CN108445228A - SCD40L albumen is preparing the application in early diagnosing dissection of aorta kit - Google Patents
SCD40L albumen is preparing the application in early diagnosing dissection of aorta kit Download PDFInfo
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- CN108445228A CN108445228A CN201810217855.8A CN201810217855A CN108445228A CN 108445228 A CN108445228 A CN 108445228A CN 201810217855 A CN201810217855 A CN 201810217855A CN 108445228 A CN108445228 A CN 108445228A
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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
- G01N33/68—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection
Abstract
The invention belongs to diagnostic kit molecular biology fields, and in particular to sCD40L albumen is preparing the application in early diagnosing dissection of aorta kit.The diagnostic reagent is that the expression quantity using sCD40L albumen as marker to it in blood plasma is detected, the risk that early diagnosis dissection of aorta occurs, can rapidly, effectively diagnose dissection of aorta, and with small, the simple and practicable feature of wound.
Description
Technical field
The invention belongs to diagnostic kit molecular biology fields, and in particular to sCD40L albumen is preparing early diagnosis master
Application in artery dissection kit.
Background technology
The disease that a kind of state of an illness of aorta clamp series of strata is dangerous, the death rate is high, aorta enhanced CT, Magnetic resonance imaging
(MRI) etc. imageological examinations are the major way of current diagnosis dissection of aorta, but there are Delay in Diagnosis, price are high for above method
The shortcomings of expensive, complicated for operation, and be invasive inspection, current diagnosis dissection of aorta still lacks specific biomarkers.With
Confirm that some are expected to the biological markers of early diagnosis dissection of aorta toward research, but its sensibility, specificity are poor.Cause
This, urgent need finds some specific biological markers and carrys out quick diagnosis dissection of aorta.
CD40L is a kind of transmembrane glycoprotein of tumor necrosis factor (TNF-α) family, is mainly expressed in the CD4+T of activation
Lymphocyte and mast cell surface provide costimulatory signal necessary to bone-marrow-derived lymphocyte activation.In addition it is also expressed in congee
Sample spot monocyte in the block, macrophage, B cell, endothelial cell and smooth muscle cell, can promote matrix metalloproteinase
(MMPs) and the release of oxygen radical.
Previously research thinks that sCD40L albumen plays a crucial role in the occurrence and development of artery sclerosis, and less studies have reported that
The relationship of sCD40L albumen and dissection of aorta.
Invention content
In view of the above-mentioned problems, the diagnosis valence that the purpose of the present invention is to provide sCD40L albumen in dissection of aorta patient
Value, relates generally to expression of the sCD40L albumen in dissection of aorta patient tissue and blood plasma；The present invention provides
SCD40L albumen is preparing the application in early diagnosing dissection of aorta kit.
In order to realize that the above problem, present invention offer adopt the following technical scheme that：
Application of the sCD40L albumen in preparing dissection of aorta early diagnosis kit, the diagnostic reagent be with
SCD40L albumen is detected its expression quantity in blood plasma as marker, is early diagnosed to dissection of aorta.
The diagnostic kit is with including：Washing lotion, dilution, titer, CD40L antibody, horseradish peroxidase-labeled chain
Mould Avidin, TMB luminous substrates, terminate liquid.
The diagnostic kit is mainly the content for detecting sCD40L albumen in blood plasma.
The application method of the diagnostic kit includes the following steps：
The acquisition of step 1, blood plasma：Research object extracts its venous blood at once after being admitted to hospital, and is received with the anticoagulant tube of vacuum containing EDTA
Collect blood specimen 5ml.
The processing of step 2, sample：In venous blood samples 10 minutes, anticoagulant tube 3000 turns/min centrifugal treating 10min, point
From blood plasma, the blood plasma handled well is stored in -80 DEG C of refrigerators and is preserved, is detected with detection kit of the present invention and is selected in research object blood
Starch sCD40L concentration；Promote solidifying pipe blood sample collection and send clinical laboratory of the court at once, detects its hs-CRP, d-dimer concentration.
SCD40L expression contents in step 3, ELISA method detection blood plasma, using experimental port and the average value of secondary orifices as examining
Survey the measured value of object.
The standardization of step 4, data：SCD40L standard concentrations are prepared according to kit specification, according to explanation
Book requires to sequentially add standard items by concentration gradient, establishes standard curve.
The detection of step 5, blood plasma marker object：It is detected with latex immunoturbidimetry and is selected in object blood plasma hs-CRP concentration；With
The detection of NycoCard methods is selected in object plasma D-dimer content concentration.
The expression of sCD40L in step 6, dissection of aorta patients blood plasma：In conjunction with Aortography as a result, according to survey
The level of sCD40L, hs-CRP, d-dimer in the blood plasma obtained, observe it and change over time, the variation of biomarker, and
Judge more accurate biomarker according to AUC area under the curve.
Step 7, Immunohistochemical detection sCD40L dissection of aorta row aorta displacement technique patient sustainer group
It knits and the expression of normal control aortic tissue.
