CN105886628B - Application of the SPRR1A gene in preparation osteoarthritis diagnostic products - Google Patents

Application of the SPRR1A gene in preparation osteoarthritis diagnostic products Download PDF

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CN105886628B
CN105886628B CN201610281744.4A CN201610281744A CN105886628B CN 105886628 B CN105886628 B CN 105886628B CN 201610281744 A CN201610281744 A CN 201610281744A CN 105886628 B CN105886628 B CN 105886628B
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gene
osteoarthritis
sprr1a
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expression
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CN105886628A (en
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肖刻
翁习生
吴志宏
邱贵兴
朱乾坤
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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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

Abstract

The invention discloses application of the SPRR1A gene in preparation osteoarthritis diagnostic products.The invention discloses a kind of gene SPRR1As relevant to osteoarthritis, and further confirm that the expression albumen and its segment of the SPRR1A gene or the gene, analogs and derivatives express downward in osteoarthritic tissue.It can not only quickly and effectively accomplish early detection using the genetic test osteoarthritis, and provide therapy target and important evidence for clinical applications such as gene therapy, drug therapies.

Description

Application of the SPRR1A gene in preparation osteoarthritis diagnostic products
Technical field
The present invention relates to biomedicine fields, and in particular to SPRR1A gene answering in preparation osteoarthritis diagnostic products With.
Background technique
Osteoarthritis (osteoarthritis, OA) is the chronic disease that articular cartilage is gradually degenerated at any time, the pass Section cartilage is covered on bone end, forms the articular surface in joint.It is reported that there are many factors, and patient to be made to be susceptible to suffer from osteoarthritis, including lose Pass neurological susceptibility, obesity, accident or athletic injury, operation, drug and weight body demand force.Osteoarthritis is considered soft from joint What the damage of bone started.Most common two kinds are sports-related injury and long-term " Reusability " joint damage to the damage in joint Wound.The joint most frequently influenced by osteoarthritis is knee, hip and hand.In most cases, due to knee and the necessary load-bearing function of hip Can, the osteoarthritis in these joints more easily leads to deformity than Hand osteoarthritis.With the development of cartilage degradation, in joint and close Secondary variation occurs in its hetero-organization (including bone, muscle, ligament, meniscus and synovial membrane) around saving.Cartilaginous tissue primary declines Exhausting with the net effect of its hetero-organization secondary damage is that pain, swelling, weakness and diseased joints forfeiture function occur for patient.These diseases Shape is often developed to the degree seriously affected, is such as made patient disability or is influenced quality of life.
Articular cartilage is mainly made of cartilage cell, II Collagen Type VI, proteoglycans and water.Articular cartilage does not have blood or nerve Distribution, cartilage cell is only cell type in the tissue.Cartilage cell is responsible for manufacturing the II Collagen Type VI and egg of cartilage matrix White glycan.Thereafter the matrix has the physicochemical characteristics for allowing to be made with water matrix to be saturated again.This structure-function relationship it is net Effect is that articular cartilage has special wear resistance characteristics, and it is mobile that almost friction free occurs between articular cartilage face.Not When suffering from osteoarthritis, articular cartilage often provides lifelong painless load-bearing and unconfined under the even physical qualification of high demand Joint is mobile.
As all living tissues, articular cartilage constantly undergoes renewal process, wherein removal (catabolic activity) " old " cell and matrix components simultaneously generate (anabolic activity) " new " cell and molecule.It is organized relative to majority, articular cartilage Anabolism or catabolism turnover rate it is lower.The long-term maintenance of mature cartilage structure integrality is synthesized and is dropped dependent on matrix Balance appropriate between solution.Cartilage cell is by response in chemistry and mechanical stimulus maintenance matrix equilibrium in its environment. It is necessary to cartilage dynamic equilibrium that cartilage cell, which stimulates suitable and effective response to these,.Pass through anabolic activity deficiency Or catabolic activity surplus destroy dynamic equilibrium can cause cartilage degradation and osteoarthritis (Adams etc., 1995, Nature377Suppl:3-174).Majority, which is damaged, can start the synthesis of enhancing with the tissue of catabolic activity raising It is metabolized response, tissue is allowed to restore.Unfortunately, articular cartilage response is damaged or is consumed in cartilage matrix and raises it and synthesize generation The ability for thanking activity and raising synthetic proteins glycan and II Collagen Type VI is very limited.
