CN105572276A - Pancreatic cancer diagnostic marker combination as well as application and determination method thereof - Google Patents
Pancreatic cancer diagnostic marker combination as well as application and determination method thereof Download PDFInfo
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Abstract
The invention discloses a pancreatic cancer diagnostic marker combination as well as application and a determination method thereof. The pancreatic cancer diagnostic marker combination comprises 15 differentiated metabolites (2,5-dihydroxybenzoic acid, talopyranose, proline, glutamate, choline, 1,5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaric acid, methylguanidine, adenine, glycocholic acid, valine and 2-aminobutyric acid). The invention further provides a combination of the 15 differentiated metabolites to serve as a marker for early discovery and diagnosis of pancreatic cancer, application and a diagnostic marker determination method, and the method is a liquid-phase/gas-phase chromatography-mass spectrometry combined metabonomics analysis method based on plasma/serum of patients with pancreatic cancer. The pancreatic cancer diagnostic marker combination provided by the technical scheme of the invention has the characteristics of being high in sensitiveness and specificity, has relatively high sensitiveness and specificity on early pancreatic cancer diagnosis, can be used for early discovery of pancreatic cancer, gains time for the patients to receive treatment as soon as possible, and improves the clinical treatment effect.
Description
Technical field
The present invention relates to biological technical field, relate to the combination of a kind of diagnosis of pancreatic cancer label, application and assay method thereof.
Background technology
Cancer of pancreas is that a kind of incidence of disease is higher, the malignant tumor of digestive tract that grade malignancy is very high, its M & M obviously rises in recent years, it is one of the poorest malignant tumour of prognosis, its five year survival rate is less than 5%, is the biomarker lacking the diagnosis of cancer of pancreas disease commitment to the major obstacle improving this situation.
So far, the basic methods for the treatment of of cancer of pancreas remains based on surgical resection, but have data show to only have the patient of 20 ~ 25% in early days stage open close operation of crossing carry out suitable excision.Because having of cancer of pancreas is invisible, the early stage diagnosis rate of cancer of pancreas is not high, once the tumour making a definite diagnosis Most patients shifts, Diagnosis and Treat is all very difficult.
Traditional tumor marker CA19-9, it can reach 80% to the diagnostic sensitivity of cancer of pancreas, but is not suitable for Early pancreatic carcinoma patient for excising staged tumors because of its muting sensitivity and low specificity.TSGF (TSGF), CA242 (8), MIC-1, platelet factor 4, peanut agglutinin (PNA) is also had to be confirmed to be candidate biomarker thing in conjunction with glycoprotein, cell adhesion molecule 17.1, sero-immunity mark affinity proteomic etc. at present, regrettably, these biomarkers all have lower and low to the disease early detection precision shortcoming of the early stage sensitivity of disease.And the early detection of cancer of pancreas, early diagnosis and early treatment are the keys improving and improve cancer of pancreas prognosis, therefore find sensitivity, specific biomarker is most important for the Diagnosis and Treat of this disease.
Patent CN101942502A discloses a kind of pancreatic cancer marker and detection method, kit and biochip.The pancreatic cancer marker that this invention provides comprises stable existence in experimenter's serum/plasma and detectable 36 kinds of miRNAs.This invention additionally provides to comprise and detects the instrument of this pancreatic cancer marker or the kit of element and biochip.The combination that this invention provides, method, kit and biochip can be used in the Diagnosis and differential diaggnosis of auxiliary cancer of pancreas, the prediction of the generation of disease complications and recurrence, therapeutic evaluation, and the aspect such as the screening of active constituents of medicine, evaluating drug effect, have and detect the advantages such as pedigree is wide, highly sensitive, testing cost is low, draw materials conveniently, sample is easy to store.But this patent has lower and low to the disease early detection precision shortcoming of the early stage sensitivity of disease.
