CN108117591A - A kind of biomarker for Diagnosis of Epilepsy - Google Patents

Abstract

The invention discloses a biomarker or its biodegradation product for epilepsy prediction, diagnosis, treatment and prognosis evaluation. The protein name is: Hemoglobin subunit alpha, and the coding gene is HBB. In particular, the present invention relates to proteomic screening and detection methods of Hemoglobin subunit beta protein or its biodegradation products. The results obtained by this method showed that the content of Hemoglobin subunit beta or its degradation products in the serum of epileptic patients before and after surgery for primary epilepsy resection showed significant differences (Abstract Figure 1). The biomarker protein Hemoglobin subunit beta or its biodegradation product proposed by the present invention has far-reaching significance in the fields of epilepsy research, prediction, diagnosis, treatment and prognosis evaluation.

Description

A biomarker for the diagnosis of epilepsy

technical field

The invention relates to the field of biomarkers for disease diagnosis and prediction. In particular, the present invention relates to the use of Hemoglobin subunit beta protein and its biodegradation products as biomarkers in epilepsy prediction, diagnosis, treatment and prognosis assessment, as well as proteomics screening and detection methods. It also relates to the application of Hemoglobin subunit beta protein or its biodegradation product in making epilepsy detection kit.

Background technique

Epilepsy is a syndrome of acute brain dysfunction caused by the abnormally synchronized discharge of neurons in the brain. According to epidemiological statistics, at present, epilepsy affects about 1% of the world's population, and its incidence rate is second only to neurovascular diseases. According to the latest epidemiology in my country, the incidence of epilepsy in my country is 7‰-1%.

Epilepsy has an extremely serious impact on the neurological function of patients, especially for infants and young children in the early stages of growth and development, even catastrophic. The 3 years after birth is the golden period for the development of the human nervous system. During this period, the nervous system must gradually form a normal neural network to gradually realize functions such as movement, speech, memory, and comprehension. However, due to epilepsy The repeated attacks of the disease lead to the inability of the children's nervous system to establish normal connections, and then the development of neuromotor function is delayed, which seriously affects the prognosis and future life of the children. For adults, the impact of epilepsy on neurological function cannot be underestimated. Due to repeated epileptic seizures, patients suffer from memory loss, cognitive decline, and mental disorders. Very serious mental burden. Therefore, epilepsy has become a serious economic and social burden both for patients and their families. Being able to diagnose and intervene in the early stage of epilepsy is of great help to the improvement of neurological function and prognosis of patients.

At present, there are three main ways to treat epilepsy, drugs, surgery and neuromodulation therapy. 70% of the patients are still treated with drugs, but about 1/3 of the patients are still ineffective to drug therapy. Epilepsy that doesn't work is called drug-resistant epilepsy. According to the more popular diagnostic criteria, the diagnosis of drug-refractory epilepsy is mainly based on two or more appropriate antiepileptic drugs taken orally for two years, and the average seizure day per month is greater than or equal to one day. Clinically, the more common drug-resistant epilepsy includes: temporal lobe epilepsy, epilepsy secondary to trauma, tumor, hemorrhage and other factors, and temporal lobe epilepsy accounts for about 50% of drug-resistant epilepsy, and drug-resistant epilepsy Treatment options for curative epilepsy include excisional surgery, palliative surgery, and neuromodulation surgery.

Clinically, for different refractory epilepsy patients, it is necessary to comprehensively judge which surgical treatment method to choose based on EEG, imaging, ictal symptoms, and brain function damage, but the prognosis of treatment varies greatly. . Sometimes, the same type of epilepsy, such as: medial temporal lobe epilepsy, adopts the same surgical method (hippocampus, amygdala and temporal lobe resection), and the treatment effect of some patients is satisfactory, but some patients still have seizure symptoms after surgery . Moreover, the resection surgery itself needs to remove part of the normal brain tissue during the resection of the lesion, so neurological deficits will inevitably occur after the operation. More importantly, there may only be one chance of surgery. Therefore, it is necessary and urgent to find the factors that determine the difference in treatment effect, so as to select the best surgical plan based on the difference factors and predict the effect of surgical treatment, so as to avoid neurological damage that is difficult to recover. At present, there are still no effective predictive methods and methods for the clinical treatment of epilepsy.

