CN104498498A - siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA - Google Patents
siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA Download PDFInfo
- Publication number
- CN104498498A CN104498498A CN201410827650.3A CN201410827650A CN104498498A CN 104498498 A CN104498498 A CN 104498498A CN 201410827650 A CN201410827650 A CN 201410827650A CN 104498498 A CN104498498 A CN 104498498A
- Authority
- CN
- China
- Prior art keywords
- seq
- single strand
- sirna
- rna single
- double
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and an application of the siRNA. The invention discloses a chemically-modified dual-chain siRNA molecule, and particularly relates to a dual-chain siRNA molecule which is formed by chemically modifying and supplementing (as shown in (1) to (13)) at least one chain in the siRNA molecule indicated by A. The siRNA can be used as a treatment drug for arthritis and relevant inflammations, and the siRNA and preparations of the siRNA can be locally injected through a bone joint cavity to inhibit the inflammation factor expression so as to realize the treatment for the arthritis.
Description
Technical field
The present invention relates to a kind of siRNA and the application thereof that suppress ADAM17 gene, belong to biological technical field.
Background technology
Osteoarthritis (Osteoarthritis, OA) is a kind of serious harm human health, still lacks the chronic degenerative osteoarthrosis of effective treatment means at present, in the urgent need to working out the novel method effectively preventing and treating osteoarthritis.One of osteoarthritis clinical pathologic characteristic is cartilage destruction and hFLS extracellular matrix degradation, and the destruction of cartilage finally all derives from the proteolysis of extracellular matrix.Therefore multiple protein hydrolytic enzyme activities increases and causes the degraded of secondary cartilage epimatrix (extracellular matrix, ECM) to be the immediate cause of cartilage degeneration extremely, finally causes the cartilage destruction covering articular bone surface.Il-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) have the effect promoting hFLS cell catabolism and degradation of cell epimatrix, there are some researches show IL-1 and the TNF-α finding high density in the synovial fluid of arthritic, they are considered to be in the pro-inflammatory cytokine played a crucial role in arthritic mechanism.
ADAM17 (disintegrin-metalloprotease 17) is a member of ADAMs family (adam protein), ADAMs family is the cell surface glycoprotein family that a class of discovered in recent years has several functions, their jointly participate in the various physiological processes such as being adhered of cell and cell, cell and matrix, the degraded of cytogamy, extracellular matrix and intracellular signaling, as leukocytic migration.In addition, they also take part in the pathologic processes such as tumour formation, propagation and transfer.The TNF-α of film mating type cuts off by ADAM17, produces the TNF-α of sequestered, is therefore called TNF-α converting Enzyme (TACE).The TNF-α of sequestered causes inflammation the obstacle etc. of the excessive secretion of the sexual cell factor, apoptosis, Cellular Signaling Transduction Mediated, cause various disease, comprise rheumatic arthritis (RA), systemic lupus erythematous (SLE), multiple sclerosis, acute infection disease, asthma, atopic dermatitis, psoriasis etc.ADAM17 is outside substrate divided by TNF-α, and macrophage colony stimulating factor or chemokine FKN are also by the adjustment of ADAM17.Therefore, think that the compound of suppression ADAM17 is expected to the curative as inflammatory disease.But Metalloproteinase familv high conservative, develops optionally micromolecular inhibitor and has been proved to be and has very large challenge.The previous use more test of the inhibitors of metalloproteinase of wide spectrum has been proved to be and has had tissue toxicity, and the ADAM17 inhibitor (Moss, 2008) therefore developing high selectivity is the problem needing to solve.
Daniel Craig plum Lip rivers in 1998 and Andrew Fa Er have found gene silencing phenomenon, Tuschl and his colleague find the siRNA (siRNA) of 19-25 base of chemosynthesis in mammalian cell subsequently, can the reticent said target mrna of differential high efficient.From then on siRNA is widely used in gene functional research, disease treatment.SiRNA can be special the said target mrna of same complementary combine, and make it degrade.The double-stranded RNA of long segment is cut into 21-23 bases longs short-movie section RNA by Dicer enzyme.Article two, the chain combined with said target mrna in chain is called antisense strand, and another chain is called positive-sense strand or messenger strand.The siRNA of iii vitro chemical synthesis enters and plays RNA interference effect equally after cell, and effectively reduces the immune response that long-chain RNA causes.But can design multiple siRNA for same gene different fragments position, silencing efficiency has notable difference.
Summary of the invention
The object of this invention is to provide a kind of siRNA and the application thereof that suppress ADAM17 gene.
The invention provides a kind of Double-stranded siRNA molecules of chemically modified, is by the Double-stranded siRNA molecules complementary after any chemically modified shown in following (1)-(13) of at least one chain in the siRNA molecule shown in following A:
A, a kind of siRNA molecule are following 1) or 2) shown in:
1) Double-stranded siRNA molecules that the RNA single strand shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2 is complementary;
2) RNA single strand shown in SEQ ID No.2 and the Double-stranded siRNA molecules that has the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.1;
Or, the RNA single strand shown in SEQ ID No.1 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.2;
Or, the Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and have the RNA single strand of more than 70% homology complementary with the RNA single strand shown in SEQ ID No.1;
(1) thiophosphoric acid of phosphate backbones is modified;
The P-S key that described thiophosphoric acid is modified to phosphate backbones replaces P-OH key;
(2) 2 '-methoxyl group of ribose or ribodesose is modified;
(3) 2 '-fluorine of ribose or ribodesose is modified;
(4) lock nucleic acid to modify;
Described lock nucleic acid is modified to 2 '-O position of ribose or ribodesose and 4 '-C position and forms ring texture by shrink effect;
(5) open loop nucleic acid is modified;
Described open loop nucleic acid is modified to the C-C bond rupture in ribose or ribodesose between 2 '-C and 3 '-C;
(6) indoles is modified;
Described indoles is modified to base and is substituted by indoles;
(7) 5-methylcytosine of base is modified;
(8) 5-ethinyluracil of base is modified;
(9) strand 5 ' end cholesterol is modified;
(10) strand 3 ' terminal galactose is modified;
(11) strand 5 ' terminal polypeptide is modified;
It is the polypeptide of Arg-Gly-Asp that described polypeptide is specially the sequence of holding C to hold from N;
(12) strand 5 ' terminal phosphateization is modified;
(13) strand 5 ' Terminal fluorescent labels is modified;
Described fluorescent mark is specially Cy line fluorescent mark;
Described have the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and have the complementary Double-stranded siRNA molecules of the RNA single strand of more than 70% homology to be specially the Double-stranded siRNA molecules of the single-stranded complementary shown in the strand shown in SEQ IDNo.3 and SEQ ID No.4 with the RNA single strand shown in SEQ ID No.1.
