CN110144350A - The siRNA and its application in inhibition scar is formed that one species specificity inhibits CTGF gene expression - Google Patents
The siRNA and its application in inhibition scar is formed that one species specificity inhibits CTGF gene expression Download PDFInfo
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- CN110144350A CN110144350A CN201910404445.9A CN201910404445A CN110144350A CN 110144350 A CN110144350 A CN 110144350A CN 201910404445 A CN201910404445 A CN 201910404445A CN 110144350 A CN110144350 A CN 110144350A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Abstract
The siRNA for inhibiting CTGF gene expression the invention discloses a species specificity and its application in inhibition scar is formed.The siRNA includes the positive-sense strand and antisense strand of complete complementary, and the nucleotides sequence of the siRNA positive-sense strand is classified as any one in SEQ ID NO:1,3,5,7,9,11,13,15,17；The nucleotides sequence of the siRNA antisense strand is classified as corresponding with positive-sense strand SEQ ID NO:2,4,6,8,10,12,14,16,18.SiRNA molecule of the invention being capable of the specific expression for inhibiting CTGF gene in humans and animals cell, inhibition or the formation for reducing scar.
Description
Technical field
The present invention relates to field of biotechnology, a more specifically species specificity inhibit CTGF gene expression siRNA and
Its application in inhibition scar is formed.
Background technique
China sees a doctor because of wound be up to 62,000,000 people every year, disfiguring keliod patients up to 12,000,000 people, the whole nation 2014
Hospitalisation for surgery person-time is 4382.92 ten thousand person-times；Human skin wound/postwar wound, incised injury, burn, skin infection, surgery hand
Necessarily lead to scar during the skin wound healings such as art, scar be normal skin tissue's mode of appearance caused by above-mentioned reason and
Tissue pathologies change.Wound healing process abnormal reaction, hyperplasia form pathologic scar.And neck surface and extremities joint
The pathologic scar at position not only influences appearance and even disfeatures, it is also possible to cause dysfunction, be often accompanied by itch, pain, infection
Etc. complication, be to burn one of most common complication after (wound) wound, operation, bring great damage to patient's physical and mental health, be to create
One of the problem that traumatology, plastic surgery and dermatology face.
Currently, effective single therapy scheme has not been established in treatment scar or prevention hand postoperative scar growth, it is existing
Treatment method mainly have physical therapy, operation excision and the common drug treatments such as steroids/chemotherapeutics/interferon and
Growth factor associated medication therapies.Physical therapy is to inhibit and treat scar by the modes such as such as electricity, light, magnetic, sound, pressure, is treated
Effect and risk are simultaneously deposited.Operation excision uses when often influencing dysfunction for pathologic scar, and recurrence rate is higher；Steroids is swashed
For the drug therapies such as element, long-time service Patients Treated with Steroid will cause corticosteroid increase disease, pigmentation, skin and burst
The adverse reactions such as ulcer, and high recurrence rate；Chemotherapeutics toxicity can lead to anaemia, thrombopenia and leukopenia etc..
Different degrees of negative effect can be all brought to patient for above-mentioned four kinds for the treatment of methods and treatment is often after cicatrization, curative effect compared with
Difference.And growth factor associated medication therapies are then the generations by blocking the conduction of cytokine signalling pathways to inhibit scar,
It plays a role before cicatrization, to reduce the formation of scar, plays the role for the treatment of.Such as fibroblast growth factor
(FGF), a- tumor necrosis factor (TNF-a), gamma interferon (IFN-γ), epidermal growth factor (EGF), β transforming growth factor
(TGF-β) etc..Though thering is artificial synthesized transforming growth factor TGF-β avotermin (avotermin) to enter the III phase at present to face
The bed experimental stage, but growth factor related drugs are still less in the application study of scar treatment.
