CN105572276B - The combination of diagnosis of pancreatic cancer marker, application and its measuring method - Google Patents
The combination of diagnosis of pancreatic cancer marker, application and its measuring method Download PDFInfo
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Abstract
The invention discloses the combination of diagnosis of pancreatic cancer marker, application and its measuring methods, including 15 species diversity metabolins (2,5-dihydroxybenzoic acid, talopyranose, proline, glutamate, choline, 1,5-anhydro-D-glucitol, tryptophan, glutamine, betaine, 2-oxoglutaric acid, methylguanidine, adenine, glycocholic acid, valine, 2-aminobutyric acid) and combinations thereof as cancer of pancreas early detection and Marker, application and its diagnostic marker measuring method of diagnosis are that the plasma/serum based on Pancreas cancer patients carries out liquid/vapor combined gas chromatography mass spectrometry metabonomic analysis methods.The diagnostic marker combination of technical solution of the present invention has the characteristics that sensitivity and specificity are high, for Early pancreatic carcinoma diagnosis also sensitivity and specificity with higher, it can be used for the early detection of cancer of pancreas, race against time for patient, start to treat as early as possible, improves clinical therapeutic efficacy.
Description
Technical field
The present invention relates to field of biotechnology, it is related to a kind of diagnosis of pancreatic cancer marker combination, application and its measuring method.
Background technique
Cancer of pancreas is that a kind of disease incidence is higher, the very high malignant tumor of digestive tract of grade malignancy, morbidity and mortality
It is obvious in recent years to rise, it is worst one of the malignant tumour of prognosis, five year survival rate is less than 5%, to improvement this case
Major obstacle is a lack of the biomarker of cancer of pancreas disease early stage diagnosis.
So far, the basic treatment method of cancer of pancreas is still based on surgical resection, but there is data to suggest that only
There is 20~25% patient to carry out excision appropriate in early stage open close surgical operation of crossing.There is concealment due to cancer of pancreas
Property, the diagnosis rate of cancer of pancreas early stage is not high, once the tumour for making a definite diagnosis Most patients has shifted, diagnosing and treating is all very
It is difficult.
Traditional tumor marker CA19-9 can reach 80% to the diagnostic sensitivity of cancer of pancreas, but for that can cut off rank
Section tumour is not suitable for Early pancreatic carcinoma patient because of its muting sensitivity and low specificity.At present there are also tumour-specific growth because
Sub (TSGF), CA242 (8), MIC-1, platelet factor 4, peanut agglutinin (PNA) combine glycoprotein, cell adhesion molecule
17.1, sero-immunity label affinity proteomic etc. is confirmed to be candidate biomarker, regrettably, these biomarkers
Object all has lower and low to the disease early detection precision disadvantage of disease early stage sensitivity.And early detection, the morning of cancer of pancreas
Phase diagnosis and early treatment are the key that the biomarkers for improving and improving cancer of pancreas prognosis, therefore find sensitive specificity
It is most important for the diagnosing and treating of the disease.
Patent CN101942502A discloses a kind of pancreatic cancer marker and its detection method, kit and biochip.
Pancreatic cancer marker provided by the invention includes to be stabilized and detectable 36 kinds of small ribose in experimenter's serum/blood plasma
Nucleic acid.The invention additionally provides the kit and biochip of the tool or element including detecting the pancreatic cancer marker.The hair
Combination provided by bright, method, kit and biochip can be used in the Diagnosis and differential diaggnosis for assisting cancer of pancreas, disease simultaneously
The generation of disease and prediction, therapeutic evaluation and the screening of active pharmaceutical ingredient, evaluating drug effect etc. of recurrence are sent out, there is inspection
The advantages that pedigree is wide out, high sensitivity, testing cost is low, materials are convenient, sample is easy to store.But the patent has disease early stage spirit
Lower and low to the disease early detection precision disadvantage of sensitivity.
