CN109797215A - Marker is used in major depressive disorder diagnosis - Google Patents

Abstract

The invention discloses major depressive disorder diagnosis marker, the marker is MRPS18C and ZER1.Present invention firstly discloses MRPS18C and ZER1 to be presented high expression in major depressive disorder patient, and the diagnosis of major depressive disorder may be implemented in the expression by detecting MRPS18C and ZER1.

Description

Marker is used in major depressive disorder diagnosis

Technical field

The present invention relates to fields of biomedicine, are related to major depressive disorder diagnosis marker, are specifically related to marker MRPS18C and ZER1.

Background technique

Major depressive disorder (major depressive disorder, MDD) is by genetic system in individual patients (gene) There are a kind of mood sex dysfunctions caused by the great change of exception or acquired environment, are with lasting the depressed of spontaneity A series of main depressive symptoms.Its typical clinical characters and symptom include: the row to the experience of once interested or pleasant sensation To be unable to lift interest, such as sexual behaviour；Loss of appetite (apositia) is along with weight loss or that eats excessive increase along with weight Add；Emotional poverty；Lasting depression and anxiety or inanition；Despair, pessimism, compunction, imperceptible existing value；Social energy Power shrinks back, uncommon fatigue, lassitude, slow in reacting；Insomnia, early awakening or drowsiness；Spirit cannot concentrate, failure of memory Or it is difficult to make a decision；Usually fidgety or irritated movability；It generates suicidal thought or suicide occurs.

Depression is a kind of serious, recurrent exerbation mental disease, not only influences the social function of patient, matter of living Amount, and increase medicine morbidity and mortality.Depression is classified as the fourth-largest disability disease in the whole world by the World Health Organization.According to pre- It surveys, arrives the year two thousand twenty, depression will become the second largest mental disease.The whole world has 3.4 hundred million people to suffer from depression at present, in annual 1-2 Million people has in the actor of conamen about 70% to suffer from depression.

The complex disease that MDD is induced as a kind of polygenes, pathogenesis are related to many factors, now think that DNA is polymorphic Property, epigenetic factor and environmental factor be the main factor for influencing MDD and occurring, the collective effect of three determines MDD. It there is no special Diagnosis of Depression tool at present.Clinically used symptom measuring scale has 3 kinds: Hamilton depression grading scale (HAMD), Beck Depression self-appraisal questionnaire (BD1) and depression self-rating scale (SDS) can be used to reliably evaluate the degree of depression, It may be major depression that HAMD total score, which is more than or equal to 35 points,.The pathogenesis for illustrating MDD, for the prevention of MDD, related drugs Design and reasonable effective clinical treatment are of great significance, and have become field of biomedicine major issue urgently to be resolved. With the development of biology techniques, research of the gene in disease is in widespread attention, the severe suppression based on gene level The research of strongly fragrant disease pathogenesis and biomarker gradually causes the concern of scholars.Find gene relevant to major depressive disorder Applied to the clinical diagnosis of major depressive disorder, the pathogenesis of major depressive disorder is disclosed, the life quality for improving patient has weight The meaning wanted.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide marker relevant to major depressive disorder, It is diagnosed the illness using marker with timeliness, specificity and sensitivity, patient can know risk, needle in depression early stage To risk height, corresponding prevention and treatment measure is taken, to improve the quality of living.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides the horizontal reagents of gene and its expression product in preparation diagnosis major depressive disorder product Using the gene includes MRPS18C or ZER1.

Further, the product includes passing through sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies, protein immunization skill Art detects the reagent of MRPS18C or ZER1 level.

Further, the reagent includes:

The probe of specific recognition MRPS18C or ZER1 gene；Or

The primer of specific amplification MRPS18C or ZER1 gene；Or

Specifically bind the antibody or ligand of the albumen of MRPS18C or ZER1 coding.

Further, the primer sequence of the specific amplification MRPS18C is as shown in NO.1~2 SEQ ID, specific amplification The primer sequence of ZER1 gene is to as shown in IDNO.3~4 SEQ.

The present invention provides a kind of testing product, the product include detection sample in MRPS18C or ZER1 gene and its The reagent of expression product level.

Further, the product includes chip, kit or nucleic acid film item.

