CN102053159A - Related protein group with differential expression in early pancreatic cancer model and application thereof - Google Patents
Related protein group with differential expression in early pancreatic cancer model and application thereof Download PDFInfo
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Abstract
In the invention, differential expression of proteins in tissues of normal pancreas and the pancreas of a rat with pancreatic intraepithelial neoplasia and early pancreatic cancer can be respectively dynamically detected by establishing a 7,12-dimethyl benzophenanthrene (DMBA)-induced rat pancreatic intraepithelial neoplasia and pancreatic cancer model and applying the DIGE (difference gel electrophoresis) protein analysis technology, bioinformatics software is utilized for screening out the proteins which synchronously significantly vary with expression of cancerization degree of the pancreatic cancer, and the mass spectrometry technology is further utilized for performing identification so as to determine the specific proteins with the differential expression during the occurrence of the early pancreatic cancer. The invention discloses a related protein group with the differential expression in the pancreas tissues in an early pancreatic cancer model, the related proteins have strong specificity, and the related proteins can be utilized as protein markers for early diagnosis of the pancreatic cancer, thereby providing an effective method for clinical early diagnosis of the pancreatic cancer. A PMF (peptide mass fingerprinting) map of differential protein points can be searched in a database of National Center for Biotechnology Information (NCBI) by utilizing Mascot software.
Description
Technical field
The present invention relates to the Different Proteomics technology, more particularly, relate to the related protein and the application thereof of one group of early stage cancer of pancreas model differential expression.
Background technology
Cancer of pancreas has the onset concealment, shifts in early days, and the grade malignancy higher organism is learned characteristic, causes cancer of pancreas early diagnosis difficulty, and clinical therapeutic efficacy is poor.Set up comparatively ideal cancer of pancreas early diagnosis tumor marker, by normal or people at highest risk are carried out screening, the early detection doubtful case all has crucial meaning to the clinical early diagnosis level that improves cancer of pancreas and result of treatment etc.
Diagnostic method commonly used at present is also very low to the diagnosis of early stage cancer of pancreas, present method mainly comprises (1) iconography and endoscopy: computed tomography (computed tomography, CT) judging cancer of pancreas by stages, prediction excision rate aspect has certain superiority, but in the cancer of pancreas of diagnosis diameter less than 1cm, aspect accuracys such as the interior little metastasis of diagnosis lymph node infiltration metastasis and liver descend, along with the improvement of multi-detector CT performance, diagnosis efficiency increases in recent years.Magnetic resonance imaging (magnetic resonance imaging, MRI) in the diagnosis liver, be better than CT aspect the metastasis, but it is poor than CT that lymph node is soaked into diagnosis effect, and nuclear magnetic resonance ERCP (magnetic resonance cholangio-pancreatography MRCP) and nuclear magnetic resonance (NMR) vessel radiography can provide the pancreas bile duct unusually and the information of aspect such as vascular wall infiltration.(endoscopic ultrasound is the higher diagnostic method of a kind of potency ratio EUS) to EUS, can find less pancreatic neoplasm, can assess tumour size and the lymph node situation of getting involved more accurately than CT.Fine needle under the EUS guiding attracts biopsy can finish the pathological diagnosis of primary tumo(u)r, lymph node and DISTANT METASTASES IN kitchen range.Antidromicity ERCP (endoscopic cholangio-pancreatography ERCP) is a kind of wound property inspection method that has, the pathology situation that can reflect main pancreatic duct and branch thereof in detail, and can finish the pancreatic duct biopsy, cytobrush cytolgical examination, and pancreatic juice collection carrying out cytology and genetic test.Laparoscope and laparoscopy ultrasound can be found metastasis in the liver that the peritonaeum metastasis hidden and additive method fail to pinpoint a disease in diagnosis, but assessment is checked before at present art of recommending to be used for cancer of pancreas more.
(2) serum tumor marker: serum tumor marker has long development course, the at present clinical more kinds such as CA19-9, CA50, CA242, Span-1, MIC-1, Dupan-2 that have commonly used, but its sensitivity and specificity are all not very good, and be more unsatisfactory to the diagnosis effect of early stage cancer of pancreas.CA19-9 be up to now report to the highest mark of cancer of pancreas susceptibility, but early stage or tumour hour can be normal in cancer of pancreas.
(3) detection in Gene Mutation: the diagnosis that genomics develops into cancer of pancreas provides new means.The K-ras gene mutation is that the most frequent genetic mutation takes place cancer of pancreas, has higher K-ras gene mutation in the peripheral blood of Pancreas cancer patients, pancreatic juice, duodenal juice, ight soil and fine needle aspiration or the ductus pancreaticus brush inspection sample.The K-ras gene mutation also appears in the cancer of pancreas precancerous lesion such as DH, although reduced the specificity of diagnosis, for more early better diagnosis prediction provide may.Other detection in Gene Mutation of carrying out also have tumor suppressor gene P53, P16, Smad4 etc.
(4) protein label: in recent years along with the particularly rise of Study on Different Proteomics of proteomics, for the research of cancer of pancreas early diagnosis label provides a kind of new research method.