Patent 103667444A discloses a kind of tumor marker relevant to cancer of pancreas and application thereof, and this mark is the combination of CystatinSN (CST1), CystatinS (CST4), CystatinSA (CST2).This mark and primer thereof, antibody can be used for diagnostic kit application, for the auxiliary diagnosis of cancer of pancreas.But this patent also has lower and low to the disease early detection precision shortcoming of the early stage sensitivity of disease.
Summary of the invention
In view of this, the invention provides the combination of a kind of diagnosis of pancreatic cancer label, application and assay method thereof.
For achieving the above object, concrete technical scheme is as follows:
On the one hand, provide the combination of a kind of diagnosis of pancreatic cancer label, be blood plasma or serum otherness metabolin, comprise phosphoamide (Urea), choline (Choline), methylguanidine (Methylguanidine), creatinine (Creatinine), 3-amino-2-piperidones (3-Amino-2-piperidone), 2-amino-butyric acid (2-Aminobutyricacid), betaine (betaine), valine (Valine), 2,4-diaminobenzoic acid (2,4-Diaminobutyricacid), glutamine (Glutamine), tryptophane (Tryptophan), proline (Proline), glutamic acid (Glutamate), N-acetylglutamat (N-Acetylglutamine), heteroauxin (Indoleaceticacid), 1,3,7-trimethyl-uric acid (1,3,7-Trimethyluricacid), uric acid (Uricacid), indole acrylic acid (Indoleacrylicacid), adenine (Adenine), single isobutyl phthalic acid (Monoisobutylphthalicacid), DHB (2,5-dihydroxybenzoicacid), p-hydroxybenzene acrylic acid (2-Hydroxycinnamicacid), 1,5-AG (1,5-Anhydro-D-glucitol), talose (Talopyranose), propionyl carnitine (Propionylcarnitine), lecithin (LysoPC (14:0)), galactitol (Galactitol), glycocholic acid (Glycocholicacid), NAMN (Nicotinicacidmononucleotide), a-KG (2-Oxoglutaricacid), one or more combination in 2-methyl-3-oxopropanoic acid (2-Methyl-3-oxopropanoicacid).
Preferably, described label comprises choline (choline), 1, 5-dewatered grape sugar alcohol (1, 5-anhydro-D-glucitol), 2, 5-dihydroxy-benzoic acid (2, 5-dihydroxybenzoicacid), talose (talopyranose), proline (proline), glutamate/glutamate (glutamate), tryptophane (tryptophan), glutamine (glutamine), betaine/betaine (betaine), 2-oxoglutaric acid (2-oxoglutaricacid), methylguanidine (methylguanidine), adenine (adenine), glycocholic acid (glycocholicacid), valine (valine), one or more combination in 2-amino-butyric acid (2-aminobutyricacid).
On the other hand, the application of diagnosis of pancreatic cancer label combination is provided, for the kit of diagnosis of pancreatic cancer.
Preferably, the diagnosis sample of diagnostic marker is blood plasma or serum.
Preferably, also comprise a regression model, as follows:
, when the Probability value in model is greater than 50%, can disease break as cancer of pancreas.
Preferably, applying clinical diagnosis performance curve evaluation otherness metabolin, when threshold value is 0.4272, diagnostic marker combination has the highest sensitivity and specificity.
On the other hand, provide the assay method of diagnosis of pancreatic cancer label combination, comprise the following steps:
Step 1, gets Patients with Pancreatic Cancer clinical blood or serum sample and normal controls blood plasma or serum sample;
Step 2, by the preliminary otherness metabolin of combined gas chromatography mass spectrometry metabonomic analysis methods Analysis and Identification Patients with Pancreatic Cancer clinical blood or serum sample and normal controls blood plasma or serum sample;
Step 3, under variable weight (VIP) value of multidimensional OPLS-DA model is greater than 1 and the non-P value tested of engaging in an inspection is less than the choice criteria of 0.05, obtains further otherness metabolin in preliminary otherness metabolin;
Step 4, carries out Logic Regression Models and verifies, obtain otherness metabolin.
