CN107064519A - A kind of chemotherapeutics correlation molecule specific proteins chip and preparation method and application - Google Patents
A kind of chemotherapeutics correlation molecule specific proteins chip and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of chemotherapeutics correlation molecule specific proteins chip, fixed on the protein chip is the antibody prepared for tumor drug resistance gene, the tumor drug resistance gene be the cell-signaling pathways information that is participated in by the molecule related to reactivity, the cytotoxicity of the chemotherapeutics to the tumour receive life cycle after chemotherapy it is related differential gene progress it is overlapping and obtain.Application the invention also discloses the preparation method of the protein chip and its in detection and analysis obtained resistance correlation molecule.The present invention is by building chemotherapeutics correlation molecule specific proteins chip, tumor drug resistance cell line is detected, and difference molecule is analyzed by bioinformatics method, so as to further investigate the molecular mechanism of chemotherapy of tumors resistance, contribute to exploitation for the molecular targeted therapy of drug resistance of tumor cell target spot, improve the overall therapeutic level of tumour.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of chemotherapeutics correlation molecule specific proteins chip and system
Preparation Method and application.
Background technology
Biochip technology conventionally comprises genetic chip and protein chip, and the former is by the complementarity principle of nucleotides in core
Hydrogen bond is formed between fixed cDNA probes and DNA to be measured (or RNA) double-strand on piece, passes through fluorescence after hybridization reaction and hybridization
The analysis of intensity obtains the relative expression quantity of specific gene in sample to be tested, so as to carry out semidefinite to the sample from different condition
Amount compares, so as to obtain the gene expression spectrum signature of relevant disease.Protein chip is that protein, antibody or peptide chain are fixed on into glass
On piece or nitrocellulose membrane, the testing protein sample of fluorescence labeling is attached on protein chip, by mutual between protein-protein
Effect, which is formed, to be combined, and then carries out laser scanning to chip, so that the specific protein content to different samples carries out sxemiquantitative point
Analysis is compared and analyzed.First is delivered with the protein chip of antibody construction in nineteen eighty-three, and obtains United States Patent (USP) US 4591570.
For technically, protein chip derives from DNA chip technology, but overcomes the latter because the expression of nucleic acid such as the mRNA that is surveyed can not
The defect that reacting cells internal protein changes.The physiological function of normal cell or the abnormal phenotype of tumour cell, are by albumen
The interaction of matter is come what is realized, and except the relative expression quantity of acquisition cell protein level, protein chip can also be to protein translation
Decorating state afterwards, such as phosphorylation, ubiquitination, which change, to be analyzed, and the exception of the apparent modification of these albumen, is often swollen
The mark of oncocyte vicious transformation.Except the advantage compared to genetic chip, protein chip also solves traditional based on electrophoresis
Immunoblot assay and thin layer chromatography method time and effort consuming based on molecular physical chemistry characteristic, the low shortcoming of sensitivity.
Chemotherapeutics drug resistance is a great problem that oncotherapy faces.Glioblastoma, particularly WHO IV grades colloids
The standard care of blastoma (GBM) includes the surgery excision of maximum magnitude and postoperative putting therapeutic alliance, but GBM is normal
Because occurring recurring to the resistance of chemotherapeutics and being in progress.The development of biological micro-array chip technology, is from genome range
The resistance correlated expression spectrum of interior research glioma provides strong instrument.Biochip research theory thinks most gliomas
With molecular heterogeneity, glue can more be accurately reflected by carrying out molecule parting and Index for diagnosis to glioma using molecular biology method
In matter knurl pathophysiological mechanism, can be more objective and illustrate tumours of chemotherapeutic sensitiveness exactly and between prognosis judges
Relation.Chemoresistance can be divided into internality and the external class of (acquisition) property two in mechanism, the former refer to tumour cell by
The hereditary capacity that change possesses in itself at the beginning of chemotherapy, such as the glue of high expression O6-Methylguanine DNA-Methyltransferase (MGMT)
Matter knurl just can produce resistance because of the reparation tolerance of the genetoxic to Temozolomide (TMZ).And acquired resistance mechanism is then
Be tumour cell during by dealed with medicine went, the mutation of the series of genes that gradually produces, apparent modification, albumen point
Son changes to obtain the gene expression that drug-resistant phenotype, including the change of transmembrane transport, apoptosis, DNA damage repair mechanism etc. are related
Change.Relative to inherent drug resistance, acquired resistance is clinically more common, because many patients with gliomas are in the beginning of chemotherapy
The reaction to medicine can be shown, but reactivity greatly lowers or even answering for tumour occurs after the treatment cycle in several cycles
Hair.Thus the molecular mechanism of research glioma acquired resistance is so as to find treatment water of the therapeutic targets to raising glioblastoma
It is flat significant.
