CN105755148B - A kind of kit and its method detecting osteoarthritis - Google Patents
A kind of kit and its method detecting osteoarthritis Download PDFInfo
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Abstract
The invention discloses the kits that a kind of detection individual suffers from osteoarthritis possibility, and the kit includes the primer and specification of specific amplification osteoarthritis related gene transcript, and the osteoarthritis related gene is UPK3A.The invention also discloses a kind of molecular marked compound for detecting osteoarthritis, the molecular marked compound is UPK3A gene or the expression product of the gene.The invention discloses a kind of gene UPK3As relevant to osteoarthritis, and further confirm that the expression albumen and its segment of the UPK3A gene or the gene, analogs and derivatives express up-regulation in osteoarthritic tissue.It can not only quickly and effectively accomplish early detection using the genetic test osteoarthritis, and provide therapy target and important evidence for clinical applications such as gene therapy, drug therapies.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of kit and its method for detecting osteoarthritis.
Background technique
Osteoarthritis (osteoarthritis, OA) is the chronic disease that articular cartilage is gradually degenerated at any time, the pass
Section cartilage is covered on bone end, forms the articular surface in joint.It is reported that there are many factors, and patient to be made to be susceptible to suffer from osteoarthritis, including lose
Pass neurological susceptibility, obesity, accident or athletic injury, operation, drug and weight body demand force.Osteoarthritis is considered soft from joint
What the damage of bone started.Most common two kinds are sports-related injury and long-term " Reusability " joint damage to the damage in joint
Wound.The joint most frequently influenced by osteoarthritis is knee, hip and hand.In most cases, due to knee and the necessary load-bearing function of hip
Can, the osteoarthritis in these joints more easily leads to deformity than Hand osteoarthritis.With the development of cartilage degradation, in joint and close
Secondary variation occurs in its hetero-organization (including bone, muscle, ligament, meniscus and synovial membrane) around saving.Cartilaginous tissue primary declines
Exhausting with the net effect of its hetero-organization secondary damage is that pain, swelling, weakness and diseased joints forfeiture function occur for patient.These diseases
Shape is often developed to the degree seriously affected, is such as made patient disability or is influenced quality of life.
Articular cartilage is mainly made of cartilage cell, II Collagen Type VI, proteoglycans and water.Articular cartilage does not have blood or nerve
Distribution, cartilage cell is only cell type in the tissue.The appearance of cartilage cell's responsibility system at cartilage matrix II Collagen Type VI
And proteoglycans.Thereafter the matrix has the physicochemical characteristics for allowing to be made with water matrix to be saturated again.This structure-function relationship
Net effect be that articular cartilage has special wear resistance characteristics, and it is mobile that almost friction free occurs between articular cartilage face.
When being not suffering from osteoarthritis, articular cartilage often provides lifelong painless load-bearing and unlimited under the even physical qualification of high demand
The joint of system is mobile.
As all living tissues, articular cartilage constantly undergoes renewal process, wherein removal (catabolic activity)
" old " cell and matrix components simultaneously generate (anabolic activity) " new " cell and molecule.It is organized relative to majority, articular cartilage
Anabolism or catabolism turnover rate it is lower.The long-term maintenance of mature cartilage structure integrality is synthesized and is dropped dependent on matrix
Balance appropriate between solution.Cartilage cell is by response in chemistry and mechanical stimulus maintenance matrix equilibrium in its environment.
It is necessary to cartilage dynamic equilibrium that cartilage cell, which stimulates suitable and effective response to these,.Pass through anabolic activity deficiency
Or catabolic activity surplus destroy dynamic equilibrium can cause cartilage degradation and osteoarthritis (Adams etc., 1995,
Nature377Suppl:3-174).Majority, which is damaged, can start the synthesis of enhancing with the tissue of catabolic activity raising
It is metabolized response, tissue is allowed to restore.Unfortunately, articular cartilage response is damaged or is consumed in cartilage matrix and raises it and synthesize generation
The ability for thanking activity and raising synthetic proteins glycan and II Collagen Type VI is very limited.