Patent 103667444A discloses one kind tumor marker relevant to cancer of pancreas and its application, the marker are
The combination of CystatinSN (CST1), CystatinS (CST4), CystatinSA (CST2).The marker and its primer, antibody
It can be used for diagnostic kit application, the auxiliary diagnosis for cancer of pancreas.But the patent also have disease early stage sensitivity lower and
The disadvantage low to disease early detection precision.
Summary of the invention
In view of this, the present invention provides a kind of combination of diagnosis of pancreatic cancer marker, application and its measuring methods.
In order to achieve the above objectives, specific technical solution is as follows:
On the one hand, a kind of diagnosis of pancreatic cancer marker combination is provided, is blood plasma or serum otherness metabolin, including carbon
Amide (Urea), choline (Choline), methylguanidine (Methylguanidine), creatinine (Creatinine), 3- amino -2- piperazine
Pyridine ketone (3-Amino-2-piperidone), 2-amino-butyric acid (2-Aminobutyric acid), glycine betaine (betaine), figured silk fabrics
Propylhomoserin (Valine), 2,4- diaminobenzoic acid (2,4-Diaminobutyric acid), glutamine (Glutamine), color
Propylhomoserin (Tryptophan), proline (Proline), glutamic acid (Glutamate), N-acetylglutamat (N-
Acetylglutamine), heteroauxin (Indoleacetic acid), 1,3,7- trimethyl-uric acid (1,3,7-
Trimethyluric acid), uric acid (Uric acid), indole acrylic acid (Indoleacrylic acid), adenine
(Adenine), single isobutyl group phthalic acid (Monoisobutylphthalic acid), 2,5- dihydroxy-benzoic acid (2,5-
Dihydroxybenzoic acid), p-hydroxybenzene acrylic acid (2-Hydroxycinnamic acid), the Portugal 1,5- dehydration-D-
Grape sugar alcohol (1,5-Anhydro-D-glucitol), talose (Talopyranose), propionyl carnitine
(Propionylcarnitine), lecithin (LysoPC (14:0)), galactitol (Galactitol), glycocholic acid
(Glycocholic acid), nicotinic acid single nucleotide (Nicotinic acid mononucleotide), a-KG
One of (2-Oxoglutaric acid), 2- methyl -3- oxopropanoic acid (2-Methyl-3-oxopropanoic acid)
Or a variety of combination.
Preferably, the marker includes choline (choline), 1,5- dewatered grape sugar alcohol (1,5-anhydro-D-
Glucitol), 2,5- dihydroxy-benzoic acid (2,5-dihydroxybenzoic acid), talose (talopyranose), dried meat
Propylhomoserin (proline), glutamate/glutamate (glutamate), tryptophan (tryptophan), glutamine
(glutamine), glycine betaine/betaine (betaine), 2-oxoglutaric acid (2-oxoglutaric acid), methylguanidine
(methylguanidine), adenine (adenine), glycocholic acid (glycocholic acid), valine (valine),
One of 2-amino-butyric acid (2-aminobutyric acid) or a variety of combinations.
On the other hand, the application of diagnosis of pancreatic cancer marker combination, the kit for diagnosis of pancreatic cancer are provided.
Preferably, the diagnosis sample of diagnostic marker is blood plasma or serum.
It preferably, also include a regression model, as follows:
, when the Probability value in model be greater than 50% when, can disease break as cancer of pancreas.
Preferably, applying clinical diagnosis performance curve evaluation otherness metabolin, when threshold value is 0.4272, diagnostic flag
Object combination has highest sensitivity and specificity.
On the other hand, the measuring method of diagnosis of pancreatic cancer marker combination is provided, comprising the following steps:
Step 1, Patients with Pancreatic Cancer clinical blood or serum sample and normal controls blood plasma or serum sample are taken；
Step 2, by combined gas chromatography mass spectrometry metabonomic analysis methods analyze and identify Patients with Pancreatic Cancer clinical blood or
The preliminary otherness metabolin of serum sample and normal controls blood plasma or serum sample；
Step 3, variable weight (VIP) value of multidimensional OPLS-DA model be greater than 1 and the non-P value tested of engaging in an inspection less than 0.05
Selection criteria under, further otherness metabolin is obtained in preliminary otherness metabolin；
Step 4, Logic Regression Models are carried out to be verified, obtains otherness metabolin.