Further, the reagent includes with reverse transcription PCR, real-time quantitative PCR, in situ hybridization, immune detection or chip inspection Survey the reagent of the gene and its expression product level.It is described to detect the gene and its expression product level with reverse transcription PCR Reagent include at least the primer of a pair of of specific amplified MRPS18C or ZER1 gene；It is described to detect the base with real-time quantitative PCR The reagent of cause and its expression product level includes at least the primer of a pair of of specific amplified MRPS18C or ZER1 gene；It is described with exempting from It includes: in conjunction with MRPS18C or ZER1 protein-specific that epidemic disease detection, which detects the gene and its reagent of expression product level, Antibody；The reagent for detecting the gene and its expression product level in situ hybridization includes: and MRPS18C or ZER1 gene Nucleic acid array hybridizing probe；The reagent that the gene and its expression product level are detected with chip includes: protein Chip and genetic chip；Wherein, protein-chip includes the antibody in conjunction with MRPS18C or ZER1 protein-specific, gene core Piece includes the probe with the nucleic acid array hybridizing of MRPS18C or ZER1 gene.

Further, the reagent for detecting the gene expression dose with real-time quantitative PCR includes at least a pair of of specificity The primer of the gene is expanded, shown in NO.1~2 primer sequence SEQ ID of the specific amplification MRPS18C, specificity expands Increase the primer sequence of ZER1 as shown in NO.3~4 SEQ ID.

In the present invention, " sample " is can therefrom to obtain nucleic acid, polypeptide or other analyses comprising cell or cellular material The substance of object.Preferably, the sample is blood.

The present invention provides application of the product recited above in preparation diagnosis major depressive disorder tool.

The present invention provides application of the MRPS18C or ZER1 in the computation model of building prediction major depressive disorder.

The present invention provides application of the MRPS18C or ZER1 in the pharmaceutical composition of preparation treatment major depressive disorder.

Further, described pharmaceutical composition is the inhibitor of MRPS18C or ZER1, and the inhibitor can reduce The expression of MRPS18C or ZER1.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection MRPS18C or ZER1 gene in major depressive disorder patient, wherein Figure A is the expression figure of MRPS18C, and figure B is the expression figure of ZER1.

Specific embodiment

The present invention utilize high-flux sequence method combination bioinformatic analysis, by detection major depressive disorder patient with The gene of differential expression in normal person finds the marker that can be used for characterizing major depressive disorder.The present invention is by analysis, for the first time It has found that differential expression is presented in MRPS18C or ZER1 in major depressive disorder patient, MRPS18C or ZER1 is prompted to can be used as inspection Survey the clinical diagnosis that index is applied to major depressive disorder.

MRPS18C or ZER1

In the present invention, MRPS18C (gene ID:51023) includes people MRPS18C gene and its encoded albumen, It is taken positioned at 2 area 1 of No. 4 chromosome long arms of people.There are four presently disclosed MRPS18C transcripts, and sequence is respectively such as NM_ 001297767.1, shown in NM_001297769.1, NM_001297770.1, NM_016067.4, encoded protein sequence Respectively as shown in NP_001284696.1, NP_001284698.1, NP_001284699.1, NP_057151.1, a kind of representativeness MRPS18C gene order as shown in NM_001297767.1.

ZER1 (gene ID:10444) includes people ZER1 gene and its encoded albumen, and it is long to be located at No. 9 chromosomes of people 3 area 4 of arm takes.A kind of nucleotide sequence of representative ZER1 such as ZER1 in current international public nucleic acid database GeneBank (NM_006336.4) shown in, encoded protein sequence is as shown in NP_006327.2.

Practicability of the invention be not limited to the gene expression to any specific variants of target gene of the invention into Row is quantitative.One skilled in the art will appreciate that when carrying out bioinformatic analysis, it will usually by sequencing sequence and known gene into Row compares, as long as related sequence can compare on related gene, so that it may regard the expression of the gene as, therefore, refer to When the gene of differential expression, different transcripts, saltant type or its segment of the gene are also included in the present invention.

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.