CD40L is the II type transmembrane glycoprotein of 33~40k of relative molecular weight D, is made of 266 amino acid, and base is encoded
Because being positioned on X chromosome galianconism, has compared with high homology on amino acid sequence with tumor necrosis factor (TNF), therefore belong to
TNF superfamily members.CD40L is a kind of transmembrane glycoprotein, the main T lymphocytic cell surfaces expressed in CD4+, with its receptor
After CD40 is combined, the activation of mediate B cell, proliferation and promote antibody tormation, promote the generation of humoral immunity, be divided into it is soluble and
Membrane-binding amphitypy.Soluble CD 40 L (sCD40L) has strong bioactivity, can be with bone-marrow-derived lymphocyte, macrophage, endothelium
CD40 interactions on cell, vascular smooth muscle cells, stimulate these cells to generate a large amount of bioactie agents:As E- is selected
Element, adhesion molecule, cell factor, matrix metalloproteinase etc., previously research shows that matrix metalloproteinase can degrade including
Extracellular matrix including collagen and elastin causes extensive matrix destruction, to destroy the recombination of vascular wall normal configuration,
Therefore, there are certain correlativities for the occurrence and development of sCD40L and dissection of aorta.
Applicant passes through the study found that sCD40L great expressions at the breach of dissection of aorta patient, illustrate sCD40L
There are correlativities for formation with dissection of aorta；SCD40L is equal at dissection of aorta acute stage aorta cut and in blood plasma
Great expression.Therefore, blood plasma sCD40L can accurately be diagnosed to be dissection of aorta in early days.
Compared with prior art, the present invention has the advantages that：Can rapidly, effectively diagnose dissection of aorta, and
Feature small with wound, simple and practicable.
Description of the drawings
Fig. 1 is that ELISA detects expression of the sCD40L in blood plasma.
1-1 is control group and expression of the dissection of aorta group sCD40L in blood plasma.
1-2 is the expression of control group, acute coronary syndrome, dissection of aorta group sCD40L in blood plasma.
1-3 be control group, aorta penetrating ulcer, false aneurysm, Acute Aortic intramural hematoma, aortic aneurysm,
Expression of the dissection of aorta group sCD40L in blood plasma.
1-4 is control group, chronic dissection of aorta, subacute dissection of aorta, Aortic Dissection sCD40L in blood
Expression in slurry.
Wherein：control：Control group；PAU:Aorta penetrating ulcer；PSA:False aneurysm；AIMH:Acute active
Arteries and veins intramural hematoma；AA:Aortic aneurysm.
Fig. 2 is that ELISA detects the expression of sCD40L, hs-CRP, d-dimer in different time points in blood plasma.
2-1 is that the phase of blood plasma sCD40L concentration sexually revises.
2-2 is that the phase of blood plasma hs-CRP concentration sexually revises.
2-3 is that the phase of plasma D-dimer content concentration sexually revises.
2-4 blood plasma sCD40L, hs-CRP, d-dimer three's concentration phase sexually revise.
Fig. 3 be dissection of aorta patients blood plasma sCD40L, hs-CRP, d-dimer concentration ROC curve figure.
3-1 is the ROC curve figure of dissection of aorta patients blood plasma's sCD40L concentration.
3-2 is the ROC curve figure of dissection of aorta patients blood plasma's hs-CRP concentration.
3-3 is the ROC curve figure of dissection of aorta patients blood plasma's d-dimer concentration.
The ROC curve that 3-4 is dissection of aorta patients blood plasma sCD40L, hs-CRP, d-dimer concentration three is common.
Fig. 4 is that sCD40L suffers from control group, chronic dissection of aorta, subacute dissection of aorta, Aortic Dissection
Expression in person's tissue.
Wherein：A–D：Control group aorta；E–H：Chronic dissection of aorta；I–L：Subacute dissection of aorta；M–P：It is anxious
Property dissection of aorta；HE：The high power fields of A/E/I/M-× 25；The high power fields of B/F/J/N-× 100；Immunohistochemistry：C/G/K/O–
× 25 high power fields；The high power fields of D/H/L/P-× 100.
Fig. 5 is dissection of aorta serial section row immunohistochemical staining, to Healthy People, chronic dissection of aorta, subacute
Dissection of aorta, patients with acute aortic dissection tissue carry out sCD40L, SMA, CD68, MPO, caspase3, MMP9 immune group
Change dyeing, observes its expression.
Wherein：HE is dyed：The high power fields of I-IV × 25；Immunohistochemical staining：Control group：The high powers of A/E/I/M/Q/U × 100
The visual field；Chronic dissection of aorta：The high power fields of B/F/J/N/R/V × 100；Subacute dissection of aorta：C/G/K/O/S/M×
100 high power fields；Aortic Dissection：SCD40L immunohistochemical stainings：A-D；SMA immunohistochemical stainings:E-H；CD68 exempts from
Epidemic disease histochemical staining:I-L；MPO immunohistochemical stainings：M-P；Caspase3 immunohistochemical stainings：Q-T；MMP9 immunohistochemical stainings：
U-X。
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
The Research foundation of the present invention.