Growth factor plays an important role in Process of Forming Scar, and TGF-β 1 is fibrotic disease occurrence and development process
In most important virulence factor.Connective Tissue Growth Factor (connective tissue growth factor, CTGF) is one
Kind promotees the growth factor of fibrosis, passes through and promotes cell Proliferation, adjusts the biological effect of the effects of Extracellular Matrix Gene Expression performance
It answers, can be seldom detected in adult normal skin histology.CTGF is the direct downstream effect medium of TGF-β 1, is stimulated into fibre
Dimension cell Proliferation and the extrtacellular matrix deposition based on collagen, the fibroblast of proliferation constantly secrete CTGF, eventually lead to
Fibrosis promotes pathologic scar to be formed.And the biological function of CTGF is more single, specificity is high, its expression is blocked to be not easy to draw
Play some problems such as wound healing delay, inflammatory reaction.Sisco M's in 2008 etc. studies have shown that in CTGF experiment in vitro
90%mRNA is suppressed, the experiment in internal cicatricial tissue, and 55%CTGF mRNA expression is suppressed, and substantially reduces CTGF albumen
Expression 20%, reduces the tendon fibroblasts quantity of the about 75% α-SMA positive, while significantly inhibiting Col I, Col III,
Fibronectin (Fn) and TIMP-1mRNA expression, and finally limitation inhibits cicatrization, while not influencing wound healing.
SiRNA (small interfering RNA, siRNA) is the short sequence double-stranded RNA point of 21~25nt size
Son.Transfection and the homologous siRNA of target gene to target cell, are combined by complementary with the single-stranded mRNA of target gene, start target base
Because of program of degrading, the mRNA of target sequence is induced to degrade, blocks expression of target gene, the defect phenotype of cells show specific gene.With
Conventional antisense nucleic acid is compared, and siRNA is technically simple easy, has gene inhibition rate is high, high specificity and concentration are low etc.
Advantage is widely used in gene functional research field, provides base for tumour, genetic disease and virus infection etc.
Because of the new way for the treatment of.
Summary of the invention
The present invention is exactly that in order to solve the above-mentioned technical problem, the species specificity proposed inhibits CTGF gene expression
SiRNA and its application in inhibition scar is formed.
The present invention is realized according to following technical scheme.
The siRNA of one species specificity inhibition CTGF gene expression, it is characterised in that: the siRNA includes complete complementary
The nucleotides sequence of positive-sense strand and antisense strand, the siRNA positive-sense strand is classified as SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID
Any one in NO:37；The nucleotides sequence of the siRNA antisense strand is classified as SEQ ID NO corresponding with positive-sense strand:
22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID
NO:34、SEQ ID NO:36、SEQ ID NO:38。
Further, at least one 3 ' single-stranded ends are also connected with 1 to 3 in the positive-sense strand and antisense strand of the siRNA
A additional nucleotide, to form at least one after siRNA positive-sense strand and antisense strand complementary pairing by 1 to 3 nucleotide
3 ' the jags constituted.
Further, the 3 ' jag is continuous 2 uridylate UU.
Further, the siRNA positive-sense strand and antisense strand all contain 3 ' jags.
One species specificity inhibits the siRNA of CTGF gene expression, and the siRNA includes the positive-sense strand and antisense of complete complementary
The nucleotides sequence of chain, the siRNA positive-sense strand is classified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
It is NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, any in SEQ ID NO:17
One；The nucleotides sequence of the siRNA antisense strand be classified as SEQ ID NO:2 corresponding with positive-sense strand, SEQ ID NO:4,
SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、
SEQ ID NO:18。
It is a kind of it is above-mentioned specificity inhibit CTGF gene expression siRNA inhibit scar formed in purposes.
Further, the siRNA inhibits the expression of CTGF gene in humans and animals cell by specificity, to inhibit
Or reduce the formation of scar.
Present invention obtains following beneficial effects.
The invention discloses the nucleotide sequences (siRNA) of nine groups of RNA interferings for CTGF gene expression, being capable of target
To inhibition CTGF gene expression and then it is used to treat disease relevant to CTGF gene unconventionality expression.In addition, of the present invention
SiRNA sequence has high homology in different plant species, due to this high homology, on the one hand, between different plant species
Test can use mutually homotactic siRNA, reduce the mistake for constituting and synthesizing different sequence siRNA according to the gene of different plant species
Journey can greatly shorten experiment process when test different animals model, various preclinical tests are rapidly completed；Another party
Face, also can avoid using brought by different sequence siRNA to corresponding animal CTGF mRNA inhibit efficiency, stability not really
It is qualitative, so as to accelerate Subsequent pharmacological to research and develop process.