Temozolomide (TMZ) belongs to second generation alkylating agent, because having higher blood-brain barrier (BBB) transmitance can be in brain
Higher concentration is formed in tumor tissues, its CDCC is mainly methylated by oncocyte DNA guanylic acid O6 sites, is caused
G-T mispairing occurs during duplication to cause DNA damage and Apoptosis.Temozolomide is the First-line chemotherapy of current Treatment for Glioma
Medicine, research glioma has to the chemotherapy resistance mechanism of Temozolomide to strengthening its cytotoxic effect increase therapeutic sensitivity
It is significant.
The research to tumor drug resistance molecular mechanism mainly has different chemosensitivities by genechip detection at present
Tumor sample, this research meanses have two kinds of limitations, and the on one side, and made genetic chip often includes full base
Because of group information, the differential gene detected be usually take part in it is a variety of pernicious with tumor development, propagation, invasion and attack, migration etc.
Biological process, but these biological functions and path might not be relevant to the reactivity of specific chemotherapy with tumour cell, base
In these interest genes expression and functional study to the Index for diagnosis and special chemosensitization of glioma chemotherapy side effect
Perhaps, improvement has no greater significance.Furthermore, even if these differential genes take part in resistance of the tumour to certain chemotherapeutics really,
Due in the acquisition of clinical samples and to cannot be distinguished by such a difference for causing drug resistance be due to that tumour cell heredity has in itself,
Or the secondary acquisition in chemotherapy process, this also causes resulting interest genes can not really react the change of tumour cell
Treat countermeasures.Second aspect, genetic chip is the biological micro-array detection platform built based on nucleic acid molecules such as cDNA, inspection
Survey object be DNA, mRNA etc. reacted cytogenetics, transcriptional information, and really embody cell tissue biological function be
Protein, in many cases, the change of cellular genome and transcript profile and the change of protein groups are not consistent, this also constrain with
Express spectra based on genetic chip detects the conspicuousness in tumor drug resistance Study on Molecular Mechanism.Further, since thin to tumour
The research of born of the same parents' acquired resistance mechanism is obtained difficulty by clinical sample and limited, and the research of the past is mainly in vitro by right
The tumor cell line of stable passage carries out the Sustained drug effect of various concentrations, so that the cell clone of tolerance medicine is obtained, and
The identification of gene and characterization of molecules is carried out on the model of this mdr cell, the analysis means taken are except foregoing gene core
Beyond piece, also PCR (PCR), Western blot (Western Blotting) are used to detect mdr cell
Molecular changes.Such research meanses can obtain the model of obtained resistance, but point of follow-up characterization of molecules spectrum
The shortcomings of poor efficiency and low sensitivity that the limitation and routine protein that analysis can not still depart from genetic chip are determined.
The content of the invention
In view of the drawbacks described above of prior art, one aspect of the present invention provides a kind of chemotherapeutics correlation molecule specificity
Protein chip, on the other hand the preparation method there is provided the protein chip and its detection and analysis obtained resistance correlation point
Application in son.
Technical scheme is as follows：
The present invention is provided on a kind of chemotherapeutics correlation molecule specific proteins chip, the protein chip in first aspect
Fixed is the antibody prepared for tumor drug resistance gene, and the tumor drug resistance gene is by reactive, thin with the chemotherapeutics
The cell-signaling pathways information that the related molecule of cellular toxicity is participated in and the tumour difference base that to receive life cycle after chemotherapy related
Obtained because carrying out overlapping.
In a preferred embodiment of the invention, above-mentioned tumour is glioma.
Preferably, above-mentioned chemotherapeutics is selected from Semustine (CCNU), BCNU (BCUN) and Temozolomide (TMZ)
One or more.
