CN108117591A - A kind of biomarker for Diagnosis of Epilepsy - Google Patents
A kind of biomarker for Diagnosis of Epilepsy Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/2857—Seizure disorders; Epilepsy
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Abstract
The invention discloses a kind of biomarkers or its biodegradable product, protein name for epileptic prediction, diagnosis, treatment and prognosis evaluation to be：Hemoglobin subunit alpha, encoding gene HBB.Particularly, the present invention relates to the screening of the proteomics of Hemoglobin subunit beta albumen or its biodegradable product and detection methods.By the method obtain the result shows that, before and after epileptic implements epilepsy primary tumor resection operation, significant difference (Figure of abstract 1) is presented in Hemoglobin subunit beta or its content of degradation products in serum.Biomarker protein Hemoglobin subunit beta or its biodegradable product proposed by the present invention, have far-reaching significance in fields such as the research of epileptic condition, prediction, diagnosis, treatment and prognosis evaluations.
Description
Technical field
The present invention relates to the biomarker fields of medical diagnosis on disease and prediction.Particularly, the present invention relates to
Hemoglobin subunit beta albumen and its biodegradable product are as biomarker in epileptic prediction, diagnosis, treatment
And purposes in prognosis evaluation and protein science screening and detection method.Further relate to Hemoglobin subunit beta albumen
Or application of its biodegradable product in epilepsy detection kit is made.
Background technology
Epilepsy is the syndrome of acute brain malfunction, is as caused by the extremely synchronized electric discharge of cerebral neuron.According to
Epidemiology statistics, at present, epilepsy influence the population in the whole world about 1%, are the diseases that incidence is only second to neurovascular disease
Kind.Display is learned according to China's cri dernier cri disease, the epilepsy invasion rate in China is 7 ‰ -1%.
Epilepsy is extremely serious on the nervous function influence of patient, particularly with infant's even calamity of growth and development early stage
Difficulty.It is the gold period of human nervous system's development in people is postnatal 3 years, in this period, nervous system will
Normal neutral net is gradually formed to be done step-by-step for example：The functions such as movement, speech, memory, comprehension, and due to epilepsy
Recurrent exerbation, causes the nervous system of infant can not establish normal contact, neuromotor function developmental lag then occurs, sternly
Ghost image rings the prognosis of infant and the life in future.And for adult, influence of the epilepsy to nervous function is also that can't neglect
, due to epileptic attack repeatedly, patient is caused decrease of memory occur, cognitive ability declines, and phrenoblabia etc., patient can not
Normal work, life, study, very serious mental burden is caused to patient.Therefore, no matter for patient or for
For household, epilepsy has become a kind of serious economic and burden on society.It can be diagnosed early stage epilepsy is formed, and
Intervene as early as possible, improve for neurological and prognosis has larger help.
At present, there are mainly three types of three kinds of mode, drug, operation and neuromodulation treatment, 70% trouble for the treatment of epilepsy
Person still has that about 1/3 patient is invalid to drug therapy still based on drug therapy, this kind of epilepsy invalid to drug therapy
It is referred to as medically intractable epilepsy.According to popular diagnostic criteria, the diagnosis basis of medically intractable epilepsy is mainly mouth
2 kinds or two or more suitable Antiepileptic Drugs 2 years are taken, are greater than or equal to one day the epileptic attack day of average every month.
Clinically, relatively conventional medically intractable epilepsy includes：Temporal epilepsy, the secondary epilepsy of the factors such as wound, tumour, bleeding
Deng, and the ratio that temporal epilepsy accounts in medically intractable epilepsy about 50%, medically intractable epilepsy can select in the treatment
Including therapies such as the operation of excision property, palliative operation, neuromodulation operations.
Clinically, for different treatment of intractable epilepsy, it is necessary to according to electroencephalogram, iconography, semiotics stage of attack,
The comprehensive descisions such as injury of brain function situation select the mode of which kind of operative treatment, but treatment prognosis has very big difference.Sometimes, together
For the epilepsy of a type, such as：Medial temporal lobe type epilepsy, takes same operation method (hippocampus amygdala temporal lobe excision), and one
Some patientss therapeutic effect is satisfied with, but still some patient is postoperative is still present with epileptic attack symptom.And excision property operation
Itself is inevitably present with nervous function and lacks after surgery due to needing cut-out normal cerebral tissue during removal of lesions
Damage.The chance more importantly performed the operation may only once.Therefore, find determine therapeutic effect difference factor be it is necessary and
Urgent, to select optimal operation plan by foundation of variance factor, and the effect of operative treatment is predicted, so as to avoid being difficult to
The nervous function damage of recovery.At present, the clinically treatment for epilepsy, still without effective predicting means and method.
