CN107703216A - A kind of method of the HG of quick detection tumor tissues 2 accumulations - Google Patents
A kind of method of the HG of quick detection tumor tissues 2 accumulations Download PDFInfo
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- CN107703216A CN107703216A CN201610637196.4A CN201610637196A CN107703216A CN 107703216 A CN107703216 A CN 107703216A CN 201610637196 A CN201610637196 A CN 201610637196A CN 107703216 A CN107703216 A CN 107703216A
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- accumulations
- tumor tissues
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F16/00—Information retrieval; Database structures therefor; File system structures therefor
- G06F16/90—Details of database functions independent of the retrieved data types
- G06F16/903—Querying
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention belongs to biological technical field, it is related to a kind of method of the HG of 2 hydroxyl glutaric acid of quick detection tumor tissues carcinogenic metabolites 2 accumulations, will obtains neoplasmic tissue sample in this method, and be placed in centrifuge tube, add the pure methanol of precooling, 14000 rpm are centrifuged after refiner grinding is stood, and take supernatant to be transferred in vial, and vacuum drying centrifuge adds derivatization reagent MTBSTFA after fully drying, 70 DEG C of placements, room temperature cools down, upper machine, Agilent 7890A 5975C；Chromatographic column is HP 5MS, and carrier gas is high-purity helium, and 2 HG are identified by the way of NIST database search and standard items checking double combination.Testing result is shown, is detected using the detection method of the present invention in art in brain tissue with the presence or absence of 2 HG high level accumulations, is helped to realize the rapid differential diagnosis of IDH1 mutation gliomas, and provide important guiding information for postoperative individualized treatment.
Description
Technical field
The invention belongs to biological technical field, is related to carcinogenic metabolites 2- hydroxyl glutaric acids（2-HG）, and in particular to it is a kind of
The method of quick detection tumor tissues 2-HG accumulations, accumulated by detecting 2-HG in tumor tissues, contribute to IDH1 to be mutated fast
Speed diagnosis.
Background technology
Prior art discloses glioma（glioma）It is the most common tumour of central nervous system, wherein glioblastoma
Cure rate is low, high recurrence rate, poor prognosis, more Temozolomides for adding 6 courses for the treatment of using Post operation Synchronous chemoradiotherapy clinical at present
(TMZ) chemotherapy, its mean survival time (MST) are only 12-15 months.Therefore, the problem that need to be solved for glioblastoma clinical at present is
Find the method that early diagnosis is early controlled.
At present in clinical practice, need to carry out stereo-directional puncture biopsy, typical case for the true focus of some acatalepsias
Person is easier to diagnose, but often frozen section pathology and is not true to type in art in practical operation, and Pathologis can only be observed
Gliosis, and gliosis may occur in which in Low grade glioma, normal cerebral tissue, inflammation, part lymthoma etc..It is although prominent
Modification IDH1 immunohistochemical stainings can differentiate glioma and other situations, but generally require 5-7 days, and time-consuming, and can not realize
Judge in art, even need to puncture repeatedly sometimes, increase patient's burden and operation risk.
Research discloses, often along with isocitric acid dehydrogenase gene in most of diffusivity human gliomas
(isocitrate dehydrogenase gene) 1 (IDH1) mutation clinical practices show there is this glioma being mutated
Patient clinically has good outcome, and more sensitive to chemotherapy and targeted therapy., therefore IDH1 mutation are glue
The important molecule parting mark of matter knurl one；IDH is catalyzed isocitrate oxidation decarboxylation, generates a -one glutaric acids（a-KG）, mutation
Type IDH is while its normal enzymatic activity is lost, and using a-KG as substrate, generates carcinogenic metabolites 2- hydroxyl glutaric acids（2-
HG）.Generally, 2-HG is horizontal very low in normal cerebral tissue, and the high accumulation in the glioma of IDH1 mutation, correlative study table
Bright, IDH1 mutant cells rely on 2-HG and maintain its carcinogenic nature.Therefore, IDH1 is mutated quick detection, it will help antidiastole
Glioma, and it is great beneficial to the formulation and optimization of individualized treatment scheme, clinical meaning.Based on this, present inventor intends
A kind of method of quick detection tumor tissues 2-HG accumulations is provided, is accumulated by detecting 2-HG in tumor tissues, IDH1 will be realized
The quick diagnosis of mutation.
The content of the invention
It is the present situation for prior art, there is provided a kind of side of quick detection tumor tissues 2-HG accumulations the mesh of the present invention
Method, accumulated by detecting in tumor tissues 2-HG, to realize that the quick diagnosis of IDH1 mutation provides foundation and is postoperative individual
Change treatment and important guiding information is provided.
Specifically, the method for the quick detection tumor tissues 2-HG accumulations of the present invention, it includes step：
1. obtaining neoplasmic tissue sample, it is placed in centrifuge tube；
2. the pure methanol of precooling is added into above-mentioned centrifuge tube；
3. refiner grinds 1 min, a moment is stood；
4.14000rpm centrifugation；
5. taking supernatant, it is transferred in vial, vacuum drying centrifuge is fully dried；
6. add derivatization reagent MTBSTFA, 70 DEG C of placements；
7. room temperature cools down, upper machine（Agilent 7890A-5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.
2-HG is identified by the way of NIST database search and standard items checking double combination.
