagent used in table 1, the building high coagulation animal model of rat
Instrument and consumptive material used in table 2, the building high coagulation animal model of rat
| Instrument consumptive material | Company |
| Hard neutral density glass point sample capillary | Instrument plant, Huaxi Medical Univ |
| DK-S22 type electric-heated thermostatic water bath | The upper macro experimental facilities Co., Ltd of Nereid |
| Dry-type thermostat | Hangzhou Ao Sheng Instrument Ltd. |
| Centrifuge 5417R | Eppendorf company |
| Electronic balance | Shenyang Teng Long projection electronic weighing instrument plant |
The foundation of the high coagulation model of embodiment 1, rat acute
One, the high coagulation animal model of rat is constructed based on etamsylate
1, experimental method
20 Male Wistar Rats are randomly divided into two groups, every group 10, are weighed.Wherein one group of rats by intraperitoneal injection phenol
Sulphur ethamine injection (250mg/kg weight), is denoted as experimental group；Another group of rats by intraperitoneal injection respective volume physiological saline
Liquid is denoted as control group.Injection time first time is denoted as 0h, and primary every 3h intraperitoneal injection, co-injection 3 times (i.e. in 0h, 3h and 6h
It is each primary).9h (i.e. the 3h after third time injection) after first time injects, angular vein is taken a blood sample immediately, and detects the blood coagulation of rat
Function determines that high coagulation rat animal model forms specification (flow chart is as shown in Figure 2).
The change of rat coagulation function is assessed by two important indicators of APTT and CT.APTT, i.e. activated partial blood coagulation
Movable enzyme time is to be carried out by full terms to intrinsic coagulation in-vitro simulated, the time of the clotting of plasma is measured, to reflect
Whether endogenous coagulation factor normally exercises biological function out.CT, i.e. clotting time refer to that blood leaves blood vessel, send out in vitro
Time needed for raw solidification, for detecting in intrinsic coagulation pathway, the quantity and function of various coagulation factors normally whether, together
When also can detect whether extra anticoagulant substances.
2, experimental result
By above-mentioned experimental procedure, after giving clot promoting drug etamsylate, the activated partial of rat as shown in Figure 3 is solidifying
Blood movable enzyme time (APTT) and clotting time (CT) testing result show, the APTT and CT of wistar rat significantly shorten (p <
0.01), show the normal coagulation function multilated of rat, be in high coagulation, model successfully.
Two, the high coagulation animal model of rat is constructed based on tranexamic acid
1, experimental procedure
Male Wistar Rats 20, it is divided into two groups at random, every group 10, weighs.Experimental group rat oral gavage ammonia first ring
Sour (315mg/kg), control group stomach-filling respective volume physiological saline.6h is primary, and totally 2 times.Stomach-filling time first time is denoted as 0h,
Second of stomach-filling time is then 6h.12h after first time stomach-filling, angular vein is taken a blood sample immediately, and detects the coagulation function of rat, really
Fixed high coagulation rat animal model forms specification (flow chart is as shown in Figure 2).
2, experimental result
By above-mentioned experimental procedure, after being acted on through tranexamic acid, the activated partial thromboplastin of rat as shown in Figure 3
Time (APTT) and clotting time (CT) testing result show that the APTT and CT of Wistar rat significantly shorten (p < 0.01),
Show that stomach-filling tranexamic acid changes rat coagulation function, blood is in high coagulation, and the solidifying states model of height models successfully.
Three, the high coagulation animal model of rat is constructed based on aminocaproic acid
1, experimental method
Male Wistar Rats 20, it is divided into two groups at random, every group 10, weighs.Experimental group rat oral gavage amino oneself
Sour (4.2g/kg), control group stomach-filling respective volume physiological saline.6h is primary, and totally 2 times.Stomach-filling time first time is denoted as 0h, the
The secondary stomach-filling time is then 6h.12h after first time stomach-filling, angular vein is taken a blood sample immediately, and detects the coagulation function of rat, is determined
High coagulation rat animal model forms specification (flow chart is as shown in Figure 2).
2, experimental result
By above-mentioned experimental procedure, after being acted on through aminocaproic acid, the activated partial thromboplastin of rat as shown in Figure 2
Time (APTT) and clotting time (CT) testing result show that the APTT and CT of wistar rat significantly shorten (p < 0.01),
Show that the intervention of aminocaproic acid changes the coagulation function of rat, blood is in high coagulation, and the solidifying states model of height models successfully.
The above method has been successfully established big based on different coagulant objects (etamsylate, tranexamic acid and aminocaproic acid)
The high coagulation animal model of mouse is based on this high coagulation rat model, can carry out related urine protein group and correlation
The research work of biomarker establishes important basis to be relevant to the high coagulation correlative study of rat.
