CN105203683B - Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application - Google Patents
Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application Download PDFInfo
- Publication number
- CN105203683B CN105203683B CN201510587062.1A CN201510587062A CN105203683B CN 105203683 B CN105203683 B CN 105203683B CN 201510587062 A CN201510587062 A CN 201510587062A CN 105203683 B CN105203683 B CN 105203683B
- Authority
- CN
- China
- Prior art keywords
- cell lung
- lung cancer
- blood plasma
- mark
- small molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention belongs to analytical chemistry and clinical medicine domain, it is related to blood plasma metabolism small molecule mark and its application that human non-small cell lung cancer is related.The related blood plasma metabolism small molecule mark of non-small cell lung cancer, the mark is one or more in cortisol, cortisone and 4 methoxyphenylacetic acids.Described blood plasma metabolism small molecule mark, the mark is made up of cortisol, cortisone and 4 methoxyphenylacetic acids.Above-mentioned three kinds of mark content ranges（95% confidential interval）Respectively, cortisol（0.00018~0.00067）, cortisone（0.000029~0.00010）, 4 methoxyphenylacetic acids（0.000015~0.000022）, metabolin can point out the generation of tumour within the above range.The corresponding horizontal extent of normal group is cortisol（0.0030~0.0037）, cortisone（0.00044~0.00056）, 4 methoxyphenylacetic acids（7.39E‑07~2.09E‑06）.Blood plasma metabolism small molecule be a kind of new biomarkers, compared to traditional protein biomarker its associate with disease outcome it is stronger, it is not only stable, minimally invasive, be easy to detection, and quantitatively accurately.
Description
Invention field
The invention belongs to analytical chemistry and clinical medicine domain, the blood plasma metabolism for being related to human non-small cell lung cancer related is small
Molecular marker and its application.
Background technology
Lung cancer is the whole world most common reason of masculinity and femininity cancer related mortality.According to estimates, global lung cancer in 2012
New cases 18,000,000, account for the 12.9% of whole tumour new cases, and death 16,000,000 accounts for whole tumour associated deaths
19.4%.Lung cancer is divided into non-small cell lung cancer and ED-SCLC (15%) according to cell type, and non-small cell lung cancer has two
Main hypotype is gland cancer (40%), squamous cell carcinoma (30%), is treated according to different its of type and prognosis is also different.
The Image Examination for being clinically used for diagnosing at present has：Chest X-ray, CT (computed
Tomography, CT), PET-CT (Positron Emission Tomography/
Computer Tomography, Positron emission computed tomography), these check there are some defects, radiate to body
Influence and financial burden higher.Clinically conventional invasive inspection method has：Lung bioplsy and CT draw under bronchoscope
Lower Drug eluting stent is led, these are checked often needs to consume the regular hour, while bringing certain wound to patient
Risk.Except the above inspection, the related tumor-marker analyte detection of lung cancer is also more often used in peripheral blood, but these indicate
The sensitiveness and specificity of thing are relatively low.Therefore in the urgent need to seek one it is non-radiative, minimally invasive, not expensive and meanwhile it is extremely sensitive and
Special diagnostic method.
Metabolism group (Metabolomics/Metabonomics) be after genomics, transcription group, proteomics it
The new branch of science for emerging rapidly afterwards, it has also become the important component of systems biology.It is research living things system by outer
The science of portion's endogenous metabolism thing entirety machine Changing Pattern produced after stimulating or disturbing, is to verify living things system metabolism way
The research method in footpath.The research object of metabolism group is the small-molecule substance of below relative molecular mass 1000Da, including：Sugar
Class, amino acid, lipid, organic acid, carnitine etc..
Organism under normal condition is a complete system, and the metabolin in biological fluid, cell and tissue is in
One poised state of stabilization.Body there occurs pathological change due to heredity or the reason day after tomorrow, and this balance is just broken, generation
Thank product and metabolic process and also generate corresponding change.In the generation and evolution of tumour, tumour cell is subject to the external world
The stimulation of environment, can cause metabolin " multiplier effect " more than the minor variations that its interior environment occurs, and metabolin occur different
Ordinary change.Understand change of the metabolism small molecule in lysis by metabonomic analysis, people can be helped to find
Relevant biomarker (biomarker) can be with the diagnosis of aided disease, it is also possible to help people to pass through small-molecule substance sheet
The metabolic pathway that body is related to understands the pathogenesis of disease and for medicament research and development provides specific target.Metabolism group occurs certainly
Since, the great interest of scientists from all over the world is caused, developed rapidly over short more than ten year.Metabolism group is explained in pathology
State, drug design exploitation, medical diagnosis on disease, the field such as parting and disease prognosis show great potential.