In the Double-stranded siRNA molecules of above-mentioned chemically modified, positive-sense strand and the antisense strand of Double-stranded siRNA molecules complementary after described chemically modified have the sequence shown in following B1 and C1 respectively:
B1、5'-K’-L’P’M’UCAUGUAUCUGAA P’M’M’dTdT-3’；
C1、5'-R’-Q’Q’L’UUCAGAUACAUGA Q’L’P’dTdT-3’；
Described K ' is that 5 ' end cholesterol is modified;
Described R ' is without modifying or the modification of 5 ' terminal phosphateization;
Described dT is thymine deoxyribotide;
The guanine deoxyribonucleotide that 2 '-methoxyl group that described L ', M ', P ' and Q ' are respectively ribodesose is modified, VITAMIN B4 deoxyribonucleotide, the cytosine(Cyt) deoxyribonucleotide of 2 '-methoxyl group modification of ribodesose and the uracil ribonucleotide of 2 '-methoxyl group modification of ribose that 2 '-methoxyl group of ribodesose is modified;
Or,
Described L ', M ', P ' and Q ' are respectively guanine deoxyribonucleotide, VITAMIN B4 deoxyribonucleotide, cytosine(Cyt) deoxyribonucleotide and uracil ribonucleotide;
Described antisense strand is the strand that same ADAM17 (disintegrin-metalloprotease 17) mRNA combines, and described positive-sense strand is the strand combined with described antisense strand complementation.
A kind of siRNA molecule also belongs to protection scope of the present invention, shown in following (1) or (2):
(1) Double-stranded siRNA molecules that the RNA single strand shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2 is complementary;
(2) RNA single strand shown in SEQ ID No.2 and the Double-stranded siRNA molecules that has the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.1;
Or, the RNA single strand shown in SEQ ID No.1 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.2;
Or, the Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and have the RNA single strand of more than 70% homology complementary with the RNA single strand shown in SEQ ID No.1;
Described siRNA molecule is applied to the product that preparation prevents and/or treats inflammation;
Described inflammation is specially arthritis, then is specially osteoarthritis.
Carrier containing above-mentioned siRNA molecule also belongs to protection scope of the present invention;
Described carrier is cationic-liposome, chitosan nano, polypeptide, polymer materials.
In above-mentioned siRNA molecule, the described Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and be the single-stranded complementary shown in the strand shown in SEQ ID No.3 and SEQ ID No.4 with the Double-stranded siRNA molecules that the RNA single strand shown in SEQ ID No.1 has the RNA single strand of more than 70% homology complementary.
A kind of DNA molecular that can produce above-mentioned siRNA molecule also belongs to protection scope of the present invention.
In above-mentioned DNA molecular, described siRNA molecule is the Double-stranded siRNA molecules of the RNA single strand complementation shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2;
Described DNA molecular is the DNA molecular containing 38-56 position Nucleotide from 5 ' end in SEQ ID No.5, be specially the DNA molecular containing the complementary double-stranded DNA of the DNA single chain shown in the DNA single chain shown in SEQ ID No.5 and SEQ ID No.6, sequence between BamHI and the HindIII restriction enzyme site that the molecule being specially the complementary double-stranded DNA of DNA single chain shown in the DNA single chain shown in SEQ ID No.5 and SEQ ID No.6 again replaces pGCsi-H1/Neo, the constant restructuring siRNA expression plasmid obtained of all the other sequences of pGCsi-H1/Neo.
A kind of test kit also belongs to protection scope of the present invention, and this test kit comprises the Double-stranded siRNA molecules of above-mentioned arbitrary described chemically modified, above-mentioned arbitrary described siRNA molecule and/or above-mentioned arbitrary described DNA molecular;
Described test kit is also containing the operation instruction be documented on readable carrier, and this operation instruction contents is as follows: the Double-stranded siRNA molecules that the RNA molecule that the siRNA molecule obtained through chemically modified again in the Double-stranded siRNA molecules of the above-mentioned arbitrary described chemically modified of the injection location that is inflamed or its preparation, above-mentioned arbitrary described siRNA molecule or its preparation and/or above-mentioned arbitrary described DNA molecular produce obtains through chemically modified again or its preparation.
The Double-stranded siRNA molecules of above-mentioned arbitrary described chemically modified, above-mentioned arbitrary described siRNA molecule and/or above-mentioned arbitrary described DNA molecular and/or mentioned reagent box also belong to protection scope of the present invention in the preparation application prevented and/or treated in the product of inflammation;
Described inflammation is specially arthritis, then is specially osteoarthritis.
The Double-stranded siRNA molecules of above-mentioned arbitrary described chemically modified, above-mentioned arbitrary described siRNA molecule and/or above-mentioned arbitrary described DNA molecular and/or mentioned reagent box be prepared as follows D1-D4 arbitrary shown in product in application also belong to protection scope of the present invention:
D1, the Fibrotic product of suppression articular surface;
The product of D2, suppression cartilage erosion;
D3, prevent and/or treat the product of synovitis;
The product of D4, protection cartilage and/or synovial membrane.
The Double-stranded siRNA molecules of above-mentioned arbitrary described chemically modified, above-mentioned arbitrary described siRNA molecule and/or above-mentioned arbitrary described DNA molecular and/or mentioned reagent box be prepared as follows E1-E6 arbitrary shown in product in application also belong to protection scope of the present invention:
E1, prevent and/or treat the product of rheumatoid arthritis;
E2, prevent and/or treat the product of systemic lupus erythematous;
E3, prevent and/or treat the product of multiple sclerosis;
E4, prevent and/or treat the product of acute infection disease;
E5, prevent and/or treat the product of atopic dermatitis;
E6, prevent and/or treat psoriasic product.
SiRNA provided by the invention as the medicine of sacroiliitis and related inflammation, can realize the treatment to arthritis by osteoarthrosis chamber local injection siRNA or its preparation inflammation-inhibiting factor expression.
Accompanying drawing explanation
Fig. 1 is the effective siRNA screening suppressing ADAM17 gene.
Fig. 2 is siRNA-AD-08 immunoblot experiment.
Fig. 3 is that siRNA-AD-08 lowers inflammatory factor.