Existing osteoarthritis treatment method include movement, drug, rest and joint care, operation, pain relief techniques,
Replacement therapy and body weight control.Treating common drug in osteoarthritis includes nonsteroidal anti-inflammatory such as aspirin, brufen
Deng；It is directly used in the topical pain-relieving creams, rubber and spray etc. of skin.It can be performed the operation so that bone surface reconstruction, bone
Reset and replacement joint.Although various medicinal treatments have been used for treating disease, they are nothings to long-term control and prevention
Effect.Further, since osteoarthritis hidden occur and develop slowly, therefore osteoarthritis often disease develop advanced stage
It is just identified, rather than potential treatment may more effective early in disease development.Therefore prevention, change or treatment osteoarthritis
Further development in lysis depends critically upon the early diagnosis marker of identification disease, so that early stage be allowed to interfere.
Summary of the invention
In order to realize the early detection of osteoarthritis, early intervention, the purpose of the present invention is to provide a kind of new bone passes
Save scorching related gene and expression albumen and its segment, the analogs and derivatives of the gene.
A further object of the invention is to provide the method using osteoarthritis related gene detection osteoarthritis.
To achieve the above object, present invention detection individual a kind of first suffers from the kit of osteoarthritis possibility, described
Kit includes the primer and specification of specific amplification osteoarthritis related gene, and the osteoarthritis related gene is
UPK3A。
Preferably, the primer has primer shown in SEQ ID NO:1 and SEQ ID NO:2.
Further, the present invention provides the kit that a kind of detection individual suffers from osteoarthritis possibility, the reagents
Box further includes the antibody and specification for specifically binding to the expression product of osteoarthritis related gene.
Further, the present invention provides a kind of kit for detecting osteoarthritis, the kit includes that detection bone closes
The chip and specification of scorching related gene are saved, the chip is selected from the group:
(1) DNA chip, the chip have substrate and point sample in the point sample DNA, the point sample DNA in point sample area on substrate
Probe including specifically binding to osteoarthritis related gene transcript, the osteoarthritis related gene is UPK3A；
(2) protein-chip, the chip have substrate and point sample in the deposited protein in point sample area on substrate, the point sample
Albumen includes the antibody for specifically binding to osteoarthritis related gene expression product, and the osteoarthritis related gene is
UPK3A。
Further, the present invention provides provide a kind of molecular marked compound for detecting osteoarthritis, the molecular marked compound
For UPK3A gene or the expression product of the gene.The expression product of the UPK3A gene or the gene is in Osteoarthritis group
Knit middle expression up-regulation.
Further, the present invention provides the molecular marked compounds of the osteoarthritis suffers from preparation for determining that test is individual
There is a purposes in the drug of the possibility of osteoarthritis, the molecular marked compound is UPK3A gene or the expression product of the gene,
The expression product of the UPK3A gene or the gene expresses up-regulation in Osteoarthritis tissue.
Drug of the invention can also be with the drug combination of other treatment osteoarthritis, and a variety of Drug combinations can be significantly
Improve the success rate for the treatment of.
Drug of the invention further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but being not limited to): diluent,
Excipient, adhesive, disintegrating agent, surfactant, Humectant, lubricant.
Drug of the invention can be used different additives and be prepared, for example, stabilizer, fungicide, buffer, etc.
Penetration enhancer, chelating agent, pH controlling agent and surfactant.
Further, the present invention provides a kind of preparation for treating Osteoarthritis, containing inhibiting UPK3A in the preparation
The reagent or compound of transcription or the expression of gene.
Further, the suppressor containing activation UPK3A, activation inhibit in the preparation of the treatment Osteoarthritis
The albumen of UPK3A expression, the siRNA for importing inhibition UPK3A transcription or expression, activation promote UPK3A mRNA to degrade
MicroRNA, the molecule for promoting UPK3A protein degradation, the expression for inhibiting the factor for promoting UPK3A to express and albumen are imported.
Preferably, the preparation of the treatment Osteoarthritis contains one group or several groups of siRNA:SEQ ID in following
NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.It is furthermore preferred that
It is SEQ ID NO.5 and SEQ ID NO.6 that the preparation of the treatment Osteoarthritis, which contains siRNA sequence,.
