CN106310292A - circRNA-CER gene inhibitor and application thereof - Google Patents

Abstract

The invention discloses a circRNA-CER gene inhibitor and application thereof, belonging to the field of bioengineering. The inhibitor can inhibit the expression of the circRNA-CER gene so as to lower the circRNA-CER gene expression product level. The nucleotide sequence of the circRNA-CER gene is disclosed as SEQ ID NO:1. The inhibitor capable of inhibiting circRNA-CER gene expression can prevent the degradation of the extracellular matrix in the cartilaginous tissue due to the expression of the circRNA-CER gene, and has important meanings in preventing and treating osteoarthritis cartilage injury.

Description

The inhibitor of a kind of circRNA-CER gene and application thereof

Technical field

The present invention relates to bioengineering field, particularly to a kind of circRNA-CER gene inhibitor and Application.

Background technology

RNA interference (RNAinterference, RNAi) refers to endogenous or exogenous double-stranded RNA (dsRNA) There is selective degradation in the intracellular mRNA of mediation, thus causes the expression silencing of target gene, and produces phase The phenomenon of the function phenotype disappearance answered.After double-stranded RNA enters cell, by Dicer enzyme (RNAase III Double-stranded RNA is had specific enzyme by race) combine and cut, form the product that length is about 20-25bp, And 3 ' ends of every chain have 2 nucleotide siRNA molecule (small interference RNA, siRNA).One chain of siRNA is incorporated into RNA-and induces silencing complex (RNA-induced silencing Complex, RISC) in, with the sequence pairing of complementary RNA.First RISC mediates siRNA double-strand solution Rotation, then, is combined with said target mrna in sequence-specific mode with the siRNA of the strand of RISC coupling, Said target mrna is cut afterwards by the catalyst component of RISC.The said target mrna of cutting can suppress it to translate, and The final expression suppressing this gene.Visible, siRNA can participate in regulating cell function, and has proven to it There is in numerous disease important regulating and controlling effect, be a kind of preferably therapy target and intervention means.

Osteoarthritis (Osteoarthritis, OA) cartilage injury is the arthritis caused by wound or chronic injury Disease is reacted.Under Biomechanical, chondrocyte produces matrix metalloproteinase MMP13 (matrix Metalloproteinase-13), this enzyme especially can cause the II Collagen Type VI of extracellular matrix in cartilaginous tissue (Collagen Type II, COL2) and the degraded of aggrecan (AGGRECAN), so that The level obtaining chondrocyte synthetic cell epimatrix declines, and strengthens the synthesis difficulty of extracellular matrix further, Cause vicious cycle, cause cartilage destruction and cause cartilage injury.Visible, the extracellular of chondrocyte The degraded of substrate is pathological change main in cartilage injury.

Therefore, develop a kind of extracellular matrix degradation by suppressing chondrocyte, do especially by RNA The method of the extracellular matrix degradation disturbing suppression chondrocyte is the most necessary.

Summary of the invention

Embodiment of the present invention technical problem to be solved is, it is provided that a kind of circRNA-CER gene Inhibitor and application thereof.Concrete technical scheme is as follows:

First aspect, embodiments provides a kind of inhibitor, described inhibitor suppression circRNA-CER The expression of gene, to reduce the level of the expression product of described circRNA-CER gene, described The nucleotide sequence of circRNA-CER gene is as shown in SEQ ID NO:1.

Specifically, as preferably, described inhibitor is siRNA molecule, the positive-sense strand of described siRNA molecule Sequence as shown in SEQ ID NO:2, the sequence of the antisense strand of described siRNA molecule such as SEQ ID NO:3 Shown in.

Further, described siRNA molecule is adorned siRNA molecule, described adorned siRNA Molecule makes the expression silencing of described circRNA-CER gene.

Specifically, it is modified to ribose modification, base modification or phosphate backbones described in modify.

Further, the nucleotide of described siRNA molecule is partially replaced or increases and decreases, and nucleotide is partly replaced SiRNA molecule after changing or increasing and decreasing makes the expression silencing of described circRNA-CER gene.

Second aspect, embodiments provide a kind of any one above-mentioned inhibitor preparation prevention or Application in treatment osteoarthritis drugs.

Second aspect, embodiments provides a kind of drug regimen for preventing or treat osteoarthritis Thing, described pharmaceutical composition includes the described inhibitor of therapeutically effective amount.

Specifically, as preferably, described pharmaceutical composition also includes other medicine classes with described inhibitor compatibility And pharmaceutically acceptable carrier and/or adjuvant.

Specifically, the dosage form of described pharmaceutical composition is selected from solution, suspension agent, Emulsion, powder, control Releasing agent or extended release preparation.

