CN104678003A - Biomarker for evaluating liver cancer disease by using urine - Google Patents
Biomarker for evaluating liver cancer disease by using urine Download PDFInfo
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- CN104678003A CN104678003A CN201410090856.2A CN201410090856A CN104678003A CN 104678003 A CN104678003 A CN 104678003A CN 201410090856 A CN201410090856 A CN 201410090856A CN 104678003 A CN104678003 A CN 104678003A
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- putrescine
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Abstract
The invention relates to a new biomarker for evaluating liver cancer disease and provides application of putrescine and acylated putrescine in human urine as diagnostic markers of liver cancer. The invention relates to application of concentration ratio of putrescine to acylated putrescine as the diagnostic marker of the liver cancer. The invention relates to a method applying the concentration ratio of putrescine to acylated putrescine for evaluating the lung cancer. The method comprises the following steps: (1) respectively collecting urine samples from a patient with the liver cancer and a person to be evaluated; (2) respectively detecting the concentration of putrescine and acylated putrescine in the urine samples in the patient with the lung cancer and the person to be evaluated; and (3) performing clustering analysis by using the concentration ratio of putrescine to acylated putrescine, which is measured in the step (2). The clustering analysis is a systemic clustering analysis method of SPSS (statistical product and service solutions software) software (version number: 19.0). The new biomarker can be used for diagnosis, prognosis and curative effect evaluation of the cancer.
Description
Technical field
The present invention relates to the neoformation label for assessment of hepatic cancer disease, described biomarker is more responsive to pathological change in liver cancer, particularly at i or I commitment.In addition, the present invention relates to the risk suffering from liver cancer for assessment of people.
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.
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.Current research fails to confirm which kind of polyamines is the biomarker of cancer, and also fail to find the specific marker thing of liver cancer, this have impact on the application of polyamines as aspects such as lung cancer auxiliary 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 of liver cancer, this label is putrescine and acidylate putrescine in human urine, is specially the ratio of putrescine and acidylate Putrescine concentration.
The present invention adopts UHPLC-MS/MS method to detect in Healthy People and cancer patient urine specimen 1 respectively; 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), the concentration of 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 polyamines in urine, remove the exceptional sample because sample collection or error at measurment etc. cause.And then be 1 and 2 as dependent variable to liver cancer patient and Healthy People assignment respectively, carry out binary logical regression analysis with 14 kinds of determinands for independent variable, the variable putrescine obtained and acidylate Putrescine concentration can as the foundations distinguishing liver cancer and Healthy People.Finally verify with the method for this variable for index employing cluster analysis.Result shows with the ratio of putrescine and acidylate Putrescine concentration for liver cancer patient and Healthy People can make a distinction by index.
Object of the present invention also comprises provides putrescine and acidylate Putrescine concentration ratio in application urine to carry out the method for liver cancer assessment, and its step comprises:
(1) urine specimen of liver cancer patient and people to be assessed is collected respectively;
(2) concentration of putrescine and acidylate putrescine in liver cancer patient and human urine sample to be assessed is detected respectively;
(3) putrescine recorded by step (2) and the ratio of acidylate Putrescine concentration carry out cluster analysis.
If finally can gather is a class, then can assesses people to be measured and suffer from liver cancer and have a big risk.System Cluster Analysis in the preferred SPSS software of clustering method (version 19.0).
Accompanying drawing explanation
Fig. 1 is after removing exceptional value data in embodiment 3, with liver cancer patient putrescine and acidylate putrescine in Healthy People urine
Concentration proportion is the Cluster tendency of index.Result shows
Liver cancer patient and Healthy People can make a distinction by index.(in figure, HU01-HU50 is Healthy People data,
Wherein exceptional value eliminates HU14, HU35, HU43, HU44 and HU45; GU01-GU50 is liver
Cancer patient data, wherein exceptional value eliminates GU05, GU30, GU39, GU40, GU45 and GU50).
