CN110272996A - Biomarker relevant to glaucoma occurrence and development and its application - Google Patents
Biomarker relevant to glaucoma occurrence and development and its application Download PDFInfo
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- CN110272996A CN110272996A CN201910698259.0A CN201910698259A CN110272996A CN 110272996 A CN110272996 A CN 110272996A CN 201910698259 A CN201910698259 A CN 201910698259A CN 110272996 A CN110272996 A CN 110272996A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/168—Glaucoma
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Abstract
The invention discloses biomarker relevant to glaucoma occurrence and development and its applications.Present invention firstly discovers that SPATA33 and WDR61 expresses up-regulation in glaucoma patient, based on this, the invention discloses SPATA33 or WDR61 preparation diagnosis glaucoma product in application, and comprising detect SPATA33 or WDR61 expression reagent diagnosis glaucoma product.
Description
Technical field
The present invention relates to fields of biomedicine, are related to one kind biomarker relevant to glaucoma occurrence and development and its answer
With the biomarker is SPATA33 or WDR61.
Background technique
Glaucoma is one group using characteristic optic atrophy and defect of visual field as the disease of common trait, pathologic intraocular pressure liter
Height is its Major Risk Factors.It is blinding eye disease second-biggest-in-the-world now, occupies first of irreversible blindness.According to system
Meter, the whole world in 2013 have 6.43 hundred million people with glaucoma, will suffer from glaucoma to the year two thousand twenty about 7.60 hundred million people, the year two thousand forty is green
Light eye number of patients will rise to 11.18 hundred million people (Tham YC, Li X, Wong TY, Quigley HA, Aung T, Cheng
CY.Global prevalence of glaucoma and projections of glaucoma burden through
2040:a systematic review and meta-analysis[J].Ophthalmology 2014；121:2081-
2090.).Glaucoma seriously threatens the quality of life of the mankind with high incidence and high blind rate.
Glaucoma is usually clinically divided into primary glaucoma, secondary glaucoma, children (development) property glaucoma three
Seed type.The pathogenesis of all types of glaucomas is imperfectly understood at present.Most scholars think glaucoma eyeground view mind at present
Damaged it is largely related to the raising of pathologic intraocular pressure, intraocular pressure increase after can mechanical pressure optic nerve fiber, or make to regard
Retinal vasculature hypoxic-ischemic eventually leads to the apoptosis of retinal ganglial cells.The morbidity of glaucoma and mode of inheritance are very complicated,
It may be that the interaction of the interaction and gene and environment between several genes causes jointly.Glaucoma may be with family
The risk factors such as history, characteristic structural, age, race, environment, heredity are related, and wherein inherent cause occupies importantly wherein
Position.Clinic is clarified a diagnosis glaucoma at present, and for the course of disease of glaucoma not in early stage, glaucoma is irreversible blinding disease, so
Early diagnosis, early treatment are of great significance.
With the development of gene studies technology, more and more Genes Associated with Glaucoma, site and dependency basis are found
Because of mutation, for further clarifying the Molecular pathogenesis of glaucoma, early gene intervention, people at highest risk's sieve are carried out to crowd
It looks into, preclinical diagnosis and gene therapy, improves Quality of Life of Glaucoma Patients, or even radical cure glaucoma has great importance.
Summary of the invention
In order to make up for the deficiencies of the prior art, the effect the present invention is based on inherent cause in the occurrence and development of glaucoma,
Biomarker relevant to glaucoma occurrence and development is studied, so that the diagnosing and treating for glaucoma provides new means.
The present invention provides application of the reagent of detection biomarker in the product of preparation diagnosis glaucoma, the lifes
Object marker is selected from SPATA33 or WDR61.
Further, be compared with normal people, when SPATA33 or WDR61 on the expression in subject's sample timing, then
Subject is with glaucoma or there is the risk for suffering from glaucoma.Wherein sample includes but is not limited to tissue or fluid, such as group
Knit, blood, blood plasma, serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its component or
Treated material.
Further, the sample is tissue, and further, the tissue is trabecular meshwork.
Further, the reagent includes:
The probe of specific recognition SPATA33 or WDR61 gene；
The primer of specific amplification SPATA33 or WDR61 gene；Or
Specifically bind the bonding agent of the albumen of SPATA33 or WDR61 coding.
