CN105256040B - Purposes of the KHDC1L genes as disease diagnosis marker - Google Patents
Purposes of the KHDC1L genes as disease diagnosis marker Download PDFInfo
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Abstract
The invention discloses the molecular marker that KHDC1L genes and KHDC1L albumen can be diagnosed as osteoarthritis, the present invention utilize it was proved that：Compared with normal person, KHDC1L genes and KHDC1L albumen expression quantity in the synovial tissue of Human Osteoarthritis are high, and difference has statistical significance.The invention also discloses the drug that KHDC1L genes and KHDC1L albumen can prepare treatment osteoarthritis, cell proliferation experiment proves interference KHDC1L genes or inhibits KHDC1L protein actives that can inhibit synovial cell proliferation.The achievement in research of the present invention provides a kind of new Bones and joints methods for clinical diagnosis, while provide a kind of drug new target drone for treating Bones and joints.
Description
Technical field
The present invention relates to biotechnology, more particularly to people KHDC1L genes in the diagnosis, treatment of osteoarthritis
Purposes.
Background technology
China has gradually striden into aging society, influence degrees and caused medical expense of the OA to health
Increase year by year with surprising rapidity.There is data to show, 50% crowd has OA's on X-ray film in the population of 60 years old or more
Performance, wherein 35%~50% has clinical symptoms, more than 80% symptom for having an OA in the crowd of 75 years old or more, this is one non-
Often fearful ratio.Now, OA, which has become, is only second to heart disease and makes the second principal disease of patient's disablement, gives
Family and society bring heavy financial burden, so as to cause the extensive concern of domestic and international professional person.
At present, it is defined as follows in osteoarthritis diagnosis and treatment guide about OA：OA refers to causes articular cartilage fine by many factors
Joint disease caused by dimensionization, chap, ulcer, depigmentation.The cause of disease is still not clear, and occurs and age, obesity, inflammation, wound
And inherent cause etc. is related.Its pathological characteristic is articular cartilage degeneration destruction, Subchondral bone sclerosis or cystis degeneration, joint margins bone
Matter hyperplasia, synovial hyperplasia, capsular ligament contracture, laxity of ligament or contracture, muscular atrophy are powerless etc..It is common with middle-older patient, female
Property be more than male.The disability rate of the disease may be up to 53%.OA is apt to occur in joint big, more than activity of bearing a heavy burden, such as knee, backbone (cervical vertebra
And lumbar vertebrae), hip, ankle, the joints such as hand, have certain relationship with heredity and physical factors.
OA initial stages are no manifest symptoms, may be along with intermittent arthralgia and tenderness or pass
Phenomena such as stiff, joint is powerless, moving obstacle is saved, many people take chances, it is believed that since without influence on daily life,
Just it is not really serious disease.But it when causing the function of joint to lose with progression of the disease serious consequence, is just likely to disable.It is right
This, clinically opinion always is all early diagnosis, early prevention, early treatment.
Invention content
In order to make up for the deficiencies of the prior art, the purpose of the present invention is 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
Marker come diagnose osteoarthritis have promptness, specificity and sensitivity, so as to make patient 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：
Application in the tool for diagnosing osteoarthritis in preparation the present invention provides the product of detection KHDC1L gene expressions.
Further, it is described detection KHDC1L gene expressions product include detection KHDC1L gene mRNA levels product,
And/or the product of detection KHDC1L protein levels.
Further, the product of the detection KHDC1L gene expressions includes：Pass through RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detect KHDC1L gene expressions to diagnose the product of osteoarthritis.
Further, the product with RT-PCR diagnosis osteoarthritis includes at least a pair of of specific amplified KHDC1L genes
Primer；The product with real-time quantitative PCR diagnosis osteoarthritis includes at least the primer of a pair of of specific amplified KHDC1L genes；
The product with immune detection diagnosis osteoarthritis includes：The antibody combined with KHDC1L protein-specifics；The original position
The product of hybridization diagnosis osteoarthritis includes：With the probe of the nucleic acid array hybridizing of KHDC1L 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 KHDC1L protein-specifics
Antibody, genetic chip include the probe with the nucleic acid array hybridizing of KHDC1L genes.
