CN105510603A - Method for detecting drug-resistant mechanism of gefitinib - Google Patents
Method for detecting drug-resistant mechanism of gefitinib Download PDFInfo
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- CN105510603A CN105510603A CN201610068206.7A CN201610068206A CN105510603A CN 105510603 A CN105510603 A CN 105510603A CN 201610068206 A CN201610068206 A CN 201610068206A CN 105510603 A CN105510603 A CN 105510603A
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- gefitinib
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- protein signaling
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis
Abstract
The invention discloses a method for detecting a drug-resistant mechanism of gefitinib and belongs to the field of medicines. The method comprises the following steps: (1) performing high throughput screening on protein signaling molecules participating in gefitinib drug resistance by adopting iTRAQ quantification (isotope labeling relative or absolute quantification) proteomics technology; (2) screening to obtain differential expression protein signaling molecules of gefitinib sensitive lung cancer cell lines and gefitinib rug-resistant cell lines by adopting western blot (western immunoblotting test) technical identification; and (3) proving expression of the protein signaling molecules discovered by interference by adopting MTT (MTT cytotoxicity assay). The sensitivity of gefitinib can be improved, so that the expression level change of the newly discovered protein signaling molecules is proved to be a drug-resistant mechanism of gefitinib. The method contributes to detecting new protein signaling molecules participating in gefitinib drug resistance, and tolerance-resistant drugs are designed by virtue of the new detected protein signaling molecules.
Description
Technical field
The invention belongs to field of medicaments, particularly relate to a kind of Ji Feini that detects for the method for resistance mechanism.
Background technology
Lung cancer is all the malignant tumour that the incidence of disease is the highest, mortality ratio is the highest in China and global range.Have 80% in lung cancer for non-small cell lung cancer, Gefitinib (Gefitinib) is the active drug for the treatment of advanced Non-small cell lung.Research display, compared with chemotherapy, Gefitinib has following advantage: oral administration; Treatment toxic and side effect is little; Can obtain similar to chemotherapy, even be better than the curative effect of chemotherapy.But patients with advanced NSCLC there will be resistance after taking the Gefitinib 10-12 month, cause the consequence that patient on medication's drug effect reduces.Although have certain understanding to the resistance mechanism of Gefitinib at present, but, do not find the reason causing drug resistance, particularly go back the method that neither one detects drug resistance, the resistance mechanism of new Gefitinib is found by this detection data research, thus for developing the therapeutic strategy delaying or overcome gefitinib resistant and provide new.
Summary of the invention
The object of the invention is to detect Ji Feini for resistance mechanism effective ways for there is no at present, finding out this situation of resistance mechanism reason, propose a kind of Ji Feini that detects for resistance mechanism effective ways, for further Effect of Anti drug resistance or method provide reference frame.
A kind of Ji Feini that detects is for Resistance Mechanism method, and this technical scheme comprises:
adopt iTRAQ quantitatively (isotope labeling relatively or absolute quantitation) proteomic techniques high flux screening is carried out to the protein signal molecule participating in Gefitinib resistance;
adopt the test of westernblot(protein immunoblotting) technical identification, the differentially expressed protein signaling molecule of the gefitinib lung cancer cell line that screening obtains and Gefitinib drug-resistant cell strain;
adopt MTT(MTT cytotoxicity assay) method, prove the expression disturbing the protein signal molecule found, the susceptibility of Gefitinib can be increased, thus prove that the change of newfound protein signal developed by molecule level is the one mechanism of Gefitinib resistance.
The present invention has following remarkable result: by the enforcement of technical solution of the present invention, contribute to the protein signal molecule new participation Gefitinib resistance being detected, set forth Gefitinib resistance new mechanism, design resistance medicine by the new protein signal molecule detected, reach the object reversing Gefitinib resistance basis.
Accompanying drawing explanation
Fig. 1 is iTRAQ quantitative proteomics analytical technology process flow diagram.
Fig. 2 is westernblot technology for detection result display figure.
Fig. 3 is MTT technology for detection result display figure.
Embodiment
The specific embodiment of the present invention is as follows:
1, the screening of Gefitinib drug-resistant protein signaling molecule is participated in.
