CN105154540A - PAEP gene and PAEP gene expression product as osteoarthritis diagnosis and treatment target - Google Patents
PAEP gene and PAEP gene expression product as osteoarthritis diagnosis and treatment target Download PDFInfo
- Publication number
- CN105154540A CN105154540A CN201510548624.1A CN201510548624A CN105154540A CN 105154540 A CN105154540 A CN 105154540A CN 201510548624 A CN201510548624 A CN 201510548624A CN 105154540 A CN105154540 A CN 105154540A
- Authority
- CN
- China
- Prior art keywords
- paep
- gene
- osteoarthritis
- chip
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses a molecular marker, namely a PAEP gene used for osteoarthritis early stage diagnosis. Experiments prove that the mRNA level of the PAEP gene in synovial tissues of an osteoarthritis patient is obviously lower than that in the normal synovial tissues. Therefore whether a subject has the osteoarthritis risk or not can be judged through testing the expression level of the PAEP gene in the synovial tissues of the subject, or whether the subject has the osteoarthritis or not can be judged. The PAEP gene can be used for preparing medicine for osteoarthritis patients or people with high osteoarthritis risk, so that the treatment on the osteoarthritis or the prevention on the occurrence of the osteoarthritis can be realized. The invention provides a new diagnosis method for clinically diagnosing the osteoarthritis, and provides new candidate medicine for treating the osteoarthritis.
Description
Technical field
The present invention relates to biological technical field, relate to the purposes of people PAEP gene in the diagnosis, treatment of osteoarthritis particularly.
Background technology
Osteoarthritis (Osterarthritis, OA) being one group, what caused by many reasons take Articular cartilage degeneration as the clinical syndrome of major pathologic features, a kind of common disease, multiple be born in the middle age after, be a kind of chronic, progressivity arthropathy.Mainly occur in joint cartilage.It shows mainly joint cartilage and is damaged, and cartilage surface loses uniformity, interruption, patch depression, fracture and ulcer.Recently some researchs show that the generation of osteoarthritis is relevant with inherited genetic factors.Synovial membrane for the normal function maintaining joint, and plays an important role in the generation evolution of osteoarthritis disorders.
At present clinically for the diagnosis of osteoarthritis mainly based on the inspection of iconography.To be mainly joint space narrow in x-ray performance, subchondral bony sclerosis and cystis degeneration, and joint margins spur is formed, and articulum withers, be out of shape and subluxation of joint etc.MRI can show the pathology of the articulation structures such as early stage cartilage, meniscus, is conducive to early diagnosis.CT is used for the diagnosis of discopathy, is better than x-ray.Joint space narrows, Subchondral bone sclerosis, and marginality spur spinal joint is linked to be bone bridge.Also visible bone cyst and deformity.As found, these changes can be used as the foundation of diagnosis and estimate the degree of joint injury.OA does not generally have in early days or seldom has symptom, and only in inflammation secondary, pain or when affecting joint movable, patient just looks for a doctor.Now, joint injury occurs for a long time.Because the method that exploitation is used for the diagnosis of early stage osteoarthritis is problem demanding prompt solution.
Summary of the invention
In order to make up the deficiencies in the prior art, one is the object of the present invention is to provide to can be used for the molecular marker of osteoarthritis (Osterarthritis, OA) early diagnosis.Compare the diagnostic method of traditional osteoarthritis, what use gene marker to diagnose osteoarthritis has promptness, specificity and susceptibility, thus make patient just can know disease risks in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of people PAEP gene and the application of expression product in the product of preparation diagnosis osteoarthritis thereof.
Further, the diagnostic products mentioned above comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection PAEP gene and expression product thereof to diagnose the product of osteoarthritis.
Further, described RT-PCR diagnoses the product of osteoarthritis at least to comprise the primer of a pair specific amplified PAEP gene; The product of described real-time quantitative PCR diagnosis osteoarthritis at least comprises the primer of a pair specific amplified PAEP gene; The product of described immunodetection diagnosis osteoarthritis comprises: the antibody be combined with PAEP protein-specific; The product of described in situ hybridization diagnosis osteoarthritis comprises: with the probe of the nucleic acid array hybridizing of PAEP gene; The product of described chip diagnosis osteoarthritis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with PAEP protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of PAEP gene.