Preferably, described step 1 comprises the Patients with Pancreatic Cancer clinical blood of the different group in different regions or serum sample and normal controls blood plasma or serum sample.
Preferably, the combined gas chromatography mass spectrometry metabonomic analysis methods in described step 2 comprises liquid/vapor combined gas chromatography mass spectrometry metabonomic analysis methods.
Preferably, in described step 2, the chromatographic condition of gas chromatography combined with mass spectrometry test comprises: Rxi-5ms capillary column: fill 5% phenylbenzene/95% dimethyl polysiloxane, carrier gas: ultrapure helium, flow: 1.0mL/min, injector temperature: 260 DEG C, transmission line temperature: 260 DEG C, ion source temperature: 210 DEG C, sample size: 1uL, input mode: Splitless injecting samples, heating schedule: from 80 DEG C and continue 2min, 220 DEG C are risen to the programming rate of 10 DEG C/min, 240 DEG C are risen to afterwards with the programming rate of 5 DEG C/min, 290 DEG C are risen to again with the programming rate of 25 DEG C/min, last at 290 DEG C of lasting 8min, mass ion source: EI source, electronics bombarding energy: 70eV, scanning of the mass spectrum scope: m/z, 40-600, full scan mode.
Preferably, in described step 2, the chromatographic condition of liquid chromatography mass coupling test comprises: AgilentZORBAXEclipseXDB-C18 post (4.6 × 150mm, 5 μm), column temperature: 30 DEG C.Mobile phase A: water (0.1% formic acid), B: acetonitrile (0.1% formic acid), mobile phase gradient is 0-25min:1-100%B, flow velocity: 0.4mL/min, sample size: 10 μ L.Flight time mass spectrum optimal conditions is: (1) positive ion mode (ES+), capillary voltage 3500V, sprayer 45psig, dry gas temperature 325 DEG C, exsiccator flow velocity 11L/min; (2) negative ion mode (ES-), capillary voltage 3000V, other parameters are consistent with positive ion mode.When metabolite profiling analyses, data collection form is that plot and centroid carries out simultaneously, and acquisition quality scope is 50-1000Da.
Preferably, in described step 2, the serum sample pre-treatment of gas chromatography combined with mass spectrometry test comprises: get 50 μ L serum, add 10ul chlorophenylalanine (0.1mg/mL, water-soluble) and 10 μ L margaric acids (1mg/mL, alcohol is molten) carry out monitor sample reappearance as interior mark; Add 175 μ L chloroform methanol mixed solvent (1:3, v/v) again, vortex oscillation 30s; Put centrifuge tube and place 10min to promote albumen precipitation in-20 DEG C; Then the centrifugal 10min of 13000rpm, get supernatant 200 μ L and reclaim in sample injection bottle in height, ambient temperature in vacuum is dry.
Preferably, in described step 2, sample product use two-step approach to derive after draining, first 50 μ L methoxamine (15mg/mL are added, pyridine is molten), vortex oscillation 30s, at 30 DEG C, react 90min, and then add 50 μ LBSTFA (containing 1%TMCS) at 70 DEG C of reaction 60min, after leaving standstill, carry out GC-TOFMS analysis.
Preferably, in described step 2, the serum sample pre-treatment of liquid chromatography mass coupling test comprises: get 50ul serum sample and 200ul and contain chlorophenylalanine (5ug/mL, water-soluble) methanol acetonitrile mixed liquor (5:3, v/v) mix, vortex oscillation 2min, after leaving standstill 10min, with the centrifugal 20min of 13000rpm, get supernatant.
