CN107300613A - A kind of biomarker, the method for sampling, modeling method and application thereof - Google Patents
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Abstract
It is an object of the present invention to provide a kind of biomarker, the method for sampling, modeling method and application thereof.Label is SFTPB, pro SFTPB, N1, N12Diacetyl spermine (N1, N12Diacetylspermine any or its combination).The method of sampling selects different detection methods based on SFTPB, pro SFTPB respectively.Mathematical modeling is：P=1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk)).Beneficial effect：The sensitivity and accuracy of the present invention is significantly improved, and can solve the problem that existing pulmonary cancer diagnosis label is single, the problem of sensitivity and specificity are not good.
Description
Technical field
The invention belongs to technical field of biological, and in particular to biomarker, the method for sampling, modeling method and its use
On the way.
Background technology
Lung cancer is one of current Chinese incidence of disease highest malignant tumour, and its death rate also remains high.According to Chinese medicine
Old ten thousand green grass or young crops in institute of oncology of tumour hospital of the academy of sciences etc. is published in internal authority medical journals magazine《CA-Cancer JClin》's
《Cancer in China statistics in 2015》, China in 2015 there are about 429.2 ten thousand cancer new cases, 281.4 ten thousand cancer mortality cases.Its
In, lung cancer is with new cases 73.3 ten thousand (accounting for overall 17.1%), death 610,000 (accounting for overall 21.1%), as China
People suffers from cancer or because of the lethal biggest threat of cancer.
In clinical practice, lung cancer early diagnosis is always difficult point, early detection effectively the controlling to cancer patient of cancer
Treatment is very important.Lung cancer survival rate is highly dependent on the stage that diagnosis is found, early diagnosis, will so as to early treatment
It is greatly improved 5 years survival rates.Diagnosis Main Basiss clinical symptoms, iconography at present for cancer is detected and histopathology
Check etc., but have the clinical symptoms appearance of many cancers later, and living body sampling detection is also difficult, has had a strong impact on cancer
Early diagnosis and the prognosis of patient.Relative to tissue biopsy, the sample such as blood plasma, sputum, bronchoalveolar lavage fluid is readily available, and to disease
People is also without wound.At present, in the circulating of periphery, the abnormal expression of specific protein, specific gene aberrant DNA methylation, certain
A little miRNA unconventionality expressions etc. are all probably caused by cancer.Therefore analysis and detection to these specific factors is also lung
The early diagnosis of cancer provides help.
Peripheral blood is the junction last eventually of the most albumen of body, the factor etc., and any one of body is damaged or should
Swash and be likely to cause many albumen, factor expression unstable, therefore a certain factor pair lung cancer in single detection peripheral blood
The sensitivity and specificity of diagnosis are barely satisfactory.Clinically with the single of macromolecular cancer markers or work is used in combination at present
Research for cancer diagnosis means is more, and small molecule class cancer markers are lacked with the deep discussion of system.
SFTPB (surfactant protein B) precursor pro-SFTPB, is by 2 type alveolar parietal cells and non-cilium
Bronchiolar cell synthesizes a hydrophilic 42kD albumen.In building-up process, pro-SFTPB is in endoplasmic reticulum rapidly by half Guang
Serine protease proteolytic cleavage, obtains the 9kD non-collegenous dormain hydrophobicity SFTPB of a secreting type, this is SFTPB maturation
Functional form.Lung carcinoma cell, especially adenocarcinoma of lung, generally entail that SFTPB synthesizes dysregulation, precursor, which is changed into, maturation
The process of hydrophobic form albumen slow down, cause pro-SFTPB overexpression.Research confirms to examine in Patients with Non-small-cell Lung
Measure the ripe SFTPB levels of circulation and be higher than normal person.SFTPB, Pro-SFTPB can examining as detection non-small cell lung cancer
Disconnected target.
Metabolism group is an important component in systems biology, and research object is the terminal small molecule in body
The science of the changing rule of metabolin (being often referred to molecular weight less than 1000Da).It is on the whole to a certain organism component or thin
Born of the same parents a specific physiological periods or under the conditions of all metabolites simultaneously carry out qualitative and quantitative analysis, instrument searches out target difference
Metabolin.Available for disease early diagnosis, drug target discovery, pathogenic mechanism research and medical diagnosis on disease etc..Metabonomic analysis
Sample be mainly urine and blood, its gatherer process is to human body fanout free region.Polyamine compounds are small cationic molecules, are led to
Often it is considered related to normal cell and cancer cell regulation key metabolic enzymes.Polyamines micromolecular include 1,3- propane diamine, putrescine,
Cadaverine, spermine, acylated spermine, spermidine, acylated spermidine etc..In normal mammalian cell this kind of small-molecule substance be must not
Can be less, and its content is by the network regulation of a series of complex, including synthesis, degraded and transport.The polyamines in epithelial tumour
Class compound also receives the regulation of proto-oncogene and tumor suppressor gene.Can not yet there is ample evidence to confirm which kind of polyamines is cancer at present
The biomarker of disease, also without lung cancer specific marker thing, this leverages lung cancer auxiliary diagnosis, prognosis and curative effect evaluation and referred to
Mark etc..
Can not yet there is ample evidence to confirm SFTPB, pro-SFTPB and N at present1, N12- diacetyl spermine is in cancer prediction
Meaning, research of the only single biomarker to cancer detection, and its sensitivity and specificity is not high.The present invention this
It is individual to combine the characteristic for combining biomarker, optimum organization, so as to provide help for cancer prediction.
At present only to SFTPB, pro-SFTPB and N1, the wherein a certain biomarker of N12- diacetyl spermine is carried out
The research of detection, the detection gimmick of each index is single, it is impossible to meet the demand of a variety of sample types, and its result is to the pre- of cancer
The sensitivity and specificity of survey are not good.
The content of the invention
Single it is an object of the invention to solve existing pulmonary cancer diagnosis label, sensitivity and specificity are not good to be showed
There is provided new biomarker thing, the method for sampling, modeling method and application thereof for shape.
A kind of biomarker, the label is SFTPB (surfactant protein B, surface protein B), pro-
SFTPB (pro-surfactant protein B, surface protein B precursor), N1, N12- diacetyl spermine (N1, N12-
Diacetylspermine any or its combination).
Furtherly, when label is SFTPB and/or pro-SFTPB, using ELISA method, chemoluminescence method, albumen
Chip method, immunoturbidimetry or immunochromatographic method carry out the detection of contents level in blood plasma.
Furtherly, when label is N1, N12- diacetyl spermine (N1, N12- diacetylspermine) when, use
UHPLC-MS/MS, ELISA and chemoluminescence method, chip method, immunoturbidimetry or immunochromatographic method carry out contents level in blood plasma
Detection.
Furtherly, the combination of the blood plasma biomarker is determined using mathematical modeling.The mathematical modeling is：
P=p (E)=eb0+b1x1+b2x2+b2x3+…+bkxk/(1+eb0+b1x1+b2x2+b2x3+…+bkxk)
=1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))
Wherein, p is probability.E is the individual of analysis.For analysis individual positive probability occurs for p (E).E is natural number, about etc.
In 2.718281828459.B0 is regression coefficient.Bk is the coefficient of k-th of factor.K span is 1 to n integer.
Furtherly, lung cancer assessment models are built using biomarker, its step includes：
Step (1)：Collect the plasma sample of object to be checked.
Step (2)：SFTPB, pro-SFTPB, N in plasma sample are detected respectively1, N12The content of-diacetyl spermine.
Step (3)：The data measured with step (2) carry out Mathematical Models, determine that blood plasma biomarker is combined.Its
In, mathematical modeling is p==1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))。
Furtherly, mathematical modeling changes for the combined horizontal of biomarker, and the p value after combination can improve sensitivity
(70-90%) and accuracy (70-90%).
Furtherly, this detection method and mathematical modeling can be used for the detection and assessment of lung cancer.
Furtherly, it is an object of the invention to the wound according to present in existing pulmonary cancer diagnosis technology it is big, find the state of an illness
Late the problem of, there is provided a kind of method of new lung cancer auxiliary diagnosis.
The beneficial effect that technical solution of the present invention is brought
Invention solve existing pulmonary cancer diagnosis be use label it is single caused by sensitivity and specificity it is not good
Present situation.The present invention not only provides one group of new lung cancer specific marker thing combination, and sensitivity and the accuracy of the product
It is significantly increased (70-90%).
Furtherly, disclosed by the invention is one group of new biomarker thing, and establishes label group on this basis
Close, the judgement data of pinpoint accuracy are provided the diagnosis of lung cancer for clinician.
The present invention is by the detection of one group of biomarker and is combined as judging the disease state offer method by inspection people, changes
The problems such as existing lung cancer early screening accuracy is low, method is complicated, round of visits is long is entered.
The plasma sample that the present invention is used, materials are simple, overcome the problem of traditional detection method tissue biopsy wound is big,
Also eliminate simultaneously due to the drawbacks of Tumor Heterogeneity easily produces false negative result.
The detection of biomarker and built-up pattern can improve the accuracy of pulmonary cancer diagnosis and ageing, be clinician
Determine the state of an illness and treatment offer scientific basis is provided.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
A kind of blood plasma biomarker, the label is SFTPB, pro-SFTPB, N1, N12Any of-diacetyl spermine
Plant or its combination.
Furtherly, SFTPB schematic diagram is：
Furtherly, SFTPB fragment can be SEQ ID1, SEQ ID2, SEQ ID3, SEQ ID4, SEQID5, SEQ
ID6, SEQ ID7 or SEQ ID8：
SEQ ID1：LSEQQFPIPLPYCWLCRALIKRIQA.
SEQ ID2：PKGALAVAVAQVCRVVPLVAGGICQCLAERYSVILLDT.
SEQ ID3：LGRMLPQLVCRLVLRC.
SEQ ID4：RDSECHLCMSVT.
SEQ ID5：EQAIPQAMLQACVGS.
SEQ ID6：KCKQFVEQHTPQLLTLVPR.
SEQ ID7：AHTTCQALGVCGT.
SEQ ID8：SSPLQCI.
Furtherly, pro-SFTPB fragment is SEQ ID9, SEQ ID10, SEQ ID11, SEQ ID12, SEQID13
Or SEQ ID14：
SEQ ID9：AESHLLQWLLLLLPTLCGP.
SEQ ID10：TTSSLACAQ.
SEQ ID11：PEFWCQSLEQALQCRALGHCLQEVWGHVGADDLCQECEDIVHIL.
SEQ ID12：LEQECNVLPLKLLMPQCNQVLDDYFPLVIDY.
SEQ ID13：GICMHLGLCKS.
SEQ ID14：DPLPKPLRDPLPDPLLDKLVLPVLPGALQAR.
The method of sampling of blood plasma biomarker, when label be SFTPB and/or pro-SFTPB when, using ELISA method,
Chemoluminescence method, protein chip, immunoturbidimetry or immunochromatographic method carry out the detection of contents level in blood plasma.
The method of sampling of blood plasma biomarker, when label is N1, N12- diacetyl spermine (N1, N12-
When diacetylspermine), using UHPLC-MS/MS, ELISA and chemoluminescence method, chip method, immunoturbidimetry or immune
Chromatography carries out the detection of contents level in blood plasma.
The modeling method of the described method of sampling, the combination of the blood plasma biomarker is determined using mathematical modeling.
The mathematical modeling is：
P=p (E)=eb0+b1x1+b2x2+b2x3+…+bkxk/(1+eb0+b1x1+b2x2+b2x3+…+bkxk)
=1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))
Wherein, p is probability.E is the individual of analysis.For analysis individual positive probability occurs for p (E).E is certainly
So number, is approximately equal to 2.718281828459.B0 is regression coefficient.Bk is the coefficient of k-th of factor.K's
Span is 1 to n integer.
The purposes of described modeling method, lung cancer assessment models are built using blood plasma biomarker, and its step includes：
Step (1)：Collect the plasma sample of object to be checked.
Step (2)：SFTPB, pro-SFTPB, N in plasma sample are detected respectively1, N12The content of-diacetyl spermine.
Step (3)：The data measured with step (2) carry out Mathematical Models, determine that blood plasma biomarker is combined.Its
In, mathematical modeling is p==1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))。
The purposes of modeling method, mathematical modeling changes for the combined horizontal of biomarker, and the p value sensitivity after combination is carried
High 70-90%, accuracy improves 70-90%.
The purposes of modeling method, this detection method and mathematical modeling can be used for the detection and assessment of lung cancer.
Embodiment
The detection of biomarker in experimenter's sample
Step 1：Lung cancer and human normal plasma sample collection method
Collect and turn out to be patients with lung cancer 55, wherein male 30 through pathocytology, women 25, age 28-76 Sui,
Average age 52 years old.Control group 55, is normal adults, is confirmed using routine inspections such as blood routine, routine urinalysis, wherein man
Property 30, women 25, age 27-66 Sui, average 51 years old.8mL patients with lung cancer and Healthy People venous blood are gathered in EDTA anti-freezings
Guan Zhong, centrifuges 12min in 1500rcf immediately.Heparin tube is taken out from centrifuge, blood plasma is transferred to using disposable pipette
In one 15mL centrifuge tube, many transfer blood plasma but cellular layer should not be touched as far as possible.Centrifuge 15mL centrifuge tubes, 1500rcf from
Heart 12min.15mL centrifuge tubes are taken out from centrifuge, using disposable pipette by 3.5mL blood plasma be transferred to one it is new
In 15mL centrifuge tubes.
Step 2：Lung cancer and N in human normal plasma1, N12- diacetyl spermine detection method
The blood plasma obtained to being collected in step 1 carries out N1, N12- diacetyl spermine immunoturbidimetry is analyzed, and detection method is such as
Under：
(1) R1 reagents are N1, N12The reagent of-diacetyl spermine antibody coupling rear addition protection liquid on nano particle.R2
Reagent is N1, N12- diacetyl spermine be coupled at by coupling reagent on carrier protein after reagent.
(2) 50mM pH6.0 MES buffer solutions are prepared, final concentration of 1%W/V activates nano particle, obtains nano particle
Suspension.
(3) every milliliter of nano particle suspension is included in 20mg EDC, is incubated at room temperature 20min.
(4) 12000rpm centrifuges 30min, and with isometric coating buffer solution, (100mM Hepes pH7.0 contain N1, N12- two
The μ g/mL of acetyl spermine antibody 100) suspend, it is incubated at room temperature 2-4 hours.2.5 μ L monoethanolamines, room are added in every milliliter of reaction solution
Temperature stirring is incubated 10min.
(5) 12000rpm centrifuges 30min, goes after supernatant with protection liquid suspension.
(6) standard items are the N being diluted with water1, N12- diacetyl spermine, concentration gradient be followed successively by 0nM, 250nM, 500nM,
5 concentration of 750nM and 1000nM.
(7) by R1, R2 reagent and N1, N12- diacetyl spermine standard items are placed on Biochemical Analyzer relevant position, make mark
Directrix curve.
(8) with the obtained μ L of plasma sample 5 in automatic clinical chemistry analyzer detecting step 1, R1 reagents 150 μ L, R2 are added
The μ L of reagent 40, using the 22nd point as first point detection, the 45th point is the second detection, using 2 differences as detected value, passes through standard
Curve draws N in sample1, N12- diacetyl spermine concentration.
N in 55 patients with lung cancer and 55 human normal plasmas is determined in the method set up1, N12- diacetyl spermine contains
Amount, each sample test is once.According to standard curve, N in plasma sample is calculated1, N12- diacetyl spermine concentration.
Method for detecting protein content can be ELISA method, chemoluminescence method, protein chip, immunoturbidimetry in sample
Method or immunochromatographic method.
Step 4：SFTPB content detections in sample
With Full-automatic chemiluminescence immunoassay analysis meter (NPD projects are biological, Maglumi 2000plus) for detection instrument, side
Science of law pattern is the coated carboxylic of SFTPB monoclonal antibodies that double antibody sandwich method, i.e. instrument sequentially add 50 μ L sample, 50 μ L
After the coated acridinium esters of SFTPB of the magnetic particle of base and 50 μ L, reaction 20min, Magneto separate is carried out, instrument, which will react, to be mixed
Thing sends into darkroom, sequentially adds luminous substrate A liquid (H2O2) and B liquid (NaOH) carries out luminescence-producing reaction, finally record luminous value.Root
Standard curve is drawn according to standard items, SFTPB contents in sample are calculated.
Step 5：Pro-SFTPB content detections in sample
With Full-automatic chemiluminescence immunoassay analysis meter (NPD projects are biological, Maglumi 2000plus) for detection instrument, side
Science of law pattern is the pro-SFTPB monoclonal antibodies coating that double antibody sandwich method, i.e. instrument sequentially add 50 μ L sample, 50 μ L
Carboxylated magnetic particle and the 50 μ L coated acridinium esters of pro-SFTPB, reaction 20min after, carry out Magneto separate, instrument will
Reactant mixture sends into darkroom, sequentially adds luminous substrate A liquid (H2O2) and B liquid (NaOH) carries out luminescence-producing reaction, finally record
Luminous value.Standard curve is drawn according to standard items, pro-SFTPB contents in sample are calculated.
The above-mentioned factor is combined
This research is entered using Receiver Operating Characteristics (receiver operator characteristic, ROC) curve
Row is evaluated.ROC curve is a kind of important tool for evaluating mark.Available two leading indicators include：It is sensitive
It is True Positive Rate to spend (sensitivity), evaluates its performance in selection specified disease patient, general to require there is high sensitivity
To exclude the people without disease.Specific (specifictiy) is Kidney-Yin dangerous rate, indicates that it is correctly selecting the people without disease
Ability.It is general in diagnosis to require there is high specific to obtain relatively low false positive rate.These factors are carried out with ROC respectively bent
Line analysis, wherein ordinate are sensitivity, and abscissa is 1- specificity.
Utilize mathematical modeling p=p (E)=eb0+b1x1+b2x2+b2x3+…+bkxk/(1+eb0+b1x1+b2x2+b2x3+…+bkxk(the 1+e of)=1/(b0+b1x1+b2x2+b2x3+…+bkxk)) calculating is combined to the above-mentioned factor.
The data that doctor can be drawn using combination, with reference to other clinical diagnostic methods, the ill shape of comprehensive descision subject
Condition.
Claims (10)
1. a kind of biomarker, it is characterised in that the label is SFTPB, pro-SFTPB, N1, N12- diacetyl spermine
It is any or its combination.
2. blood plasma biomarker according to claim 1, it is characterised in that SFTPB schematic diagram is：
3. biomarker according to claim 1, it is characterised in that SFTPB fragment can be SEQ ID1, SEQ
ID2, SEQ ID3, SEQ ID4, SEQ ID5, SEQ ID6, SEQ ID7 or SEQ ID8：
SEQ ID1：LSEQQFPIPLPYCWLCRALIKRIQA；
SEQ ID2：PKGALAVAVAQVCRVVPLVAGGICQCLAERYSVILLDT；
SEQ ID3：LGRMLPQLVCRLVLRC；
SEQ ID4：RDSECHLCMSVT；
SEQ ID5：EQAIPQAMLQACVGS；
SEQ ID6：KCKQFVEQHTPQLLTLVPR；
SEQ ID7：AHTTCQALGVCGT；
SEQ ID8：SSPLQCI.
4. biomarker according to claim 1 or 2, it is characterised in that pro-SFTPB fragment is SEQ ID9,
SEQ ID10, SEQ ID11, SEQ ID12, SEQ ID13 or SEQ ID14：
SEQ ID9：AESHLLQWLLLLLPTLCGP；
SEQ ID10：TTSSLACAQ；
SEQ ID11：PEFWCQSLEQALQCRALGHCLQEVWGHVGADDLCQECEDIVHIL；
SEQ ID12：LEQECNVLPLKLLMPQCNQVLDDYFPLVIDY；
SEQ ID13：GICMHLGLCKS；
SEQ ID14：DPLPKPLRDPLPDPLLDKLVLPVLPGALQAR.
5. the method for sampling of the biomarker described in claim 1, it is characterised in that when label is SFTPB and/or pro-
During SFTPB, blood plasma is carried out using ELISA method, chemoluminescence method, protein chip, immunoturbidimetry or immunochromatographic method and included
The detection of amount level.
6. the method for sampling of the biomarker described in claim 1, it is characterised in that when label is N1, N12- diacetyl essence
Amine (N1, N12- diacetylspermine) when, using UHPLC-MS/MS, ELISA and chemoluminescence method, chip method, immune ratio
Turbid method or immunochromatographic method carry out the detection of contents level in blood plasma.
7. the modeling method of the method for sampling described in claim 5 or 6, it is characterised in that determined using mathematical modeling described
The combination of blood plasma biomarker；The mathematical modeling is：
P=p (E)=eb0+b1x1+b2x2+b2x3+…+bkxk/(1+eb0+b1x1+b2x2+b2x3+…+bkxk)
=1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))
Wherein, p is probability；E is the individual of analysis；For analysis individual positive probability occurs for p (E)；E is natural number, is approximately equal to
2.718281828459；B0 is regression coefficient；Bk is the coefficient of k-th of factor；K span is 1 to n integer.
8. the purposes of the modeling method described in claim 7, it is characterised in that build lung cancer using blood plasma biomarker and assess
Model, its step includes：
Step (1)：Collect the plasma sample of object to be checked；
Step (2)：SFTPB, pro-SFTPB, N in plasma sample are detected respectively1, N12The content of-diacetyl spermine；
Step (3)：The data measured with step (2) carry out Mathematical Models, determine that blood plasma biomarker is combined；Wherein,
Mathematical modeling is p==1/ (1+e(b0+b1x1+b2x2+b2x3+…+bkxk))。
9. the purposes of modeling method according to claim 7, it is characterised in that mathematical modeling is the combination of biomarker
Level changes, and the p value sensitivity after combination improves 70-90%, and accuracy improves 70-90%.
10. the purposes of modeling method according to claim 7, it is characterised in that this detection method and mathematical modeling are available
In the detection and assessment of lung cancer.
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| CN108226483A (en) * | 2018-01-19 | 2018-06-29 | 辽宁迈迪生物科技股份有限公司 | A kind of DAS detection kits and its preparation method and application |
| CN108226483B (en) * | 2018-01-19 | 2020-06-26 | 辽宁迈迪生物科技股份有限公司 | DAS detection kit and preparation method and application thereof |
| CN111667918A (en) * | 2020-05-29 | 2020-09-15 | 杭州广科安德生物科技有限公司 | Method for constructing mathematical model for in vitro detection of lung cancer and application |
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