Is that the Separation of Proteins authenticate technology route of representative is classical proteomics core technology with the two-phase electrophoresis in conjunction with mass spectrum, protein dissimilar in the protein example can separate by the two-phase electrophoresis, its principle is first different and separate with isoelectric focusing by the isoelectric point of protein, second then separates with SDS-PAGE by the difference of molecular weight mutually, thereby the protein in the complicated protein mixture is separated on two dimensional surface.Mass-spectrometric technique is with fastest developing speed in the present proteomics research, one of technology of also most active and potentiality, and the PMF of its high flux ground mensuration protein by sensitivity differentiates the kind of protein, thereby obtains the qualitative data of protein.Therefore the subject matter of two-phase electrophoresis is that reproducibility of results is poor, and poor stability causes the real difference of compartment system sum of errors sample room effectively, requires a great deal of time and work is carried out repetition in the hope of obtaining quantitative result more accurately.Difference gel electrophoresis (DIGE) technology combines the method for multi-fluorescence labeled analysis on the basis of traditional two-phase electrophoretic techniques, the fluorescent dye that is used for protein labeling comprises three kinds, is respectively Cy2, Cy3 and Cy5.Similar on these fluorescent marker chemical constitutions, molecular weight is approaching, have identical activated group-NHS fat, can specific marker on the ε of lysine residue amino, because three kinds of dyestuffs all have a positive charge, molecular weight and electric charge mate, and the protein behind the mark should not have the change on the isoelectric point, it is minimum that change on the molecular weight also keeps substantially, and the molecular weight change that different fluorescent dye causes should be consistent.Because different fluorescence labeling samples has different excitation wavelengths, can write down non-interfering glue figure result by different optical filters.The multicolor fluorescence mark makes to separate and analyze a plurality of samples in same glue becomes possibility, effective like this systematic error of having avoided between different glue.
The DIGE technology has been introduced interior mark for the first time in the two-phase electrophoresis, the interior mark of DIGE is that all samples in the test is got mixed in equal amounts, separately with a kind of fluorescent dye (normally Cy2) mark, with all samples electrophoresis together, this means that the protein spots in all samples all can have corresponding interior mark, by interior mark can be easily that sample between the sample of different fluorochrome labels in the glue and glue is carried out normalization is quantitative, and the coupling between the different gel becomes very light.Tradition two-phase electrophoretic techniques can be respectively separated protein group on isoelectric point and molecular weight both direction, yet is on the quantitative accuracy and not fully up to expectations in the 3rd direction.In be marked on introducing in the two-phase electrophoresis, greatly improved result's quantitative accuracy, reliability and repeatability, effectively reduce systematic error, guaranteed the high confidence level of experimental result.
Utilize the DIGE technology in recent years, the researcher has carried out multinomial Study on Different Proteomics to cancer of pancreas, and discovery is fallen ill with cancer of pancreas to expect, transfer is relevant, or has the protein label of diagnostic value.After Yu KH etc. and Kakisaka T etc. utilize high performance liquid chromatography and immunoabsorption to remove the serum high-abundance proteins respectively, the serum differential protein group of utilized the comparative studies of DIGE connexus spectral technology normal person and Pancreas cancer patients has found that some express protein of significant difference in cancer of pancreas.In addition, the DIGE technology also is used to study the protein group in normal person and the ductal adenocarcinoma of pancreas patient pancreatic juice, finds and verified MMP-9, and DJ-1 and AlBG albumen have significant difference expresses.Cancer of pancreas has the advantages that early stage generation is shifted, there is research to utilize high metastatic and low metastatic pancreas cancer cell strain respectively, and lymphatic metastasis and no lymphatic metastasis cancer of pancreas tissue are arranged, utilize the discovery of DIGE connexus spectral technology some differential proteins may to attack metastasis closely related with cancer of pancreas.
The animal model that foundation meets the human pancreatic adenocarcinoma principal character is an important foundation of carrying out cancer of pancreas research in a deep going way, and these features should comprise the following aspects usually: (1) is the pancreatic duct origin of cell of pancreatic duct phenotype and supposition; (2) connective tissue reactive hyperplasia widely; (3) progression of disease can cause blocking of biliary tract and stomach; (4) with neural invasion and peritonaeum, the early stage aggressive of liver shifts is feature; (5) have higher K-ras gene, P16 gene and P53 gene mutation.The method of setting up the animal model of cancer of pancreas at present mainly comprises: transplanted tumor in nude mice model, genetic engineering mouse model and chemical carcinogen lure the cancer model.Therefore different types of cancer of pancreas animal model has its merits and demerits respectively, can be used for the experimental study of various objectives at characteristics separately.The cancer of pancreas transplantation model of nude mice passes through in nude mice hypodermis implanting tissue or clone, can realize the heterotopic transplantation of cancer of pancreas, but the tumour that produces is confined to local growth usually and seldom shifts, and repeatedly going down to posterity owing to pancreatic cancer cell, the instability of gene increases, the gene alteration that some change the primary tumo(u)r feature may occur, this model is used for studying cancer of pancreas biology and medication effect etc. more.Genetic engineering cancer of pancreas model is mainly transgene mouse model, creates the cancer of pancreas model by transfection cancer of pancreas gene or growth factor gene overexpression, is used for the research that cancer of pancreas transforms and makes progress more.The number of chemical carcinogenic substance has been employed induces the cancer of pancreas animal model, mainly includes azaserine, nitroso-amines and DMBA etc.What azaserine was induced generation is the pancreatic acinar cell cancer, the hyperplasia and the change of pancreatic duct do not occur, is different from the cancer of pancreas of human conduit origin; Although and the cancer of pancreas that nitroso-amines is induced can be expressed vessel cell keratin label, and has the K-ras gene mutation, but nitroso-amines is induced specificity and the selectivity of shortage to pancreas, easily induces other internal organs tumours when inducing cancer of pancreas.