The iTRAQ quantitative proteomics technology of generally acknowledging in the world is at present adopted to screen the protein signal molecule participating in Gefitinib resistance.Concrete steps comprise: with human lung carcinoma cell line PC-9(normal lung cancer cell, responsive to treated with gefitinib) be control group, with human lung carcinoma cell line PC-9R(, PC-9 cell line is adopted long-time low dosage treated with gefitinib, the Gefitinib drug-resistant cell strain obtained) be experimental group, adopt the protein signal molecule of iTRAQ quantitative proteomics technology screening PC-9 and PC-9R cell line differential expression.The concrete techniqueflow of iTRAQ quantitative proteomics technology is shown in Fig. 1.
2, whether newfound protein signal molecule there are differences the checking of expression in PC-9 and PC-9R.
Find screening, compared with PC-9, the protein signal molecule of up-regulated in PC-9R, adopts westernblot technology to verify its actual expression.The key step of westernblot technology comprises: protein extraction, determination of protein concentration, gel electrophoresis, albumen transferring film, antibody incubation and protein expression level detection etc.
3, new discovery protein signal molecule participates in the checking of Gefitinib resistance.
To PC-9R and PC-9R of interference new discovery protein signal developed by molecule itself, carry out treated with gefitinib, mtt assay is adopted whether to compare interference new discovery protein signal molecule, Gefitinib is on the impact of the PC-9R rate of increase, thus confirm interference new discovery protein signal developed by molecule, the susceptibility of PC-9R to Gefitinib can be increased, and then explanation, the numerator mediated Gefitinib resistance of newfound protein signal, has the potential value as drug design target spot.
As shown in Figure 1: iTRAQ quantitative proteomics analytical technology flow process is: the first step, extracts albumen respectively from PC-9 and PC-9R cell sample; Second step, carry out reductive alkylation process to the protein sample after extracting, opened disulfide bond is so that the abundant enzymolysis protein of subsequent step; 3rd step, carries out the concentration determination of albumen by Coomassie Brilliant Blue (Brandford) method, polyacrylamide gel electrophoresis (SDS-PAGE) detects, and equal protein got by each sample, carries out trypsin digestion; 4th step, by iTRAQ reagent mark peptide section; 5th step, carries out mixed in equal amounts by the peptide section after mark; 6th step, uses strong cation exchange chromatography (StrongCationExchangechromatography, SCX) to carry out pre-separation to mixed peptide section; 7th step, carries out liquid phase tandem mass spectrum (liquidchromatographycoupledwithtandemmassspectrometry, LC-MS/MS) analysis; 8th step, data analysis: first for the source document of machine under mass spectrum, carries out peak identification, obtains peak list.Next sets up reference database, carries out the qualification of peptide section and protein.The finally relation of the relative content of more each albumen between each sample, thus obtain target protein.
As shown in Figure 2: AnnexinA1 expresses higher than PC-9(color more black in PC-9R, and expression is higher) β-actin is internal reference, for demarcating the benchmark of protein expression.The molecular weight of AnnexinA1 is the molecular weight of 38.5kDa, β-actin is 45kDa.
As shown in Figure 3: research object adopts the PC-9R cell to Gefitinib resistance, because it is to Gefitinib resistance, so along with the increase of Gefitinib concentration, the appreciation rate of PC-9R has reduction, but not obvious.If first disturb the AnnexinA1 expression of PC-9R cell, and then adopt treated with gefitinib, along with the increase of Gefitinib concentration, the rate of increase of PC-9R cell can be subject to obvious suppression.Illustrate, AnnexinA1 take part in the resistance of Gefitinib, is a kind of new mechanism of Gefitinib resistance., also imply that, AnnexinA1 can as the target spot reversing Gefitinib medicine-resistant medicine meanwhile.
PC-9 and the PC-9R differentially expressed protein signaling molecule that table 1 obtains through screening
| Identification sequence number | Protein signal molecule | Molecular weight (KDa) | Coverage rate | Rise rate | |
| P04083 | Annexin A1 | 38.5 | 59.2 | 1.963666667 |
Note: according to rise rate rank, AnnexinA1 is positioned at the 3rd.In view of the protein signal molecule of rise rate front two has bibliographical information, this programme selects AnnexinA1 to declare as the signaling molecule of new participation Gefitinib resistance.
Rise rate a: part for iTRAQ quantitative proteomics result, thinks that the expression of AnnexinA1 in PC-9R is higher than the expression in PC-9, and has raised 1.96366667 times.
Above-mentioned accompanying drawing illustrates and in table 1: PC-9 is human lung adenocarcinoma PC-9 cell; PC-9R is the human lung adenocarcinoma PC-9 cell cell of resistance to Gefitinib; SCX strong cation exchange is; NM is that nanomole often rises Gefitinib concentration; AnnexinA1 is Annexin A1, and express higher than PC-9(color more black in PC-9R, expression is higher).