A pair of of specific amplified KHDC1L genes that the product with real-time quantitative PCR diagnosis osteoarthritis includes at least
Primer is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection KHDC1L gene expressions can be the reagent for detecting KHDC1L gene expressions, can also be packet
Kit, chip, test paper containing the reagent etc. or the high-flux sequence platform for using the reagent.
The tool of the diagnosis osteoarthritis includes but not limited to chip, kit, test paper or high-flux sequence platform；
High-flux sequence platform is a kind of tool of special diagnosis osteoarthritis, with the development of high throughput sequencing technologies, to one
The structure of the gene expression profile of people, which will become, very easily to work.By the gene expression for comparing Disease and normal population
Spectrum, the exception for easily analyzing which gene are related to disease.Therefore, the exception of KHDC1L genes is known in high-flux sequence
The purposes for also belonging to KHDC1L genes related to osteoarthritis, equally within protection scope of the present invention.
The present invention also provides a kind of tool for diagnosing osteoarthritis, the tool includes detection KHDC1L gene expressions
Reagent；The reagent includes the primer of detection KHDC1L gene mRNAs and/or probe, the antibody for detecting KHDC1L albumen.
The tool includes but not limited to chip, kit, test paper or high-flux sequence platform.
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 detecting being directed to for KHDC1L gene transcription levels
The oligonucleotide probe of KHDC1L genes；The protein-chip includes solid phase carrier and is fixed on the KHDC1L of solid phase carrier
The specific antibody of albumen；The genetic chip can be used for multiple genes of the detection including KHDC1L genes (for example, and bone
The relevant multiple genes of arthritis) expression.The protein-chip can be used for detection including KHDC1L albumen
The expression of multiple protein (such as with the relevant multiple protein of osteoarthritis).By by multiple with osteoarthritis mark
Will object detects simultaneously, is greatly improved the accuracy rate of osteoarthritis diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting KHDC1L gene transcription levels；The protein immunization detection kit includes KHDC1L albumen
Specific antibody.Further, the reagent includes the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or core
Reagent needed for piece method detection KHDC1L gene expression dose processes.Preference, the reagent are included for KHDC1L bases
The primer and/or probe of cause.It is easily designed according to the nucleotide sequence information of KHDC1L genes and can be used for detecting KHDC1L
The primer and probe of gene expression dose.
The test paper includes the reagent of detection KHDC1L gene expressions.
The high-flux sequence platform includes the reagent of detection KHDC1L gene expressions.
Probe with the nucleic acid array hybridizing of KHDC1L genes can be DNA, RNA, DNA-RNA chimera, PNA or other
Derivative.There is no limit for the length of the probe, 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 genes.Since 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, and longest does not surpass generally
30 base-pairs are crossed, it is best with 15-25 base-pair with the length of purpose nucleotide sequence complementation.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 KHDC1L albumen includes monoclonal antibody, polyclonal antibody.The KHDC1L
The specific antibody of albumen include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with KHDC1L albumen.For protein water
During the preparation of flat antibody well known to a person skilled in the art, and the present invention may use any method it is described anti-to prepare
Body.
In specific embodiments of the present invention, the primer of the detection KHDC1L gene mRNAs includes SEQ ID NO.3
With the primer pair shown in SEQ ID NO.4.
The present invention also provides the inhibitor of KHDC1L genes and/or its expression product in the medicine for preparing treatment osteoarthritis
Application in object.The inhibitor includes the reagent for inhibiting KHDC1L gene expressions, and/or inhibits KHDC1L gene expression products
Reagent.