Embodiment 2, high coagulation urine protein group credit analysis and biomarker research
One, experimental method
1, ethanol precipitation extracts urine protein group
Rat based on different coagulant objects (etamsylate, tranexamic acid and aminocaproic acid) building in Example 1
The urine sample of high coagulation animal model is made as experimental group sample, while with the urine sample of the control group in each model
For control sample, and extract the protein group in urine sample.Specific extracting method is as follows: taking urine sample 8mL in cleaning
50mL centrifuge tube in, centrifugation: 2500g, 30min, 4 DEG C.Supernatant is shifted into clean 50mL centrifuge tube, centrifugation:
12000g, 30min, 4 DEG C.Supernatant volume is measured, is transferred in clean 50mL centrifuge tube, -20 DEG C of three times volume are added
The dehydrated alcohol of pre-cooling, shakes up, and precipitates 2 hours in -20 DEG C.After the completion of precipitating, centrifugation: 12000g, 30min, 4 DEG C.Supernatant is abandoned,
It is air-dried at room temperature precipitating (about 20min).Appropriate 25mM NH is added to precipitating4HCO3(about 600 μ L) redissolves precipitating, and liquid-transfering gun is blown repeatedly
It beats, is completely dissolved precipitating.Acquired solution is shifted into clean 1.5mL EP pipe, ultrasonic 3min is subsequently placed in rotation and mixes
4 DEG C of rotations mix 2h on instrument.Later, be centrifuged: 12000g, 30min, 4 DEG C shift supernatant in clean EP pipe, as extract
Obtained urine protein group.
2, Bradford method surveys protein concentration
1 μ g/ μ L bovine serum albumin(BSA) (BSA) is prepared as standard protein solution, then by standard protein solution,
Bradford working solution and ddH2O is mixed according to the volume in table 3, and final volume is 300 μ L, then with enzyme mark in 96 orifice plates
Instrument detects absorbance value at 595nm, draws standard curve.
The preparation of table 3, various concentration standard protein solution
For urine sample, respectively take 1 μ L urine sample, then with 285 μ L Bradford working solutions and 14 μ L
ddH2O is uniformly mixed, and same to detect absorbance value at 595nm, according to gained absorbance value, reference standard curve obtains urine
The protein concentration of sample dispenses sample, -80 DEG C freeze later.
3, SDS-PAGE Preliminary detection urine protein group
1) SDS-PAGE detects urine protein group
(1) sample treatment before electrophoresis: each sample applied sample amount is 20 μ g, by sample and 5 × SDS-PAGE sample-loading buffer
It is uniformly mixed according to the volume ratio of 4:1,10min is heated in boiling water makes protein denaturation.
(2) offset plate is installed according to specification, ultrapure water is injected into gap, detects whether leak after 10min.
(3) according to shown in table 4, glue is concentrated in preparation 12%SDS-PAGE separation gel and 5%SDS-PAGE.Separation gel is poured into
In the gap of two glass plates, speed is slow, tries not to generate bubble, covers sealing with dehydrated alcohol immediately after, and room temperature is put
Set 20min.Dehydrated alcohol is removed after gelling to be separated is solid, residual liquid is blotted with filter paper, slightly dries, then pour into concentration
Glue plugs comb, is stored at room temperature 20min, and glue to be concentrated is waited to polymerize.
Glue is concentrated in table 4,12%SDS-PAGE separation gel and 5%SDS-PAGE
(4) after glue polymerization completely to be concentrated, gel slab is fixed on electrophoretic apparatus, interior outer groove is separately added into 1 × SDS electricity
Swimming buffer, carefully takes out comb.
(5) according to predetermined order loading, wherein albumen Marker is added in leftmost side duct.
(6) electrophoresis: carrying out electrophoresis in concentration glue with 80V constant pressure, when bromophenol blue indicator reaches point of concentration glue and separation gel
When boundary line, adjusts voltage and reach gel bottom in separation gel with 120V constant pressure electrophoresis to 120V to bromophenol blue indicator, stop electricity
Swimming.
(7) after electrophoresis, gel is carefully removed, with ultrapure water repeated flushing, removes the electrophoretic buffer of gel surface.
2) coomassie brilliant blue staining
(1) gel is put into glass culture dish, appropriate Coomassie brilliant blue dye liquor is added, room temperature is slowly shaken on decolorization swinging table
2h is swung, is dyed.
(2) dye liquor is abandoned, ultrapure water rinses for several times, and destainer is added, and room temperature slow oscillation is stayed overnight on decolorization swinging table, and midway is more
Destainer is changed 2-3 times, until decoloration causes clear protein band occur, and background color is shallower.
(3) destainer is abandoned, ultrapure water rinses for several times, immerses in ultrapure water, be imaged in gel imager later.