Relative to prior art, technical scheme of the present invention proposes 2 first, 5-dihydroxybenzoicacid, talopyranose, proline, glutamate, choline, 1, 5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaricacid, methylguanidine, adenine, glycocholicacid, valine, this 15 species diversity metabolin of 2-aminobutyricacid and the application of combining as diagnosis of pancreatic cancer label thereof, this diagnostic marker and combination thereof have higher sensitivity and specificity, can be used for differentiating pancreatic cancer patient and normal person, for the early detection of cancer of pancreas, for patient races against time, start treatment as early as possible, improve clinical therapeutic efficacy.Simultaneously, by using LC-TOFMS and GC-TOFMS, the full analysis of spectrum test of metabolin is carried out to the blood plasma of Patients with Pancreatic Cancer and normal person or serum, identify 15 species diversity metabolins, biomarker as cancer of pancreas combines, may be used for early detection and the diagnosis of cancer of pancreas, improve the clinical therapeutic efficacy of cancer of pancreas.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Figure 1A is the OPLS-DA figure of the Patients with Pancreatic Cancer of the embodiment of the present invention and the metabolin of normal control plasma sample LC-TOFMS and GC-TOFMS detection;
Figure 1B is the OPLS-DA figure of the Patients with Pancreatic Cancer of the embodiment of the present invention and the metabolin of normal control serum sample LC-TOFMS and GC-TOFMS detection;
Fig. 2 A is that the ROC curve map of the blood plasma biomarker analyses and prediction cancer of pancreas of the cancer of pancreas sample of the embodiment of the present invention (comprises 15 blood plasma biomarkers: 2,5-dihydroxybenzoicacid, talopyranose, proline, glutamate, choline, 1,5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaricacid, methylguanidine, adenine, glycocholicacid, valine, 2-aminobutyricacid);
Fig. 2 B is the ROC curve map of the serum biomarker thing analyses and prediction cancer of pancreas of the cancer of pancreas sample of the embodiment of the present invention;
Fig. 3 A ~ Fig. 3 B is that the diagnostic model of application 15 metabolins foundation of the embodiment of the present invention is to predict the distribution plan of the diagnostic value of pancreas (plasma sample and serum sample) and normal control.Cancer of pancreas S1:TNM is first phase patient by stages; Cancer of pancreas S2:TNM is the second stage of patient by stages; Cancer of pancreas S3:TNM is three phase patients by stages.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
It should be noted that, when not conflicting, the embodiment in the present invention and the feature in embodiment can combine mutually.
Below with reference to accompanying drawing, concrete explaination is done to embodiments of the invention.
Embodiments of the invention application hydrolysis and condensation, detects the metabolin of serum or plasma sample, finds the diagnostic marker of cancer of pancreas.Specifically preferably include:
Collect the plasma/serum sample of Patients with Pancreatic Cancer and normal person, detect through chromatographic mass spectrometry after process and analyze, different by the metabolism spectral difference shown between Patients with Pancreatic Cancer and normal person with setting up multidimensional statistics model visualization, obtain the otherness metabolin between two groups, and Preliminary Identification is carried out to these materials.
Assay method of the present invention comprehensively, synthetically can embody the variation situation of the metabolic product between Patients with Pancreatic Cancer and normal person, finds the diagnostic marker of cancer of pancreas, for the early diagnosis of cancer of pancreas and prognosis provide favourable technical support.
One, experiment material and method of testing:
1. plasma/serum sample situation
(1) plasma sample is from 94 routine Patients with Pancreatic Cancers and the normal controls plasma sample 100 corresponding with its age and sex example.
(2) serum sample is from 96 routine Patients with Pancreatic Cancers and the normal human control sera sample 98 corresponding with its age and sex example.