Further, it is described that reagent of the reagent of KHDC1L gene expressions including suppressor transcription, suppressor is inhibited to turn over
The reagent translated；It is described that the reagent of KHDC1L gene expression products is inhibited to include inhibiting the reagent of KHDC1L gene mRNAs, inhibit
The reagent of KHDC1L albumen.It is described that the reagent of KHDC1L gene mRNAs is inhibited to include inhibiting the reagent of mRNA stability, inhibit
The reagent of mRNA translation activity.It is described that the reagent of KHDC1L albumen is inhibited to include inhibiting the reagent of KHDC1L protein stabilities, suppression
The reagent of KHDC1L protein actives processed, the reagent for inhibiting KHDC1L protein functions.
Further, the reagent of KHDC1L gene mRNAs is inhibited to include the double stranded RNA for being directed to KHDC1L gene mRNAs；
The reagent of KHDC1L protein functions is inhibited to include the tumor vaccine of KHDC1L antigen proteins, inhibit the anti-of KHDC1L protein functions
Body.The antibody can be polyclonal antibody or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for KHDC1L gene mRNAs is siRNA.
In order to ensure KHDC1L genes can be rejected efficiently or silence, siRNA spies are devised according to the mRNA sequence of KHDC1L genes
Specific fragment.The design of siRNA is according to general design principle (the Elbashir et.al 2001, Schwarz delivered
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, which is：siRNASelectionProgram of
Whitehead Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/
) and BLOCK-iTTM RNAi Designer ofINVITROGEN (winner of the 2004Frost＆ siRNAext/
Sullivan Excellence in Research Award, https://rnaidesigner.invitrogen.com/
sirna/).In order to be designed for the advantages of further improving the validity of siRNA segments, integrate two Photographing On-line tools
The siRNA segments of screening.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve siRNA pieces
Disconnected specificity and the undershooting-effect for reducing RNAi interference.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.11 and SEQ ID NO.12.
The present invention also provides a kind of for treating the pharmaceutical composition of osteoarthritis, described pharmaceutical composition is included above
The KHDC1L genes and/or the inhibitor of its expression product.
The pharmaceutical composition of the present invention further includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but and unlimited
In)：Diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin
And polyvinylpyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient is quaternized
Close object；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and
Magnesium, polyethylene glycol etc..
The pharmaceutical composition of the present invention can also be with the drug combination of other treatment osteoarthritis, and a variety of Drug combinations can
To mention the success rate for the treatment of significantly.
In the context of the present invention, " KHDC1L genes " is any including people KHDC1L genes and people's KHDC1L genes
The polynucleotides of functional equivalent.KHDC1L genes include in the public GenBank GeneBank in the current world
KHDC1L genes (NC_000006.12) DNA sequence dna has more than 70% homology, and encodes the DNA sequences of identical function protein
Row；
Preferably, the coded sequence of KHDC1L 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) DNA sequence dna that limits hybridizes and encodes the DNA sequence dna of identical function protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical work(
The DNA molecular of energy protein.
In specific embodiments of the present invention, the coded sequence of the KHDC1L genes is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, KHDC1L gene expression products include people KHDC1L albumen and people's KHDC1L albumen
Partial peptide.The partial peptide of the KHDC1L albumen contains and the relevant functional domain of osteoarthritis.
Any functional equivalent of " KHDC1L albumen " including people KHDC1L albumen and people's KHDC1L albumen.The function
Equivalent includes people KHDC1L 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 the DNA hybridization of people KHDC1L coded by
Protein.
Preferably, KHDC1L albumen is the protein for having following amino acid sequences：
(1) protein being made 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 a, 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 KHDC1L albumen is the amino acid having 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 to indivedual additions of amino acid sequence,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein generates 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 KHDC1L albumen
Hop protein.For the peptide or protein with KHDC1L protein fusions, there is no limit as long as the fusion protein of gained retains
The biological activity of KHDC1L albumen.
The KHDC1L 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 KHDC1L albumen.It dashes forward in such modification protein
The amino acid number of change is typically 10 either less such as 6 either less such as 3 or less.
