CN106222183A - The siRNA of targeted human IRAK1 gene and application thereof - Google Patents
The siRNA of targeted human IRAK1 gene and application thereof Download PDFInfo
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- CN106222183A CN106222183A CN201610589804.9A CN201610589804A CN106222183A CN 106222183 A CN106222183 A CN 106222183A CN 201610589804 A CN201610589804 A CN 201610589804A CN 106222183 A CN106222183 A CN 106222183A
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Abstract
The present invention relates to biological technical field, the sequences of small interfering RNAs of a kind of targeted human IRAK1 gene treating dental caries and application thereof, described siRNA includes positive-sense strand and antisense strand, and the nucleotide sequence of described positive-sense strand is as shown in SEQ ID NO:2；The nucleotide sequence of described antisense strand is as shown in SEQID NO:3.The sequences of small interfering RNAs that the present invention obtains is not found in the disclosedest report, can disturb after the transcribing of IRAK1 gene, thus specificity reduces the protein expression of IRAK1, the expression of NF κ B in inflamed human dental pulp stem cell can be reduced, reduce the generation of dental pulp stem cell inflammatory factor tumor necrosis factor, the development of suppression inflammation, promotes the one-tenth dentin differentiation of dental pulp stem cell, provides new therapeutic scheme for dental caries.
Description
Technical field
The present invention relates to biological technical field, specifically, be targeted human IRAK1 gene sequences of small interfering RNAs and
Application.
Background technology
Dental caries (dental caries) is that the dental hard tissue caused by many factors compound action in oral cavity occurs
Chronic, Progressive symmetric erythrokeratodermia disease damage, its sickness rate is high, Crowds Distribute is wide, is one of commonly encountered diseases and the frequently-occurring disease in oral cavity.Traditional mechanical removes dental caries
Method is the main method of Dental treatment the most clinically, and the dissection of main application admantine drill removes the bad tissue of dental caries, but
Easy damage healthy tissue of tooth in application, and excitation easy to cold stimulation when the heat of drill bit cutting generation and liquid cooling
Pulp tissue, causes poor prognosis.Therefore, finding new alternative medicine, the treatment for dental caries has great importance.
IRAK1 (interleukin-1 receptor-associated kinase 1) is IRAK family member, passes through
Its kinase activity and the effect of linkers, mediation includes Toll-like receptor (Toll like receptor, TLR) signal path
In interior a series of signal transduction, ultimately result in the isogenic expression of Pro-inflammatory mediator, participate in intracellular signal network
Regulation and control and inflammatory reaction.The components such as the LPS of G-bacterium by activate TLR-MyD88-IRAK1 activation NF-κ B mediate downstream inflammation because of
Son release, the TNF-α secreted downstream and RANKL are broken bone factor, stimulate differentiation of osteoclast, activation, cause bone to lure and split
Formed, closely related with the pathogenesis such as pulpitis, periodontitis, dental caries.
That RNA interference (RNA interference, RANi) refers to be highly conserved during evolution, by double-stranded RNA
(double-stranded RNA, dsRNA) induce, a kind of biological phenomena of the efficient selective degradation of homologous mRNA, can be special
The expression of different interference target gene.Its mechanism of action is: the Dicer enzyme of rnase iii family, is combined with dsRNA, is cut
It is cut into the siRNA (Small interfering RNA, siRNA) that 21-25nt and 3 ' distal process goes out, siRNA and RNA subsequently
Induction silencing complex (RNA-induced silencing complex, RISC) combines, and untwists into strand, the RISC of activation
Become the siRNA of strand to guide by, be attached to target messenger RNA (mRNA) sequence-specific and above and cut off, cause target
The specific cleavage of mRNA, thus suppress corresponding gene to express.An independent technology, action target spot specificity are become at present
High, little to cytotoxicity, comparatively safe, cause the broad interest of all research fields in biomedical boundary.
Summary of the invention
It is an object of the invention to up-to-date IRAK1 gene order as target spot, it is provided that a kind of new spy treating dental caries
The sequences of small interfering RNAs of opposite sex suppression people's IRAK1 gene expression and application thereof.
A first aspect of the present invention, it is provided that the target gene of the siRNA of a kind of targeted human IRAK1 gene, described
The nucleotides sequence of target gene is classified as 5 '-CCGAAGAAAGTGATGAATT-3 ', as shown in SEQ ID NO:1.
A second aspect of the present invention, it is provided that the siRNA of a kind of targeted human IRAK1 gene, by following positive-sense strand with anti-
Justice chain composition:
Positive-sense strand: 5 '-GCCCGAAGAAAGUGAUGAAUU-3 ' (SEQ ID NO:2)；
Antisense strand: 5 '-AAUUCAUCACUUUCUUCGGGC-3 ' (SEQ ID NO:3).
A third aspect of the present invention, it is provided that the target gene of the siRNA of above-mentioned targeted human IRAK1 gene is controlled in preparation
Treat the application in dental caries medicine.
A fourth aspect of the present invention, it is provided that the siRNA of above-mentioned targeted human IRAK1 gene is at preparation treatment dental caries medicine
In application.
The siRNA specificity of described targeted human IRAK1 gene reduces the protein expression of IRAK1, suppression inflammation
Development, promotes the one-tenth dentin differentiation of dental pulp stem cell.
A fifth aspect of the present invention, it is provided that a kind of pharmaceutical composition treating dental caries, described pharmaceutical composition includes
State the siRNA of targeted human IRAK1 gene and pharmaceutically acceptable carrier.
Described pharmaceutically acceptable carrier is pharmaceutically acceptable excipient, suspending agent, filler and/or dilution
Agent.