Specifically, the administering mode of described pharmaceutical composition is administered selected from drug administration by injection or perfusion.

The technical scheme that the embodiment of the present invention provides has the benefit that

Inventor studies discovery, and circRNA-CER gene is the weight of chondrocyte skeleton in regulation and control articular cartilage Wanting the factor, it is expressed and can Induction of chondrocytes framing structure change, and promotes the generation of MMP13, causes soft Extracellular matrix degradation in osseous tissue, and then cause cartilage tissue damage.The embodiment of the present invention is by providing one Planting the inhibitor that can suppress circRNA-CER gene expression, it selectively targeted can act on circular rna, The degraded of extracellular matrix in the cartilaginous tissue that can block the expression by circRNA-CER gene and cause, For preventing and treat cartilage of osteoarthritis damage to have great importance.

Accompanying drawing explanation

For the technical scheme being illustrated more clearly that in the embodiment of the present invention, institute in embodiment being described below The accompanying drawing used is needed to be briefly described, it should be apparent that, the accompanying drawing in describing below is only the present invention Some embodiments, for those of ordinary skill in the art, on the premise of not paying creative work, Other accompanying drawing can also be obtained according to these accompanying drawings.

Fig. 1 is that the IL-1 that is utilized respectively that the embodiment of the present invention 1 provides stimulates chondrocyte different with TNF-α Time after, relative expression levels's schematic diagram of circRNA-CER gene in chondrocyte；

Fig. 2 be the embodiment of the present invention 1 provide utilize IL-1 to stimulate chondrocyte different time after, cartilage Relative expression levels's schematic diagram of circRNA-CER gene and MMP13 in cell；

Fig. 3 is the negative control chondrocyte of the embodiment of the present invention 2 offer and tests in chondrocyte, COL2, The expression schematic diagram of the mRNA of MMP13, Aggrecan；

Fig. 4 a is the negative control chondrocyte of the embodiment of the present invention 3 offer and tests in chondrocyte, The expression schematic diagram of MMP13；

Fig. 4 b is the negative control chondrocyte of the embodiment of the present invention 3 offer and tests in chondrocyte, The expression schematic diagram of circRNA-CER；

Fig. 4 c is the negative control chondrocyte of the embodiment of the present invention 3 offer and tests in chondrocyte, Relative expression levels's schematic diagram of MMP13 albumen；

Fig. 4 d is the negative control chondrocyte of the embodiment of the present invention 3 offer and tests in chondrocyte, COL2 Relative expression levels's schematic diagram of albumen.

Detailed description of the invention

Unless otherwise defined, all technical terms used by the embodiment of the present invention are respectively provided with and people in the art The identical implication that member is generally understood that.Before embodiment of the present invention is described further in detail, right Understand that some terms of the embodiment of the present invention provide definition.

(1), in the embodiment of the present invention, described " therapeutically effective amount " refers to produce human or animal body Desired treatment function or therapeutic activity, and can be from the amount that physiologically can be accepted by human or animal.

(2), in the embodiment of the present invention, described " pharmaceutically acceptable carrier and/or adjuvant " refers to fit For human or animal, and without excessive bad side reaction (such as toxicity, stimulation and allergy), there is conjunction The carrier of the benefit/risk ratio of reason and/or adjuvant.

For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing to the present invention Embodiment is described in further detail.

Inventor studies discovery, and the generation of osteoarthritis (Osteoarthritis, OA) and development include several heavy Want pathological change: cartilaginous tissue regression, Subchondral bone sclerosis, synovial membrane inflammation and hyperosteogeny are formed.Wherein, soft Osseous tissue regression is initially to produce in osteoarthritis, and current treatment means cannot reverse, and is also most important disease Reason changes.Cartilaginous tissue major part is by chondrocyte and extracellular matrix (extracellular matrix, ECM) Composition.Chondrocyte is unique cell type in cartilaginous tissue, and it can synthesize and decompose extracellular matrix. The extracellular matrix of cartilaginous tissue is mainly by II Collagen Type VI (Collagen type II, COL2) and collectin Polysaccharide (Aggrecan) forms.Chondrocyte and extracellular matrix jointly maintain the structure of articular cartilage and its The stable state of extracellular environment.When there is OA, this stable state is broken, and shows as the fall of extracellular matrix Solution, the regression etc. of chondrocyte.During OA, regression and the extracellular matrix degradation of chondrocyte mainly divide Two stages: biosynthesis stage and degradation period.In the biosynthesis stage, chondrocyte is attempted going to repair The cartilage cell epimatrix of damage.But, owing to the gene expression dose of synthetic cell epimatrix is the lowest, no Can effectively synthesize enough extracellular matrixs.In degradation period, under the effect of inflammatory factor, cartilage is thin Born of the same parents produce the matrix metalloproteinase MMP13 (matrix making extracellular matrix degradation Metalloproteinase-13), extracellular matrix degradation is caused.Meanwhile, inflammatory factor can also cause synthesis The gene expression of extracellular matrix declines further, and the synthesis of extracellular matrix is more not enough, causes pernicious following Ring, causes the drastically destruction of cartilaginous tissue.Therefore, extracellular matrix synthesis deficiency and extracellular matrix Degraded be pathological change main in OA cartilage injury.