In the context of the present invention, " diagnosis osteoarthritis " had both included judging whether subject has suffered from Bones and joints
Inflammation also includes 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 present invention studies the therapeutic effect of KHDC1L gene pairs osteoarthritis using the synovial cell of in vitro culture.Ability
Field technique personnel are it is found that the important physiological characteristic that osteoarthritis occurs is that the proliferation of synovial cell is accelerated, and secretion is more
More factors deposits into the occurrence and development of osteoarthritis, therefore the present invention studies KHDC1L using the proliferation experiment of synovial cell
The relationship of gene and osteoarthritis treatment.
The advantages of the present invention：
Present invention firstly discovers that KHDC1L gene expressions are related to osteoarthritis, by detecting in subject synovial tissue
The expression of KHDC1L, it can be determined that whether subject suffers from osteoarthritis or judge that subject whether there is with Bones and joints
Scorching risk, so as to which clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-KHDC1L 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.
Description of the drawings
Fig. 1 shows the expression in synovial tissue using high-flux sequence detection KHDC1L genes；
Fig. 2 shows the expression in synovial tissue using QPCR detection KHDC1L genes；
Fig. 3 shows the expression in synovial tissue using Western blot detection KHDC1L albumen；
Fig. 4 shows the jamming effectiveness to KHDC1L genes using QPCR detection siRNA.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in or according to the normal condition proposed by manufacturer.
The differential expression of KHDC1L genes in 1 OA synovial tissues of embodiment and normal synovial tissue
The synovial tissue of 8 OA patients comes from BJ Union Hospital's orthopaedics row knee prosthesis or the OA of villusectomy
Patient.Case used meets the diagnostic criteria about OA of Altam propositions.8 normal synovial tissues come from Beijing consonance doctor
Institute's orthopaedics 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
For use.Clinical sample used in this research to patient know and informs and pass through through Ethics Committee of the court.
1st, the extraction of synovial tissue's total serum IgE
It freezes to be put into tissue in the mortar being pre-chilled after liquid nitrogen, taking-up after collection sample and be ground, treat tissue sample
This is into after powdered：
1. Trizol is added in, room temperature preservation 5min；
2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, abundant mixing places 5min-10min at room temperature；
3. being drawn after 12000rpm high speed centrifugations 15min in upper strata aqueous phase (inhaling 70%) to another new centrifuge tube, pay attention to not
The protein substance being drawn onto between two layers of water phase.New pipe is moved into, adds in the pre- cold isopropanol of isometric -20 DEG C, it is fully reverse mixed
It is even, it is placed in 10min on ice；
4. 12000rpm adds in 75% at a high speed from supernatant is carefully discarded after 15min in the ratio of 1ml/ml Trizol
DEPC ethyl alcohol washing precipitation (4 DEG C of preservations), washing precipitate, oscillation mixes, 12000rpm high speed centrifugations 5min at 4 DEG C；
5. discarding ethanol liquid, 5min is placed at room temperature fully to dry precipitation, is added in the processed water dissolutions of DEPC and is sunk
It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometers, freeze in -70 DEG C.
2nd, the quality analysis (NanoDrop1000 spectrophotometers) of RNA sample
NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is
1.8-2.2。
3rd, the quality analysis (2100 Bioanalyzer of Agilent Technologies) of RNA sample
2100 Bioanalyzer of Agilent Technologies detect RNA sample quality, observation 28S rRNA and
18S rRNA master tapes are apparent, cDNA texts are sequenced without degradation, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement
The requirement of library structure, can be used for library construction and sequencing.
4th, high-throughput transcript profile sequencing
(1) RNA-seq reads position
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens reference genes groups (hg19) are matched, the index built in advance of H.sapiens UCSC hg19 editions
It is downloaded from TopHat homepages, and as with reference to genome, when being matched using TopHat with genome, allows each read (acquiescence
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signals
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat methods.
(2) transcript abundance is assessed
The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to matching in every 1,000,000 sequencing segment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated values is calculated by Bayesian inference method.
The GTF comment files for the reference that Cufflinks is used download (Homo_ from Ensembl databases
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
Cuffdiff is transferred to by the Ensembl GTF files of download and by the matched original documents of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table
It reaches.The only q values ＜ 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.