2. plasma/serum Sample pretreatment:
(1) gas chromatograph-mass spectrometer (GC-TOFMS) tests plasma/serum Sample pretreatment
Get 50 μ L blood plasma or serum in the centrifuge tube of 1.5mL, add 10ul chlorophenylalanine (0.1mg/mL, water-soluble) and 10 μ L margaric acids (1mg/mL, alcohol is molten) carry out monitor sample reappearance as interior mark respectively.Add 175 μ L chloroform methanol mixed solvent (1:3, v/v) again, vortex oscillation 30s; Put centrifuge tube and place 10min to promote albumen precipitation in-20 DEG C.Then the centrifugal 10min of 13000rpm, get supernatant 200 μ L and reclaim in sample injection bottle in height, ambient temperature in vacuum is dry.Sample product use two-step approach to derive after draining, first 50 μ L methoxamine (15mg/mL, pyridine is molten) are added, vortex oscillation 30s, at 30 DEG C, react 90min, and then add 50 μ LBSTFA (containing 1%TMCS) at 70 DEG C of reaction 60min.Reaction product carries out GC-TOFMS analysis after at room temperature leaving standstill 1h.
(2) liquid chromatography mass combined instrument (LC-TOFMS) tests blood plasma or serum sample pre-treatment
Get 50ul plasma/serum sample and 200ul to mix containing the methanol acetonitrile mixed liquor (5:3, v/v) of chlorophenylalanine (5ug/mL, water-soluble), vortex oscillation 2min, after leaving standstill 10min, with 13, the centrifugal 20min of 000rpm, gets supernatant and analyzes for LC-TOFMS.
3. analytical instrument test:
(1) gas chromatograph-mass spectrometer (GC-TOFMS) test
GC-TOFMS:LecoPegasusHT gas chromatography time-of-flight mass spectrometry (Leco Corporation, the U.S.), chromatographic column: Rxi-5ms capillary column (fills 5% phenylbenzene/95% dimethyl polysiloxane, Restek, the U.S.), carrier gas: ultrapure helium, flow: 1.0mL/min, injector temperature: 260 DEG C, transmission line temperature: 260 DEG C, ion source temperature: 210 DEG C, sample size: 1uL, input mode: Splitless injecting samples, heating schedule: from 80 DEG C and continue 2min, 220 DEG C are risen to the programming rate of 10 DEG C/min, 240 DEG C are risen to afterwards with the programming rate of 5 DEG C/min, 290 DEG C are risen to again with the programming rate of 25 DEG C/min, last at 290 DEG C of lasting 8min.Mass ion source: EI source, electronics bombarding energy: 70eV, scanning of the mass spectrum scope: m/z, 40-600, full scan mode.Data Analysis Services uses ChromaTOF software (v4.33, Leco Corporation, the U.S.).
(2) liquid chromatography mass combined instrument (LC-TOFMS) test
LC-TOFMS: Agilent Ultra Performance Liquid Chromatography 1200 system (Agilent company, the U.S.), is equipped with binary solvent controller and sample manager.Mass spectrophotometry adopts Agilent 6220MSD type time of-flight mass spectrometer (Agilent company, the U.S.), is equipped with two electron spray ionisation source.Utilize the tuning standard items mixed liquor of Agilent ESI-L low concentration, ionize pattern (ES-) pattern by system tunning to optimum sensitivity and resolution at positive ionization electrospray ionization pattern (ES+) and negative electrospray respectively.Chromatographic column: AgilentZORBAXEclipseXDB-C18 post (4.6 × 150mm, 5 μm), column temperature: 30 DEG C.Mobile phase A: water (0.1% formic acid), B: acetonitrile (0.1% formic acid), mobile phase gradient is 0-25min:1-100%B, flow velocity: 0.4mL/min, sample size: 10 μ L.Flight time mass spectrum optimal conditions is: (1) positive ion mode (ES+), capillary voltage 3500V, sprayer 45psig, dry gas temperature 325 DEG C, exsiccator flow velocity 11L/min; (2) negative ion mode (ES-), capillary voltage 3000V, other parameters are consistent with positive ion mode.When metabolite profiling analyses, data collection form is that plot and centroid carries out simultaneously, and acquisition quality scope is 50-1000Da.