DMBA began to be used to induce rat pancreas gland cancer at first in 1975, although the cancer of pancreas that DMBA induces that studies have shown that at that time has the conduit phenotype, but because Bockman inferred by experiment that the tumour that DMBA induces was not the conduit origin afterwards, but the result that acinar cells dedifferentes, acinar cells is through dedifferenting the form that presents conduit, and this result's hypothesis makes DMBA induce the method for cancer of pancreas model to be given a cold shoulder.Until 1997, DMBA induces the effect of cancer of pancreas model to be reappraised, studies confirm that the DMBA pancreatic tissue is implanted into or the interior injection of conduit can induce cancer of pancreas reliably, the tumour of inducing on the Histopathology shows as between significant proliferation of fibrous tissue has irregular aggressive body of gland in the matter, hyperplasia, atrophy and the atypical hyperplasia that simultaneously can observe pancreatic duct have pointed out DMBA to induce the back pancreatic duct by the progressively transformation of normal epithelial to invasive carcinoma until multiple pathological changes such as invasive carcinomas.The cancer of pancreas that DMBA induces is carried out the SABC detection discovery of conduit and acinar cells marker representation, cytokeratin 19, the 20 marker representation positives such as vessel cell such as grade and acinar cells label chymotrypsin are expressed negative, and same prompting cytokeratin 19 is expressed the positive and chymotrypsin expression feminine gender in the precancerous lesion tissue after DMBA implanted for 2 weeks, but the normal pancreatic tissue chymotrypsin is expressed positive on every side, cancer of pancreas and human pancreas cancer that these research promptings DMBA induces are similar, may actually be the pancreatic duct origin, and be not the pancreatic acini origin that Bockman thinks.
After the PanIN hierarchy system is released foundation, induce the cancer effect that lures of generation to analyze discovery according to this grade scale to local plantation of DMBA mice pancreatic, in the short period in January, DMBA can induce the abundant PanIN of rank and early stage cancer of pancreas pathology change, and do not induce other internal organs tumours, shortened in the previous experiment DMBA greatly and induced macroscopic pancreatic neoplasm and need the very long wait more than 9 months consuming time.DMBA induces the concrete mechanism of cancer of pancreas to understand at present not deeply, but discover, PanIN that DMBA induces and cancer of pancreas have the K-ras gene mutation up to 90%, and smad 4, and expression and the human pancreas cancer of cyclin Dl and p53 etc. are similar.The expression of Notch-1 also is positive in the process that DMBA induces, and by suppressing the growth that the Notch signal path can reduce pancreatic cancer cell, thinks that therefore DMBA induces the mechanism of cancer of pancreas may be relevant with the activation of Notch signal path.DMBA is because the speciality of himself thereby become and set up the important carcinogen that the cancer of pancreas chemistry lures the cancer model, in recent years, cancer of pancreas that DMBA induces and PanIN model have been applied to determining of cancer of pancreas carcinogen, the pancreatic tumour image-forming diagnose is in the researchs such as The Molecular Biology Mechanism of cancer of pancreas morbidity.
Summary of the invention
The related protein that the purpose of this invention is to provide one group of early stage cancer of pancreas model differential expression.
Second purpose of the present invention provides the application of described related protein as cancer of pancreas early diagnosis label.
The present invention is by setting up 7, pancreas in rat intraepithelial neoplasia (cin) that 12-dimethylbenzanthracene (DMBA) is induced and cancer of pancreas model, use DIGE protein analysis technology, the differential expression of protein in difference detection of dynamic Normal Pancreas, pancreas intraepithelial neoplasia (cin) and the early stage cancer of pancreas pancreas in rat tissue, utilize bioinformatics software therefrom to filter out the protein that reaches synchronous generation marked change with cancer of pancreas range kilsyth basalt, and utilize mass-spectrometric technique that it is identified, the concrete protein of differential expression during clear and definite cancer of pancreas in early days takes place.
Described related protein is:
1, enolase 1 (enolase 1);
2、TPTl；
3, pancreas elastoser 3B (pancreatic elastase 3B, ELA3B);
4, phosphoenolpyruvic acid carboxylase 2 (phosphoenolpyruvate carboxykinase 2);
5, Carboxylesterase (carboxyl ester lipase, CEL);
6, heterogeneity ribonucleoprotein A2/B1 (heterogeneous nuclear ribonucleoprotein, hnRNP, A2/B1);
7, the chromatin territory untwist enzyme dna in conjunction with albumen 3 (chromodomain helicase DNA binding protein 3, CHD3);
8, the cell transfer Profilin (non-metastatic cells 2, protein (NM23B) expressed in, NME2);
9、necdin；
10、Hbp23；
11、unnamed?protein?product；
12、retinoid?x?receptor?interacting?protein；
13、guanine?nucleotide?binding?protein(G?protein)，beta?polypeptide?2-like?1(Gnb211)；
14、glycine?C-acetyltransferase。
The PMF of described differential protein particle desires to make money or profit with Mascot software at (the National center for biotechnology information of American National biotechnology information center, NCBI) database (on November 30th, 2007 version, 5678482sequences; Search the storehouse 1961803296residues).