Since Warburg is assumed because the metabolism that mitochondrial defects change is to the specificity of cancer cell, metabolism biological mark
Will thing is interested to researchers, is a kind of diagnosis to early-stage cancer and the effective biomarker of prognosis.From that time, perhaps
The identifications for making great efforts to be devoted to metabolism biological marker in oncology more.Only have two metabolism biological markers to have been enter into until now
Clinical practice, is respectively metanephrine and methoxyepinephrine, as prediction pheochromocytoma morbid state
Mark.It is critical only that for metabolism group research is fast and accurately analyzed and identified to a large amount of small molecule metabolites, this
It is largely dependent upon the progress of correlation technique.The development and combination of the technologies such as nuclear magnetic resonance, mass spectrum, chromatogram so that generation
Thank to group extensive use learned to be possibly realized.
At present, the detection and analysis technology of metabolin integral level includes liquid chromatograph mass spectrography (LC-MS), meteorological color
Spectrum-mass spectrometry (GC-MS) and nuclear magnetic resonance technique (NMR).NMR features are without destruction, Sample pretreatment letter to component to be measured
It is single, but sensitivity is relatively low, detect narrow dynamic range；GC-MS has preferable sensitivity and reappearance, but typically will be using derivative
Change method carries out pre-treatment to sample so that experimental procedure becomes complicated.For GC-MS, LC-MS can analyze it is highly polar and
Molecular weight compound relatively higher, more suitable for the detection of complicated metabolite and potential mark in metabolism group biological specimen
The identification of thing.Additionally, the characteristics of sample process is simple, and sensitivity is high, Clinical practicability is strong is had more, so entered using LC-MS
The metabonomic analysis of row metabolism small molecule, if the special blood plasma metabolism related to non-small cell lung cancer morbidity of stabilization can be found
Small molecule researches and develops the LC-MS detection methods of the metabolism small molecule mark of corresponding disease as biomarker, not only in the neck
Domain is in first place in the world, can create the economic benefit for attracting people's attention, early detection, early stage to China's non-small cell lung cancer
Diagnosis also will be once strong promotion.
Maeda etc. has inquired into amino acid profile by studying the Amino Acid profile of non-small cell lung cancer and healthy person
As the possibility of non-small cell screening index, and find 6 Sensitivity and Specificities diagnosis marker higher.Duarte
Deng to not yet having carried out network analysis by the initial stage patients with lung cancer of radiation and chemotherapy and the plasma sample of healthy volunteer, it is believed that
The change of endogenous metabolism thing appears in the initial stage of disease, thereby increases and it is possible to related to cancer biochemical process, such as the sugar of enhancement
Glycolysis and gluconeogenesis and the tricarboxylic acid cycle being suppressed and lipid metaboli.Carrola etc. is to patients with lung cancer and the urine of healthy person
Liquid has carried out metabolism group research, as a result finds hippurate and the reduction of trigonelline content in lung cancer urine, and beta-hydroxy is different
Valeric acid, AHIB, N- acetyl-glutamines and creatinine relative comparison group are raised.Fan T etc. are utilized13C stable isotopes
Indication difference group analytic approach " SIRM ", is studied Patients with Non-small-cell Lung cancerous tissue with cancer side non-cancer tissue.Both
The metabolic disorder of tumour is pointed out toward research, and has been found that the metabolin of otherness in various degree, can be used as prediction tumour
The mark of development.But, there are several keys in metabolism group research, in clinical practice except technology is in itself to grinding
Outside the influence studied carefully, the influence of different physiological status, such as sex, age, diet, day-night change, culture to metabolism group is also more next
More it is taken seriously.Therefore these influence factors are paid particular attention in the screening of disease or treatment mark, additionally needs knot
The method of mathematics is closed to exclude these influence factors, to be preferably applied for clinic.