4, digestion is assisted on the film of protein
1) digestion is assisted on albuminous membranae
(1) now match UA solution (8M urea, 0.1M Tris/HCl, pH 8.5) and ABC solution (25mM NH4HCO3Solution).
(2) it cleans super filter tube: 200 μ L of UA solution, centrifugation: 12 000g, 10min, 18 being added in each 10kD super filter tube
DEG C, abandon lower layer's filtered solution.Repeated washing is three times.
(3) 200 μ g of protein example is respectively taken to be added on filter membrane, then addition UA solution, supplement total volume to 200 μ L, from
The heart: 12 000g, 40min, 18 DEG C abandon lower layer's filtered solution.
(4) 200 μ L of UA solution is added on filter membrane, is blown and beaten repeatedly with liquid-transfering gun, is centrifuged: 12 000g, 40min, 18 DEG C,
Abandon lower layer's filtered solution.This step is in triplicate.
(5) 200 μ L of ABC solution, vortex oscillation 30s are added on filter membrane.Be added DTT to its final concentration of 4.5mM (i.e. plus
Enter the 1M DTT of 0.9 μ L), vortex oscillation 30s, after mixing well, 50 DEG C water-bath 1 hour.
(6) sample is taken out from water-bath, is cooled to room temperature, and the IAA now matched, which is added, makes its final concentration of 10mM, vortex oscillation
1min, after mixing well, room temperature is protected from light effect 35min.
(7) being centrifuged: 12 000g, 40min, 18 DEG C makes liquid sufficiently from dry, abandoning lower layer's filtered solution.
(8) 200 μ L of ABC solution is added on filter membrane, liquid-transfering gun is blown and beaten repeatedly, and protein on film is resuspended sufficiently, it is centrifuged:
12 000g, 40min, 18 DEG C abandon lower layer's filtered solution.Repeat this step twice.
(9) 200 μ L of ABC solution is added on filter membrane, is that pancreatin is added in 50:1 by the mass ratio of protein and pancreatin, is vortexed
1min.Then, it is put into oscillation vortex mixer and mixes: 37 DEG C, 400rpm, 30min.Digestion 14h is stayed overnight in 37 DEG C of waters bath with thermostatic control.
(10) take out super filter tube, be added formic acid only its final concentration of 0.1%, to terminate endonuclease reaction.The ultrafiltration more renewed
Pipe outer tube, centrifugation: 12 000g, 18 DEG C, until transfer filtered solution to new EP is managed close to being completely dried.
(11) 200 μ L of ABC solution is added into super filter tube again, is blown and beaten repeatedly with liquid-transfering gun, vortex 30s, is centrifuged: 12
000g, 18 DEG C, centrifugation is to being completely dried.Filtered solution is mixed with gained filtered solution in (10), as the digestion products of albumen.
2) digestion products desalination
Digestion products desalination is carried out using Oasis HLB solid-phase extraction column desalination method.Specific step is as follows:
(1) activation of Oasis HLB solid-phase extraction column: pillar successively is activated with 1mL methanol, 1mL acetonitrile.Pay attention to making filtrate
Naturally it drips.
(2) balance of Oasis HLB solid-phase extraction column: pillar is balanced with 1mL 0.l% formic acid/2% acetonitrile.This step weight
Again three times, it drips naturally.
(3) loading: the sample in 4.4.1 after gained digestion is added, it is allowed to drip naturally.
(4) it washing and desalting: is washed desalination 5 times with 1mL 0.l% formic acid/2% acetonitrile.
(5) peptide fragment elutes: 90% acetonitrile solution of 1mL elution extraction peptide fragment is added, collects eluent with 1.5mL centrifuge tube.
Sample drips naturally.
(6) vacuum drains sample, -80 DEG C freeze it is spare.
5, the Mass Spectrometric Identification of urine albumen
1) BCA method surveys peptide fragment concentration
(1) 1 μ g/ μ L angiotensins to 0.1 μ g/ μ L, 0.2 μ g/ μ L, 0.5 μ g/ μ L is diluted with 0.1% formic acid.
(2) with the peptide fragment sample cut of 0.1% formic acid weight lyase, weight solution product is 20 μ L, with 0.1% formic acid when measurement
10 times of dilution.
(3) prepare BCA working solution: A liquid is uniformly mixed with B liquid according to volume ratio 50:1.
(4) standard curve making method: according to shown in table 5, liquid is added in 96 orifice plates, is mixed.
Table 5, the concentration and volume for making standard curve agents useful for same
| Angiotensins concentration (μ g/ μ L) | 0 | 0.1 | 0.2 | 0.5 | 1.0 |
| Vasotonia pixel volume (μ L) | 10 | 10 | 10 | 10 | 10 |
| BCA working solution volume (μ L) | 200 | 200 | 200 | 200 | 200 |
(5) the peptide fragment sample after 10 μ L dilution, 200 μ L BCA working solutions are added in sample well.