Screen and identify by protein, help to disclose the mechanism of cancer of pancreas, and provide novel targets for the therapeutic intervention of cancer of pancreas from protein level to early stage cancer of pancreas animal model differential expression.Related protein sensitivity height disclosed by the invention, high specificity can be used as the protein marker of cancer of pancreas early diagnosis, for clinical early diagnosis cancer of pancreas provides effective ways.
Description of drawings
Below in conjunction with Figure of description, the present invention is described in more detail.
Fig. 1 is the protein point diagram of expressing significant difference between Normal Pancreas, pancreas intraepithelial neoplasia (cin) and the cancer of pancreas.
The differential expression protein point diagram that Fig. 2 selects pending mass spectrum to identify.
Fig. 3 is that the expression of differential protein particle 1402 in Normal Pancreas (A), PanIN (B) and cancer of pancreas (C) tissue changes.
Fig. 4 is differential protein particle 1205 is identified peptide hydrolysis through MALDI-TOF MS a PMF collection of illustrative plates.
Fig. 5 is the expression of enolase 1 albumen in human pancreas cancer and cancer beside organism.(A: cancer of pancreas is expressed positive, and * 200; B: cancer of pancreas is expressed positive, and * 400; C: cancer beside organism expresses weak positive, and * 200; D: cancer beside organism expresses weak positive, and * 400).
Fig. 6 is that human pancreas cancer and the enolase of cancer beside organism 1 express the positive cell average positive rate.
Embodiment
One, the foundation of early stage cancer of pancreas model and the acquisition of pancreatic tissue sample
1. animal and grouping
60 of male cleaning level SD rats, body weight 100-110g is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center (production licence number is SCXK (Shanghai) 2003-0003, occupancy permit number be SYXK (Shanghai) 2007-0007).Under the feeding standard condition, raise.Rat is divided into model group in January (20), model group in February (20) and normal control group (20) at random.
2. operation method
The operation rat plays fasting morning on the same day and prohibits water, ketalar 100mg/kg intramuscular anesthesia, middle upper abdomen preserved skin sterile drape, belly median incision is opened abdomen, carefully cut off capsula pancreatis at the pancreas body after fully exposing pancreas, will the place of cutting off capsula pancreatis purse string suture on every side.The model group rat is tightened up ligation with pocket after being implanted into the DMBA particle (Sigma company) of 10mg/100g body weight dosage at pocket, confirms that no DMBA spills the back and closes abdomen.
3. gross examination of skeletal muscle and organize HE dyeing
The model group rat is respectively at the execution of operation back 1 month and 2 months, rats in normal control group and February model group put to death simultaneously.Cut open the belly and observe pancreatic surgery position and surrounding tissue adhesion situation, form, the quality of pancreas DMBA implantation place.After from the abdominal cavity, downcutting pancreatic tissue PBS rinsing, pancreatic tissue around the model group clip DMBA embedding plantation, a small amount of normal pancreatic tissue of normal control group clip is fixed with 4% paraformaldehyde immediately, and paraffin embedding, serial section are carried out conventional H E dyeing.All the other pancreatic tissues of leaving and taking after pathology is identified are inserted-80 ℃ of refrigerators preservations.
4. pancreas intraepithelial neoplasia (cin) and cancer of pancreas classification
HE dyeing histotomy not knowing to test under the situation of grouping, according to the PanIN grade scale, carries out classification to the pancreatic tissue pathology by the same professional pancreas pathology doctor in position, is divided into 4 grades of PanIN-1, PanIN-2, PanIN-3 and cancers of pancreas.All with the highest level pathology that occurs in the section as diagnosis.
5. pathology and classification results
The rat operative incision infects, and healing is good.Model group rat hair color dimness, weight increase is slower.In the postoperative experimental observation phase, January model group, February, the mortality ratio of model group was respectively 15% (3/20), 30% (6/20).Dead rat does not count the Histopathology statistical study.
The model group rat forms the spherical enclosed mass of the about 0.3-0.6cm of diameter at pancreas DMBA implant site, cuts the residual DMBA particle of enclosed mass minority visible yellow color.The pancreatic tissue quality of enclosed mass wall is harder, and is obviously different with normal pancreatic tissue, and the organ adhesion is obvious around pancreas and stomach, the intestinal tube etc.All have in each routine pancreatic tissue of model group tangible inflammatory cell infiltration and widely between the matter connective tissue proliferation; Except that 2 examples in the January model group are the normal ducts epithelium, other all and in various degree ductal epithelium hyperplasia occurs, catheter segment epithelial cell special-shaped hyperplasia is obvious, breaks through basilar memebrane and forms cancer of pancreas.Confirm to form cancer of pancreas 11 examples altogether PanIN18 example, PanIN-1 level 4 examples wherein, PanIN-2 level 5 examples, PanIN-3 level 9 examples through pathological grading.