MRPS18C or ZER1 of the invention uses multiple nucleic acids known to persons of ordinary skill in the art and protein techniques It is detected, these technologies include but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and dye Expect terminator sequencing.Those skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcription at DNA before sequencing.

The another exemplary non-limiting example of Nucleic acid sequencing techniques includes that (deep sequencing/high pass measures for next-generation sequencing Sequence), high throughput sequencing technologies are a kind of sequencing technologies in synthesis based on unimolecule cluster, based on proprietary reversible termination chemistry Reaction principle.The random fragment of the DNA of genome is attached to optically transparent glass surface when sequencing, these DNA fragmentations warp After crossing extension and bridge amplification, hundreds of millions of clusters is formed in glass surface, each cluster is the list with thousands of parts of same templates Then molecular cluster utilizes four kinds of special deoxyribonucleotides with fluorophor, skill is sequenced in synthesis by reversible Template DNA to be measured is sequenced in art.

The exemplary, non-limitative example of nucleic acid hybridization technique include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with Position tissue a part or slice (original position) are the spy in entire tissue (full organization embedding ISH) if tissue is sufficiently small The hybridization of anisotropic DNA or RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring and positioning group Knit the mRNA and other transcripts (for example, ncRNA) in slice or full organization embedding.Usually to sample cell and tissue at Reason increases the entrance of probe with fixation in situ target transcript.Probe hybridizes with target sequence at high temperature, then by extra spy Needle is washed off.Use autoradiograph, fluorescence microscopy or immunohistochemistry respectively, in tissue with radiation, fluorescence or antigen The probe of the kilobase marker of label is positioned and is quantified.Two or more can also be used by radioactivity by ISH or other are non- The probe of radioactive label substance markers, to detect two or more transcripts simultaneously.

The exemplary, non-limitative embodiment of nucleic acid amplification technologies includes but is not limited to: polymerase chain reaction (PCR), inverse Transcriptional polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification based on nucleic acid sequence (NASBA).Those skilled in the art are it will be recognized that certain amplification technique (examples Such as, PCR) it needs RNA reverse transcription before amplification at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

Chip, kit, nucleic acid film item

Chip includes genetic chip, protein chip in the present invention；The genetic chip includes solid phase carrier；And orderly The oligonucleotide probe being fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to MRPS18C or Sequence some or all of shown in ZER1.The protein chip includes solid phase carrier, and is fixed on solid phase carrier The antibody or ligand of the albumen of MRPS18C or ZER1 coding.The solid phase carrier includes inorganic carrier and organic carrier, the nothing Airborne body includes but is not limited to have silicon carrier, glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, Buddhist nun Imperial film etc..

" antibody " clearly covers monoclonal antibody, polyclonal antibody, from least two herein with broadest use The multi-specificity antibody (such as bispecific antibody) and antibody fragment that kind of complete antibody is formed, if they show it is desired Biological activity.

The present invention provides a kind of kit, the kit can be used for detecting MRPS18C or ZER1 gene or albumen Expression, comprising for MRPS18C or ZER1 detection and/or quantitative primer, oligonucleotide probe, ligand, and/or core Piece.One or more substances selected from the group below: container, operation instructions, positive control, negative control object, buffer, auxiliary agent Or solvent.

In kit of the invention can also have kit operation instructions, be described how using kit into Row detection, and how disease development to be judged using testing result, therapeutic scheme is selected.

The component of kit can pack in the form of aqueous medium or in the form of freeze-drying.Container appropriate in kit It include typically at least a kind of bottle, test tube, flask, PET bottle, syringe or other containers, wherein a kind of component can be placed, and And preferably, suitably equal part can be carried out.There are more than one group timesharing in kit, generally also will include in kit Second, third or other additional containers, wherein being positioned separately additional component.However, the component of various combination can be wrapped It is contained in a bottle.Kit of the invention will include generally also a kind of container for being used to accommodate reactant, seal to be used for Commercial distribution.This container may include the plastic containers of injection molding or blowing mould, wherein can retain required bottle.

In the present invention, nucleic acid film item include substrate and the specific recognition MRPS18C that is fixed in the substrate or The probe of ZER1；The substrate can be any substrate for being suitable for fixed probe, such as nylon membrane, nitrocellulose filter, poly- third Alkene film, sheet glass, silica gel chip, miniature magnetic bead etc..

