CN109628588B - Osteoarthritis disorders screening gene PRXL2A and ACTR8 and application thereof - Google Patents
Osteoarthritis disorders screening gene PRXL2A and ACTR8 and application thereof Download PDFInfo
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Abstract
The invention discloses osteoarthritis disorders screening gene PRXL2A and ACTR8 and application thereof, and in particular to osteoarthritis disorders screening gene PRXL2A and ACTR8 and its application in preparation osteoarthritis testing product.Inventor has found the molecular marker PRXL2A and ACTR8 gene closely related with osteoarthritis, the result shows that they have certain guidance meaning to the diagnosis of osteoarthritis, additionally, it is observed that they can also distinguish between osteoarthritis and rheumatoid arthritis, the Combining diagnosis of the two has Overlay.PRXL2A and ACTR8 gene is expected to become the candidate markers of osteoarthritis molecular diagnosis and differentiation osteoarthritis and rheumatoid arthritis in clinic or human health screening.
Description
Technical field
The present invention relates to medical diagnosis on disease fields, more particularly it relates to osteoarthritis (OA) disorder in screening gene
PRXL2A and ACTR8 and its application in preparation osteoarthritis testing product, the invention further relates to the two to distinguish bone in preparation
Application in arthritis and rheumatoid arthritis (RA) testing product.
Background technique
Osteoarthritis (osteoarthritis, OA) is one kind with articular cartilage retrogression pathological changes and secondary osteoproliferation
For the chronic joint diseases of characteristic.The inspection of iconography is clinically based primarily upon for the diagnosis of osteoarthritis: x-ray performance is main
Narrow for joint space, subchondral bony sclerosis and cystis degeneration, joint margins spur are formed, and articular surface withers, deforms and joint
Subluxation etc.；MRI can show the lesion of the joint structures such as early stage cartilage, meniscus, be conducive to early diagnose；CT is mainly used for intervertebral
The diagnosis of disk disease.Osteoarthritis waits until inflammation secondary, pain or the activity for influencing joint in early stage general few symptoms, patient
When, just go to a doctor.At this point, joint injury occurs for a long time, so, urgently exploitation clinical at present is diagnosed for early stage osteoarthritis
Method.
With the rapid development of the related disciplines such as molecular biology, immunology, protein and genomics, to osteoarthritis
The research of pathogenesis has new understanding, it has now been found that its morbidity has close pass with aging, inflammation and metabolic disorder
Connection.In terms of molecular mechanism that researches show that osteoarthritis is related to TGF-β/Smad signal path, have researches show that TGF-β can
Promote the synthesis and secretion of cartilage cell and synovial cell's extracellular matrix secretion ingredient relevant enzyme, such as MMP-1, MMP-9 with
MMP-13 promotes the destruction of arthritis reaction and cartilage of osteoarthritis matrix.In terms of patent, patent 201610271798.2,
201510548635.X, 2015105495645 etc. disclose some molecular markers that can be used for osteoarthritis diagnosis.But
It is that the requirement in precisely medical treatment to detection mark number quantity research is also much not achieved in existing research, the application is intended to provide
The biomarker of new osteoarthritis diagnosis provides Research foundation for clinical gene detection.
It is rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of slow characterized by arthrosynovitis
Property is systemic itself to exempt from class epidemic disease disease.The lasting recurrent exerbation of synovitis can lead to the destruction of intra-articular cartilage and bone, joint function
Energy obstacle, in addition it is maimed.RA is a kind of chronic systemic inflammation disease that the cause of disease is not yet clear, with chronic, symmetry, mostly sliding
Lesion is main clinical manifestation outside syndesis inflammation and joint, belongs to autoimmune inflammatory disease.Osteoarthritis and rheumatoid arthrosis
Inflammation is all systemic disease, and big Minor articulus can be involved, but the two is two different diseases, it is necessary to be distinguished, but not have at present
It is found the extraordinary molecular marker that can distinguish the two.