Two, the Study on Different Proteomics of pancreas intraepithelial neoplasia (cin) and early stage cancer of pancreas model
1. Zu Zhi acquisition and cracking
It is 3 groups that tissue specimen is divided into, and is respectively normal control group (A group), pancreas intraepithelial neoplasia (cin) group (B group, PanIN-2 level), cancer of pancreas group (C group), every group of 3 samples (1,2,3).Pancreatic tissue about 300mg is cut into the square fritter of 3mm, wash 3 times, add the about 1ml of DIGE lysate (DIGE lysate and enzyme inhibitor mix with volume ratio at 50: 1), use the homogenate of Dounce ' s homogenizer with PBS.Change in the centrifuge tube ultrasonication cell (80W 5 times, each 10 seconds, 15 seconds at interval, finishes) on ice.4 ℃ are centrifugal, and 15,000g, 45 minutes, supernatant Bradford standard measure, every group of interior 5 routine sample mixed in equal amounts are distributed into 100 μ g, one pipe, place 500 μ l centrifuge tubes, cryopreservation (80 ℃).Pilot study, sample 100 μ g on each sample, 13cm pH3-10NL makees 3 clotting glue, judgement sample 2D feasibility.
2. two-phase electrophoresis trial test
The first phase isoelectric focusing is carried out on the Ettan of Amersham IPGphor isoelectric focusing system instrument, and applied sample amount is 100ug, adopts the non-linear adhesive tape of the long pH3-10 of 13cm, the isoelectric focusing electrophoresis condition is: 30V 12hrs, 500V 1hr, 1000V 1hr, 8000V 8hrs, 500V 4hrs.After isoelectric focusing finished, adhesive tape was taken out at 50mM Tris-HCl, 6M urea, and 30% glycerine, 2%SDS, balance 15min in the equilibrium liquid of 1%DTT and trace bromophenol blue, the back is balance 15min in the identical equilibrium liquid of 2.5% iodo-acetamide replacement 1%DTT.Then adhesive tape is moved to the linear glue of second phase SDS upper end, 0.5% agarose binds, and the second phase SDS-PAGE adopts the gel of 1mm thick 12.5%, applies electric current 15mA/ gel electrophoresis 30min, after use electric current 30mA/ glue instead, electrophoresis arrives until bromophenol blue and ends apart from 0.5cm place, glue lower edge.Give much attention in the whole process and want lucifuge.Row argentation dyeing behind the electrophoresis, concrete grammar is: with SDS-PAGE glue Milli-Q water rinse 5min, fixing 1hr in the immobile liquid that contains 40% ethanol and 10% acetate, 5% ethanol and 5% acetate fixedly spend the night, washing 20min totally 2 times, 5% glutaraldehyde soaks 1hr, washing 30min totally 4 times, silver-colored dye liquor incubation 30min after washing 3min totally 3 times.(5% acetate stops showing 15min to developer solution for 50mg/L citric acid, 0.5ml 36% formaldehyde/L) develop to the background appearance.The scanning of UMax2110XL scanning densitometer obtains collection of illustrative plates, judges each sample 2D feasibility.
3.DIGE test:
3.1 sample dye marker
Each sample concentration all is diluted to 5ug/ul, with all samples mixed in equal amounts, standard specimen product in the 50ug/10ul packing gets then.Sample thief and each 50ug of interior mark regulate the pH value between the 8.0-9.0, respectively according to table 1 mark sample, add DIGE dyestuff Cy2 respectively then, Cy3, and each 1ul of Cy5, mark 30 minutes places on ice lucifuge to carry out.Mark adds 1ul 10mM lysine cessation reaction 10 minutes after finishing again.
3.2 two-phase electrophoresis
The sample that above-mentioned mark is good is by the form sample on carrying out on five glue respectively that distributes, the first phase isoelectric focusing is carried out on the Ettan of Amersham IPGphor isoelectric focusing system instrument, applied sample amount is 100ug, adopt the non-linear adhesive tape of the long pH3-10 of 13cm, the isoelectric focusing electrophoresis condition is: 30V 12hrs, 500V 1hr, 1000V 1hr, 8000V 8hrs, 500V 4hrs.After isoelectric focusing finished, adhesive tape was taken out at 50mM Tris-HCl, 6M urea, and 30% glycerine, 2%SDS, balance 15min in the equilibrium liquid of 1%DTT and trace bromophenol blue, the back is balance 15min in the identical equilibrium liquid of 2.5% iodo-acetamide replacement 1%DTT.Then adhesive tape is moved to the linear glue of second phase SDS upper end, 0.5% agarose binds, and the second phase SDS-PAGE adopts the gel of 1mm thick 12.5%, applies electric current 15mA/ gel electrophoresis 30min, after use electric current 30mA/ glue instead, electrophoresis arrives until bromophenol blue and ends apart from 0.5cm place, glue lower edge.Give much attention in the whole process and want lucifuge.
3.3 scanning
Use the Typhoon scanner, use three kinds of different wavelength 488nm (Cy2) respectively, 532nm (Cy3), 633nm (Cy5) scanning obtains collection of illustrative plates.