Preferably, described product comprises chip, test kit.
Present invention also offers the application of people PAEP gene in high-flux sequence platform.Along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work very easily.By contrasting the gene expression profile of Disease and normal population, easily analyze exception and the disease-related of which gene.Therefore, in high-flux sequence, the exception of the people PAEP gene purposes that also belong to people PAEP gene relevant to osteoarthritis is known, equally within protection scope of the present invention.
Present invention also offers people PAEP gene and the application of expression product in the medicine of preparation treatment osteoarthritis thereof.
Further, described medicine comprises: the medicine that the carrier of the medicine containing people PAEP gene, carrier PAEP gene or host cell are formed, people PAEP pharmaceutical grade protein or other can promote the medicine of PAEP genetic expression.Medicine of the present invention may be used for disappearance or the deficiency of supplementary endogenic people PAEP albumen, by improving the expression of people PAEP albumen, thus the osteoarthritis that treatment causes because of people PAEP hypoproteinosis.
The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, comprises plasmid, clay, phage, virus etc.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
Further, medicine of the present invention also comprises pharmaceutically acceptable carrier, carrier, and this kind of carrier comprises (but being not limited to): thinner, vehicle are if water etc., weighting agent are as starch, sucrose etc.; Tackiness agent is as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent is as glycerine; Disintegrating agent is as agar, calcium carbonate and sodium bicarbonate; Absorption enhancer quaternary ammonium compound; Tensio-active agent is as cetyl alcohol; Absorption carrier is as kaolin and soap clay; Lubricant is as talcum powder, calcium stearate and magnesium, polyoxyethylene glycol etc.
The mode of drugs delivery tissue of the present invention or cell can be divided into the mode in external or body.Vitro formats comprises by the medicine containing people PAEP gene or containing in the drugs delivery cell of people PAEP protein, then by Transplanted cells or feed back in body.In body, mode comprises directly by the medicine containing people PAEP gene or containing in the infusion of medicine in-vivo tissue of people PAEP protein.
Medicine of the present invention also can with the drug combination of other treatment osteoarthritis, multi-medicament conbined usage can mention the success ratio for the treatment of greatly.
Present invention also offers a kind of chip diagnosing osteoarthritis, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for PAEP gene for detecting PAEP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of PAEP albumen of solid phase carrier.
Further, described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to osteoarthritis multiple genes) comprising people PAEP gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to osteoarthritis multiple protein) comprising people PAEP albumen.By being detected by multiple mark with osteoarthritis simultaneously, the accuracy rate of osteoarthritis diagnosis greatly can be improved.
The invention provides a kind of test kit diagnosing osteoarthritis, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting PAEP gene transcription level; Described protein immunization detection kit comprises the specific antibody of PAEP albumen.
Further, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection PAEP gene expression dose process.Preference, described reagent comprises primer for PAEP gene and/or probe.The primer and probe that may be used for detecting PAEP gene expression dose is easily designed according to the nucleotide sequence of PAEP gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of PAEP gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
Further, the specific antibody of described PAEP albumen comprises monoclonal antibody, polyclonal antibody.The specific antibody of described PAEP albumen comprise complete antibody molecule, any fragment of antibody or modification (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with PAEP albumen.Well known to a person skilled in the art during preparation for the antibody of protein level, and the present invention can use any method to prepare described antibody.
In the context of the present invention, " PAEP gene " comprises the polynucleotide of any function equivalent of people PAEP gene and people PAEP gene.PAEP gene comprises and has more than 70% homology with PAEP gene (NC_000009.12) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of PAEP gene comprises any DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described PAEP gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, PAEP gene expression product comprises the partial peptide of people PAEP albumen and people PAEP albumen.The partial peptide of described PAEP albumen contains the functional domain relevant to osteoarthritis.