Beneficial effects of the present invention are as follows:
The invention discloses a kind of gene UPK3As relevant to osteoarthritis, and further confirm the UPK3A gene or be somebody's turn to do
Expression albumen and its segment, the analogs and derivatives of gene express up-regulation in osteoarthritic tissue.Utilize the genetic test
Osteoarthritis can not only quickly and effectively accomplish early detection, and provide for clinical applications such as gene therapy, drug therapies
Therapy target and important evidence.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
Inventor is obtained using differential expression genes in cDNA microarray technology screening osteoarthritis and normal population
The expression map of ten thousand genes.The gene expression profile of normal person synovial tissue and the synovial tissue of Human Osteoarthritis is carried out pair
Than having found 3208 gene expression up-regulations, both greater than 4 times of the expression up-regulation of these genes by comparing analysis.It is of the invention public
The UPK3A gene opened expresses up-regulation in 50% or more sample.
UPK3A gene of the invention is known before making the present invention, and essential information is as follows:
Genbank accession number: NM_001167574 derives from human genome.
The present invention also uses RT-PCR method and MTT method to detect said gene in Human Osteoarthritis and normal population
Expression, and demonstrate the gene be osteoarthritis expression up-regulation gene.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker
Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Mtt assay is also known as MTT colorimetric method, is a kind of method for detecting cell survival and growth.Its testing principle is living cells
Succinate dehydrogenase in mitochondria can make exogenous MTT be reduced to bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) of water-insoluble simultaneously
It is deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, be examined with enzyme linked immunological
It surveys instrument and measures its absorbance value at 490nm wavelength, can reflect living cells quantity indirectly.Within the scope of certain cell number, MTT knot
The amount that crystalline substance is formed is directly proportional to cell number.This method is widely used in the Activity determination, large-scale of some bioactie agents
Screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity measurement etc..Its feature is high sensitivity, economy.
The nucleotide full length sequence or its segment of UPK3A gene of the invention usually can with PCR amplification method, recombination method or
Artificial synthesized method obtains.It, can be according to published related nucleotide sequence, especially open reading for PCR amplification method
Frame sequence carrys out design primer, and with the commercially available library cDNA or by cDNA prepared by conventional method well known by persons skilled in the art
As template, expands and obtain related sequence.When sequence is longer, it is often necessary to which progress expands twice or repeatedly, then again will
The segment repeatedly amplified is stitched together by proper order.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, by first synthesizing
Then multiple small fragments are attached the very long segment of available sequence again.
At present, it is already possible to encode albumen of the invention (or its segment, or derivatives thereof) by chemical synthesis completely
DNA sequence dna.Then the DNA sequence dna can be introduced into various DNA moleculars (such as carrier) and cell in this field.In addition, also
Mutation can be introduced into protein sequence of the present invention by chemical synthesis.
The segment of albumen of the present invention also can be used solid phase technique to pass through direct synthetic peptide other than available recombination method generates
Produced (Stewart et al., (1969) Soliod-Phase Peptide Synthesis, WH Freeman Co., San
Francisco；Merrifield J.(1963)J.Am Chem.Soc85:2149-2154).Synthetic proteins matter can be in vitro
By hand or automatically it carries out.For example, can with the 431A type peptide synthesizer of Applied Biosystems (Foster City,
CA) it is automatically synthesized peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then chemically connected to produce
The molecule of raw overall length.
The particular form of terms used herein " osteoarthritis " articulations digitorum manus inflammation, especially articular cartilage are gradually moved back at any time
The chronic disease of change, the articular cartilage are covered on the bone end to form articular surface.