5th, result
RNA-seq results show (as shown in Figure 1), and normal person compares, KHDC1L bases in Human Osteoarthritis synovial tissue
The mRNA level in-site of cause dramatically increases, and difference has statistical significance (P<0.05).
2 large sample of embodiment verifies difference expression gene
The synovial tissue of 40 OA patients comes from BJ Union Hospital's orthopaedics row knee prosthesis or villusectomy
Patient OA.Case used meets the diagnostic criteria about OA of Altam propositions.40 normal synovial tissues come from Beijing consonance
Hospital orthopedics are 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
It deposits for use.Clinical sample used in this research to patient know and informs and pass through through Ethics Committee of the court.
1st, the differential expression of KHDC1L genes is detected on transcriptional level
1.1 extraction synovial tissue total serum IgEs
Step is the same as embodiment 1.
1.2 reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample
1 μ g total serum IgEs is taken to be separately added into following components in PCR pipe as template ribonucleic acid：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
1.3QPCR amplifications are examined
Using 25 μ l reaction systems, each sample sets 3 parallel pipes.Prepare following reaction system：SYBR Green gather
12.5 μ l of polymerase chain reaction system, 1 μ l of forward primer (5 μM), reverse primer (5 μM) 2.0 μ l of 1 μ l, template cDNA, no enzyme
8.5 μ l of water；The forward primer sequence for expanding KHDC1L genes is 5 '-TTCATTCTCCAATGGTGTT-3 ' (SEQ ID NO.3),
Reverse primer sequences are 5 '-GAAGGTACGTGTCATCAA-3 ' (SEQ ID NO.4)；Expand the forward primer sequence of GAPDH genes
It is classified as 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5), reverse primer sequences 5 '-
GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6), operations are carried out on ice.Amplification program is：95℃
10min, (95 DEG C of 15s, 60 DEG C of 60s) * 47 cycles.It is glimmering in Light Cycler using SYBR Green as fluorescent marker
PCR reactions are carried out on light real-time PCR, purpose band are determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out
Relative quantification.
1.4 statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
1.5 result
The results are shown in Figure 2, compared with normal synovial tissue, Human Osteoarthritis synovial tissue KHDC1L gene expression amounts
It dramatically increases, difference has statistical significance (P<0.05).
2nd, the differential expression of KHDC1L genes is detected on protein level
The extraction of 2.1 synovial tissue's total proteins
Use the total protein extraction kit (article No. of Beijing Puli's lema gene Technology Co., Ltd.：P1250 synovial membrane) is extracted
Total protein in tissue, concrete operation step is referring to specification.
2.2 Western blot
Total protein Brandford standard measures take to mix with sample buffer in right amount and boil 5min, cool down 5min；It takes
30pg albumen is loaded to 15% polyacrylamide gel prepared, carries out electrophoresis, starts to be set as 80V constant pressures, see Marker
After increase to 120V；Glue after electrophoresis is taken out, 50min is shifted in 100V using the half-dried transferring systems of Bio.Rad；Transferring film finishes
Afterwards, it is washed once with 1xPBS, immerses confining liquid, 40C is overnight；Confining liquid is outwelled, adds in Western cleaning solutions washing 5-10min,
Add in primary antibody shaking table room temperature hybridization 2h；It is diluted in Block buffer, is incubated with Western secondary antibody diluents according to proper proportion
60min；Film washing liquid is washed 3 times, each 10min；Developed using ECL reagents, fixing detection protein expression.
2.3 statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by KHDC1L eggs
The gray value of informal voucher band is normalized.Result data is represented in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
2.4 result
The results are shown in Figure 3, compared with normal synovial tissue, the table of KHDC1L albumen in Human Osteoarthritis synovial tissue
It is dramatically increased up to amount, difference has statistical significance (P<0.05).