1, enzyme-linked immunosorbent assay detection dissection of aorta patients blood plasma sCD40L expression variations.
1.1, research object.
Continuously it is selected in May, 2015 in March, 2017 because pectoralgia is gone to a doctor in General Hospital, Shenyang Military Command's Internal Medicine-Cardiovascular Dept., through master
Artery enhanced CT is diagnosed as the patient totally 152 of arotic disease, wherein dissection of aorta patient 116, aorta penetrability
Ulcer 8, false aneurysm 12, Acute Aortic intramural hematoma 7, aortic aneurysm 9.Dissection of aorta patient is grouped：
With pectoralgia most significantly for the initial onset time, morbidity is acute stage within 7 days, and morbidity 7 days to 14 days (including 14 days) is sub- anxious
Property the phase, more than 14 days be chronic phase.
1.2 experiment flow.
1.2.1 dissection of aorta patient 116 is selected from above-mentioned 152 arotic disease patients, taken a blood sample after being admitted to hospital
Slurry samples.Blood sample to be checked is sent to clinical laboratory detection hs-CRP simultaneously, the concentration of d-dimer is expressed, and uses the present invention's
Kit detects the concentration expression of sCD40L in patients blood plasma.
Diagnostic kit of the present invention is with including：Washing lotion, dilution, titer, CD40L antibody, horseradish peroxidase mark
Remember Streptavidin, TMB luminous substrates, terminate liquid.
Diagnostic kit of the present invention is mainly the content for detecting sCD40L albumen in blood plasma.
The application method of diagnostic kit of the present invention includes the following steps：
The acquisition of step 1, blood plasma：Research object extracts its venous blood at once after being admitted to hospital, and is received with the anticoagulant tube of vacuum containing EDTA
Collect blood specimen 5ml.
The processing of step 2, sample：In venous blood samples 10 minutes, anticoagulant tube 3000 turns/min centrifugal treating 10min, point
From blood plasma, the blood plasma handled well is stored in -80 DEG C of refrigerators and is preserved, is detected with detection kit of the present invention and is selected in research object blood
Starch sCD40L concentration；Promote solidifying pipe blood sample collection and send clinical laboratory of the court at once, detects its hs-CRP, d-dimer concentration.
SCD40L expression contents in step 3, ELISA method detection blood plasma, using experimental port and the average value of secondary orifices as examining
Survey the measured value of object.
The standardization of step 4, data：SCD40L standard concentrations are prepared according to kit specification, according to explanation
Book requires to sequentially add standard items by concentration gradient, establishes standard curve.
The detection of step 5, blood plasma marker object：It is detected with latex immunoturbidimetry and is selected in object blood plasma hs-CRP concentration；With
The detection of NycoCard methods is selected in object plasma D-dimer content concentration.
The expression of sCD40L in step 6, dissection of aorta patients blood plasma：In conjunction with Aortography as a result, according to survey
The level of sCD40L, hs-CRP, d-dimer in the blood plasma obtained, observe it and change over time, the variation of biomarker, and
Judge more accurate biomarker according to AUC area under the curve.
Step 7, Immunohistochemical detection sCD40L dissection of aorta row aorta displacement technique patient sustainer group
It knits and the expression of normal control aortic tissue.
1.2.2 inquire research object medical history, it is carried out conventional physical examination and blood routine, blood biochemical, Analysis of blood lipid,
The clinical examination of the routine such as hepatic and renal function.
1.2.3 Aortography inspection：Selected research object is admitted to hospital emergency treatment or inherent General Hospital, Shenyang Military Command's heart of selecting a time
Internal medicine conduit room receives Aortography inspection.All patients in operation morning day fasting water, give intravenous glucose liquid fluid infusion,
Preoperative routine is for blood 800mL.Surgical procedure is carried out in Internal Medicine-Cardiovascular Dept. conduit room, is assisted using caudal anaesthesia, lung ventilator
Breathing punctures left radial artery, routinely gives heparin.Through " pigtail type " galactography, position, the number of aortic tunica intima cut are specified
Mesh and involve range, and measures arch of aorta reference vessel diameter.In femoral artery back side row surgical sectioning, " pig is sent through seal wire
Tail type " conduit exchanges stiffened seal wire to aorta ascendens, through conduit, and pushes overlay film frame (in conjunction with Aortography result and CTA
As a result the size of overlay film frame, the selection of Conventional coverlay film stent diameter is selected to be more than aortic arch diameter with proximal segment stent diameter
15-20%), be accurately positioned, control systolic pressure, discharge holder section by section, after holder release completely, gradually rise blood pressure, check
The clear holder of radiography is adherent and interior leakage situation.