Fig. 4 is the impact of siRNA structure on target gene silencing efficiency.
Fig. 5 is the modification position view of thiophosphoric acid (P-S key).
Fig. 6 is that chemically modified strengthens nucleic acid oligomer serum stability.
Fig. 7 is rat tissue's pathological section analysis.
Fig. 8 is rat articular liquid Inflammatory Factors Contents.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
HFLS cell (human desmocyte sample synovial cell) is Cell Applications product, and catalog number is 408-05a.
293T cell is ATCC product, and catalog number is CRL-3216.
MCF-7 cell is ATCC product, and catalog number is HTB-22.
Human IL-1 β immunoassay detection kit is AssayPro product, and catalog number is EI2200-1.
PGCsi-H1/Neo document " Ji Guozhong; Zhang Faming; the structure of the .Smad4/DPC4 gene children purpura nephritis plasmid expression vectors such as Huang Shu and qualification [J]. medical graduate students journal; 2006; 19 (11): 973-977 " in be disclosed, the public can obtain from Guangzhou Ribo Bio Co., Ltd..
Lipofectamine2000 test kit is Invitrogen product.
Male SD rat (220 ± 20g) is Guangdong Medical Lab Animal Center's product.
SiRNA in following embodiment is Double-stranded siRNA molecules, and the solvent of each injection liquid of injection rat is PBS.
The screening of effective nucleic acid oligomer of embodiment 1, suppression ADAM17 gene mRNA expression
One, carry out siRNA design to determine that target is in the siRNA of ADAM17, and carry out bioinformation screening, guarantee that sequence is specific for ADAM17 sequence and is not specific for the sequence from any other gene.The blast search engine that target sequence uses NCBI to provide is checked relative to the sequence in GenBank, 8 effective siRNA are filtered out, respectively called after siRNA-AD-01, siRNA-AD-02, siRNA-AD-03, siRNA-AD-04, siRNA-AD-05, siRNA-AD-06, siRNA-AD-07, siRNA-AD-08 through preliminary experiment.Above siRNA is the siRNA for the design of ADAM17 gene order different positions.
Two, cell transfecting
Experiment is divided into 10 groups, and be respectively siRNA-AD-01 to siRNA-AD-08 experimental group, Notarget (NTC) is negative control group, NC is blank group.
The method to set up of siRNA-AD-01 to siRNA-AD-08 experimental group is as follows:
By hFLS cell with 0.25% pancreatin digest, making concentration with DMEM substratum is 1 × 10
4the cell suspension of individual/ml, be inoculated in 12 well culture plates, every hole 500ul, when hFLS Growth of Cells is to logarithmic phase (i.e. growth reach 80% fusion in blocks), according to the specification sheets of Lipofectamine2000 test kit, by the final concentration transfection hFLS cell of the siRNA of each correspondence according to 50nM.
No target (NTC) negative control group: the siRNA of experimental group is replaced with random non-specific siRNA, and all the other steps are constant.Wherein, random non-specific siRNA is not the special siRNA be directed to designed by target gene (ADAM17 gene):
Positive-sense strand: 5 '-AGUAUGCCACAUAAGCAUC dTdT-3 ';
Antisense strand: 5 '-GAUGCUUAUGUGGCAUACU dTdT-3 '.
NC blank group: do not add siRNA, all the other steps are consistent with experimental group.
Three, after transfection 24h, collect each group of hFLS cell, in 1000rpm centrifugal 5 minutes, remove supernatant, Trizol method extracts the RNA of each group.
Four, be cDNA by the RNA reverse transcription of each group, with the cDNA of each group for template, with ADAM17-F1 and ADAM17-R1 for primer, carry out real-time fluorescence quantitative PCR, detected result as shown in Figure 1, with β-actin for reference gene.
ADAM17-F1:5’-GGACCAGGGAGGGAAATA-3’
ADAM17-R1:3’-TTGCTGTGGACGACGTTG-5’
Fig. 1 shows, in 8 effective siRNA that early stage, screening obtained, the silencing efficiency of siRNA-AD-08 to ADAM17 is best, inhibits the gene expression amount of 86%.
Wherein the sequence of siRNA-AD-08 positive-sense strand is as shown in SEQ ID No.1, and the sequence of antisense strand is as shown in SEQ ID No.2.
SiRNA-AD-08 positive-sense strand: 5'-GCAUCAUGUAUCUGAACAA-3 ' (SEQ ID No.1)
SiRNA-AD-08 antisense strand: 5'-UUGUUCAGAUACAUGAUGC-3'(SEQ ID No.2)
Five, Western blot detects
Get the hFLS cell of siRNA-AD-08 experimental group, discard cell culture fluid, with PBS washed cell 2 times, outwell PBS, add 2 × Lysis Buffer of appropriate precooling, scrape with cell and cell is scraped, be placed in abundant lysing cell 30min on ice, in refrigerated centrifuge 4 DEG C, 12000g, centrifugal 15min, get supernatant liquor, measure protein concentration by Bradford method, finally the final concentration of sample protein is all adjusted to 2 μ g/ μ l, for subsequent use in-80 DEG C of Refrigerator stores.Get the sample of 12 μ g total protein concentrations respectively, add isopyknic 2X loading buffer sample-loading buffer.After the two fully mixing, in boiling water, boil bath 10 minutes, deposit for subsequent use for 4 DEG C.According to the glue (the SDS-PAGE separation gel of 10% and the concentrated glue of 5%) of target protein molecular size range preparation respective concentration, after preparing in glue, comb is taken out rear electrophoresis buffer solution for cleaning loading hole, to ready sample loading before, every hole adds protein sample, carries out electrophoresis.After electrophoresis terminates, use electrophoretic blotting device, at 4 DEG C, under 400mA constant current conditions, electricity turns 2 hours, by protein delivery on pvdf membrane.Carry out subsequently developing the color and exposure analysis.
Carry out above-mentioned experiment in contrast with NC blank group and No target (NTC) negative control group simultaneously.Result as shown in Figure 2.
In Fig. 2, Control is NC blank group, and no target is No target (NTC) negative control group, and siRNA is siRNA-AD-08 experimental group.
Fig. 2 shows, siRNA-AD-08 significantly suppress the protein expression of ADAM17, and the follow-up siRNA-AD-08 of selecting is further analyzed.