The osteoarthritis sample collected in clinic is carried out high-flux sequence by inventor, is filtered out and the close phase of osteoarthritis
The molecular marker of pass is simultaneously verified, and the diagnosis of PRXL2A and ACTR8 gene pairs osteoarthritis, which has, as the result is shown centainly refers to
Lead meaning.In addition, being analyzed by the data in existing GEO database, discovery PRXL2A and ACTR8 gene can be with area
Divide osteoarthritis and rheumatoid arthritis (RA), the Combining diagnosis of the two has Overlay.PRXL2A and ACTR8 gene has
Hope the candidate mark for becoming osteoarthritis molecular diagnosis and differentiation osteoarthritis and rheumatoid arthritis in clinic or human health screening
Will object.
Summary of the invention
The object of the present invention is to provide osteoarthritis testing product, PRXL2A in the testing product detection sample
And/or the expression of ACTR8 gene or albumen.
When PRXL2A and/or ACTR8 gene expression amount is low, sample is with higher to suffer from osteoarthritis risk.
Preferably, it is detected as quantitative detection.
Further, testing product passes through quantitative fluorescent PCR, Southern hybridization, Northern hybridization, dot blot, fluorescence
In situ hybridization, DNA microarray, ASO method, high-flux sequence method, immunization detection sample in PRXL2A and/or ACTR8 gene or
The expression of albumen.
Further, immunization includes ELISA method, radioimmunoassay, immunohistochemical method, western blot method
Deng.
It include the primer and/or probe for capableing of specific amplification PRXL2A and/or ACTR8 gene in testing product；Or
The antibody of PRXL2A and/or ACTR8 albumen can be specifically bound.
Further, the primer sequence of specific amplification PRXL2A gene such as SEQ ID NO.1 and SEQ ID NO.2；Specifically
Property amplification ACTR8 gene primer sequence such as SEQ ID NO.3 and SEQ ID NO.4.
It detects sample and uses the tissue sample or fluid for example obtained from biopsy subject.Sample is not particularly limited, only
It is wanted to be suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma, serum, lymph, urine, serous cavity liquid,
Spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material.Preferably, detection sample is tissue or outer
All blood.
Application of the above-mentioned testing product in preparation osteoarthritis diagnostic tool.
The reagent for detecting PRXL2A and/or ACTR8 gene or albumen distinguishes osteoarthritis and rheumatoid joint in preparation
Application in scorching diagnostic tool.
Further, reagent passes through quantitative fluorescent PCR, Southern hybridization, Northern hybridization, dot blot, fluorescent in situ
Hybridization, DNA microarray, ASO method, high-flux sequence method, immunization detect PRXL2A and/or ACTR8 gene or albumen in sample
Expression.
Further, immunization includes ELISA method, radioimmunoassay, immunohistochemical method, western blot method
Deng.
It include the primer and/or probe for capableing of specific amplification PRXL2A and/or ACTR8 gene in reagent；Or it can
Specifically bind the antibody of PRXL2A and/or ACTR8 albumen.
Further, the primer sequence of specific amplification PRXL2A gene such as SEQ ID NO.1 and SEQ ID NO.2；Specifically
Property amplification ACTR8 gene primer sequence such as SEQ ID NO.3 and SEQ ID NO.4.
The primer for including in product can by being prepared by chemical synthesis, by using those skilled in the art will know that
Method be suitably designed with reference to Given information, and prepared by chemical synthesis.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody fragment
Refer to and retains peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment
It may include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization
The area V (double antibody) or the peptide containing CDR.The product of specific binding PRXL2A and/or ACTR8 albumen of the invention can wrap
The isolated nucleic acid of the amino acid sequence of encoding antibody or Encoding Antibody Fragment, the carrier comprising the nucleic acid are included, and carries the load
The cell of body.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It finally can be by using PRXL2A and/or ACTR8 albumen for being used as antigen or part thereof to obtaining
The antibody obtained is implemented antigentic specificity and is purified to obtain the monoclonal antibody for PRXL2A and/or ACTR8 albumen.It can be as follows
It prepares polyclonal antibody: with antigen-immunized animal same as above, blood sample is collected from by immune animal, from blood
In isolate serum, then using above-mentioned antigen to serum implement antigentic specificity purifying.It can be by being obtained with enzymatic treatment
Antibody obtains antibody fragment by using the sequence information of the antibody of acquisition.