Further, above-mentioned chemotherapeutics is Temozolomide.
The present invention provides a kind of preparation method of chemotherapeutics correlation molecule specific proteins chip, bag in second aspect
Include following steps：
Step 1, the cell-signaling pathways for being participated in the molecule related to reactivity, the cytotoxicity of above-mentioned chemotherapeutics
Information and above-mentioned tumour receive the related differential gene of life cycle after chemotherapy and carry out overlapping, filter out tumor drug resistance base interested
Cause；
Step 2, for the tumor drug resistance gene obtained in step 1 prepare human monoclonal antibody；
Step 3, the human monoclonal antibody prepared in step 2 is fixed on scribbles Bovine serum albumin and poly holder
Slide on, be made protein chip；
Step 4, obtained protein chip in step 3 is soaked in Bovine serum albumin buffer solution, to eliminate non-specific knot
Close.
In a preferred embodiment of the invention, above-mentioned tumour is glioma.
Preferably, above-mentioned chemotherapeutics is Temozolomide.
It is swollen in detection and analysis that the present invention provides above-mentioned chemotherapeutics correlation molecule specific proteins chip in the third aspect
Application in knurl acquired resistance correlation molecule, comprises the following steps：
Step 1, with above-mentioned induced by chemotherapeutic agents culture tumor drug resistance cell line, separately set up parent's tumour cell as control
Group；
Step 2, Protein Detection is carried out to above-mentioned tumor drug resistance cell line and above-mentioned parent's tumour cell, including protein is taken out
Carry and mark, chip hybridization and scanning chip obtain differential protein；
Step 3, analysis of biological information.
In a preferred embodiment of the invention, when chemotherapeutics is Temozolomide, Temozolomide correlation molecule is special
Property protein chip detection and analysis obtained resistance correlation molecule in application comprise the following steps：
Step 1, with the Temozolomide Fiber differentiation tumour cell line of resistance to Temozolomide, separately set up parent's tumour cell as right
According to group；
Step 2, Protein Detection, including egg are carried out to the above-mentioned tumour cell line of resistance to Temozolomide and above-mentioned parent's tumour cell
White matter is extracted and mark, chip hybridization and scanning chip obtain differential protein；
Step 3, analysis of biological information.
In a preferred embodiment, above-mentioned tumour is glioma, and the cell of culture is glioma U251 cells.
Present invention mainly solves problems with：
1st, using protein chip carry out tumour especially glioma resistance mechanism analyze so that the molecule and path of acquisition compared with
The more notable functional meaning of gene aspect, Late Stage Verification experiment in select negative molecule through PCR detection discovery rna level
With significant difference, show that the molecule have passed through certain and modify in translation skill and cause final expression in persister and parent
Maintain an equal level between strain, therefore the resistance mechanism that the present invention is detected is evidentiary stronger；
2nd, resistance mechanism is studied using protein chip, it is than the detection technique such as traditional immunization blotting efficiency faster, sensitive
Property is higher, segment chip difference molecule is verified with Western blot (Western Blotting), acquired results and core
Piece result has statistics reduction compared to expressing quantity difference, shows that the detection efficiency of the present invention is higher；
3rd, the gene information obtained by being studied using premenstruum (premenstrua) tumor drug resistance, with reference to newest chemosensitivity pertinent literature number
According to library inquiry, build comprehensively, targetedly protein antibodies microarray, concentrate on numerous and diverse mechanism and logical than the past protein chip
Road is studied, and the present invention is more specific, more results in the achievement in research closely related with chemotherapy resistance；
4th, the detection object in a preferred embodiment of the invention is the glioma drug-resistant cell strain induced through Temozolomide
With the parent plant of its not resistance, the developed by molecule difference of two samples represents multidrug-resisting (MDR) phenotype of tumour cell
Difference, such a detection model is closer to the resistance pattern of clinical glioma chemotherapy, and illustrated molecular mechanism is in relation tumour
The mechanism of even more important acquired resistance in the molecular mechanism of chemotherapy；
5th, the present invention carries out progressively analysis of biological information to the difference molecule obtained by protein chip detection, passes through molecular signal
Network is intuitively shown, the tune of certain specific passageways (such as reparation of apoptosis, DNA, cell dryness) can be shown in network
Control information.