Embodiment 2, nucleic acid oligomer are to the suppression of inflammatory factor
One, experiment is divided into following each group:
HFLS-siRNA-AD-08 experimental group: original cuiture hFLS cell to 6 orifice plate, when cell density about 50%, according to Lipofectamine2000 test kit specification sheets, by the final concentration transfection hFLS cell of siRNA-AD-08 according to 50nM.
MCF-7-siRNA-AD-08 experimental group: original cuiture MCF-7 cell to 6 orifice plate, when cell density about 50%, according to Lipofectamine2000 test kit specification sheets, by the final concentration transfection MCF-7 cell of siRNA-AD-08 according to 50nM.
HFLS-No target (NTC) negative control group: the siRNA of hFLS-siRNA-AD-08 experimental group is replaced with random non-specific siRNA, and all the other steps are constant.Wherein, random non-specific siRNA is not the special siRNA be directed to designed by target gene (ADAM17 gene):
Positive-sense strand: 5 '-AGUAUGCCACAUAAGCAUC dTdT-3 ';
Antisense strand: 5 '-GAUGCUUAUGUGGCAUACU dTdT-3 '.
MCF-7-No target (NTC) negative control group: the siRNA of MCF-7-siRNA-AD-08 experimental group is replaced with random non-specific siRNA, and all the other steps are constant.Wherein, random non-specific siRNA is not the special siRNA be directed to designed by target gene (ADAM17 gene):
Positive-sense strand: 5 '-AGUAUGCCACAUAAGCAUC dTdT-3 ';
Antisense strand: 5 '-GAUGCUUAUGUGGCAUACU dTdT-3 '.
HFLS-NC blank group: do not add siRNA in hFLS-siRNA-AD-08 experimental group, all the other steps are consistent with hFLS-siRNA-AD-08 experimental group.
MCF-7-NC blank group: do not add siRNA in MCF-7-siRNA-AD-08 experimental group, all the other steps are consistent with MCF-7-siRNA-AD-08 experimental group.
Two, transfection is after 24 hours, changes non-serum starved cultivation each group of cell 24 hours.
Three, in each group of cell, add IL 1-α, make its final concentration be 10ng/ml, stimulate 24 hours.
Four, the RNA of each group of cell is extracted and reverse transcription is cDNA, with the cDNA of each group for template, respectively with TNF-F and TNF-R for primer, with cox2-F and cox2-R for primer, with IL-1 β-F and IL-1 β-R for primer, carry out real-time fluorescence quantitative PCR, the expression level of corresponding detection TNF, COX-2 and IL-1 β gene, with β-actin for reference gene.
TNF-F:5’-CGAGTGACAAGCCTGTAGCC-3’；
TNF-R:5’-TGAAGAGGACCTGGGAGTAGAT-3’。
cox2-F：5'-CAGGGTTGCTGGTGGTAGGA-3'；
cox2-R：5'-GCATAAAGCGTTTGCGGTAC-3'。
IL-1β-F：5'-ACGAATCTCCGACCACCA-3'；
IL-1β-R：5'-GGACCAGACATCACCAAGC-3'。
Result is as shown in A in Fig. 3.
What the NTC group in Fig. 3 A all represented is the NTC group adding above-mentioned IL 1-α in step afterwards.
Collect the supernatant liquor of each group of cell, utilize Human IL-1 β immunoassay detection kit to detect the secretion level of each group of cell IL-1 β.
Wherein, above-mentioned NTC group establishes again following each group respectively:
HFLS-No target (NTC+) negative control group: hFLS-No target (NTC) negative control group adds above-mentioned IL 1-α in step afterwards.
HFLS-No target (NTC-) negative control group: hFLS-No target (NTC) negative control group does not add above-mentioned IL 1-α in step afterwards.
MCF-7-No target (NTC+) negative control group: MCF-7-No target (NTC) negative control group adds above-mentioned IL 1-α in step afterwards.
MCF-7-No target (NTC-) negative control group: MCF-7-No target (NTC) negative control group does not add above-mentioned IL 1-α in step afterwards.
Result is as shown in B in Fig. 3.
Fig. 3 shows, respectively compared with NTC or NTC+, siRNA-AD-08 all effectively can suppress the gene expression amount of COX-2 and IL-1 β inflammatory factor in MCF-7 and hFLS cell, and suppresses the secretion of IL-1 β, wherein in hFLS cell, reaches 88% to the gene inhibition rate of IL-1 β.In MCF-7 cell, after transfection siRNA-AD-08, the gene expression amount of TNF is higher than NTC group, may be to be caused by the cell function more complicated of TNF.
Embodiment 3, homooligomeric nucleic acid are to the checking of ADAM17 gene inhibition effect
For the impact that checking homology ratio presses down the ADAM17 potency of gene to siRNA-AD-08, carry out following three groups of experiments:
One, first group of experiment
The siRNA antisense strand of first group is " 5'-UUGUUCAGAUACAUGAUGC-3' "
, positive-sense strand is the homologous sequence of " 5'-GCAUCAUGUAUCUGAACAA-3' ", as shown in table 1.
Table 1 antisense strand group
Note: S=positive-sense strand, AS=antisense strand.Positive-sense strand selects 11nt, 15nt, 23nt, 27nt, mispairing respectively.
According to the method for embodiment 1 by each siRNA transfection hFLS cell shown in table 1, and detect its suppression efficiency to ADAM17 gene mRNA expression.
Two, second group of experiment
The siRNA positive-sense strand of second group is " 5'-GCAUCAUGUAUCUGAACAA-3' ", and antisense strand is the homologous sequence of " 3'-CGUAGUACAUAGACUUGUU-5' ", as shown in table 2.
Table 2 positive-sense strand group
Note: S=positive-sense strand, AS=antisense strand.
According to the method for embodiment 1 by each siRNA transfected hFLS cell shown in table 2, detect its suppression efficiency to ADAM17 gene mRNA expression.
Three, the 3rd group of experiment
The siRNA positive-sense strand of the 3rd group and antisense strand are the combination of above two groups, as shown in table 3.
Table 3 combination group
Note: S=positive-sense strand, AS=antisense strand.
According to the method for embodiment 1 by each siRNA transfected hFLS cell shown in table 3, detect its suppression efficiency to ADAM17 gene mRNA expression.
Each group experiment arranges No target (NTC) negative control group and NC blank group according to the method for embodiment 1 above.
Result as shown in Figure 4.