Existing osteoarthritis treatment method include movement, drug, rest and joint care, operation, pain relief techniques, Replacement therapy and body weight control.Treating common drug in osteoarthritis includes nonsteroidal anti-inflammatory such as aspirin, brufen Deng；It is directly used in the topical pain-relieving creams, rubber and spray etc. of skin.It can be performed the operation so that bone surface reconstruction, bone Reset and replacement joint.Although various medicinal treatments have been used for treating disease, they are nothings to long-term control and prevention Effect.Further, since osteoarthritis hidden occur and develop slowly, therefore osteoarthritis often disease develop advanced stage It is just identified, rather than potential treatment may more effective early in disease development.Therefore prevention, change or treatment osteoarthritis Further development in lysis depends critically upon the early diagnosis marker of identification disease, so that early stage be allowed to interfere.
Summary of the invention
In order to realize the early detection of osteoarthritis, early intervention, the purpose of the present invention is to provide a kind of new bone passes Save scorching related gene and expression albumen and its segment, the analogs and derivatives of the gene.
A further object of the invention is to provide the osteoarthritis related gene and its expression product in detection osteoarthritis Application.
To achieve the above object, the answering in preparation osteoarthritis diagnostic products present invention firstly provides SPRR1A gene With.
Further the study found that the expression albumen and its segment of the SPRR1A gene or the gene, analog and Derivative expresses downward in Osteoarthritis tissue.
Further, the diagnostic products include the diagnostic products of gene level and the diagnostic products of protein level.
Preferably, the diagnostic products of the gene level be by real-time PCR, Northern blot, Southern blot, genetic chip, in situ hybridization or RNA enzyme Protection method detect the expression of SPRR1A gene； The diagnostic products of the protein level are to detect SPRR1A base by immune detection, Western blotting or protein chip Because of the expression of expression product.
Preferably, the product includes chip or kit.
It is furthermore preferred that in order to enhance the accuracy of detection, the chip is genetic chip.
It is furthermore preferred that the kit includes SYBR Green polymerase chain reaction system, for expanding SPRR1A base The primer pair of cause and house-keeping gene.
It is furthermore preferred that it is described amplification SPRR1A gene primer pair as shown in SEQ ID NO:1 and SEQ ID NO:2, institute The primer pair of amplification house-keeping gene is stated as shown in SEQ ID NO:3 and SEQ ID NO:4；The SYBR Green polymerase chain Reaction system includes PCR buffer, dNTPs, SYBR Green fluorescent dye；The PCR buffer includes 25mM KCL, 2.5mM MgCL2, 200mM (NH4)2SO4。
In a preferred embodiment, the kit further includes reverse transcription system, and the reverse transcription system includes: reverse transcription Reaction solution, M-MLV reverse transcriptase, T repeat oligonucleotides Oligo dT, RNase inhibitor, dNTPs；The reverse transcription reaction liquid Tris-HCL, 375mM KCL, 15mM MgCL comprising 250mM pH8.32、50mM DTT。
In another preferred embodiment, the kit further includes that RNA extracts reagent, and the RNA extracts reagent and includes Trizol, chloroform, isopropanol and 75% ethyl alcohol.
Further, the application the invention discloses the product of the diagnosis osteoarthritis in osteoarthritis diagnosis.
Specifically, applying step is as follows:
(1) expression for detecting osteoarthritis related gene in sample to be tested, wherein the osteoarthritis related gene It is SPRR1A gene；
(2) expression of osteoarthritis related gene in cell to be measured is compared with a control, SPRR1A gene expression It lowers, shows that research object is Human Osteoarthritis.
Preferably, the standard that judgement is lowered in step (2) is that the expression of osteoarthritis related gene compares normal population The reduction of the expression at least 10% or more of middle osteoarthritis related gene.
Preferably, the standard of judgement up-regulation is that the expression of osteoarthritis related gene compares normal population in step (2) The expression low 60% of middle osteoarthritis related gene.
Preferably, the standard of judgement up-regulation is that the expression of osteoarthritis related gene compares normal population in step (2) The expression low 90% of middle osteoarthritis related gene.
Beneficial effects of the present invention are as follows:
The invention discloses a kind of gene SPRR1As relevant to osteoarthritis, and further confirm the SPRR1A gene or Expression albumen and its segment, the analogs and derivatives of the gene express up-regulation in osteoarthritic tissue.It is examined using the gene Early detection can not only quickly and effectively be accomplished by surveying osteoarthritis, and be mentioned for clinical applications such as gene therapy, drug therapies Therapy target and important evidence are supplied.
Detailed description of the invention
SPRR1A down regulation of gene expression in Human Osteoarthritis in embodiment 4 in Fig. 1 present invention.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, reagent used can be commercially available.
Test method without specific conditions in embodiment, usually conventional method in that art, such as according to normal conditions Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to Condition proposed by reagent manufacturing firm.
The present inventor carries out high-flux sequence to 10 Osteoarthritis samples and 4 check samples, in conjunction with life Object Informatics Method carries out genescreen, picks out candidate gene SPRR1A, and there is no SPRR1A and bone to close in existing research The scorching relevant report of section, further, inventor has carried out molecular biology method verifying, it was confirmed that SPRR1A is in Osteoarthritis It expresses and lowers in tissue, related preparations can be used for treating Osteoarthritis.
