CN105039577B - Application of the CYP2F1 genes in osteoarthritis diagnosis and treatment - Google Patents
Application of the CYP2F1 genes in osteoarthritis diagnosis and treatment Download PDFInfo
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Abstract
The invention discloses the molecular marker that CYP2F1 genes can early diagnose as osteoarthritis.The present invention proves that compared with normal synovial tissue the CYP2F1 gene expressions in Human Osteoarthritis synovial tissue significantly raise using QPCR experiments, and proves that CYP2F1 can influence the propagation of synovial cell by RNA interference experiments.According to the achievement in research of the present invention, the medicine that can suppress CYP2F1 gene expressions or CYP2F1 gene expression product functions can be suppressed can be researched and developed, so as to realize the clinically prevention and treatment for osteoarthritis.
Description
Technical field
The present invention relates to biological technical field, more particularly to people CYP2F1 genes in the diagnosis, treatment of osteoarthritis
Purposes.
Background technology
Osteoarthritis (Osterarthritis, OA) is also referred to as degenerative arthritis, be in the elderly and overweight people most
Universal disease.OA is the disease in joint, but is different from rheumatoid arthritis (RA), and this disease is not systematic, is led to
Often only influence one or several joints.This disease causes total destruction of articular cartilage, the hardening of lower section bone, and spur shape
Into, cause locomitivity lose and pain.Final result often needs total joint replacement.
Demography trend shows, to the year two thousand twenty, the crowd influenceed by joint disease will be added to 50%, and the whole world will
There are 5.7 hundred million people to be perplexed by osteoarthritis.Chinese society progresses into aging society, therefore we will face osteoarthritis
The generally popular period of morbidity.It is particularly significant and urgent to the sick research.
God in articular cartilage, synovial membrane, subchondral bone, ligament or periarticular can occur for the primary exception of osteoarthritis
Through musculature.The infringement of articular cartilage is that osteoarthritis most significantly changes.Articular cartilage forfeiture is the key of osteoarthritis
Lesion.With the decline of proteoglycan content, articular cartilage is gradually slowly degraded.Due to the proteoglycans of Human Osteoarthritis,
Collagen and hyaluronic acid synthesis rate increase so that the catabolic activity of tissue is especially high.Generally, it is considered that protein resolvase
With metalloprotease osteoarthritic joint cartilage loss in be most important reason.There is no blood vessel in articular cartilage tissue
And nerve, the reaction to wound, inflammation are situated between by such as cell factor of the protein in articular cartilage, synovial tissue or joint fluid etc.
Lead.Synovial membrane plays during the occurrence and development of osteoarthritis disorders important for maintaining the normal function in joint
Effect.
The diagnosis clinically for osteoarthritis is based primarily upon the inspection of iconography at present.Between x-ray performance predominantly joint
Gap is narrow, and subchondral bony sclerosis and cystis degeneration, joint margins spur are formed, and articular surface withers, deforms and subluxation of joint etc..
MRI can show the lesion of the articulation structures such as early stage cartilage, meniscus, be advantageous to early diagnose.CT is used for the diagnosis of discopathy, excellent
In x-ray.Joint space narrows, Subchondral bone sclerosis, and marginality spur spinal joint is linked to be bone bridge.Also visible bone cyst and abnormal
Shape.As found, these changes can be as the foundation of diagnosis and the degree of estimation joint injury.OA is typically no or seldom in early stage
There is symptom, only in the activity of inflammation secondary, pain or influence joint, patient just looks for a doctor.Now, joint injury occurs
Long.Because it is urgent problem to be solved to develop for the method for early stage osteoarthritis diagnosis.
The content of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide one kind can be used for osteoarthritis
The molecular marker of (Osterarthritis, OA) early diagnosis.Compared to the diagnostic method of traditional osteoarthritis, gene is used
Mark has promptness, specificity and sensitivity diagnose osteoarthritis so that patient in disease early stage with regard to that can know
Disease risks, for risk height, take corresponding prevention and treatment measure.
