CN101363837A - Method for estimating curative effect of pancreatic cancer chemotherapy medicine - Google Patents
Method for estimating curative effect of pancreatic cancer chemotherapy medicine Download PDFInfo
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Abstract
The invention provides a method for evaluating the therapeutic effect of a chemotherapeutic drug for treating pancreatic cancer. The composition difference of serum protein is analyzed by utilizing the surface-enhancement laser desorption ionization-flight time mass-spectrometric technique, and a serum protein finger print of pancreatic cancer is established. The tumor totem of pancreatic cancer with high diagnostic efficiency is screened by utilizing the SELDI-TOF-MS technique and the bioinformatics method, and a mass spectrometric model is formed by the serum protein with two mass/charge ratios at 3879Da and 4772Da. The method is objective and effective for evaluating the curative treatment and prognoses of pancreatic cancer, has high sensitivity, specificity and accuracy, and can improve and modify the protein finger print technique. The method has a reasonable design, and is used for reducing the national fatality rate of pancreatic cancer, improving the curative ratio of pancreatic cancer and evaluating the therapeutic effect of drugs for treating pancreatic cancer.
Description
Technical field
The invention belongs to biotechnology, relate to a kind of serum that utilizes proteomics to detect cancer of pancreas chemotherapy model, thereby estimate the method for pancreatic cancer chemotherapy medicine curative effect.
Background technology
Cancer of pancreas is low with morbidity concealment, grade malignancy height, excision rate, the prognosis extreme difference is a feature, is one of the highest tumour of grade malignancy.Now occupy the 6th of cancer mortality in China, its 5 years survival rates only are 1%-4%.Surgical radical treatment is the unique method that definite curative effect is arranged of cancer of pancreas for the treatment of at present, the postoperative combined with chemotherapy, and its 5 years survival rates can reach 15%-40%.But the patient who has only 10%-15% clinically can obtain the chance of capable radical excision operation, 85% left and right sides Pancreas cancer patients when making a definite diagnosis because of occurring shifting etc. former thereby losing the chance of surgical radical treatment.Therefore chemotherapy has the effect of not replacing in the treatment of pancreatic cancer late.Gemcitabine is widely accepted as " goldstandard " for the treatment of the advanced pancreatic cancer chemotherapy.
At present, the effect assessment to the treatment cancer of pancreas remains in certain difficulty.Though the iconography technology can provide certain help, but pancreas is positioned at posterior peritoneum, add that postoperative pancreas bed changes, CT, MRI etc. often is difficult to accurately differentiate cancer of pancreas and normal structure boundary and objective measurement tumour size, and the DISTANT METASTASES IN kitchen range also is difficult to monitor fully in addition.Present clinical benefit curative effect (clinical benefit response, CBR) comprise that stomachache, physical situation, 3 measurement index trends of body weight replacement tumour size become main pancreatic cancer chemotherapy index, but still lack the objective susceptibility as tumor markers.The blood serum tumor markers of present commonplace application comprises CA19-9, CA50, CEA etc., though certain susceptibility and specificity are arranged, is used for early detection and diagnosis more.Up to now, do not find a kind of index that result of treatment and prognosis is had certain evaluation meaning as yet.
In the past few years, proteomics has become one of important means of detection and examination tumor marker.At present, novel mass-spectrometric technique is widely used in the research of tumor marker screening with advantages such as its high flux, hypersensitivity, sampling are convenient.It is a kind of new protein science research method that just grows up recent years that laser desorption ionisation-flight time mass spectrum technology is strengthened on the surface, and it can directly obtain protein profiling from undressed biological sample.With this technology is that the serial protein-chip of foundational development can be non-specific or specifically in conjunction with the range protein in the tested sample, when in mass spectrometer, being subjected to laser bombardment, various conjugated proteins can be excited and form the gasification ion, because the time that the ion of different mass-to-charge ratioes (m/z) flies in electric field is different in size, therefore receiving trap can show with position, strong and weak different peak intuitively according to the different of protein mass-to-charge ratio and the number of measuring, and then the formation collection of illustrative plates is used for analyzing.It is little that this method has amount of samples, easy and simple to handle, highly sensitive, and advantages such as high flux can detect fmol (10
-15Mol) trace of albumin of the order of magnitude, and thousands of protein datas in can a certain sample of disposable acquisition.
