CN102565253A - Micromolecular metabolite atlas as well as producing method and application thereof - Google Patents
Micromolecular metabolite atlas as well as producing method and application thereof Download PDFInfo
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- CN102565253A CN102565253A CN201010591347XA CN201010591347A CN102565253A CN 102565253 A CN102565253 A CN 102565253A CN 201010591347X A CN201010591347X A CN 201010591347XA CN 201010591347 A CN201010591347 A CN 201010591347A CN 102565253 A CN102565253 A CN 102565253A
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Abstract
The invention relates to the technical field of medical diagnostics, in particular to a blood micromolecular metabolite atlas for identifying papillary thyroid cancer, benign goiter or normal person, and a producing method thereof. The micromolecular metabolite comprises two blood metabolism markers, namely sphinganine 1-phosphate and hydroxybutyric acid which respectively have molecular weight of 453.2855 and 569.3481 as well as corresponding ions detected by mass spectroscopy of 454.2928 and 570.3547. Clinical experiment proves that the atlas has thyroid cancer judgment accuracy rate of 82% and non-cancer (benign goiter and normal person) judgment accuracy rate of 75.3%. The method provided by the invention has the advantages of high sensitivity and high flux, overcomes the defect that a non-invasive diagnosis method for diagnosing benign and malignant thyroid tubercle is lacking clinically at present, and is suitable for screening and assistant diagnosis of thyroid cancer.
Description
Technical field
The present invention relates to the medical diagnosis field that learns a skill, is a kind of blood micromolecule metabolin collection of illustrative plates of differentiating papillary thyroid carcinoma, benign goiter and normal person and preparation method thereof.
Background technology
Metabolism group has obtained significant progress in recent years as important techniques means in the genome times afterwards comprehensively systems biology field.The starting point of metabolism group be that pathology stimulates or genetic modification after, cell, tissue, organ carry out system description through the variation of metabolin for the incidence and development of disease from whole angle in the dynamic change of interior all metabolin amounts.All metabolins in the body fluid can be used as the pointer of life system state, not only can reflect the result of various physiological regulation, also can develop into the strong instrument of auxiliary clinical diagnosis.At present, in the research that comprises diseases such as cancer, diabetes, angiocardiopathy, found corresponding diagnosis metabolic markers.And the development that the metabolism spectrum of using one group of blood micromolecule metabolin formation is carried out method of diagnosing to disease is in a developing stage fast in the world, possibly produce great influence in the clinical diagnosis to disease in future.
Thyroid cancer is first tumour occurred frequently of incidence, and the incidence of disease of thyroid cancer significantly increases in recent years, has become one of fastest-rising malignant tumour of the incidence of disease.With the U.S. is example, and the incidence of disease of nearly 30 years thyroid cancers has just increased 2-3 doubly, and thyroid cancer has accounted for the 7th of women's malignant tumour.The new cases of the annual thyroid cancer of the U.S. are up to 3-4 ten thousand people at present.
Thyroid tumors can be divided into two types of benign tumour and malignant tumours.The generation of the pernicious thyroid cancer of the overwhelming majority is from follicular epithelium, and minority can be from parafollicular cell, and only a few is from thyroid matter.Often be divided into thyroid cancer clinically: four kinds of papillary carcinoma, folliculus shape cancer, end differentiation cancer and cephalomas.Wherein papillary carcinoma is modal kind, accounts for 75%.
Thyroid tumors often only show as thyroid nodule clinically, therefore often mix thyroid tumors mutually with thyroid nodule.In fact tubercle only is the description of form, and it comprises agglomerate and the caused thyroid gland lump of other diseases that tumour, tumour, normal tissues constitute.Be difficult to confirm the character of thyroid nodule clinically, though pathological biopsy, thyroid adenoma and nodular hyperplasia sometimes, benign tumour and malignant tumour also are difficult for clearly recognizing.At present still lack the good pernicious judgement that mark can be used for thyroid nodule, also do not see and adopt blood micromolecule metabolin collection of illustrative plates to differentiate papillary thyroid carcinoma, optimum thyroid nodule or normal person's relevant report.
Summary of the invention
The objective of the invention is to theory and the method system new, set up a kind of collection of illustrative plates of forming by blood micromolecule metabolin and preparation method thereof according to this system biological medical domain of metabolism group.2 blood metabolic markers; Be 1-phosphoric acid sphingol (Sphinganine) and hydroxybutyric acid (Hydroxybutyric acid); Their molecular weight is respectively: 453.2855 and 569.3481, and detected corresponding ion is respectively 454.2928 and 570.3547 on the mass spectrum.