Below with reference to accompanying drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technique effect.
Brief description of the drawings
Fig. 1 shows the schematic flow sheet of protein chip detection testing protein molecule in preferred embodiment of the present invention；
Fig. 2 shows that protein chip is (right with extracting from persister (experimental group) and parent plant in preferred embodiment of the present invention
According to group) chip scanning image after the protein hybridizations of U251 glioma cells；
Fig. 3 shows the analysis of biological information result in preferred embodiment of the present invention；Wherein Fig. 3 a show differential gene
The P values of the GO classification of participation, and it is used as using-log10 (P values) the enrichment value of the molecule；Fig. 3 b show differential protein molecule pair
The signal path of gene is answered, the P values per signal paths is calculated, the enrichment value of the molecule is used as using-log10 (P values)；
Fig. 4 shows the related signal network figure of the Temozolomide resistance in preferred embodiment of the present invention.
Embodiment
Glioblastoma acquired resistance phase is detected with the protein chip built for Temozolomide chemosensitivity below
Close exemplified by molecule, elaborate the present invention.It should be understood that these embodiments are merely to illustrate the present invention rather than the limitation present invention
Scope, the scope of the present invention will be limited only by the claims which follow.
Build alkylating agent correlation molecule specific proteins chip
1st, retrospective analysis the present inventor early stage is by being followed by 4 by Semustine (CCNU) chemotherapy to tumor resection
The Affymetrix Oligo Microarray U133 containing 54000 nucleic acid probes that the tumor specimen of GBM patient makes
2.0 genetic chips, chip is divided into two groups by 4 patient survival length, after being detected to two groups of chip through statistical check (with
Machine Tobin's mean variance model t is examined, FDR<0.05, P<0.01) 2018 genes with statistically-significant difference altogether are obtained, wherein
934 gene upregulations, 1084 gene deregulation (Zhao Z, Liu Y, He H, Chen X, Chen J, Lu YC.Candidate
genes influencing sensitivity and resistance of human glioblastoma to
Semustine.Brain Res Bull.2011Oct 10；86(3-4):189-94.).
2nd, on the basis of this 2018 genes, NCBI pubmed literature queries (https is passed through://
Www.ncbi.nlm.nih.gov/pubmed, retrieval type " semustine " [MeSH Terms] OR " temozolomide "
[Supplementary Concept] AND " neoplasms " [MeSH Terms]), find and with Semustine, Temozolomide
It is the related molecule of alkylating agent reactivity, the cytotoxicity of representative Deng a line glioma chemotherapeutics, then in KEGG
(http://www.genome.jp/kegg/pathway.html) cell signal that obtains the participation of these molecules in database leads to
Road information, then the differential gene progress related to GBM life cycles are overlapping, obtain the interest genes in 1358 present invention.
3rd, human monoclonal antibody is prepared for 1358 glioma filtered out drug resistant genes, then uses high precision machine
The needle-like point sample pipette tips of tool hand sessile antibody, point on the slide for scribbling Bovine serum albumin (BSA) and poly holder
Chip is placed in warm bath 3 hours again after sample and Bovine serum albumin buffer solution is soaked in eliminate on non-specific binding, chip
Each antibody for being used to detect repeats point sample three times, and another individually point sample GAPDH, Actin are as internal reference, in chip edge point sample
Cy3 labelled antibodies are as positive internal control, and negative control is BSA, and blank control is without any composition and is used as chip background signal.
Two duplicate protein chips are built altogether, are respectively used to the detection of persister and parent plant sample.
The Fiber differentiation glioma U251 cell lines of resistance to Temozolomide
1st, the preparation of Temozolomide medicine
At room temperature, electronic balance weighs 50mg Temozolomide powder, is dissolved in 4ml DMSO, and about 30 were shaken every 5 minutes
Second, observation medicine dissolving situation, until complete drug dissolution, room temperature brown vial avoid light place is stand-by after filtering.For not azoles
Amine mother liquid concentration is 12.5mg/ml.
