CN108241054A - G protein coupled receptor 18 is preparing septicemia monitoring and is judging to apply in reagent - Google Patents
G protein coupled receptor 18 is preparing septicemia monitoring and is judging to apply in reagent Download PDFInfo
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- CN108241054A CN108241054A CN201711427770.4A CN201711427770A CN108241054A CN 108241054 A CN108241054 A CN 108241054A CN 201711427770 A CN201711427770 A CN 201711427770A CN 108241054 A CN108241054 A CN 108241054A
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Abstract
The invention discloses g protein coupled receptors 18 to express the application in Diagnosis of Septicemia and course of disease monitoring and Index for diagnosis reagent is prepared, and confirms that 18 expression of g protein coupled receptor is lowered in the expression of septicemia neutrophils surface.The method of the neutrophils surface g protein coupled receptor 18 detected in peripheral blood is applied in diagnosis septicemia and serious bacterial infections, cell surface g protein coupled receptor 18 is expressed lower in especially finding for the first time, septicemia and serious bacterial infections patient's multiple organ injury's degree are heavier, and prognosis is poorer.This provides approach that is more easy, sensitive, quick, stablizing for the sick human or animal and monitoring progression of disease of clinical diagnosis septicemia or serious bacterial infections.
Description
Technical field
The present invention relates to biomedical and medical diagnosis on disease technical field, specially g protein coupled receptor 18 is preparing sepsis
Disease monitors and judges to apply in reagent.
Background technology
Septicemia or septic shock are clinically common critical illnesses, at present, although the development of medicine is maked rapid progress,
It is that septicemia is still world-class problem.G protein coupled receptor 18 is expressed in human body in various kinds of cell and tissue.As
Cell surface receptor can be by multiple ligands, such as anandamide, peanut acylglycine, cannboid, recession element
Activation participates in reproduction, immune, metabolism and the regulation and control of nervous system.It recent studies have shown that g protein coupled receptor 18 in septicemia inflammation
It plays an important role in disease response regulatory, gene, which knocks out the 18 mouse septicemia death rate of g protein coupled receptor, significantly to be increased, huge
Phagocyte inflammatory factor secretes showed increased, while g protein coupled receptor 18 is expressed in neutrophils surface height, and regulation and control are neutral
The chemotaxis of granulocyte.Recession element can activate 18 receptor of neutrophils surface g protein coupled receptor, reduce mouse septicemia
Severity.In addition research shows that the expression of neutrophils surface receptor and the severity of septicemia are closely related, such as
The expression of complement receptors C5aR and the injuries of tissues and organs degree of septicemia and case fatality rate positive correlation, the expression of C5aR is lower, group
It is more serious to knit organ damage, case fatality rate is higher.Therefore, the expression of detection neutrophils surface associated receptor is to the morning of septicemia
Phase diagnoses and Index for diagnosis has important value.The inspection of septicemia or serious bacterial infections is mainly by bacteriology side at present
The degree of method, the blood routine especially prompting bacterium infection such as the inspection of white blood cell count(WBC) and classification, c reactive protein detection, passes through
The gene amplification method of method or applied molecular biology technology for doing blood Bacteria Culture of taking a blood sample is determined whether for infection.It is but thin
Mycology method has that time-consuming and the weakness of false positive, to diagnosing in time and medication generates great limitation, and in vitro culture
It cannot reflect the severity of bacterium infection.Thus, the auxiliary septicemia or severe bacterium that development is easy, sensitive, quick, stable
The diagnosis of infection or the new method of monitoring progression of disease and Index for diagnosis, it is most important for clinical timely diagnose and treat, because
This monitors in preparation septicemia we have proposed g protein coupled receptor 18 and to judge to apply in reagent.
Invention content
The purpose of the present invention is to provide g protein coupled receptors 18 to prepare septicemia monitoring and judge to apply in reagent,
To solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical solution：G protein coupled receptor 18, which is expressed, is preparing septicemia
Application in diagnosis and course of disease monitoring and Index for diagnosis reagent.
Preferably, the g protein coupled receptor 18 is neutrophil leucocyte biomarker.
Preferably, the g protein coupled receptor 18 is expressed in neutrophils surface and is lowered.
Preferably, diagnostic reagent of the diagnostic reagent for cell membrane surface protein level, the cell membrane surface protein
Horizontal diagnostic reagent includes the expression that 18 albumen of g protein coupled receptor is detected by immunological method.
