CN102323362A - Model used for diagnosing lung cancer based on UPLC-MS technology - Google Patents
Model used for diagnosing lung cancer based on UPLC-MS technology Download PDFInfo
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- CN102323362A CN102323362A CN201010545401A CN201010545401A CN102323362A CN 102323362 A CN102323362 A CN 102323362A CN 201010545401 A CN201010545401 A CN 201010545401A CN 201010545401 A CN201010545401 A CN 201010545401A CN 102323362 A CN102323362 A CN 102323362A
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Abstract
The invention relates to the technical field of biology, and relates to a model used for diagnosing lung cancers. According to the invention, an ultrahigh performance liquid chromatography-mass spectrometry system is adopted, peripheral serum samples of normal subjects and lung cancer patients are detected, specific metabolites which are substantially different from those of the cancer patients are found, and a diagnostic model is established according to the concentrations of the metabolites and an average value of the content of normal subjects. When in use, the concentrations of three corresponding metabolites of a subject requiring detection are compared with the model provided by the invention, and the analyzed results can be used in the primary diagnosis of lung cancer.
Description
Technical field
The invention belongs to biological technical field, is a kind of model that can be used for diagnosing, for the diagnosis of lung cancer provides experiment basis.
Background technology
Lung cancer betides the tunica mucosa bronchiorum epithelium and also claims lung bronchogenic carcinoma.Lung cancer generally refers to the cancer of pulmonary parenchyma portion; Usually the mesoderm tumour (mesothelioma) that does not comprise other pleura origins; Perhaps other malignant tumours such as carcinoid (carcinoid), malignant lymphoma (malignant lymphoma), or shift tumour from other sources.Therefore our said lung cancer below is meant the malignant tumour that comes from bronchus (bronchial) or bronchiole (bronchiolar) epidermal cell (epithelial cell), has accounted for the 90-95% of pulmonary parenchyma malignant tumour.
Lung cancer is the first place of the whole world cancer cause of the death at present.Lung cancer originates from the tunica mucosa bronchiorum epithelium, is confined to that the person is called the carcinoma in situ cancerous swelling in the basilar memebrane, can be in bronchial lumen or/and the lung tissue of closing on growth, and can be through lymph blood capable or shift diffusion through bronchus.The cancer knurl speed of growth and the situation that shifts diffusion have certain relation with biological characteristicses such as the histological type of cancer knurl, differentiation degrees.
The distribution situation right lung of lung cancer is more than left lung, and last leaf all cancerous swelling can take place more than inferior lobe from the main bronchus to the bronchiole.The lung cancer that originates from main bronchus, lobar bronchi, the position is called central type carcinoma of lung near the hilus pulumonis person; Originate from the lung cancer below the segmental bronchi, the position is called peripheral type carcinoma of lung peripheral part person of lung.
Metabolism group (metabonomics) is a research idea of imitateing genomics and proteomics; All metabolins in the biosome are carried out quantitative test; And the research mode of the relativeness of searching metabolin and physiological and pathological variation, be the ingredient of systems biology.Its research object mostly is that relative molecular mass 1000 is with interior small-molecule substance.Computational analysis methods such as identification of advanced analysis detection technique binding pattern and expert system are the basic skills of metabolism group research.UPLC-MS (Ultra Performance Liquid Chromatography-mass spectrometry) technology is one of analytical technology of metabolism group.Chromatogram mainly is to let potpourri carried the capillary column that scribbles very thin one deck liquid film (liquid stationary phase) through a long inwall by moving phase.Because the different component of potpourri is different with the binding ability of stationary phase, thus each component in the terminal potpourri of post one by one wash-out come out and reach the purpose of separation.Mass spectrum then is earlier with substance ionization, becomes the gas ion potpourri, press the mass-to-charge ratio separation of ion, measures the intensity at various ionic spectrums peak then and realizes analysis purpose.Have higher sensitivity and specificity, express-analysis and evaluation when can realize to a plurality of compound.
The present invention utilizes this technology of UPLC-MS in the metabolism group, has set up a kind of model that can be used for diagnosing, for the diagnosis of lung cancer provides experiment basis.
Summary of the invention
The present invention utilizes Waters
UPLC-MS system; The metabolin profile of detection of lung cancer patient and healthy subjects serum; Utilize the software analysis lung cancer metabolic product different with normal human serum; Find new lung cancer correlativity organism, be used for diagnosing.
The present invention utilizes the UPLC-MS of Waters company to go up machine testing respectively lung cancer and healthy subjects serum, obtains raw data, from the subsidiary mass spectral database of machine, finds out the compound that is complementary.Adopt the peak area method for normalizing to carry out the adjustment of data; The statistical method of utilization is calculated every kind of compound in the conspicuousness of difference in the sample on the same group not after the adjustment of data; With p value 0.05 is threshold value; Judge that the p value is the compound that detection level has significant difference less than the compound of threshold value, filters out the Peak that there were significant differences in two groups.Carry out PCA then and analyze, PCA can focus on the statistical analysis technique on certain several overall target (major component) with the metabolism finger-print information that is dispersed on the n dimension variable.Through the preliminary relation between the judgement sample of this analysis, and the data that can judge whether individual samples depart from big and needs are disallowable falls.The significant metabolic peak of determining difference at last has 3, and its mass-to-charge ratio is respectively m/z203, m/z250, m/z996.Utilize this 3 kinds of metabolins, the mean concentration of the concentration of being examined respective substance in the human serum and model of the present invention is compared one by one, the diagnosis that can be lung cancer provides guidance.
The detection method of model is following:
1. sample collecting
Gather patients with lung cancer and normal control peripheric venous blood respectively, centrifugal after, get supernatant and place-80 ℃ of preservations subsequent use.