In addition, after medically intractable epilepsy operative treatment, under normal circumstances, it is necessary to continue oral Antiepileptic Drugs
At least 2 years, according to the prognosis situation of patient and the information summary that breaks out judge whether that drug withdrawal object can be subtracted afterwards.But facing
On bed, side effect caused by Long-term Oral antiepileptic, such as：Obesity, mental symptom, hepatic and renal function damage situations such as and
Economic factor is clinician's admitted facts, and that therefore, after operation shortens antiepileptic as far as possible takes the phase, in mitigation
The influence for stating side effect is extremely important.
At present, clinically still without a kind of prediction of serum or cerebrospinal fluid regarding to the issue above, diagnosis, treatment and
The method and reagent kit product of prognosis evaluation.Technology of the present invention according to proteomics, by suffering to medically intractable epilepsy
The research of person's serum, research, which is found that, relatively stable can be used as the albumen of epileptic condition biomarker or its biodegradation
Product clinically has more wide application prospect.
The content of the invention
The method that the present invention uses proteomics using the serum of medically intractable epilepsy patient as research object, is found
Relatively stable biomarker is Hemoglobin subunit beta albumen or its biodegradable product, encodes base
Because HBB.The biomarker has stable in the preoperative serum of medically intractable epilepsy patient, especially patients with temporal lobe epilepsy
Height expression, but after surgery the follow-up of different time node when the serum that gathers in expression quantity be decreased obviously, this result provides
A kind of new epileptic prediction, diagnosis, the idea and method for the treatment of and prognosis evaluation.
Therefore, the present invention relates to it is a kind of for epileptic prediction, diagnosis, treatment and prognosis evaluation biomarker,
For the Hemoglobin subunit beta albumen of encoding gene HBB or its biodegradable product.Its amino acid sequence such as sequence
Shown in table SEQ ID No.1.
In a first aspect, screening and detection method the present invention provides the biomarker, are included in biological sample
In, it is screened and is detected using the technology of proteomics.
Further, the biological sample is body fluid or tissue, is preferred from the sample of serum or cerebrospinal fluid.
Further, wherein the technology of the proteomics includes：Sample preparation, high performance liquid chromatography (HPLC) removal
High-abundance proteins, the digestion of sample, desalination, mark, high performance liquid chromatography (HPLC) peptides separation, mass spectrum (MS) detection, analysis
Deng.
In second aspect, the albumen or its biodegradable product are being prepared for epileptic prediction, diagnosis, treatment and in advance
Purposes in the biomarker assessed afterwards.
Further, biomarker of the present invention, Hemoglobin subunit beta albumen or its biology drop
Product is solved, the excitatoty index of epileptic neuron can be used as.
Further, biomarker of the present invention, Hemoglobin subunit beta albumen or its biology drop
Product is solved, can be used for prediction and the classification diagnosis of medically intractable epilepsy, such as temporal epilepsy.
Further, biomarker of the present invention, Hemoglobin subunit beta albumen or its biology drop
Solve the index of product, the prognosis evaluation postoperative available for medically intractable epilepsy and reduction dose or drug withdrawal (subtracting drug withdrawal object).
In the third aspect, the present invention relates to a kind of Hemoglobin subunit beta albumen or its biodegradable products
Examination and detecting system, including detect subject body fluid or tissue in Hemoglobin subunit beta albumen or
The expression of its biodegradable product, the body fluid or tissue are preferred from the sample of serum or cerebrospinal fluid.
Further, the examination and detecting system, including the albumen described in this patent or its biodegradable product list
Solely application or the use in conjunction with other biomarkers in epileptic condition detection.
In fourth aspect, the biomarker is being prepared for epileptic prediction, diagnosis, treatment and the examination of prognosis evaluation
Application in agent box.
Further, the kit include encoding gene be HBB Hemoglobin subunit beta albumen or
The antibody of its biodegradable product.
Hemoglobin subunit beta albumen or its biodegradable product detected by the present invention are to pass through albumen
The method of matter group is found in medically intractable epilepsy patients serum, in the different clinical samples operation consents detected
Variation afterwards has high consistency and stability, can be used as a kind of biomarker of medically intractable epilepsy
In including assessing epileptic condition state, i.e. epileptic neuron excitability, the classification diagnosis of medically intractable epilepsy, drug refractory
Epilepsy is postoperative to subtract drug withdrawal object foundation etc..However, the present invention method and biomarker purposes be not limited only to it is above-mentioned
Several aspects.
The description as described in term in the present invention, should be to be described as if different from description well known in the art in the application
It is accurate.