2nd, the determination of Temozolomide concentration
With reference to the medicine preliminary experiment done before previous literature and formal inducing cell culture so that it is determined that optimal induced drug
The activity of Temozolomide, starting least concentration is 1 μ g/ml, and double the μ g/ml of concentration 2,4 μ g/ml, 8 μ g/ml, 16 μ afterwards
G/ml, 32 μ g/ml are until the μ g/ml of maximum concentration 64.
3rd, cell induction process
Drug-resistant cell strain is set up to induce using the method for interruption multiplication Temozolomide drug concentration in the present embodiment.U251
The initial concentration of Temozolomide is 1 μ g/ml in cell culture medium, when U251 cells enter exponential phase, begins to increase
Plus gradually the drug concentration of the Temozolomide in multiplication culture medium so that most of death of neoplastic cells and the U251 cells survived
Slow growth.So medicine multiplication induces continuous in vitro culture after 5 months repeatedly, makes Temozolomide in mdr cell culture medium
Ultimate density be 64 μ g/ml, Temozolomide glioblastoma cells strain at this moment is named as U251/TMZ, parent is separately set up
This U251 cells are control group.
Protein Detection
1st, the extracting of sample protein matter and mark
A, protein extracting
1) persister (experimental group) and parent plant (control group) U251 glioma cells, are collected.
2), using vortice concussion 1min at a high speed, then stand 10min on ice, repeat that (this step is in 1 hour 3-5 times
Complete).4 DEG C, 10,000xg (14000rpm) centrifugation 15min suct clear in a new centrifuge tube, repeated centrifugation 4-6 times.
3), last time is centrifuged, and supernatant is moved into a clean test tube, carries out next step experiment or -80 DEG C
Storage.
B, determination of protein concentration
Use BCA kit measurement protein concentrations.
C, protein labeling
1), biotin reagent (Biotin Reagent) is simply centrifuged using preceding, added per 1mg biotin reagents
100uL DMF, final concentration of 10ug/uL, labeled as Biotin/DMF.
2), each sample respectively takes 50ug protein samples to be marked, and often pipe adds 3uL biotins/DMF, final volume 75uL；
Mixed at room temperature is uniform, at room temperature concussion reaction 2h.
3), add 35uL and stop liquid, at room temperature concussion reaction 30min.
4) sample, is stored in -80 DEG C or next step is carried out.
| No. | Sample names | Concentration (g/L) | Volume (μ L) | Total amount (μ g) |
| 1 | U251/TMZ | 2.45 | 100 | 245 |
| 2 | U251 | 2.21 | 100 | 221 |
The Protein Extraction information of table 1
2nd, chip hybridization
A. chip is closed
1), protein chip is taken out from refrigerator, equilibrium at room temperature 45min.
2) 30ml lock solutions, are added in 100x15mm culture dishes, protein chip is totally submerged.Determine chip surface
With bar code upwards, culture dish is put on shaking table, room temperature 55rpm closings 45min.
3), using Milli-Q water according to below step full scale wash：
A) chip, is put into the circular centrifuge tubes of 50ml, 45ml Milli-Q is added, covers tightly lid；
B), turned upside down with hand or shake pipe 10s, outwell Milli-Q；
C) new Milli-Q, is changed in centrifuge tube, pipe 10s is shaked, outwells Milli-Q；
D), it is repeated 10 times.
4) the unnecessary Milli-Q water of chip surface, is got rid of, next step reaction is immediately entered.
B, target protein and antibody hybridization
1), in a test tube, 6ml conjugate solutions (Coupling Solution) are added, the egg of biotin labeling is added
(50ug) is simply mixed uniformly in vain, labeled as albumen coupling mixture (Protein Coupling Mix).
2), chip is put into clean culture dish, chip surface is upward, slowly by 6ml albumen coupling mixtures
(Protein Coupling Mix) is added to chip surface.Chip is totally submerged, coupling chamber (Coupling is covered
Chamber)；Room temperature reacts 2h on shaking table with 35rpm.
3) chip, is moved on into a 100x15mm culture dish for filling 30ml 1X cleaning solutions (Wash Solution)
In, room temperature reacts 10min on shaking table with 55rpm, outwells washing lotion, repeats this step 3 times.