The content of the invention
Small molecule mark is metabolized it is an object of the invention to provide a kind of related blood plasma of non-small cell lung cancer.
The purpose of the present invention is realized by following technical measures：
The related blood plasma metabolism small molecule mark of non-small cell lung cancer, the mark is cortisol, cortisone and 4- first
One or more in epoxide phenylacetic acid.
Described blood plasma metabolism small molecule mark, the mark is by cortisol, cortisone and 4- methoxyphenylacetic acid structures
Into.
Above-mentioned three kinds of mark content ranges (95% confidential interval) are respectively, cortisol (0.00018~0.00067),
Cortisone (0.000029~0.00010), 4- methoxyphenylacetic acids (0.000015~0.000022), metabolin is in above range
The interior generation that can point out tumour.The corresponding horizontal extent of normal group is cortisol (0.0030~0.0037), cortisone
(0.00044~0.00056), 4- methoxyphenylacetic acids (7.39E-07~2.09E-06).
The present invention is described in detail as follows：
The present inventor gathers standard compliant blood sample, the complete crowd of systematic collection with S.O.P. (SOP)
Back ground Information and clinical data, and employ the metabolism group method based on UPLC/MS and be analyzed.
The experimental technique specifically studied mainly includes following components：
First, research object selection and packet foundation
A groups：Healthy control group (n=112,56 people screening, 56 people's independence crowds checking)：
1. the age is between 42 years old to 80 years old；
2. tumour is had not been diagnosed as through physical examination, previously without tumour medical history；
3. without other general major diseases；
4. the chronic medical history without long-term prescription.
B groups：Non-small cell lung cancer group (n=99,53 people screening, 46 people's independence crowds checking)：
1. the age is between 42 years old to 83 years old；
2. underwent operative or biopsy first visit is non-small cell lung cancer；
3. without other general major diseases；
4. the chronic medical history without long-term prescription.
2nd, UPLC-MS metabonomic analysis and Diagnosis of Non-Small Cell Lung are screened and verified with metabolism small molecule
1. Sample pretreatment
1.1 new bloods are centrifuged 5min in centrifuge 3000rpm, take the μ l of supernatant 100 and dispense to clean 1.5ml EP pipes
In.
1.2 100 μ l blood plasma are with 300 μ l methanol extraction albumen.
1.3 draw supernatant, are divided into 2 parts, are dried up again with vacuum drying with nitrogen.
1.4 are used with 50 μ L water (containing 0.1% formic acid) a dried object (acidic extraction thing) of dissolving, another dried object
The water dissolves (alkaline extraction) of ammonium bicarbonate pH=8s of the 50 μ L containing 6.5mM.
2. instrument detection
2.1 analytical instrument：UPLC series connection ThermoFisher LTQ-FT using Waters Acquity companies are linear
Ion trap synchrometer.
2.2 liquid-phase conditions：
2.2.1 liquid-phase chromatographic column is 1.7 μm of 100mm chromatographic columns of Waters BEH C18, and column temperature is 40 degree.
2.2.2 acidic extraction thing：The mobile phase for the using first of water and (B) containing 0.1% formic acid containing 0.1% formic acid for (A)
Alcohol, flow velocity is 350 μ L/min.1.2 instrument gradients are：0-4min 0%B to 70%B, 4-4.5min70%-98%B, 4.5-
5.4min 98%B.
2.2.3 alkaline extraction：Mobile phase is the water of the ammonium bicarbonate pH=8 of (A) containing 6.5mM, and (B) is containing 6.5mM weight carbon
The methyl alcohol (methyl alcohol compares 95/5 with ammonium bicarbonate liquor capacity) of sour ammonium.
2.2.4 instrument gradient is：0-4min 0%B to 70%B, 4-4.5min 70%-98%B, 4.5-
5.4min98%B.
2.3 sample introduction patterns：The μ l of volume 5, using the pattern sample introduction of 2 × overfill.
2.4 Mass Spectrometry Conditions
2.4.1 it is analyzed with ESI ionization sources.
2.4.2 acid extract uses positive ion mode, alkaline extraction to be detected using negative ion mode.