To achieve these goals, the present invention adopts the following technical scheme that：
The invention provides a kind of people CYP2F1 genes and its expression product in the product for preparing diagnosis osteoarthritis
Using.
Further, diagnostic products mentioned above include：Pass through RT-PCR, real-time quantitative PCR, immune detection, original position
Hybridization or the expression of chip detection CYP2F1 genes and its expression product are to diagnose the product of osteoarthritis.
Further, the product that osteoarthritis is diagnosed with RT-PCR comprises at least a pair of specific amplified CYP2F1 genes
Primer；The primer of the product including at least a pair of specific amplified CYP2F1 genes that osteoarthritis is diagnosed with real-time quantitative PCR；
The product that osteoarthritis is diagnosed with immune detection includes：The antibody combined with CYP2F1 protein-specifics；The original position
The product of hybridization diagnosis osteoarthritis includes：With the probe of the nucleic acid array hybridizing of CYP2F1 genes；It is described to diagnose bone with chip
Arthritic product includes：Protein chip and genetic chip；Wherein, protein chip includes what is combined with CYP2F1 protein-specifics
Antibody, genetic chip include the probe with the nucleic acid array hybridizing of CYP2F1 genes.
Preferably, the product includes chip, kit.
Present invention also offers application of the people CYP2F1 genes in high-flux sequence platform.With high throughput sequencing technologies
Development, the structure of the gene expression profile of a people will be turned into and very easily worked.By contrasting Disease and normal
The gene expression profile of crowd, the exception for easily analyzing which gene are related to disease.Therefore, people is known in high-flux sequence
The exception of the CYP2F1 genes purposes for falling within people's CYP2F1 genes related to osteoarthritis, equally in protection scope of the present invention
Within.
Present invention also offers people CYP2F1 genes and its expression product answering in the medicine for preparing treatment osteoarthritis
With.
The main active of " medicine for the treatment of osteoarthritis " of the present invention includes suppressing CYP2F1 gene expressions
Material, suppress the material of CYP2F1 gene expression product stability, and/or suppress the thing of CYP2F1 gene expression products activity
Matter.
Further, the medicine for the treatment of osteoarthritis of the present invention includes：CYP2F1 gene tables are suppressed by RNA interfering
The double stranded RNA reached, or the tumor vaccine based on CYP2F1 antigen proteins or the egg for suppressing CYP2F1 protein actives
White matter.
Present invention also offers a kind of pharmaceutical composition for being used to treat osteoarthritis, described pharmaceutical composition includes
CYP2F1 genes and/or its expression product inhibitor.The inhibitor includes suppressing the material of CYP2F1 gene expressions, suppressed
The material of CYP2F1 gene expression product stability, and/or the material for suppressing CYP2F1 gene expression products activity.
Further, inhibitor of the present invention includes：Suppress the double-strand ribose core of CYP2F1 gene expressions by RNA interfering
Acid, or the tumor vaccine based on CYP2F1 antigen proteins or the protein for suppressing CYP2F1 protein actives.
Present invention also offers above-mentioned CYP2F1 genes and/or its expression product inhibitor to prepare treatment osteoarthritis medicine
Application in thing.
In the present invention, the RNA interference (RNA interference, RNAi) refers to be highly conserved during evolution
, induced by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Make
With RNAi technology can with specific depletion or close specific gene expression, the technology have been widely used for explore gene function and
The field of gene of communicable disease and malignant tumour.RNAi based on cell is screened in terms of functional gene research
With many advantages, RNAi methods can be used by being mainly manifested in most cell types, and is easier to lower or is sunk relatively
The expression of silent any target gene.