It preferably, include the other Patients with Pancreatic Cancer clinical blood of different regions difference group or serum sample in the step 1
Sheet and normal controls blood plasma or serum sample.
Preferably, the combined gas chromatography mass spectrometry metabonomic analysis methods in the step 2 include liquid/vapor chromatographic mass spectrometry
It is combined metabonomic analysis methods.
Preferably, the chromatographic condition that gas chromatography combined with mass spectrometry is tested in the step 2 includes: 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 volume: 1uL, input mode: Splitless injecting samples, temperature program:
Since 80 DEG C and continue 2min, rise to 220 DEG C with the heating rate of 10 DEG C/min, later with the heating rate liter of 5 DEG C/min
290 DEG C are risen to 240 DEG C, then with the heating rate of 25 DEG C/min, finally continues 8min at 290 DEG C, mass ion source: the source EI,
Electron bombardment energy: 70eV, scanning of the mass spectrum range: m/z, 40-600, full scan mode.
Preferably, the chromatographic condition of liquid chromatography mass combination test includes: Agilent ZORBAX in the step 2
Eclipse XDB-C18 column (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 are 0-25min:1-100%B, flow velocity: 0.4mL/min, sample volume: 10 μ L.Flight
Time mass spectrum optimal conditions are as follows: (1) positive ion mode (ES+), capillary voltage 3500V, sprayer 45psig, dry temperature degree
325 DEG C, drier flow velocity 11L/min；(2) negative ion mode (ES-), capillary voltage 3000V, other parameters and cation mould
Formula is consistent.In metabolite profile analysis, data collection form is that plot and centroid is carried out simultaneously, and acquisition quality range is
50-1000Da。
Preferably, the serum sample pre-treatment that gas chromatography combined with mass spectrometry is tested in the step 2 includes: to take 50 μ L blood
Clearly, 10ul chlorophenylalanine (0.1mg/mL, water-soluble) is added and 10 μ L heptadecanoic acids (1mg/mL, alcohol are molten) are used as internal standard to monitor sample
This reproducibility；Add 175 μ L chloroform methanol mixed solvents (1:3, v/v), vortex oscillation 30s；Centrifuge tube is set to put in -20 DEG C
10min is set to promote albumen precipitation；Then 13000rpm is centrifuged 10min, takes 200 μ L of supernatant in height recycling sample injection bottle, room temperature
Lower vacuum drying.
Preferably, sample product are derived after draining using two-step method in the step 2, are firstly added 50 μ L methoxamine
(15mg/mL, pyridine are molten), vortex oscillation 30s react 90min at 30 DEG C, then add 50 μ L BSTFA (containing 1%
TMCS GC-TOFMS analysis) is carried out after 70 DEG C of reaction 60min, standing.
Preferably, the serum sample pre-treatment of liquid chromatography mass combination test includes: to take 50ul serum in the step 2
The methanol acetonitrile mixed liquor (5:3, v/v) of sample and 200ul containing chlorophenylalanine (5ug/mL, water-soluble) mixes, vortex oscillation
2min after standing 10min, is centrifuged 20min with 13000rpm, takes supernatant.