Clinical trial shows that the accuracy rate that this collection of illustrative plates thyroid cancer is judged is 82%, and the accuracy rate that non-cancer (benign goiter and normal person) is judged is 75.3%.。The inventive method has high sensitivity, high-throughout advantage, has remedied the deficiency of the good pernicious diagnostic method of the thyroid nodule that lacks Noninvasive now clinically, is applicable to the examination and the auxiliary diagnosis of thyroid cancer.
The method for making of collection of illustrative plates of the present invention is following.
The present invention is that the method with metabolism group filters out the metabolic markers group that can be used for the good pernicious diagnosis of thyroid nodule from the serum of 3 groups of samples of normal person, simplex goiter and papillary thyroid carcinoma, constitutes the diagnosing cancer of liver metabolite profile.Wherein, blood metabolin acquisition methods is following:
1) obtains the research object blood sample.
Blood sample is a fasting blood before the meal in early morning, gathers the back and leaves standstill under half an hour, the 9000g condition in the refrigerator that is stored in-80 ℃ after getting supernatant in centrifugal 15 minutes immediately subsequent use in 4 ℃.
2) blood sample pre-service
The sample room temperature is thawed.Get 50 μ L blood samples and add 200 μ L acetonitriles, left standstill 10 minutes in 4 ℃ behind the concuss, under 4 ℃ centrifugal 10 minutes then, get supernatant 4 μ L sample introductions with 15000g.
3) sample analysis
What stratographic analysis was adopted is that Agilent 1200 series are differentiated liquid chromatography fast, and what liquid-phase chromatographic column adopted is the C18 post, and what mass spectrophotometry was adopted is Agilent 6510 quadrupole rods-flight time mass spectrum.
4) blood micromolecule metabolin profile obtains
Utilize software from raw data, to extract compound information, calculate accurate molecular weight and do the chromatographic peak coupling.Data process normalization after the coupling is to reduce systematic error.
The screening process of micromolecule metabolite profile that is used for the good pernicious diagnosis of thyroid cancer is following:
Each group through the clear and definite thyroid benign tubercle of pathological diagnosis and pernicious papillary thyroid carcinoma and normal person; With statistical method; Seek the specific metabolic thing like the multifactor analysis of variance, Z value etc., the screening papillary thyroid carcinoma has significant difference with optimum thyroid nodule and normal group people metabolin.Further filter for the difference metabolin, to take the least possible metabolin, reaching better effect is prerequisite, confirms that finally 2 metabolins constitute the diagnosing cancer of liver metabolite profile.This group metabolin is 82% to the accuracy rate of the judgement of papillary thyroid carcinoma, and the accuracy rate of optimum thyroid nodule is 75.3%.
This method has good extendability, can improve the corresponding judgment ability through adding new metabolic markers.This method is applicable to the exploitation of other disease early diagnosis marker simultaneously, and simultaneously, the mark group of discovery has the prospect that exploitation becomes diagnostic kit.
Description of drawings
Fig. 1 is the susceptibility and the specific analysis chart of papillary thyroid carcinoma for micromolecule metabolin of the present invention is combined in the diagnosis thyroid nodule.
Fig. 2 is the application of micromolecule metabolin collection of illustrative plates of the present invention in differentiating papillary thyroid carcinoma and simplex goiter and normal person.The control group comprises 50 routine normal human serum samples, and the cyst group comprises 68 routine simplex goiter serum samples, and the cancer group is 40 routine papillary thyroid carcinoma serum samples.
Embodiment
Screen the metabolin collection of illustrative plates that can be used for the good pernicious antidiastole of thyroid nodule totally 3 groups of serum samples from normal person, simple property thyroid nodule and papillary thyroid carcinoma.
1, serum sample collection and processing
Research object is mainly because of thyroid nodule swollen, and the underwent operative of being admitted to hospital first excision back pathological diagnosis is respectively two groups of patients of simple property thyroid nodule and papillary thyroid carcinoma.In these two groups second day early morning of not carrying out any treatment after being admitted to hospital, vein is taken a blood sample on an empty stomach.Another group research object is normal medical examiner's like age and sex and above-mentioned two categories a blood sample.Above blood sample prepares serum by conventional method.
Blood serum sample pre-service: before blood serum sample is stored in and extremely analyzes in-80 ℃ of refrigerators.Palilate thyroid gland group 40 examples, simple property thyroid nodule group 68 examples, normal control group 50 examples.Before the analysis, sample is taken out from refrigerator, room temperature is thawed.Get 50 μ L serum respectively and add 200 μ L acetonitriles, left standstill 10 minutes with 4 ℃ behind the concuss, under 4 ℃ centrifugal 10 minutes then, get supernatant 4 μ L sample introductions with 15000g.