Terms used herein " expression up-regulation " refer to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
It is bright, it is determined in the individual for having identified morbid state different with osteoarthritis by stages from from normal individual or from by osteoarthritis
Same gene in isolated biological sample is compared, and the gene is from suffering from osteoarthritis or determined by osteoarthritis by stages
Osteoarthritis identified the expression in the biological sample separated in the individual of morbid state increase.According to the present invention,
" expression up-regulation " refer to intensity for hybridization measurement by the method for the invention at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10% or higher expression increases, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
It is high or be higher than 1 times, up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or higher.For example, up-regulation sequence include with
The biological sample for being isolated from normal individual is compared, and is to suffer from slight, moderate, significant or severe OA individual being isolated from feature
The increased sequence of expression in cartilage or blood.Up-regulation sequence may also include compared with other osteoarthritis stages, be isolated from
Feature is the increased sequence of expression (such as significant OA degree of specific gravity in the biological sample for the individual for suffering from some osteoarthritis stages
OA)。
Used in the context of terms used herein " protein substance " (such as protein, polypeptide, peptide and antibody)
Term " analog " refers to such protein substance: its have the function of with second of protein substance it is similar or identical,
But not necessarily comprising the amino acid sequence similar or identical with second of protein substance or have and second of albumen
The similar or identical structure of matter substance.Protein substance with similar amino acid sequence refers at least one of satisfaction or less
Second of protein substance:
(a) have with the amino acid sequence at least 30% of second of protein substance, at least 35%, at least 40%, extremely
Few 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, the protein substance of the amino acid sequence of at least 90%, at least 95% or at least 99% consistency；
(b) by nucleotide sequence coded protein substance, the nucleotides sequence is listed under high stringency conditions and coding the
At least five continuous amino acid residue, at least ten continuous amino acid residue, at least 15 Continuance ammines of two kinds of protein substances
Base acid residue, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous hydrogen-based acid are residual
Base, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least
80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, at least 125 companies
The nucleotide sequence hybridization of continuous amino acid residue or at least 150 continuous amino acid residues；
(c) by nucleotide sequence coded protein substance, second of protein of the nucleotide sequence and coding
The nucleotides sequence of substance shows at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, extremely
Few 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least
99% consistency.Protein substance similar with second of protein substance structure, which refers to, to be had and second of protein-based object
The protein substance of the similar second level of matter, three or four structure.
Terms used herein " protein ", " polypeptide " and " protein substance " are used interchangeably, and are referred to and are connected by peptide bond
The amino acid chain being connected together optionally may include natural or non-natural amino acids.Optionally, protein or polypeptide may include removing
Other molecules other than amino acid.The chain can be any length.In a specific embodiment, protein is by passing through peptide
Key connection together less than 200, less than 175, less than 150, less than 125, less than 100, less than 50, be less than
45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, be less than 10 or less than 5
Amino acid composition.In another embodiment, protein by linked together by peptide bond at least 200, at least 250,
At least 300, at least 350, at least 400, at least 450, at least 500 or more compositions.
Terms used herein " biological sample " include but is not limited to blood, serum, saliva, urine, synovia, cartilage, synovial membrane
The samples such as tissue.Preferably, Patient Sample A is synovial tissue.
Terms used herein " expression " refer to through the measurement of known to those skilled in the art and method described herein
Given nucleic acid or protein can measure.It is related to corresponding RNA, hnRNA, mRNA or mRNA montage of biomarker of the present invention
When variant, expression can be measured by hybridization or more, for example including use SYBR green, TaqMan and point
The quantitative real-time RT PCR of sub- beacon technique.
" control " used herein refer to do not show any OA symptom (including arthralgia) and have not been diagnosed as joint injury or
The individual or group of individuals of OA.Preferably, it is described control individual be not used influence OA drug, and have not been diagnosed as with it is any its
His disease.It is highly preferred that control individual has similar gender, age and Body Mass Index (BMI) compared with test sample.According to
The present invention, " control " also refer to the sample for being isolated from normal individual, total serum IgE or mRNA including being isolated from normal individual.It is derived from pair
Sample according to individual may include the RNA for being isolated from synovial tissue, and wherein RNA is isolated from synovial tissue, synovial tissue's separation
Self-organizing has not been diagnosed as OA and does not show the individual of any OA symptom when removing.In one embodiment of the invention, it is
Meniscus injury and cruciate ligament carry out the patient of arthrocsopic surgery treatment.