Summary of the invention
The purpose of this invention is to provide a kind of proteomics of utilizing and detect cancer of pancreas chemotherapy model serum, obtaining a kind of tumor markers combination of pointing out cancer of pancreas chemotherapy curative effect, for the drug therapy and the prognostic evaluation of cancer of pancreas provides objective effective method.Application surface of the present invention is strengthened the difference of the haemocyanin composition of laser desorption ionisation-flight time mass spectrum technical Analysis cancer of pancreas before and after the gemcitabine treatment, set up the serum protein fingerprint of pancreatic cancer chemotherapy, explore the tumor markers combination of prompting cancer of pancreas chemotherapy curative effect.Specifically realize by following steps:
(1) preparation blood serum sample: get the serum supernatant, centrifugal 10 minutes, get supernatant serum, in 10 μ lU9 serum treating fluids, add serum 5 μ L, vibration makes abundant mixing on the shaking table, serum after with Binding Buffer (binding buffer liquid) U9 being handled is diluted to 200 μ l and is used for sample, get and above-mentionedly handle and the serum 100 μ l that diluted are added on the CM10 type protein-chip through U9, chip and serum fully reacted after 1 hour removes blood serum sample, every hole adds the corresponding Binding Buffer of 200 μ l, 5 minutes (600rpm of vibration on the shaking table, room temperature) removes liquid after, repeat this process 2 times, water (purity HHPLC level) 200 each hole of μ l Rapid Cleaning 1 time, firmly dry and Bio-Processor (96 porin matter chip operation support) is tipped upside down on the thieving paper of cleaning and pat to remove unnecessary water, chip is taken out from Bio-processor, air dry, every hole point adds 50% saturated SPA (energy absorption molecule) solution 1 μ l, treat to repeat a little to add 1 time after its air dry, air dry can be gone up machine testing.
(2) utilize the surface to strengthen laser desorption ionisation-flight time mass spectrum technology (Surfance EnhancedLaser Desorption/Ionization Time of Flight Mass Spectrometry, SELDI-TOF-MS) serum sample of detection step (1), obtain the haemocyanin mass spectrum: mass spectrometer parameter setting: laser intensity 165, sensitivity 7, data aggregation scope 2,000 one 30000m/z (protein quality and electric charge ratio, be mass-to-charge ratio), collect before the data with standard protein chip calibration molecule amount at every turn.
(3) data aggregation and bioinformatic analysis: raw data is proofreaied and correct with Proteinchip Software3.0 software earlier, make total ionic strength adjustment buffer degree and molecular weight reach homogeneous, and filtering noise, initial noise filtering value 5, secondary noise filtering value 2 is that the minimum value of explaining is carried out cluster with 10%.After above-mentioned data pre-service, serum proteins mass spectrometric data (being finished by the Biomarker Wizard3.2.0 software that Proteinchip Software3.0 carries) is respectively organized in the Mann-Whitney rank test, seeks between each group and expresses discrepant protein peak.Each organizes SVM, the discriminatory analysis software of serum proteins mass spectrum data importing Matlab6.5 data processing software relatively.Select between two groups the most significant 10 the protein peak data of difference to analyze.Filter out the highest sum of ranks of Youden index and make up, set up model by stages with SVM leaving-one method and discriminatory analysis leaving-one method than the peak.Every group analysis sample is made as n, then randomly draws one 1 samples of n in all samples and is made as training set and is used to set up model, remains 1 sample and is made as test set and carries out cross validation.So sampling, training and proof procedure repeat n time, to obtain the result near actual value.The last result who exports SVM, original differentiation and the two cross validation simultaneously.And carry out statistical study with the SPSS13.0 statistical package, relatively the discriminatory analysis result of different proteins peak permutation and combination selects the optimal arrangement combination;
(4) result shows: whether two protein peak values that mass-to-charge ratio is respectively 3879Da, 4772Da raise accurately to judge through tumour after the chemotherapy whether dwindle, thereby estimate the curative effect of chemotherapeutic.
The present invention utilizes proteomics to detect cancer of pancreas chemotherapy model serum, to obtain a kind of tumor markers combination of pointing out cancer of pancreas chemotherapy curative effect, for the drug therapy and the prognostic evaluation of cancer of pancreas provides objective effective method, have higher sensitivity, specificity and accuracy.And can improve and the improved protein fingerprint pattern technology, reduce inspection charge, make it more appropriate to clinical practice.For the treatment evaluation of cancer of pancreas medicine provides the zoopery basis.
Description of drawings
Fig. 1 is that karyoplasmic ratio (m/z) is positioned at 3879. cancer of pancreas model group and the contrast of gemcitabine group nude mouse serum protein fingerprint.
Fig. 2 is that karyoplasmic ratio (m/z) is positioned at 4772. cancer of pancreas model group and the contrast of gemcitabine group nude mouse serum protein fingerprint.
Embodiment
The present invention will be described further in conjunction with specific embodiments, and these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.