(6) 37 DEG C of water-bath 30min.
(7) absorbance value at 562nm is measured, standard curve is drawn, finds out peptide fragment concentration.
(8) peptide fragment concentration is diluted to 0.5 μ g/ μ L.After dilution, each sample respectively takes 20 μ L, centrifugation: 12000g, 30min, 4
DEG C, it is careful to take out, take supernatant to wait mass spectrum loading.
2) liquid chromatogram separation tandem mass spectrum identification (LC-MS/MS)
With EASY-nLC 1200HPLC system loading, sample is the last gained peptide fragment of 4.5.1, and each sample applied sample amount is
500ng.Autopipette by sample be added trapping column (trap column) (75 μ m 2cm, 3 μm, C18,) after, with
The flow velocity of 2.5 μ L/min rinses 5min and further removes impurity, carries out reversed analytical column later with the flow velocity of 0.25 μ L/min and washes
De- (50 μ m 150mm, 2 μm, C18,).Gradient be 5-28% Mobile phase B (mobile phase A are as follows: 0.1% formic acid+
99.9% water；Mobile phase B are as follows:+20% water of+79.9% acetonitrile of 0.1% formic acid), time 60min.
The polypeptide eluted is detected using Orbitrap Fusion Lumos MS mass spectrograph.Spray voltage 2.1kV, from
300 DEG C of sub- transfer tube temperature.Parent ion m/z scanning range is 350-1550, scanning resolution 120000.At full throttle mould
Formula obtains MS/MS map, and ion fragmentation mode is that HCD daughter ion m/z scanning range is 250-1800, and scanning resolution is
60000, dynamic excludes the time as 30s.
Each sample repetitive identified 2 times.It is separated by the cleaning procedure of 30min twice between different samples, to reduce sample
Between pollute.
3) data are analyzed
(1) data retrieval
Second order ms result carries out database retrieval with MASCOT 2.4.0 software.Database used is Swiss-prot
Rat Database.Search condition are as follows: pancreatin digestion allows maximum 2 leakage enzyme sites, and peptide fragment retrieval parent ion error is
10ppm, daughter ion allowable error are 0.02Da, and peptide fragment false positive rate (FDR) < 1% will only identify more than or equal to 2 peptides
The protein of section is included in analysis.
(2) differential protein screens
The screening operation of differential protein is carried out using spectrogram number sizing technique.Screening criteria: control group spectrogram number average value and
The average of experimental group spectrogram number carries out T- inspection, p < 0.05；1.5 times of multiple of variation, i.e. experimental group are increased or are dropped than control group
It is low >=1.5 times；Maximum spectrogram number in minimum spectrogram number > reduction group in raising group.
Two, experimental result
(1) proteome analysis and biology mark of the high coagulation animal model of rat based on etamsylate building
Will object
1, the SDS-PAGE analysis of the high coagulation model urine protein of etamsylate
Influence with SDS-PAGE preliminary analysis clot promoting drug etamsylate intervention to urine albumen, the loading of each sample
Amount is 20 μ g.SDS-PAGE is shown: the urine protein sample after etamsylate effect, compared with control group (physiological saline group),
Most bands are essentially identical, without apparent protein degradation (Fig. 4).Illustrate that technology repeatability is good, protein example
Quality is good, can be used for subsequent experimental.SDS-PAGE result no significant difference illustrates that the protein of variation may be low abundance
Albumen has exceeded the resolution ratio of SDS-PAGE, can not differentiate high blood coagulation shape in this case with conventional detection technology, method
The protein difference of state and control group, needs finer detection means to carry out examination judgement.
2, the high coagulation model urine protein group Mass Spectrometric Identification result of etamsylate
The present invention identifies altogether obtains 533 protein, wherein physiological saline group (control group) 530, etamsylate group
(experimental group) 504, two groups identify 501 jointly.
(1) the high coagulation model difference protein classes of etamsylate
According to screening criteria, the protein with significant difference shares 20, and specifying information is as shown in table 6.And control group
It compares, DNA enzymatic -2- β, proteoglycan aggregates core protein, height in the rat urine of the high coagulation model of etamsylate
Dictyosome protein I, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3, ribalgilase 4, neutrophil leucocyte gelatinase correlation rouge
The content of matter transporter, zinc-α -2- glycoprotein and I class loading compatibility antigen significantly improves, chloride ion channel protein intracellular
1, biglycan, Rab GDP dissociate inhibitor β, glutathione S-transferase α -3, phosphoglyceric kinase 1, Huang Qu
Mould toxin B1 aldehyde reductase member 2, Histidine Triad nucleotide binding protein 1, myosin light chain 6, L- xylulose reduc-tase
The content of enzyme, Apolipoprotein A-IV and hemoglobin β-chain -1 is substantially reduced.