4), using Milli-Q water according to below step full scale wash：
A) chip, is put into the circular centrifuge tubes of 50ml, 45ml Milli-Q is added, covers tightly lid；
B), turned upside down with hand or shake pipe 10s, outwell Milli-Q；
C) new Milli-Q, is changed in centrifuge tube, pipe 10s is shaked, outwells Milli-Q；
D), it is repeated 10 times；
5) the unnecessary Milli-Q water of chip surface, is got rid of, next step reaction is immediately entered.
C, signal detection
1), detected to 60ml and 60mL Cy3- Streptavidins (0.5mg/ is added in buffer solution (Detection Buffer)
ml)。
2) 30ml Cy3- Streptavidins, are added in a 100x15mm culture dish.
3) chip, is submerged into Cy3- solution of streptavidin.Room temperature lucifuge, 55rpm reacts 20min on shaking table.
4) chip, is moved into a new 100x15mm for filling 30ml 1X cleaning solutions (Wash Solution) to cultivate
In ware；Room temperature 55rpm on shaking table reacts 10min.Repeat this step 3 time.
5), using Milli-Q water according to below step full scale wash：
A) chip, is put into the circular centrifuge tubes of 50ml, 45ml Milli-Q is added, covers tightly lid；
B), turned upside down with hand or shake pipe 10s, outwell Milli-Q；
C) new Milli-Q, is changed in centrifuge tube, pipe 10s is shaked, outwells Milli-Q；
D), it is repeated 10 times.
6), centrifugal drying chip surface.
7) chip, is scanned.
| Title | Producer |
| 4 degree of centrifuge Fresco 17 | Sai Mo flies generation that (Thermo Scientific), the U.S. |
| Normal temperature centrifuge Pico21 | Sai Mo flies generation that (Thermo Scientific), the U.S. |
| ELIASA PE Victor | PerkinElmer (PerkinElmer), the U.S. |
| Turbula shaker Vortex 5 | Its woods AT＆T Labs instrument, China |
| Shaking table TS-2 | Its woods AT＆T Labs instrument, China |
| Chip compact centrifuge ChipMate PMC-082 | Soup rice (Tomy), Japan |
| Chip compact centrifuge ChipMate PMC-082 | Shanghai day energy |
| Chip scanner GenePix 4000B | The gloomy instrument of Acker (Axon Instruments), the U.S. |
The array experiment instrument of table 2
3rd, scanning chip obtains differential protein
1) chip after, hybridizing is carried out using GenePix 4000B scanners, and the result that scanning is obtained is shown in Fig. 2.
2), the chip image obtained to scanning, is used the softwares of GenePix Pro 6.0 (Axon Instruments, USA)
Initial data is read, and is further analyzed.Every kind of antibody is repeated twice in experiment and two chips of control group, is calculated first
Average, standard deviation SD and the coefficient of variation CV repeated twice.For the screening of two inter-sample difference albumen, select in internal reference
Signal is metastable and this signal between two groups of samples ratio close to 1.According to above principle, from GAPDH as internal reference,
Therefore protein expression ratio is the average value of each antibody and GAPDH ratio.
| Sample ID | Background signal | Positive internal control signal | Beta-actin | GAPDH |
| U251/TMZ | 810.5 | 3500.0 | 654.0 | 673.0 |
| U251 | 973.0 | 6228.5 | 943.0 | 808.5 |
The chip internal reference data of table 3
3), experimental group and control group differential protein calculation formula：
(being notable upregulated protein ,≤0.50 for notable down-regulation protein with difference Bei Shuo≤1.50)
Analysis of biological information
1st, Gene Ontology (GO) are analyzed：The present invention utilizes Gene ontology databases (http://
) and online query instrument DAVID (http www.geneontology.org/://david.abcc.ncifcrf.gov/) carry out
The functional annotation of differential protein correspondence gene and enrichment are analyzed, and are accurately examined using double tail Fisher's and are obtained inquired difference
The P values for the GO classification that allogene is participated in, and using-log10 (P values) as the enrichment value of the molecule, as shown in Figure 3 a.Matlab
(http://www.mathworks.com) and Mysql (http://www.mysql.com/) it is calculating platform.
2nd, resistance path analysis：Based on KEGG (http://www.genome.jp/kegg/)and NCBI(http://
Www.ncbi.nlm.nih.gov/) database, the signal path of inquiry differential protein molecule correspondence gene, is calculated per bars
The P values of path, using-log10 (P values) as the enrichment value of the molecule, as shown in Figure 3 b, equally with Matlab (http://
) and Mysql (http www.mathworks.com://www.mysql.com/) it is calculating platform.