Fig. 4 shows, the siRNA of three groups of designs all serves the effect of the mrna expression of silencing of target genes ADAM17, the RNA single strand shown in SEQ ID No.2 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.1; Or, the RNA single strand shown in SEQ ID No.1 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.2; Or, there is the RNA single strand of more than 70% homology with the RNA single strand shown in SEQID No.1 and have the complementary Double-stranded siRNA molecules of the RNA single strand of more than 70% homology all can disturb the expression of ADAM17 gene with the RNA single strand shown in SEQ ID No.2.The siRNA-AD-13 interference effect that wherein with the addition of the 21nt hanging base is best, and it is 88% to the suppression efficiency of ADAM17 gene mRNA expression; Only the siRNA-AD-20 interference effect of 11nt complementation is the poorest, and it is 20% to the suppression efficiency of ADAM17 gene mRNA expression, but also serves the effect of interference target gene ADAM17 expression.
Embodiment 4, plasmid target gene silencing efficiency affect
One, according to ADAM17 full length sequence, design is containing the Double stranded oligonucleotide acid sequence of siRNA-AD-08 sequence, as shown in table 4.
Table 4 is containing the double-strand of siRNA-AD-08 sequence
Note: the dashed part of SEQ ID No.5 and SEQ ID No.6 is base complementrity region, in SEQ ID No.5, from 5 ' end, 38-56 position Nucleotide is the DNA sequence dna of RNA single strand (siRNA-AD-08 antisense strand) correspondence shown in SEQ ID No.2.In SEQ ID No.6, from 5 ' end, 8-26 position Nucleotide is the DNA sequence dna of RNA single strand (siRNA-AD-08 positive-sense strand) correspondence shown in SEQ ID No.1.
Two, the oligonucleotide of his-and-hers watches 4 forms double-strand after annealing, sequence between BamHI and the HindIII restriction enzyme site of replacement siRNA expression plasmid pGCsi-H1/Neo carrier, all the other sequences remain unchanged, obtain interference fragment expression vector (restructuring siRNA expression plasmid), send order-checking by interference fragment expression vector, result is correct.
Three, experiment is divided into following each group:
Experimental group: one day before infection, hFLS cell good for growth conditions is inoculated in 6 orifice plates and carries out transfection, according to the specification sheets of Lipofectamine2000 test kit, by the final concentration transfection of interference fragment expression vector according to 50nM, 48h collecting cell after transfection.Its suppression efficiency to ADAM17 gene mRNA expression is detected according to the method for step 4 in embodiment 1.
No target (NTC) negative control group: the siRNA of experimental group is replaced with unrelated sequences:
Positive-sense strand: 5 '-AGUAUGCCACAUAAGCAUC dTdT-3 ';
Antisense strand: 5 '-GAUGCUUAUGUGGCAUACU dTdT-3 '.
All the other steps are constant.
NC blank group: do not add interference fragment expression vector, all the other steps are consistent with experimental group.
Result is as shown in table 5
The relative expression quantity of ADAM17 gene mRNA respectively organized by table 5
Table 5 shows, with the DNA transfectional cell of transcribing siRNA-AD-08 sequence, can disturb the expression of target gene ADAM17mRNA equally.
Embodiment 5, chemically modified are on the impact of ADAM17 inhibition
Different chemically modifieds and combination modification thereof are carried out to siRNA-AD-13, to improve siRNA stability, promotes interference effect.Chemically modified comprises the halogen of ribose and modifies (2 '-F modifies), methoxyl group modification (2 '-OMe), thio-modification, cholesterol modification etc., and modification kind is as shown in table 6, and the sequence after modification is as shown in table 7.
Kind modified by table 6
In table 6, the modification position of thiophosphoric acid (P-S key) as shown in Figure 5, lock nucleic acid (LNA) is modified to 2 '-O position of ribose and 4 '-C position and forms ring texture by shrink effect, polypeptide is that (sequence of holding C to hold from N is Arg-Gly-Asp to RGD, for sigma product), A represents certain Nucleotide.
Table 7 chemically modified is on the impact of siRNA silencing efficiency
Note: S=positive-sense strand, AS=antisense strand.
According to the method for embodiment 1 by each siRNA transfected hFLS cell shown in table 7, and detect its suppression efficiency to ADAM17 gene mRNA expression.When wherein carrying out transfection with the siRNA that cholesterol, polypeptide, semi-lactosi are modified, do not add transfection reagent and directly carry out transfection.
Result is as shown in table 7.
Table 7 shows, the siRNA-AD-13 modifier obtained after all kinds of suitable chemically modified all serves the effect that reticent goal gene ADAM17 expresses.
Embodiment 6, chemically modified are on the impact of nucleic acid oligomer serum stability
Carry out serum stability detection to some chemically modified nucleic acid molecule of embodiment 5, step is as follows:
Each siRNA molecule is added after being diluted to 5 μMs without RNA enzyme water isopyknic fresh rat serum (for Yuan Mu bio tech ltd, Shanghai product), then hatch 30 minutes at 37 DEG C, the integrity of the different siRNA of electrophoresis observation is carried out in sampling.
Result as shown in Figure 6.
Fig. 6 shows, unmodified siRNA-AD-13 obvious degradation after 30 minutes, and modification of nucleic acids siRNA-AD-26, siRNA-AD-39, siRNA-AD-40 in 30 minutes without obviously decomposing.
Embodiment 7, osteoarthritis rat pathological section are tested
One, the structure of inflammatory model rat
Arthritic formation is promoted as inductor with ox Type Ⅱ collagen.Ox Type Ⅱ collagen (Sigma product) is expelled to the male SD rat joint cavity of 220 ± 20g, ox Type Ⅱ collagen concentration is 4mg/mL, disposablely gives 200 μ L, 100 μ L/ legs.
Two, following each group is arranged:
After injection ox Type Ⅱ collagen 3d, inflammatory model rat is divided into 2 groups at random, often organizes 8.One group is PBS group, and one group is siRNA-AD-26 experimental group.The wherein siRNA-AD-26 solution of siRNA-AD-26 experimental group every rat injection 10nmol/ leg at every turn, volume injected 100 μ L, 50 μ L/ legs, PBS group at every turn every rat injects isopyknic PBS, often organize equal Per-Hop behavior 2 times, successive administration 2 weeks, administration time is consistent.
Three, put to death animal respectively at second day after the 2nd, 4 administration process to draw materials, cut off skin, getting knee joint is soaked in tissue preserration liquid, through fixing, decalcification, paraffin embedding, section, Hematoxylin-eosin (HE) and toluidine blue (TB) dyeing, examine under a microscope histopathology performance, result as shown in Figure 7.