In order to ensure CYP2F1 genes can be rejected efficiently or silence, devised according to the mRNA sequence of CYP2F1 genes
SiRNA specific fragments.SiRNA design according to delivered general design principle (Elbashir et.al 2001,
Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004,
Ui-Tei et.al 2004), by online tool complete design, the online tool is：
SiRNASelectionProgram of Whitehead Institute (BingbingYuan et.al 2004,
http://jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTM RNAi Designer ofINVITROGEN
(winner of the 2004Frost＆Sullivan Excellence in Research Award, https://
rnaidesigner.invitrogen.com/sirna/).It is comprehensive two in order to further improve the validity of siRNA segments
The advantages of Photographing On-line instrument, is designed for the siRNA segments of screening.Finally, by sequence analysis (NCBI BLAST) come
SiRNA sequence is filtered, to improve the specificity of siRNA segments and reduce the effect of missing the target of RNAi interference.
The medicine of the present invention also includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but being not limited to)：It is dilute
Release agent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and poly- second
Alkene pyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium acid carbonate；Sorbefacient quaternary ammonium compound；Table
Face activating agent such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium, poly- second
Glycol etc..
The medicine of the present invention can also be with the drug combination of other treatment osteoarthritis, and multi-medicament is used in combination can be significantly
Mention the success rate for the treatment of.
Present invention also offers a kind of product for diagnosing osteoarthritis, the product includes but is not limited to chip, kit.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes being used to detect being directed to for CYP2F1 gene transcription levels
The oligonucleotide probe of CYP2F1 genes；The protein-chip includes solid phase carrier and is fixed on the CYP2F1 of solid phase carrier
The specific antibody of albumen；The genetic chip can be used for detection including people's CYP2F1 genes multiple genes (for example, with
The related multiple genes of osteoarthritis) expression.The protein-chip includes people's CYP2F1 albumen available for detection and existed
The expression of interior multiple protein (such as multiple protein related to osteoarthritis).By by multiple and osteoarthritis
Mark detect simultaneously, be greatly improved osteoarthritis diagnosis accuracy rate.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes being used for the reagent for detecting CYP2F1 gene transcription levels；The protein immunization detection kit includes CYP2F1 albumen
Specific antibody.Further, the reagent is including the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or core
Reagent needed for during piece method detection CYP2F1 gene expression doses.Preference, the reagent include being directed to CYP2F1 bases
The primer and/or probe of cause.Easily designed according to the nucleotide sequence information of CYP2F1 genes and can be used for detecting CYP2F1
The primer and probe of gene expression dose.
Probe with the nucleic acid array hybridizing of CYP2F1 genes can be DNA, RNA, DNA-RNA chimera, PNA or other
Derivative.The length of the probe does not limit, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, most long not surpass typically
30 base-pairs are crossed, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequence
Row are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the CYP2F1 albumen includes monoclonal antibody, polyclonal antibody.The CYP2F1
The specific antibody of albumen include complete antibody molecule, antibody any fragment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the fragment can retain the binding ability with CYP2F1 albumen.For protein water
During the preparation of flat antibody well known to a person skilled in the art, and the present invention can be prepared using any method it is described anti-
Body.
In the context of the present invention, " CYP2F1 genes " includes any of people CYP2F1 genes and people's CYP2F1 genes
The polynucleotides of functional equivalent.CYP2F1 genes include with the public GenBank GeneBank in the current world
CYP2F1 genes (NC_000019.10) DNA sequence dna has more than 70% homology, and encodes identical function protein DNA sequence
Row；
Preferably, the coded sequence of CYP2F1 genes includes following any DNA molecular：
(1) DNA sequence dna in sequence table shown in SEQ ID NO.1；
(2) under strict conditions with 1) the DNA sequence dna hybridization that limits and coding identical function protein DNA sequence；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical work(
Can protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of the CYP2F1 genes is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, CYP2F1 gene expression products include people CYP2F1 albumen and people's CYP2F1 albumen
Partial peptide.The partial peptide of the CYP2F1 albumen contains the functional domain related to osteoarthritis.
" CYP2F1 albumen " includes any functional equivalent of people CYP2F1 albumen and people's CYP2F1 albumen.The function
Equivalent includes people CYP2F1 albumen conservative variation protein or its active fragment, or its reactive derivative, allelic variation
Body, natural mutation, induced mutants, under high or low stringent condition can with the DNA of people CYP2F1 DNA hybridization coded by
Protein.