1.2.4 coronary artery revasualization：Selected research object 1 week inherent General Hospital, Shenyang Military Command's Cardiological of being admitted to hospital is led
Pipe room receives coronary artery revasualization.Using Judkins methods, more position rows or so coronarography is selected.According to coronary disease
Sick diagnostic criteria, under the external membrane of heart in coronary artery LM, LAD, LCX, RCA any any narrow persons of Zhi Junwu be considered as it is completely normal,
It is meaningless pathology lesion as diameter stenosis is less than 50%, if diameter stenosis is the lesion for having pathology sense more than 50%.By having
The clinician of experience assesses coronary stenosis degree and coronary artery number of stenosed coronary vessel respectively.
Collect above three groups of crowd's general information, the above each group age, gender, smoking history, hypertension, diabetes medical history,
The laboratory examinations such as total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, hemoglobin, blood platelet, creatinine
Data difference is not statistically significant, and difference is statistically significant in terms of leucocyte, sCD40L, hs-CRP, d-dimer,
(P equal ＜ 0.05), refers to table 1.
1. control group of table is compared with the general Baseline Data of dissection of aorta group
Note：1. continuous normally distributed variable is indicated with mean ± standard deviation；2. classified variable number of cases (percentage %) table
Show；3.§：Represent median；4.hs-CRP：Hs-CRP；5.D-Dimer：D-dimer.
The result shows that in age, gender, smoking history, hypertension, diabetes medical history, the matched crowd of blood fat, actively
The expression of arteries and veins interlayer patient's inflammatory factor is compared with control group apparent increase, and difference is statistically significant, the results show that selected pair
According to site concentration in the sCD40L in group and dissection of aorta group research object blood plasma be respectively 435.60 (717.19) mg/L,
1386.09(1577.72)mg/L.SCD40L concentration is apparently higher than Normal group (P in dissection of aorta group research object blood plasma
<0.001) difference is statistically significant, illustrates that the variation of sCD40L concentration has correlativity with dissection of aorta.
Embodiment 2
2.1, the acquisition of sample and the processing of sample.
2.1.1, the acquisition of sample：It is selected in 152 research objects, extracts its venous blood after being admitted to hospital at once, with true containing EDTA
Empty anticoagulant tube collects blood specimen 5ml；
2.1.2, the processing of sample：In venous blood samples 10 minutes, anticoagulant tube 3000 turns/min centrifugal treating 10min, point
From blood plasma, the blood plasma handled well is stored in -80 DEG C of refrigerators and is preserved, using double antibody sandwich enzyme immunologic test (ELISA
Method) detection blood plasma sCD40L concentration；Promote to coagulate pipe blood sample collection, send clinical laboratory of the court at once, detect its hs-CRP, d-dimer
Concentration.
2.2, the measurement of sCD40L：Using the detection kit of the present invention, component is as follows：Washing lotion, dilution, standard
Product, CD40L antibody, horseradish peroxidase-labeled Streptavidin, TMB luminous substrates, terminate liquid；Its detection range is 0-
6000pg/ml, detection sensitivity 6pg/ml.
Following concrete operations are carried out by sample specification：Establish standard curve：SCD40L is prepared according to kit specification
Standard concentration, standard concentration gradient be respectively 6000pg/ml, 2000pg/ml, 666.7pg/ml, 222.2pg/ml,
74.07pg/ml, 24.6pg/ml, 8.23pg/ml, 0pg/ml require to sequentially add standard items by concentration gradient according to specification,
After 3 times of test plasma sample dilutions, microplate item is taken out.100 μ L standard items, reference substance and sample, viscosity are added per hole and pastes lid.
4 DEG C of overnight incubations.Liquid is abandoned in suction, 300 μ L washing lotions board-washing is added per hole 4 times.Each step all as possible all removes liquid.Finally
One step removes the washing lotion of all remainings as possible when cleaning.100 μ L CD40L antibody are added per hole, change a new viscosity patch, room
Temperature is incubated 1h.Repeat board-washing 4 times.100 μ L horseradish peroxidase-labeled Streptavidins are added per hole and are incubated at room temperature 45min.
Repeat board-washing 4 times.100 μ L TMB luminous substrates are added per hole, are protected from light incubation at room temperature 30min.50 μ L terminate liquids are added per hole, gently
Tapping is floged to be uniformly mixed.Microplate reader is read as a result, 450nm wavelength readings, 540nm or 570nm wavelength school in 30min
Just.Experiment reading takes 3 every time, this experiment is repeated 3 times.
2.3, the detection of blood plasma predictive factor：It is detected with latex immunoturbidimetry and is selected in object blood plasma hs-CRP concentration；With
The detection of NycoCard methods is selected in object plasma D-dimer content concentration；Range of normal value is：hsCRP≤2mg/L；D-dimer ＜
0.3mg/L。
2.4.ELISA expression of the sCD40L in control group and dissection of aorta patients blood plasma is measured.