According to the research to OA pathological process, in order to slow down the symptom of OA, and effectively reverse or stop bone Arthritis, invention further contemplates the gene played an important role in the pathological process of OA, CircRNA-CER gene be a kind of regulate and control chondrocyte skeleton important factor, its expression can induce cartilage thin The framing structure of born of the same parents changes, and promotes chondrocyte to produce MMP13, causes extracellular matrix degradation.Cause This, by blocking or suppressing expressing of circRNA-CER gene will block or reduce the expression of MMP13 also Reduce the degraded of extracellular matrix in cartilaginous tissue.

Based on above-mentioned, first aspect, embodiments provide a kind of inhibitor, this inhibitor can press down The expression of circRNA-CER gene processed, to reduce the level of the expression product of circRNA-CER gene, tool The nucleotide sequence of body ground circRNA-CER gene is as shown in SEQ ID NO:1.

Specifically, the nucleotide sequence of this circRNA-CER gene is as follows:

gttgatatgt cagatctctc tccagaagag caatggaggg tcgagcacgc acgcatgcat 60

gccaagcacc gtggccatga agctatgcat gctgaaatgg tcctcatcct catcgcaacc 120

ttggtggtgg cccagctgct cctggtgcag tggaagcaga ggcacccacg ctcctacaat 180

Visible, the embodiment of the present invention by provide a kind of inhibitor that can suppress circRNA-CER gene expression, Maintained the matrix morphology of chondrocyte by the expression of suppression circRNA-CER gene, suppress this skeleton weight Structure, and suppress the expression of MMP13, thus block the degraded of extracellular matrix in cartilaginous tissue, for prevention And treat cartilage of osteoarthritis damage have great importance.

It is understood that for can suppress circRNA-CER gene expression or can be with The inhibitor kind that the expression product of circRNA-CER gene combines has no particular limits, as long as can realize Expressing or the merit promoting lipid to generate of suppression circRNA-CER gene of reticent circRNA-CER gene Can.Such as, this can suppress the inhibitor of circRNA-CER gene expression can include but not limited to little Disturb nucleotide, antisense oligonucleotide, antibody, active organic compounds and can have suppression circRNA-CER Gene expression or other inhibitor kinds that can combine with the expression product of circRNA-CER gene.

For example, the inhibitor suppressing circRNA-CER gene expression in the embodiment of the present invention can be one Plant or multiple antisense oligonucleotide, antisense oligonucleotide and phase in the nucleotide sequence shown in SEQ ID NO:1 The nucleotide sequence of pairing has the homogeneity of more than 90%.

In further example, the inhibitor suppressing circRNA-CER gene expression in the embodiment of the present invention can Thinking one or more antibody, this antibody can be monoclonal antibody or polyclonal antibody, both can be Mus Endogenous antibody can also be humanized antibody.From structure, both can be that single-chain antibody can also be for it Its antibody analog, such as it can include but not limited to nucleic acid aptamer etc..

The most for example, the embodiment of the present invention suppresses the inhibitor of circRNA-CER gene expression Can be to suppress circRNA-CER gene expression or can be combined with the expression product of circRNA-CER gene One or more reactive compounds.

As one preferred embodiment, the embodiment of the present invention suppresses circRNA-CER gene expression Inhibitor is a kind of siRNA molecule, the sequence of the positive-sense strand of this siRNA molecule such as SEQ ID NO:2 institute Showing, the sequence of the antisense strand of this siRNA molecule is as shown in SEQ ID NO:3.Shown in specific as follows:

Positive-sense strand (5'-3') 5 ' CCCACGCUCCUACAAUGUU dTdT 3 '

Antisense strand (3'-5') 3 ' dTdT GGGUGCGAGGAUGUUACAA 5 '

Wherein, A adenine, G guanine, C cytosine, U uracil, dTdT is siRNA molecule End pendency, have strengthen siRNA molecule stability effect.