In the application, quick detection is carried out to the in vitro glioma brain tissue obtained in art, testing result is shown, is adopted
With the detection method of the present invention, detected in art in brain tissue with the presence or absence of 2-HG high level accumulations, help to realize that IDH1 dashes forward
Become the rapid differential diagnosis of glioma, and important guiding information is provided for postoperative individualized treatment.
The advantages of method of the detection tumor tissues 2-HG accumulations of the present invention, has：
1, can be provided while detection to 2-HG for diagnosis IDH1 mutation has specific reference data；
2, it need not be punctured repeatedly for the true focus of some acatalepsias, hence it is evident that reduce patient's danger risk；
3rd, detection time is quick, and preliminary judgement can be obtained in art, need to only determine whether that 2-HG accumulates, and then carry out discriminating to be
No is glioma, or the glioma of IDH1 mutation, and important information is provided for clinical judgment.
In order to make it easy to understand, the method for the present invention will be described in detail by specific embodiment below.Need
It is emphasized that instantiation is merely to explanation, it is clear that one of ordinary skill in the art can according to illustrating herein,
Various modifications and variations are made to the present invention in the scope of the present invention, the model of the present invention is also included in these modifications and variations
In enclosing.
Embodiment
Embodiment 1
Quick detection has been carried out to the in vitro glioma brain tissue obtained in glioma art, neoplasmic tissue sample is cut into 25-
30 μ g, are placed in 1.5ml centrifuge tubes；The 100 pure methanol of μ l precoolings are added into the centrifuge tube；Hand-held refiner grinding 1min,
Stand a moment；14000rpm centrifuges 10min；The μ l of supernatant 40 are taken, are transferred in the GC special glass bottles of V-type bottom, vacuum drying centrifugation
Machine is fully dried, and takes about 25min；The μ l MTBSTFA of derivatization reagent 40 are added in ventilating kitchen, cover tightly bottle cap, 70 DEG C put
Put 25min；Room temperature natural cooling, upper machine, Agilent 7890A-5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.
2-HG is identified by the way of NIST database search and standard items checking double combination.
Testing result is shown, using the detection method of the present invention, is detected in art in brain tissue and be whether there is 2-HG Gao Shui
Flat accumulation, help to realize the rapid differential diagnosis of IDH1 mutation gliomas, and important finger is provided for postoperative individualized treatment
Lead information.
Claims (7)
- A kind of 1. method of quick detection tumor tissues 2-HG accumulations, it is characterised in that it includes step： 1）Obtain tumor group Tissue samples, it is placed in centrifuge tube；2）The pure methanol of precooling is added into above-mentioned centrifuge tube；3）Refiner grinds 1min, stands a moment；4）14000rpm is centrifuged；5）Supernatant is taken, is transferred in vial, vacuum drying centrifuge is fully dried；6）Add derivatization reagent MTBSTFA, 70 DEG C of placements；7）Room temperature cools down, upper machine, Agilent 7890A-5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.
- 2. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that in this method, adopt 2-HG is identified with the mode of NIST database search and standard items checking double combination.
- 3. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 1） In, neoplasmic tissue sample is 25-30 μ g, is placed in 1.5ml centrifuge tubes.
- 4. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 2） In, the 100 pure methanol of μ l precoolings are added into centrifuge tube.
- 5. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 3） In, refiner grinding 1min is held, stands a momentThe method of quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 4） In, 14000rpm centrifugations 10min.
- 6. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 5） In, the μ l of supernatant 40 are taken, are transferred in the GC special glass bottles of V-type bottom, vacuum drying centrifuge dries 25min.
- 7. the method for the quick detection tumor tissues 2-HG accumulations as described in claim 1, it is characterised in that described step 6） In, the μ l MTBSTFA of derivatization reagent 40 are added in ventilating kitchen, cover tightly bottle cap, 70 DEG C of placement 25min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022165917A1 (en) * | 2021-02-05 | 2022-08-11 | 山东大学 | Transcriptional regulation factor specifically responsive to d-2-hydroxyglutaric acid and application thereof |
| CN115248272A (en) * | 2022-06-08 | 2022-10-28 | 深检集团(深圳)医学检验实验室 | Method for detecting alpha-ketoglutaric acid and chiral 2-hydroxyglutaric acid |
Citations (3)
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| JP2011247869A (en) * | 2010-04-27 | 2011-12-08 | Kobe Univ | Inspection method of specific disease using metabolome analysis method |
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| WO2016161525A1 (en) * | 2015-04-10 | 2016-10-13 | Gregor Reid | Diagnosis of bacterial vaginosis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011247869A (en) * | 2010-04-27 | 2011-12-08 | Kobe Univ | Inspection method of specific disease using metabolome analysis method |
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| WO2016161525A1 (en) * | 2015-04-10 | 2016-10-13 | Gregor Reid | Diagnosis of bacterial vaginosis |
Non-Patent Citations (2)
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| 姚卫峰等: "细胞代谢组学及其应用", 《中国临床药理学与治疗学》 * |
| 张辰辰 等: "基于代谢组学的细胞内源性代谢物研究进展", 《药学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022165917A1 (en) * | 2021-02-05 | 2022-08-11 | 山东大学 | Transcriptional regulation factor specifically responsive to d-2-hydroxyglutaric acid and application thereof |
| CN115248272A (en) * | 2022-06-08 | 2022-10-28 | 深检集团(深圳)医学检验实验室 | Method for detecting alpha-ketoglutaric acid and chiral 2-hydroxyglutaric acid |
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