CN107255711B - Osteopontin is used to prepare or screens the purposes of acute-on-chronic liver failure diagnostic reagent - Google Patents
Osteopontin is used to prepare or screens the purposes of acute-on-chronic liver failure diagnostic reagent Download PDFInfo
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- CN107255711B CN107255711B CN201710326873.5A CN201710326873A CN107255711B CN 107255711 B CN107255711 B CN 107255711B CN 201710326873 A CN201710326873 A CN 201710326873A CN 107255711 B CN107255711 B CN 107255711B
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- osteopontin
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Abstract
The invention belongs to biomedical and medical diagnosis on disease fields, and in particular to osteopontin is used to prepare or screens the purposes of acute-on-chronic liver failure diagnostic reagent.Biomarker whether can be as differentiation acute-on-chronic liver failure the invention firstly discloses osteopontin (diagnosis effect is up to 0.9454), be apparently higher than M30(0.7167), M65(0.8319) and M30/M65(0.8100), and almost maintain an equal level with clinically common counter MELD(0.9317).
Description
Technical field
The invention belongs to biomedical and medical diagnosis on disease fields, and in particular to osteopontin is used to prepare or screens slow urgent
The purposes of property hepatic failure diagnostic reagent.
Background technique
Osteopontin (Osteopontin, OPN) is a kind of multifunctional protein, is distributed widely in Various Tissues and cell,
Immune, angiogenesis, fiber generate and tumour row at etc. during play an important role.Osteopontin with it by matching
Body integral protein, CD44, lipopolysaccharides, vimentin and MyD88 effect are sent out with during intercellular signal transduction in the cell
Important function is waved, to adjust cell Proliferation, migration, inflammation, fibrosis and canceration.A large amount of research reports bone bridge at present
Albumen is sent out in the disease of acute liver damage, nonalcoholic fatty liver, alcoholic liver, hepatitis, Hepatic fibrosis and cirrhosis and liver cancer
Crucial signal is played during hair tonic exhibition goes to effect.
Acute-on-chronic liver failure (Acute-on-Chronic Liver Failure, ACLF) is on the basis of chronic liver disease
The acute hepatic decompensation of appearance.This is different from chronic liver function decompensation, and the latter refers on liver cirrhosis basis, liver function
It with ascites or portal hypertension, coagulation disorders and hepatic encephalopathy etc. is the chronic liver function mainly showed caused by progressive decline
It can decompensation.Slow urgent type hepatic failure is mainly shown as multiple organ failure, and along with 30% or more the death rates on the 28th and
60% or more the death rates on the 90th (Bernal W, Jalan R, Quaglia A, Simpson K, Wendon J, Burroughs
A. Acute-on-chronic liver failure.Lancet 2015.).Therefore, early stage occurs for acute-on-chronic liver failure
Timely diagnosing and treating is most important for survival of patients.The diagnostic criteria of acute-on-chronic liver failure is still disputable at present, CLIF-
C OF scoring is one of current Main Diagnosis standard.There is research by there is sub- massive necrosis region on pathological section from decompensation
Acute-on-chronic liver failure patient is distinguished in liver cirrhosis patient.But the above diagnostic method or complicated and time consumption or invasive, for
Diagnosis has significant limitation in time.Thus, develop new diagnosis that is sensitive, fast and efficiently assisting acute-on-chronic liver failure
Or the method for monitoring progression of disease, it is most important for clinical timely diagnosing and treating.
Summary of the invention
In order to overcome the defects of the prior art, it is used to prepare or screens the purpose of the present invention is to provide osteopontin and is slow
The purposes of extra urgaent dispatch hepatic failure diagnostic reagent.
Another object of the present invention is to provide the reagents of specific recognition osteopontin to prepare acute-on-chronic liver failure
Purposes in diagnostic kit.
Another object of the present invention is to provide a kind of acute-on-chronic liver failure diagnostic kits.
Purposes another object of the present invention is to provide osteopontin as acute-on-chronic liver failure biomarker.
Another object of the present invention is to provide a kind of acute-on-chronic liver failure diagnostic methods.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention provides the use that osteopontin was used to prepare or screened acute-on-chronic liver failure diagnostic reagent
On the way.
Preferably, the osteopontin is as biomarker.
More preferably, the osteopontin is as serum biomarkers.
Preferably, osteopontin is used to prepare or screens acute-on-chronic liver failure diagnostic reagent, including both sides content:
First, osteopontin is used to prepare acute-on-chronic liver failure diagnostic reagent, refer to using osteopontin as slow urgent
Property hepatic failure diagnosis index be applied to acute-on-chronic liver failure diagnostic reagent preparation.
It in some embodiments, can be using osteopontin as standard items or positive control, for the bone in sample serum
The detection of pontin protein level.