Description of the drawings
The proteomic techniques experiment flow figure of Fig. 1 present invention；
Fig. 2 high performance liquid chromatography (HPLC) separates the liquid phase figure of high-abundance proteins；
The liquid phase figure of Fig. 3 high performance liquid chromatography (HPLC) peptides separation；
The mass spectrum (MS) of Fig. 4 HBB peptide fragments is quantitatively schemed.
Fig. 5 screening experiments high performance liquid chromatography (HPLC) removes high-abundance proteins peak area
9 groups of sample high performance liquid chromatography (HPLC) removal high-abundance proteins peak area average values of Fig. 6 confirmatory experiments
Specific embodiment
The present invention will be further described With reference to embodiment, and the embodiment is the solution to the present invention
It releases rather than limits.
Embodiment 1
1. sample collection
The patient of medically intractable epilepsy is clinically diagnosed as, totally 6 (table 1), is temporal epilepsy, carried out same
Surgical intervention：Preceding temporal lobe adds hippocampus amygdala resection.Postoperative carry out follow-up, during follow-up, passes through electroencephalogram and clinical episodes
The verification of situation, this 6 patients meet the standard of clinical cure.According to the peripheral vein of different timing node acquisition patients
Blood or arterial blood, time point be respectively：Preoperative, Follow-up After half a year, Follow-up After 1 year.Peripheral blood 5ml is extracted every time, with
4000rpm/min is centrifuged 20 minutes, takes supernatant (serum), -80 DEG C of preservations afterwards.
1. clinical sample basic condition of table
I cases are screening experiment sample, and II-VI cases are confirmatory experiment sample
2. experimental technique flow (Fig. 1)-screening experiment
2.1. sample preparation
(1) by the preoperative serum " I-PRE " of I cases and postoperative half a year serum " I-POST-HALF ", room temperature after taking-up
It thaws, it is each to extract serum 20, it is separately added into " Buffer A " reagent (Agilent companies of the U.S.) 60 μ l, mixing.
(2) after the completion of matching, 2 groups of samples (each 80 μ l) are with 16000g centrifugation 15min (20 DEG C).
2.2. high performance liquid chromatography (HPLC) removal high-abundance proteins experiment (Fig. 2)
(1) 2 groups of samples for completing above-mentioned preparation are transferred to high performance liquid chromatograph (U.S. Thermo-Ultimate
3000) in the chassis of autopipette.
(2) U.S.'s Agilent MARC liquid-phase chromatographic columns are selected, and separable programming (LC method) is set.
(3) 2 groups of samples recycle after going high-abundance proteins.
2.3. the digestion of sample
(1) above-mentioned 2 groups of samples are separately added into dithiothreitol (DTT) (DTT), react 30 minutes.
(2) every group of sample adds in iodacetyl ammonia (IAA) after reacting, and is protected from light 30 minutes.
(3) according to 1:20-1:The ratio of 100 (m/m) adds in Trypsin digestions, 37 DEG C of incubator overnights into every group of sample.
2.4. the desalination of sample, mark, drain
(1) 2 pieces of SPE desalting columns (Waters HLB Cartridge) are taken, are soaked respectively with 1ml ACN.
(2)ddH2Each 1ml of O balance 2 pieces of desalting columns respectively.
(3) 2 groups of samples are separately added into 2 pieces of desalting columns.
(4)ddH2Each 1ml of O wash 2 pieces of desalting columns for being loaded with sample respectively.
(5) each 1ml of PBS wash 2 pieces of desalting columns for being loaded with sample respectively.
(6) 2 pieces of desalting columns are respectively with the reagent of the mark again (CD of each 1ml2O reagent (CH) and is gently marked2O) wash, and respectively wash 5 times.
" I-PRE ", which attaches most importance to, to be marked, and " I-POST-HALF " is light mark mark.
(7)ddH2Each 1ml of O wash 2 pieces of desalting columns of the sample that carrying symbols are crossed respectively.
(8) 2 groups of samples are eluted using each 500 μ l of TFA, by labeled " I-PRE " and " I-POST-HALF " sample after elution
This merging, the sample number into spectrum after merging：1.
(9) sample after merging is drained machine using U.S.'s Thermo vacuum and is drained.
2.5. high performance liquid chromatography (HPLC) peptides separation is tested
(1) sample after draining adds in A phases (water phase) 50 μ l dissolvings, and centrifuge 16000g, centrifuges 15min by 20 DEG C.
(2) above-mentioned sample is transferred to the automatic sample of high performance liquid chromatograph (U.S. Thermo-Ultimate 3000)
In the chassis of device.
(3) U.S.'s Waters BEH C18 liquid-phase chromatographic columns are selected, and separable programming (LC method) is set.