The urine differential protein of the high coagulation model of table 6, etamsylate
(2) human homology's property analysis of the high coagulation model urine differential protein of etamsylate
The present invention is by constructing high coagulation rat model, to probe into high blood coagulation state to mankind's urine protein group
It influences, and then provide certain clue to find high coagulation urine protein group biomarker.Different plant species it is homologous
Albumen has similar structure and function.Sequence is carried out by the website Uniprot (http://www.uniprot.org/blast/)
Column compare, and find human homology's albumen of the high coagulation differential protein of rat.The present invention will be by Swiss-prot database
Verifying, and human homology's albumen of homology >=80% is believable homologous protein.It was found that the high blood coagulation of etamsylate rat
In 20 differential proteins that state model obtains, there are 13 people's class homologous proteins, specifying information is as shown in table 7.By this 13 people
Class homologous protein is compared with mankind's core proteinuria data library (Human core urinary proteome), finds it
In to have 9 be mankind core urine protein member, prompt under high coagulation, mankind's core urine protein group may become
Change.
Table 7, etamsylate cause human homology's albumen of the high coagulation urine differential protein of rat
(3) etamsylate causes the tissue expression analysis of high coagulation model difference albumen
By human homology's albumen of differential protein in step (2) and human protein's expression database (Human Protein
Atlas, http://www.proteinatlas.org) it compares, the organization type that expression quantity is High is filtered out, it can be with
Determine there are 7 differential protein height to be expressed in 29 tissues, tissue expression analysis situation is as shown in table 8 and Fig. 5.Wherein, kidney
In highly expressed protein content it is most, which imply that internal body, the organ most sensitive for high blood coagulation blood state response are
Kidney, the high coagulation variation of kidney response, has carried out maximum adjustment and has been adapted in terms of protein expression.In addition, disappearing
Highly expressed albumen is also more in change system and nervous system.
The urine differential protein tissue expression analysis of the high coagulation model of table 8, etamsylate
(2) proteome analysis and biology mark of the high coagulation animal model of rat based on tranexamic acid building
Will object
1, the SDS-PAGE analysis of the high coagulation model urine protein of tranexamic acid
Influence with SDS-PAGE Preliminary detection coagulant object tranexamic acid intervention to urine albumen, each sample it is upper
Sample amount is 20 μ g.SDS-PAGE is as the result is shown: urine protein band is complete, and protein is not degraded significantly.With control group phase
Than (physiological saline group), the urine albumen of tranexamic acid group does not show significant change on PAGE gel.May be
Because the protein of variation is low-abundance protein (Fig. 4).
2, the high coagulation model urine protein group Mass Spectrometric Identification result of tranexamic acid
The present invention identifies altogether obtains 582 protein.Wherein, physiological saline group (control group) 568, tranexamic acid group
(experimental group) 563, two groups identify 549 jointly.
(1) the high coagulation model difference albumen of tranexamic acid
According to screening criteria, the protein with significant difference shares 28, and specifying information is as shown in table 9.And control group
It compares, cytosol aminopeptidase, SMR1 albumen, ribonucleoside triphosphote diphosphonic acid in the rat urine of the high coagulation model of tranexamic acid
Hydrolase 5, guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanylic acid
Binding protein G (k) α subunit, CD302 antigen, guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -2 subunit, blood vessel are tight
The content for opening 2 homologue of plain converting Enzyme, Nuvance α subunit, nestin -2 and Urine proteins 1 significantly improves, three leaves
The factor 1, WAP tetrasulfide Core domain albumen 2, superoxide dismutase [Cu-Zn], D- dopamine decarboxylase enzyme, amyloid
Albumen 2, Pu Luosha star (sulfated glycoprotein 1), calbindin, fumarylacetoacetase, myosin light chain 6, volume
Bent albumen 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1, pancreatic stone protein and calmodulin (CaM) contain
Amount is substantially reduced.
The urine differential protein of the high coagulation model of table 9, tranexamic acid
(2) human homology's property analysis of the high coagulation model urine differential protein of tranexamic acid
According to human homology's albumen of the method analysis differential protein in (2) in the 2 of step (1).Tranexamic acid causes big
The high coagulation of mouse in 28 differential proteins identified, has 18 someone's class homologous proteins, wherein 10 are mankind's core
The member of heart urine protein group, specifying information are as shown in table 10.Wherein, coagulation function GAP-associated protein GAP Apoliprotein M also belongs to people
Class core urine protein group.