The product that major depressive disorder is diagnosed in the present invention can be used for detecting multiple including MRPS18C or ZER1 gene The expression of gene (for example, multiple genes relevant to major depressive disorder), simultaneously by multiple markers of major depressive disorder It is detected, is greatly improved the accuracy rate of major depressive disorder diagnosis.

The present invention provides application of the MRPS18C or ZER1 in the computation model of building prediction major depressive disorder, can be with It is implemented in various ways and realizes the step of marker levels and certain possibility or risk association are got up.Preferably, in number The measurement concentration of composite marker object and one or more other markers on, and combined value is associated with basic diagnosis problem Get up.It can be combined the measurement of marker levels by any suitable prior art mathematical method.

Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a Body associates about the risk of rectal adenocarcinoma or with the other intentional diagnostic uses for helping to assess rectal adenocarcinoma patient.With one Kind preferred mode, such logarithmic function obtain as follows: individual segregation a) being entered group, such as normal person, has rectal adenocarcinoma The individual of risk, the patient with rectal adenocarcinoma etc., b) significant difference between these groups is identified by univariate analysis Marker, c) logarithmic regressions analysis to be to assess the independent difference values that can be used for assessing these difference groups of marker, and d) structure It builds logarithmic function and carrys out composition independency difference value.In such analysis, marker is no longer independent, but represents one Marker combination.

In the present invention, " sample " " is the nucleic acid of can therefrom obtain comprising cell or cellular material, polypeptide or other points Analyse the sample of object.The example of biological sample includes: urine, blood, serum, blood plasma, celiolymph, liquor pleurae, branch in non-limiting manner Lavage of trachea, phlegm, peritoneal fluid, bladder irrigation, secretion (for example, mammal gland secretion), dentilave, swab are (for example, oral cavity is wiped Son), separation cell, tissue samples, touching prepare object and fine needle puncture object.In a specific embodiment of the present invention, the sample This is blood.

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, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to major depressive disorder

1, sample collection

4 normal human bloods and major depressive disorder blood samples of patients sample are collected respectively, and the acquirement of above-mentioned all samples obtains The approval of Ethics Committee.

The inclusion criteria of major depressive disorder patient:

The diagnostic criteria of Americanism medical diagnosis on disease and statistic handbook fourth edition (DSM- IV) depression；

Hamilton depressive scale scores >=34 points.

Exclusion criteria:

Previously or at present with Diseases such as severe physical, nervous system, endocrine system, immune systems；

There are previously or at present psychoactive drug substance, alcohol abuse or relies on history person；

Previously or at present with other psychotic disorders in addition to depression；

There are the clear psychotic disorder or the nervous system disease family history in addition to depression, head injury history；

Significant wound event, secondary to drug, organic disease or depression caused by postpartum；

2, the preparation and quality analysis of RNA sample

(1) blood is taken, 3 times of volume erythrocyte cracked liquids are added, 10min, 10,000rpm centrifugations are placed at room temperature for after mixing 1min inhales and abandons supernatant, collects leukocyte cell pellet；

(2) Trizol is added, is placed at room temperature for 5min, 4 DEG C of 12,000rpm are centrifuged 10min, take supernatant；

(3) 200 μ l chloroforms are added in every 1ml Trizol, are placed at room temperature for 3-5min after shaking vigorously and mix well, and 4 DEG C 12, 000rpm is centrifuged 10-15min, water phase being transferred in new pipe carefully；

(4) isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min, 4 DEG C of 12,000rpm centrifugations 10min abandons supernatant；

(5) 75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in RNA precipitate, mildly vibrates centrifuge tube, suspends Precipitating；

(6) 4 DEG C of 8,000rpm are centrifuged 1-2min, abandon supernatant；Of short duration rapid centrifugation is carefully inhaled with pipettor and abandons supernatant, room Temperature places 2min and dries precipitating；

(7) 50 μ l RNase-free water are added in precipitating, flick tube wall, sufficiently to dissolve RNA, -70 DEG C of preservations.

(8) extracted RNA concentration and purity are detected using Nanodrop2000.