The antisense strand of this siRNA molecule can be combined with the mRNA of circRNA-CER gene, and degraded should MRNA, thus rear translation process is transcribed in interference, reduces the degraded of extracellular matrix in cartilaginous tissue, protects soft Osteocyte skeleton, and then the generation of suppression MMP13, reach to treat the purpose of cartilage injury.

Further, this siRNA molecule is adorned siRNA molecule, this adorned siRNA molecule Make the expression silencing of circRNA-CER gene.It will be appreciated by those skilled in the art that to be, due to siRNA The less stable of molecule, the most easily by nuclease degradation, is difficult to be absorbed by tissue.So, this Bright embodiment is by being chemically modified above-mentioned siRNA molecule, and first the siRNA molecule after being modified should When making the expression silencing of circRNA-CER gene, i.e. there is the ability of suppression circRNA-CER gene expression, Additionally, the siRNA molecule after this is modified has the stability of increase, it is easier to manipulation in vitro and internal Tissue resorption.Specifically, above-mentioned modification can be ribose modification, base modification or phosphoric acid commonly used in the art Backbone modification, or the modification of alternate manner.

Or, the nucleotide of this siRNA molecule is partially replaced or increases and decreases, and nucleotide is partially replaced or increases SiRNA molecule after subtracting makes the expression silencing of circRNA-CER gene, i.e. has suppression circRNA-CER The ability of gene expression.

Second aspect, embodiments provides any one inhibitor above-mentioned at preparation prevention or treatment bone Application in arthritis drug.Prevent or treatment osteoarthrosis by being used for preparing by any one inhibitor above-mentioned Scorching medicine, can effectively prevent or improve cartilage of osteoarthritis injury symptoms.

The third aspect, embodiments provides a kind of drug regimen for preventing or treat osteoarthritis Thing, this pharmaceutical composition includes any one inhibitor above-mentioned of therapeutically effective amount.

Containing the above-mentioned inhibitor of therapeutically effective amount in the pharmaceutical composition that the embodiment of the present invention provides, it can The expression of suppression circRNA-CER gene, suppresses the expression of MMP13 further, thus is used for treating bone Arthritis and relevant disease.

Specifically, as preferably, this pharmaceutical composition also include other medicine classes with above-mentioned inhibitor compatibility with And pharmaceutically acceptable carrier and/or adjuvant.

When aforementioned pharmaceutical compositions is used for preventing or treating osteoarthritis, it can be used alone, it is also possible to With other compatible medicine classes and pharmaceutically acceptable carrier and/or adjuvant with the use of.Wherein, made Carrier or adjuvant be the common prior art in this area.For example, this carrier can be nanometer Grain, liposome, cholesterol, chitosan and virus etc., and adjuvant can be protective agent, excipient, infiltration Pressure regulator etc..The consumption of carrier or adjuvant is had no particular limits, as long as pharmaceutical composition can be made It is able to maintain that and/or strengthens its prevention or the effect for the treatment of osteoarthritis.

Further, the dosage form of this pharmaceutical composition can be various pharmaceutical dosage forms commonly used in the art, as long as It is suitable for the administration of corresponding disease, and is properly maintained circRNA-CER gene inhibitor, especially go up State the activity of the siRNA molecule of suppression circRNA-CER gene expression.For example, drug regimen The dosage form of thing can be solution, suspension agent, Emulsion, powder (such as lyophilized powder), Controlled release formulation or hold Continuous delivery formulations.

Correspondingly, the administering mode of pharmaceutical composition can also be insecticide-applying way commonly used in the art, and citing comes Saying, it can be selected from drug administration by injection (such as, intravenous injection, articular cavity local injection or intramuscular injection etc.) Or perfusion is administered, or can also oral administration etc..

It will be appreciated by persons skilled in the art that the osteoarthritis described in the embodiment of the present invention can include Primary arthritis, secondary osteoarthritis, tissue engineering bone/cartilage or chondrocyte cell transplantation treatment joint are soft The osteoarthritis of Cranial defect generation in long term or other disease relevant to cartilage injury, the embodiment of the present invention Concrete restriction is not made at this.

Hereinafter will be further described through the present invention by specific embodiment.

In specific examples below, the unreceipted condition person of involved operation, all according to normal condition or The condition of manufacturer's suggestion is carried out.Raw materials used unreceipted production firm and specification person are can be by commercial The conventional products obtained.

Wherein, in following example, Trizol reagent is purchased from Invitrogen company；M-MLV reverse transcription Purchased from Promega company；5 × M-MLV Buffer is purchased from Promega company；Taq DNA polymerase is purchased From Promega company；RNase inhibitor (RNasin) has purchased from prestige lattice Lars biotechnology (Beijing) Limit company；DNTP is purchased from Promega company；Realtime PCR Mater Mix is purchased from TOYOBO company.