In Fig. 7, siRNA group is siRNA-AD-26 experimental group.1W and 2W represents the modeling of inflammatory model rat after one week and two weeks respectively.
Fig. 7 shows, the modeling of inflammatory model rat is after one week, and PBS group occurs meniscal fibrosis and ossify, few fibersization tissue invades cartilage layers, chondrocyte's arrangement disorder, collagenous portion runs off, and obvious fibrosis and inflammatory cell have also appearred in intraarticular reticular tissue; SiRNA-AD-26 experimental group articulum is level and smooth, and cartilage layers cell arrangement is orderly, and only local occurs that the ossified sex change of slight chondrocyte and extracellular collagen run off.Modeling is after two weeks, the hamartoplasia of PBS group articulum multifilament, a large amount of fibrosed tissue in joint cavity, and meniscus ossify and covers multi-layer fiber hyperplastic tissue, and part cartilage layers is ossify and occurred fragmentation, the sex change of cartilage layers cell disorder, and collagen runs off in a large number; Although siRNA-AD-26 experimental group meniscus occurs ossify and fibroplasia in local, joint and meniscus still keep normal morphology, and articulum is level and smooth, and cartilage layers cell is normal, and all obviously comparatively PBS group is slight for every extent of damage.
Result shows, siRNA-AD-26 can suppress the disease process of the rat suffering from osteoarthritis, can be used as the arthritis treatment medicine of potential improvement disease.
The detection of embodiment 8, rat articular liquid Inflammatory Factors Contents
One, according to method establishment PBS group, siRNA-AD-26 experimental group, siRNA-AD-39 experimental group, the siRNA-AD-40 experimental group of embodiment 7.Wherein, siRNA-AD-26 is only replaced with siRNA-AD-40, siRNA-AD-39 by siRNA-AD-40 experimental group, siRNA-AD-39 experimental group, and wherein siRNA-AD-40 is wrapped up by chitosan nano, and all the other steps are identical.
Two, within second day after the 4th administration process, put to death animal to draw materials: after peeling off skin and organizing, get knee joint, in the mortar pouring liquid nitrogen into, fully be ground to osseous tissue powdered, according to Rneasy Mini kit (for Guangzhou Ji Taixin unravels silk bio tech ltd's product, catalog number is 217004) specification sheets, extracting RNA reverse transcription is cDNA.With each cDNA for template, according to step 4 in embodiment 1, detect the relative expression quantity of ADAM17 gene, detect the relative expression quantity of TNF, COX-2 and IL-1 β gene according to the method for step 4 in embodiment 2.
Carry out above-mentioned experiment with healthy male SD rat for contrasting simultaneously.
The relative expression quantity statistics of ADAM17 and inflammatory factor as shown in Figure 8.
In Fig. 8, Normal is healthy male SD rat group, Model is PBS group.
Fig. 8 shows; compared with healthy male SD rat group; in PBS group, inflammation-related gene ADAM17, TNF, COX-2, IL-1 β expresses and raises; and siRNA-AD-26, siRNA-AD-39, siRNA-AD-40 significantly can lower the expression of ADAM17, TNF, COX-2, IL-1 β in the different process of rat inflammation disease; serve protection cartilage; improve the effect of inflammation, show that siRNA molecule of the present invention is the potential medicine preventing or treat inflammation.
Embodiment 9, cell proliferation experiment
SiRNA group: the hFLS cell DMEM nutrient solution substratum of 10% foetal calf serum is mixed with 4 × 10
4the concentration of individual/ml, it is added to 96 orifice plates with the amount in 100ul/ hole, according to the specification sheets of CCK-8 test kit (for Yeasen Products), be each porocyte of amount transfection of 50nM with final concentration by siRNA-AD-13, change serum-free after transfection 24h into and do synchronization without dual anti-DMEM substratum, serum-free is without changing perfect medium into after dual anti-DMEM culture medium culturing 24h and adding that IL-1a stimulates (with incite inflammation) in partial hole, another part hole does not add, and IL-1a uses the proliferative conditions of CCK-8 kit detection cell after stimulating 48h and 72h.
NTC group: the siRNA-AD-13 in above-mentioned siRNA group is replaced with random non-specific siRNA, and all the other steps are constant.Wherein, random non-specific siRNA is not the special siRNA be directed to designed by target gene (ADAM17 gene):
Positive-sense strand: 5 '-AGUAUGCCACAUAAGCAUC dTdT-3 ';
Antisense strand: 5 '-GAUGCUUAUGUGGCAUACU dTdT-3 '.
Result is as shown in table 8.
Cell proliferative conditions after table 8 siRNA disturbs
(in table 8, IL-1a+ representative adds IL-1a stimulates, and IL-1a-representative does not add IL-1a to stimulate)
Result shows, compared with NTC group, the quantity adding the cell of siRNA-AD-13 has slight propagation, shows siRNA-AD-13 no cytotoxicity of the present invention, and may have the effect of repair cell.
Claims (10)
1. a Double-stranded siRNA molecules for chemically modified is by the Double-stranded siRNA molecules complementary after any chemically modified shown in following (1)-(13) of at least one chain in the siRNA molecule shown in following A:
A, a kind of siRNA molecule are following 1) or 2) shown in:
1) Double-stranded siRNA molecules that the RNA single strand shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2 is complementary;
2) RNA single strand shown in SEQ ID No.2 and the Double-stranded siRNA molecules that has the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.1;
Or, the RNA single strand shown in SEQ ID No.1 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.2;
Or, the Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and have the RNA single strand of more than 70% homology complementary with the RNA single strand shown in SEQ ID No.1;
(1) thiophosphoric acid of phosphate backbones is modified;
(2) 2 '-methoxyl group of ribose or ribodesose is modified;
(3) 2 '-fluorine of ribose or ribodesose is modified;
(4) lock nucleic acid to modify;
(5) open loop nucleic acid is modified;
(6) indoles is modified;
(7) 5-methylcytosine of base is modified;
(8) 5-ethinyluracil of base is modified;
(9) strand 5 ' end cholesterol is modified;
(10) strand 3 ' terminal galactose is modified;
(11) strand 5 ' terminal polypeptide is modified;
(12) strand 5 ' terminal phosphateization is modified;
(13) strand 5 ' Terminal fluorescent labels is modified.