Terms used herein " primer " refer to the oligonucleotides for being present in the restrictive digestion content of purifying or being synthetically produced,
When being placed in induction and synthesizing under conditions of the primer extension product complementary with nucleic acid chains, that is, there is nucleotide and inducer, (such as DNA gathers
Synthase) and temperature appropriate and when pH, the oligonucleotides can be as the starting point of synthesis.Primer can be single-stranded or double
Chain, and must have enough length, to cause the synthesis of required extension products in the presence of inducer.The definite length of primer
Degree can depend on many factors, including temperature, Primer Source and the method used.For example, depending on target for diagnostic application
The complexity of sequence, Oligonucleolide primers typically contain the nucleotide of 15-25 or more, although can also contain less nucleosides
Acid.Factor involved in the determination of primer appropriate length is known to those of ordinary skill in the art.In general, according to ability
Standard method known to domain designs and selects primer of the invention, refering to Dieffenbach, C.W., Lowe, T.M.J.,
Dveksler, G.S. (1995) General Concepts for PCR Primer Design. " PCR Primer, A
Laboratory Manual " (Dieffenbach, CW and Dveksler, G.S. are edited) Cold Spring Harbor
Laboratory Press, New York, 133-155.
Embodiment 1 establishes gene differential expression spectrum
1, material
Human Osteoarthritis synovial tissue and control synovial tissue are chosen, Human Osteoarthritis meets knee joint OA diagnosis mark
Standard carries out knee joint artificial joint replacement patient；Control is that meniscus injury and cruciate ligament carry out arthrocsopic surgery
The patient for the treatment of.
2, RNA is extracted
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experiment behaviour
Make to carry out by product description.
RNA uses Nanodrop2000 ultraviolet specrophotometer measurement RNA purity and concentration after extracting, freeze in -70 DEG C.
RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram have clearly 28S,
18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
Preparation of the total serum IgE after denaturing formaldehyde agarose electrophoretic examinations is without price reduction, for mRNA.Primary first-order equation 200ug
The separation of total serum IgE separation preparation mRNA, mRNA use Oligotex mRNA min kit (Qiagen, Hilden, Germany)
Kit.
3, the micro- array of cDNA
Incyte Genimic company is purchased from using the micro- array film of LifeGrid 1.0, which contains about 8,000 4 hundred cDNA
PCR product and 27 control, according to two point mode, be imprinted on the nylon membrane of a 12*22cm.From osteoarthritis and normally
The mRNA of tissue is respectively in a-33Under the conditions of P dCTP is existing, generated with reverse transcription33The cDNA probe of P- label.Label incorporation
Percentage is not less than 40%.The condition that each cDNA probe hybridizes with micro- array is shown in the micro- array specification of LifeGrid 1.0.It is miscellaneous
Film after handing over overnight, room temperature are dried, and are exposed and are scanned, scanning result, that is, hybridization signal by phosphorus screen instrument (Phosphor image)
Computer analysis is interpreted to be completed by Incyte company.
4, result
According to the computer analysis results that Incyte company provides, it is poor to obtain expression in osteoarthritis and health adult tissue
Allogene obtains the expression map of 8400 genes.Normal person synovial tissue and the synovial tissue of Human Osteoarthritis
Gene expression profile compares, and removal all gene of expression deletion in osteoarthritis and health adult tissue removes identical cDNA
Two differences are greater than 2.5 times and one 2 times less than background of point of gene.Finally, having found 3208 bases by comparing analysis
Because expression is raised, both greater than 3 times of the expression up-regulation of these genes.UPK3A gene disclosed by the invention is in 50% or more sample
Expression up-regulation.
2 Human Osteoarthritis of embodiment and control synovial tissue UPK3A expression conditions
One, material and method
1, material
39 Human Osteoarthritis synovial tissues and 8 control synovial tissues are chosen, it is grouped and is numbered.Case
Group meets knee joint OA diagnostic criteria, carries out knee joint artificial joint replacement patient；Control group is meniscus injury and friendship
Pitch the patient that ligament injury carries out arthrocsopic surgery treatment.
2, method
The extraction of 2.1 Human Osteoarthritis and synovial tissue's total serum IgE of control
With embodiment 1
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044)
CDNA reverse transcription is carried out, experimental implementation is carried out by product description.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- Δ Δ CT method.
2.3.2 design of primers
Using online primer-design software, gene order is referring to NCBI:NM_001167574 (UPK3A), interior participation in the election
GAPDH is synthesized by invitrogen company after design of primers.Specific primer sequence is as follows:
1 primer sequence of table
Operating process is as follows:
2 Real Time reaction system of table+
| Component | Additional amount |
| 2×mix | 10μL |
| Upstream primer (10uM) | 0.5μL |
| Downstream primer (10uM) | 0.5μL |
| Template | 2μL |
| Sterile purified water is added | To 25 μ L |
(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No.