The results show that sCD40L expresses apparent increase compared with normal person in dissection of aorta patients blood plasma, difference has statistics
Meaning (P ＜ 0.05), shows that the expression of sCD40L and dissection of aorta have correlativity；Dissection of aorta patient's blood
The expression of sCD40L is significantly higher than control group and acute coronary syndrome group in slurry, but between acute coronary syndrome and control group
SCD40L differential expressions are not notable；SCD40L is compared with control group, aorta penetrating ulcer, false in dissection of aorta patients blood plasma
Expression in aneurysm, Acute Aortic intramural hematoma, aortic aneurysm blood plasma is significantly raised, statistically significant (the P ＜ of difference
0.05)；But sCD40L is in control group, aorta penetrating ulcer, false aneurysm, Acute Aortic intramural hematoma, aorta
Differential expression in tumor blood plasma is not statistically significant, shows the occurrence and development of sCD40L and dissection of aorta there are correlativity,
But with other arotic diseases without correlativity；The chronicer dissection of aorta of sCD40L and Asia are anxious in Aortic Dissection blood plasma
Property dissection of aorta expression it is significantly raised, difference is statistically significant (P ＜ 0.05), but in blood plasma sCD40L control group with it is slow
Property dissection of aorta, subacute dissection of aorta between differential expression it is not statistically significant, show sCD40L participate in aorta
The occurrence and development of interlayer, it is more notable in acute stage.Refer to Fig. 1.
2.5.ELISA sCD40L, hs-CRP, the expression of d-dimer in different time points are detected.
As a result, it has been found that finding in selected dissection of aorta patient, sCD40L is in dissection of aorta morbidity 0.5d blood plasma
In concentration obviously increase, S100A12 concentration reaches peak value in morbidity 0.5-1d its blood plasma, may persist to morbidity 2d, then
SCD40L plasma concentrations are begun to decline, until morbidity 7-14d blood plasma sCD40L concentration is down to normally substantially；Hs-CRP concentration is in master
The 0.5-1d or so of artery dissection morbidity increases, and hs-CRP concentration reaches peak value in its blood plasma of morbidity 1-2d, may persist to morbidity
2-4d, morbidity 4-7d are down to normal range (NR) substantially；Concentration of the d-dimer in dissection of aorta falls ill 0.5-1d blood plasma is apparent
Increase, d-dimer concentration reaches peak value in its blood plasma of morbidity 1-2d, may persist to morbidity 2-4d, then d-dimer blood plasma is dense
Degree is begun to decline, until morbidity 7-14d plasma D-dimer contents concentration is down to normally substantially.Early stage dissection of aorta is fallen ill,
Blood plasma sCD40L concentration increases more early.Using selected control group research object as negative reference group, dissection of aorta patient
As positive reference group, draw respectively 0.5d, 1d after dissection of aorta morbidity, 2d, 4d, 7d, 14d, 30d,>30d blood
The ROC curve of slurry sCD40L, hs-CRP, d-dimer, refer to Fig. 2.
It is shown by ROC curve analysis result, blood plasma sCD40L, hs-CRP, d-dimer after the onset of dissection of aorta
Diagnose dissection of aorta ROC curve under Line Integral not Wei 0.807,0.704,0.686, show and hs-CRP, d-dimer phase
Than sCD40L diagnostics in the diagnosis of dissection of aorta are higher, refer to Fig. 3.
Embodiment 3.
3.1, the collection of normal human aorta and dissection of aorta patient sustainer sample.
It collects without acute coronary syndrome, arotic disease medical history, because of 1, young postmortem person's aorta of unexpected death；
Dissection of aorta sample obtains when being replaced by department of cardiovascular surgery row aorta, the grouping of dissection of aorta patient tissue：Most with pectoralgia
It significantly it is the initial onset time, morbidity is acute stage within 7 days, and morbidity 7 days to 14 days (including 14 days) is subacute stage, is more than
14 days are chronic phase；Control group is no acute coronary syndrome, arotic disease medical history, because of the young postmortem person master of unexpected death
Artery；All aortas are put into 10% paraformaldehyde fixed；Selected sample is both needed to by General Hospital, Shenyang Military Command's ethics
The committee is agreed to, and signs informed consent form.
3.2, the preparation of paraffin section.
Materials：Dissection of aorta patient tissue and control group blood clot tissue are placed in 4% paraformaldehyde solution overnight,
And trim tissue.
Dehydration：Particular order is as follows：4h-95% in 70% alcohol 2h-80% alcohol 2h-90% alcohol 2h-95% alcohol I
1.5h in 1.5h-100% alcohol II in -100% alcohol I overnight in alcohol II.
It is transparent：Tissue block is put into dimethylbenzene I and impregnates 1h, is put into after taking-up in dimethylbenzene II and impregnates 1h.
Waxdip：Particular order is as follows：1h in 1h- paraffin III in overnight-paraffin II in paraffin I.
Embedding：Tissue block is embedded using paraffin, is placed at room temperature for.
Slice：Tissue block is sliced using paraffin slicing machine, thickness is 3 μm, is affixed on glass slide.