Table 10, tranexamic acid cause human homology's albumen of the high coagulation urine differential protein of rat
(3) tissue expression analysis of the high coagulation model difference albumen of tranexamic acid
The tissue expression of human homology's albumen of differential protein is determined according to the method in (3) in the 2 of step (1).Most
Determine there are 8 differential protein height to be expressed in 19 tissues, specifically as shown in table 11 and Fig. 5 eventually.It can be seen that these albumen
Matter is largely the high expression in urinary system and digestive system.
The urine differential protein tissue expression analysis of the high coagulation model of table 11, tranexamic acid
| Tissue | It is horizontal | Uniprot ID | Title |
| Liver | High | P28838 | Cytosol aminopeptidase |
| Stomach | High | P28838 | Cytosol aminopeptidase |
| Kidney | High | P28838 | Cytosol aminopeptidase |
| Annex | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Liver | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Rectum | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Stomach | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Duodenum | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Colon | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Bladder bladder | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Kidney | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Prostate | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Breast | High | O75356 | Ribonucleoside triphosphote diphosphonic acid hydrolase 5 |
| Gall-bladder | High | P62873 | Guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -1 subunit |
| Cerebral cortex | High | P62873 | Guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -1 subunit |
| Salivary gland | High | P62873 | Guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -1 subunit |
| Kidney | High | P62873 | Guanine-nucleotide-binding protein G (I)/G (S)/G (T) β -1 subunit |
| Annex | High | Q14019 | Actin sample albumen |
| Tonsillotome | High | Q14019 | Actin sample albumen |
| Lymph node | High | Q14019 | Actin sample albumen |
| Spleen | High | Q14019 | Actin sample albumen |
| Liver | High | P00441 | Superoxide dismutase [Cu-Zn] |
| Cerebellum | High | P05937 | Calbindin |
| Kidney | High | P05937 | Calbindin |
| Liver | High | P16930 | Fumarylacetoacetase |
| Kidney | High | P16930 | Fumarylacetoacetase |
| Skeletal muscle | High | P02144 | Myoglobins |
| Cardiac muscle | High | P02144 | Myoglobins |
| Nasopharynx | High | Q13228 | Selenium Binding Protein 1 |
| Bronchus | High | Q13228 | Selenium Binding Protein 1 |
| Liver | High | Q13228 | Selenium Binding Protein 1 |
| Rectum | High | Q13228 | Selenium Binding Protein 1 |
| Annex | High | Q13228 | Selenium Binding Protein 1 |
| Thyroid gland | High | Q13228 | Selenium Binding Protein 1 |
| Colon | High | Q13228 | Selenium Binding Protein 1 |
(3) proteome analysis and biology mark of the high coagulation animal model of rat based on aminocaproic acid building
Will object
1, the SDS-PAGE analysis of the high coagulation model urine protein of aminocaproic acid
Influence with the intervention of SDS-PAGE Preliminary detection clot promoting drug aminocaproic acid to urine protein group, each sample
Applied sample amount be 20 μ g.On PAGE gel, it can be seen that (physiological saline group) compared with the control group, aminocaproic acid group
Urine albumen show certain variation.The protein that molecular weight is about 55kDa, aminocaproic acid group are expressed than physiological saline group
Amount will less, but the protein that molecular weight is about 60kDa or so, and aminocaproic acid group is one more than physiological saline group expression quantity
A bit.Molecular weight is two protein bands of 15kDa or more, and aminocaproic acid group is more less slightly than the expression quantity of physiological saline group, but
Molecular weight is the protein band of 15kDa, and aminocaproic acid group is more slightly higher than physiological saline group expression quantity.Two groups of urine protein bands are equal
Completely, protein is without significantly degrading (Fig. 4).
2, the high coagulation model urine protein group Mass Spectrometric Identification result of aminocaproic acid
The present invention identifies altogether obtains 423 protein.Wherein, physiological saline group (control group) 417, aminocaproic acid group
(experimental group) 291, two groups identify 285 jointly.