Preferably, the 18 streaming antibody of g protein coupled receptor or 18 antibody of g protein coupled receptor composition kit, institute
State the secondary antibody that 18 streaming antibody of g protein coupled receptor and 18 antibody of g protein coupled receptor carry fluorescence.
Compared with prior art, the beneficial effects of the invention are as follows：The g protein coupled receptor 18 prepare septicemia monitoring and
Judge to apply in reagent, the method for the middle cell surface g protein coupled receptor 18 detected in peripheral blood is applied to diagnosis sepsis
In disease and serious bacterial infections, cell surface g protein coupled receptor 18 expresses lower, septicemia and severe in especially finding for the first time
Bacterial infection patients multiple organ injury's degree is heavier, and prognosis is poorer.This is clinical diagnosis septicemia or the disease of serious bacterial infections
Human or animal and monitoring progression of disease provide approach that is more easy, sensitive, quick, stablizing.
Description of the drawings
Fig. 1 is the neutrophil leucocyte subgroup figure of flow cytometer delineation of the present invention；
Fig. 2 is the block diagram that flow cytomery g protein coupled receptor 18 of the present invention is expressed；
Fig. 3 is 18 expression figure of normal person of the present invention and septicaemia patient's neutrophils surface g protein coupled receptor；
Fig. 4 is the expression of septicaemia patient neutrophils surface g protein coupled receptor 18 of the present invention and SOFA scoring relationships
Figure；
Fig. 5 is 18 expression and c reactive protein relationship of septicaemia patient neutrophils surface g protein coupled receptor of the present invention
Figure；
Fig. 6 is that septicaemia patient neutrophils surface g protein coupled receptor 18 of the present invention is expressed and Prognostic Significance；
Fig. 7 is critical value of the expression of the present inventor's neutrophils surface g protein coupled receptor 18 as diagnosis of sepsis disease
Confirm figure；
Fig. 8 is the expression of neutrophils surface g protein coupled receptor 18 variation and time phase in septicemia mouse of the present invention
Guan Xingtu；
Fig. 9 is that LPS of the present invention stimulates the normal expression of human neutrophil rear surface g protein coupled receptor 18 variation and time
Dependency graph；
Figure 10 is that various concentration LPS of the present invention stimulates normal human neutrophil rear surface g protein coupled receptor 18 to change
Figure.
Specific embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, the every other reality that those of ordinary skill in the art are obtained without making creative work
Example is applied, shall fall within the protection scope of the present invention.
- 10 are please referred to Fig.1, the present invention provides a kind of technical solution, and experimentation is as follows：
First, one is tested
1st, experiment packet：67 peripheral blood samples for making a definite diagnosis septic patient are collected as experimental group, collect 29 health
The peripheral blood sample of people is as a control group.For each part Blood Sample Collection in anticoagulant tube, volume is 2ml or so, and room temperature is preserved, taken
Stream measuring neutrophils surface receptors G protein coupled receptor 18 is expressed on the day of blood, at the same monitor death number and when
Between.It is still necessary to acquire following information by all subjects：Age, gender, with disease and medicining condition, vital sign readings, qSOFA
Scoring, SOFA scorings, position, type, degree, the type of pathogenic microorganism and the drug sensitivity tests of infection, therapeutic scheme.Exclude mark
It is accurate：Gestation, blood disease, cardiogenic shock, HIV infection, the law of acceptance glucocorticoid in nearly 6 months, immunosuppressor and thin
Born of the same parents' toxitherapy, progression of disease is quickly expected may died in 24 hours.
2nd, diagnostic criteria：The infection made a definite diagnosis meets one of them：(1) by cultivating, dyeing or the certified diseases of PCR
Pathogenic microorganism infects.(2) clinical specificity infection sign, with reference to radiological evidence (such as：Abdominal X-ray or CT scan are visible free
The bowel perforation evidence of gas；The visible sheet shadow prompting pneumonia of rabat；Organs infection that abdominal ultrasonic or iconography confirm etc.).
3rd, multiple organ injury's degree evaluation standard：Intensive care unit sepsis is assessed using Sequential organ failure assessment score
Disease patient multiple organ injury degree has received and has reflected relatively accurate amount in patient's severity by most experts now
Table.SOFA standards of grading such as following table：SOFA scorings are each system scoring summations, i.e., are first scored according to standard each system,
Finally each system is scored and is added, concentrated expression patient organ's degree of injury.