Further, the osteoarthritis diagnostic tool includes but is not limited to chip, kit, test paper or high-flux sequence
Platform.High-flux sequence platform is a kind of tool of special diagnosis osteoarthritis, right with the development of high throughput sequencing technologies
The building of the gene expression profile of one people will become very easily work.By the gene for comparing Disease and normal population
Express spectra, the exception for being easy to analyze which gene are related to disease.Therefore, know in high-flux sequence PRXL2A and/or
The exception of the ACTR8 gene purposes for also belonging to PRXL2A and/or ACTR8 related to osteoarthritis, equally in protection of the invention
Within the scope of.
Anti- PRXL2A and/or ACTR8 antibody used in testing product of the invention, diagnostic tool or its segment are identified
The number of amino acid be not particularly limited, as long as antibody can combine PRXL2A and/or ACTR8.
The present invention also provides a kind of screening techniques of osteoarthritis drugs, can be by adding test medicine to osteocyte
The table of some period measurement PRXL2A and/or ACTR8 gene or albumen after object or after applying testing drug to animal pattern
Hyanalgese-D object is measured up to horizontal improves the effect of inflammation prognosis.
More specifically, when the expression of PRXL2A and/or ACTR8 gene or albumen is adding or applying testing drug
When increasing afterwards or when restoring normal level, the drug may be selected as the therapeutic agent for improving osteoarthritis prognosis.
In the context of the present invention, " diagnosis osteoarthritis " include judge subject whether suffered from osteoarthritis,
Judge that subject whether there is suffer from the risk of osteoarthritis, judge whether Human Osteoarthritis has recurred and shifted, judged
Human Osteoarthritis is to the reactivity of drug therapy or judges the prognosis situation of Human Osteoarthritis.
The advantages of the present invention:
Of the invention has found a kind of molecular marker for diagnosing osteoarthritis, can be closed in bone using the molecular marker
Saving the scorching early stage occurred can be used as judging, realizes and early finds early treatment.
Of the invention has found a kind of molecular marker compositions for distinguishing osteoarthritis and rheumatoid arthritis, uses
The molecular marker compositions can be with two kinds of similar arthritis diseases of supplementary globe.
Detailed description of the invention
Fig. 1 is the statistical chart for detecting gene differential expression situation in mRNA level in-site using quantitative fluorescent PCR；
Fig. 2 is the ROC curve that PRXL2A gene and ACTR8 gene individually distinguish osteoarthritis and rheumatoid arthritis；
Fig. 3 is the ROC curve that PRXL2A gene and ACTR8 gene association distinguish osteoarthritis and rheumatoid arthritis.
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, 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.
The high-flux sequence of embodiment 1 OA synovial tissue and normal synovial tissue
Take the synovial tissue of 3 comminuted fracture patients as control, the synovial membrane group of 4 Human Osteoarthritis in this hospital
It knits as case group, constructs the library mRNA after extracting sample rna, then use llumina hiseq x-ten second generation high pass
MRNA is sequenced in amount sequencing technologies.By the processes such as removing connector, going low quality, depollute complete the processing of data, obtain
Sequencing data.
MRNA, which is carried out, using software cuffquan, cuffdiff expresses quantitative and Differential expression analysis.Cuffquant is
It is specifically used to carry out expression quantity assessment and standardized tool in Cufflinks external member, cuffdiff is in Cufflinks external member
For calculating a tool of differential expression, cuffdiff utilizes the more each gene/transcript of quantitative result of cuffquant
Differential expression between two groupings.Significant difference mRNA screening conditions: P<0.05, | log2FC |>1.In the gene of downward
PRXL2A gene and ACTR8 gene differential expression are significant, carry out subsequent authentication as target gene.
In addition, carrying out GO function to difference expression gene using DAVID 6.8 (https: //david.ncifcrf.gov/)
It can enrichment and the enrichment analysis of KEGG function.P value is calculated using Hypergeometric test method.Screening criteria are as follows: FDR <
The enrichment display of 0.05GO function, difference expression gene significant enrichment is in innate immune response (innate immune
Response), virus defense (defense response to virus), blood platelet threshing (platelet
The bioprocess such as degranulation), extracellular body (extracellular exosome), cell liquid (cytosol), cell membrane
Cellular components such as (plasma membrane), receptor active (receptor activity), protein binding (protein
Binding), carbohydrate combines in molecular functions such as (carbohydrate binding).