(1) the high coagulation model difference albumen of aminocaproic acid
According to screening criteria, the protein with significant difference shares 65, and specifying information is as shown in table 12.And control group
It compares, cell adhesion molecule 3, Thy-1 membrane glycoprotein, serine egg in the rat urine of the high coagulation model of aminocaproic acid
It is white enzyme inhibitor A3M, complement C9, cathepsin S, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin, Complement Factor D, preceding
Column gland basic protein, thioredoxin, CD59 glycoprotein, seralbumin, CD44 antigen, secretion phosphoprotein 24, β -2 microballoon egg
White and neutrophil gelatinase-associated lipocalin content significantly improves, and α -1 inhibitor 3, peptidyl prolyl are along anti-
Isomerase A, gamma glutamyl transpeptidase, ABHD14B albumen, ethyldopa A β subunit, Complement C_3, frizzled protein 4, α-enolization
Enzyme, anthrax toxin acceptor 1, cystatin GAP-associated protein GAP 2, connection adhesion molecule A, frizzled protein 2, sulfydryl oxygen
Change enzyme 1, attract element, monocyte differentiation antigen CD14, Neuroplastin, CD166 antigen, clusterin, glutathione S-
Transferase P, albumen FAM151A, alkaline phosphatase, glutathione S-transferase α -3,14-3-3 albumen ζ/δ, prostatein,
β-hexosaminidase β subunit, quinolone oxidoreducing enzyme, D- dopamine decarboxylase enzyme, Rho GDP- dissociate inhibitor 1, peroxide
Compound reductase -6, transcobalamin -2, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, sufficient glycocalicin,
E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, histidine three
Body nucleotide binding protein 1, inhibin-β C chain, connection adhesion molecule C, this calcium element -1, Glutamate-cysteine ligase are urged
Change subunit, N (G), N (G)-Dimethylarginine dimethylaminohydro-lase 1, alpha-N-Acetylgalactosaminidase, fumaryl second
Ethyl acetoacetic acid enzyme, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene dioxygenase, selenium Binding Protein 1, kynurenin/Amicar
The content that transaminase (mitochondria), chondroitin sulfate proteoglycan 4 and Na (+)/H (+) exchange adjust co-factor NHE-RF3 is obvious
It reduces.
The urine differential protein of the high coagulation model of table 12, aminocaproic acid
(2) human homology's property analysis of the high coagulation model urine differential protein of aminocaproic acid
According to human homology's albumen of the method analysis differential protein in (2) in the 2 of step (1).Aminocaproic acid causes big
The high coagulation of mouse in 65 differential proteins identified, has 34 someone's class homologous proteins, wherein 16 are mankind's core
The member of heart urine protein group, specifying information are as shown in table 13.
Table 13, aminocaproic acid cause human homology's albumen of the high coagulation urine differential protein of rat
(3) tissue expression analysis of the high coagulation model difference albumen of aminocaproic acid
The tissue expression of human homology's albumen of differential protein is determined according to the method in (3) in the 2 of step (1).Point
Determine there are 9 differential protein height to be expressed in 36 tissues, specifically as shown in table 14 and Fig. 5 after analysis.It can be seen that these eggs
White matter has higher expression in urinary system, digestive system and respiratory system.
The urine differential protein tissue expression analysis of the high coagulation model of table 14, aminocaproic acid
It therefore, can be according to the high coagulation marker that above-mentioned each model discrimination arrives according to as follows in practical application
(1) any method judges whether subject is in high coagulation in-(3):
(1) if the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribose core
The content of sour enzyme 4 is above at least one times of Healthy People, and chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is below at least one times of Healthy People, then
Subject is in or candidate is in high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate induction institute
Cause high coagulation；
If the Golgi apparatus protein I of subject's urine, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3 and ribonucleic acid
The content of enzyme 4 is not above at least one times of Healthy People or chloride ion channel protein 1 intracellular, biglycan, Rab GDP
Dissociate inhibitor, phosphoglyceric kinase 1, aflatoxin B1 aldehyde reductase member 2, Histidine Triad nucleotide combination egg
White 1, the content of myosin light chain 6, L- xyloketose reductase and hemoglobin β-chain is not below at least one times of Healthy People,
Then subject is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to etamsylate
High coagulation caused by inducing.
(2) if the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanylic acid combine
Protein G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanine
The content of nucleotide binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is above health
At least one times of people, and superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase,
Myosin light chain 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is below at least one times of Healthy People, then subject is in or candidate is in high coagulation；And this high blood coagulation
The Forming Mechanism of state is relevant to high coagulation caused by tranexamic acid induces.
If the cytosol aminopeptidase of subject's urine, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanine-nucleotide-binding protein
G (I)/G (S)/G (T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanosine
The content of acid binding protein G (I)/G (S)/G (T) β -2 subunit and angiotensin converting enzyme2 homologue is not above Healthy People
At least one times or superoxide dismutase [Cu-Zn], amyloid protein 2, calbindin, fumarylacetoacetase, flesh
Immunoglobulin light chains 6, frizzled protein 2, Apoliprotein M, myoglobins, ephrins B1, selenium Binding Protein 1 and calmodulin
(CaM) content is not below at least one times of Healthy People, then subject is not at or candidate is not at high coagulation；And it is this
The Forming Mechanism of high coagulation is relevant to high coagulation caused by tranexamic acid induces.
(3) if the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also
The content of albumen (Trx) is above at least one times of Healthy People, and peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is below at least one times of Healthy People, then subject
It is in or candidate in high coagulation；And the Forming Mechanism of this high coagulation is relevant to Gao Ning caused by aminocaproic acid induces
Blood state.