Further, the primer sequence of the specific amplification SPATA33 or WDR61 gene to respectively as SEQ IDNO.1~
Shown in IDNO.3~4 2 and SEQ.
The present invention provides a kind of product for diagnosing glaucoma, the product includes detecting SPATA33 or WDR61 level
Reagent.
Further, the product includes nucleic acid film item, chip or kit.
Further, nucleic acid film item includes substrate and the specific recognition SPATA33 or WDR61 that are fixed in the substrate
Oligonucleotide probe；The substrate can be any substrate suitable for immobilized oligonucleotide probe, such as nylon membrane, cellulose nitrate
Plain film, polypropylene screen, sheet glass, silica gel chip, miniature magnetic bead etc..
Further, the chip includes genetic chip, protein-chip, wherein genetic chip include for SPATA33 or
The primer or oligonucleotide probe of WDR61, protein-chip include the bonding agent for specifically binding SPATA33 or WDR61 albumen.
Wherein, specific binding agent is such as receptor of protein s PATA33 or WDR61, conjugated protein SPATA33 or WDR61
Agglutinin, for the antibody of protein s PATA33 or WDR61, for the peptide antibody of protein s PATA33 or WDR61
(peptidebody), the agent of bispecific dual combination or bispecific antibody form.
The example of specific binding agent is peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repetition
Albumen, Kunitz type domain, antibody, single domain antibody and monovalent antibody fragments.In a specific embodiment of the present invention, the specificity
Bonding agent is SPATA33 or WDR61 specific antibody.
Further, the kit includes gene detecting kit and protein detection kit, the gene detection reagent
Box includes primer, oligonucleotide probe or the chip that specificity is directed to SPATA33 or WDR61；The protein detection kit packet
Include the bonding agent of specific binding SPATA33 or WDR61 albumen.Kit includes one or more sterile chambers, such appearance
Device can be box, ampoule, bottle, phial, pipe, bag, pouch, blister package or other suitable containers known in the art
Form.Such container can be by plastics, glass, laminated paper, metal foil or other materials suitable for container drug.
Further, specificity is for the primer sequence of SPATA33 or WDR61 as shown in IDNO.1~4 SEQ.
The present invention provides a kind of methods for diagnosing glaucoma, comprising steps of
1) sample is obtained；
2) RNA in sample is extracted；
3) expression of the SPATA33 or WDR61 in RNA are detected；
4) multilevel iudge is compared with normal people, when the expression of SPATA33 or WDR61 significantly increases, then subject
With glaucoma.
The present invention provides application of the SPATA33 or WDR61 in the pharmaceutical composition of preparation treatment glaucoma.
Further, described pharmaceutical composition includes the inhibitor of SPATA33 or WDR61.Wherein, the inhibitor can be
Transcription or translation skill reduce the expression of SPATA33 or WDR61.
In the present invention, (gene I/D: 124045) including people SPATA33 gene and its encoded albumen to SPATA33.Make
For unrestricted example, the gene order of SPATA33 such as NM_001271907.1, NM_001271908.1, NM_
001271909.1, shown in any transcript of NM_001271910.1, NM_153025.2, corresponding amino acid sequence such as NP_
001258836.1, shown in NP_001258837.1, NP_001258838.1, NP_001258839.1, NP_694570.1.Its
In, a kind of gene order of representative SPATA33 is as shown in NM_001271907.1, corresponding amino acid sequence such as NP_
Shown in 001258836.1.
(gene I/D: 80349) including people WDR61 gene and its encoded albumen to WDR61.As unrestricted reality
Example, the gene order of WDR61 as shown in any transcript of NM_001303247.2, NM_001303248.2, NM_025234.3,
Its corresponding amino acid sequence is as shown in NP_001290176.1, NP_001290177.1, NP_079510.1.In this application,
A kind of gene order of representative WDR61 is as shown in NM_001134870.2, amino acid sequence such as NP_001290176.1 institute
Show.
It will be appreciated by those skilled in the art that the means of measurement gene expression are not importances of the invention.The present invention
It can use the expression of any method known in the art measurement gene.
SPATA33 or WDR61 of the invention uses multiple nucleic acids known to persons of ordinary skill in the art and albumen skill
Art is detected, these technologies include but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.