2.4.3 mass spectrographic interface capillary temperature is 350 degree, and shealth gas flow velocitys are 40 (arbitrary
Units), aux gas flow are 5 (arbitrary units), and Spray voltages positive ion mode is 4.5kv, negative ion mode
It is 3.75kv.The molecular weight ranges of instrument scanning are 99-1000m/z.Sweep speed is that 6 times per second (3 times MS and 3 time MS/MS sweeps
Retouch).
3. statistical data analysis and biomarker screening
Multiple metabolin information that each plasma sample is detected are imported in the softwares of SIMCA-P 12.0 and carry out multivariable
Statistical analysis, using OPLS-DA (orthogonal to partial least squares discriminant
Analysis, the orthogonal partial least squares discriminant analysis method of supervision) it is modeled analysis.OPLS-DA multivariate statistical analysis can
Provide the VIP values (variable importance in projection, variable importance projection index) of each variable, VIP
Variable of the value more than 1 is considered as larger to model packet contribution.
Difference thing compares between two groups and uses t-tests, and carries out multiple statistical using FDR and compare p value control, according to generation
Thank to a group data and set up OPLS-DA models, with reference to metabolism group clinical research standard, 0.001, and VIP are less than with p value after control>
1, potential source biomolecule mark 28 (being shown in Table 1) is filtered out, screening criteria is further limited, non-small cell lung cancer group becomes than control group
Change multiple to raise or lower more than 2.5 times, carry out the screening of blood plasma diagnosis difference small molecule, three potential marks are found altogether
Meet above-mentioned condition.
4. material is qualitative
Chemspider databases (http is searched for by Information in Mass Spectra://www.chemspider.com/), it is preliminary to judge
Above-mentioned chemicals are cortisol, cortisone and 4- methoxyphenylacetic acids.Chemicals final qualitative use cortisol, cortisone and 4-
Cortisol, cortisone and 4- methoxyphenylacetic acid stable isotope internal standards that the standard items and carbon 13 of methoxyphenylacetic acid are marked
Thing, compares using Chromatographic information (retention time) and Information in Mass Spectra (accurate molecular weight, IP and MS/MS fragments letter
Breath).
5. the difference and diagnostic significance of small molecule are metabolized in healthy control group, non-small cell lung cancer group plasma sample
The normal healthy controls having differences and Patients with Non-small-cell Lung blood plasma the metabolism small molecule 4- methoxybenzenes for detecting
Content of the acetic acid in Patients with Non-small-cell Lung is substantially less than healthy control group, and cortisol and cortisone are in non-small cell lung
Content in cancer patient is significantly higher than healthy control group, and these measurers of metabolism small molecule in blood plasma have stability.Adopt
Above-mentioned metabolism small molecule diagnosing non-small cell lung cancer is applied with independent crowd, sensitivity is 87.00%, and specificity is 98.20%,
Area is 0.946 under ROC curve, with diagnostic value higher.
Beneficial effects of the present invention：
The present inventor is by using small point of metabolism in the healthier controls of UPLC-MS and Patients with Non-small-cell Lung blood plasma
Son, it was found that exist in blood plasma and can be used to assess whether that the blood plasma with diagnostic value with non-small cell lung cancer is metabolized small point
Sub- mark.
The present invention is advantageous in that using blood plasma metabolism small molecule as the mark of non-small cell lung cancer evaluation：
(1) cortisol is most important glucocorticoid in human body.Existing research and inquirement plasma total cortisol concentration and
Relation between the human peripheral lymphocyte DNA repair abilities of in vitro culture, as shown by data, the cortisol of high concentration is inhibited
The reparation of human peripheral lymphocyte DNA.Day and night Secretion Rhyme can predict the Deaths of lung cancer to stable cortisol, and
It is its independent prognostic indicator.Tumor cell suspension is injected intravenously to Swiss mice, while give cortisol can be oncogenic
Transfer extensively, the main mechanism being related to is probably that influence of the cortisol to reticuloendothellium result in specificity can not be produced anti-
Body.Previously research shows that cortisol serves the effect for promoting tumor development in non-small cell lung cancer develops.Cortex
Ketone is still not clear with the relation of tumour, and research shows, mice plasma corticosterone content changes after psychological stress.4- methoxies
Base phenylacetic acid is intermediate important during medicine synthesizes, and can be synthesized with anti-angiogenic exception by raw material of 4- methoxyphenylacetic acids
It is also that synthesis has raising immune with the medicine of tumor promotion, strengthens myocardial contractive power, protect cardiac muscle cell, reduce blood pressure, resists
The raw material of the medicine Puerarin of the effects such as platelet aggregation.This research by by patients with lung cancer and normal healthy controls person according to the age,
Sex, BMI are divided into screening group and validation group after matching, each group carries out statistics screening respectively：P value is less than 0.001, and VIP>
1, meeting the condition can draw 28 otherness metabolins simultaneously in screening group and validation group, by the result that two steps draw,
The reliability and accuracy of this 28 otherness metabolins are improve, further limit standard, non-small cell lung cancer group is compared
Raise or lower according to a group change multiple and be more than 2.5 times, obtained 3 final metabolins, at the same have sensitiveness higher and
Specificity, and can preferably be clinical practice service.