Two, result:
All experiment samples carry out the full analysis of spectrum test of metabolin by LC-TOFMS and GC-TOFMS above.Test data result processes and metabolite identification by analysis, blood plasma or serum protein moteblites is utilized to establish orthogonal inclined minimum variance discriminatory analysis (OPLS-DA) model, can well identify in cancer of pancreas plasma sample and differentiating pancreatic cancer patient and normal person's (as shown in Figure 1A), this result obtains good checking (as shown in fig. 1b) in different pancreatopathy cancer serum samples.
Under variable weight (VIP) value of multidimensional OPLS-DA model is greater than 1 and the non-P value tested of engaging in an inspection is less than the choice criteria of 0.05, obtains and comprise phosphoamide (Urea), choline (Choline), methylguanidine (Methylguanidine), creatinine (Creatinine), 3-amino-2-piperidones (3-Amino-2-piperidone), 2-amino-butyric acid (2-Aminobutyricacid), betaine (betaine), valine (Valine), 2,4-diaminobenzoic acid (2,4-Diaminobutyricacid), glutamine (Glutamine), tryptophane (Tryptophan), proline (Proline), glutamic acid (Glutamate), N-acetylglutamat (N-Acetylglutamine), heteroauxin (Indoleaceticacid), 1,3,7-trimethyl-uric acid (1,3,7-Trimethyluricacid), uric acid (Uricacid), indole acrylic acid (Indoleacrylicacid), adenine (Adenine), single isobutyl phthalic acid (Monoisobutylphthalicacid), DHB (2,5-dihydroxybenzoicacid), p-hydroxybenzene acrylic acid (2-Hydroxycinnamicacid), 1,5-AG (1,5-Anhydro-D-glucitol), talose (Talopyranose), propionyl carnitine (Propionylcarnitine), lecithin (LysoPC (14:0)), galactitol (Galactitol), glycocholic acid (Glycocholicacid), NAMN (Nicotinicacidmononucleotide), a-KG (2-Oxoglutaricacid), 2-methyl-3-oxopropanoic acid (2-Methyl-3-oxopropanoicacid) otherness metabolic product (is mainly amino acid, carbohydrates, lipid, nucleosides, organic acid and assorted poly-ring aromatic compounds etc.).
Utilize Logic Regression Models to verify, find wherein have 15 difference metabolins particularly important as the effect of pancreatic cancer marker, respectively: 2,5-dihydroxybenzoicacid, talopyranose, proline, glutamate, choline, 1,5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaricacid, methylguanidine, adenine, glycocholicacid, valine, 2-aminobutyricacid.Use these metabolins, we establish following regression model:
Applying clinical diagnosis performance curve (ROC curve) is evaluated at cancer of pancreas plasma sample tumor marker subsequently.When threshold value 0.4272, find that ROC area under curve reached for 0.960 (95% fiducial interval 0.931 ~ 0.990) (as shown in Figure 2 A).Same diagnostic model is adopted to verify pancreatopathy cancer serum sample, under same threshold condition, find that ROC area under curve reaches 0.817 (95%CI, 0.756 – 0.877) (as shown in Figure 2 B), its sensitivity and specificity be respectively 77.4% and 74.7%, there is good accuracy equally, show that the biomarker found is as cancer of pancreas clinical diagnosis mark, has certain reference value.More than find to confirm, 15 plasma/serum metabolins are used for the effect of diagnosis of pancreatic cancer as a kind of AT biomarker.