3.4 software analysis differential protein particle
Adopt the GE DeCyder of company
TMAnalysis software is analyzed, and adopts One-Way ANOVA method to carry out normal control group (A group), and PanIN organizes (B group), the variance analysis between 3 groups of samples of cancer of pancreas group (C group), with P＜0.01 for having significant difference.
The mass spectrum of 4 differential protein particles is identified
4.1 preparation type two-phase electrophoresis
Preparation type two-phase deposition condition and two-phase deposition condition are basic identical, get respectively to organize and go up sample about sample 1mg, and electrophoresis finishes back coomassie brilliant blue staining method dyeing, downcuts protein spots with blade along differential protein point edge to be identified.
4.2 enzymolysis in the glue
The blob of viscose that contains differential protein particle to be identified that downcuts adds 30% acetate/100mM NH
4HCO
3Decolouring, chopping is dry then, and gel pieces is soaked in the NH of the 40mM that contains Trypsin
4HCO
3Solution was done the interior enzymolysis of glue 20 hours, 0.1%TFA/60%ACN extracting peptide hydrolysis 2 times, cryopreservation after the peptide solution freeze drying that obtains.
4.3MALDI-TOF or MALDI-TOF-TOF mass spectrophotometry
Dry peptide section is dissolved among 0.1% the FA, the ZipTip desalination, and the peptide section under the 0.1%TFA/60%ACN eluant solution is mixed with matrix solution HCCA (5mg/m1).Use the Bruker Bruker-Daltonics AutoFlex TOF-TOF LIFT of company mass spectrometer to carry out the peptide section and detect, detection mode is a positive ion, and reflection detects, flight pipe range 2.7M, accelerating potential 20KV, reflected voltage 23KV.Finally obtain each sample polypeptide quality fingerprinting collection of illustrative plates (Peptide mass fingerprint, PMF).
4.4 database search
The PMF of each the differential protein particle that is obtained by MALDI-TOF MS desires to make money or profit with Mascot software at (the National center for biotechnology information of American National biotechnology information center, NCBI) database (on November 30th, 2007 version, 5678482sequences; 1961803296 residues) search the storehouse in, kind is chosen as rat (Rattus, 67989 sequences), proteinase is elected trypsase as, the restriction enzyme site that maximum allows to lose is 1, the carboxymethylation that fixedly is modified to halfcystine of protein (Carbamidomethyl (C)), and selective modification is the oxidation (Oxidation (M)) of methionine, use monoisotopic peak (Monoisotopic), the quality error of peptide section is ± 100ppm.
Table 1DIGE goes up the sample form
5. result
5.1 the feasibility of sample two-phase electrophoresis
By to three groups of normal control groups (A group), PanIN group (B group, PanIN-2 level), cancer of pancreas group (C group), every group of 3 samples (1,2,3) carry out the trial test of two-phase electrophoresis, and the collection of illustrative plates show sample protein site migration that is obtained is basicly stable normal, can carry out next step DIGE and formally test.
5.2DIGE proteomic map
By adopting 488nm (Cy2) respectively, 532nm (Cy3), three kinds of each running gels of different length scannings of 633nm (Cy5), the collection of illustrative plates that is obtained shows that Gell gel protein particle quantity is 1445, Gel2 gel protein particle quantity is 1469, Gel3 gel protein particle quantity is 1380, and Gel4 gel protein particle quantity is 1512, and Gel5 gel protein particle quantity is 1637.
5.3 three groups of sample room protein group differential expressions change
Adopt GE DeCyder
TMAnalysis software, adopt One-Way ANOVA method to normal control group (A group), PanIN organizes (B group), protein group between 3 groups of samples of cancer of pancreas group (C group) is carried out the statistical analysis of quantitative differential expression, and we find that having 155 expression of protein spots between three groups of samples has significant difference (P＜0.01).Wherein compare with the normal control group, the protein spots that PanIN group and PC group expression intensity significantly raise has 89, and the protein spots of remarkable downward modulation has 56; Wherein control group, PanIN group and PC group expression intensity are 31 of protein spots that increase progressively trend step by step, and expression intensity is step by step that the protein spots of decline trend has 17; PanIN group and PC group expression intensity change irregular or cause the protein spots that can't judge to have 10 because of protein spots in certain group detects disappearance in addition.As shown in Figure 3, protein spots with label 1402 is that example illustrates its differential expression situation in 3 groups of samples, 1402 albumen expression in normal pancreatic tissue (A group) is the highest, and in PanIN group (B group) and PC group (C group), expression then significantly descends step by step.