Compared with the existing technology, technical solution of the present invention is put forward for the first time 2,5-dihydroxybenzoic acid,
talopyranose,proline,glutamate,choline,1,5-anhydro-D-glucitol,tryptophan,
glutamine,betaine,2-oxoglutaric acid,methylguanidine,adenine,glycocholic
This 15 species diversity metabolin of acid, valine, 2-aminobutyric acid and combinations thereof is used as diagnosis of pancreatic cancer marker
Application, the diagnostic marker and combinations thereof sensitivity and specificity with higher, can be used for differentiating pancreatic cancer patient with just
Ordinary person races against time for the early detection of cancer of pancreas for patient, starts to treat as early as possible, improves clinical therapeutic efficacy.Meanwhile
Divided by being composed entirely with blood plasma or serum progress metabolin of the LC-TOFMS and GC-TOFMS to Patients with Pancreatic Cancer and normal person
Analysis test, identifies 15 species diversity metabolins, and the biomarker as cancer of pancreas combines, and can be used for the early stage of cancer of pancreas
It was found that and diagnosis, improve the clinical therapeutic efficacy of cancer of pancreas.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Figure 1A is the Patients with Pancreatic Cancer and normal control plasma sample LC-TOFMS and GC-TOFMS of the embodiment of the present invention
The OPLS-DA of the metabolin of detection schemes；
Figure 1B is the Patients with Pancreatic Cancer and normal control serum sample LC-TOFMS and GC-TOFMS of the embodiment of the present invention
The OPLS-DA of the metabolin of detection schemes；
Fig. 2A is the ROC curve of the blood plasma biomarker analysis prediction cancer of pancreas of the cancer of pancreas sample of the embodiment of the present invention
Figure (it include 15 blood plasma biomarkers: 2,5-dihydroxybenzoic acid, talopyranose, proline,
glutamate,choline,1,5-anhydro-D-glucitol,tryptophan,glutamine,betaine,2-
oxoglutaric acid,methylguanidine,adenine,glycocholic acid,valine,2-
aminobutyric acid)；
Fig. 2 B is the ROC curve of the serum biomarker object analysis prediction cancer of pancreas of the cancer of pancreas sample of the embodiment of the present invention
Figure；
Fig. 3 A~Fig. 3 B is the diagnostic model of application 15 metabolins foundation of the embodiment of the present invention to predict pancreas (blood plasma
Sample and serum sample) and normal control diagnostic value distribution map.A cancer of pancreas S1:TNM phase patient by stages；Cancer of pancreas
S2:TNM the second stage of patient by stages；Cancer of pancreas S3:TNM three phase patient by stages.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
Specific explaination is done to the embodiment of the present invention below with reference to attached drawing.
The embodiment of the present invention application hydrolysis and condensation detects the metabolin of serum or plasma sample, finds
The diagnostic marker of cancer of pancreas.Specifically preferably include:
Patients with Pancreatic Cancer and the plasma/serum sample of normal person are collected, is tested and analyzed after processing through chromatographic mass spectrometry, by building
It shows to vertical multidimensional statistics model visualization the metabolism spectrum difference between Patients with Pancreatic Cancer and normal person, obtains the difference between two groups
Anisotropic metabolin, and Preliminary Identification is carried out to these substances.
Measuring method of the invention can comprehensively, synthetically embody the metabolite between Patients with Pancreatic Cancer and normal person
Variation situation finds the diagnostic marker of cancer of pancreas, provides advantageous technical support for the early diagnosis and prognosis of cancer of pancreas.
One, experimental material and test method:
1. plasma/serum sample situation
(1) plasma sample comes from 94 Patients with Pancreatic Cancer and normal controls blood plasma sample corresponding with its age and gender
This 100.
(2) serum sample comes from 96 Patients with Pancreatic Cancer and normal human control sera's sample corresponding with its age and gender
This 98.
2. plasma/serum Sample pretreatment:
(1) gas chromatography-mass spectrometry (GC-TOFMS) tests plasma/serum Sample pretreatment
Take 50 μ L blood plasma or serum in the centrifuge tube of 1.5mL, be separately added into 10ul chlorophenylalanine (0.1mg/mL,
It is water-soluble) and 10 μ L heptadecanoic acids (1mg/mL, alcohol are molten) carry out the reproducibility of monitor sample as internal standard.Add 175 μ L chloroform methanols
Mixed solvent (1:3, v/v), vortex oscillation 30s；Centrifuge tube is set in -20 DEG C of placement 10min to promote albumen precipitation.Then
13000rpm is centrifuged 10min, takes 200 μ L of supernatant in height recycling sample injection bottle, is dried in vacuo at room temperature.Sample product make after draining
Derived with two-step method, be firstly added 50 μ L methoxamine (15mg/mL, pyridine are molten), vortex oscillation 30s reacts at 30 DEG C
Then 90min adds 50 μ LBSTFA (containing 1%TMCS) in 70 DEG C of reaction 60min.After reaction product stands 1h at room temperature
Carry out GC-TOFMS analysis.