Dry piece and roasting piece：Glass slide is placed in 60 DEG C to dry on piece machine, places 1h；Glass slide is put in 65 DEG C of ovens later
In, place 48h.
3.3, tissue HE dyeing.
Slice dewaxing：Particular order is as follows：In dimethylbenzene I in 20min- dimethylbenzene II in 20min-95% alcohol I
15min-90% alcohol 10min-80% alcohol 5min-70% alcohol 5min- distilled water 30min in 15min-95% alcohol II.
Nuclear targeting：Paraffin section is placed and dyes 10min in haematoxylin, flowing water rinses.
Differentiation：Paraffin section is put in 1% hydrochloride alcohol and carries out differentiation 30s, flowing water rinses.
Return indigo plant：Paraffin section is put in progress nucleus in ammonium hydroxide and returns blue 30s, flowing water rinses.
Cytoplasm dyes：Paraffin section is put in water-soluble Yihong solution and dyes 5min, flowing water rinses.
It is transparent：Paraffin section is put in 5min-100% alcohol in 80% alcohol 5min-90% alcohol 5min-100 alcohol I
5min in 5min- dimethylbenzene II in 5min- dimethylbenzene I in II.
Mounting：Paraffin section is put in draught cupboard and is dried, mounting is carried out using resinene.
3.4, tissue immunohistochemistry dyes.
(1) paraffin section de-waxing：It is dyed with HE.
(2) antigen retrieval：Paraffin section is put in 200ml antigen retrieval buffers, 100 DEG C are boiled 40min, Temperature fall.
(3) it is added dropwise in 50 μ l reagent As to paraffin section, is incubated at room temperature 10min, PBS rinses 3 times/5min.
(4) it is added dropwise on 50 μ l reagents B to paraffin section, is incubated at room temperature 10min, remove serum.
(5) it is added dropwise (1 on the primary antibody solution to paraffin section after 50 μ l dilutions:100 dilutions, are diluted with PBS), 4 DEG C
It is incubated overnight.
(6) paraffin section is carried out rewarming 30min by next day at ambient temperature；PBS rinses 3 times/5min.
(7) it is added dropwise (1 on 50 μ l reagent C solution to paraffin section:100 dilutions, are diluted with PBS), incubation at room temperature
10min；3 times/5min is rinsed with PBS.
(8) it is added dropwise on 50 μ l reagent D solution to paraffin section, is incubated at room temperature 10min；3 times/5min is rinsed with PBS.
(9) it develops the color using DAB solution, microscopically observation coloring.
(10) nuclear targeting, step are dyed with HE.
The result shows that the relatively normal group of dissection of aorta patient sustainer pathological tissues sCD40L expression is significantly raised, especially with
Acute stage, is more notable, and subacute stage takes second place；Dissection of aorta patient sustainer pathological tissues inflammatory Cytokines Expression is significantly raised,
Acute stage, is the most notable, and subacute stage, chronic phase increase, but is less than acute stage.
SCD40L participates in the inflammatory reaction of dissection of aorta pathogenesis.Most significantly it is with pectoralgia by active surface interlayer patient
Initial onset time, morbidity were acute stage within 7 days, fell ill 7 days to 14 days (including 14 days) for subacute stage, were more than 14 days
Chronic phase；Collect age, the relevant aorta penetrating ulcer of gender, false aneurysm, Acute Aortic intramural hematoma patient
As a contrast；
Immunohistochemistry detects the expression of sCD40L in control group aorta and dissection of aorta patient sustainer tissue：HE contaminates
Color and Showed by immune group result, the expression of sCD40L is relatively low in control group aorta, dissection of aorta patient actively
It can be seen that the expression of sCD40L is apparently higher than Normal aorta tissue specimen in arteries and veins tissue.In dissection of aorta patient sustainer
SCD40L great expressions around tearing port, especially with acute stage be very, therefore dissection of aorta occur acute stage sCD40L largely gather
Collection, while it being released into blood, refer to Fig. 4.
Expression of the Immunohistochemical detection sCD40L in dissection of aorta patient's diseased region：Immunohistochemistry is found
SMA, CD68, MPO, caspase3, MMP9 are apparently higher than control group in dissection of aorta patient's lesion vascular expression, cut acute
Phase is more notable, illustrates that, in the acute stage that dissection of aorta occurs, dissection of aorta breach can assemble a large amount of sCD40L and inflammation
Sex factor shows that the raising of dissection of aorta patient sCD40L is related with inflammatory factor, refers to Fig. 5.
4. statistical method：
Statistical procedures apply 17.0 software packages of SPSS to handle.Statistical check is all made of two-sided test, and inspection level is
0.05；The demographic and clinical variable of each group are counted；Quantitative target is indicated using mean, standard deviation；Using at
Group t is examined or single argument ANOVA carries out comparison among groups；Two sample averages of Non-Gaussian Distribution compare using quartile；It is qualitative
Index is indicated using number of cases and percentage；It is examined using Chi-square Test or exact method and carries out comparison among groups；Two comparison among groups,
Using two-sided test (two-side test), α=0.05 is taken；P≤0.05, it is believed that statistically significant；All credibility intervals
Confidence level use 95%.