The detection of embodiment 2 OA and normal population PRXL2A gene and ACTR8 expression
1, sample collection and extraction
Sample collection chooses peripheral blood 25 that this hospital is diagnosed as osteoarthritis patients, compares the healthy population for physical examination
Peripheral blood 33, the RNA in serum, specific steps reference book are extracted using QIAGEN blood rna extracts kit.
2, reverse transcription:
1) the total serum IgE template of 10pg-1 μ g and 2 10 × buffers of μ l, 2 μ l dATP (10mM), 0.5 μ l polyA is more
Poly- enzyme, 0.5 μ l ribalgilase (RNase) inhibitor and deoxyribonuclease water (RNase free water) mixing, volume
It is finally 20 μ l, 37 DEG C of incubation 1h.
2) 1 μ l 0.5 μ g/ μ l Oligo (dT) specific RT primer, 70 DEG C of incubation 5min are added in reaction tube.
3) it is incubated at least 2min on ice immediately, interrupts the secondary structure of RNA and primer.
4) by above-mentioned 20 μ l reaction mixture and 45 × buffers of μ l, 1 μ l dNTP (10mM), 0.5 μ l M-MLV is reversed
Record enzyme, 0.5 μ l ribalgilase (RNase) inhibitor, 10 μ l polyA reaction mixtures and 4 μ l deoxyribonuclease water
(RNase free water) mixing, 42 DEG C of incubation 1h.
3、QPCR
(1) design of primers
It is designed according to PRXL2A gene (NM_001243778.1) in Genbank and ACTR8 gene (NM_022899.5)
QPCR amplimer, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and internal reference selects GAPDH.Specific primer sequence
It arranges as follows:
PRXL2A gene:
Forward primer is 5 '-GGAGAATCACTTGAACCT-3 ' (SEQ ID NO.1)
Reverse primer is 5 '-TAATGAGGCACAGCAATC-3 ' (SEQ ID NO.2)
Amplification length 129bp.
ACTR8 gene:
Forward primer is 5 '-CTCCTACAATAAGCAGAT-3 ' (SEQ ID NO.3)
Reverse primer is 5 '-CAGTGGATTAACATACAAG-3 ' (SEQ ID NO.4)
Amplification length 121bp.
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
1 PCR reaction system of table
| Reagent | Volume (μ l) |
| Upstream primer | 1 |
| Downstream primer | 1 |
| SYBR Green polymerase chain reaction system | 12.5 |
| Template | 2 |
| Deionized water | Filling-in is to 25 μ l |
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 10s, 58 DEG C of 55s) × 35 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries out relative quantification.
1.5 statistical method
Experiment is all to be repeated 3 times according to each sample to complete, and result data is all the side with mean+SD
Formula indicates, using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that works as P
There is statistical significance when < 0.05.
1.6 result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2-
Δ Ct × 100%.As a result as shown in Figure 1, compared with Healthy People group, ACTR8 gene expression amount has significant decrease in illness group,
Only 0.1 times of Healthy People group, difference have statistical significance (P < 0.05)；Also there is PRXL2A gene expression amount in illness group
It is substantially reduced, is 0.44 times of Healthy People group, difference has statistical significance (P < 0.05).It is average with 33 Healthy People groups
Value is detection baseline, in 25 Human Osteoarthritis, 21 ACTR8 down regulation of gene expression, 2 ACTR8 gene expression differences without
Statistical significance, 2 ACTR8 gene expression up-regulations；In 25 osteoarthritis patients, 20 PRXL2A down regulation of gene expression, 4
PRXL2A gene expression difference is not statistically significant, 1 PRXL2A gene expression up-regulation.Show PRXL2A gene and ACTR8 base
Because the diagnosis to osteoarthritis has certain guidance meaning, it is expected to become osteoarthritis molecular diagnosis in clinic or human health screening
Candidate markers.