" PAEP albumen " comprises any function equivalent of people PAEP albumen and people PAEP albumen.Described function equivalent comprises people PAEP albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people PAEP under high or low stringent condition.
Preferably, PAEP albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described PAEP albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is PAEP albumen.Peptide or protein with PAEP protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of PAEP albumen.
PAEP albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of PAEP albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis osteoarthritis " had both comprised and had judged whether experimenter has suffered from osteoarthritis, also comprised and judge whether experimenter exists the risk suffering from osteoarthritis.
In the context of the present invention, " treatment osteoarthritis " divides from the change of state of disease, can comprise the healing completely of the alleviation of disease, disease.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention PAEP genetic expression is relevant to osteoarthritis, by detecting the expression of PAEP in experimenter synovial tissue, can judge whether experimenter suffers from osteoarthritis or judge whether experimenter exists the risk suffering from osteoarthritis, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-PAEP gene, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of osteoarthritis can be realized, thus reduce the mortality ratio of osteoarthritis.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of PAEP gene in synovial cell;
Fig. 2 display utilizes Westernblot to detect the expression of PAEP albumen in synovial cell;
What Fig. 3 display utilized QPCR to detect PAEP gene transcribes situation;
Fig. 4 display utilizes Westernblot to detect the process LAN situation of PAEP albumen;
Fig. 5 shows the proliferation function of synovial fluid to OA fibroblast-like synoviocyte;
Fig. 6 shows the restraining effect of PAEP gene overexpression to the OA fibroblast-like synoviocyte propagation under incentive condition;
Fig. 7 shows the restraining effect that PAEP gene overexpression is bred OA fibroblast-like synoviocyte.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
The differential expression of PAEP gene in embodiment 1OA synovial tissue and normal synovial tissue
The synovial tissue of 60 routine OA patients comes from patient OA of the capable knee prosthesis of BJ Union Hospital's orthopaedics or villusectomy.Case used meets the Case definition about OA that Altam proposes.40 routine normal synovial tissue come from BJ Union Hospital's orthopaedics, are trauma surgery patient synovial tissue of joint.Get supernatant after OA patient's synovial fluid (SF) high speed centrifugation ,-80 DEG C of storages are stand-by.Clinical sample used in this research, all carries out knowing the inside story to patient and informs and pass through through Ethics Committee of the court.
1, the detection of PAEP gene transcription level
1.1 synovial tissue's cell injuring model
By the synovial tissue of aseptic acquisition with after PBS cleaning, the tissue block into about 1mmx1mmx1mm is repeatedly sheared with aseptic operation scissors, after adding collagenase II (0.5mg/ml) 37 DEG C, digestion 2h, filter through 200 order gauzes, centrifugal remove supernatant after, cell is resuspended in DMEM nutrient solution, is placed in 37 DEG C, 5%CO
2cultivate in cell culture incubator.After cell grows up to fusiformis and be in blocks, carry out Secondary Culture.After cell reached for the 3rd generation, add respectively FITC mark mouse anti human CD3, CD14, CD19 and PE mark mouse anti human CD11b mark, flow cytomery identify.It is that fibroblast-like synoviocyte (Fibroblast-likeSynoviocytes, FLS) is for this research that above-mentioned 4 kinds of marks are negative cell.
1.2 Total RNAs extraction
The tissue/cell total RNA extraction reagent box of QIAGEN company is utilized to extract the total serum IgE of OA patient synovial tissue cell and normal synovial tissue cell.
1.3 reverse transcription
The Reverse Transcriptase kit of TAKARA company is utilized to carry out the reverse transcription of RNA.
1.4QPCR
(1) design of primers
According to the encoding sequence design QPCR amplimer of PAEP gene and GAPDH gene in Genbank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
PAEP gene:
Forward primer is 5 '-GCTGTGTTGAGAAGAAGGT-3 ' (SEQIDNO.3);
Reverse primer is 5 '-TTCGCCACCGTATAGTTG-3 ' (SEQIDNO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQIDNO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 46 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
1.5 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
1.6 result
As shown in Figure 1, compared with normal synovial tissue, in Osteoarthritic Synovium histocyte, PAEP gene expression amount significantly reduces result, and difference has statistical significance (P<0.05).