(2) liquid chromatography mass combined instrument (LC-TOFMS) test blood plasma or serum sample pre-treatment
50ul plasma/serum sample and 200ul is taken to contain the methanol acetonitrile mixed liquor of chlorophenylalanine (5ug/mL, water-soluble)
(5:3, v/v) mixing, vortex oscillation 2min after standing 10min, are centrifuged 20min with 13,000rpm, take supernatant for LC-
TOFMS analysis.
3. analysis instrument is tested:
(1) gas chromatography-mass spectrometry (GC-TOFMS) is tested
GC-TOFMS:LecoPegasusHT gas-chromatography time-of-flight mass spectrometry (Leco Corporation, the U.S.), chromatographic column:
Rxi-5ms capillary column (filling 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 volume: 1uL,
Input mode: temperature program: Splitless injecting samples since 80 DEG C and continue 2min, rise to 220 with the heating rate of 10 DEG C/min
DEG C, 240 DEG C are risen to the heating rate of 5 DEG C/min later, then rise to 290 DEG C with the heating rate of 25 DEG C/min, finally 290
DEG C continue 8min.Mass ion source: the source EI, electron bombardment energy: 70eV, scanning of the mass spectrum range: m/z, 40-600, full scan side
Formula.Data Analysis Services use ChromaTOF software (v4.33, Leco Corporation, the U.S.).
(2) liquid chromatography mass combined instrument (LC-TOFMS) is tested
LC-TOFMS: 1200 system of Agilent ultra performance liquid chromatography (agilent company, the U.S.) is equipped with binary solvent control
Device and sample manager processed.Mass spectral analysis uses Agilent 6220MSD type time of-flight mass spectrometer (agilent company, the U.S.),
It is equipped with double electrospray ionisation sources.Standard items mixed liquor is tuned using Agilent ESI-L low concentration, respectively in positive ionization electrospray electricity
Mode (ES-) mode is ionized by system tunning to optimal sensitivity and resolution ratio from mode (ES+) and negative electrospray.Color
Compose column: Agilent ZORBAX Eclipse XDB-C18 column (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 are 0-25min:1-100%B, flow velocity: 0.4mL/
Min, sample volume: 10 μ L.Flight time mass spectrum optimal conditions are as follows: (1) positive ion mode (ES+), capillary voltage 3500V, spray
Day with fog 45psig, dry 325 DEG C of temperature degree, drier flow velocity 11L/min；(2) negative ion mode (ES-), capillary voltage
3000V, other parameters are consistent with positive ion mode.In metabolite profile analysis, data collection form is plot and centroid
It carries out simultaneously, acquisition quality range is 50-1000Da.
Two, result:
All of above experiment sample carries out the full spectrum analysis test of metabolin by LC-TOFMS and GC-TOFMS.Test data
As a result processing and metabolite identification by analysis establish orthogonal inclined minimum variance using blood plasma or serum protein moteblites and differentiate point
(OPLS-DA) model is analysed, Patients with Pancreatic Cancer and normal person (such as Figure 1A can be identified and distinguished among well in cancer of pancreas plasma sample
Shown in), this result has obtained good verifying (as shown in fig. 1b) in different pancreatopathy cancer serum samples.