5.4 the evaluation of part differential expression protein point
Select 21 protein spots to carry out MALDI-TOF or MALDI-TOF-TOF mass spectrophotometry from 155 protein spots of differential expression, protein spots numbering to be identified is followed successively by 90,230,780,1205,1402,329,1132,1066,1272,226,1405,755,1259,298,1585,1030,1057,993,1040,1470,658.The differential protein particle main standard that selection is identified is: (1) in normal control group (A group), PanIN organizes (B group), and differential expression is more remarkable between three groups of cancer of pancreas groups (C group); (2) expression has the trend that changes step by step with the pathology classification; (3) the different sample room coefficient of variation are less in the group.In 21 kinds of differential protein particles to be identified, protein spots 1259 uses the MALDI-TOF-TOF mass spectrophotometry, and other protein spots are the MALDI-TOF mass spectrophotometry.Mass spectrometry results utilizes Mascot software to search the storehouse in ncbi database, with protein spots 1205 is example, Fig. 4 is the PMF collection of illustrative plates of 1205 protein spots peptide hydrolysis, in ncbi database, search the storehouse with Mascot software, the possibility judgment basis Mowse integration of protein coupling, surpassing 61 with integration is divided into and has significance,statistical (P＜0.05), the result shows, 1205 protein spots and oncoprotein translation regulatory factor 1 (tumor protein-translationally-controlled 1, TPT 1) the Mowse integration that is consistent is 98 minutes, thereby identification of protein point 1205 is a TPT1 albumen.
Differential protein particle 1205 is as follows in Mascot database retrieval result:
User ：Zhaoyang?Liu
Email ：zyliu@sibs.ac.cn
Search?title?：P070811205
Database ：NCBInr?20071130(5678482?sequences；1961803296residues)
Taxonomy ：Rattus(67989sequences)
Timestamp ：3?Dec?2007?at?08:43:01?GMT
Top?Score ：98?for?gi|6678437，tumor?protein，translationally-controlled?1[Mus?musculus]
Prob?ability?Based?Mowse?Score
Protein?score?is-10*Log(P)，where?P?is?the?probability?that?the?observed?match?is?a?random?event.
Protein?scores?greater?than?61?are?significant(p＜0.05).
Protein?View
Match?to：gi|6678437?Score：98?Expect：1.1e-05
tumor?protein，translationally-controlled?1[Mus?musculus]
Nominal?mass(M
r)：19564；Calculated?pI?value：4.76
NCBI?BLAST?search?of?
gi|6678437?againstnr
Unformatted?
sequence?string?for?pasting?into?other?applications
Taxonomy：
Mus?musculus
Fixed?modifications：Carbamidomethyl(C)
Variable?modifications：Oxidation(M)
Cleavage?by?Trypsin：cuts?C-term?side?of?KR?unless?next?residue?is?P
Number?of?mass?values?searched：10
Number?of?mass?values?matched：7
Sequence?Coverage：37％
Matched?peptides?shown?in?Bold?Red
1MIIYRDLISH?DELFSDIYKI?REIADGLCLE?VEGKMVSRTE?GAIDDSLIGG
51NASAEGPEGE?GTESTVVTGV?DIVMNHHLQE?TSFTKEAYKK?YIKDYMKSLK
101GKLEEQKPER?VKPFMTGAAE?QIKHILANFN?NYQFFIGENM?NPDGMVALLD
151YREDGVTPFM?IFFKDGLEME?KC
No?match?to：1526.7061，1716.7813，1964.9606
Search the differential protein particle that the storehouse identifies and specifically comprise enolase 1 (enolase 1); TPT1; Pancreas elastoser 3B (pancreatic elastase 3B, ELA3B); Phosphoenolpyruvic acid carboxylase 2 (phosphoenolpyruvate carboxykinase 2); Carboxylesterase (carboxyl ester lipase, CEL); Heterogeneity ribonucleoprotein A2/B 1 (heterogeneous nuclear ribonucleoprotein, hnRNP, A2/B1); The chromatin territory untwist enzyme dna in conjunction with albumen 3 (chromodomain helicase DNA binding protein 3, CHD3); The cell transfer Profilin (non-metastatic cells 2, protein (NM23B) expressed in, NME2); Necdin; Hbp23; Unnamed protein product; Retinoid x receptor interacting protein; Guanine nucleotide binding protein (Gprotein), beta polypeptide 2-like 1 (Gnb211) and glycine C-acetyltransferase (table 2).Wherein enolase 1, pancreas ELA3B, and necdin, Hbp23, CHD3, hnRNP A2/B 1, retinoid x receptor interacting protein, Gnb211 albumen raises with the canceration degree expression; CEL, TPT 1, NME2, phosphoenolpyruvic acid carboxyl kinases 2, unnamed protein product, glycine C-acetyltransferase albumen is reduced with the canceration degree expression.
Table 2 differential protein particle mass spectrum qualification result
Three, the checking of differential protein enolase 1 expression in human pancreas cancer.
1. sample is prepared
Human pancreas cancer organization chip (numbering OD-CT-DgPan03-002), 62 points of always counting, several 30 examples of total example comprise 30 routine human pancreas cancers and 30 routine corresponding human pancreas cancer cancer beside organisms, formalin fixed, array spot diameter 2mm, slice thickness are 4mm.Human pancreas cancer sample details see Table 3.