The detection of 2, PAEP genetic expression--protein level
2.1 steps: use conventional westernblot to carry out the detection of PAEP protein level.
2.2 statistical procedures
Use ImageJ software to analyze the gray-scale value of protein band, with β-actin for internal reference, the gray-scale value of PAEP protein band is normalized.Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
2.3 result
As shown in Figure 2, compared with normal synovial tissue, in Osteoarthritic Synovium histocyte, the expression amount of PAEP albumen significantly reduces result, and difference has statistical significance (P<0.05).
Embodiment 3PAEP gene expression plasmid builds
1, the process LAN of PAEP gene
The structure of 1.1PAEP expression vector
According to encoding sequence (as shown in the SEQIDNO.1) design of amplification primers of PAEP gene, primer sequence is as follows: forward primer is 5 '-ATGCTGTGCCTCCTGCTCA-3 ' (SEQIDNO.7), and reverse primer is 5 '-ACGGCTCTTCCATCTGTTTCA-3 ' (SEQIDNO.8).From cDNA library (the clontech company becoming Human fetal spleen, article No.: the encoding sequence of the PAEP gene of amplification total length 638831), above-mentioned cDNA sequence is inserted in the eukaryotic expression vector pcDNA3.1 of restriction enzyme BamHI and XhoI double digestion after restriction enzyme BamHI and XhoI double digestion, connects the recombinant vectors pcDNA3.1-PAEP obtained and is used for subsequent experimental.
1.2 transfection
OA fibroblast-like synoviocyte is divided into two groups, is respectively control group (transfection pcDNA3.1 empty carrier) and PAEP process LAN group (transfection pcDNA3.1-PAEP).Use liposome 2000 to carry out the transfection of carrier, the instruction to specifications of concrete transfection method is carried out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-PAEP is 0.5 μ g/ml.
1.3QPCR detect
Concrete steps are with embodiment 2.
1.4Western detect
Concrete steps are with embodiment 2.
2, result
As shown in Figure 3, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-PAEP, the mRNA level in-site of PAEP significantly raises, and difference has statistical significance (P<0.05); As shown in Figure 4, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-PAEP, the protein level of PAEP significantly raises, and difference has statistical significance (P<0.05).
OA fibroblast-like synoviocyte proliferation experiment under embodiment 4 incentive condition
1, OA fibroblast-like synoviocyte proliferation experiment under incentive condition
1.1 step
OA fibroblast-like synoviocyte is inoculated in 96 porocyte culture plates, every hole 2x10
3cells/ hole/200 μ l, 37 DEG C, 5%CO
2after incubator hatches 12 hours, add (1:5 dilution) in synovial fluid, continue to hatch 24 hours, add
3h-TdR (1 μ Ci/ hole), then cultivate 24 hours, collecting cell, add liquid scintillation solution, β calculating instrument detects cpm value.
1.2 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
1.3 result
Result as shown in Figure 5, and does not add compared with control group that synovial fluid stimulates, and the FLS propagation that adding synovial fluid stimulates obviously is accelerated, and difference has statistical significance (P<0.05).
Embodiment 5PAEP gene overexpression is on the impact of OA fibroblast-like synoviocyte propagation under incentive condition
1, step
Carry out cell cultures and plasmid transfection according to the method for embodiment 3, transfection, after 12 hours, adds (1:5 dilution) in synovial fluid, continues to hatch 24 hours, add
3h-TdR (1 μ Ci/ hole), then cultivate 24 hours, collecting cell, add liquid scintillation solution, β calculating instrument detects cpm value.
2, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, employing SPSS13.0 statistical software carries out statistical study, difference between PAEP gene overexpression group and control group adopts t to check, and thinks to have statistical significance as P<0.05.
3, result
Result such as Fig. 6 shows, and compared with the cell of transfection pcDNA3.1 empty carrier, the cell proliferation under transfection pcDNA3.1-PAEP inhibits synovial fluid to stimulate, difference has statistical significance (P<0.05).