It is greater than 1 and non-selection of the P value less than 0.05 tested of engaging in an inspection in variable weight (VIP) value of multidimensional OPLS-DA model
Under standard, obtain including carbamide (Urea), choline (Choline), methylguanidine (Methylguanidine), creatinine
(Creatinine), 3- amino -2- piperidones (3-Amino-2-piperidone), 2-amino-butyric acid (2-Aminobutyric
Acid), glycine betaine (betaine), valine (Valine), 2,4- diaminobenzoic acid (2,4-Diaminobutyric
Acid), glutamine (Glutamine), tryptophan (Tryptophan), proline (Proline), glutamic acid
(Glutamate), N-acetylglutamat (N-Acetylglutamine), heteroauxin (Indoleacetic acid), 1,3,
7- trimethyl-uric acid (1,3,7-Trimethyluric acid), uric acid (Uric acid), indole acrylic acid
(Indoleacrylic acid), adenine (Adenine), single isobutyl group phthalic acid (Monoisobutylphthalic
Acid), 2,5- dihydroxy-benzoic acid (2,5-dihydroxybenzoic acid), p-hydroxybenzene acrylic acid (2-
Hydroxycinnamic acid), 1,5- dewatering-D-glucitol (1,5-Anhydro-D-glucitol), talose
(Talopyranose), propionyl carnitine (Propionylcarnitine), lecithin (LysoPC (14:0)), galactitol
(Galactitol), glycocholic acid (Glycocholic acid), nicotinic acid single nucleotide (Nicotinic acid
Mononucleotide), a-KG (2-Oxoglutaric acid), 2- methyl -3- oxopropanoic acid (2-Methyl-
3-oxopropanoic acid) otherness metabolite (predominantly amino acid, carbohydrate, lipid, nucleosides, organic acid and
Miscellaneous poly-ring aromatic compounds etc.).
It is verified using Logic Regression Models, discovery wherein has work of 15 difference metabolins as pancreatic cancer marker
With particularly important, it is respectively: 2,5-dihydroxybenzoic acid, talopyranose, proline, glutamate,
choline,1,5-anhydro-D-glucitol,tryptophan,glutamine,betaine,2-oxoglutaric
acid,methylguanidine,adenine,glycocholic acid,valine,2-aminobutyric acid.It uses
These metabolins, we establish regression model below:
Subsequent applying clinical diagnosis performance curve (ROC curve) comments tumor marker in cancer of pancreas plasma sample
Valence.In threshold value 0.4272, it is found that area reaches 0.960 (95% confidence interval 0.931~0.990) (such as Fig. 2A under ROC curve
Shown in).Pancreatopathy cancer serum sample is verified using same diagnostic model, under same threshold condition, finds ROC
Area under the curve reaches 0.817 (95%CI, 0.756-0.877) (as shown in Figure 2 B), its sensitivity and specificity is respectively
77.4% and 74.7%, equally there is good accuracy, show the biomarker found as cancer of pancreas clinical diagnosis mark
Will object has certain reference value.The above discovery confirmation, a kind of biology of 15 plasma/serum metabolins as hurtless measure
Marker is used for the effect of diagnosis of pancreatic cancer.
As shown in figures 3 a and 3b, this 15 kinds of metabolic marker objects of the present embodiment and combinations thereof (2,5-
dihydroxybenzoic acid,talopyranose,proline,glutamate,choline,1,5-anhydro-D-
glucitol,tryptophan,glutamine,betaine,2-oxoglutaric acid,methylguanidine,
Adenine, glycocholic acid, valine, 2-aminobutyric acid) it is good cancer of pancreas early diagnosis mark
Remember object, 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, subtract
The pain of hypopathia people improves the survival rate of clinical patient.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (11)
1. a kind of combination of diagnosis of pancreatic cancer marker, which is characterized in that it is blood plasma or serum otherness metabolin, including choline,
1,5- dewatered grape sugar alcohol, 2,5- dihydroxy-benzoic acid, talose, proline, glutamate, tryptophan, glutamine, beet
Alkali, 2-oxoglutaric acid, methylguanidine, adenine, glycocholic acid, valine, 2-amino-butyric acid；Wherein glutamate can use glutamate
Instead of.
2. diagnosis of pancreatic cancer marker combination according to claim 1, which is characterized in that further include carbamide, creatinine, 3-
Amino -2- piperidones, 2,4- diaminobenzoic acid, N-acetylglutamat, heteroauxin, 1,3,7- trimethyl-uric acid, uric acid, Yin
Diindyl acrylic acid, single isobutyl group phthalic acid, p-hydroxybenzene acrylic acid, propionyl carnitine, lecithin, galactitol, niacin list
Nucleotide, 2- methyl -3- oxopropanoic acid.
3. the application of the combination of diagnosis of pancreatic cancer marker described in claims 1 or 2, which is characterized in that be used to prepare diagnosis
The kit of cancer of pancreas.
4. the application of diagnosis of pancreatic cancer marker combination according to claim 3, which is characterized in that the kit is examined
Disconnected sample is blood plasma or serum.
5. the measuring method of diagnosis of pancreatic cancer marker combination of any of claims 1 or 2, which is characterized in that including following step
It is rapid:
Step 1, Patients with Pancreatic Cancer clinical blood or serum sample and normal controls blood plasma or serum sample are taken；
Step 2, Patients with Pancreatic Cancer clinical blood/serum sample is analyzed and identified by combined gas chromatography mass spectrometry metabonomic analysis methods
With the preliminary otherness metabolin of normal controls plasma/serum sample；
Step 3, it is greater than 1 and non-selection of the P value less than 0.05 tested of engaging in an inspection in the variable weight VIP value of multidimensional OPLS-DA model
Under standard, further otherness metabolin is obtained；
Step 4, Logic Regression Models are carried out to be verified, obtains otherness metabolin.
6. measuring method as claimed in claim 5, which is characterized in that include that difference group in different regions is other in the step 1
Patients with Pancreatic Cancer clinical blood/serum sample and normal controls plasma/serum sample.
7. measuring method as claimed in claim 5, which is characterized in that the combined gas chromatography mass spectrometry metabolism group in the step 2
Analysis method includes liquid/vapor combined gas chromatography mass spectrometry metabonomic analysis methods.
8. measuring method as claimed in claim 7, which is characterized in that gas chromatography combined with mass spectrometry is tested in the step 2
Chromatographic condition includes: Rxi-5ms capillary column: filling 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 volume: 1 μ L,
Input mode: Splitless injecting samples, temperature program: since 80 DEG C and continue 2min, rise to 220 with the heating rate of 10 DEG C/min
DEG C, 240 DEG C are risen to the heating rate of 5 DEG C/min later, then rise to 290 DEG C with the heating rate of 25 DEG C/min, finally 290
DEG C continue 8min, mass ion source: the source EI, electron bombardment energy: 70eV, scanning of the mass spectrum range: m/z, 40-600, full scan side
Formula.
9. measuring method as claimed in claim 7, which is characterized in that liquid chromatography mass combination test in the step 2
Chromatographic condition includes: 4.6 × 150mm of Agilent ZORBAX Eclipse XDB-C18 column, and 5 μm, column temperature: 30 DEG C.Mobile phase
A: water, wherein including 0.1% formic acid；B: acetonitrile, wherein including 0.1% formic acid, mobile phase gradient is 0-25min:
1-100%B, flow velocity: 0.4mL/min, sample volume: 10 μ L.Flight time mass spectrum optimal conditions are as follows: (1) positive ion mode ES+,
Capillary voltage 3500V, sprayer 45psig, dry 325 DEG C of temperature degree, drier flow velocity 11L/min；(2) negative ion mode
ES-, capillary voltage 3000V, other parameters are consistent with positive ion mode.In metabolite profile analysis, data collection form is
Plot and centroid is carried out simultaneously, and acquisition quality range is 50-1000Da.