Embodiment 1

Step 1, cultured cartilage cell

Aseptically, with shears, cartilaginous tissue is shredded to less than 1cm³Size, and utilize containing dual anti- PBS rinses repeatedly.Then, utilize the pancreatin of amount of 10 times of cartilaginous tissue quality at 37 DEG C Digest half an hour, inhale and abandon enzyme liquid.Then the concentration utilizing the amount of 10 times of cartilaginous tissue quality is 0.2% (g/ml) The PBS of II Collagenase Type continues stirring digestion 4 hours at 37 DEG C, after screen filtration decontamination, Will process after cartilaginous tissue fragment plant in base area be 10cm²New culture dish in, culture fluid is low Sugar DMEM complete medium, the CO in 37 DEG C, containing 5% volumetric concentration₂The CO of saturated humidity₂Constant temperature is trained Support case to cultivate.

Step 2, utilize inflammatory factor stimulate chondrocyte

The chondrocyte obtained in step 1 is separately added into inflammatory factor: interleukin-1 (IL-1) and Tumor necrosis factor α (TNF-α), and stimulate 4,6 and 12h respectively, obtain different inflammatory cartilages thin Born of the same parents.

Step 3, the extraction of RNA of chondrocyte

In experiment, centrifuge tube used and pipettor gun head are through HIGH PRESSURE TREATMENT, use after drying；Water used is through removing RNase (ribonuclease) process (adding mass concentration is that 0.1%DEPC processes water and acutely shakes and make fully to mix, 37 DEG C of reactions, next day, high pressure removed DEPC (diethypyrocarbonate, coke diethyl phthalate)；Quality is dense Degree is the water preparation after the ethanol DEPC process of 75%.Remaining operates with reference to Trizol test kit description, Specifically comprise the following steps that

3.1, cracking chondrocyte: respectively the chondrocyte of cultivation is placed in the culture dish of 60mm specification, And in culture dish, add the Trizol reagent of 1ml respectively, after repeatedly aspirating with pipettor gun head, quiet at 4 DEG C Put 20 minutes, obtain the chondrocyte that nucleoprotein dissociates.

3.2, liquid phase separation: continuing to add 0.2ml chloroform in culture dish, shake up, room temperature stands 18 minutes After, with the relative centrifugal force 15 minutes of 12000g at 4 DEG C.

3.3, precipitation RNA: the upper water obtained in 3.1.2 is moved into mutually new pipe, and adds the isopropyl of 0.5ml Alcohol, stands 15 minutes.Then with the relative centrifugal force 10 minutes of 12000g at 4 DEG C.

3.4, discard the supernatant obtained in 3.3, and addition 1ml mass concentration is the second of 75% in precipitation Alcohol, shakes up, with the relative centrifugal force 5 minutes of 10000g at 4 DEG C.

3.5, put in fume hood through 3.4 products obtained and be dried 10 minutes, add appropriate through DEPC process After water dissolution, obtain extract RNA.

The brightness of 3.6,3 bands of 5s, 18s and 28s seen from RNA electrophoresis, and 28s band is 18s band 1 times, illustrate that extracted RNA purity is higher.

3.7, ultraviolet spectrophotometer quantitative: from the RNA extracted, draw 1 μ l, be diluted to 100 μ After l, ultraviolet spectrophotometer is utilized to measure it respectively at the absorbance (OD260) of 260nm and 280nm Absorbance (OD280) at wavelength, so detection obtain RNA concentration.Result shows, OD260/OD280 Ratio more than 1.8, the RNA that this explanation is extracted is purer, without protein and DNA remnants.By extract RNA preserves at 80 DEG C.

Step 4, reverse transcription synthesis cDNA

4.1, RT PCR (reverse transcription-PCR, reverse transcription PCR) reaction system 1

Reaction system 1, the RNA extracted including the random primer of 2 μ l, the step 3 of 2 μ g, through DEPC Water after process complements to 11 μ l.The most each composition is mix homogeneously in EP pipe, then carries out EP pipe gently Degree is centrifugal, and puts in the water-bath of 70 DEG C 10 minutes.Wherein, random primer is purchased from Promega company, Catalog number (Cat.No.) is C1181.

4.2, reverse transcription PCR reaction system 2

By the M-MLV 5 × buffer of 5 μ l, the dNTP (10Mm) of 2 μ l, the RNasin (20-40u/ml) of 0.5 μ l And 0.5 M-MLV enzyme mix homogeneously of μ l, above each composition mix homogeneously in EP pipe, the most right EP pipe carries out mild centrifugation, and puts in the water-bath of 42 DEG C 60 minutes, 10min in the water-bath of 70 DEG C. Place at 4 DEG C or store at-20 DEG C standby.