In addition, after surgery for drug-refractory epilepsy, it is generally necessary to continue oral antiepileptic drug therapy for at least 2 years, and then judge whether the drug can be reduced or stopped based on the patient's prognosis and seizure status. However, clinically, the side effects of long-term oral antiepileptic drugs, such as: obesity, mental symptoms, liver and kidney function damage, etc., as well as economic factors are recognized by clinicians. Therefore, the use of antiepileptic drugs should be shortened as much as possible after surgery. Period, is very important to reduce the impact of the above side effects.

At present, there is still no method and kit product for the prediction, diagnosis, treatment and prognosis evaluation of serum or cerebrospinal fluid for the above-mentioned problems in clinical practice. Based on the technology of proteomics, the present invention discovers a relatively stable protein or its biodegradation product that can be used as a biomarker of epilepsy through the research on the serum of patients with drug-refractory epilepsy, which has a relatively broad clinical application prospect.

Contents of the invention

The present invention adopts the method of proteomics, takes the serum of patients with drug-refractory epilepsy as the research object, and finds a relatively stable biomarker, which is Hemoglobin subunit beta protein or its biodegradation product, and the coding gene is HBB. This biomarker has stable and high expression in preoperative serum of patients with drug-refractory epilepsy, especially temporal lobe epilepsy, but the expression level in serum collected at different time points after surgery is significantly decreased. This result provides A new idea and method for epilepsy prediction, diagnosis, treatment and prognosis evaluation.

Therefore, the present invention relates to a biomarker for epilepsy prediction, diagnosis, treatment and prognosis assessment, which is Hemoglobin subunit beta protein encoding gene HBB or its biodegradation product. Its amino acid sequence is shown in the sequence table SEQ ID No.1.

In the first aspect, the present invention provides the screening and detection method of the biomarker, which includes screening and detection using proteomics technology in biological samples.

Further, the biological sample is body fluid or tissue, preferably a sample from serum or cerebrospinal fluid.

Further, the proteomics technology includes: sample preparation, high-performance liquid chromatography (HPLC) to remove high-abundance proteins, sample digestion, desalting, labeling, high-performance liquid chromatography (HPLC) peptide separation, mass spectrometry (MS) detection, analysis, etc.

In the second aspect, the use of the protein or its biodegradation product in preparing biomarkers for epilepsy prediction, diagnosis, treatment and prognosis assessment.

Furthermore, the biomarker involved in the present invention, Hemoglobin subunit beta protein or its biodegradation product, can be used as an index of epileptic neuron excitability.

Furthermore, the biomarker involved in the present invention, Hemoglobin subunit beta protein or its biodegradation product, can be used for the prediction and typing diagnosis of drug-refractory epilepsy, such as temporal lobe epilepsy and the like.

Furthermore, the biomarker involved in the present invention, Hemoglobin subunit beta protein or its biodegradation product, can be used for postoperative prognosis assessment of drug-refractory epilepsy, as well as an indicator for reducing drug dose or stopping (reducing drug withdrawal).

In a third aspect, the present invention relates to a screening and detection system for a Hemoglobin subunit beta protein or its biodegradation product, which includes detecting the expression of the Hemoglobin subunit beta protein or its biodegradation product in the body fluid or tissue of a subject, the The body fluid or tissue is preferably a sample from serum or cerebrospinal fluid.

Further, the screening and detection system includes the single application of the protein or its biodegradation products described in this patent or the combined application with other biomarkers in the detection of epilepsy diseases.

In the fourth aspect, the application of the biomarker in the preparation of a kit for epilepsy prediction, diagnosis, treatment and prognosis assessment.