4367659) it is expanded, experimental implementation is carried out by product description.
Amplification program are as follows: 95 ° of 5min, (95 DEG C of 15sec, 62 DEG C of 45sec) × 35 circulations.
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ L to make template after dilution,
It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion
Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.
(3) sample Real TimePCR is detected
2 μ L will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into
Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.
Two, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2-
Δ Ct × 100% compares expression of the UPK3A gene in osteoarthritic tissue and control tissue.As the result is shown: qRT-
PCR amplification result is stablized, and wherein expression of the UPK3A gene in osteoarthritic tissue is 3 times of control tissue, the above knot
Fruit demonstrates the result that UPK3A gene expresses up-regulation in Human Osteoarthritis.
The separation and culture of 3 cartilage cell of embodiment
One, experimental method
(1) it will be immediately placed in PBS sterile petri dish, remove after sterile knee cartilage sample that traumatic amputation is removed
All soft tissues around articular cartilage obtain kneed full-thickness cartilage piece；
(2) cartilage is trimmed to by about 3mm × 1mm × 1mm size tissue block with aseptic operation knife, shreds cartilaginous tissue,
After the PBS buffer solution of these cartilage fragments dual anti-liquid containing Benzylpenicillin sodium salt/gentamicin is rinsed for several times later, it is put into and fills
In the sterile petri dish of 12 culture medium of DMEM/F；
(3) the cartilage fragment of these trimmings is moved into centrifuge tube, is centrifuged about 5 points under low temperature with 1000 revs/min of speed
Clock；
(4) 0.25% trypsase of 5ML is added after discarding supernatant, digests 30-50 on 37 DEG C of warm blenders
Minute, then gently piping and druming discards supernatant liquid, and D-Hanks liquid rinses 3-5 times；
(5) be added 5ML 0.15% II Collagenase Type, be put into constant temperature and shake in case, swayed at 37 DEG C digestion 6 hours, every 3
Cell of a hour receipts；
(6) 200 mesh net filtration postdigestive cell suspensions discard again after low-temperature centrifugation 5 minutes (about 2800 revs/min)
Then supernatant washs 3 times in 12 culture solution of DMEM/F, the DMEM/F12 culture medium for containing 10% fetal calf serum is added, with suction
Pipe is gently blown and beaten, and so that cell suspension is evenly distributed, to obtain the cell suspension containing cartilage cell；
(7) by cell with 1 × 108/ L is inoculated in 25CM2In culture bottle (5ML/ bottles), it is placed in 37 DEG C of constant temperature, 5%CO2Saturation
Culture in humidified incubator, the frequency for replacing culture solution is every two days 1 time, is observed and is shone using inverted phase contrast microscope
Phase starts to pass on as long as cartilage cell converges after in blocks and adherent rate reaches 85%-90%；
(8) before passage starts, culture solution is first cleaned, is then rinsed cell 2-3 times with HANKS liquid；
(9) 0.05% trypsase 1ML is added in culture bottle, is exhausted after bottom of bottle is uniformly distributed after it, so
0.05% tryptic digestive juice is added again afterwards, is limited with covering bottom of bottle, is placed it in 37 DEG C of constant incubators later
2-3 minutes；
(10) it is observed with inverted microscope, when cell starts to bounce back, and space between cells starts to increase, then shows cartilage
Cell starts to dissociate；
(11) digestion in HANKS liquid termination culture bottle is added, featheriness bottom of bottle cartilage cell generally requires repeatedly more
It is secondary just adherent cartilage cell to be made to completely fall off；
(12) cell suspension is centrifuged with the speed of 1200rpm, after ten minutes discards supernatant；
(13) cell suspension is made and counts, then carries out secondary culture according to the ratio of 1:3.