3, construction cDNA library

(1) rRNA in total serum IgE is removed using the Ribo-Zero kit of Illumina；

(2) building of cDNA library is carried out using the Truseq RNA sample Prep Kit of Illumina.

4, upper machine sequencing

CDNA library is sequenced using Hiseq4000 microarray dataset, concrete operations by specification carries out.

5, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, merges P value method using R packet metaMA, as P < 0.05, it is believed that The expression of mRNA significant difference.

6, result

Sequencing result is shown, is compared with normal people, table of the MRPS18C or ZER1 gene in major depressive disorder blood samples of patients It is dramatically increased up to amount.

The differential expression of embodiment 2QPCR sequence verification MRPS18C or ZER1 gene

1, MRPS18C or ZER1 gene is selected to carry out large sample QPCR verifying according to the testing result of high-flux sequence.It presses Major depressive disorder blood samples of patients and normal human blood each 28 are selected according to the sample collection mode in embodiment 1.

2, RNA extraction step is the same as embodiment 1.

3, it reverse transcription: is operated using the reverse transcription reagent box (Takara code:DRR047A) of TAKARA company, instead Answer system and reaction condition as shown in table 1.

1 reverse transcription reaction system of table and reaction condition

4, QPCR is expanded

(1) design of primers

QPCR is designed according to the coded sequence of MRPS18C or ZER1 gene in Genebank and house-keeping gene GAPDH gene Amplimer, wherein the gene for having different transcripts is used, design of primers is carried out to its common region, synthesized by raw work.

The primer sequence of table 2MRPS18C and ZER1

(2) QPCR amplification is examined

WithPremix Ex Taq^TMII (Takara Code:DRR081) kit configures PCR reaction system, Reagent and reaction system are as shown in table 3, in Thermal CyclerPCR is carried out on Real Time System amplification instrument Amplification, confirms the amplification curve and solubility curve of Real Time PCR after reaction, and Δ Δ CT method carries out relative quantification.

Table 3QPCR amplification reaction system and reaction condition

5, result

As a result as shown in Figure 1, compared with normal human blood, MRPS18C or ZER1 gene is in major depressive disorder blood samples of patients In expression up-regulation, difference have statistical significance (P < 0.05), prompt MRPS18C and ZER1 gene can be used as gene marker Diagnosis applied to major depressive disorder.

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

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Claims (10)

1. the horizontal reagent for detecting gene and its expression product diagnoses the application in major depressive disorder product, feature in preparation It is, the gene includes MRPS18C or ZER1.

2. reagent according to claim 1, which is characterized in that the product includes hybridizing skill by sequencing technologies, nucleic acid Art, nucleic acid amplification technologies, protein immunization technology detect MRPS18C or the reagent of ZER1 level in sample.

3. application according to claim 1 or 2, which is characterized in that the reagent includes:

The probe of specific recognition MRPS18C or ZER1 gene；Or

The primer of specific amplification MRPS18C or ZER1 gene；Or

Specifically bind the antibody or ligand of the albumen of MRPS18C or ZER1 coding.

4. application according to claim 3, which is characterized in that the primer sequence such as SEQ of the specific amplification MRPS18C Shown in NO.1~2 ID, the primer sequence of specific amplification ZER1 gene is to as shown in SEQIDNO.3~4.

5. a kind of testing product, which is characterized in that the product includes MRPS18C or ZER1 gene and its expression in detection sample The reagent of product level.

6. product according to claim 5, which is characterized in that the product includes chip, kit or nucleic acid film item.

7. product according to claim 5, which is characterized in that the reagent includes passing through reverse transcription PCR, real-time quantitative PCR, in situ hybridization, immune detection or chip detect the reagent of the gene and its expression product level.

8. product according to claim 7, which is characterized in that described to detect the gene expression water with real-time quantitative PCR Flat reagent includes at least the primer of gene described in a pair of of specific amplification, the primer sequence of the specific amplification MRPS18C Shown in NO.1~2 SEQ ID, the primer sequence of specific amplification ZER1 is as shown in NO.3~4 SEQ ID.

9. according to the described in any item products of claim 5-8, which is characterized in that the sample is blood.

Application of the 10.MRPS18C or ZER1 in the computation model of building prediction major depressive disorder.