Further, the kit includes an antibody encoding a Hemoglobin subunit beta protein whose gene is HBB or a biodegradation product thereof.

The Hemoglobin subunit beta protein or its biodegradation products detected in the present invention are found in the serum of patients with drug-refractory epilepsy through the method of proteomics, and the changes of the detected samples of different patients before and after surgery have a high degree of Consistency and stability, can be used as a biomarker for drug-resistant epilepsy, and can be used to include the evaluation of epileptic disease states, that is, the excitability of epileptic neurons, the classification of drug-resistant epilepsy, and the diagnosis of drug-resistant epilepsy Postoperative reduction and withdrawal of drug basis and so on. However, the methods and biomarkers of the present invention are not limited to the above aspects.

Regarding the description of the terms in the present invention, if it is different from the well-known description in the art, the description in this application shall prevail.

Description of drawings

Fig. 1 proteomics technology experiment flowchart of the present invention;

Fig. 2 The liquid phase diagram of high-performance liquid chromatography (HPLC) separation high-abundance protein;

Fig. 3 is a liquid phase diagram of high performance liquid chromatography (HPLC) peptide segment separation;

Figure 4 Mass spectrometry (MS) quantification of HBB peptides.

Figure 5 Screening experiment High-performance liquid chromatography (HPLC) to remove high-abundance protein peak area

Fig. 6 Validation experiment 9 groups of samples High-performance liquid chromatography (HPLC) removes the average value of the peak area of high-abundance proteins

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the described embodiments are explanations of the present invention rather than limitations.

Embodiment 1

1. Sample Collection

There were 6 patients clinically diagnosed as drug-refractory epilepsy (Table 1), all of whom were temporal lobe epilepsy. They all underwent the same surgical treatment: anterior temporal lobe plus hippocampal amygdala resection. Follow-up was carried out after operation. During the follow-up, the 6 patients were all in line with the standard of clinical cure through the verification of EEG and clinical seizures. The peripheral venous blood or arterial blood of the patients was collected according to different time points, the time points were: preoperative, postoperative follow-up for half a year, and postoperative follow-up for one year. 5ml of peripheral blood was drawn each time, centrifuged at 4000rpm/min for 20 minutes, and then the supernatant (serum) was collected and stored at -80°C.

Table 1. Basic information of clinical samples

Case I is the screening experimental sample, and cases II-VI are the verification experimental samples

2. Experimental technical process (Figure 1)-screening experiment

2.1. Sample preparation

(1) The preoperative serum "I-PRE" and postoperative serum "I-POST-HALF" of case I were taken out and thawed at room temperature, 20% of each serum was extracted, and "Buffer A" reagent (Agilent, USA) was added to them respectively. company) 60μl, mix well.

(2) After the proportioning is completed, the two groups of samples (80 μl each) are centrifuged at 16000 g for 15 min (20° C.).

2.2. High-performance liquid chromatography (HPLC) to remove high-abundance protein experiment (Figure 2)

(1) The two groups of samples prepared above were transferred to the chassis of the autosampler of the high performance liquid chromatograph (Thermo-Ultimate 3000, USA).

(2) Select the American Agilent MARC liquid chromatography column, and set the separation program (LC method).

(3) Two groups of samples were recovered after removal of high-abundance proteins.

2.3. Digestion of samples

(1) Add dithiothreitol (DTT) to the above two groups of samples respectively, and react for 30 minutes.

(2) After the reaction, add iodoacetamide (IAA) to each group of samples and react in the dark for 30 minutes.

(3) Add Trypsin digestion to each group of samples at a ratio of 1:20-1:100 (m/m), and shake overnight at 37°C.

2.4. Desalting, labeling and drying of samples

(1) Take two SPE desalting columns (Waters HLB Cartridge) and wet them with 1ml ACN respectively.

(2) Equilibrate 2 desalting columns with 1ml each of ddH ₂ O.