SOFA grade forms：
Table explanation：
MAP：Mean arterial pressure；
Catecholamine dosage is given at least 1 hour by ug/kg/min；
Glasgow stupor grade scorings：3-15 points of range, score is higher to show that nervous function is better.
Glasgow stupor scales：
Table explanation
Slightly：13-15；Moderate：9-12；Severe：4-8；Brain death：3.
4th, flow cytometry
Take Healthy People or patient whole blood, washed 2-3 times with PBS, with 10% serum close 10min, carry out G-protein coupling by
18 antibody of body dyes, while sets Isotype control group, adds colleague's control antibodies dyeing.And it is contaminated with the donkey anti-rabbit secondary antibody with fluorescence
Color, erythrocyte cracked liquid cracking 10min, after removing red blood cell, flow cytometer delineation neutrophil leucocyte group (Fig. 1), homotype pair
According to a series of paintings door, while detect g protein coupled receptor 18 on neutrophil cell membrane receptor and (scheme in the expression of neutrophils surface
2)。
5th, experimental result and analysis
67 make a definite diagnosis septic patient and 29 normal healthy people neutrophils surface g protein coupled receptors 18 express into
Row compares, and the expression of g protein coupled receptor 18 of normal healthy people and septic patient has apparent difference, as shown in figure 3, with
Healthy People ratio, septic patient neutrophils surface g protein coupled receptor 18 express apparent downward, prompt neutrophil leucocyte table
18 expression of face g protein coupled receptor can be as diagnosis septicemia or the reliable method of serious bacterial infections.
Meanwhile we carry out SOFA scorings to 67 patient organ's degree of injury, standard as shown above, and analyzes G eggs
The correlation that white 18 expression of coupled receptor scores with SOFA, is as a result shown in Fig. 4, as shown in figure 4,18 table of g protein coupled receptor
There is correlation with SOFA scorings up to horizontal, the lower patient of 18 expression of g protein coupled receptor, SOFA scorings are generally all
Higher, i.e., organ damage is more serious；In addition, c reactive protein is present clinically another index of the detection pyemia state of an illness, with this
We analyze 18 expression of g protein coupled receptor and the correlation of CRP, as a result see Fig. 5, as shown in figure 5, G-protein coupling by
18 expression of body has negative correlation with CRP, prompts neutrophils surface GPR18 receptor expression levels that can be used as monitoring
Septicemia or the reliable method of serious bacterial infections severity and disease progression.
In addition, we record 67 death situations, and 18 expression of g protein coupled receptor is analyzed with suffering from
The correlation of person's existence, is as a result shown in Fig. 6, as shown in fig. 6,18 expression of g protein coupled receptor and survival of patients are with related
Property, its prognosis possibility of the lower patient of 18 expression of g protein coupled receptor is poorer；We determine G-protein idol with ROC curve
Join the critical value of receptor 18 expression diagnosis of sepsis disease, as a result see Fig. 7, as shown in fig. 7, according to statistical analysis, G-protein coupling
(susceptibility 83%, 47%) specificity is, is less than to the best critical value of receptor 18 expression diagnosis of sepsis disease for 43.7%
Critical value patient death rate apparent increase in 28 days prompts 18 expression of neutrophils surface g protein coupled receptor that can make
To judge the reliable method of septicemia or serious bacterial infections prognosis.
Experiment two
1st, experimental animal
Experimental animal is C57BL/6 mouse, is randomly divided into 2 groups, i.e. sham-operation group and model group, every group 30, experiment is adopted
With ligaturing and mouse cap end of perforating, septicemia animal model is established.3h, 6h after modeling, for 24 hours, 48h and 72h take
Blood stream measuring neutrophils surface GPR18 receptor expression levels, are as a result shown in Fig. 6.
2nd, experimental result and analysis
CLP mouse models are the classical models for simulating Clinical sepsis, research shows that septicemia is with the time caused by CLP
Extension gradually aggravate, if do not treated, it is final most can be dead, therefore we check modeling after different time G-protein be coupled
Receptor 18 is expressed, and reacts its relationship changed with coincident with severity degree of condition, finds 3h and 6h after modeling, 18 table of g protein coupled receptor
Do not change up to level, as shown in fig. 6, modeling is begun to decline afterwards for 24 hours, with the extension of time, measuring neutrophils surface G
18 expression of G-protein linked receptor continuously decreases, and further neutrophils surface g protein coupled receptor 18 is prompted to express water
Putting down can be as monitoring septicemia or the reliable method of serious bacterial infections severity and disease progression.