SPRR1A gene of the invention is known before making the present invention, and essential information is as follows:
Genbank accession number: NCBI GeneID:6698 derives from human genome.
The present invention also uses RT-PCR method and method for gene chip detection said gene in Human Osteoarthritis and normally Expression in crowd, and demonstrating the gene is that osteoarthritis expresses down-regulated gene.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation. The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition The good, high sensitivity of property, high specificity, result are clear.
The prototype of genetic chip (genechip) (also known as DNA chip, biochip) is that the mid-80 proposes.Gene The sequencing principle of chip is sequencing by hybridization method, i.e., by carrying out nucleic acid sequence survey with the nucleic acid probe hybridization of one group of known array Fixed method secures the probe of target nucleotide known to sequence in one piece of substrate surface.When having fluorescent marker in solution Nucleic acid sequence TATGCAATCTAG, it is glimmering by determination and on genetic chip when the nucleic acid probe complementary matching of generation of corresponding position The strongest probe location of luminous intensity obtains the probe sequence of one group of sequence complete complementary.The sequence of target nucleic acid can be recombinated out accordingly.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types: 1) be fixed on poly- The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used Gene is hybrid with it, and is detected by radiography technology.The advantages of this method is required detection device and current molecule Radiography technology used in biology is consistent, relatively mature.But probe density is not high on chip, sample and reagent Demand is big, and there are more problems for quantitative detection.2) fix DNA probe array on a glass with point sample method, by with it is glimmering The target gene hybridization of signal is detected.This method reticular density can be greatly improved, and each probe is on the surface Binding capacity still has the difficulty being not easily overcome in terms of standardizing with mass production also than more consistent.3) in hard such as glass The oligonucleotide probe array synthesized directly on a surface hybridizes with the target gene of fluorescent marker and is detected.This method is micro- electricity Sub-light lithography is combined with DNA chemical synthesising technology, and the probe density of genetic chip can be made to greatly improve, reduce reagent Dosage realizes standardization and mass large-scale production, there is highly important development potentiality.
Biochip technology mainly includes four bare bones: the building of chip square matrix, the preparation of sample, biomolecule are anti- It should be with the detection of signal.1, prepared by chip, is first surface-treated sheet glass or silicon wafer, then makes DNA fragmentation or protein Molecule is arranged in order on chip.2, sample preparation, biological sample are often extremely complex biomolecule mixture, except few Outside number particular sample, generally cannot directly it be reacted with chip.Sample can be subjected to biological treatment, obtain protein therein or DNA, RNA, and marked, to improve the sensitivity of detection.3, biomolecule is reacted, between the biomolecule on chip Reaction is the key that chip detects a step.By reaction between selecting suitable reaction condition to make biomolecule in optimum In, reduce the mispairing ratio between biomolecule.4, chip signal detects, and common chip signal detection method is to set chip Enter in chip scanner, related biological information is obtained by scanning.
The nucleotide full length sequence or its segment of SPRR1A gene of the invention can usually use PCR amplification method, recombination method Or artificial synthesized method obtains.For PCR amplification method, can be read according to published related nucleotide sequence, especially opening Frame sequence carrys out design primer, and with the commercially available library cDNA or by prepared by conventional method well known by persons skilled in the art CDNA is expanded as template and is obtained related sequence.When sequence is longer, it is often necessary to which progress expands twice or repeatedly, then The segment repeatedly amplified is stitched together by proper order again.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, by first synthesizing Then multiple small fragments are attached the very long segment of available sequence again.
At present, it is already possible to encode albumen of the invention (or its segment, or derivatives thereof) by chemical synthesis completely DNA sequence dna.Then the DNA sequence dna can be introduced into various DNA moleculars (such as carrier) and cell in this field.In addition, also Mutation can be introduced into protein sequence of the present invention by chemical synthesis.
The segment of albumen of the present invention also can be used solid phase technique to pass through direct synthetic peptide other than available recombination method generates Produced (Stewart et al., (1969) Soliod-Phase Peptide Synthesis, WH Freeman Co., San Francisco；Merrifield J.(1963)J.Am Chem.Soc85:2149-2154).Synthetic proteins matter can be in vitro By hand or automatically it carries out.For example, can with the 431A type peptide synthesizer of Applied Biosystems (Foster City, CA) it is automatically synthesized peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then chemically connected to produce The molecule of raw overall length.
The particular form of terms used herein " osteoarthritis " articulations digitorum manus inflammation, especially articular cartilage are gradually moved back at any time The chronic disease of change, the articular cartilage are covered on the bone end to form articular surface.