4.1 control groups are compared with the general Baseline Data of dissection of aorta group.Above three groups of crowd's general information are collected, it is above
Each group age, gender, smoking history, hypertension, diabetes medical history, total cholesterol, triglycerides, low-density lipoprotein, high density
The laboratory examinations data difference such as lipoprotein, hemoglobin, blood platelet, creatinine is not statistically significant, and leucocyte,
SCD40L, hs-CRP, difference is statistically significant in terms of d-dimer, (P equal ＜ 0.05).
The result shows that：In age, gender, smoking history, hypertension, diabetes medical history, the matched crowd of blood fat, actively
The expression of arteries and veins interlayer patient's inflammatory factor is compared with control group apparent increase, and difference is statistically significant, the results show that selected pair
According to site concentration in the sCD40L in group and dissection of aorta group research object blood plasma be respectively 435.60 (717.19) mg/L,
1386.09(1577.72)mg/L.SCD40L concentration is apparently higher than Normal group (P in dissection of aorta group research object blood plasma
<0.001) difference is statistically significant, illustrates that the variation of sCD40L concentration has correlativity with dissection of aorta.
4.2.sCD40L the inflammatory reaction of dissection of aorta pathogenesis is participated in.Active interlayer patient is most notable with pectoralgia
For the initial onset time, morbidity was acute stage within 7 days, fell ill 7 days to 14 days (including 14 days) for subacute stage, more than 14 days
For chronic phase；Collect age, the relevant aorta penetrating ulcer of gender, false aneurysm, Acute Aortic intramural hematoma trouble
Person is as a contrast；1-1.ELISA measures expression of the sCD40L in control group and dissection of aorta patients blood plasma；1-2.sCD40L
Expression in control group, acute coronary syndrome group, dissection of aorta group blood plasma；1-3.sCD40L is in control group, aorta
Expression in penetrating ulcer, false aneurysm, Acute Aortic intramural hematoma, aortic aneurysm and dissection of aorta blood plasma；1-
Expression of the 4.sCD40L in control group, chronic dissection of aorta, subacute dissection of aorta, Aortic Dissection blood plasma.
The results show that sCD40L expresses apparent increase compared with normal person in 1-1. dissection of aorta patients blood plasmas, difference has system
Meter learns meaning (P ＜ 0.05), shows that the expression of sCD40L and dissection of aorta have correlativity；1-2. aorta clamp
The expression of sCD40L is significantly higher than control group and acute coronary syndrome group in layer patients blood plasma, but acute coronary syndrome with it is right
It is not notable according to the sCD40L differential expressions between group；SCD40L is worn compared with control group, aorta in 1-3. dissection of aorta patients blood plasmas
Expression in permeability ulcer, false aneurysm, Acute Aortic intramural hematoma, aortic aneurysm blood plasma is significantly raised, and difference has system
Meter learns meaning (P ＜ 0.05)；But sCD40L is in control group, aorta penetrating ulcer, false aneurysm, Acute Aortic wall
Differential expression in hemotoncus, aortic aneurysm blood plasma is not statistically significant, shows that the occurrence and development of sCD40L are deposited with dissection of aorta
In correlativity, but with other arotic diseases without correlativity；SCD40L is chronicer in 1-4. Aortic Dissection blood plasma
Dissection of aorta and the expression of subacute dissection of aorta are significantly raised, and difference is statistically significant (P ＜ 0.05), but in blood plasma
Differential expressions of the sCD40L between control group and chronic dissection of aorta, subacute dissection of aorta is not statistically significant, table
Bright sCD40L participates in the occurrence and development of dissection of aorta, more notable in acute stage.
4.3.ELISA sCD40L, hs-CRP, the expression of d-dimer in different time points are detected.2-1.ELISA detection
The expression of sCD40L in different time points；The expression of 2-2. test in laboratory hs-CRP in different time points；It examines in the laboratories 2-3.
Survey d-dimer expression in different time points, 2-4.sCD40L, hs-CRP, d-dimer expression quantity in different time points, (<
0.5d indicates that 0.5-1d is indicated with 2 with 1, and 1-2d is indicated with 3, and 2-4d are indicated with 4, and 4-7d are indicated with 5, and 7-14d are indicated with 6,
14-30d indicate with 7,>30d is indicated with 8).