In 3 OA fibroblast-like synoviocyte antibody of embodiment and test
1st, step
1.1 synovial tissue's cell injuring models
After the synovial tissue of sterile acquisition is cleaned with PBS, about 1mm x 1mm x are cut into repeatedly with aseptic operation scissors
The tissue block of 1mm after adding in 37 DEG C of clostridiopetidase A II (0.5mg/ml), digestion 2h, is filtered through 200 mesh gauzes, after supernatant is removed in centrifugation,
Cell is resuspended in DMEM culture solutions, is placed in 37 DEG C, 5%CO2It is cultivated in cell incubator.When cell grows up to fusiformis and in blocks
Afterwards, secondary culture is carried out.After cell reached for 3 generation, it is separately added into mouse anti human CD3, CD14, CD19 and PE of FITC labels
The mouse anti human CD11b of label is marked, flow cytomery identification.Above-mentioned 4 kinds of labels be negative cell be into
Fiber-like synovial cell (Fibroblast-like Synoviocytes, FLS) is for this research.
In 1.3 antibody and test
Experiment packet：
Experimental group 1：Human Osteoarthritis synovial cell adds in unrelated monoclonal antibody (1：25)；
Experimental group 2：Human Osteoarthritis synovial cell adds in people KHDC1L monoclonal antibodies (1:25).
Fiber-like synovial cell is seeded in 96 well culture plates, per 100 μ l of hole, 3 multiple holes are set per experimental group, it will be single
Resist according to 1：25 ratio adds in mixing in synovial fluid, after being cultivated 48 hours with synovial cell, respectively in required detection
10 μ l CK-8 solution are added in culture hole per hole, continues to be incubated 1h in cell incubator, measures each hole absorbance at 450nm
It is worth (OD values).
2nd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
3rd, result
The results are shown in Table 1, and compared with adding in unrelated monoclonal antibody, the synovial cell of Human Osteoarthritis adds in KHDC1L monoclonal antibodies
Cell Proliferation slows down afterwards, and difference has statistical significance (P<0.05).It is above-mentioned the experimental results showed that, the suppression of KHDC1L protein actives
System can slow down the proliferation of the synovial cell of Human Osteoarthritis.
1 synovial cell's OD values of table
| Experimental group | OD values (optical density) |
| Osteoarthritis+unrelated monoclonal antibody | 0.2825±0.005 |
| Osteoarthritis+KHDC1L monoclonal antibodies | 0.1547±0.004 |
Embodiment 4 interferes the expression of KHDC1L genes
1st, siRNA designs synthesis
For the siRNA sequence of KHDC1L：
siRNA1-KHDC1L：
Positive-sense strand is 5 '-AUGUCUUUCUCACCAAAAGCG-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CUUUUGGUGAGAAAGACAUGA-3 ' (SEQ ID NO.8),
siRNA2-KHDC1L：
Positive-sense strand is 5 '-ACUUUCUUCAGUGUUUUUCAU-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-GAAAAACACUGAAGAAAGUUU-3 ' (SEQ ID NO.10),
siRNA3-KHDC1L：
Positive-sense strand is 5 '-AAUAACACUUCUAACACUCAG-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GAGUGUUAGAAGUGUUAUUGG-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 for 24 hours, in without DMEM culture mediums dual anti-, containing 10%FBS, is transfected and is tried according to liposome transfection in incubator
The specification transfection of agent 2000 (be purchased from Invitrogen companies), experiment is divided into, negative control group and experimental group (20nM),
In, the sequence of negative control group siRNA and KHDC1L genes transfects respectively without homology, a concentration of 20nM/ holes.
2nd, the jamming effectiveness of detection siRNA is tested using QPCR
2.1 extraction cell total rnas
Cell total rna is extracted using the tissue/cell RNA extracts kits of QIAGEN companies.
2.2 reverse transcriptions and QPCR
Step is the same as embodiment 2.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, the difference between KHDC1L gene expression panels and control group is interfered to adopt
It is examined with t, it is believed that work as P<There is statistical significance when 0.05.