Terms used herein " expression is lowered " refer to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount It is bright, it is determined in the individual for having identified morbid state different with osteoarthritis by stages from from normal individual or from by osteoarthritis Same gene in isolated biological sample is compared, and the gene is from suffering from osteoarthritis or determined by osteoarthritis by stages Osteoarthritis identified the expression in the biological sample separated in the individual of morbid state reduction.According to the present invention, " expression lower " refer to intensity for hybridization measurement by the method for the invention at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more expression reduces, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more It is low.
Used in the context of terms used herein " protein substance " (such as protein, polypeptide, peptide and antibody) Term " analog " refers to such protein substance: its have the function of with second of protein substance it is similar or identical, But not necessarily comprising the amino acid sequence similar or identical with second of protein substance or have and second of albumen The similar or identical structure of matter substance.Protein substance with similar amino acid sequence refers at least one of satisfaction or less Second of protein substance:
(a) have with the amino acid sequence at least 30% of second of protein substance, at least 35%, at least 40%, extremely Few 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, the protein substance of the amino acid sequence of at least 90%, at least 95% or at least 99% consistency；
(b) by nucleotide sequence coded protein substance, the nucleotides sequence is listed under high stringency conditions and coding the At least five continuous amino acid residue, at least ten continuous amino acid residue, at least 15 Continuance ammines of two kinds of protein substances Base acid residue, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous hydrogen-based acid are residual Base, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, at least 125 companies The nucleotide sequence hybridization of continuous amino acid residue or at least 150 continuous amino acid residues；
(c) by nucleotide sequence coded protein substance, second of protein of the nucleotide sequence and coding The nucleotides sequence of substance shows at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, extremely Few 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% consistency.Protein substance similar with second of protein substance structure, which refers to, to be had and second of protein-based object The protein substance of the similar second level of matter, three or four structure.
Terms used herein " protein ", " polypeptide " and " protein substance " are used interchangeably, and are referred to and are connected by peptide bond The amino acid chain being connected together optionally may include natural or non-natural amino acids.Optionally, protein or polypeptide may include removing Other molecules other than amino acid.The chain can be any length, if the protein by peptide bond as being linked together Less than 200, less than 175, less than 150, less than 125, less than 100, less than 50, less than 45, less than 40, Less than 35, less than 30, less than 25, less than 20, less than 15, less than 10 or less than 5 amino acid composition.Another In one embodiment, protein by linked together by peptide bond at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500 or more compositions.
Terms used herein " expression " refer to through the measurement of known to those skilled in the art and method described herein Given nucleic acid or protein can measure.It is related to corresponding RNA, hnRNA, mRNA or mRNA montage of biomarker of the present invention When variant, expression can be measured by hybridization or more, for example including use SYBR green, TaqMan and point The quantitative real-time RT PCR of sub- beacon technique.
" control " used herein refer to do not show any OA symptom (including arthralgia) and have not been diagnosed as joint injury or The individual or group of individuals of OA.Preferably, it is described control individual be not used influence OA drug, and have not been diagnosed as with it is any its His disease.It is highly preferred that control individual has similar gender, age and Body Mass Index (BMI) compared with test sample.According to The present invention, " control " also refer to the sample for being isolated from normal individual, total serum IgE or mRNA including being isolated from normal individual.It is derived from pair Sample according to individual may include the RNA for being isolated from sample tissue, and wherein RNA is isolated from sample tissue, the sample tissue separation Self-organizing has not been diagnosed as OA and does not show the individual of any OA symptom when removing.In one embodiment of the invention, it is Meniscus injury and cruciate ligament carry out the patient of arthrocsopic surgery treatment.
Terms used herein " primer " refer to the oligonucleotides for being present in the restrictive digestion content of purifying or being synthetically produced, When being placed in induction and synthesizing under conditions of the primer extension product complementary with nucleic acid chains, that is, there is nucleotide and inducer, (such as DNA gathers Synthase) and temperature appropriate and when pH, the oligonucleotides can be as the starting point of synthesis.Primer can be single-stranded or double Chain, and must have enough length, to cause the synthesis of required extension products in the presence of inducer.The definite length of primer Degree can depend on many factors, including temperature, Primer Source and the method used.For example, depending on target for diagnostic application The complexity of sequence, Oligonucleolide primers typically contain the nucleotide of 15-25 or more, although can also contain less nucleosides Acid.Factor involved in the determination of primer appropriate length is known to those of ordinary skill in the art.In general, according to ability Standard method known to domain designs and selects primer of the invention, refering to Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S. (1995) General Concepts for PCR Primer Design. " PCR Primer, A Laboratory Manual " (Dieffenbach, CW and Dveksler, G.S. are edited) Cold Spring Harbor Laboratory Press, New York, 133-155.