Blood plasma metabolism small molecule is a kind of new biomarkers, compared to traditional protein biomarker itself and disease outcome
Association is stronger, not only stable, minimally invasive, be easy to detection, and it is quantitative accurately, the sensitiveness of Diagnosis of Non-Small Cell Lung will be greatly improved
And specificity, the successful exploitation of the micromolecular biomarker is overturning to the traditional biological mark based on albumen,
Brand-new situation will be started for the preventing and treating of inborn defect, be that the development of other diseases biomarker is offered reference.
(2) the blood plasma metabolism small molecule mark that the present invention is provided can be used for Diagnosis of Non-Small Cell Lung mark, can keep away
Exempt from invasive diagnosis, and the risk of non-small cell lung cancer can be obtained by invasive manner in early stage, so as to be clinician
Further testing in depth testing provides foundation, is quick and precisely to grasp the morbid state and coincident with severity degree of condition of patient, take more in time
Have personalized control prece and support is provided, delay and prevent progression of disease.
(3) present invention use meets non-small cell lung cancer and the sample of normal healthy controls crowd is verified, it was demonstrated that this is several
There is significant difference between group and there is stability in marker levels, to illustrate that the mark has sensitivity and specificity,
Can be used as mark.
(4) present invention uses tight, multistage checking and appraisement system, and the initial stage screens various blood plasma generations by preliminary experiment
Thank to small molecule, independent crowd's checking is carried out using UPLC-MS, it is ensured that the blood plasma metabolism biological marker has reliable reference
Value, for the clinical diagnosis of non-small cell lung cancer provides preferable auxiliary information.
(5) UPLC-MS technologies sample process is simple, and Instrumental Analysis is accurate rapidly, with clinical detection practicality valency higher
Value.
Brief description of the drawings
Fig. 1 pathology figures, a adenocarcinomas of lung, b lung squamous cancers.
Fig. 2 chest CT imageological examinations, a lung windows, the vertical diaphragm windows of b.
Fig. 3 is metabolized horizontal fluctuation of the small molecule in different time points.
ROC curve (cortisol) between Fig. 4 Normal groups and non-small cell lung cancer group.
ROC curve (cortisone) between Fig. 5 Normal groups and non-small cell lung cancer group.
ROC curve (4- methoxyphenylacetic acids) between Fig. 6 Normal groups and non-small cell lung cancer group.
ROC curve (cortisol associated cortex ketone) between Fig. 7 Normal groups and non-small cell lung cancer group.
ROC curve (cortisol joint 4- methoxyphenylacetic acids) between Fig. 8 Normal groups and non-small cell lung cancer group.
ROC curve (cortisone joint 4- methoxyphenylacetic acids) between Fig. 9 Normal groups and non-small cell lung cancer group.
(cortisol associated cortex ketone combines 4- first to ROC curve between Figure 10 Normal groups and non-small cell lung cancer group
Epoxide phenylacetic acid).
Specific embodiment
The invention will be further elaborated by the following examples.
The research object of embodiment 1 is selected and packet foundation
The present inventor conforms to between in June, 2014 in October, 2011 from No.1 Attached Hospital, Nanjing Medical Univ collection
The Patients with Non-small-cell Lung asked and normal healthy controls person's blood sample, by the arrangement to sample data, therefrom have selected and meet
It is required that 112 normal healthy controls (average ages：58.5 years old, scope 42-80 Sui), 99 non-small cell lung cancer patients it is (average
Age：59.5 years old, scope 42-83 Sui) the screening experiment object of small molecule biomarker is metabolized as non-small cell lung cancer.Tool
The sample group standard of body is as follows：
A groups：Healthy control group (n=112,56 people screening, 56 people's independence crowds checking)：
1. the age is between 42 years old to 80 years old；
2. tumour is had not been diagnosed as through physical examination, previously without tumour medical history；
3. without other general major diseases；
4. the chronic medical history without long-term prescription.