Preferably, CYP2F1 albumen is the protein for having following amino acid sequences：
(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；
(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the amino acid sequence shown in SEQ ID NO.2 have identical function as the ammonia shown in SEQ ID NO.2
Protein derived from base acid sequence.The number of the amino acid of substitution, missing or addition is usually 1-50, preferably 1-30
It is individual, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2,
It is highly preferred that the homology with the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%,
98%th, the polypeptide that the amino acid sequence of 99% homology is formed.
In specific embodiments of the present invention, the CYP2F1 albumen is that have the amino acid shown in SEQ ID NO.2
The protein of sequence.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein.
Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or indivedual additions to amino acid sequence,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by the protein for adding an amino acid or more amino acid modification is melting for CYP2F1 albumen
Hop protein.Do not limited for the peptide or protein with CYP2F1 protein fusions, as long as the fusion protein of gained retains
The biological activity of CYP2F1 albumen.
The CYP2F1 albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, only
The protein that pass through modification remains able to the biological activity for retaining CYP2F1 albumen.Dashed forward in such modifying protein
The amino acid number of change is typically 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis osteoarthritis " had both included judging whether subject suffers from Bones and joints
Inflammation, also include judging that subject whether there is the risk with osteoarthritis.
In the context of the present invention, " treatment osteoarthritis " divides from the state change of disease, can include disease
Alleviate, the complete healing of disease.
The advantages of the present invention：
Present invention firstly discovers that CYP2F1 gene expressions are related to osteoarthritis, by detecting in subject synovial tissue
CYP2F1 expression, it can be determined that whether subject, which suffers from osteoarthritis or judge that subject whether there is, suffers from Bones and joints
Scorching risk, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-CYP2F1 genes, compared to traditional detection means, gene diagnosis
More in time, it is more special, sensitiveer, the early diagnosis of osteoarthritis can be realized, so as to reduce the death rate of osteoarthritis.
Brief description of the drawings
Fig. 1 shows the expression in synovial cell using QPCR detection CYP2F1 genes；
Fig. 2 shows the expression in synovial cell using Western blot detection CYP2F1 albumen；
Fig. 3 shows proliferation function of the synovial fluid to OA fibroblast-like synoviocytes；
Fig. 4 shows the inhibitory action that CYP2F1 protein antibodies are bred to OA fibroblast-like synoviocytes；
Fig. 5 shows the jamming effectiveness to CYP2F1 genes using QPCR detection siRNA；
The inhibitory action that Fig. 6 display interference CYP2F1 gene expressions are bred to OA fibroblast-like synoviocytes.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The differential expression of CYP2F1 genes in embodiment 1OA synovial tissues and normal synovial tissue
The synovial tissue of 60 OA patients comes from BJ Union Hospital's orthopaedics row knee prosthesis or villusectomy
Patient OA.Case used meets the diagnostic criteria on OA of Altam propositions.40 normal synovial tissues come from Beijing consonance
Hospital orthopedics, it is trauma surgery patient synovial tissue of joint.Supernatant, -80 DEG C of storages are taken after OA patient's synovial fluid (SF) high speed centrifugation
Deposit stand-by.Clinical sample used in this research, patient know and inform and pass through through Ethics Committee of the court.
1st, the detection of CYP2F1 gene transcription levels
1.1 synovial tissue's cell injuring models
After synovial tissue's PBS of sterile acquisition, about 1mm x1mm x are cut into repeatedly with aseptic operation scissors
1mm tissue block, after adding 37 DEG C of clostridiopetidase A II (0.5mg/ml), digestion 2h, filtered through 200 mesh gauzes, after supernatant is removed in centrifugation,
Cell is resuspended in DMEM nutrient solutions, is placed in 37 DEG C, 5%CO2Cultivated in cell culture incubator.When cell grows up to fusiformis and in blocks
Afterwards, Secondary Culture is carried out.After cell reached for 3 generation, mouse anti human CD3, CD14, CD19 and PE of FITC marks are separately added into
The mouse anti human CD11b of mark is marked, flow cytomery identification.Above-mentioned 4 kinds of marks be negative cell be into
Fiber-like synovial cell (Fibroblast-like Synoviocytes, FLS) is used for this research.