(3) Put the two groups of samples into two desalting columns respectively.

(4) Wash 2 desalting columns loaded with samples with 1ml each of ddH ₂ O.

(5) Wash 2 desalting columns loaded with samples with 1ml each of PBS.

(6) The two desalting columns were washed with 1ml of heavy label reagent (CD ₂ O) and light label reagent (CH ₂ O) respectively, and washed 5 times each. "I-PRE" is a heavy mark, and "I-POST-HALF" is a light mark.

(7) 1ml each of ddH ₂ O was used to wash the 2 desalting columns loaded with the labeled samples.

(8) The two groups of samples were eluted with 500 μl of TFA each. After elution, the labeled "I-PRE" and "I-POST-HALF" samples were combined. The combined sample number: 1.

(9) The combined samples should be dried with a Thermo vacuum dryer in the United States.

2.5. High performance liquid chromatography (HPLC) peptide separation experiment

(1) Add 50 μl of phase A (aqueous phase) to the dried sample to dissolve, centrifuge at 16000 g, 20 ° C, for 15 min.

(2) The above samples were transferred to the chassis of the autosampler of the high performance liquid chromatograph (Thermo-Ultimate 3000, USA).

(3) Select the American Waters BEH C18 liquid chromatography column, and set the separation program (LC method).

(4) After the peptides of the samples were separated (Figure 3), they were recovered into 24 recovery bottles.

(5) The recovered samples were combined into 12 bottles according to the principle of "the first bottle + the 13th bottle, the second bottle + the 14th bottle...".

(6) The combined samples should be dried with a Thermo vacuum dryer in the United States.

2.6. Mass spectrometry detection and analysis experiments

(1) Add 10 μl of 1% acetic acid (AA) to each of the 12 samples after draining to dissolve, and mix well.

(2) 12 samples (10 μl each) were centrifuged at 16,000 g at 20°C for 15 min.

(3) Transfer the above sample to the liquid phase part of a liquid phase mass spectrometer (Thermo-LTQ Orbitrap Elite, USA).

(4) Set up the mass spectrometry program, collect data, search the database, and analyze (Fig. 4).

3. Result Analysis

Through the above experiment process, it is obtained that

3.1 The peak area of the total protein to be tested obtained in step 2.2 is very different between the sample "I-PRE" and the sample "I-POST-HALF" (Figure 5). This shows that compared with the preoperative sample, the amount of total protein to be tested in the serum decreased significantly after the postoperative sample.

3.2 Hemoglobin subunit beta protein or its degradation product, the encoding gene is HBB, which changed significantly before and after the operation of case I, and the ratio before and after the operation was "PRE/POST-HALF, H/L=563.32" (Table 2), that is, after operation The expression level of this protein was 563.32 times higher than that after operation.

Table 2. Screening experiment results

Embodiment 2

Verification experiment based on the above results to expand the sample

The experimental technical process is the same as the screening experimental technical process (Figure 1)

The case numbers of the verification experimental samples are II-VI, and they are cured cases of temporal lobe epilepsy like the screening experimental case I.

The reagents, proportioning ratio, reaction time, light-heavy labeling and other methods used in the verification experiment are the same as those in the screening experiment.

Table 3. Sample grouping and labeling methods for verification experiments

Labeled sample number Remark (H) Light (L) 2 II-PRE II-POST-HALF 3 III-PRE III-POST-HALF 4 IV-PRE IV-POST-HALF 5 IV-PRE IV-POST-ONE 6 V-PRE V-POST-HALF 7 V-PRE V-POST-ONE 8 VI-PRE VI-POST-HALF 9 VI-PRE VI-POST-ONE

Result analysis

After the above verification experiment process, it is concluded that:

1. The total protein peak area "POST" sample (n=8) obtained after the step "high-performance liquid chromatography (HPLC) to remove high-abundance protein experiment" is compared with the "PRE" sample (n=8) There was a significant decrease (Figure 6). This shows that the expression level of total protein in serum has a significant decrease after operation compared with before operation.

