CN107703216A - A kind of method of the HG of quick detection tumor tissues 2 accumulations - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to a method for rapidly detecting the accumulation of tumor tissue carcinogenic metabolite 2-hydroxyglutaric acid 2-HG. In the method, a tumor tissue sample is obtained, placed in a centrifuge tube, and pre-cooled pure methanol is added , centrifuge at 14000 rpm after the homogenizer grinds and stands still, take the supernatant and transfer it to a glass bottle, add the derivatization reagent MTBSTFA after the vacuum drying centrifuge is fully dried, place at 70 ° C, cool at room temperature, and put it on the machine, Agilent 7890A-5975C; The column is HP-5MS, the carrier gas is high-purity helium, and the 2-HG is identified by the combination of NIST database search and standard verification. The test results show that using the detection method of the present invention to detect whether there is a high level of 2-HG accumulation in the brain tissue during the operation will help realize the rapid differential diagnosis of IDH1 mutant gliomas and provide important information for postoperative individualized treatment. guidance information.

Description

A method for rapid detection of 2-HG accumulation in tumor tissue

technical field

The invention belongs to the field of biotechnology, and relates to carcinogenic metabolite 2-hydroxyglutarate (2-HG), in particular to a method for rapidly detecting the accumulation of 2-HG in tumor tissue. By detecting the accumulation of 2-HG in tumor tissue, it helps For the rapid diagnosis of IDH1 mutations.

Background technique

The prior art discloses that glioma is the most common tumor of the central nervous system, among which the cure rate of malignant glioma is low, the recurrence rate is high, and the prognosis is extremely poor. At present, synchronous radiotherapy and chemotherapy after surgery plus 6 courses of treatment are mostly used clinically Temozolomide (TMZ) chemotherapy, the average survival period is only 12-15 months. Therefore, the current clinical problem to be solved for malignant glioma is to find a method for early diagnosis and early treatment.

In current clinical practice, stereotaxic biopsy is required for some lesions that are not clearly diagnosed. Typical cases are easier to diagnose. However, in practice, the pathology of frozen sections during surgery is often not typical, and pathologists can only observe gliosis. Gliosis can occur in low-grade glioma, normal brain tissue, inflammation, and some lymphomas. Although mutant IDH1 immunohistochemical staining can differentiate glioma from other conditions, it usually takes 5-7 days, which takes a long time, and intraoperative judgment cannot be realized, and sometimes even repeated punctures are required, which increases the burden on patients and the risk of surgery.

Studies have disclosed that most diffuse human gliomas are often accompanied by mutations of isocitrate dehydrogenase gene 1 (IDH1). Clinical practice shows that patients with gliomas with this mutation have It has a good prognostic effect and is more sensitive to chemotherapy and targeted therapy. , therefore. IDH1 mutation is an important molecular typing marker for glioma; IDH catalyzes the oxidative decarboxylation of isocitrate to generate a-ketoglutarate (a-KG), while the mutant IDH loses its normal enzyme activity, Using α-KG as a substrate, the carcinogenic metabolite 2-hydroxyglutarate (2-HG) is generated. Normally, 2-HG is low in normal brain tissue but highly accumulated in IDH1-mutant gliomas. Related studies have shown that IDH1-mutant cells rely on 2-HG to maintain their oncogenic properties. Therefore, the rapid detection of IDH1 mutation will help in the differential diagnosis of glioma, and facilitate the formulation and optimization of individualized treatment plan, which is of great clinical significance. Based on this, the inventors of the present application intend to provide a method for rapid detection of 2-HG accumulation in tumor tissue, by detecting the accumulation of 2-HG in tumor tissue, the rapid diagnosis of IDH1 mutation will be realized.

Contents of the invention

The purpose of the present invention is to provide a method for rapid detection of 2-HG accumulation in tumor tissue in view of the status quo of the prior art. By detecting the accumulation of 2-HG in tumor tissue, it provides a basis for the rapid diagnosis of IDH1 mutation and provides a basis for postoperative diagnosis. Individualized treatment provides important guiding information.

Specifically, the method for rapidly detecting tumor tissue 2-HG accumulation of the present invention comprises the steps of:

1. Obtain tumor tissue samples and place them in centrifuge tubes;

2. Add pre-cooled pure methanol to the above centrifuge tube;

3. Grind with a homogenizer for 1 min and let stand for a while;

4. 14000rpm centrifugal;

5. Take the supernatant, transfer it to a glass bottle, and fully dry it in a vacuum drying centrifuge;

6. Add the derivatization reagent MTBSTFA and place at 70°C;

7. Cool at room temperature, and put it on the machine (Agilent 7890A-5975C; the chromatographic column is HP-5MS, and the carrier gas is high-purity helium.

2-HG was identified by a combination of NIST database search and standard verification.

In this application, rapid detection was performed on the isolated glioma brain tissue obtained during the operation, and the detection results showed that, by using the detection method of the present invention, it is possible to detect whether there is a high level of 2-HG accumulation in the brain tissue during the operation. It helps to realize the rapid differential diagnosis of IDH1 mutant glioma, and provides important guidance information for postoperative individualized treatment.

The advantages of the method for detecting tumor tissue 2-HG accumulation of the present invention are:

1. While detecting 2-HG, it can provide specific reference data for the diagnosis of IDH1 mutation;

2. Repeated puncture is not required for some lesions with unclear diagnosis, which significantly reduces the risk of patients;

3. The detection time is fast, and a preliminary judgment can be obtained during the operation. It only needs to judge whether there is 2-HG accumulation, and then distinguish whether it is a glioma or a glioma with an IDH1 mutation, which provides important information for clinical judgment.

For ease of understanding, the method of the present invention will be described in detail below through specific examples. It should be pointed out that the specific examples are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein, and these amendments and changes are also incorporated into within the scope of the present invention.

detailed description

Example 1

The isolated glioma brain tissue obtained during glioma surgery was rapidly detected, and 25-30 μg of the tumor tissue sample was excised and placed in a 1.5ml centrifuge tube; 100 μl of pre-cooled pure Methanol; Grind with a hand-held homogenizer for 1 min, and let stand for a while; Centrifuge at 14,000 rpm for 10 min; Take 40 μl of the supernatant, transfer it to a V-bottom GC special glass bottle, and fully dry it in a vacuum drying centrifuge, which takes about 25 min;. In a fume hood Add 40 μl of derivatization reagent MTBSTFA, tightly cap the bottle, and place it at 70°C for 25 minutes; let it cool naturally at room temperature, and put it on the machine, Agilent 7890A-5975C; the chromatographic column is HP-5MS, and the carrier gas is high-purity helium. 2-HG was identified by a combination of NIST database search and standard verification.

The test results show that using the detection method of the present invention to detect whether there is a high-level accumulation of 2-HG in the brain tissue during the operation is helpful to realize the rapid differential diagnosis of IDH1 mutant glioma, and provides a basis for individualized treatment after the operation. Important Guidance Information.

Claims (7)

1. 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. 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. 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. 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. 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 moment

   The 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. 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. 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.