Second, osteopontin refers to for screening acute-on-chronic liver failure diagnostic reagent using osteopontin as slow urgent
Property hepatic failure identification target sieving specific recognition osteopontin reagent, thus as acute-on-chronic liver failure diagnose try
Agent, to detect acute-on-chronic liver failure.
In some embodiments, the antibody of osteopontin is specifically bound based on the osteopontin screening or matched
Body, thus as acute-on-chronic liver failure diagnostic reagent.For example, osteopontin enzyme linked immunological kit (is purchased from R&D Systems
Company, article No.: DOST00) in reagent can be with specific recognition osteopontin, therefore can be used as acute-on-chronic liver failure and examine
Disconnected reagent.
The second aspect of the present invention provides the reagent of specific recognition osteopontin and examines preparing acute-on-chronic liver failure
Purposes in disconnected kit.
In some embodiments, the reagent of the specific recognition osteopontin can be specific binding osteopontin
Antibody or ligand.The antibody can be monoclonal antibody or polyclonal antibody.
For example, the reagent in osteopontin enzyme linked immunological kit (being purchased from R&D Systems company, article No.: DOST00)
Can be with specific recognition osteopontin, therefore can be used as and prepare acute-on-chronic liver failure diagnosis examination box.
In the third aspect of the present invention, a kind of acute-on-chronic liver failure diagnostic kit is provided, in the kit
Reagent at least containing specific recognition osteopontin.
In some embodiments, the reagent of the specific recognition osteopontin can be specific binding osteopontin
Antibody or ligand.The antibody can be monoclonal antibody or polyclonal antibody.
In some embodiments, also contain in the kit: immune to combine (such as antigen-antibody combination) reagent；Or
Enzyme linked immunosorbent detection (ELISA) reagent.For example, osteopontin enzyme linked immunological kit (it is purchased from R&D Systems company, article No.:
DOST00)。
In the fourth aspect of the present invention, purposes of the osteopontin as acute-on-chronic liver failure biomarker is provided.
Preferably, the biomarker is serum biomarkers.
In the fifth aspect of the invention, a kind of method for diagnosing acute-on-chronic liver failure, including detection sample blood are provided
The level of osteopontin in clear.
Compared with prior art, the beneficial effects of the present invention are:
The invention firstly discloses osteopontins (to examine as biomarker whether distinguishing acute-on-chronic liver failure
0.9454) disconnected effect is up to, hence it is evident that be higher than M30 (0.7167), M65 (0.8319) and M30/M65 (0.8100), and with clinically
Common counter MELD (0.9317) almost maintains an equal level.
Detailed description of the invention
Fig. 1: osteopontin is in acute-on-chronic liver failure patient, Decompensated liver cirrhosis patient, patients with hepatocellular carcinoma and normal
The detection of protein expression level in human serum.
Fig. 2: M30, egg of the M65 and M30/M65 in acute-on-chronic liver failure patient and Decompensated liver cirrhosis patients serum
The detection of white expression.
Fig. 3: osteopontin, MELD, M30, M65 and M30/M65 work for diagnosing the subject of acute-on-chronic liver failure
The drafting of feature (ROC) curve.
Specific embodiment
The present invention after extensive and in-depth study, lose with Healthy People, patients with hepatocellular carcinoma and cirrhosis by discovery for the first time
Compensatory patient compares, and osteopontin content significantly increases in acute-on-chronic liver failure peripheral blood in patients serum, with End-stage liver disease
Model (MELD), total bilirubin, creatinine level are positively correlated, and related to disease and coincident with severity degree of condition.Osteopontin
A kind of diagnosis marker that can be used as acute-on-chronic liver failure facilitates susceptibility analysis, the diagnosis of acute-on-chronic liver failure
And treatment.According to the drug and diagnostic techniques of human osteopontin design, it can be used for the slow extra urgaent dispatch liver failure of the diagnosing and treating mankind
It exhausts.The present invention is completed on this basis.
Osteopontin and application thereof
Full text in the present invention, the osteopontin are known albumen, and English is Osteopontin, abbreviation OPN.People's bone bridge
The protein sequence of albumen can refer to GenBank:BAA03554.1.
In the prior art, there are no using osteopontin as the related report of the molecular labeling of acute-on-chronic liver failure.
The present invention is found through experiments that osteopontin expression quantity in acute-on-chronic liver failure patients serum is significantly higher than just
Ordinary person's group.Therefore, the purposes the present invention provides a kind of osteopontin as acute-on-chronic liver failure marker.As the present invention
Preferred embodiment, osteopontin as acute-on-chronic liver failure serum detect marker.Serum detection materials are convenient, operation is simple
List is convenient for check, and patient is easy to receive, and assesses prognosis, guiding treatment.