B groups：Non-small cell lung cancer group (n=99,53 people screening, 46 people's independence crowds checking)：
1. the age is between 42 years old to 83 years old；
2. the treatments such as first visit patient, non-underwent operative, chemotherapy, radiotherapy, be non-small cell lung cancer through pathological diagnosis；
3. without other general major diseases；
4. the chronic medical history without long-term prescription.
The research object main bases of embodiment 2
Research non-underwent operative, is put for punctures, biopsy, the patient that cytology pathology ID is non-small cell lung cancer
Treatment, the patient of chemotherapy.Main bases are：(1) chest CT Radiologic imaging：See Fig. 2；(2) puncture, biopsy, cytology disease
Reason inspection result：See Fig. 1.
There is its limitation in above method of clinical analysis, wherein chest CT diagnosing has false negative higher and vacation
Positive findings, and radiologic method has certain radiation injury to patients with lung cancer.Puncture, biopsy, cytology are more smart
Really, but invasive diagnostic method is belonged to, and patient has been in middle and advanced stage when diagnosing, it is impossible to early detection disease.Clinical position
In the method made a definite diagnosis of more patients be postoperative pathological examination to be carried out to operation Operated Specimens, it is impossible to operator's row bronchoscope
Lower lung bioplsy or CT Guided Percutaneous Transthoracic Needle Aspiration Biopsies.
The UPLC-MS metabolism group non-small cell lung cancers biomarker of embodiment 3 is screened
1. Sample pretreatment
1. new blood is centrifuged 5min in centrifuge 3000rpm, takes the μ l of supernatant 400 and dispenses into clean 1.5ml EP pipes.
2. 400 μ l blood plasma are with 1200 μ l methanol extraction albumen.
3. supernatant is drawn, 2 parts are divided into, is dried up again with vacuum drying with nitrogen.
4. with 50 μ L water (containing 0.1% formic acid) a dried object (acidic extraction thing) of dissolving, 50 μ of another dried object
The water dissolves (alkaline extraction) of ammonium bicarbonate pH=8s of the L containing 6.5mM.
2. instrument detection
2.1 analytical instrument：UPLC series connection ThermoFisher LTQ-FT using Waters Acquity companies are linear
Ion trap synchrometer.
2.2 liquid-phase conditions：
2.2.1 liquid-phase chromatographic column is 1.7 μm of 100mm chromatographic columns of Waters BEH C18, and column temperature is 40 degree.
2.2.2 acidic extraction thing：The mobile phase for the using first of water and (B) containing 0.1% formic acid containing 0.1% formic acid for (A)
Alcohol, flow velocity is 350 μ L/min.1.2 instrument gradients are：0-4min 0%B to 70%B, 4-4.5min70%-98%B, 4.5-
5.4min 98%B.
2.2.3 alkaline extraction：Mobile phase is the water of the ammonium bicarbonate pH=8 of (A) containing 6.5mM, and (B) is containing 6.5mM weight carbon
The methyl alcohol (methyl alcohol compares 95/5 with ammonium bicarbonate liquor capacity) of sour ammonium.
2.2.4 instrument gradient is：0-4min 0%B to 70%B, 4-4.5min 70%-98%B, 4.5-
5.4min98%B.
2.3 sample introduction patterns：The μ l of volume 5, using the pattern sample introduction of 2 × overfill.
2.4 Mass Spectrometry Conditions
2.4.1 it is analyzed with ESI ionization sources.
2.4.2 acid extract uses positive ion mode, alkaline extraction to be detected using negative ion mode.
2.4.3 mass spectrographic interface capillary temperature is 350 degree, and shealth gas flow velocitys are 40 (arbitrary
Units), aux gas flow are 5 (arbitrary units), and Spray voltages positive ion mode is 4.5kv, negative ion mode
It is 3.75kv.The molecular weight ranges of instrument scanning are 99-1000m/z.Sweep speed is that 6 times per second (3 times MS and 3 time MS/MS sweeps
Retouch).