3rd, three are tested
1st, cell experiment
Normal healthy people whole blood is taken, GE companies Percol separating liquid Separation of Neutral granulocytes are stimulated with the LPS of 20 μ g/mL
Different time, 18 expression variation of (10min, 20min, 30min, 1h) detection cell surface g protein coupled receptor, is as a result shown in
Fig. 7 detects cell surface after in addition stimulating 30min with various concentration LPS (100ng/mL, 1 μ g/mL, 10 μ g/mL, 20 μ g/mL)
18 expression of g protein coupled receptor changes, and as a result sees Fig. 8.
2nd, experimental result and analysis
The LPS that Gram-negative bacteria generates is the main medium for leading to septic, therefore we are pierced with the LPS of 20 μ g/mL
Swash normal human neutrophil observation g protein coupled receptor 18 to express, as shown in fig. 7,30min, as stimulation time extends, by
Body surface is up to continuously decreasing, and after 30min, expression of receptor is not changing, and g protein coupled receptor 18 is sent out rapidly after may being stimulated by LPS
Raw internalization prompts 18 expression of neutrophils surface g protein coupled receptor that can be used as quick diagnosis septicemia or severe
The reliable method of bacterium infection severity and disease progression.In addition we stimulate neutrophil leucocyte with the LPS of various concentration, such as
Shown in Fig. 8, the concentration increase of stimulation, the expression of g protein coupled receptor 18 is lower, prompts the coupling of neutrophils surface G-protein
Receptor 18 expression and septicemia or serious bacterial infections severity positive correlation can be used as quick monitoring septicemia or again
The reliable method of disease bacterium infection severity and disease progression.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1.G G-protein linked receptors 18 express the application in Diagnosis of Septicemia and course of disease monitoring and Index for diagnosis reagent is prepared.
2. g protein coupled receptor 18 according to claim 1 is preparing septicemia monitoring and is judging to apply in reagent, special
Sign is：The g protein coupled receptor 18 is neutrophil leucocyte biomarker.
3. g protein coupled receptor 18 according to claim 1 is preparing septicemia monitoring and is judging to apply in reagent, special
Sign is：The g protein coupled receptor 18 is expressed in neutrophils surface and is lowered.
4. g protein coupled receptor 18 according to claim 1 is preparing septicemia monitoring and is judging to apply in reagent, special
Sign is：The diagnostic reagent is the diagnostic reagent of cell membrane surface protein level, and the cell membrane surface protein is horizontal to examine
Disconnected reagent includes the expression that 18 albumen of g protein coupled receptor is detected by immunological method.
5. g protein coupled receptor 18 according to claim 1 is preparing septicemia monitoring and is judging to apply in reagent, special
Sign is：The 18 streaming antibody of g protein coupled receptor or 18 antibody of g protein coupled receptor composition kit, the G-protein are even
Join the secondary antibody that receptor 18 streaming antibody and 18 antibody of g protein coupled receptor carry fluorescence.
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| CN201711427770.4A CN108241054B (en) | 2017-12-26 | 2017-12-26 | Application of reagent for detecting G protein-coupled receptor 18 in preparation of sepsis diagnosis, disease course monitoring and prognosis judgment reagent |
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Citations (6)
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|---|---|---|---|---|
| CN1592625A (en) * | 2001-08-10 | 2005-03-09 | 惠氏公司 | G protein-coupled receptor assay |
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| CN106344922A (en) * | 2016-09-14 | 2017-01-25 | 安徽济人药业有限公司 | Application of G protein coupling receptor 18 agonist in preparation of infection drugs |
-
2017
- 2017-12-26 CN CN201711427770.4A patent/CN108241054B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1592625A (en) * | 2001-08-10 | 2005-03-09 | 惠氏公司 | G protein-coupled receptor assay |
| CN104630192A (en) * | 2002-10-02 | 2015-05-20 | 催化剂生物科学有限公司 | Methods of generating and screenign for porteases with altered specificity |
| WO2006117194A2 (en) * | 2005-05-03 | 2006-11-09 | Novartis Ag | Gpr18 as a biomaker for th1 mediated immune response |
| WO2007045197A2 (en) * | 2005-10-21 | 2007-04-26 | Justus Liebig Universität Giessen | Expression profiles for predicting septic conditions |
| CN102552306A (en) * | 2010-12-13 | 2012-07-11 | 马鞍山中美德康生物科技有限公司 | Method for treating septicemia with human neutrophil peptide (HNP) blocking agents |
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