2. Hemoglobin subunit beta protein or its degradation product, the coding gene is HBB, its changes in the other 8 groups of samples are still very obvious, combined with the results of the screening experiment, a total of 9 groups, the comprehensive T-test statistical results show that Hemoglobin subunit beta protein There is a significant difference in the changes of its degradation products before and after the operation, and the statistical results are statistically significant (P<0.05=(Table 4).

Table 4. Experimental results and statistical analysis

Embodiment 3

Kit: a kit for detecting Hemoglobin subunit beta protein or its degradation products in body fluids or tissues such as serum or cerebrospinal fluid, which contains antibodies against Hemoglobin subunit beta protein or its degradation products.

The steps of using the above kit to detect the Hemoglobin subunit beta protein or its degradation products in the patient's serum: collect the patient's serum according to the method of embodiment 1, react with the antibody of the Hemoglobin subunit beta protein or its degradation products, and effectively detect the Hemoglobin subunit beta protein or its degradation products of the present invention The expression levels of biomarkers can effectively detect whether the subject has epilepsy, assist in the classification and diagnosis of drug-refractory epilepsy, and determine whether the drug can be reduced or discontinued after surgery for drug-refractory epilepsy.

The purpose of the above-mentioned embodiment is to further describe the specific implementation of the present invention, and not to limit the present invention. Within the scope of protection of the technical solution of the invention.

SEQUENCE LISTING

<110> Peking University

<120> A biomarker for the diagnosis of epilepsy

<160> 1

<210> 1

<211> 147

<212> PRT

<213> Homo sapiens

<400> 1

Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp

1 5 10 15

Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu

20 25 30

Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp

35 40 45

Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His

50 55 60

Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp

65 70 75 80

Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys

85 90 95

Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val

100 105 110

Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln

115 120 125

Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His

130 135 140

Lys Tyr His

145

Claims (8)

1. a kind of biomarker for epileptic prediction, diagnosis, treatment and prognosis evaluation is that encoding gene is HBB's Hemoglobin subunit beta albumen or its biodegradable product.

The full length amino acid sequence of the albumen Hemoglobin subunit beta is as shown in SEQ ID No.1 in sequence table.

2. the biomarker in claim 1 is for the intact proteins of Hemoglobin subunit beta or including part SEQ The biodegradable product of ID No.1 sequences.

3. albumen according to claim 1 or its biodegradable product for the prediction of epileptic condition, diagnosis, treatment and Purposes in prognosis condition predicting as biomarker.

4. application of the biomarker in preparing for detection kit according to any one of claim 2.

5. the screening of biomarker described in a kind of claim 1 is with detection method in epilepsy biomarker screens and detects Commercial use.It is included in biological sample, is screened and is detected using the technology of proteomics, the spy of screening technique Sign comprises the following steps：

(1) pre-treatment is carried out to sample, sample adds in high performance liquid chromatography by treated, and uses Agilent MARC liquid phases High-abundance proteins in chromatographic column removal sample.

(2) sample is digested, desalination and mark.

(3) treated sample is added in into high performance liquid chromatography, and using Waters BEH C18 liquid-phase chromatographic columns to sample into Row subdivision.

(4) sample after subdivision is drained, is redissolved with 1% acetic acid, be transferred in liquid phase mass spectrograph and be detected.

(5) potential biomarker is selected in gathered data, database retrieval, analysis.

6. screening technique according to claim 5, it is characterised in that Agilent MARC liquid chromatograies are selected in step (1) High-abundance proteins in column removal epilepsy sample.

7. screening technique according to claim 5, it is characterised in that Waters BEH C18 liquid phase colors are selected in step (3) Spectrum column is finely divided the ingredient in pretreated epilepsy sample.

8. screening technique according to claim 5, it is characterised in that wherein described biological sample is the body fluid of epileptic Or organize, it is preferred from the body fluid of serum or cerebrospinal fluid.