Detailed description of the invention
Fig. 1 is the Alexa that inverted fluorescence microscope of the present invention transfects triumphant peptideRed Fluorescent
Transfected condition figure of the Oligo in three kinds of cell lines；
Fig. 2 is influence diagram of the transfection reagent of the present invention to three kinds of Apoptosis；
Fig. 3 is present invention difference CTGF siRNA (CT1, CT2, CT3, CT9) and its concentration (1,10,100nM) to CTGF
The influence diagram of expression.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and embodiments.
Embodiment 1siRNA sequence information
With CTGF mRNA, (Genebank number of registration: the template that (NM_001901) designs for siRNA obtains three to the present invention
The conservative siRNA of species, sequence information are shown in Table 1.Meanwhile positive-sense strand nucleotide sequence is set as shown in sequence 19 and antisense
Chain the nucleotide sequence siRNA as shown in sequence 20, number NC.NC is the nothing with CTGF mRNA without corresponding target action site
Sequence is closed, as negative control.
The siRNA information of 1: three, table targeting CTGF
The positive-sense strand of listed siRNA and antisense strand are synthesized by Shanghai Ji Ma Bioisystech Co., Ltd in table 1.Wherein,
Sequence SEQ ID NO:1-SEQ ID NO:18 is corresponding in turn to for the 3 ' end sequence SEQ ID NO:21-SEQ ID NO:38 to be drawn
Enter 2 uridylates and is formed.Sequence SEQ ID NO:1-SEQ ID NO:18 is equimolar with annealing salt solution
Positive-sense strand and antisense strand mixture, for subsequent conventional annealing to form siRNA double-strand, it is 3 ' prominent to be respectively provided with UU for the both ends of double-strand
Outlet.
Embodiment 2: transfection reagent verifying
Triumphant peptide gene transfection reagent (Kai Er Bioisystech Co., Ltd, Shanxi China Telecom) is transfection reagent used in the present invention, this
Invention first verifies the transfection efficiency and cytotoxicity of triumphant peptide transfection siRNA, excludes influence of the transfection reagent to experiment.
(1) cell transfecting efficiency
Human lung carcinoma cell (maxicell lung cancer) (H460 is purchased from ATCC, BNCC102112), gland cancer alveolar substrate epithelium is thin
Born of the same parents' (A549 cell) (be purchased from ATCC, BNCC100258) and human bronchial epithelial like cell (HBE, purchased from ATCC,
BNCC338600) cell inoculation is in 24 orifice plates, inoculum density 105Cells/well, with the cell containing F12+10% fetal calf serum
Culture medium is placed in 37 DEG C of 5%CO2Culture.Cell transfecting illustrates the BLOCK- for transfecting 50nM respectively according to triumphant peptide gene transfection reagent
iTTMAlexaRed Fluorescent Oligo (be purchased from invitrogen) arrives H460, A549 and HBE cell, and 37 DEG C
Culture.Pass through inverted fluorescence microscope and flow cytomery cell transfecting efficiency afterwards for 24 hours, the results showed that in three kinds of cells
The transfection efficiency of triumphant peptide transfection siRNA is all larger than 90% (Fig. 1) in system.
(2) cytotoxicity
By H460, A549 and HBE cell inoculation in 24 orifice plates, inoculum density 105Cells/well, with containing F12+10%
The cell culture medium of fetal calf serum is placed in 37 DEG C of 5%CO2Overnight incubation.It is greater than 90% triumphant peptide reagent amount with transfection efficiency respectively
Handle A549, H460 and HBE cell, 37 DEG C of 5%CO2Cultivate 72h.Respectively for 24 hours, 48h and 72h pass through Annexin V-
EGFP/PI Apoptosis Detection Kit (be purchased from Beijing Quanshijin Biotechnology Co., Ltd) and fluorescence microscope or
Flow cytomery cell survival rate.Negative control is the cell culture medium of F12+10% fetal calf serum, F12 cell culture fluid
(being purchased from GIBCO), fetal calf serum (are purchased from Biological Industries), and reference reagent is Lipofectaine
RNAiMAX (is purchased from Invitrogen).The result shows that cell and untransfected of the cell transfecting efficiency in 90-99%, after transfection
Cell compare, Apoptosis there are no significant difference shows triumphant peptide transfection reagent almost no cytotoxicity (Fig. 2).