3. biomarker screening
Difference thing compares between two groups and uses t-tests, and carries out multiple statistical using FDR and compare p value control, according to generation
Thank to a group data and set up OPLS-DA models, with reference to metabolism group clinical research standard, 0.001, and VIP are less than with p value after control>
1, potential source biomolecule mark 28 (being shown in Table 1) is filtered out, screening criteria is further limited, non-small cell lung cancer group becomes than control group
Change multiple to raise or lower more than 2.5 times, carry out the screening of blood plasma diagnosis difference small molecule, three potential marks are found altogether
Meet above-mentioned condition.
4. material is qualitative
Chemspider databases (http is searched for by Information in Mass Spectra://www.chemspider.com/), it is preliminary to judge
Above-mentioned chemicals are cortisol, cortisone and 4- methoxyphenylacetic acids.Chemicals final qualitative use cortisol, cortisone and 4-
Cortisol, cortisone and 4- methoxyphenylacetic acid stable isotope internal standards that the standard items and carbon 13 of methoxyphenylacetic acid are marked
Thing, compares using Chromatographic information (retention time) and Information in Mass Spectra (accurate molecular weight, IP and MS/MS fragments letter
Breath).
The non-small cell lung cancer biomarker information of table 1
Note：4- methoxyphenylacetic acids (case/control) value 0.121 i.e. 0.121/1, equivalent to 8.26 times of downward.
The individual blood plasma of embodiment 4 is metabolized the stability analysis of small molecule
The stability of 10 blood plasma metabolism small molecule levels of adult is evaluated using the method for embodiment 3.And reality
Apply same continuous three blood plasma of acquisition method collection research object of example 3 (interval time is without disease in 2 weeks, interval).Knot
Fruit shows that cortisol, cortisone and 4- methoxyphenylacetic acids level relatively stablize (Fig. 3) in blood plasma.These have all pointed out individual blood
The level of slurry metabolism small molecule is relatively stable, possesses the characteristic as diagnosis/monitoring mark.
Embodiment 5 is metabolized evaluation capacity of the small molecule combinatorial to non-small cell lung cancer
According to above-mentioned UPLC-MS metabolism group method, the present inventor is by the case of independent crowd 46 and 56 blood for compareing
Slurry samples detection cortisol, cortisone and 4- methoxyphenylacetic acids, sensitivity and spy that ROC curve carrys out assessment prediction are drawn with this
Evaluation capacity of this 3 metabolism small molecule levels to non-small cell lung cancer in the opposite sex, and then assessment detection blood plasma.
The sensitivity of cortisol is 87.00%, and specificity is 100.00%, and area is 0.932 under ROC curve；Cortisone
Sensitivity is 87.00%, and specificity is 98.00%, and area is 0.955 under ROC curve；The sensitivity of 4- methoxyphenylacetic acids is
91.30%, specificity is 80.40%, and area is 0.919 under ROC curve.
Combination cortisol and cortisone, sensitivity is 87.00%, and specificity is 100.00%, and area is under ROC curve
0.948；Combination cortisol and 4- methoxyphenylacetic acids, sensitivity is 87.00%, and specificity is 98.20%, below ROC curve
Product is 0.930；Combination cortisone and 4- methoxyphenylacetic acids, sensitivity is 87.00%, and specificity is 96.40%, ROC curve
Lower area is 0.948.
Combination cortisol, cortisone and 4- methoxyphenylacetic acids, sensitivity is 87.00%, and specificity is 98.20%, ROC
TG-AUC is 0.946.
3 metabolism small molecules of the above are individually or two or two combinations can come non-small cell lung cancer group with healthy group differentiation.