Two, experimental result
Normal chondrocyte is generally in 24 hours or so beginning adherent growths, and attached cell can achieve after 48 hours
90% or so, bottom of bottle can be covered within 4 to 7 days under normal circumstances, and adherent cartilage cell has endochylema abundant, flat in polygonal
Shape can have multiple protrusions, the karyon that cell space center is round or oval, it is seen that the cartilage of 2 to 3 kernels, some regions is thin
Born of the same parents are in colony growth.When culture to the third generation, cartilage cell starts to dedifferente, and this trend dedifferented can be with algebra
Increase and enhance, the variation of fibrocyte sample is gradually appeared when to forth generation, is embodied in: form is in spindle shape；Edge
Protrusion is more and elongated；Endochylema increases but nucleus offset from center and kernel are loose.
4 RNAi of embodiment interferes UPK3A expression and the influence to cartilage cell
One, material
(1) cell origin
The cartilage cell that embodiment 3 is cultivated.
(2) siRNA design and synthesis
According to Photographing On-line software siDirect version 2.0 (http://design.rnai.jp/), according to gene
Sequence designs corresponding siRNA, particular sequence is shown in Table 3 referring to NCBI:NG_016203.1 (UPK3A).Synthesis is sent to after design
Company's synthesis.
3 siRNA sequence list of table
Two, experimental method
(1) expression of RNA AF panel cartilage cell UPK3A gene
Cell grouping and transfection
1. cell is grouped
C group: blank control group；C1 group: transfection liposome group；C2 group: nonspecific siRNA group is transfected；S1, S2, S3
Group: the siRNA group of specificity is transfected.
2. transfection
According to LipofectamineTMThe step of 2000 Transfection Reagent are provided carries out.
(1) cartilage cell synchronizes: the day before transfection takes first generation cartilage cell to test, and is added into 5%
In FBS DMEM in high glucose culture medium, continue in 37 DEG C of 5%CO2It is inside cultivated, so that all cartilage cells be made to be in synchronous shape
State reduces test interference；
(2)5×104For cell inoculation in 6 orifice plates, these are used for the cell quantity of initial vaccination, should be able to be in 24 hours
Cell confluency is set to reach 70%；
(3) prepare siRNA-LipofectamineTM2000 compounds:
A. 5ul LipofectamineTM 2000 is diluted with 250ul Opti-MEM, mixed gently, be incubated for 5 points at room temperature
Clock.
B. experiment each group takes 7.5ul siRNA to be added in 250ul Opti-MEM I respectively and is diluted, and gently shakes
It is mixed；
C. it is incubated for after five minutes, by diluted siRNA and LipofectamineTM20 are incubated at room temperature after 2000 mixing
Minute.
(4) cell, culture medium and siRNA-Lipofectamine is added in each hole in culture plateTM2000 is multiple
Close object.Then culture plate is gently shaken, them are sufficiently mixed；
(5) 37 DEG C are placed on, CO2It is incubated for 48 hours in incubator, in fluorescence microscopy microscopic observation cell transfecting quantity, inspection
Survey transfection efficiency；
(6) transfection efficiency is the ratio of the cell quantity in fluorescope and the light microscopic visual field, and the transfection efficiency of cell reaches
90% or more, it can just carry out subsequent experiment.Calculation formula is as follows:
Cell quantity × 100% under the transfection efficiency=cell that fluoresces quantity/same field of view
3. the variation of application Real-time PCR method detection transfection front and back UPK3A gene expression
(1) building of standard curve: being chosen at 1 bottle of cartilage cell normally cultivated in 50mI culture bottle, extracts RNA, surveys
Determine RNA concentration and purity, carry out reverse transcription reaction, ten times of the DNA profiling dilutions that reaction is generated obtain being equivalent to 104-
100The DNA profiling of copies/ul is separately added into UPK3A gene primer and internal control primer, prepares 25ul reaction system, uses
Real-time PCR amplification instrument carries out pcr amplification reaction.Obtain the standard curve of UPK3A and internal reference.
(2) variation of Real-time PCR method detection transfection front and back UPK3A gene expression: group of cells is extracted
RNA measures RNA concentration and purity, carries out reverse transcription reaction, every group of DNA profiling is simultaneously into the Real-time of UPK3A and internal reference
PCR reaction, experiment is in triplicate.
(3) agarose gel electrophoresis is carried out to PCR product.