If the cell adhesion molecule 3 of subject's urine, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin and sulphur oxygen also egg
The content of white (Trx) is not above at least one times of Healthy People or peptidyl prolyl cis-trans isomerase A, ABHD14B albumen, curling
Albumen 4, anthrax toxin acceptor 1, frizzled protein 2, attracts element, Neuroplastin, CD166 antigen, gluathione at α-enolase
Peptide S- transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxidoreducing enzyme, Rho GDP- dissociate inhibitor 1, mistake
It is oxidoreductase -6, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, preceding
Column parathyrine F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, Histidine Triad nucleotide binding protein 1, connection are viscous
Attached molecule C, this calcium plain -1, Glutamate-cysteine ligase catalytic subunit, N (G), N (G)-diethylarginine dimethylamino
Base hydrolase 1, alpha-N-Acetylgalactosaminidase, fumarylacetoacetase, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene
The content of dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4 is not below at least one times of Healthy People, then tested
Person is not at or candidate is not at high coagulation；And the Forming Mechanism of this high coagulation is relevant to aminocaproic acid induction institute
Cause high coagulation.
Claims (3)
1. detecting application of the system of X content in the product of preparation diagnosis or auxiliary diagnosis people or the high coagulation of rat；
Or, application of the system of detection X content in the product of preparation diagnosis or auxiliary diagnosis people or rat suppository disease；
The X content is X content in urine；
The system of the detection X content is reagent and/or instrument needed for the detection X content；
The X be following X1) X2) or X3):
X1 be) Golgi apparatus protein I, carbonic anhydrase I, Monophosphoinositideproteoglycans proteoglycans-3, ribalgilase 4, chloride ion it is intracellular
Channel protein 1, biglycan, Rab GDP dissociation inhibitor, phosphoglyceric kinase 1, the reduction of aflatoxin B1 aldehyde
Enzyme member 2, Histidine Triad nucleotide binding protein 1, myosin light chain 6, L- xyloketose reductase and hemoglobin β are sub-
Base；
It X2) is cytosol aminopeptidase, ribonucleoside triphosphote diphosphonic acid hydrolase 5, guanine-nucleotide-binding protein G (I)/G (S)/G
(T) β -1 subunit, actin sample albumen, guanine-nucleotide-binding protein G (k) α subunit, guanine-nucleotide-binding protein G
(I)/G (S)/G (T) β -2 subunit, angiotensin converting enzyme2 homologue, superoxide dismutase [Cu-Zn], amyloid protein
2, calbindin, fumarylacetoacetase, myosin light chain 6, frizzled protein 2, Apoliprotein M, myoglobins, liver
With protein B 1, selenium Binding Protein 1 and calmodulin (CaM)；
It X3) is cell adhesion molecule 3, precollagen C- endopeptidase reinforcing agent 1, ceruloplasmin, thioredoxin (Trx), peptidyl dried meat
Aminoacyl cis-trans isomerase A, ABHD14B albumen, α-enolase, anthrax toxin acceptor 1, frizzled protein 2, attract frizzled protein 4
Element, Neuroplastin, CD166 antigen, glutathione S-transferase P, alkaline phosphatase, 14-3-3 albumen ζ/δ, quinolone oxygen
Change reductase, Rho GDP- dissociates inhibitor 1, peroxiredoxin -6, polyphosphoinositide phosphatase 1, G-6-P
Isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative growth factor, chloride ion channel protein 4 intracellular, group
Propylhomoserin triplet nucleotide binding protein 1, connection adhesion molecule C, this calcium element -1, Glutamate-cysteine ligase catalysis are sub-
Base, N (G), N (G)-Dimethylarginine dimethylaminohydro-lase 1, alpha-N-Acetylgalactosaminidase, fumaryl acetyl second
Sour enzyme, 1,2- dihydroxy -3- ketone -5- methyl mercapto amylene dioxygenase, selenium Binding Protein 1 and chondroitin sulfate proteoglycan 4；
The alkaline phosphatase is tissue non-specific alkaline phosphatase.
2. X described in claim 1.
3. X described in claim 1 is diagnosed using the X described in claim 1 as high coagulation marker in preparation
Or the application in the product of auxiliary diagnosis people or the high coagulation of rat；
Or, X described in claim 1 or using the X described in claim 1 as high coagulation marker preparation diagnose
Or the application in the product of auxiliary diagnosis people or rat suppository disease.
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| CN101111604A (en) * | 2004-10-06 | 2008-01-23 | 新加坡科技研究局 | Methods, systems, and arrays based on correlating p53 status with gene expression profiles, for classification, prognosis, and diagnosis of cancers |
| CN102834116A (en) * | 2009-09-21 | 2012-12-19 | 杜克大学 | Treatment of WNT/frizzled-related diseases |
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