2, blood serum metabolic profiling analytical approach
2.1 liquid chromatography mass analysis
(1) chromatographic condition: employing be that Agilent 1200 series is differentiated liquid chromatographies fast, specification is 10cm * 2.1mm 1.8 μ m ZORBAX TM SB-AQ C18,50 ℃ of column temperature.Mobile phase A is the water that contains 0.1% formic acid, and Mobile phase B is an acetonitrile.Gradient is that 2%B is initial, keeps in 4 minutes, rising to 30% after 1.5 minutes, reaches 100% at 25 minutes immediately.Keep carrying out column equilibration 5 minutes after 4 minutes.Flow is 0.4mL/min.Sample size is 4 μ L.
(2) mass spectrum condition: the mass spectrum condition is: what mass spectrophotometry was adopted is Agilent 6510 quadrupole rods-flight time mass spectrum.Mass spectrum carries out data acquisition under positive ion mode.The mass spectrum capillary voltage is made as 4000V, and the dry gas flow is 11L/min, and temperature is 350 ℃.Atomisation pressure is made as 45psig.Fragmentor voltage and skimmer voltage are made as 230V and 65V respectively.The potpourri of purine and six phosphine piperazines (hexakis phosphazine) is used for keeping the precision and the stability of mass number measurement as correcting fluid.Under positive ion mode they to produce mass-to-charge ratio respectively be 121.0508 and 922.0097 ion.The data acquisition scope is mass-to-charge ratio 80-1000, with the barycenter type collection.Acquisition rate is 500 milliseconds.When carrying out automatic second order ms analysis, collision voltage is made as 10V and 30V respectively.
3, the pre-service of metabolism group data
Utilize software from raw data, to extract compound information, calculate accurate molecular weight and do the chromatographic peak coupling.The following normalization process of data process after the coupling is to reduce systematic error: at first, all 0 values are made as 0.01, then data are carried out normalization with central value, then adopt the average of all measurements for mass number.Adopt classification 80% principle in the processing of missing values, promptly, can be used when in a certain class sample of an ion 80% when all non-vanishing.
4, the screening of metabolin group
To all metabolins, at first the method through ANOVA filters out the metabolin that has significant difference between group, and filters out the metabolin of the interior difference of group greater than group difference.Pour data into SIMCA-P software then and analyze,, find the maximum ion of each group difference, and carry out the grade cluster analysis, guarantee that the ion that will not come from same metabolin repeats to be selected into according to the result of variable importance analysis.Finally, selected 2 kinds of metabolins to be used for antidiastole papillary thyroid carcinoma, simple property thyroid nodule and normal person.Comprise: 1-phosphoric acid sphingol (Sphinganine) and hydroxybutyric acid (Hydroxybutyric acid).
5, diagnosis metabolin group is used for the judgement of serum sample
Through these metabolins, carry out discriminatory analysis with SPSS software, the method that returns with binary logic is to papillary thyroid carcinoma, and simple property thyroid nodule and normal person judge.This group metabolin is 82% to the accuracy rate of the judgement of papillary thyroid carcinoma as a result, and simple property thyroid nodule and normal person's accuracy rate is 75.3%.The result shows, 1-phosphoric acid sphingol and hydroxybutyric acid metabolin group can be preferably carried out antidiastole to thyroid nodule good pernicious.
Claims (3)
1. micromolecule metabolin collection of illustrative plates; It is characterized in that said micromolecule metabolin is 1-phosphoric acid sphingol and 2 blood metabolic markers of hydroxybutyric acid; Their molecular weight is respectively: 453.2855 and 569.3481, and detected corresponding ion is respectively on the mass spectrum: 454.2928 and 570.3547.
2. the method for making of the said micromolecule metabolin of claim 1 collection of illustrative plates, step is following:
1) with 1-phosphoric acid sphingol and hydroxybutyric acid, adopt Obitrap respectively, positive ion mode is gathered metabolite concentration information down;
2) adopt reverse-phase chromatographic column to carry out micromolecule metabolin compartment analysis;
3) the metabolism group information of being obtained reduces the error in the sample analysis process through normalized method, thereby obtains the relative concentration information of metabolin;
4) according to the relative concentration value of these two blood metabolin components, the method that returns through binary logic is judged the variation tendency of metabolite profile, constitutes the metabolin spectral pattern.
3. the application of the said micromolecule metabolin of claim 1 collection of illustrative plates in differentiating papillary thyroid carcinoma, benign goiter or normal person.
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| CN111413396A (en) * | 2020-04-21 | 2020-07-14 | 四川大学 | Application of ferric oxide nano material and MA L DI-TOF MS in detection of small molecule metabolites |
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Application publication date: 20120711 |