Step 5, Real-time PCR (real-time PCR) react

5.1, real-time PCR reactions system

The SYBR Green Real-time PCR Master Mix test kit using TOYOBO company exists Real-time PCR reactions is carried out on MX3005P type quantitative PCR apparatus.

The overall reaction system of 15 μ L, including cDNA template, the SYBR Green Real-time of 7.5 μ l of 1 μ l PCR Master Mix, primer 1 μ l, surplus are aquesterilisa.

Wherein, the sequence of this primer is as follows:

Forward:5'-CTGGTGCAGTGGAAGCAGAG-3',

Reverse:5'-CGACCCTCCATTGCTCTTCT-3'.

5.2, real-time PCR reactions condition

Degeneration 5 minutes at 94 DEG C, then carry out degeneration, anneal, extend circulation, and condition is at 94 DEG C 3 points Clock degeneration, at 94 DEG C 45 seconds, at 60 DEG C 30 seconds, at 72 DEG C 45 seconds, totally 40 circulations, at 72 DEG C Extend 8 minutes.This real-time PCR reactions is carried out in realtime quantitative inspection instrument.

5.3, calculate

Real-time PCR reactions terminates rear reading, calculates.With 2-Δ Δ Ct methods analyst gene expression difference.Tool Body method is as follows: measure each sample Ct (Cycle threshold) value, compares by calculating 2-Δ Δ Ct The differential expression of specific gene between different samples.The expression of detection COL2, MMP13, Aggrecan, Internal reference is GAPDH.In order to reduce systematic error and sample error, each sample all sets three parallel multiple holes, It is the PCR of genes of interest and reference gene simultaneously；These three meansigma methods Ct values as this sample answering holes, Then 2-Δ Δ Ct is calculated according to the following equation out.Test the most in triplicate, with the shape of mean+SD Formula represents experimental result in block diagram.

Wherein, Δ Ct experimental group=Ct experimental group gene-Ct experimental group internal reference

Δ Ct matched group=Ct matched group gene-Ct matched group internal reference

Δ Δ Ct=Δ Ct experimental group-Δ Ct matched group

2-Δ Δ Ct=2-(Δ Ct experimental group-Δ Ct matched group).

Experimental result is distinguished the most as depicted in figs. 1 and 2, as shown in Figure 1, along with IL-1 and TNF-α are to cartilage The prolongation of cytositimulation time, in chondrocyte, the expression of circRNA-CER gene also increases, this table Bright inflammatory factor L-1 and TNF-α can promote the expression of circRNA-CER gene.As shown in Figure 2, with The IL-1 prolongation to chondrocyte stimulation time, circRNA-CER gene in chondrocyte in chondrocyte Expression also increase, and the expression of MMP13 also increases, and the trend that both increase keeps one Causing, the expression this demonstrating circRNA-CER gene can promote the expression of MMP13.In sum, originally Case verification circRNA-CER gene can make the chondrocyte inflammatory symptoms increase the weight of, and then induce and increase the weight of bone Arthritis.Visible, that the embodiment of the present invention the provides inhibitor that can suppress circRNA-CER gene expression Prevention and/or treatment osteoarthritis are had great importance.

Embodiment 2

Step 1, cultured cartilage cell: the incubation of chondrocyte and step in embodiment 1 in the present embodiment The cultured chondrocytes process of 1 is identical.

Step 2, the in-vitro transfection of siRNA molecule

Negative control siRNA: selected negative control siRNA is the Guangzhou limited public affairs of sharp rich biotechnology The siRNA molecule of the siN05815122147-1-5 of department's product sold number.

Experiment siRNA: use siCatch^TMSiRNA design intelligent design targeting circRNA-CER gene SiRNA sequence, the siRNA sequence of circRNA-CER gene expression can be suppressed, its sequence is as follows:

Positive-sense strand (5'-3') 5 ' CCCACGCUCCUACAAUGUU dTdT 3 '

Antisense strand (3'-5') 3 ' dTdT GGGUGCGAGGAUGUUACAA 5 '

2.1, day before transfection, inoculates 2 × 10⁵The chondrocyte cultivated in individual step 1 is cultivated to 6 porocytes In plate, every hole adds the culture medium without antibiotic, enables cell density during transfection to reach 80% Degrees of fusion.

2.2, for each transfection sample, plasmid-lipo2000 mixed liquor is prepared as follows:

2.2.1, dilution experiment siRNA and negative control siRNA: utilize 250 μ l without blood serum medium Above-mentioned each siRNA of Opti-MEM dilution, mixes, incubated at room 5 minutes gently, obtains plasmid dilution Liquid.Wherein, the amount of the siRNA that every 250 μ l Opti-MEM culture medium are diluted is 100pmol.