Above-mentioned new discovery based on the present inventor, the mark using osteopontin as diagnosis acute-on-chronic liver failure
Object: the antidiastole and/or susceptible analysis of (i) progress acute-on-chronic liver failure；(ii) the slow extra urgaent dispatch of correlated crowd is assessed
Hepatic failure therapeutic agent, curative effect of medication, patient's prognosis, and the suitable treatment method of selection；(iii) assessment correlated crowd adds slowly
Acute hepatic failure risk, detection simultaneously carry out early prevention and treatment.
Therefore, it the present invention provides the purposes of osteopontin, is used to prepare or the slow extra urgaent dispatch liver of Screening Diagnosis (or detection)
The reagent or kit of failure.
Detection reagent
Various techniques known in the art can be used to detect presence or absence and the expression of osteopontin, these technologies
It is all contained in invention.For example, prior art such as Southern blotting, Wester blotting etc. can be used, these methods can be tied
It closes and uses.
The present invention also provides the reagents of presence or absence and expression for detecting osteopontin in analyte.It is excellent
Choosing, when carrying out the detection of protein level, the antibody of specific recognition osteopontin can be taken to determine depositing for osteopontin
Whether.
It is also come the method for testing and analyzing osteopontin expression situation in object using the antibody for specifically binding osteopontin
Technology well known in the art.
The antibody that bind osteopontin can be prepared by various technologies known to those skilled in the art, can also be used
Commercialized antibody.For example, the osteopontin of purifying, can be applied to animal to induce polyclonal antibody to generate.Similarly
, the cell for expressing osteopontin can be used to immune animal to generate antibody.The antibody of this discovery is also possible to monoclonal antibody.
Such clonal antibody can use hybridoma technology to prepare (see Kohler et al., Nature256；495,1975；Kohler etc.
People, Eur.J.Immunol.6:511,1976；Hammerling et al., In Monoclonal Antibodies and T
Cell Hybridomas, Elsevier, N.Y., 1981).
Animal, such as rabbit, mouse, rat can be immunized in the production of polyclonal antibody with osteopontin.A variety of adjuvants are available
It is immunoreacted in enhancing, including but not limited to Freund's adjuvant etc..
The detectable signal that specificity is marked in monoclonal antibody or polyclonal antibody is also known to those skilled in the art
Technology, for example, can be by horseradish root peroxidase conjugate in the antibody of the specific binding osteopontin, so as to pass through
Whether generation of the chromogenic reaction to learn antigen-antibody binding reaction.
A kind of method that whether there is osteopontin in detection sample to be tested, be using osteopontin specific antibody into
Row detection, it includes: to contact sample with Effect of Osteopontin-specific antibody；It sees whether to form antibody complex, form anti-
Nanocrystal composition means that there are osteopontins in sample.
Detection kit
The present invention also provides the presence or absence for detecting osteopontin in analyte with the kit of expression,
The kit includes: the antibody or ligand for specifically binding osteopontin.
In addition, may also include in the kit immune in conjunction with (antigen-antibody combination) reagent or enzyme linked immunosorbent detection
(such as ELISA) reagent；And for the various reagents needed for hybridizing, develop the color etc., including but not limited to: hybridization solution, enzyme, right
According to liquid, developing solution, cleaning solution etc..
Main advantages of the present invention are:
The present invention is disclosed by research and facing for acute-on-chronic liver failure is diagnosed and monitored using Serum osteopontin content
Bed purposes.The method that the present invention will test osteopontin content in peripheral blood for the first time is applied to diagnosis acute-on-chronic liver failure,
Especially the content of osteopontin is slower than M30, M65, M30/M65 tri- that early period, other people had found in the peripheral blood of discovery detection for the first time
The serum mark of extra urgaent dispatch hepatic failure is sensitiveer, and then verifies a kind of method for detecting osteopontin content in peripheral blood, makees
For the kit of preparation diagnosis acute-on-chronic liver failure.This supervises the timely diagnosis of clinical acute-on-chronic liver failure patient and the state of an illness
Survey provides more sensitive, efficient, stable approach.And these innovative discoveries of the present inventor, are slow extra urgaent dispatch liver failure
The quick diagnosis and monitoring exhausted provides theoretical basis, new detection method, has to basic research and clinical application important
Meaning.
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Disclosed content understands further advantage and effect of the invention easily.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLEC μ LAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLEC μ LAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLEC μ LAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
1. experimental method
Collect 70 confirmation acute-on-chronic liver failure patients (ACLF+), 67 Decompensated liver cirrhosis patients (ACLF-), 50
Example patients with hepatocellular carcinoma (HCC), 30 normal persons (Health) peripheral blood sample, each part Blood Sample Collection is in coagulation vessel
In, volume is 5ml or so, and 5000rpm is centrifuged 10min, draws the clear serum in upper layer.Serum is detected by ELISA kit
Middle osteopontin, M30, M65 concentration (osteopontin enzyme linked immunological kit be purchased from R&D Systems company, article No.:
DOST00；M30, M65 enzyme linked immunological kit are purchased from VLVbio company, and article No. is respectively nr.10011 and nr.10040),
MELD, total bilirubin and creatine concentration are obtained by patient history data.ROC is carried out using 6.0 software of GraphPad Prism
Drawing of Curve examines comparison among groups with Mann-Whitney, to correlation analysis Spearman correlation test.P<0.05
Think that there were significant differences.