1.2 Total RNAs extraction
Utilize tissue/cell total RNA extraction reagent box extraction OA patient's synovial tissue's cell of QIAGEN companies and normal
The total serum IgE of synovial tissue's cell.
1.3 reverse transcription
RNA reverse transcription is carried out using the Reverse Transcriptase kit of TAKARA companies.
1.4QPCR
(1) design of primers
QPCR amplimers are designed according to the coded sequence of CYP2F1 genes and GAPDH genes in Genbank, given birth to by Shanghai
Work biotechnology Services Co., Ltd synthesizes.Specific primer sequence is as follows：
CYP2F1 genes：
Forward primer is 5 '-TCACCATTATCCGCCTTA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGAAGATGTCGTACAACTC-3 ' (SEQ ID NO.4),
GAPDH genes：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction systems are prepared according to table 1：
Wherein, SYBR Green PCRs system is purchased from Invitrogen companies.
Table 1PCR reaction systems
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green PCR systems | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions：95 DEG C of 12min, (95 DEG C of 20s, 60 DEG C of 55s) * 45 circulations.Using SYBR Green as
Fluorescent marker, it is true by melt curve analysis analysis and electrophoresis in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instruments
Determine purpose band, Δ Δ CT methods carry out relative quantification.
1.5 statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
1.6 result
As a result as shown in figure 1, compared with normal synovial tissue, CYP2F1 gene expressions in Osteoarthritic Synovium histocyte
Amount dramatically increases, and difference has statistical significance (P<0.05).
2nd, the detection of CYP2F1 gene expressions -- protein level
2.1 step：The detection of CYP2F1 protein levels is carried out using the western blot of routine.
2.2 statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by CYP2F1 eggs
The gray value of informal voucher band is normalized.Experiment is all completed according to being repeated 3 times, and result data is all with average value
The mode of ± standard deviation represents that carry out statistical analysis using SPSS13.0 statistical softwares, difference between the two uses t
Examine, it is believed that work as P<There is statistical significance when 0.05.
2.3 result
As a result as shown in Fig. 2 compared with normal synovial tissue, the table of CYP2F1 albumen in Osteoarthritic Synovium histocyte
Dramatically increased up to amount, difference has statistical significance (P<0.05).
In embodiment 2OA fibroblast-like synoviocytes proliferation experiment and antibody and test
1st, OA fibroblast-like synoviocytes proliferation experiment
1.1 step
FLS is inoculated in 96 porocyte culture plates, per hole 2x103Cells/ holes/200 μ l, 37 DEG C, 5%CO2Incubator
After being incubated 12 hours, add (1 in synovial fluid：5 dilutions), continue to be incubated 24 hours, add3H-TdR (1 μ Ci/ holes), is further cultured for
24 hours, cell is collected, adds liquid scintillation solution, β calculating instruments detection cpm values.
1.2 statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
1.3 result
As a result as shown in figure 3, compared with being not added with the control group of synovial fluid stimulation, the OA that synovial fluid stimulates is added to be slided into fiber-like
Theca cell propagation is obvious to be accelerated, and difference has statistical significance (P<0.05).
2nd, in fibroblast-like synoviocyte antibody and test
2.1 step
OA fibroblast-like synoviocytes are seeded in 96 well culture plates, by anti-human CYP2F1 monoclonal antibodies according to 1：25 ratio
Add in synovial fluid and mix, while unrelated monoclonal antibody (1 is set：25) it is control, is added after being cultivated 24 hours with synovial cell3H-TdR (1 μ Ci/ holes), is further cultured for 24 hours, collects cell, adds liquid scintillation solution, β calculating instruments detection cpm values.