Claims (4)
1. detect a method for Gefitinib resistance mechanism, it is characterized in that: the method comprises:
adopt iTRAQ quantitatively (isotope labeling relatively or absolute quantitation) proteomic techniques high flux screening is carried out to the protein signal molecule participating in Gefitinib resistance;
adopt the test of westernblot(protein immunoblotting) technical identification, the differentially expressed protein signaling molecule of the gefitinib lung cancer cell line that screening obtains and Gefitinib drug-resistant cell strain;
adopt MTT(MTT cytotoxicity assay) method, prove the expression disturbing the protein signal molecule found, the susceptibility of Gefitinib can be increased, thus prove that the change of newfound protein signal developed by molecule level is the one mechanism of Gefitinib resistance.
2. detect the method for Gefitinib resistance mechanism according to claim 1, it is characterized in that: the concrete steps that protein signal molecule carries out high flux screening comprise: with human lung carcinoma cell line PC-9(normal lung cancer cell, responsive to treated with gefitinib) be control group, with human lung carcinoma cell line PC-9R(, PC-9 cell line is adopted long-time low dosage treated with gefitinib, the Gefitinib drug-resistant cell strain obtained) be experimental group, adopt the protein signal molecule of iTRAQ quantitative proteomics technology screening PC-9 and PC-9R cell line differential expression.
3. detect the method for Gefitinib resistance mechanism according to claim 1, it is characterized in that: the key step of westernblot technical identification comprises: protein extraction, determination of protein concentration, gel electrophoresis, albumen transferring film, antibody incubation and protein expression level detect.
4. according to claim 1 or 2, detect the method for Gefitinib resistance mechanism, it is characterized in that: iTRAQ quantitative proteomics analytical technology flow process is: the first step, extracts albumen respectively from PC-9 and PC-9R cell sample; Second step, carry out reductive alkylation process to the protein sample after extracting, opened disulfide bond is so that the abundant enzymolysis protein of subsequent step; 3rd step, carries out the concentration determination of albumen by Coomassie Brilliant Blue (Brandford) method, polyacrylamide gel electrophoresis (SDS-PAGE) detects, and equal protein got by each sample, carries out trypsin digestion; 4th step, by iTRAQ reagent mark peptide section; 5th step, carries out mixed in equal amounts by the peptide section after mark; 6th step, uses strong cation exchange chromatography (StrongCationExchangechromatography, SCX) to carry out pre-separation to mixed peptide section; 7th step, carries out liquid phase tandem mass spectrum (liquidchromatographycoupledwithtandemmassspectrometry, LC-MS/MS) analysis; 8th step, data analysis: first for the source document of machine under mass spectrum, carries out peak identification, obtains peak list.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546754A (en) * | 2016-12-09 | 2017-03-29 | 新疆医科大学 | Yimusake table acts on abnormal mucus cross-examination with sexual impotence Syndrome model target point protein and its screening technique |
| CN112485442A (en) * | 2020-11-12 | 2021-03-12 | 中国药科大学 | Small molecule target screening method based on chemical proteomics and application thereof |
Citations (2)
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| US20050272083A1 (en) * | 2004-06-04 | 2005-12-08 | Somasekar Seshagiri | EGFR mutations |
| CN102321733A (en) * | 2010-11-15 | 2012-01-18 | 上海聚类生物科技有限公司 | Method for analyzing iTRAQ (isobaric Tags for Relative and Absolute Quantitation) data |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050272083A1 (en) * | 2004-06-04 | 2005-12-08 | Somasekar Seshagiri | EGFR mutations |
| CN102321733A (en) * | 2010-11-15 | 2012-01-18 | 上海聚类生物科技有限公司 | Method for analyzing iTRAQ (isobaric Tags for Relative and Absolute Quantitation) data |
Non-Patent Citations (2)
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| 孙传玉: "基于前列腺癌蛋白质组学研究筛选出的Periostin在前列腺癌中的表达及功能研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 蔡习强: "基于iTRAQ的胃癌多药耐药相关蛋白筛选研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546754A (en) * | 2016-12-09 | 2017-03-29 | 新疆医科大学 | Yimusake table acts on abnormal mucus cross-examination with sexual impotence Syndrome model target point protein and its screening technique |
| CN112485442A (en) * | 2020-11-12 | 2021-03-12 | 中国药科大学 | Small molecule target screening method based on chemical proteomics and application thereof |
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