" nucleic acid " used herein, is often referred to any polybribonucleotide and polydeoxyribonucleotide, can be Unmodified RNA or DNA or modified RNA or DNA or any combination thereof." nucleic acid " includes but is not limited to single-stranded and double-strand core Acid.Terms used herein " nucleic acid " further include the DNA or RNA as described above containing one or more modified bases.Therefore, In order to which DNA or RNA that stability or other reasons have modified skeleton are " nucleic acid ".Terms used herein " nucleic acid " include nucleic acid These chemistry, enzymes or metabolism modification form and virus and cell (including such as simple cell and complex cell) DNA and RNA The chemical species of feature." nucleic acid " or " nucleic acid sequence " may also include single-stranded or double-stranded RNA or DNA region or its any group It closes, and may include the EST (EST) of some embodiments of the invention.EST is by the area reverse transcription mRNA to generate CDNA and a part of expressed sequence (i.e. " label " of sequence) of gene generated.
" nucleic acid probe " used herein include can by the interaction of one group of Non-covalent binding (including complementary base is matched Interaction) nucleic acid in conjunction with the complementary series of nucleic acid member on array.Nucleic acid probe used herein may include it is natural (i.e. A, G, C or T) or modified base (7- denitrogenation guanosine, inosine etc.).In addition, the base in nucleic acid probe can pass through di-phosphate ester Bond other than key is closed, if it does not interfere hybridization (i.e. the nucleic acid probe still in the case where standard is rigorous or selective cross condition and Its complementary series specific binding).Therefore, nucleic acid probe can be peptide nucleic acid, wherein composition base passes through peptide bond rather than phosphorus Acid diesters bond is closed.
Terms used herein " biological sample " include but is not limited to blood, serum, saliva, urine, synovia, cartilage, synovial membrane The samples such as tissue.
Biological sample of the invention
For biological sample of the invention derive from any subject any tissue sample (such as cartilage, synovial membrane, synovia or Blood sample) or cell sample (such as cartilage cell or blood cell samples) can use according to the method for the present invention.It can therefrom obtain These samples and the use of subject's example of these samples includes but is not limited to according to the method for the present invention asymptomatic subject, performance Or more the subjects of osteoarthritis symptoms, clinical diagnosis be subject with osteoarthritis, be susceptible to suffer from the tested of osteoarthritis Person (if any the subject of osteoarthritis family history, has the subject of osteoarthritis genetic predisposition and life style to make it easily Sense osteoarthritis or improve suffer from osteoarthritis possibility subject), suspect with osteoarthritis subject, just receiving bone pass It is the subject of the scorching treatment of section, true with osteoarthritis and the non-subject for receiving osteoarthritis treatment, practitioner (such as doctor) It is set to health or bone-free arthritic subject (i.e. normal), the subject that has cured osteoarthritis, is controlling its Bones and joints Scorching subject and the subject for having not been diagnosed as osteoarthritis.In a specific embodiment, it can get sample and use Subject to suffer from hand, foot, backbone, knee, hip and/or articulatio intercarpea scorching.
Cartilage, synovial membrane
In an aspect, from the normal individual of existence or after death 14 according to method that is known in the art and being described below Cartilaginous tissue within hour obtains cartilage samples or synovial samples.It is obtained using any known method from adult normal pass Cartilage is saved, receives the individual of arthrocsopic surgery or total knee replacement as cartilage or synovial membrane derive from, sample saves for use in liquid nitrogen. Since ethics considers, true normal cartilage or synovial membrane generally can not be sampled extensively from live donor.It is therefore preferred that 14 Normal cartilage or synovial samples are obtained from deceased donor after stopping to the perfusion of sampling joint in the after death window of hour, so as to see The RNA degradation observed is minimum.In another aspect, cartilage or synovial membrane derive from the subject for being diagnosed as osteoarthritis.Using any Known technology obtains person joint's sample from osteoarthritis individual.Preferably, joint sample is stored in liquid nitrogen for use.? In one specific embodiment, separation at least 0.05g cartilage or synovial samples, to obtain 2 μ g Total RNAs extraction objects.It can be according to this It invents the cartilage used or synovial samples is the amount for being enough to detect one or more nucleic acid sequences of the present invention or amino acid sequence.
Synovia
In an aspect, synovial fluid samples are obtained from subject using method well known in the art.For example, can be closed Section punctures.In arthrocentesiS, synovia is removed from joint using sterile needle.Can be used arthrocentesiS from knee, elbow, wrist, refer to, Synovia is collected in hip, backbone or any other joint.In a specific embodiment, synovia is collected from suffering from osteoarthritis or bosom Doubt the joint for suffering from osteoarthritis.Synovia, which is available from, to be suffered from OA, be not suffering from the subject of OA or derive from test subject.