The product that glaucoma is diagnosed in the present invention can be used for detecting multiple bases including SPATA33 or WDR61 gene
Because of the expression of (for example, multiple genes relevant to glaucoma), multiple markers of glaucoma are detected simultaneously, it can
Greatly improve the accuracy rate of diagnosis of glaucoma.
The advantages of the present invention:
Present invention firstly discovers that SPATA33 or WDR61 gene expression in glaucoma patient raises, pass through detection
Whether the expression of SPATA33 or WDR61 gene can suffer from glaucoma with auxiliary diagnosis subject or suffer from glaucoma disease
Risk, so that doctor be instructed to provide prevention or therapeutic scheme.
Detailed description of the invention
Fig. 1 is the expression figure using QPCR detection SPATA33 in glaucoma patient, wherein figure A is SPATA33
Expression figure, figure B are the expression figures of WDR61.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.
Embodiment QPCR detects expression of the SPATA33 and WDR61 in glaucoma
1, sample collection
Trabecular tissue 19 of trabecular tissue during operation for glaucoma 31 and normal wound, exclusion keratonosus,
The patient of uveitis, entophthamia and retinopathy and systemic disease.
2, the preparation and quality analysis of RNA sample
The total serum IgE in tissue is extracted using Trizol method
Tissue plus liquid nitrogen grinding are taken, Trizol lysate (Invitrogen company, the U.S.) and chloroform (Trizo1 is added
Lysate: chloroform=5:1), oscillation mixes 15s, 37 DEG C of incubation 2min, 12 000r/min at 4 DEG C.It is centrifuged 15min, in absorption
Layer colourless liquid is added isometric isopropanol and mixes into clean sterile centrifugation tube, 37 DEG C of incubation 10min, 12 at 4 DEG C
000r/min is centrifuged 10min, abandons supernatant, cleans RNA precipitate with 75% ethyl alcohol of volume fraction, dries 5~10min at room temperature, uses
The sterile water of no RNA enzyme dissolves RNA.Ultraviolet absorption method detects RNA purity, A260/A280Ratio 1.8~2.1.
3, reverse transcription:
It is operated using the reverse transcription reagent box (Takara code:DRR047A) of TAKARA company.
1) genomic DNA is removed
5 × gDNA Eraser B μ ffer, 2.0 μ l, gDNA Eraser 1.0 μ l, 1 μ g of total serum IgE are added in test tube,
Add Rnase Free ddH2O makes total volume to 10 μ l, 42 DEG C of heating 2min in water-bath.
2) reverse transcription reaction
It will2 4.0 μ l of Buffer,RT Enzyme Mix I 1.0 μ l, RT
1.0 μ l, RNase Free ddH of Primer Mix24.0 μ l of O, which is added in above-mentioned test tube, is mixed together totally 20 μ l, in water-bath
37 DEG C of 15min, 85 DEG C of 5s.
4, real-time fluorescence quantitative PCR
1) design of primers
According to the gene order design primer of SPATA33, WDR61 and GADPH, specific primer sequence is as shown in table 1:
1 primer sequence of table
2) real-time fluorescence quantitative PCR amplification is examined
WithPremix Ex TaqTMII (Takara Code:DRR081) kit configures PCR reaction system,
Thermal Cycler PCR amplification is carried out on Real Time System amplification instrument, amplification system and reaction condition are such as
Shown in table 2.The amplification curve and solubility curve of Real Time PCR are confirmed after reaction, and Δ Δ CT method carries out relative quantification.
2 real-time fluorescence quantitative PCR amplification system of table and reaction condition
5, result
As shown in Figure 1, compared with normal tissue, SPATA33 and WDR61 are expressed significantly QPCR result in glaucoma patient
Up-regulation, SPATA33 raise about 7.46 times, and WDR61 raises about 5.37 times, and difference has statistical significance (P < 0.05), wherein 31
There are 26 SPATA33 significantly to raise in example glaucoma patient, there are 29 WDR61 significantly to raise, prompts SPATA33 and WDR61 can
Diagnosis applied to glaucoma.