2. chromatographic condition optimization
Comprise the selection of chromatogram flow phase, the optimization of eluent gradient condition, and the optimization of chromatogram column temperature and optimum flow rate are to reach optimal separating efficiency.
3. mass spectrum condition optimizing
Comprise the selection of type ion source and the setting of parameter thereof, make peak figure optimize more.
4. preliminary experiment and upward appearance detection
The blood serum sample of getting after the dilution injects sample holes, carries out the setting of related coefficient.
5. the analysis of test report
The raw data that obtains is after the adjustment of data, and the utilization statistical method is found out the material of content notable difference, and utilization PCA analyzes, and will depart from bigger material and weed out, and obtains significant difference metabolin at last.
Description of drawings
Three peak heights of peak in each sample of Fig. 1
Grey straight line left side is a normal group, and grey straight line right side is a lung cancer group.Peak2.409 representes the metabolin of m/z203, and peak2.78 representes the metabolin of m/z250, and peak9.526 representes the metabolin of m/z996.
Three characteristic peaks that Fig. 2 finds are normal group to circle in the classification chart of normal group (normal) and lung cancer group (hepatitis B), and prismatic is a patients with lung cancer.
Embodiment
1. material
1.1 sample collecting
Use anticoagulant tube to gather normal group and lung cancer group venous blood 2ml respectively, centrifugal after in 4 ℃ of refrigerators, leaving standstill 30min (4000rpm, 5min). get the 200ul supernatant and place the low centrifuge tube that adsorbs, preserve in-80 ℃ of refrigerators, subsequent use.
1.2 required main agents and instrument
Methyl alcohol, ammonium acetate solution, formic acid, acetone etc. are all available from Shanghai chemical reagents corporation.
UPLC-MS system is available from Waters company.
2. method
2.1 the removal of high-abundance proteins
Melt under the sample room temperature, add the methyl alcohol ultrasonic extraction 2min of 1.0ml.The centrifugal again 14000rpm of suspending liquid that forms, 10min, 10 ℃.Get the 0.2ml supernatant at last and place LC-MS sample introduction bottle, add 1.0ml water, place-20 ℃ for use.
2.2 sample dries up
Placing liquid nitrogen to dry up appearance in the sample introduction bottle dries up.
2.3 preliminary experiment
Get the sample size of 20ul and carry out preliminary experiment, the adjustment related coefficient obtains best mass spectrogram.
2.4 formal experiment
Sample is injected the UPLC-MS system singly, get collection of illustrative plates, obtain raw data.
2.5 data analysis
Raw data is proofreaied and correct; P value with 0.05 is a threshold value, and the utilization statistical method is found out the material of obvious difference, utilizes PCA to analyze; The material that deviation is bigger weeds out, and obtains 3 kinds of significant difference metabolins at last: be respectively m/z203, m/z250, m/z996.Utilize this 3 kinds of materials, the concentration of being examined respective substance in the human serum and model of the present invention are compared one by one, the diagnosis that can be lung cancer provides auxiliary and instructs.
Experiment embodiment
Get person under inspection's serum; Obtain its metabolic product collection of illustrative plates through check; Obtain the content that mass-to-charge ratio is respectively the special metabolin of m/z203, m/z250, m/z996, compare with the average content of counter sample in this experiment, if content is approximately 2 times of this model average content; Then be lung cancer group, otherwise be normal group.
More than be the description of this invention and non-limiting, based on other embodiment of inventive concept, all among protection scope of the present invention.
Claims (1)
1. the model that is used for pulmonary cancer diagnosis; It is characterized in that metabolin collection of illustrative plates by Ultra Performance Liquid Chromatography-mass spectrometry system detection; Analysis obtains the concentration of a plurality of special metabolins of patients with lung cancer through software statistics, and the mass-to-charge ratio of said special metabolin is respectively m/z203, m/z250, m/z996.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033580A (en) * | 2013-01-06 | 2013-04-10 | 浙江中烟工业有限责任公司 | Detecting method for lung cancer characteristic metabolite fingerprint spectrum in urine |
| CN103616450A (en) * | 2013-11-29 | 2014-03-05 | 湖州市中心医院 | Serum specificity metabolite spectrum for patient with lung cancer, and building method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101769910A (en) * | 2008-12-30 | 2010-07-07 | 中国科学院大连化学物理研究所 | Method for screening malignant ovarian tumor markers from blood serum metabolic profiling |
| CN101832977A (en) * | 2009-03-09 | 2010-09-15 | 复旦大学附属妇产科医院 | Ovarian tumor serum marker |
-
2010
- 2010-11-15 CN CN201010545401A patent/CN102323362A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101769910A (en) * | 2008-12-30 | 2010-07-07 | 中国科学院大连化学物理研究所 | Method for screening malignant ovarian tumor markers from blood serum metabolic profiling |
| CN101832977A (en) * | 2009-03-09 | 2010-09-15 | 复旦大学附属妇产科医院 | Ovarian tumor serum marker |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033580A (en) * | 2013-01-06 | 2013-04-10 | 浙江中烟工业有限责任公司 | Detecting method for lung cancer characteristic metabolite fingerprint spectrum in urine |
| CN103033580B (en) * | 2013-01-06 | 2014-07-23 | 浙江中烟工业有限责任公司 | Detecting method for lung cancer characteristic metabolite fingerprint spectrum in urine |
| CN103616450A (en) * | 2013-11-29 | 2014-03-05 | 湖州市中心医院 | Serum specificity metabolite spectrum for patient with lung cancer, and building method thereof |
| CN103616450B (en) * | 2013-11-29 | 2015-12-30 | 湖州市中心医院 | A kind of Serum of Patients with Lung Cancer specific metabolic production spectra and method for building up thereof |
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Application publication date: 20120118 |