Synovial fluid samples that can be used according to the invention are to be enough to detect one or more nucleic acid sequences of the present invention or amino acid The amount of sequence.In a specific embodiment, the amount range of synovial fluid samples that can be used according to the invention from 0.1mL to 20mL, 0.1mL to 15mi, 0.1mL to 10mL, 0.1mL to 5mL, 0.1 to 2mL, 0.5mL to 20mL, 0.5mL to 15mL, 0.5mL is to 10mL, 0.5mL to 5mL or 0.5mL to 2mL.
In some embodiments of the present invention, using cell present in methods known in the art separation synovia, and It uses according to the method for the present invention.The visible following cell usually in synovia: lymphocyte (B and T lymphocyte), monocyte, Neutrophil cell, synovial cell and macrophage.May be used also in the synovia of the patient from pathological state (such as osteoarthritis) See following cell: cartilage cell, osteoblast and osteoclast.It can separate in the method in accordance with the invention and thin using these Born of the same parents.In a specific embodiment, lymphocyte (B and T lymphocyte) and according to the present invention side are separated from synovial fluid samples Method uses.In another embodiment, monocyte or neutrophil cell and according to the present invention side are separated from synovial fluid samples Method uses.It, can be by standard technique freezing separation from the cell of synovia for before the method for the present invention.
Blood
In one aspect of the invention, blood sample is obtained from subject according to method well known in the art.It can be used Technology well known by persons skilled in the art (especially known blood-sampling method) from any physical feeling of subject (such as finger, hand, Wrist, arm, leg, foot, ankle, stomach and neck) take blood.In a specific embodiment, venous blood is obtained from subject and according to this Inventive method uses.Vacuum tube, syringe or butterfly needle can be used and take blood.
Blood sampling volume will depend on the comfort level of blood sampling site, the amount that the method for the present invention needs and subject and change.Such as exist In multiple specific embodiments, from subject collect 0.001mL, 0.005mL, 0.01mL, 0.05mL, 0.1mL, 0.15mL, 0.2mL, 0.25mL, 0.5mL, 0.75mL, 1mL, 1.5mL, 2mL, 3mL, 4mL, 5mL, 10mL, 15mL or more.Blood is protected There are in K3/EDTA pipe.
In an aspect, according to blood-sampling method well known in the art from normal individual or from being diagnosed as suffering from osteoarthritis Or suspect that the individual for suffering from osteoarthritis takes whole blood.Whole blood includes the blood that can be used directly, and including according to it is well known that Method (such as being preferably mildly centrifuged 5-10 minutes in 300-800 × g) except serum deprivation or blood plasma and from remaining blood sample The middle blood for having separated RNA or mRNA.In a specific embodiment, by derive from subject whole blood (not being classified blood) with Lysis buffer (such as lysis buffer (1L): 0.6g EDTA；1.0g KHCO2,8.2g NH4Cl is adjusted with NaOH to arriving PH7.4 it) mixes, Centrifuge A sample simultaneously retains cell precipitation, extracts RNA or mRNA (" cracking blood ") according to method well known in the art (refering to such as Sambrook etc.).It is preferable to use whole bloods, because of the step of it avoids cell type in costly and time consuming separation blood (Kimoto, 1998, Mol.Gen.Genet258:233-239；Chelly J etc., 1989, Proc.Nat.Acad.Sci.USA86:2617-2621；Chelly J etc., 1988, Nature333:858-860).
In some embodiments of the present invention, it will be classified collected from subject whole blood and (be separated into component). It is thin from the whole blood separation blood collected from subject using techniques known in the art in one embodiment of the invention Born of the same parents.For example, Ficoll-Hypaque (Pharmacia) gradient centrifugation can be carried out to the blood collected from subject.This centrifugation Red blood cell (erythrocyte) is separated with various types of karyocytes and blood plasma.Specifically, Ficoll-Hypaque gradient Centrifugation can be used for separating peripheral white blood cells (PBL), they can be used according to the method for the present invention.Other whole blood separation methods according to Method well-known to those skilled in the art is operated.
According to the method for the present invention before use, the biological sample of collection optionally (but preferably) is stored in low temperature such as 4 At DEG C.In some embodiments, in a part for using sample according to the method for the present invention at the first time, and will be one or more Remaining sample part saves a period of time for future use.This time can be 1 hour or longer, 1 day or longer, 1 week or Longer, January or it is longer, 1 year it is longer or uncertain.For long-term preservation, store method well known in the art can be used, Such as it is saved in cryogenic temperature (as being lower than -80 DEG C).It in some embodiments, will as the supplement or substitution for saving sample Isolated nucleic acid or protein save a period of time (such as 1 hour or longer, 1 day or longer, 1 week or longer, January or it is longer, 1 Year is longer or uncertain) for future use.