10. measuring method as claimed in claim 7, which is characterized in that gas chromatography combined with mass spectrometry is tested in the step 2
Plasma/serum Sample pretreatment includes: to take 50 μ L plasma/serums, and the chlorophenylalanine water that 10 μ l concentration are 0.1mg/mL is added
The alcoholic solution for the heptadecanoic acid that solution and 10 μ L concentration are 1mg/mL carrys out the reproducibility of monitor sample as internal standard；Add 175 μ L
Chloroform methanol mixed solvent, wherein the volume ratio of chloroform and methanol is 1:3, vortex oscillation 30s；Centrifuge tube is set to place in -20 DEG C
10min is to promote albumen precipitation；Then 13000rpm is centrifuged 10min, takes 200 μ L of supernatant in height recycling sample injection bottle, at room temperature
Vacuum drying obtains sample product.
11. measuring method as claimed in claim 10, which is characterized in that sample product use two after draining in the step 2
Footwork is derived, and 50 μ L concentration are firstly addedThe methoxamine pyridine solution of 15mg/mL, vortex oscillation 30s are anti-at 30 DEG C
90min is answered, 50 BSTFAs of the μ L containing 1%TMCS is then added and carries out GC-TOFMS analysis after 70 DEG C of reaction 60min, standing.
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| CN109946467B (en) * | 2017-12-21 | 2021-05-28 | 中国医学科学院北京协和医院 | Biomarker for ossification diagnosis of thoracic vertebra ligamentum flavum |
| CN109187805A (en) * | 2018-10-22 | 2019-01-11 | 嘉兴迈维代谢生物科技有限公司 | A kind of metabolin liquid chromatograph mass spectrography detection method |
| CN113640420B (en) * | 2021-08-13 | 2023-05-02 | 上海市内分泌代谢病研究所 | Application of serum metabolite combination in early diagnosis of pancreatic cancer |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942502A (en) * | 2009-12-24 | 2011-01-12 | 北京命码生科科技有限公司 | Pancreatic cancer marker, and detection method, kit and biochip thereof |
| CN103038644A (en) * | 2010-06-01 | 2013-04-10 | 梅坦诺米克斯保健有限公司 | Means and methods for diagnosing pancreatic cancer in a subject |
| CN103534594A (en) * | 2011-03-04 | 2014-01-22 | 伊缪诺维亚公司 | Method, array and use thereof |
| CN103667444A (en) * | 2013-09-29 | 2014-03-26 | 中山大学附属第三医院 | Tumor markers associated with pancreatic cancer and application thereof |
| CN103837620A (en) * | 2014-03-21 | 2014-06-04 | 上海硕源健标生物医学科技有限公司 | Method for simultaneously detecting content of multiple amino acids in plasma of human body |
-
2014
- 2014-11-07 CN CN201410624200.4A patent/CN105572276B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942502A (en) * | 2009-12-24 | 2011-01-12 | 北京命码生科科技有限公司 | Pancreatic cancer marker, and detection method, kit and biochip thereof |
| CN103038644A (en) * | 2010-06-01 | 2013-04-10 | 梅坦诺米克斯保健有限公司 | Means and methods for diagnosing pancreatic cancer in a subject |
| CN103534594A (en) * | 2011-03-04 | 2014-01-22 | 伊缪诺维亚公司 | Method, array and use thereof |
| CN103667444A (en) * | 2013-09-29 | 2014-03-26 | 中山大学附属第三医院 | Tumor markers associated with pancreatic cancer and application thereof |
| CN103837620A (en) * | 2014-03-21 | 2014-06-04 | 上海硕源健标生物医学科技有限公司 | Method for simultaneously detecting content of multiple amino acids in plasma of human body |
Non-Patent Citations (2)
| Title |
|---|
| 人胰腺癌组织的差异蛋白质组学研究;汪建国 等;《临床肿瘤学杂志》;20111130;第16卷(第11期);第961-965页 * |
| 应用2D-DIGE及MALDI-TOF-MS筛选胰腺癌血清标志物;夏云连 等;《国际外科学杂志》;20120531;第39卷(第5期);第313-317页 * |
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