Three, experimental result
The siRNA and control siRNA of 3 UPK3A genes transfect first generation cartilage cell respectively, as the result is shown a large amount of soft
Green fluorescence is found in osteocyte, it was demonstrated that the transfection of siRNA has been obtained in cartilage cell, then under fluorescope and light microscopic
Cartilage cell's quantity is observed, carries out the detection of transfection efficiency, transfection efficiency reaches 90% or more as the result is shown.Real-time
PCR is the results show that transfect nonspecific siRNA group to UPK3A gene expression in cartilage cell without obvious inhibiting effect, and sky
3 UPK3A gene siRNA groups of white control group no difference of science of statistics, transfection all play UPK3A gene expression in cartilage cell
Certain inhibiting effect inhibiting rate is 56%, and wherein UPK3A-siRNA2 and UPK3A-siRNA3 inhibitory effect is most obvious, inhibiting rate
Up to 65% and 72%.
The proliferative conditions of 5 mtt assay of embodiment detection cartilage cell
One, mtt assay experimental procedure
1. being added about 1 × 10 according to every hole in 96 orifice plates4Cell, then in 37 DEG C of 5%CO2Under conditions of cultivate 24
Hour；
2. according to experimental group, (blank control group, cartilage cell add nonspecific siRNA group, cartilage cell to add
UPK3A-siRNA3 group, every group of 6 repetitions) continue culture until being suitble to detection；
3. then at 37 DEG C, 5%CO2And 100% continue under conditions of humidity to be incubated for appropriate time；
4. 5 × MTT is diluted to 1 × MTT with Dilution Buffer simultaneously；
5. 1 × MTT of 50ul is added in every hole, and it is incubated for 4 hours under the conditions of 37 DEG C；
6. after supernatant is sucked out, also needing that 150ul DMSO is added in every hole, and place it on plate shaker and carry out
It shakes up；
7. microplate reader wavelength is set as 570nm, the optical density in each hole is detected.
8. the calculating of cell proliferation rate: cell proliferation rate %=(by prospect hole OD value/control cell OD value) × 100%,
In by prospect hole OD value=instrument connection OD value-background OD value (no cell but the complete medium for having MTT), control cell OD value
For the OD value for normally cultivating cell hole.
Three, experimental result
It was sampled on node at 0,12,24,48 and 72 hour respectively, opposite cartilage cell's control group, cartilage cell Jia Feite
For anisotropic siRNA group cell Proliferation without significant difference, cartilage cell adds UPK3A-siRNA3 group to be proliferated not in 0-24 hour cell
Obviously, but as time increases, chondrocyte proliferation rate dramatically increases on 48,72 hours nodes, and difference has statistics
It learns meaning (P < 0.05), the results are shown in Table shown in 4.
The proliferation rate (%) of 4 each group cartilage cell of table
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. a kind of purposes of molecular marked compound in reagent of the preparation for the individual possibility with osteoarthritis of determining test,
The molecular marked compound is UPK3A gene or the expression product of the gene, the expression product of the UPK3A gene or the gene
Up-regulation is expressed in Osteoarthritis tissue.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102105598A (en) * | 2008-06-20 | 2011-06-22 | 代理生命科学控股公司 | Microvesicle-based compositions and methods |
| WO2013057293A1 (en) * | 2011-10-20 | 2013-04-25 | Assistance Publique - Hôpitaux De Paris | Method for quantifying renal markers by assaying urine |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102105598A (en) * | 2008-06-20 | 2011-06-22 | 代理生命科学控股公司 | Microvesicle-based compositions and methods |
| WO2013057293A1 (en) * | 2011-10-20 | 2013-04-25 | Assistance Publique - Hôpitaux De Paris | Method for quantifying renal markers by assaying urine |
Non-Patent Citations (3)
| Title |
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| UPK3A：a promising novel urinary marker for the detection of bladder cancer;Yongqing Lai等;《Urology》;20101231;第76卷(第2期);514e6-e10 |
| 膀胱癌新标志物UPK3 A的临床应用研究;来永庆等;《现代泌尿生殖肿瘤杂志》;20100831;第2卷(第4期);232-236 |
| 骨性关节炎生物标志物的研究进展;任敏;《中外医疗》;20151231(第22期);196-198 |
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