Embodiment 3: the real-time PCR of fluorescent quantitation (qRT-PCR) detects siRNA in vitro to CTGF mRNA expression
Inhibitory effect.
By A549 cell inoculation in 24 orifice plates, inoculum density 105Cells/well, the cell training of F12+10% fetal calf serum
Feeding base is placed in 37 DEG C of 5%CO2Overnight incubation.
Illustrate to transfect 4 kinds of CTGF siRNA (CT1:SEQ ID NO:1-SEQ ID respectively according to triumphant peptide gene transfection reagent
NO:2；CT2:SEQ ID NO:3-SEQ ID NO:4；CT3:SEQ ID NO:5-SEQ ID NO:6；CT9:SEQ ID NO:
17-SEQ ID NO:18) He Yiwu targeting negative control siRNA (being denoted as NC) arrive A549 cell, siRNA concentration be 1,10
And 100nM, then in 37 DEG C of 5%CO2Cultivate 48h.
After the cell 48h of culture transfection, the total serum IgE in cell is extracted with Rneasy Mini Kit (Qiagen company).
Each sample takes 2 μ g total serum IgEs, according to PrimeScriptTM1st Strand cDNA Synthesis Kit (Takara company)
Application method reverse transcription obtain cDNA, using TaqMan Assay detection kit (Applied Biosystems company) into
Row fluorescent quantitation real-time PCR reactions.PCR condition is as follows: 95 DEG C of initial denaturation 10min, and into circulation: 95 DEG C of denaturation 30s, 60 DEG C are moved back
Fiery 30s, 72 DEG C of extension 30s, after 40 recycle, 72 DEG C of extension 10min.User β 2microglobulin (B2M) is as quantitative
The reference gene (Applied Biosystems company, Catalog number:4310886E) of PCR reaction, the base of CTGF mesh
Because the Ct value of expression is standardized with the Ct value of B2M, the calculating of the multiple variation of sample amplification uses 2–△△tMethod.PCR used
It expands CTGF primer and B2M primer sequence is as shown in table 3.
Table 3: quantitative PCR detection primer sequence
| Title | Upstream (5 ' -3 ') | Downstream (5 ' -3 ') |
| People-CTGF | CACCCGGGTTACCAATGACA | TCCGGGACAGTTGTAATGGC |
The results show that the cell through handling 48h, CT9 and CT3 have significant inhibiting effect to the expression of CTGF mRNA, with
The expression of increase (1nM, 10nM, 100nM) the CTGF mRNA of its siRNA dosage gradually decrease.Such as 1nM CT9siRNA couple
CTGF expression inhibiting rate is that the processing of 36%, 100nM can be promoted to 88%, and the processing of negative control siRNA is to the table of CTGF
Up to not influencing, show that CTGF siRNA transfects the expression (Fig. 3) for successfully and effectively having suppressed CTGF.