Key instrument and reagent: CM10 type protein-chip
96 porin matter chip operation supports are Bio-proeessor
Analyze pure urea, sodium acetate, second eyeball, DTT and cHAPS buffer salt (sigma company)
Energy absorption molecule SPA,
Implementation step: get cancer of pancreas nude mice whole blood and gemcitabine group nude mice whole blood 1ml respectively, spend low-temperature centrifugations (4000 rev/mins) 10 minutes, get supernatant, centrifugal once more 10 minutes, get supernatant serum 4.Add serum 5 μ L in the 10 μ l U9 serum treating fluids, vibration makes abundant mixing on the shaking table.Serum after with Binding Buffer U9 being handled is diluted to 200 μ l and is used for sample.
Get and above-mentionedly handle and the serum 100 μ l that diluted are added on the chip through U9, chip and serum fully reacted after 1 hour removes sample.Every hole adds the corresponding Binding Buffer of 200 μ l, and vibration was removed liquid after (600rpm, room temperature) in 5 minutes on the shaking table.Repeat this process 2 times.Water (purity HPLC level) 200 each hole of μ l Rapid Cleaning 1 time firmly dry and Bio-Processor are tipped upside down on the thieving paper of cleaning and pat to remove unnecessary water.Chip is taken out air dry from Bio-processor.Every hole point adds 50% saturated SPA solution 1 μ l, treats to repeat a little to add 1 time after its air dry.Air dry can be gone up machine testing.
Machine testing on the embodiment 2:
Key instrument, software: PBS one II surface reinforcement laser desorption ionisation one time of-flight mass spectrometer (SELDI one TOF one MS),
Analysis software ProteinChipSoftware3.0 (U.S. Ciphergen company): support vector machine (supportveetor machines, SVM), discriminatory analysis (discriminant analysis) and time series analysis (time-sequence analysis) software utilization Matlab6.5 data processing software (MathworkS company) exploitation;
Implementation step: mass spectrometer parameter setting: laser intensity 165, sensitivity 7, data aggregation scope 2,000 one 30000m/z (protein quality and electric charge ratio, i.e. mass-to-charge ratio) collect before the data with standard protein chip calibration molecule amount at every turn.Utilize the surface to strengthen laser desorption ionisation-flight time mass spectrum technology for detection serum sample, obtain the haemocyanin mass spectrum.
Embodiment 3 data aggregations and bioinformatic analysis
Raw data is proofreaied and correct with Proteinchip Software3.0 software earlier, makes total ionic strength adjustment buffer degree and molecular weight reach homogeneous, and filtering noise, initial noise filtering value 5, and secondary noise filtering value 2 is that the minimum value of explaining is carried out cluster with 10%.After above-mentioned data pre-service, serum proteins mass spectrometric data (being finished by the Biomarker Wizard3.2.0 software that Proteinchip Software3.0 carries) is respectively organized in the Mann-Whitney rank test, seeks between each group and expresses discrepant protein peak.
Each organizes SVM, the discriminatory analysis software of serum proteins mass spectrum data importing Matlab6.5 data processing software relatively.Select between two groups the most significant 10 the protein peak data of difference to analyze.Filter out the highest sum of ranks of Youden index and make up, set up model by stages with SVM leaving-one method and discriminatory analysis leaving-one method than the peak.Every group analysis sample is made as n, then randomly draws one 1 samples of n in all samples and is made as training set and is used to set up model, remains 1 sample and is made as test set and carries out cross validation.So sampling, training and proof procedure repeat n time, to obtain the result near actual value.The last result who exports SVM, original differentiation and the two cross validation simultaneously.And carry out statistical study with the SPSS13.0 statistical package, relatively the discriminatory analysis result of different proteins peak permutation and combination selects the optimal arrangement combination.
Experimental result
The treatment of cancer of pancreas model group and gemcitabine group after 35 days the contrast of primary tumo(u)r quality see Table 1.
Table 1 cancer of pancreas model group and the treatment of gemcitabine group primary tumo(u)r quality contrast after 35 days
Single use gemcitabine lumbar injection, pancreatic tumour inhibiting rate be 50.14% (calculating as follows:
Tumor control rate %=(the average knurl quality of the average knurl quality/model group of 1-gemcitabine group) * 100%) and the metastases rate be starkly lower than the cancer of pancreas model group, verified the validity of gemcitabine to treatment of pancreatic cancer.