As shown in figures 3 a and 3b, this 15 kinds of metabolic marker things and combination (2 thereof of the present embodiment, 5-dihydroxybenzoicacid, talopyranose, proline, glutamate, choline, 1, 5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaricacid, methylguanidine, adenine, glycocholicacid, valine, 2-aminobutyricacid) be good cancer of pancreas early diagnosis label, can be used in clinical diagnosis, greatly improve the early detective rate of cancer of pancreas, improve the clinical therapeutic efficacy of cancer of pancreas, alleviate the misery of patient, improve the survival rate of clinical patient.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (14)
1. a diagnosis of pancreatic cancer label combination, is characterized in that, be blood plasma or serum otherness metabolin, comprise phosphoamide (Urea), choline (Choline), methylguanidine (Methylguanidine), creatinine (Creatinine), 3-amino-2-piperidones (3-Amino-2-piperidone), 2-amino-butyric acid (2-Aminobutyricacid), betaine (betaine), valine (Valine), 2,4-diaminobenzoic acid (2,4-Diaminobutyricacid), glutamine (Glutamine), tryptophane (Tryptophan), proline (Proline), glutamic acid (Glutamate), N-acetylglutamat (N-Acetylglutamine), heteroauxin (Indoleaceticacid), 1,3,7-trimethyl-uric acid (1,3,7-Trimethyluricacid), uric acid (Uricacid), indole acrylic acid (Indoleacrylicacid), adenine (Adenine), single isobutyl phthalic acid (Monoisobutylphthalicacid), DHB (2,5-dihydroxybenzoicacid), p-hydroxybenzene acrylic acid (2-Hydroxycinnamicacid), 1,5-AG (1,5-Anhydro-D-glucitol), talose (Talopyranose), propionyl carnitine (Propionylcarnitine), lecithin (LysoPC (14:0)), galactitol (Galactitol), glycocholic acid (Glycocholicacid), NAMN (Nicotinicacidmononucleotide), a-KG (2-Oxoglutaricacid), one or more combination in 2-methyl-3-oxopropanoic acid (2-Methyl-3-oxopropanoicacid).
2. diagnosis of pancreatic cancer label combination as claimed in claim 1, it is characterized in that, for blood plasma or serum otherness metabolin, comprise choline (choline), 1, 5-dewatered grape sugar alcohol (1, 5-anhydro-D-glucitol), 2, 5-dihydroxy-benzoic acid (2, 5-dihydroxybenzoicacid), talose (talopyranose), proline (proline), glutamate/glutamate (glutamate), tryptophane (tryptophan), glutamine (glutamine), betaine/betaine (betaine), 2-oxoglutaric acid (2-oxoglutaricacid), methylguanidine (methylguanidine), adenine (adenine), glycocholic acid (glycocholicacid), valine (valine), one or more combination in 2-amino-butyric acid (2-aminobutyricacid).
3. the application of the diagnosis of pancreatic cancer label combination as described in claim 1 or 2, is characterized in that, for the kit of diagnosis of pancreatic cancer.
4. the application of diagnosis of pancreatic cancer label combination as claimed in claim 1 or 2, it is characterized in that, the diagnosis sample of diagnostic marker is blood plasma or serum.
5. the application of diagnosis of pancreatic cancer label combination as claimed in claim 3, is characterized in that, also comprise a regression model, as follows:
6. the application of diagnosis of pancreatic cancer label combination as claimed in claim 4, is characterized in that, applying clinical diagnosis performance curve evaluation otherness metabolin, and when threshold value is 0.4272, diagnostic marker combination has the highest sensitivity and specificity.
7. the assay method of diagnosis of pancreatic cancer label combination as claimed in claim 1, is characterized in that, comprise the following steps:
Step 1, gets Patients with Pancreatic Cancer clinical blood or serum sample and normal controls blood plasma or serum sample;
Step 2, by the preliminary otherness metabolin of combined gas chromatography mass spectrometry metabonomic analysis methods Analysis and Identification Patients with Pancreatic Cancer clinical blood/serum sample and normal controls plasma/serum sample;
Step 3, under variable weight (VIP) value of multidimensional OPLS-DA model is greater than 1 and the non-P value tested of engaging in an inspection is less than the choice criteria of 0.05, obtains further otherness metabolin;
Step 4, carries out Logic Regression Models and verifies, obtain otherness metabolin.