1 high-flux sequence of embodiment screens difference expression gene
1, it samples
Take in October, 2012 to during in December, 2015 BJ Union Hospital's orthopaedics go to a doctor osteoarthritis patient, case Group collects 10 altogether, and the other diseases patient that control is hospitalized from same time orthopaedics collects 4 altogether.Obtain all researchs pair The synovia sample of elephant, -80 DEG C of low temperature refrigerators of number postposition save.
Case group meets knee joint OA diagnostic criteria, carries out knee joint artificial joint replacement patient；Control group is half Month dash-board injury and cruciate ligament carry out the patient of arthrocsopic surgery treatment.According to nineteen ninety-five U.S.'s rheumatism association's bone joint Scorching diagnostic criteria, is diagnosed as severe Osteoarthritis, there is the patient cases of knee prosthesis indication.
Wherein, clinical criteria is according to the prepared standard of nineteen ninety-five American Rheumatism Association:
Most of time has goinyalgia in (1) 1 month；
(2) sound when joint motion；
(3) morning stiffness is not more than 30 minutes；
(4) age is not less than 40 years old；
(5) swelling of knee joint bone is with snap；
(6) swelling of knee joint bone is not accompanied by snap.
It is minimum to there is ((1), (2), (3), (4) or (1), (2), (3), (5) or (1), (6) i.e. diagnosable OA.
2, Total RNAs extraction is carried out to synovia
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation It is carried out by product description, concrete operations are as follows:
It freezes to be put into the mortar being pre-chilled synovia after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized This is at powdered rear:
1. Trizol is added, room temperature preservation 5 minutes；
2. chlorination imitates 0.2mL, with forced oscillation centrifuge tube, mix well, places -10 minutes 5 minutes at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse It mixes, is placed in 10 minutes on ice；
4. 12000rpm high speed from 15 minutes after carefully discard supernatant, in I mL/mL Trizol ratio be added 75% DEPC ethyl alcohol washes paint precipitating (4 DEG C of preservations), washes paint sediment, oscillation mixes, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometer, freeze in -80 DEG C.RNA mass is sentenced Calibration is quasi-: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band； 70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
3, the quality analysis of RNA sample
RNA extract after agarose gel electrophoresis, from electrophoresis result can with preliminary judgement extract RNA sample it is up-to-standard with It is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometer Extract situation, the sample requirement of RNA-seq sequencing: OD260/OD280 1.8-2.2.
4, high-flux sequence
Microarray dataset is the 2500 high-flux sequence platform of HiSeq of Illumina company, carries out high-throughput transcript profile depth Sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/ after sequencing Fastqc/) software carries out total evaluation, the quality Distribution value including base, the position point of mass value to the quality of sequencing data Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.In differential genes expression analysis, according to obtaining FPKM value, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, when screening, LOG2FC>1 or<-1, FDR< 0.05.In order to better understand the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above data Analysis as a result, we have screened difference expression gene SPRR1A in conjunction with document, the gene is in Osteoarthritis sample tissue Expression is lowered.
2 RT-PCR of embodiment verifies osteoarthritis patient and control synovia SPRR1A expression conditions
1, material
42 osteoarthritis patient's cartilaginous tissues and 8 control cartilaginous tissues are chosen, it is grouped and is numbered.Disease Example group meets knee joint OA diagnostic criteria, carries out knee joint artificial joint replacement patient；Control group be meniscus injury and The patient of cruciate ligament progress arthrocsopic surgery treatment.
2, method
2.1 pairs of synovia carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) CDNA reverse transcription is carried out, experimental implementation is carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ L Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- Δ Δ CT method.
2.3.2 design of primers
Using online primer-design software, gene order is referring to NCBI:Gene ID:6698 (SPRR1A), interior participation in the election GAPDH is synthesized by invitrogen company after design of primers.Specific primer sequence is as shown in table 1:
1 primer sequence of table
Operating process is as follows:
(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No. 4367659) it is expanded, experimental implementation is carried out by product description.Amplification program are as follows: 95 ° of 5min, (95 DEG C of 15sec, 60 DEG C 45sec, 72 DEG C of 35sec) × 40 circulations.
2 RealTime reaction system of table
Component Additional amount
2×mix 10μL
Upstream primer (10 μM) 0.5μL
Downstream primer (10 μM) 0.5μL
Template 2μL
Sterile purified water is added To 25 μ L
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ L to make template after dilution, It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.
(3) sample RealTime-PCR is detected
2 μ L will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.
Two, experimental 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% compares expression of the SPRR1A gene in Osteoarthritis tissue and control tissue.As the result is shown: QRT-PCR stable amplification result, wherein expression of the SPRR1A gene in Osteoarthritis tissue is only control tissue 20%, result above demonstrates the confluence analysis SPRR1A gene of high-throughput transcript profile expression data in osteoarthritis patient Express the result lowered.