2. SABC step
Place dimethylbenzene I5min, dimethylbenzene II 5min, anhydrous alcohol 1min, 95% alcohol 1min * 2 time, 85% alcohol 1min dewaxing, aquation, entry respectively successively; PBS rinsing 3 times, each 2min; 3%H
2O
2Incubated at room 10min removes endogenous peroxydase, PBS rinsing 3 times, each 2min; 37 ℃ of effects of 0.05% trypsase 30min, PBS rinsing 3 times, each 3min; Drip 10% normal sheep serum, hatch 30min for 37 ℃; Abandon sheep blood serum, drip anti-enolase 1 antibody of dilution in 1: 400,4 ℃ of overnight incubation in the wet box; PBS rinsing 3 times, each 3min; Drip two and resist, hatch 30min for 37 ℃, PBS rinsing 5 times, each 3min; DAB (preparation in 1: 50) colour developing 2-20min, washing 5min; Haematoxylin lining transfect cell nuclear, hydrochloride alcohol differentiation, gradient alcohol dehydration, neutral resins mounting.
3. the SABC result judges
Replace one to resist with the PBS damping fluid as negative control, use single blind evaluation rat model of optical microscope and micro image analysis system and human pancreas cancer organization chip SABC result, criterion is that tumour cell endochylema or karyon have the positive cell of pale brown look tinter.Every routine human pancreas cancer tissue specimen is counted 3 high power field cells respectively, counting endochylema or karyon stained positive cell average positive rate.
4. statistical analysis
Data use the SPSS10.0 statistical software to adopt the t check to carry out comparing between two groups.
5. the expression of the other normal pancreatic tissue enolase of human pancreas cancer and cancer 1 albumen
Immunohistochemical staining shows that enolase 1 mainly is expressed in cell cytosol and karyon, be expressed in cell membrane on a small quantity, except that above-mentioned epithelial cell was expressed (being endochylema, karyon, after birth), the fiber in the matter, vascular wall flesh layer and inflammatory cell were also expressed therebetween, and the part positive staining is very strong.In 30 pairs of human pancreas cancers and pancreas cancer beside organism, enolase 1 all has expression, enolase 1 positive cell average positive rate in 30 routine human pancreas cancer tissues is 91 ± 5.6%, the positive cell average positive rate is 37 ± 22.2% in 30 routine human pancreas cancer cancer beside organisms, The positive expression rate in the human pancreas cancer tissue will be significantly higher than cancer of pancreas cancer beside organism, and difference of them has statistical significance (P＜0.05) (Fig. 5, Fig. 6).
Table 3 human pancreas cancer organization chip distributes and clinical pathologic characteristic
Claims (2)
1. the related protein of one group of early stage cancer of pancreas model differential expression is characterized in that, described related protein be following one or more: enolase 1; TPT1; Pancreas elastoser 3B; Phosphoenolpyruvic acid carboxylase 2; Carboxylesterase; Heterogeneity ribonucleoprotein A2/B1; Enzyme dna is untwisted in conjunction with albumen 3 in the chromatin territory; The cell transfer Profilin; Necdin; Hbp23; Unnamed proteinproduct; Retinoid x receptor interacting protein; Guanine nucleotide binding protein, beta polypeptide 2-like 1; Glycine C-acetyltransferase.
2. the described related protein of claim 1 is as the application of cancer of pancreas early diagnosis label.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105301082A (en) * | 2015-07-23 | 2016-02-03 | 海南师范大学 | Method for detecting differential protein produced by action between alpinetin or cardamonin and serum |
| CN111316102A (en) * | 2017-09-05 | 2020-06-19 | 温口特 | Method for diagnosing pancreatic cancer using methionyl-tRNA synthetase and pancreatic cancer diagnostic kit using same |
| CN114164268A (en) * | 2021-07-14 | 2022-03-11 | 中山大学孙逸仙纪念医院 | Application of hnRNPA1 in diagnosis, prognosis and treatment of pancreatic cancer |
| CN114217065A (en) * | 2021-12-22 | 2022-03-22 | 引加(上海)生物医药科技有限公司 | Application of combination of multiple serum tumor markers in pancreatic cancer specific early diagnosis and curative effect monitoring and detection |
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| DELTEV SUCKAU ET.AL.: "A novel MALDI LIFT-TOFTOF mass spectrometer for proteomics", 《ANAL BIOANAL CHEM》 * |
| KENNETH H.YU ET.AL.: "Characterization of Proteins in Human Pancreatic Cancer Serum Using Differential Gel Electrophoresis and Tandem Mass Spectrometry", 《JOURNAL OF PROTEOME RESEARCH》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301082A (en) * | 2015-07-23 | 2016-02-03 | 海南师范大学 | Method for detecting differential protein produced by action between alpinetin or cardamonin and serum |
| CN105301082B (en) * | 2015-07-23 | 2018-01-23 | 海南师范大学 | Alpinetin and CDK1 and the differential protein detection method of serum effect |
| CN111316102A (en) * | 2017-09-05 | 2020-06-19 | 温口特 | Method for diagnosing pancreatic cancer using methionyl-tRNA synthetase and pancreatic cancer diagnostic kit using same |
| CN114164268A (en) * | 2021-07-14 | 2022-03-11 | 中山大学孙逸仙纪念医院 | Application of hnRNPA1 in diagnosis, prognosis and treatment of pancreatic cancer |
| CN114217065A (en) * | 2021-12-22 | 2022-03-22 | 引加(上海)生物医药科技有限公司 | Application of combination of multiple serum tumor markers in pancreatic cancer specific early diagnosis and curative effect monitoring and detection |
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