The result of 3 embodiment is referring to Fig. 1-2.Horizontal ordinate is protein mass-to-charge ratio (m/z) among the figure, and ordinate is relative peak (relative content).Fig. 1 shows that karyoplasmic ratio 3879Da albumen is high expressed in gemcitabine group nude mouse serum, what wherein (A) showed is that karyoplasmic ratio is arranged in the performance of the crest of 3879Da at cancer of pancreas model group serum, and what (B) show is that karyoplasmic ratio is arranged in the performance of the crest of 3879Da at gemcitabine group serum.Fig. 2 shows that karyoplasmic ratio 4772Da albumen is low and expresses in gemcitabine group nude mouse serum, what wherein (A) showed is that karyoplasmic ratio is arranged in the performance of the crest of 4772Da at cancer of pancreas model group serum, and what (B) show is that karyoplasmic ratio is arranged in the performance of the crest of 4772Da at gemcitabine group serum.
2 protein peaks (3879,4772m/z) combination is configured to the effective forecast model of chemotherapy (table 2-3).The susceptibility 100% (19/19) of forecast model discriminating model group and gemcitabine group nude mouse serum; Specificity 95.24% (20/21); Accuracy rate 97.5% (39/40).
2 protein peaks comparisons of table 2 model group and gemcitabine group (x ± s)
Table 3 forecast model is to the cross validation result (routine number) of gemcitabine group and cancer of pancreas model group
Annotate: susceptibility 100% (19/19); Specificity 95.24% (20/21); Accuracy rate 97.5% (39/40)
Sum up above each result of experiment, drawn to draw a conclusion: whether two protein peak values that mass-to-charge ratio is respectively 3879Da, 4772Da raise can be judged accurately that whether the experiment nude mice that suffers from cancer of pancreas dwindles through primary tumo(u)r after the chemotherapy, has higher sensitivity, specificity and accuracy.This just provides objective effective method and zoopery basis for the drug therapy and the prognostic evaluation of cancer of pancreas.
Claims (2)
1. method for estimating curative effect of pancreatic cancer chemotherapy medicine is characterized in that realizing by following steps:
(1) blood serum sample preparation: get supernatant serum and add in the U9 serum treating fluid, fully mix, again with the dilution of binding buffer liquid, getting the serum that diluted is added on the CM10 type protein-chip, fully blood serum sample is removed in the reaction back, and every hole adds binding buffer liquid, removes liquid after the vibration, water cleans, dry and remove unnecessary water, take out chip, air dry, every hole point adds the energy absorption molecular solution, can go up machine testing after the air dry;
(2) utilize the surface to strengthen the serum sample of laser desorption ionisation-flight time mass spectrum technology for detection step (1), carry out the data aggregation of haemocyanin mass spectrum, laser intensity 165, sensitivity 7, data aggregation scope 2000-30000m/z collects before the data with standard protein chip calibration molecule amount at every turn;
(3) data aggregation and bioinformatic analysis: the mass spectra model that the serum proteins that are positioned at 3879Da, 4772Da by two mass-to-charge ratioes are formed, estimate the curative effect of chemotherapeutic.
2. the detection method of a kind of blood serum tumor markers according to claim 1, it is characterized in that: the described bioinformatic analysis of step (3) is that raw data is proofreaied and correct with Proteinchip Software3.0 software, with 10% is that the minimum value of explaining is carried out cluster, the Mann-Whitney rank test is the serum proteins mass spectrometric data of chemotherapy front and back relatively, set up discrimination model, use the leaving-one method cross validation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103460050A (en) * | 2011-03-24 | 2013-12-18 | 学校法人庆应义塾 | Marker for determination of sensitivity to anticancer agent |
| CN112048401A (en) * | 2020-08-28 | 2020-12-08 | 上海符贝基因科技有限公司 | Micro-fluidic chip cleaning agent and method thereof |
| CN113189346A (en) * | 2021-04-27 | 2021-07-30 | 嘉兴学院 | Serum protein markers for detecting quality of pulse-activating injection and application thereof |
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2008
- 2008-09-02 CN CNA2008101206881A patent/CN101363837A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103460050A (en) * | 2011-03-24 | 2013-12-18 | 学校法人庆应义塾 | Marker for determination of sensitivity to anticancer agent |
| US9797884B2 (en) | 2011-03-24 | 2017-10-24 | Keio University | Marker for determination of sensitivity to anticancer agent |
| US10309957B2 (en) | 2011-03-24 | 2019-06-04 | Keio University | Method for determining sensitivity to fluorouracil in a subject having colorectal cancer |
| CN112048401A (en) * | 2020-08-28 | 2020-12-08 | 上海符贝基因科技有限公司 | Micro-fluidic chip cleaning agent and method thereof |
| CN113189346A (en) * | 2021-04-27 | 2021-07-30 | 嘉兴学院 | Serum protein markers for detecting quality of pulse-activating injection and application thereof |
| CN113189346B (en) * | 2021-04-27 | 2024-03-22 | 嘉兴学院 | Serum protein markers for detecting quality of Shengmai injection and application thereof |
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