2. the Double-stranded siRNA molecules of chemically modified according to claim 1, is characterized in that: positive-sense strand and the antisense strand of Double-stranded siRNA molecules complementary after described chemically modified have the sequence shown in following B1 and C1 respectively:
B1、5'-K’-L’P’M’UCAUGUAUCUGAA P’M’M’dTdT-3’；
C1、5'-R’-Q’Q’L’UUCAGAUACAUGA Q’L’P’dTdT-3’；
Described K ' is that 5 ' end cholesterol is modified;
Described R ' is without modifying or the modification of 5 ' terminal phosphateization;
Described dT is thymine deoxyribotide;
The guanine deoxyribonucleotide that 2 '-methoxyl group that described L ', M ', P ' and Q ' are respectively ribodesose is modified, VITAMIN B4 deoxyribonucleotide, the cytosine(Cyt) deoxyribonucleotide of 2 '-methoxyl group modification of ribodesose and the uracil ribonucleotide of 2 '-methoxyl group modification of ribose that 2 '-methoxyl group of ribodesose is modified;
Or,
Described L ', M ', P ' and Q ' are respectively guanine deoxyribonucleotide, VITAMIN B4 deoxyribonucleotide, cytosine(Cyt) deoxyribonucleotide and uracil ribonucleotide.
3. a siRNA molecule, shown in following (1) or (2):
(1) Double-stranded siRNA molecules that the RNA single strand shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2 is complementary;
(2) RNA single strand shown in SEQ ID No.2 and the Double-stranded siRNA molecules that has the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.1;
Or, the RNA single strand shown in SEQ ID No.1 and the Double-stranded siRNA molecules having the RNA single strand of more than 60% homology complementary with the RNA single strand shown in SEQ ID No.2;
Or, the Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and have the RNA single strand of more than 70% homology complementary with the RNA single strand shown in SEQ ID No.1.
4. siRNA molecule according to claim 3, is characterized in that: the described Double-stranded siRNA molecules having the RNA single strand of more than 70% homology with the RNA single strand shown in SEQ ID No.2 and be the single-stranded complementary shown in the strand shown in SEQ ID No.3 and SEQ ID No.4 with the Double-stranded siRNA molecules that the RNA single strand shown in SEQ ID No.1 has the RNA single strand of more than 70% homology complementary.
5. one kind can produce the DNA molecular of siRNA molecule according to claim 3.
6. DNA molecular according to claim 5, is characterized in that: described siRNA molecule is the Double-stranded siRNA molecules of the RNA single strand complementation shown in the RNA single strand shown in SEQ ID No.1 and SEQ ID No.2;
Described DNA molecular is the DNA molecular containing 38-56 position Nucleotide from 5 ' end in SEQ ID No.5, be specially the DNA molecular containing the complementary double-stranded DNA of the DNA single chain shown in the DNA single chain shown in SEQ ID No.5 and SEQ ID No.6, sequence between BamHI and the HindIII restriction enzyme site that the molecule being specially the complementary double-stranded DNA of DNA single chain shown in the DNA single chain shown in SEQ ID No.5 and SEQ ID No.6 again replaces pGCsi-H1/Neo, the constant restructuring siRNA expression plasmid obtained of all the other sequences of pGCsi-H1/Neo.
7. a test kit, this test kit comprises the Double-stranded siRNA molecules of the chemically modified described in claim 1 or 2, the siRNA molecule described in claim 3 or 4 and/or the DNA molecular described in claim 5 or 6.
8. the Double-stranded siRNA molecules of the chemically modified described in claim 1 or 2, the siRNA molecule described in claim 3 or 4 and/or the DNA molecular described in claim 5 or 6 and/or test kit according to claim 7 are preparing the application prevented and/or treated in the product of inflammation;
Described inflammation is specially arthritis, then is specially osteoarthritis.
9. the Double-stranded siRNA molecules of the chemically modified described in claim 1 or 2, the siRNA molecule described in claim 3 or 4 and/or the DNA molecular described in claim 5 or 6 and/or test kit according to claim 7 be prepared as follows D1-D4 arbitrary shown in product in application:
D1, the Fibrotic product of suppression articular surface;
The product of D2, suppression cartilage erosion;
D3, prevent and/or treat the product of synovitis;
The product of D4, protection cartilage and/or synovial membrane.
10. the Double-stranded siRNA molecules of the chemically modified described in claim 1 or 2, the siRNA molecule described in claim 3 or 4 and/or the DNA molecular described in claim 5 or 6 and/or test kit according to claim 7 be prepared as follows E1-E6 arbitrary shown in product in application:
E1, prevent and/or treat the product of rheumatoid arthritis;
E2, prevent and/or treat the product of systemic lupus erythematous;
E3, prevent and/or treat the product of multiple sclerosis;
E4, prevent and/or treat the product of acute infection disease;
E5, prevent and/or treat the product of atopic dermatitis;
E6, prevent and/or treat psoriasic product.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410827650.3A CN104498498A (en) | 2014-12-25 | 2014-12-25 | siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA |
| JP2017552533A JP6706628B2 (en) | 2014-12-25 | 2015-12-23 | Composition for suppressing expression of ADAMTS-5 or ADAMTS and method thereof |
| US15/539,671 US10709729B2 (en) | 2014-12-25 | 2015-12-23 | Compositions and methods for inhibiting expression of ADAMTS-5 and ADAM17 |
| PCT/IB2015/002574 WO2016103042A1 (en) | 2014-12-25 | 2015-12-23 | Compositions and methods for inhibiting expression of adamts-5 and adam17 |
| DK15872041.7T DK3237619T3 (en) | 2014-12-25 | 2015-12-23 | COMPOSITIONS AND PROCEDURES TO INHIBIT EXPRESSION OF ADAMTS-5 AND ADAM17 |
| ES15872041T ES2833028T3 (en) | 2014-12-25 | 2015-12-23 | Compositions and methods to inhibit the expression of ADAMTS-5 and ADAM17 |
| EP15872041.7A EP3237619B8 (en) | 2014-12-25 | 2015-12-23 | Compositions and methods for inhibiting expression of adamts-5 and adam17 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410827650.3A CN104498498A (en) | 2014-12-25 | 2014-12-25 | siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104498498A true CN104498498A (en) | 2015-04-08 |