3 PRXL2A gene of embodiment and ACTR8 distinguish RA and OA
Method for the efficiency evaluation of individual molecule or diagnostic model is to establish Receiver Operating Characteristics (receiver
Operating characteristic, ROC) curve, judged by area under calculated curve (Area Under Curve)
The ability of diagnosis.Area value under ROC curve is between 1.0 and 0.5, and in the case where AUC > 0.5, AUC is said closer to 1
Bright diagnosis effect is better, and AUC has lower accuracy in 0.5-0.7, and AUC has certain accuracy in 0.7-0.9, and AUC is 0.9
There is high accuracy when above.
Arthritis can be divided into according to the cause of disease by Osteoarthritis, rheumatoid arthritis, ankylosing arthritis, anti-in clinic
Answering property arthritis, urarthritis etc., osteoarthritis and rheumatoid arthritis are all systemic diseases, and big Minor articulus can be by
Tired, but the two is two different diseases, currently without the good molecular marker both distinguished, inventor is under study for action not yet
It is disconnected to find, it is found in the further research to PRXL2A gene and ACTR8 gene, both opposite osteoarthritis patient groups exist
Low expression in rheumatoid arthritis patient groups, it may be possible to completely new potential differentiation osteoarthritis and rheumatoid arthritis
Molecular marker compositions.
It chooses in GEO database and is downloaded in relation to the data of osteoarthritis and rheumatoid arthritis, obtain data set (32
The mRNA data of the mRNA data of OA patient and 59 RA patients), and then analyzed, the results show that PRXL2A gene and
It is 0.731 and 0.733 (see Fig. 2), the two Combining diagnosis that ACTR8 gene, which distinguishes osteoarthritis and the AUC of rheumatoid arthritis,
The AUC for distinguishing osteoarthritis and rheumatoid arthritis is 0.815 (see Fig. 3), and the two joint is used as molecular marker compositions
There is certain Overlay for distinguishing osteoarthritis and rheumatoid arthritis.
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. application of the reagent of the expression of gene or albumen in preparation osteoarthritis testing product in quantitative detection sample,
It is characterized in that, the gene or albumen are PRXL2A or ACTR8.
2. application according to claim 1, which is characterized in that the testing product passes through quantitative fluorescent PCR, Southern
Hybridization, Northern hybridization, fluorescence in situ hybridization, DNA microarray, high-flux sequence method, immunization detect PRXL2A in sample
Or the expression of ACTR8.
3. application according to claim 2, which is characterized in that the immunization includes ELISA method, radiommunoassay
Method, immunohistochemical method, western blot method.
4. application according to claim 2, which is characterized in that including in the testing product being capable of specific amplification
The primer and/or probe of PRXL2A or ACTR8 gene；Or the antibody of PRXL2A or ACTR8 albumen can be specifically bound.
5. application according to claim 4, which is characterized in that the primer sequence of the specific amplification PRXL2A gene is
SEQ ID NO.1 and SEQ ID NO.2.
6. application according to claim 4, which is characterized in that the primer sequence of the specific amplification ACTR8 gene is
SEQ ID NO.3 and SEQ ID NO.4.
7. application according to claim 1, which is characterized in that the sample is tissue or peripheral blood.
8. application according to claim 1, which is characterized in that the osteoarthritis testing product include chip, kit,
Test paper or high-flux sequence platform.
9. the reagent of the expression of gene or albumen distinguishes osteoarthritis in preparation in quantitative detection sample and rheumatoid closes
Save the application in scorching diagnostic tool, which is characterized in that the gene or albumen are PRXL2A and/or ACTR8, and detection sample is outer
All blood.
10. application according to claim 9, which is characterized in that the reagent is miscellaneous by quantitative fluorescent PCR, Southern
Friendship, Northern hybridization, fluorescence in situ hybridization, DNA microarray, high-flux sequence method, immunization detect PRXL2A in sample
And/or the expression of ACTR8.
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| CN108642168A (en) * | 2018-03-30 | 2018-10-12 | 北京泱深生物信息技术有限公司 | The Clinics and Practices of rheumatoid arthritis and osteoarthritis based on SPAST genes |
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| WO2017208001A1 (en) * | 2016-06-03 | 2017-12-07 | Babraham Institute | Biomarkers for platelet disorders |
| CN108642168A (en) * | 2018-03-30 | 2018-10-12 | 北京泱深生物信息技术有限公司 | The Clinics and Practices of rheumatoid arthritis and osteoarthritis based on SPAST genes |
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