(4) (Fig. 3) is recycled in 24 pieces of returnable bottles after sample peptides separation.
(5) sample of recycling merges into 12 bottles of samples according to the principle of " the 1st bottle+the 13 bottle, the 2nd bottle+the 14 bottle ... ".
(6) sample after merging is drained machine using U.S.'s Thermo vacuum and is drained.
2.6. Mass Spectrometer Method, analysis experiment
(1) 12 samples after draining are separately added into 1% acetic acid (AA), 10 μ l dissolvings, mixing.
(2) 12 sample (each 10 μ l) centrifuge 16000g, 20 degree, 15min is centrifuged.
(3) above-mentioned sample is transferred to the liquid phase portion of liquid phase mass spectrograph (U.S. Thermo-LTQ Orbitrap Elite)
Point.
(4) mass spectrum program, gathered data are set, and database retrieval is analyzed (Fig. 4).
3. interpretation of result
By above-mentioned experimentation, draw
The 3.1 total protein peak areas to be measured obtained when carrying out step 2.2, sample " I-PRE " and sample " I-POST-
There is very big difference (Fig. 5) in HALF ".This illustrates postoperative sample compared with preoperative sample, under total protein concentration to be measured is notable in serum
Drop.
3.2Hemoglobin subunit beta albumen or its catabolite, encoding gene HBB, in I cases
Perioperatively variation it is apparent, perioperatively than for " PRE/POST-HALF, H/L=563.32 " (table 2), i.e., the preoperative albumen
Expression quantity be postoperative 563.32 times.
2. screening experiment result of table
Embodiment 2
Confirmatory experiment based on the above results enlarged sample
Experimental technique flow is the same as screening experiment techniqueflow (Fig. 1)
The case number of confirmatory experiment sample is II-VI, is equally all to be controlled after temporal epilepsy is performed the operation with screening experiment case I
More case.
The methods of reagent that confirmatory experiment is applied, proportioning, reaction time, light-heavy label, is same as screening experiment.
The sample packet and labeling method of 3. confirmatory experiment of table
| Sample number after mark | Mark (H) again | Light mark (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 |
Interpretation of result
By above-mentioned confirmatory experiment process, draw：
1. the total protein to be measured obtained after step " experiment of high performance liquid chromatography (HPLC) removal high-abundance proteins " is carried out
Peak area " POST " sample (n=8) is decreased significantly (Fig. 6) compared with " PRE " sample (n=8).This illustrates, total in serum
The expression of albumen, postoperative compare preoperative have significant decline.
2.Hemoglobin subunit beta albumen or its catabolite, encoding gene HBB, in other 8 groups of samples
Variation in this still clearly, with reference to screening experiment totally 9 groups as a result, carry out comprehensive T-test statistical result showeds,
Hemoglobin subunit beta albumen or its catabolite before surgery after variation there is significant difference, statistical result
Statistically significant (P ＜ 0.05=(table 4).
4. experimental result of table and statistical analysis
Embodiment 3
Kit：Detect the body fluid such as serum or cerebrospinal fluid or tissue in Hemoglobin subunit beta albumen or its
The kit of catabolite, it includes anti-Hemoglobin subunit beta albumen or the antibody of its catabolite.
Hemoglobin subunit beta albumen or its catabolite in patients serum are detected using mentioned reagent box
Step：The serum of patient is gathered in the way of embodiment 1, with Hemoglobin subunit beta albumen or its degradation
The antibody of product is reacted, the expression of the biomarker of the effective detection present invention, so as to be effectively detected
Person to be detected whether there is the morbid state of epilepsy, and the classification diagnosis and drug refractory of ancillary drug intractable epilepsy are insane
Whether epilepsy is postoperative can subtracts judgement of drug withdrawal object etc..
The purpose of embodiment as described above is to be further described the specific embodiment of the present invention, not
The present invention is imposed any restrictions, any simple modification, change and the equivalence changes that every technical solution according to the present invention is made,
It still falls in the protection domain of technical solution of the present invention.
SEQUENCE LISTING
<110>Peking University
<120>A kind of biomarker for 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.
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| CN114350777A (en) * | 2021-12-13 | 2022-04-15 | 海南医学院 | Biomarker for predicting drug resistance of epilepsy infant to valproic acid and application thereof |
| CN116813707A (en) * | 2023-07-04 | 2023-09-29 | 杭州佰倍优生物科技有限公司 | Blood protein polypeptide and application thereof |
| CN116813707B (en) * | 2023-07-04 | 2024-05-10 | 杭州佰倍优生物科技有限公司 | Blood protein polypeptide and application thereof |
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