3rd, resistance network of relation figure：Inquire about Human Protein Reference databases (http://
Www.hprd.org/), the mutual regulation relationship of the 200 significant difference molecules filtered out using protein chip, is built for not azoles
The related signal network figure of amine resistance, each node size represents level of difference, and color represents up-regulation or lowered, lines
Pattern represents regulation relationship, as shown in Figure 4.
The present embodiment is built special on the basis of existing high flux biological micro-array technology and glioma gene spectrum signature
The opposite sex is directed to the protein chip of Temozolomide chemotherapy drug susceptibility, and external evoked glioma drug-resistant cell strain is detected
And the closely related differential gene and molecule of chemotherapy side effect is found by bioinformatics method, so as to understand glioma in depth
The mechanism of acquired resistance, contributes to exploitation for the molecular targeted therapy of drug resistance of tumor cell target spot, improves glioblastoma
Overall therapeutic level.
Preferred embodiment of the invention described in detail above.It should be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technical staff in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a kind of chemotherapeutics correlation molecule specific proteins chip, it is characterised in that what is fixed on the protein chip is pin
The antibody prepared to tumor drug resistance gene, the tumor drug resistance gene is by the reactivity with the chemotherapeutics, cytotoxicity
The cell-signaling pathways information that related molecule is participated in and the tumour receive the related differential gene of life cycle after chemotherapy and entered
Row is overlapping and obtains.
2. chemotherapeutics correlation molecule specific proteins chip according to claim 1, it is characterised in that the tumour is
Glioma.
3. chemotherapeutics correlation molecule specific proteins chip according to claim 1 or 2, it is characterised in that describedization
Treat one or more of the medicine in Semustine, BCNU and Temozolomide.
4. chemotherapeutics correlation molecule specific proteins chip according to claim 1 or 2, it is characterised in that describedization
Treatment medicine is Temozolomide.
5. a kind of preparation method of chemotherapeutics correlation molecule specific proteins chip, it is characterised in that the preparation method bag
Include following steps：
Step 1, the cell-signaling pathways information for being participated in the molecule related to reactivity, the cytotoxicity of the chemotherapeutics
Receive the related differential gene of life cycle after chemotherapy to the tumour and carry out overlapping, filter out tumor drug resistance gene interested；
Step 2, for the tumor drug resistance gene obtained in step 1 prepare human monoclonal antibody；
Step 3, the human monoclonal antibody prepared in step 2 is fixed on scribbles Bovine serum albumin and poly holder
Slide on, be made protein chip；
Step 4, the obtained protein chip in step 3 is soaked in Bovine serum albumin buffer solution, to eliminate non-specific knot
Close.
6. preparation method according to claim 5, it is characterised in that the tumour is glioma.
7. preparation method according to claim 5, it is characterised in that the chemotherapeutics is Temozolomide.
8. chemotherapeutics correlation molecule specific proteins chip according to claim 1 or 2 is obtained in detection and analysis tumour
Application in property resistance correlation molecule, it is characterised in that the application comprises the following steps：
Step 1, with the induced by chemotherapeutic agents culture tumor drug resistance cell line, separately set up parent's tumour cell as a control group；
Step 2, carry out Protein Detection to the tumor drug resistance cell line and parent's tumour cell, including protein extracting and
Mark, chip hybridization and scanning chip obtain differential protein；
Step 3, analysis of biological information.
9. chemotherapeutics correlation molecule specific proteins chip according to claim 4 is resistance in detection and analysis obtained
Application in medicine correlation molecule, it is characterised in that the application comprises the following steps：
Step 1, with the Temozolomide Fiber differentiation tumour cell line of resistance to Temozolomide, separately set up parent's tumour cell as control
Group；
Step 2, Protein Detection, including protein are carried out to the tumour cell line of resistance to Temozolomide and parent's tumour cell
Extracting and mark, chip hybridization and scanning chip obtain differential protein；
Step 3, analysis of biological information.
10. application according to claim 9, it is characterised in that the tumour is glioma, and the cell of culture is glioma
U251 cells.
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