Embodiment
Embodiment 1 liver cancer and Healthy People urine sample collection method
Collect through pathocytology turn out to be liver cancer patient 50 example, wherein the male sex 30 example, women 20 example, age 45-77 year, average 57 years old.Control group 50 example, is normal adults, adopts routine blood test, and the routine inspection such as routine urinalysis confirms, wherein the male sex 30 example, women 20 example, age 27-65 year, average 52 years old.Gather liver cancer patient and normal person's urina sanguinis.After A urine sample collection device in ultra low temperature freezer lower than-80 DEG C of freezen protective.
Get urine 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.
Embodiment 2 liver cancer and Healthy People polyamines in urine detection method
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 ion 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 liver cancer patients with set up method, each sample test once.According to typical curve, calculate each testing concentration (referring to table two) in urine sample.
Table one Polyamine Metabolism thing source parameters and quantitative analysis method
Polyamine Metabolism concentration profile situation in table two 50 liver cancer patients and 50 Healthy People urines.
The mathematical statistics method of embodiment 3 liver cancer and Healthy People 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:
Average (the M of the whole data set of 1 calculating
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 | ri|>=4.5M
e|ri|
When above condition is set up, namely the numerical value of this data centralization be regarded as exceptional value.
6 routine liver cancer data and 5 routine Healthy People data are removed.And then respectively to liver cancer urine and Healthy People urine 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 acidylate putrescine in regression equation, and its regression coefficient Sig value is all less than 0.002(and refers to table three).Related coefficient according to putrescine and acidylate putrescine is-0.778, is expressed as negative correlation (referring to table four).Therefore ratio is done to the concentration of putrescine and acidylate putrescine; application SPSS software (version 19.0) carries out hierarchial-cluster analysis to the situation of putrescine and acidylate Putrescine concentration ratio in liver cancer patient and healthy population blood plasma; employing sum of squares of deviations method (Ward ' s method), using Chebyshev's law (chebychev) estimating as sample similarity.89 urine specimens are divided into 2 classes by cluster analysis, and wherein liver cancer patient is divided into one group, and healthy population is divided into another group.Prove to adopt the ratio of putrescine and acidylate Putrescine concentration in urine can distinguish liver cancer patient and Healthy People.(referring to Fig. 1)
Table three liver cancer patient and Healthy People binary logical regression analysis result
Variables in the Equation
a.Variable(s)entered on step1:NPUT.
b.Variable(s)entered on step2:PUT.
c.Variable(s)entered on step3:SPD.
d.Variable(s)entered on step4:GABA.
Table four liver cancer patient and Healthy People binary logical regression analysis result
Correlation Matrix
Claims (4)
1. in human urine putrescine and acidylate putrescine as the application of diagnosing cancer of liver label.
2. apply as claimed in claim 1, it is characterized in that the application of the ratio of putrescine and acidylate Putrescine concentration as diagnosing cancer of liver label.
3. the ratio applying putrescine and acidylate Putrescine concentration carries out a method for liver cancer assessment, and its step comprises:
(1) urine specimen of known liver cancer patient and people to be assessed is collected respectively;
(2) concentration of putrescine and acidylate putrescine in liver cancer patient and human urine sample to be assessed is detected respectively;
(3) putrescine recorded by step (2) and the ratio of acidylate Putrescine 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108333275A (en) * | 2018-04-24 | 2018-07-27 | 许昌学院 | A kind of method that LC-MS/MS measures the putrescine in soy sauce |
| CN108922630A (en) * | 2018-06-28 | 2018-11-30 | 上海森亿医疗科技有限公司 | A kind of method and device pushing knowledge |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333275A (en) * | 2018-04-24 | 2018-07-27 | 许昌学院 | A kind of method that LC-MS/MS measures the putrescine in soy sauce |
| CN108922630A (en) * | 2018-06-28 | 2018-11-30 | 上海森亿医疗科技有限公司 | A kind of method and device pushing knowledge |
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