Meanwhile significantly being raised in glaucoma according to SPATA33 and WDR61, it can design for SPATA33 and WDR61
SiRNA, shRNA of gene or the antibody for specifically binding SPATA33 and WDR61 albumen are applied to the treatment of glaucoma.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>Wenshang County institute of traditional Chinese medicine
<120>biomarker relevant to glaucoma occurrence and development and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aaagagaaac ccaggaaag 19
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaatgcttct ccatcaact 19
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cagataaagt ccatagat 18
<210> 4
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcaatactaa gaatgaat 18
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aatcccatca ccatcttcca g 21
<210> 6
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gagccccagc cttctccat 19
Claims (10)
1. detecting application of the reagent of biomarker in the product of preparation diagnosis glaucoma, which is characterized in that biological marker
Object is selected from SPATA33 or WDR61.
2. application according to claim 1, which is characterized in that be compared with normal people, when SPATA33 or WDR61 is tested
Timing on expression in person's sample, then subject suffers from glaucoma.
3. application according to claim 2, which is characterized in that the sample is tissue.
4. application according to claim 1-3, which is characterized in that the reagent includes:
The oligonucleotide probe of specific recognition SPATA33 or WDR61 gene；
The primer of specific amplification SPATA33 or WDR61 gene；Or
Specifically bind the bonding agent of the albumen of SPATA33 or WDR61 coding.
5. application according to claim 4, which is characterized in that the specific amplification SPATA33 or WDR61 gene draw
Object sequence is respectively as shown in NO.1~2 SEQ ID and NO.3~4 SEQ ID.
6. a kind of product for diagnosing glaucoma, which is characterized in that the product includes the examination for detecting SPATA33 or WDR61 level
Agent.
7. product according to claim 6, which is characterized in that the product includes nucleic acid film item, chip or kit.
8. product according to claim 7, which is characterized in that the chip includes genetic chip, protein-chip, wherein
Genetic chip includes the primer or oligonucleotide probe for SPATA33 or WDR61, and protein-chip includes specific binding
The bonding agent of SPATA33 or WDR61 albumen.
9. product according to claim 8, which is characterized in that the kit includes gene detecting kit and albumen inspection
Test agent box, the gene detecting kit include specificity for the primer of SPATA33 or WDR61, oligonucleotide probe or
Chip；The protein detection kit includes the bonding agent for specifically binding SPATA33 or WDR61 albumen.
10. product according to claim 9, which is characterized in that specificity is directed to the primer sequence of SPATA33 or WDR61
As shown in NO.1~4 SEQ ID.
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| CN201910698259.0A CN110272996A (en) | 2019-07-31 | 2019-07-31 | Biomarker relevant to glaucoma occurrence and development and its application |
| CN202010225651.6A CN111187832B (en) | 2019-07-31 | 2020-03-26 | Biomarker related to occurrence and development of glaucoma and application thereof |
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| CN201910698259.0A CN110272996A (en) | 2019-07-31 | 2019-07-31 | Biomarker relevant to glaucoma occurrence and development and its application |
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| CN202010225651.6A Active CN111187832B (en) | 2019-07-31 | 2020-03-26 | Biomarker related to occurrence and development of glaucoma and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444789A (en) * | 2021-08-27 | 2021-09-28 | 中国医学科学院北京协和医院 | Glaucoma-associated biomarkers and uses thereof |
| CN114875132A (en) * | 2022-05-09 | 2022-08-09 | 中国人民解放军总医院第三医学中心 | Diagnostic biomarkers for glaucoma and uses thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878352B (en) * | 2019-12-19 | 2022-04-29 | 青岛市海慈医疗集团 | Tool for diagnosing glaucoma and drug target for treating glaucoma |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2008263384B2 (en) * | 2007-06-13 | 2014-08-28 | Decode Genetics Ehf | Genetic variants on CHR 15Q24 as markers for use in diagnosis, prognosis and treatment of exfoliation syndrome and glaucoma |
| JP2011505579A (en) * | 2007-12-04 | 2011-02-24 | ユニバーシティ オブ マイアミ | Molecular targets for modulating intraocular pressure and distinguishing steroid responders from non-responders |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444789A (en) * | 2021-08-27 | 2021-09-28 | 中国医学科学院北京协和医院 | Glaucoma-associated biomarkers and uses thereof |
| CN114875132A (en) * | 2022-05-09 | 2022-08-09 | 中国人民解放军总医院第三医学中心 | Diagnostic biomarkers for glaucoma and uses thereof |
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| Publication number | Publication date |
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| CN111187832A (en) | 2020-05-22 |
| CN111187832B (en) | 2020-09-15 |
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Application publication date: 20190924 |