8. assay method as claimed in claim 6, is characterized in that, described step 1 comprises Patients with Pancreatic Cancer clinical blood/serum sample and the normal controls plasma/serum sample of the different group in different regions.
9. assay method as claimed in claim 7, it is characterized in that, the combined gas chromatography mass spectrometry metabonomic analysis methods in described step 2 comprises liquid/vapor combined gas chromatography mass spectrometry metabonomic analysis methods.
10. assay method as claimed in claim 8, it is characterized in that, in described step 2, the chromatographic condition of gas chromatography combined with mass spectrometry test comprises: Rxi-5ms capillary column: fill 5% phenylbenzene/95% dimethyl polysiloxane, carrier gas: ultrapure helium, flow: 1.0mL/min, injector temperature: 260 DEG C, transmission line temperature: 260 DEG C, ion source temperature: 210 DEG C, sample size: 1 μ L, input mode: Splitless injecting samples, heating schedule: from 80 DEG C and continue 2min, 220 DEG C are risen to the programming rate of 10 DEG C/min, 240 DEG C are risen to afterwards with the programming rate of 5 DEG C/min, 290 DEG C are risen to again with the programming rate of 25 DEG C/min, last at 290 DEG C of lasting 8min, mass ion source: EI source, electronics bombarding energy: 70eV, scanning of the mass spectrum scope: m/z, 40-600, full scan mode.
11. assay methods as claimed in claim 9, is characterized in that, in described step 2, the chromatographic condition of liquid chromatography mass coupling test comprises: AgilentZORBAXEclipseXDB-C18 post (4.6 × 150mm, 5 μm), column temperature: 30 DEG C.Mobile phase A: water (0.1% formic acid), B: acetonitrile (0.1% formic acid), mobile phase gradient is 0-25min:1-100%B, flow velocity: 0.4mL/min, sample size: 10 μ L.Flight time mass spectrum optimal conditions is: (1) positive ion mode (ES+), capillary voltage 3500V, sprayer 45psig, dry gas temperature 325 DEG C, exsiccator flow velocity 11L/min; (2) negative ion mode (ES-), capillary voltage 3000V, other parameters are consistent with positive ion mode.When metabolite profiling analyses, data collection form is that plot and centroid carries out simultaneously, and acquisition quality scope is 50-1000Da.
12. assay methods as claimed in claim 10, it is characterized in that, in described step 2, the plasma/serum Sample pretreatment of gas chromatography combined with mass spectrometry test comprises: get 50 μ L plasma/serum, add 10 μ l chlorophenylalanine (0.1mg/mL, water-soluble) and 10 μ L margaric acids (1mg/mL, alcohol is molten) carry out the reappearance of monitor sample as interior mark; Add 175 μ L chloroform methanol mixed solvent (1:3, v/v) again, vortex oscillation 30s; Put centrifuge tube and place 10min to promote albumen precipitation in-20 DEG C; Then the centrifugal 10min of 13000rpm, get supernatant 200 μ L and reclaim in sample injection bottle in height, ambient temperature in vacuum drying obtains sample product.
13. assay methods as claimed in claim 11, it is characterized in that, in described step 2, sample product use two-step approach to derive after draining, first 50 μ L methoxamine (15mg/mL are added, pyridine is molten), vortex oscillation 30s, reacts 90min at 30 DEG C, and then add 50 μ LBSTFA (containing 1%TMCS) at 70 DEG C of reaction 60min, carry out GC-TOFMS analysis after leaving standstill.
14. assay methods as claimed in claim 12, it is characterized in that, in described step 2, the plasma/serum Sample pretreatment of liquid chromatography mass coupling test comprises: get 50ul plasma/serum sample and 200 μ l and contain chlorophenylalanine (5 μ g/mL, water-soluble) methanol acetonitrile mixed liquor (5:3, v/v) mix, vortex oscillation 2min, after leaving standstill 10min, with the centrifugal 20min of 13000rpm, get supernatant.
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