The impact that embodiment 6PAEP gene overexpression is bred OA fibroblast-like synoviocyte
1, step
Carry out cell cultures and plasmid transfection according to the method for embodiment 3, transfection, after 12 hours, adds
3h-TdR (1 μ Ci/ hole), then cultivate 24 hours, collecting cell, add liquid scintillation solution, β calculating instrument detects cpm value.
2, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, employing SPSS13.0 statistical software carries out statistical study, difference between PAEP gene overexpression group and control group adopts t to check, and thinks to have statistical significance as P<0.05.
3, result
Result such as Fig. 7 shows, and compared with the cell of transfection pcDNA3.1 empty carrier, transfection pcDNA3.1-PAEP cell proliferation is obviously slowed down, and difference has statistical significance (P<0.05).
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
1. people PAEP gene and expression product thereof the application in the product of preparation diagnosis osteoarthritis.
2. application according to claim 1, is characterized in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection PAEP gene and expression product thereof to diagnose the product of osteoarthritis.
3. application according to claim 2, is characterized in that, described RT-PCR diagnoses the product of osteoarthritis at least to comprise the primer of a pair specific amplified PAEP gene; The product of described real-time quantitative PCR diagnosis osteoarthritis at least comprises the primer of a pair specific amplified PAEP gene; The product of described immunodetection diagnosis osteoarthritis comprises: the antibody be combined with PAEP protein-specific; The product of described in situ hybridization diagnosis osteoarthritis comprises: with the probe of the nucleic acid array hybridizing of PAEP gene; The product of described chip diagnosis osteoarthritis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with PAEP protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of PAEP gene.
4. the application according to any one of claim 1-3, is characterized in that, described product comprises chip, test kit.
5. the application of people PAEP gene in high-flux sequence platform, is characterized in that, can know that the abnormal expression of PAEP gene is to the generation of osteoarthritis with develop relevant by high-flux sequence.
6. people PAEP gene and expression product thereof the application in the medicine of preparation treatment osteoarthritis.
7. application according to claim 6, it is characterized in that, described medicine comprises: the medicine that the carrier of the medicine containing people PAEP gene, carrier PAEP gene or host cell are formed, people PAEP pharmaceutical grade protein or other can promote the medicine of PAEP genetic expression.
8. diagnose a chip for osteoarthritis, it is characterized in that, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for PAEP gene for detecting PAEP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of PAEP albumen of solid phase carrier.
9. diagnose a test kit for osteoarthritis, it is characterized in that, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting PAEP gene transcription level; Described protein immunization detection kit comprises the specific antibody of PAEP albumen.
10. test kit according to claim 9, is characterized in that, described reagent comprises primer for PAEP gene and/or probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510548624.1A CN105154540B (en) | 2015-08-31 | 2015-08-31 | The diagnosis and treatment target of PAEP gene and its expression product as osteoarthritis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510548624.1A CN105154540B (en) | 2015-08-31 | 2015-08-31 | The diagnosis and treatment target of PAEP gene and its expression product as osteoarthritis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105154540A true CN105154540A (en) | 2015-12-16 |
| CN105154540B CN105154540B (en) | 2019-03-01 |
Family
ID=54795582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510548624.1A Active CN105154540B (en) | 2015-08-31 | 2015-08-31 | The diagnosis and treatment target of PAEP gene and its expression product as osteoarthritis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105154540B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755146A (en) * | 2016-04-29 | 2016-07-13 | 北京致成生物医学科技有限公司 | Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation |
| CN106755549A (en) * | 2017-03-16 | 2017-05-31 | 天津市天津医院 | Application of the molecular marker in osteoarthritis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5039521A (en) * | 1989-01-11 | 1991-08-13 | Hyal Pharmaceutical Corporation | Immune cell proliferation inhibitors |
| WO2001032920A2 (en) * | 1999-11-03 | 2001-05-10 | Metris Therapeutics Limited | Agents implicated in endometriosis |
-
2015
- 2015-08-31 CN CN201510548624.1A patent/CN105154540B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5039521A (en) * | 1989-01-11 | 1991-08-13 | Hyal Pharmaceutical Corporation | Immune cell proliferation inhibitors |
| WO2001032920A2 (en) * | 1999-11-03 | 2001-05-10 | Metris Therapeutics Limited | Agents implicated in endometriosis |
Non-Patent Citations (1)
| Title |
|---|
| 张松英: "人子宫内膜容受性相关基因的筛选与鉴定", 《中国博士学位论文全文数据库（电子期刊）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755146A (en) * | 2016-04-29 | 2016-07-13 | 北京致成生物医学科技有限公司 | Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation |
| CN106755549A (en) * | 2017-03-16 | 2017-05-31 | 天津市天津医院 | Application of the molecular marker in osteoarthritis |
| CN106755549B (en) * | 2017-03-16 | 2019-06-25 | 天津市天津医院 | Application of the molecular marker in osteoarthritis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105154540B (en) | 2019-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132550B (en) | Detect application of the reagent of MUC21 gene expressions in preparing osteoarthritis diagnosis and treatment product | |
| CN105296623B (en) | A kind of molecular marker of diagnosis and treatment osteoarthritis | |
| CN105132575A (en) | Molecular marker for osteoporosis and application of marker | |
| CN105296656A (en) | Molecular marker for nasopharynx cancer diagnosis and treatment | |
| CN105132574B (en) | Osteoarthritis diagnosis and treatment marker and its application | |
| CN105400885A (en) | Applications of TFF2 gene as intracranial aneurysm diagnosis and treatment marker | |
| CN104975104A (en) | Alzheimer disease diagnosis and treatment marker and application thereof | |
| CN105154540A (en) | PAEP gene and PAEP gene expression product as osteoarthritis diagnosis and treatment target | |
| CN105112550B (en) | MTUS1 genes as osteoporosis diagnosis and treatment target | |
| CN105039577B (en) | Application of the CYP2F1 genes in osteoarthritis diagnosis and treatment | |
| CN105400888A (en) | Molecular marker for diagnosis and treatment of intracranial aneurysm | |
| CN105200137A (en) | CERS2 (ceramide synthase 2) gene and its expression product serving as treatment target of osteoporosis | |
| CN108103186B (en) | Molecular markers for diagnosis of rheumatoid arthritis and osteoarthritis | |
| CN108642167B (en) | BAIAP2 as a target for diagnosis and treatment of rheumatoid arthritis and/or osteoarthritis | |
| CN105400894A (en) | Intervertebral disc degenerative change diagnosis and treatment marker | |
| CN105648076A (en) | NUDT11 genes and expression products thereof serving as diagnosis targets of UL (uterine leiomyoma) | |
| CN104878104A (en) | Bile duct cancer diagnosis and treatment molecular marker and application thereof | |
| CN107034276B (en) | Diagnosis and treatment target of the ZNF669 as osteoarthritis | |
| CN105238872A (en) | Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma | |
| CN105087821B (en) | A kind of molecular marker of diagnosis and treatment osteoporosis | |
| CN105296657A (en) | Intracranial aneurism diagnosis marker | |
| CN107090501B (en) | Application of C1GA L T1C1 in osteoarthritis diagnosis and treatment | |
| CN105695621B (en) | Application of the REPS1 genes in diagnosis and treatment osteoarthritis product is prepared | |
| CN105506169A (en) | Diagnosis and treatment markers for hysteromyoma | |
| CN105256040A (en) | Application of KHDC1L gene serving as disease diagnosis marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211117 Address after: 266000 room 2503, Qianshan building, D2, Qingdao International Innovation Park Phase II, No.1 Keyuan Weiyi Road, Laoshan District, Qingdao City, Shandong Province Patentee after: Qingdao Yangshen biomedical Co.,Ltd. Address before: 100080 room 3103, cube court building, 1 good luck street, Haidian District, Beijing. Patentee before: BEIJING MEDINTELL BIOMED Co.,Ltd. |