Sequence table
<110>Jino Tai Kang biotechnology (Beijing) Co., Ltd
The siRNA and its application in inhibition scar is formed that<120>one species specificity inhibit CTGF gene expression
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ccggguuacc aaugacaacu u 21
<210> 2
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
guugucauug guaacccggu u 21
<210> 3
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
agaccugugg gaugggcauu u 21
<210> 4
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
augcccaucc cacaggucuu u 21
<210> 5
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
ccgacuggaa gacacguuuu u 21
<210> 6
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
aaacgugucu uccagucggu u 21
<210> 7
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
gcaccagcau gaagacauau u 21
<210> 8
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
uaugucuuca ugcuggugcu u 21
<210> 9
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
gaagacacgu uuggcccagu u 21
<210> 10
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
cugggccaaa cgugucuucu u 21
<210> 11
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
gcuaaauucu guggaguauu u 21
<210> 12
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
auacuccaca gaauuuagcu u 21
<210> 13
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 13
gcgaggucau gaagaagaau u 21
<210> 14
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 14
uucuucuuca ugaccucgcu u 21
<210> 15
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 15
aagcugaccu ggaagagaau u 21
<210> 16
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 16
uucucuucca ggucagcuuu u 21
<210> 17
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 17
ccuaucaagu uugagcuuuu u 21
<210> 18
<211> 21
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 18
aaagcucaaa cuugauaggu u 21
<210> 19
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 19
uucuccgaac gucgcacgu 19
<210> 20
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 20
acgugacacg uucggagaa 19
<210> 21
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 21
ccggguuacc aaugacaac 19
<210> 22
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 22
guugucauug guaacccgg 19
<210> 23
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 23
agaccugugg gaugggcau 19
<210> 24
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 24
augcccaucc cacaggucu 19
<210> 25
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 25
ccgacuggaa gacacguuu 19
<210> 26
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 26
aaacgugucu uccagucgg 19
<210> 27
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 27
gcaccagcau gaagacaua 19
<210> 28
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 28
uaugucuuca ugcuggugc 19
<210> 29
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 29
gaagacacgu uuggcccag 19
<210> 30
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 30
cugggccaaa cgugucuuc 19
<210> 31
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 31
gcuaaauucu guggaguau 19
<210> 32
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 32
auacuccaca gaauuuagc 19
<210> 33
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 33
gcgaggucau gaagaagaa 19
<210> 34
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 34
uucuucuuca ugaccucgc 19
<210> 35
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 35
aagcugaccu ggaagagaa 19
<210> 36
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 36
uucucuucca ggucagcuu 19
<210> 37
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 37
ccuaucaagu uugagcuuu 19
<210> 38
<211> 19
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 38
aaagcucaaa cuugauagg 19
Claims (7)
1. a species specificity inhibition CTGF gene expression siRNA, it is characterised in that: the siRNA include complete complementary just
The nucleotides sequence of adopted chain and antisense strand, the siRNA positive-sense strand is classified as SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID
Any one in NO:37；The nucleotides sequence of the siRNA antisense strand is classified as SEQ ID NO corresponding with positive-sense strand:
22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID
NO:34、SEQ ID NO:36、SEQ ID NO:38。
2. the siRNA that a species specificity according to claim 1 inhibits CTGF gene expression, it is characterised in that: described
At least one 3 ' single-stranded ends are also connected with 1 to 3 additional nucleotide in the positive-sense strand and antisense strand of siRNA, thus
At least one 3 ' jag being made of 1 to 3 nucleotide is formed after siRNA positive-sense strand and antisense strand complementary pairing.
3. the siRNA that a species specificity according to claim 2 inhibits CTGF gene expression, it is characterised in that: described 3 '
Jag is continuous 2 uridylate UU.
4. the siRNA that a species specificity according to claim 2 inhibits CTGF gene expression, it is characterised in that: described
SiRNA positive-sense strand and antisense strand all contain 3 ' jags.
5. a species specificity inhibition CTGF gene expression siRNA, it is characterised in that: the siRNA include complete complementary just
Adopted chain and antisense strand, the nucleotides sequence of the siRNA positive-sense strand are classified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:
5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17
In any one；The nucleotides sequence of the siRNA antisense strand is classified as SEQ ID NO:2 corresponding with positive-sense strand, SEQ
ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID
NO:16、SEQ ID NO:18。
6. specificity described in a kind of claim 1-5 inhibits purposes of the siRNA of CTGF gene expression in inhibition scar is formed.
7. a species specificity according to claim 6 inhibits the siRNA of CTGF gene expression in inhibition scar is formed
Purposes, which is characterized in that the siRNA inhibits the expression of CTGF gene in humans and animals cell by specificity, to inhibit
Or reduce the formation of scar.
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| CN114807127A (en) * | 2021-01-19 | 2022-07-29 | 陈璞 | Small interfering RNA for connective tissue growth factor and application thereof |
Citations (5)
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