2. experimental result and analysis
Fig. 1 is shown in comparison in serum between the level and each group of osteopontin.
As shown in Figure 1, in acute-on-chronic liver failure patients serum osteopontin levels it is extremely significant be higher than other three groups (P <
0.0001), the level of osteopontin in serum is prompted to can be used as blood serum designated object, for diagnosing and monitoring slow extra urgaent dispatch liver failure
Exhaust patient.
M30, M65 and M30/M65 compare in Decompensated liver cirrhosis group and acute-on-chronic liver failure group in serum sees Fig. 2.
As shown in Figure 2, M30, M65 acute-on-chronic liver failure patient be significantly higher than Decompensated liver cirrhosis group (P <
0.0001), M30/M65 is substantially less than Decompensated liver cirrhosis group (P < 0.0001) in acute-on-chronic liver failure patient.This with before
Reported in literature be consistent.
The correlation detection of osteopontin and MELD, total bilirubin, creatinine in acute-on-chronic liver failure patient, as a result such as
Shown in table 1.
Table 1
As shown in Table 1, osteopontin levels and its MELD, total bilirubin and creatinine in acute-on-chronic liver failure patients serum
Be positively correlated (P < 0.01), it is seen that osteopontin levels are related with disease severity in acute-on-chronic liver failure patients serum.
Osteopontin, MELD, M30, M65 and M30/M65 for diagnose acute-on-chronic liver failure Receiver Operating Characteristics
(ROC) drafting of curve is as shown in table 2:
Table 2
As shown in Table 2, it is diagnosed to be acute-on-chronic liver failure patient, from Decompensated liver cirrhosis patient with bone bridge egg in serum
White level is apparently higher than M30 (0.7167), M65 (0.8319) and M30/ as the diagnosis effect (0.9454) of biomarker
M65 (0.8100), and almost maintain an equal level with clinically common counter MELD (0.9317).
In conclusion the present invention provides one kind in decompensation cirrhosis quick diagnosis and to screen acute-on-chronic liver failure trouble
The method of person, to realize the timely and correct treatment of patient；The present invention can also be used in the disease progression for monitoring patient, so as to
In adjustment therapeutic strategy, prevent treatment is inappropriate not in time from causing death, great clinical value and application prospect.
Above embodiment is can not to be interpreted as in order to illustrate embodiment disclosed by the invention to limit of the invention
System.In addition, in various modifications and invention listed herein method, composition variation, do not departing from the scope of the present invention
Be obvious for those skilled in the art under the premise of spirit.Although having combined of the invention a variety of specific
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various obviously modify as described above for those skilled in the art to obtain invention all should include
Within the scope of the invention.
Claims (5)
1. osteopontin is used to prepare or screens the purposes of acute-on-chronic liver failure diagnostic reagent, institute as serum biomarkers
Diagnostic reagent is stated for being diagnosed to be acute-on-chronic liver failure patient from Decompensated liver cirrhosis patient.
2. purposes according to claim 1, which is characterized in that osteopontin is used to prepare or screens acute-on-chronic liver failure
Diagnostic reagent, including both sides content: first, osteopontin is used to prepare acute-on-chronic liver failure diagnostic reagent, refer to by
Osteopontin is applied to the preparation of acute-on-chronic liver failure diagnostic reagent as acute-on-chronic liver failure diagnosis index, can be by bone bridge
Detection of the albumen as standard items or positive control, for the osteopontin levels in sample serum；Second, osteopontin is used for
Acute-on-chronic liver failure diagnostic reagent is screened, is referred to special using osteopontin as the identification target sieving of acute-on-chronic liver failure
Property identification osteopontin reagent, thus as acute-on-chronic liver failure diagnostic reagent, to detect acute-on-chronic liver failure.
3. the reagent of specific recognition osteopontin is preparing the purposes in acute-on-chronic liver failure diagnostic kit, the diagnosis
Kit from Decompensated liver cirrhosis patient for being diagnosed to be acute-on-chronic liver failure patient.
4. purposes according to claim 3, which is characterized in that the reagent of the specific recognition osteopontin is specificity
The antibody or ligand of bind osteopontin.
5. purposes according to claim 4, which is characterized in that the antibody is monoclonal antibody or polyclonal antibody.
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