Family
ID=52939950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410827650.3A Pending CN104498498A (en) | 2014-12-25 | 2014-12-25 | siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104498498A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016103042A1 (en) * | 2014-12-25 | 2016-06-30 | Guangzhou Ribobio Co., Ltd. | Compositions and methods for inhibiting expression of adamts-5 and adam17 |
| CN113004390A (en) * | 2019-12-20 | 2021-06-22 | 中国科学院动物研究所 | Application of ADAM17 as receptor of hog cholera virus |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090035225A1 (en) * | 2007-08-03 | 2009-02-05 | Alcon Research, Ltd. | RNAi-RELATED INHIBITION OF TNFa SIGNALING PATHWAY FOR TREATMENT OF GLAUCOMA |
| TW200916117A (en) * | 2007-08-03 | 2009-04-16 | Alcon Res Ltd | RNAi-related inhibition of TNF α signaling pathway for treatment of ocular angiogenesis |
| CN101448943A (en) * | 2006-05-19 | 2009-06-03 | 爱尔康研究有限公司 | RNAi-mediated inhibition of tumor necrosis factor alpha-related conditions |
-
2014
- 2014-12-25 CN CN201410827650.3A patent/CN104498498A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101448943A (en) * | 2006-05-19 | 2009-06-03 | 爱尔康研究有限公司 | RNAi-mediated inhibition of tumor necrosis factor alpha-related conditions |
| CN103285026A (en) * | 2006-05-19 | 2013-09-11 | 爱尔康研究有限公司 | RNAi-mediated inhibition of tumor necrosis factor alpha-related conditions |
| US20090035225A1 (en) * | 2007-08-03 | 2009-02-05 | Alcon Research, Ltd. | RNAi-RELATED INHIBITION OF TNFa SIGNALING PATHWAY FOR TREATMENT OF GLAUCOMA |
| TW200916117A (en) * | 2007-08-03 | 2009-04-16 | Alcon Res Ltd | RNAi-related inhibition of TNF α signaling pathway for treatment of ocular angiogenesis |
Non-Patent Citations (3)
| Title |
|---|
| BRIAN A. JONES ET AL.: "Role of ADAM-17, p38 MAPK, Cathepsins, and the Proteasome Pathway in the Synthesis and Shedding of Fractalkine/CX3CL1 in Rheumatoid Arthritis", 《ARTHRITIS & RHEUMATISM》 * |
| JULIO C. FERNANDES ET AL.: "The role of cytokines in osteoarthritis pathophysiology", 《BIORHEOLOGY》 * |
| XIANGYU CHU ET AL.: "Protective effect of lentivirus-mediated siRNA targeting ADAMTS-5 on cartilage degradation in a rat model of osteoarthritis", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016103042A1 (en) * | 2014-12-25 | 2016-06-30 | Guangzhou Ribobio Co., Ltd. | Compositions and methods for inhibiting expression of adamts-5 and adam17 |
| US10709729B2 (en) | 2014-12-25 | 2020-07-14 | Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences | Compositions and methods for inhibiting expression of ADAMTS-5 and ADAM17 |
| CN113004390A (en) * | 2019-12-20 | 2021-06-22 | 中国科学院动物研究所 | Application of ADAM17 as receptor of hog cholera virus |
| CN113004390B (en) * | 2019-12-20 | 2022-08-26 | 中国科学院动物研究所 | Application of ADAM17 as receptor of hog cholera virus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cherubini et al. | FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition | |
| Zhu et al. | IL-13 secreted by ILC2s promotes the self-renewal of intestinal stem cells through circular RNA circPan3 | |
| Wang et al. | Intra-articular delivery of extracellular vesicles secreted by chondrogenic progenitor cells from MRL/MpJ superhealer mice enhances articular cartilage repair in a mouse injury model | |
| Peng et al. | Amelioration of experimental autoimmune arthritis through targeting of synovial fibroblasts by intraarticular delivery of microRNAs 140‐3p and 140‐5p | |
| EP2374884A2 (en) | Human miRNAs isolated from mesenchymal stem cells | |
| Xiang et al. | MiR‐142‐5p as a CXCR4‐targeted microRNA attenuates SDF‐1‐induced chondrocyte apoptosis and cartilage degradation via inactivating MAPK signaling pathway | |
| Wang et al. | Upregulation of miR-98 inhibits apoptosis in cartilage cells in osteoarthritis | |
| Liao et al. | HDAC10 upregulation contributes to interleukin 1β‐mediated inflammatory activation of synovium‐derived mesenchymal stem cells in temporomandibular joint | |
| WO2017214463A1 (en) | Compositions and methods for treating cancer and biomarkers to detect cancer stem cell reprogramming and progression | |
| CN107142310B (en) | Screening method of specific shRNA for inhibiting lung cancer cells by targeting Ang-2 gene | |
| CN104498498A (en) | siRNA for inhibiting ADAM17 (a disintegrin and metalloprotease 17) genes and application of siRNA | |
| CN110172462B (en) | Gene with promotion effect on generation and development of tumor, expression product and application thereof | |
| CN107028970A (en) | Applications of the miR 1288 in diagnosing or treating osteoarthritis disorders | |
| CN104560997A (en) | SiRNA (small interfering ribonucleic acid) composition inhibiting ADAMTS-5 (a disintegrin-like and metalloproteinase with thrombospondin type 1motifs-5) and ADAM17 (a disintegrin-like and metalloproteinase with thrombospondin type 1motifs 17) genes and application of siRNA composition | |
| US20180221393A1 (en) | Cxcr4 binding agents for treatment of diseases | |
| Xia et al. | Increased miR-381a-3p contributes to osteoarthritis by targeting IkBα | |
| CN104561000B (en) | Inhibit nucleic acid oligomer and its application of CD44 genes | |
| CN104560999B (en) | Suppress siRNA and its application of the genes of ADAMTS 5 | |
| Wu et al. | Targeting A549 lung adenocarcinoma cell growth and invasion with protease‑activated receptor‑1 siRNA | |
| Laird et al. | Identification of a novel gene involved in asexual organogenesis in the budding ascidian Botryllus schlosseri | |
| CN111569077B (en) | Use of Flot2 inhibitor for inhibiting osteoclast formation and/or osteoclast activity | |
| CN103952406B (en) | The siRNA of targeting STAT3 gene of suppression people's malignant glioma propagation and expression vector thereof and application | |
| CN110279706B (en) | Application of hnRNPC gene C1 and/or C2 subtype as drug target in screening anti-cancer drugs | |
| JP7231917B2 (en) | Immune checkpoint factor expression inhibitor in cancer cells and pharmaceutical composition for cancer treatment | |
| CN104436195B (en) | Purposes of the miR-155 in preparation prevention and treatment acute lung injury drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150408 |