As a result, it has been found that finding in selected dissection of aorta patient, sCD40L is in dissection of aorta morbidity 0.5d blood plasma
In concentration obviously increase, S100A12 concentration reaches peak value in morbidity 0.5-1d its blood plasma, may persist to morbidity 2d, then
SCD40L plasma concentrations are begun to decline, until morbidity 7-14d blood plasma sCD40L concentration is down to normally substantially；Hs-CRP concentration is in master
The 0.5-1d or so of artery dissection morbidity increases, and hs-CRP concentration reaches peak value in its blood plasma of morbidity 1-2d, may persist to morbidity
2-4d, morbidity 4-7d are down to normal range (NR) substantially；Concentration of the d-dimer in dissection of aorta falls ill 0.5-1d blood plasma is apparent
Increase, d-dimer concentration reaches peak value in its blood plasma of morbidity 1-2d, may persist to morbidity 2-4d, then d-dimer blood plasma is dense
Degree is begun to decline, until morbidity 7-14d plasma D-dimer contents concentration is down to normally substantially.
Early stage dissection of aorta is fallen ill, blood plasma sCD40L concentration increases more early.With selected control group research object
As negative reference group, dissection of aorta patient is as positive reference group, after drawing dissection of aorta morbidity respectively
0.5d、1d、2d、4d、7d、14d、30d、>The ROC curve of 30d blood plasma sCD40L, hs-CRP, d-dimer, are shown in Fig. 3.
It is shown by ROC curve analysis result, blood plasma sCD40L, hs-CRP, d-dimer after the onset of dissection of aorta
Diagnose dissection of aorta ROC curve under Line Integral not Wei 0.807,0.704,0.686, show and hs-CRP, d-dimer phase
Than sCD40L diagnostics in the diagnosis of dissection of aorta are higher, see Fig. 3.
Immunohistochemistry detects the expression of sCD40L in control group aorta and dissection of aorta patient sustainer tissue：HE contaminates
Color and Showed by immune group result, the expression of sCD40L is relatively low in control group aorta, dissection of aorta patient actively
It can be seen that the expression of sCD40L is apparently higher than Normal aorta tissue specimen in arteries and veins tissue.In dissection of aorta patient sustainer
SCD40L great expressions around tearing port, especially with acute stage be very, therefore dissection of aorta occur acute stage sCD40L largely gather
Collection, while it being released into blood, see Fig. 4.
Expression of the Immunohistochemical detection sCD40L in dissection of aorta patient's diseased region：Immunohistochemistry is found
SMA, CD68, MPO, caspase3, MMP9 are apparently higher than control group in dissection of aorta patient's lesion vascular expression, cut acute
Phase is more notable, illustrates that, in the acute stage that dissection of aorta occurs, dissection of aorta breach can assemble a large amount of sCD40L and inflammation
Sex factor shows that the raising of dissection of aorta patient sCD40L is related with inflammatory factor, sees Fig. 5.
Claims (4)
- Application of the 1.sCD40L albumen in preparing dissection of aorta early diagnosis kit, which is characterized in that the diagnosis tries Agent box is that the expression quantity using sCD40L albumen as marker to it in blood plasma is detected, and early stage is carried out to dissection of aorta Diagnosis.
- 2. application of the sCD40L albumen according to claim 1 in preparing dissection of aorta early diagnosis kit, It is characterized in that, the diagnostic kit is with including：Washing lotion, dilution, titer, CD40L antibody, horseradish peroxidase-labeled chain Mould Avidin, TMB luminous substrates, terminate liquid.
- 3. application of the sCD40L albumen according to claim 1 in preparing dissection of aorta early diagnosis kit, It is characterized in that, the diagnostic kit is mainly the content for detecting blood plasma sCD40L albumen.
- 4. application of the sCD40L albumen according to claim 1 in preparing dissection of aorta early diagnosis kit, It is characterized in that, the application method of the diagnostic kit includes the following steps：The acquisition of step 1, blood plasma：Venous blood samples collect blood sample；The processing of step 2, sample：In venous blood samples 10 minutes, centrifugal treating 10min, separated plasma, the blood plasma that will be handled well It is stored in -80 DEG C of refrigerators to preserve, sCD40L concentration is detected with dissection of aorta early diagnosis kit；Detect its hs-CRP, D- Dimerization bulk concentration；SCD40L protein expression contents in step 3, detection blood plasma：The variation for detecting sCD40L albumen concentration, with experimental port and pair Measured value of the average value in hole as detection object；The standardization of step 4, data：SCD40L standard concentrations are prepared, standard items are sequentially added by concentration gradient, are established Standard curve；The detection of step 5, blood plasma marker object：It is detected with latex immunoturbidimetry and is selected in object blood plasma hs-CRP concentration；With The detection of NycoCard methods is selected in object plasma D-dimer content concentration；The expression of sCD40L in step 6, dissection of aorta patients blood plasma：In conjunction with Aortography as a result, according to measuring The level of sCD40L, hs-CRP, d-dimer in blood plasma, observe it and change over time, the variation of biomarker, and according to AUC area under the curve judges more accurate biomarker；Step 7, Immunohistochemical detection sCD40L dissection of aorta row aorta displacement technique patient sustainer tissue and The expression of normal control aortic tissue.
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| CN111190019A (en) * | 2020-01-07 | 2020-05-22 | 南京医科大学 | Medical application of choline transporter 2 |
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