4th, result
As a result as Fig. 4 shows that compared with siRNA1-KHDC1L, siRNA2-KHDC1L, siRNA3-KHDC1L can more have
The expression of the inhibition KHDC1L genes of effect, difference have statistical significance (P<0.05) it, is carried out using siRNA3-KHDC1L follow-up
Experiment.
The inhibiting effect of 5 KHDC1L gene pairs Osteoarthritic Synovium tissue cell proliferations of embodiment
1st, cell transfecting：According to embodiment 4 method to OA fibroblast-like synoviocytes carry out siRNA3-KHDC1L and
The transfection of siRNA-NC.
2nd, by OA fibroblast-like synoviocytes with 2 × 10 after transfecting 24 hours5/ ml density is seeded in 96 well culture plates,
Per 100 μ l of hole, every group sets three parallel holes, after cultivating 48 hours, adds in 10 μ l per hole in the culture hole of required detection respectively
CK-8 solution continues to be incubated 1h in cell incubator, measures each hole absorbance value (OD values) at 450nm.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
4th, result
The results are shown in Table 2, and compared with transfecting siRNA-NC groups of cells, the cell Proliferation for transfecting siRNA3-KHDC1L subtracts
Slowly, difference has statistical significance (P<0.05).
2 synovial cell's OD values of table
| Experimental group | OD values (optical density) |
| siRNA-NC | 0.1741±0.002 |
| siRNA3-KHDC1L | 0.1097±0.004 |
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, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will be also fallen into the protection domain of the claims in the present invention.
Claims (7)
1. detect application of the product of KHDC1L gene expressions in the tool for preparing diagnosis osteoarthritis.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform KHDC1L gene expressions are to diagnose the product of osteoarthritis；
The product with RT-PCR diagnosis osteoarthritis includes at least the primer of a pair of of specific amplified KHDC1L genes；It is described to use in real time
The product of quantitative PCR diagnosis osteoarthritis includes at least the primer of a pair of of specific amplified KHDC1L genes；It is described to use immune detection
The product of diagnosis osteoarthritis includes：The antibody combined with KHDC1L protein-specifics；It is described to diagnose Bones and joints in situ hybridization
Scorching product includes：With the probe of the nucleic acid array hybridizing of KHDC1L genes；The product packet with chip diagnosis osteoarthritis
It includes：Protein chip and genetic chip；Wherein, protein chip includes the antibody combined with KHDC1L protein-specifics, genetic chip
Include the probe of the nucleic acid array hybridizing with KHDC1L genes.
3. application according to claim 2, which is characterized in that the product with real-time quantitative PCR diagnosis osteoarthritis
The primer of a pair of of specific amplified KHDC1L genes included at least is as shown in SEQ ID NO.3 and SEQ ID NO.4.
Application of the inhibitor of 4.KHDC1L genes and/or its expression product in the drug for preparing treatment osteoarthritis.
5. application according to claim 4, which is characterized in that the inhibitor includes inhibiting the examination of KHDC1L gene expressions
Agent, and/or the reagent for inhibiting KHDC1L gene expression products.
6. application according to claim 5, which is characterized in that the reagent for inhibiting KHDC1L gene expression products includes
Inhibit the antibody of the siRNA, and/or KHDC1L albumen for KHDC1L genes.
7. application according to claim 6, which is characterized in that the siRNA sequence such as SEQ for KHDC1L genes
Shown in ID NO.11 and SEQ ID NO.12.
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| Cloning of a gene highly overexpressed in cancer coding for a novel KH-domain containing protein;Friederike MuÈ eller-Pillasch,et al;《Oncogene》;19971231;2729-2733页 * |
| Differences in Gene Expression between Mouse and Human for Dynamically Regulated Genes in Early Embryo;Elo Madissoon，et al;《PLOS ONE》;20141231;第9卷(第8期);e102949页 * |
| ＲNA 测序技术在筛选食管鳞癌差异表达基因中的应用;陈友芳等;《广东医学》;20141231;第35卷(第6期);844-847页 * |
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