Claims (1)
1. application of a kind of blood plasma metabolism small molecule in detection non-small cell lung carcinoma marker is prepared, it is characterised in that described
Blood plasma metabolism small molecule is made up of cortisol, cortisone and 4- methoxyphenylacetic acids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587062.1A CN105203683B (en) | 2015-09-15 | 2015-09-15 | Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587062.1A CN105203683B (en) | 2015-09-15 | 2015-09-15 | Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105203683A CN105203683A (en) | 2015-12-30 |
| CN105203683B true CN105203683B (en) | 2017-06-20 |
Family
ID=54951474
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510587062.1A Expired - Fee Related CN105203683B (en) | 2015-09-15 | 2015-09-15 | Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105203683B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112820348B (en) * | 2021-01-06 | 2023-04-07 | 宁夏医科大学总医院 | Method for detecting interaction between plasma metabolism and skin microflora of psoriasis patient |
| CN114306365A (en) * | 2021-12-16 | 2022-04-12 | 苏州大学附属第二医院 | Extraction test device based on deoxyinosine for treating rheumatoid arthritis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080070303A1 (en) * | 2005-11-21 | 2008-03-20 | West Michael D | Methods to accelerate the isolation of novel cell strains from pluripotent stem cells and cells obtained thereby |
| CA2606658A1 (en) * | 2006-10-13 | 2008-04-13 | Mike Tyers | Compositions and methods for treating neurological disorders or damage |
| EP3702774A1 (en) * | 2011-02-07 | 2020-09-02 | Laboratory Corporation of America Holdings | Method for determining the presence or amount of testosterone in a sample |
| CN103293268B (en) * | 2013-06-18 | 2016-03-23 | 南京金域医学检验所有限公司 | A kind of people urinates the detection method of hydrocortisone |
-
2015
- 2015-09-15 CN CN201510587062.1A patent/CN105203683B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105203683A (en) | 2015-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109884302A (en) | Lung cancer early diagnosis marker and its application based on metabolism group and artificial intelligence technology | |
| KLUPCZY—SKA et al. | Metabolomics in medical sciences ń trends, challenges and perspectives | |
| CN109239210B (en) | A kind of ductal adenocarcinoma of pancreas marker and its screening technique | |
| CN108603859B (en) | Use of metabolites in urine for preparing kit used in method for evaluating cancer | |
| CN108414660B (en) | Application of group of plasma metabolism small molecule markers related to early diagnosis of lung cancer | |
| Zhang et al. | Metabolomics approaches and applications in prostate cancer research | |
| CN101769910A (en) | Method for screening malignant ovarian tumor markers from blood serum metabolic profiling | |
| Chen et al. | Study on metabonomic characteristics of human lung cancer using high resolution magic‐angle spinning 1H NMR spectroscopy and multivariate data analysis | |
| CN103616450A (en) | Serum specificity metabolite spectrum for patient with lung cancer, and building method thereof | |
| Kyriakides et al. | Metabonomic analysis of ovarian tumour cyst fluid by proton nuclear magnetic resonance spectroscopy | |
| WO2011046597A2 (en) | Biomarkers for the detection of breast cancer | |
| CN110514772A (en) | Application of the transparent clear-cell carcinoma metabolic markers in clear-cell carcinoma early screening and diagnostic products | |
| CN112183616B (en) | Diagnostic marker and kit for diagnosis of glioma, screening method and construction method of glioma diagnostic model | |
| US20190293653A1 (en) | Urine metabolite marker for childhood cancer | |
| CN106018640A (en) | Method for rapid screening and identification of tumor biomarkers and application | |
| CN106716127A (en) | Methods for detecting ovarian cancer | |
| Zennaro et al. | Metabonomics by proton nuclear magnetic resonance in human pleural effusions: A route to discriminate between benign and malignant pleural effusions and to target small molecules as potential cancer biomarkers | |
| CN106556656A (en) | A kind of blood plasma metabolism small molecule mark related to cholelithiasis and its application | |
| CN105203683B (en) | Human non-small cell lung cancer related blood plasma metabolism small molecule mark and its application | |
| Hyndman et al. | Metabolomics and bladder cancer | |
| CN110568196B (en) | Metabolic marker related to low-grade glioma in urine and application thereof | |
| CN106153739A (en) | A kind of model based on metabonomic technology diagnosing liver cancer | |
| CN110261518A (en) | A kind of screening of human body effect of low dose radiation molecular injury marker taurine and verification method | |
| Gabbani et al. | Metabolomic analysis with 1H-NMR for non-invasive diagnosis of hepatic fibrosis degree in patients with chronic hepatitis C | |
| Derveaux et al. | Diagnosis of lung cancer: what metabolomics can contribute |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170620 Termination date: 20190915 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |