CN104677999A - Biomarker for recognizing liver cancer and lung cancer through plasma - Google Patents
Biomarker for recognizing liver cancer and lung cancer through plasma Download PDFInfo
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- CN104677999A CN104677999A CN201310625538.7A CN201310625538A CN104677999A CN 104677999 A CN104677999 A CN 104677999A CN 201310625538 A CN201310625538 A CN 201310625538A CN 104677999 A CN104677999 A CN 104677999A
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Abstract
The invention relates to a novel biomarker for recognizing liver cancer and lung cancer. The novel biomarker is sensitive to pathological alteration of different cancers, and in particular sensitive in the early stage of the diseases or injury. According to the invention, a UHPLC-MS/MS (Ultra High Pressure Liquid Chromatography--Tandem Mass Spectrometry) method is carried out to respectively detect plasma samples of a patient with lung cancer and a patient with liver cancer to obtain detection data; the detected data of plasma polyamine substances is subjected to outlier detection to remove abnormal samples caused by sample collection, detection error or the like; the patient with lung cancer and the patient with liver cancer are assigned with numbers 1 and 2 as dependent variables, and 14 substances to be detected are used as independent variables to perform binary logical regression analysis to obtain the product of concentration of variables, putrescine and spermidine, as the basis for recognizing liver cancer and lung cancer; the variables are used as the indexes for verifying by a clustering analyzing method. The biomarker is sensitive to pathological alteration of cancers and can be applied to diagnosis, prognosis and treatment effect evaluation of cancers.
Description
Technical field
The present invention relates to the neoformation label for distinguishing liver cancer and lung cancer, described biomarker is more responsive to pathological change in various cancers, particularly at i or I commitment.
Background technology
Metabolism group (metabonomics/metabolomics) is the new subject of of proposing after genomics, transcription group, proteomics, is an important component part in systems biology.Living organism is when being subject to the stimulation of extraneous pathogeny or environment change, can produce multi-level, the responsing reaction of multiple organ and many tissues, the change that these responsing reactions have influence on terminal metaboilic level is the most at last studied this body just and is being subject to internal cause, during the stimulation of external cause, the science of the Changing Pattern of the terminal small molecule metabolites (be often referred to molecular weight and be less than 1000Da) in body, dynamically set forth the change procedure of response that physiology or pathological state lower body stimulate to external world and dynamic system on the whole, for the mechanism research of the cancer under complete understanding multifactor impact will provide a new visual angle.Meanwhile, investigate the physiological status of human body on the whole, more disease association information can be obtained, be conducive to the early diagnosis of cancer.In addition, sample mainly urine and the blood that metabonomic technology is analyzed, its gatherer process is to human body fanout free region or have minimum injury, and therefore the method for metabolism group will be conducive to the extensive examination of cancer.
Cancer is the general designation of a large class malignant tumour.The feature of cancer cell be unrestrictedly, hyperplasia without end, the nutriment in patient body is consumed in a large number; Cancer cell discharges multiple toxin, makes human body produce series of symptoms; Cancer cell also can be transferred to whole body growth and breeding everywhere, cause human body to be become thin, unable, anaemia, poor appetite, heating and serious organ function impaired etc., then the 26S Proteasome Structure and Function of disorganize, organ, cause downright bad hemorrhage concurrent infection, patient is finally dead due to organ failure.
Lung cancer is modal lung primary malignant tumor, be no matter the whole world or domestic in view of, lung cancer all accounts for first of tumour, and its incidence of disease and case fatality rate rise all rapidly, dies from lung cancer in the male patient of carninomatosis and ranks first.The M & M of lung cancer is rising year by year, and world's year number of patients is about 900,000, and in nearly 10 years of China, Cancer Mortality and mortality ratio increase the rapidest with lung cancer, and lung cancer has become the first cancer of China.Lung cancer is the tumour that recurrence and metastatic rate is high, even if carry out radical excision, also how dead because of postoperative recurrence transfer.The disease of lung cancer is sent out and bad habits and customs, and environmental factor and atmospheric pollution etc. are closely related.Lung cancer growth speed is slow, and the early diagnosis that the course of disease is longer and treatment can bring the chance of healing to a great extent to patient, in the diagnosis of lung cancer, the detection of tumor marker just seems very important.
According to statistics, liver cancer patient about 600,000 is newly sent out in the whole world every year, occupies the 5th of malignant tumour.Primary carcinoma of liver can be divided into Hepatocellular carcinoma, intrahepatic cholangiocarcinoma and mixed carcinoma of liver by cell typing.Nodular type, massive type and diffuse type can be divided into by the form of tumour.Primary carcinoma of liver belongs to high morbidity in China, and the general male sex is more than women.China is hepatitis B big country, and the liver cancer of China is many to be developed on the basis of hbv-liver cirrhosis, and the third hepatopathy people is also increasing gradually, also can develop into liver cancer after hepatitis B.Current China number of the infected accounts for the more than half of the whole world, accounts for 55% of global hepatocarcinoma patient, has become a large killer of serious threat our people health and lives.Because its grade of malignancy is high, disease progression fast, it is uncomfortable what patient generally do not have in early days, once occur that symptom is gone to a doctor, often belongs to middle and advanced stage.Therefore treatment difficulty is large, weak curative effect, after morbidity, life span is short.In China, have 110,000 people to die from liver cancer every year, wherein the male sex 80,000, women 30,000, account for 45% of whole world PLC mortality number.As modal a kind of malignant tumour, mortality of liver cancer only occupies high, and the whole nation has at least 120,000 people to be devitalized by liver cancer every year.
Liver cancer onset is hidden, and the liver cancer patient more than 60% enters middle and advanced stage when first going to a doctor, thus loses the chance of radical treatment, and within overall 5 years, survival rate only has about 7%.At present, diagnosing cancer of liver label conventional is clinically alpha-fetoprotein, is called for short AFP.But the susceptibility of alpha-fetoprotein diagnosing liver cancer and specificity are not very good, also may raise in the crowds such as gravid woman, acute, chronic hepatitis, gonad tumour and gastroenteric tumor.And, there is the liver cancer patient alpha-fetoprotein of 40% not raise clinically, present feminine gender.Therefore, find the molecular marked compound of specificity and all high diagnosing liver cancer, the particularly early liver cancer of susceptibility, become the Focal point and difficult point of clinical research.
More as the research of cancer diagnosis means using the single or conbined usage of large molecule cancer markers clinically at present, and the deep research of system is lacked for Small molecular class cancer markers.Modern study shows, the generation of endogenous small-molecule substance polyamine compounds and cancer is closely related.Domestic and international research all shows that in cancer patients, polyamine level is higher than polyamine level in normal population and non-tumor patient body.Polyamines is mostly is in vivo water miscible, usually completely protonated under body fluid pH environment, exists with polycation form, and the polyanionic complexs such as normal and RNA, DNA combine, and are among mobile equilibrium.Polyamines can participate in the multiple physiological activity in biosome directly, and as the copying of nucleic acid, transcribe, translate, the synthesis of protein and the stable of membrane structure etc., it is the necessary trace endogenous active substance of biology growing and cellular metabolism.Generally before RNA, DNA and protein synthesis increase, polyamine content sharply rises, and as in cancer tissue, polyamine level obviously raises, and the excessive accumulation of polyamines also can cause apoptosis and the transformation of cell, and cancer is worsened further.Polyamines comprises 1,3-propane diamine, putrescine, cadaverine, spermidine, spermine, acidylate spermidine, acidylate spermine etc.Polyamines comprises 1,3-propane diamine, putrescine, cadaverine, spermidine, spermine, acidylate spermidine, acidylate spermine etc.Current research fails to confirm which kind of polyamines is the biomarker of cancer, does not also find the specific biomarkers distinguishing various cancers.This have impact on the application of polyamines as aspects such as aided Cancer Diagnosis, prognosis and index of assessment of curative effects greatly.
Summary of the invention
The object of this invention is to provide the specificity polyamines biomarker for liver cancer and lung cancer parting, this label is putrescine in human plasma and spermidine, more precisely both product.
The present invention adopts UHPLC-MS/MS method respectively in detection of lung cancer patient and liver cancer patient plasma sample 1; 3-propane diamine (DAP); putrescine (PUT); cadaverine (CAD); spermidine (SPD); spermine (SPM); agmatine (AGM); ornithine (ORN), lysine (LYS), arginine (ARG); SAM (SAM); acidylate putrescine (NPUT), acidylate spermine (NSPM), acidylate spermidine (NSPD) and γ-aminobutyric acid (GABA) etc. 14 kinds and the closely-related material of polyamines composition and decomposition metabolism.(refer to Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography-tandem mass spectrometry method:Application to identify potential markers for human hepatic cancer.Anal Chim Acta.2013; 791:36-45) for the detection data obtained, first rejecting outliers is carried out to the data of measured blood plasma polyamines, remove the exceptional sample because sample collection or error at measurment etc. cause.And then be 1 and 2 as dependent variable to lung cancer and liver cancer patient assignment respectively, carry out binary logical regression analysis with 14 kinds of determinands for independent variable, the variable putrescine obtained and the product of spermidine concentration can as the foundations distinguishing lung cancer and liver cancer.Finally verify with the method for this variable for index employing cluster analysis.Result shows with the product of putrescine and spermidine concentration for patients with lung cancer and liver cancer patient can make a distinction by index.
Accompanying drawing explanation
Figure mono-is in embodiment 3, the Cluster tendency being index with putrescine in patients with lung cancer and liver cancer patient blood plasma and spermidine concentration product after removing exceptional value.Result shows with the product of putrescine and spermidine concentration for patients with lung cancer and liver cancer patient can make a distinction by index.(in figure, GP01-GP50 is liver cancer patient data, and wherein exceptional value eliminates GP05, GP32, GP40, GP41 and GP45; FP01-FP50 is patients with lung cancer data, and wherein exceptional value eliminates FP02, FP26 and FP30)
Embodiment
Embodiment 1 lung cancer and liver cancer plasma sample collection method
Collect through pathocytology turn out to be patients with lung cancer 50 example, wherein the male sex 30 example, women 20 example, age 27-77 year, average 53 years old.Liver cancer patient 50 example, wherein the male sex 30 example, women 20 example, age 45-77 year, average 57 years old.Gather patients with lung cancer and liver cancer patient venous blood, should immediately in 3500rpm centrifugal 10 minutes after blood sample collection, separated plasma, isolated plasma sample is placed in the test tube marked, lower than-80 DEG C of freezen protective in ultra low temperature freezer.
Get plasma sample 250 μ L in EP pipe, add the methyl alcohol that 50 μ L add blood: water (20:80, v:v) mixed solution and 50 μ L solution inner mark solutions (1,6-hexane diamine, 100ng/mL), vortex 30s.Add the centrifugal 3min of vortex 5min, 15000r/min after the methanol solution containing 0.1% acetic acid, get supernatant and flow down in air and dry up, redissolve containing 0.05% hyptafluorobutyric acid aqueous solution (20:80, v/v) containing 0.05% hyptafluorobutyric acid Jia Chun Rong Ye – with 50 μ L.Get 5 μ L supernatants to analyze for UHPLC-MS/MS.
The detection method of polyamines in embodiment 2 lung cancer and liver cancer plasma sample
The 5 μ L supernatants obtained to embodiment 1 carry out UHPLC-MS/MS analysis, and testing conditions is as follows:
(1) reagent
Standard substance 1,3-propane diamine, putrescine; cadaverine hydrochloride salt, spermidine hydrochloride, spermine hydrochloride; agmatine sulfate, acidylate putrescine hydrochloride, acidylate spermidine hydrochloride; acidylate spermine hydrochloride; ornithine hydrochloride, lysine, arginine; SAM, hyptafluorobutyric acid is Sigma Products.HPLC level methyl alcohol is Fisher Products, and it is pure that other chemical reagent is only analysis, and water is deionization redistilled water.
(2) instrument
The supper-fast high score of Prominence UFLC XR type is furnished with LC-20AD infusion pump, SIL-20AC automatic sampler, CTO-20AC column oven and DGU-20A3 degasser (Japanese Shimadzu Corporation) from liquid chromatograph; API4000 type triple quadrupole bar tandem mass spectrometer (American AB SCIEX company) and Analyst1.5.1 data handling system; AB135-S electronic balance (plum Teller-Tuo benefit Instrument Ltd. of Switzerland); Ultrasonic generator (Xiangshan of Zhejiang Province Shi Puhaitian Electronic Instruments Plant); Liquid flash mixer (Instrument Factory, Shanghai Medical Science Univ.); HC-2516 supercentrifuge (Keda Innovation Co., Ltd).
(3) chromatographic condition
Adopt Shimadzu Corporation XRLC-20AD Prominence
tMuFLC series instrument, chromatographic column: Shim-pack XR-ODS chromatographic column (75mm × 3.0mm, 2.2 μm), mobile phase: with 0.05% hyptafluorobutyric acid aqueous solution (A)-0.05% hyptafluorobutyric acid methyl alcohol (B) gradient elution, gradient elution program is as follows: 0.01-2.00min, 20%B; 2.01-4.00min, 20%B → 50%B; 4.01 – 6.00min, 50%B; 6.01 – 9.00min, 20%B.Flow velocity: 0.4mL/min; Column temperature: 40 DEG C; Sample size: 5 μ L.
(4) Mass Spectrometry Conditions
Adopt AB company QTRAPTM4000MS/MS series mass spectrometer.Ion gun: ESI; Detecting pattern: positive ion mode; Scan pattern: many reactive ions monitoring (MRM).Source temperature 500 DEG C; Gas curtain gas (nitrogen) pressure 138kPa (20psi); Atomization gas pressure 276kPa (40psi); Turbine gas pressure 276kPa (40psi); Ion spray voltage 5500V; As shown in Table 1, sweep time is 200ms for each determinand source parameters and quantitative analysis method.Measure the content of each determinand in Polyamine Metabolism profile in 50 routine patients with lung cancer and 50 routine human normal plasmas with set up method, each sample test once.According to typical curve, calculate each testing concentration in plasma sample.(data see attached list two)
Table one Polyamine Metabolism thing source parameters and quantitative analysis method
Polyamine Metabolism concentration profile situation in table two 50 patients with lung cancer and 50 liver cancer patient blood plasma
The mathematical statistics method of embodiment 3 liver cancer and lung cancer sample
First carry out test of outlier to the data that embodiment 2 records, adopt Han Peier method of inspection to analyze, its concrete steps are:
1. calculate the average (M of whole data set
e), this data set is divided into height two parts by this average.
2. according to the mean value computation r of data centralization all elements
i
R
i=(x
i-M
e) be the simple data of data centralization,
X belongs to this data set of 1--n, and n is the quantity of data centralization element, M
efor average
Calculation deviation M
e|ri|average, according to condition | r
i|>=4.5M
e|ri|
When above condition is set up, namely the numerical value of this data centralization be regarded as exceptional value.
3 routine lung cancer data and 5 routine liver cancer data are removed.And then respectively to lung cancer urine and liver cancer plasma data assignment 1 and 2 as dependent variable, 14 polyamines determinands carry out binary logical regression analysis as independent variable.Result obtains independent variable putrescine and spermidine in regression equation, and its regression coefficient Sig value is all less than 0.001(and refers to table three).Related coefficient according to putrescine and spermidine is 0.859, is expressed as positive correlation (referring to table four).Therefore product is done to the concentration of putrescine and spermidine, application SPSS software (version 19.0) carries out hierarchial-cluster analysis to the situation of putrescine and spermidine concentration product in patients with lung cancer and liver cancer patient blood plasma, employing sum of squares of deviations method (Ward ' s method), using Chebyshev's law (chebychev) estimating as sample similarity.92 plasma samples are divided into 2 classes by cluster analysis, and wherein patients with lung cancer is divided into one group, and liver cancer patient is divided into another group.Prove to adopt the product of putrescine and spermidine concentration in blood plasma can distinguish patients with lung cancer and liver cancer patient.(referring to figure mono-)
Table three patients with lung cancer and liver cancer patient binary logical regression analysis result
Variables in the Equation
a.Variable(s)entered on step1:PUT.
b.Variable(s)entered on step2:SPD.
c.Variable(s)entered on step3:NSPM.
d.Variable(s)entered on step4:GABA.
Table four patients with lung cancer and liver cancer patient binary logical regression analysis result
Correlation Matrix
Claims (4)
1. in human plasma putrescine and spermidine as the application distinguishing lung cancer and diagnosing cancer of liver label.
2. apply as claimed in claim 1, it is characterized in that the application of the product of putrescine and spermidine concentration as pulmonary cancer diagnosis label.
3. the product applying putrescine and spermidine concentration carries out the method distinguishing lung cancer and liver cancer, and its step comprises:
(1) blood plasma of known patients with lung cancer and liver cancer patient is collected respectively;
(2) putrescine respectively in detection of lung cancer patient and liver cancer patient blood plasma and the concentration of spermidine;
(3) putrescine recorded by step (2) and the product of spermidine concentration carry out cluster analysis.
4. method as claimed in claim 3, is characterized in that cluster analysis is the System Cluster Analysis in SPSS software (version number 19.0).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPWO2018079840A1 (en) * | 2016-10-31 | 2019-09-19 | 株式会社Ｐｒｅｆｅｒｒｅｄ Ｎｅｔｗｏｒｋｓ | Disease determination apparatus, disease determination method, and disease determination program |
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| CN102426210A (en) * | 2011-11-22 | 2012-04-25 | 沈阳药科大学 | Detection method of polyamine substances in blood plasma |
| CN102495148A (en) * | 2011-11-22 | 2012-06-13 | 沈阳药科大学 | Method for determining polyamines in urine |
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| CN102426210A (en) * | 2011-11-22 | 2012-04-25 | 沈阳药科大学 | Detection method of polyamine substances in blood plasma |
| CN102495148A (en) * | 2011-11-22 | 2012-06-13 | 沈阳药科大学 | Method for determining polyamines in urine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPWO2018079840A1 (en) * | 2016-10-31 | 2019-09-19 | 株式会社Ｐｒｅｆｅｒｒｅｄ Ｎｅｔｗｏｒｋｓ | Disease determination apparatus, disease determination method, and disease determination program |
| JP2022024092A (en) * | 2016-10-31 | 2022-02-08 | 株式会社Preferred Networks | Disease affection determination device, disease affection determination method and disease affection determination program |
| JP7021097B2 (en) | 2016-10-31 | 2022-02-16 | 株式会社Preferred Networks | Disease morbidity determination device, disease morbidity determination method and disease morbidity determination program |
| JP7411619B2 (en) | 2016-10-31 | 2024-01-11 | 株式会社Preferred Networks | Disease prevalence determination device, disease prevalence determination method, and disease prevalence determination program |
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