3 genetic chip of embodiment verifies osteoarthritis patient and control synovia SPRR1A expression conditions
1, the acquirement of material, with embodiment 2.
2, the extraction of total serum IgE, with the method for embodiment 1.
3, genetic chip is verified
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty People's full genome chip of expression spectrum (4x 44K gene) of Agilent company, state, 65 DEG C of hybridization 17h in chip hybridization furnace, then Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P < 0.05 difference has significant meaning.The results show that being compared with normal people, the mRNA water of SPRR1A gene in Human Osteoarthritis synovia Flat is only 21%, and difference has statistical significance (P < 0.05).
The preparation of 4 kit of embodiment
Based on the primer sets that embodiment 2 obtains, the kit of the present invention for being used to detect osteoarthritis is assembled, it is described Kit includes the primer pair of specific amplified SPRR1A gene as shown in SEQ ID NO:1 and SEQ ID NO:2 and specific amplified The primer pair of house-keeping gene (GAPDH) is as shown in SEQ ID NO:3 and SEQ ID NO:4；It further include SYBR Green polymerase Chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs.The ingredient of the PCR buffer is 25mM KCL, 2.5mM MgCL2, 200mM (NH4)2SO4。
It is final to determine that reaction system is as shown in table 3 by the optimization to primer concentration and annealing temperature:
3 PCR reaction system of table
Optimum reaction condition are as follows:
95 ° of initial denaturation 5min, (95 DEG C of denaturation 15sec, 61 DEG C of annealing 45sec, 72 DEG C of extension 35sec) × 40 circulations, 72 DEG C of extension 15min.
Performance verification of 5 kit of embodiment to osteoarthritis
1, case
Take the synovial tissue of 60 patients with osteoarthritis to be checked and 10 normal persons, the artificial Beijing association of osteoarthritic condition to be checked It goes to a doctor osteoarthritis patient with hospital orthopedics, 10 normal persons are sliding for trauma surgery patient joint from the hospital orthopedics Liquid tissue.All clinical samples of this research know to patient and inform and pass through through this Hospital Ethical Committee.
2, method
RNA is extracted using conventional method or using specific kit, reverse transcription is at cDNA, with the reagent in embodiment 4 Box carries out detection reaction.
3, result
Purpose band is determined by solubility curve analysis and electrophoresis, and Δ Δ Ct method carries out relative quantification, as a result such as Fig. 1 institute Show, compared with normal tissue, it is the 19% of normal population that SPRR1A gene is expressed only in Human Osteoarthritis, it was demonstrated that the gene Expression is lowered.So can not only quickly and effectively accomplish early detection with the genetic test osteoarthritis, and controlled for gene The clinical applications such as treatment, drug therapy provide therapy target and important evidence.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (10)

  1. Application of the detection reagent of 1.SPRR1A gene expression dose in preparation osteoarthritis diagnostic products.
  2. 2. application as described in claim 1, which is characterized in that under the SPRR1A gene is expressed in Osteoarthritis tissue It adjusts.
  3. 3. application as claimed in claim 1 or 2, which is characterized in that the diagnostic products include the diagnostic products of gene level With the diagnostic products of protein level.
  4. 4. application as claimed in claim 3, which is characterized in that the diagnostic products of the gene level are to pass through real-time PCR, Northern blot, Southern blot, genetic chip, in situ hybridization or RNA enzyme Protection method detect The expression of SPRR1A gene；The diagnostic products of the protein level be by immune detection, Western blotting or Protein chip detects the expression of SPRR1A gene expression product.
  5. 5. application as claimed in claim 4, which is characterized in that the product includes chip or kit.
  6. 6. application as claimed in claim 5, which is characterized in that the chip is genetic chip.
  7. 7. application as claimed in claim 5, which is characterized in that the kit includes SYBRGreen polymerase chain reaction System, the primer pair for expanding SPRR1A gene and house-keeping gene.
  8. 8. the use as claimed in claim 7, which is characterized in that the primer pair such as SEQ ID NO of the amplification SPRR1A gene: Shown in 1 and SEQ ID NO:2, the primer pair of the amplification house-keeping gene is as shown in SEQ ID NO:3 and SEQ ID NO:4；Institute Stating SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green fluorescent dye.
  9. 9. the use as claimed in claim 7, which is characterized in that the kit further includes reverse transcription system, the reverse transcription System includes: reverse transcription reaction liquid, M-MLV reverse transcriptase, T repeat oligonucleotides Oligo dT, RNase inhibitor, dNTPs.
  10. 10. the application as described in claim 7 or 9, which is characterized in that the kit further includes that RNA extracts reagent, described It includes Trizol, chloroform, isopropanol and 75% ethyl alcohol that RNA, which extracts reagent,.
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