2.2.2, dilution lipo2000: utilize 250 μ l to dilute 10 μ l without blood serum medium Opti-MEM Lipo2000, mixing incubated at room 5 minutes, obtain lipo2000 diluent gently.

2.2.3 the lipo2000 diluent that the plasmid diluent, by 2.2.1 obtained and 2.2.2 obtain mixes gently, Obtain mixed liquor incubated at room 20 minutes (solution there may be muddiness, but does not interferes with transfection).

2.2.4 the mixed liquor that, obtained by 2.2.3 adds containing the chondrocyte cultivated in steps 1 and culture fluid The each hole of culture plate in, mix gently.

2.2.5, the culture plate that 2.2.4 obtains is placed in the CO of 37 DEG C₂In incubator, cultivate 24 respectively little Time (after cultivating 4-6 hour, the culture medium in hole is removed, change complete medium), after being transfected (transfection has negative control for experiment chondrocyte (transfection has experiment siRNA) and negative control chondrocyte siRNA)。

Step 3, utilize method in the same manner as in Example 1, the chondrocyte after step 2 in-vitro transfection is depended on The secondary extraction carrying out total serum IgE, reverse transcription synthesis cDNA, real-time PCR reactions.

Experimental result is as it is shown on figure 3, in the experiment chondrocyte that provides of the present embodiment, owing to it has transfected this Invent the desired reticent siRNA molecule expressing circRNA-CER gene, effectively inhibit The expression of circRNA-CER gene, thus also inhibits the expression of MMP13 so that it is content reduces, and Correspondingly facilitate the expression of COL2 and Aggrecan so that it is content raises, and this is more conducive in cartilaginous tissue The synthesis of extracellular matrix also suppresses the degraded of this extracellular matrix.Visible, that the embodiment of the present invention provides tool The siRNA molecule having above-mentioned sequence for preventing and/or can treat the diseases such as cartilage of osteoarthritis damage.

Embodiment 3

The present embodiment utilizes Western Blot (western blotting) test experience chondrocyte and negative control In chondrocyte, the expression of each albumen, specifically comprises the following steps that

Step 1, preparation experiment chondrocyte and negative control chondrocyte, wherein this experiment chondrocyte and the moon Property comparison chondrocyte cultivation, transfection the most same as in Example 2.

Step 2, total protein of cell concentration measure and Western Blot experiment.Wherein, by from the cartilage cultivated The albumen extracted in cell is as protein sample.By using PBS to rinse the cultivation in chondrocyte Base 2 times, then in six orifice plates, every hole adds the protein lysate of the SDS that 200ul mass concentration is 2%, I.e. obtain protein sample.

2.1, total protein of cell concentration measures

The preparation of bovine serum albumin BSA standard substance and protein sample concentration measure with reference to Pierce test kit (BCA Protein Assay Reagent Kit) description, specifically comprise the following steps that

2.1.1 BSA standard substance 20ul and protein sample solution 20ul (18ul PBS+2ul, is taken Protein sample) add 96 orifice plates, each BSA standard substance and protein sample solution the most parallel repetition 3 hole.

2.1.2, A reagent in test kit is mixed by 50:1 with B reagent, obtain mixed liquor.

2.1.3, by the mixed liquor in 2.1.2 it is separately added in each BSA standard substance and protein sample solution, often Hole 200 μ l, mixing.

2.1.4,30 minutes are hatched at 37 DEG C.

2.1.5, BSA standard substance and the absorbance of protein sample solution are measured with microplate reader at 562nm.

2.1.6, making standard curve, obtain the protein concentration in protein sample, subpackage is frozen.

2.2, Western Blot experimental procedure

2.2.1, glue: install vertical electrophoresis plate, successively perfusion separation gel and concentration glue, separation gel and concentration Glue formula is as shown in table 1:

Table 1

Wherein, in above each composition, Tris-HCl (Tris (hydroxymethyl) aminomethaneTris-HCl) It it is three (methylol) aminomethane；SDS (sodium dodecyl sulfate, sodium salt) is dodecane Base sodium sulfate；TEMED (N, N, N', N'-Tetramethylethylenediamine) is N, N, N', N'-tetramethyl Base ethylenediamine.

2.2.2, sample treatment: take protein sample 20 μ g (calculating applied sample amount according to the protein concentration measured), Mix with 5 × sample-loading buffer of 1/4 applied sample amount, boil 5 minutes.

2.2.3, loading: sample and albumen pre-dyed Marker (6 μ l) are sequentially added into loading hole, every hole loading Volume all should be identical, and emptying aperture is with 1 × sample-loading buffer polishing volume.