2.2 statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
2.3 result
As a result as shown in figure 4, compared with being not added with the control group of people's CYP2F1 monoclonal antibodies, the OA of people's CYP2F1 monoclonal antibodies is added into fiber
Sample synovial cell proliferation substantially slows down, and difference has statistical significance (P<0.05).
Embodiment 3 disturbs the expression of CYP2F1 genes
1st, siRNA designs synthesis
For CYP2F1 siRNA sequence：
siRNA1-CYP2F1：
Positive-sense strand is 5 '-UUGAUAAGGCGGAUAAUGGUG-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CCAUUAUCCGCCUUAUCAAUG-3 ' (SEQ ID NO.8),
siRNA2-CYP2F1：
Positive-sense strand is 5 '-UGGUAAAGUUGAAAAAGGCAG-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-GCCUUUUUCAACUUUACCAAG-3 ' (SEQ ID NO.10),
siRNA3-CYP2F1：
Positive-sense strand is 5 '-AACUUUUGGGUACUUCAUGAG-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-CAUGAAGUACCCAAAAGUUCA-3 ' (SEQ ID NO.12)
Negative control siRNA sequence (siRNA-NC)：
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO.13)；
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO.14).
OA fibroblast-like synoviocytes cell is pressed 1 × 104/ hole is inoculated into 24 porocyte culture plates, 37 DEG C, 5%
CO2Cell culture 24h in incubator, in without DMEM culture mediums dual anti-, containing 10%FBS, transfect and tried according to liposome transfection
The specification transfection of agent 2000 (being purchased from Invitrogen companies), experiment is divided into, negative control group and experimental group (20nM), its
In, for the sequence of negative control group siRNA and CYP2F1 genes without homology, concentration is 20nM/ holes, while is transfected respectively.
2nd, detection siRNA jamming effectiveness method is tested with embodiment 1 using QPCR.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, disturbs the difference between CYP2F1 gene expression panels and control group to adopt
Examined with t, it is believed that work as P<There is statistical significance when 0.05.
4th, result
As a result as Fig. 5 shows that compared with siRNA2-CYP2F1, siRNA3-CYP2F1, siRNA1-CYP2F1 can more have
The expression of the suppression CYP2F1 genes of effect, difference have statistical significance (P<0.05), carried out using siRNA1-CYP2F1 follow-up
Experiment.
The inhibitory action of embodiment 4CYP2F1 gene pairs Osteoarthritic Synovium tissue cell proliferations
1st, cell transfecting：According to embodiment 3 method to OA fibroblast-like synoviocytes carry out siRNA1-CYP2F1 and
SiRNA-NC transfection.
2nd, added after transfecting 24 hours3H-TdR (1 μ Ci/ holes), is further cultured for 24 hours, collects cell, adds liquid scintillation solution,
β calculating instruments detect cpm values.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, disturbs the difference between CYP2F1 gene expression panels and control group to adopt
Examined with t, it is believed that work as P<There is statistical significance when 0.05.
4th, result
As a result such as Fig. 6 is shown, compared with siRNA-NC groups, the cell for transfecting siRNA1-CYP2F1 breeds slack-off, difference
With statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Claims (4)
1. application of the reagent of detection people CYP2F1 genes and its expression product in the product for preparing diagnosis osteoarthritis.
2. application according to claim 1, it is characterised in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization or the expression of chip detection CYP2F1 genes and its expression product are to diagnose the production of osteoarthritis
Product.
Application of the inhibitor of 3.CYP2F1 genes and/or its expression product in the medicine for preparing treatment osteoarthritis.
4. application according to claim 3, it is characterised in that the inhibitor is the siRNA for CYP2F1, described
SiRNA sense strand sequence is shown in SEQ ID NO.7, and the antisense strand sequence of the siRNA is shown in SEQ ID NO. 8.
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