2.2.4, electrophoresis: carry out electrophoresis concentrating glue with voltage stabilizing 80V, 30-45 minute, protect after entering separation gel Hold constant voltage 120V, 1 hour, until bromjophenol blue dye front is at the bottom of glue.

2.2.5, electricity turns: gel moves into electricity turn trough so that it is be in negative electrode, pvdf membrane (polyvinylidene fluoride film) The both sides being in anode, gel and pvdf membrane are filter paper and sponge respectively, inject electricity and turn buffering after clamping Liquid, turns 2 hours on ice with 200mA electricity.

2.2.6, observe electricity according to albumen pre-dyed Marker and turn the most complete.

2.2.7, closing: proceeded to by pvdf membrane in mass concentration 5% defatted milk powder solution, room temperature closes 1 Hour.

2.2.8, with the ratio of 1:1000, one anti-hatches pvdf membrane, 4 DEG C of overnight incubation or room temperature 3-4 hour.

2.2.9, with the ratio of 1:2000, two anti-hatch pvdf membrane: wash film with TBST lavation buffer solution, 10 minutes × 3 times；It is subsequently adding two anti-to hatch, room temperature 1.5 hours.

2.2.10, chemiluminescence: wash pvdf membrane with TBST lavation buffer solution, 10 minutes × 3 times；By ECL The A liquid of chemiluminescence agent, B liquid mix, and drip and hatch on pvdf membrane 1 minute, then use preservative film Wrapping, gel imaging system is exposed or light field exposure.

Result is as shown in Fig. 4 a, 4b, 4c and 4d, it is seen then that compared with negative control chondrocyte, the present invention Embodiment provide experiment chondrocyte in due to transfected suppression circRNA-CER gene expression siRNA divide Son, this siRNA molecule can suppress the expression of MMP13, and make its content reduce (seeing Fig. 4 a and 4c), Simultaneously facilitating the expression of COL2, and make its content raise (seeing Fig. 4 b and 4d), this is furtherly simultaneously (its positive-sense strand is (5'-3') to the siRNA molecule with following sequence of clear embodiment of the present invention offer 5‘CCCACGCUCCUACAAUGUU dTdT 3‘；Its antisense strand is (3'-5') 3 ' dTdT GGGUGCGAGGAUGUUACAA 5 ') can be effective to prevention and/or treatment osteoarthritis, can For preparing prevention and/or the medicine for the treatment of osteoarthritis.

It is only presently preferred embodiments of the present invention, not in order to limit the scope of the invention, all in the present invention Spirit and principle within, any modification, equivalent substitution and improvement etc. made, should be included in the present invention Protection domain within.

Claims (10)

1. an inhibitor, the expression of described inhibitor suppression circRNA-CER gene, described to reduce The level of the expression product of circRNA-CER gene, the nucleotide sequence of described circRNA-CER gene is such as Shown in SEQ ID NO:1.

Inhibitor the most according to claim 1, it is characterised in that described inhibitor is siRNA molecule, The sequence of the positive-sense strand of described siRNA molecule as shown in SEQ ID NO:2, described siRNA molecule anti- The sequence of justice chain is as shown in SEQ ID NO:3.

Inhibitor the most according to claim 2, it is characterised in that described siRNA molecule is for being modified SiRNA molecule, described adorned siRNA molecule make described circRNA-CER gene expression sink Silent.

Inhibitor the most according to claim 3, it is characterised in that described in be modified to ribose modify, alkali Base is modified or phosphate backbones is modified.

Inhibitor the most according to claim 2, it is characterised in that the nucleotide of described siRNA molecule Being partially replaced or increase and decrease, the siRNA molecule after nucleotide is partially replaced or increases and decreases makes described The expression silencing of circRNA-CER gene.

6. the inhibitor described in any one of claim 1-5 is in preparation prevention or treatment osteoarthritis drugs Application.

7., for preventing or treat a pharmaceutical composition for osteoarthritis, described pharmaceutical composition includes treatment The inhibitor described in any one of claim 1-5 of effective dose.

Pharmaceutical composition the most according to claim 7, it is characterised in that described pharmaceutical composition also wraps Include other medicine classes with described inhibitor compatibility and pharmaceutically acceptable carrier and/or adjuvant.

Pharmaceutical composition the most according to claim 8, it is characterised in that the agent of described pharmaceutical composition Type is selected from solution, suspension agent, Emulsion, powder, Controlled release formulation or extended release preparation.

Pharmaceutical composition the most according to claim 8 or claim 9, it is characterised in that described drug regimen The administering mode of thing is administered selected from drug administration by injection or perfusion.