The invention has the advantages that:
The sequences of small interfering RNAs that the present invention obtains is not found in the disclosedest report, can be laggard transcribing of IRAK1 gene
Row interference, thus specificity reduces the protein expression of IRAK1, it is possible to decrease the expression of NF-κ B in inflamed human dental pulp stem cell, reduces tooth
The generation of marrow stem cell inflammatory factor-tumor necrosis factor (TNF-α), the development of suppression inflammation, promote the one-tenth of dental pulp stem cell
Dentin breaks up, and can be that treatment dental caries provides new scheme.
Accompanying drawing explanation
Fig. 1 is the siRNA downward effect to dental pulp stem cell IRAK1 albumen.(it is illustrated as Western blot detection
IRAK1 expressing quantity, is followed successively by blank group, matched group and siRNA1, siRNA2, siRNA3, siRNA4 group；GD is internal reference.
Point out only siRNA1 can significantly reduce the expression of IRAK1 albumen, if siRNA the most hereinafter all refers to without specified otherwise
siRNA1)。
Fig. 2 is the impact that dental pulp stem cell is become dentin to break up under hyperphlogosis by western blot detection siRNA.
A is that dental pulp stem cell becomes dentin differentiation culture to become dentin label dentin after 14 days under 50ng/mL TNF-α stimulates
Saliva phosphoprotein (dentin sialophosphoprotein, DSPP), dentin extracellular matrix (dental matrix
Protein-1, DMP1) expression.B is statistical result.Result shows: siRNA can significantly improve the one-tenth tooth of dental pulp stem cell
Essence differentiation capability, particularly under 50ng/mL TNF-α stimulates, the one-tenth dentin ability of dental pulp stem cell significantly improves, and becomes
The expression of dentin label DSPP, DMP1 is substantially increased.Point out this siRNA to have and facilitate dentin differentiation capability.
Fig. 3 is the impact that ALP dyeing detection siRNA becomes dentin differentiation capability to dental pulp stem cell.A is dental pulp stem cell
The ALP dyeing after 14 days of dentin differentiation culture is become under 50ng/mL TNF-α stimulates.B is statistical result, with matched group phase
Ratio, siRNA can be obviously promoted the one-tenth dentin differentiation capability of dental pulp stem cell under height inflammation.
Fig. 4 is the impact that Alizarin red staining detection siRNA becomes dentin differentiation capability to dental pulp stem cell.A is that dental pulp is done
Cell becomes 14 days hystazarin red colourings of dentin differentiation culture under 50ng/mL TNF-α stimulates.B is statistical result, with right
Comparing according to group, siRNA can be obviously promoted the one-tenth dentin differentiation capability of dental pulp stem cell under height inflammation.
Detailed description of the invention
The detailed description of the invention provided the present invention below in conjunction with embodiment elaborates.
The experimental technique of unreceipted actual conditions in all methods of the present invention, agents useful for same is purchased from if no special instructions
Invitrogen, interference sequence is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd.Experimental technique in embodiment generally according to
Normal condition, reference molecule cloning experimentation guide (third edition, J. Pehanorm Brooker etc. writes, and yellow training hall etc. is translated, Science Press,
2002) described in condition, or carry out according to the condition proposed by manufacturer.
Embodiment 1: the acquisition of targeted human IRAK1 gene specific interference sequence
According to people IRAK1 gene order (Genbank:NM_001025243) design siRNA sequence disclosed in NCBI, specifically
Design reference American I nvitrogen company's site (its website http://rnaidesigner.invitrogen.com/
Rnaiexpress/sort.do) the RNAi Photographing On-line software provided, obtains the target gene containing 19 bases through comparison screening
Sequence: 5 '-CCGAAGAAAGTGATGAATT-3 ' (SEQ ID NO:1), corresponding siRNA interference sequence is by the lucky triumphant gene in Shanghai
Chemical technology company limited synthesizes, specific as follows:
Positive-sense strand: 5 '-GCCCGAAGAAAGUGAUGAAUU-3 ' (SEQ ID NO:2)
Antisense strand: 5 '-AAUUCAUCACUUUCUUCGGGC-3 ' (SEQ ID NO:3)
Embodiment 2: dental pulp stem cell is cultivated and Western blot detects the siRNA effect to IRAK1 protein expression
1) pulp cells in experimentation derives from the normal clinically and third molar of dental caries and because of correction needs
The second premolars pulled out, in patient age 15-25 year, entire patient is all right.Obtain into group personnel's informed consent, and through south
The Medical Ethics Committee approval of Affiliated Hospital of logical university.The tooth pulled out is immediately placed in the basic DMEM culture fluid of ice bag pre-cooling
In (containing 100U/mL penicillin/100 μ g/mL streptomycin).After super-clean bench rinses dental surface with povidone iodine, use sterile physiological
The surface of normal saline washing tooth, to without bloodstain, opens pulp cavity with the rongeur after sterilization along enamelo-cemental junction, and tweezers take out dental pulp,
Cut off the pulp tissue of root.Mixing with 1:1 ratio with the Bacillus polymyxa Neutral proteinase of 4mg/mL with 3mg/mL NTx enzyme, two kinds of enzymes are with 1:
The volume of 10 and the dental pulp mixture slaking shredded 1 hour, be placed in a centrifuge centrifugal by postdigestive tissue, 1000rpm/min
Centrifugal 5 minutes, abandoning supernatant, add complete medium, mix, blow and beat, through the cellular metal net filtration of 70 μm, it is thus achieved that cell
Inoculation of suspension liquid, in 10cm culture dish, adds complete culture solution (10% hyclone, DMEM, 1% glutamic acid, 1% penicillium sp
Element/streptomycin).Condition of culture: relative humidity 100%, 5%CO2, 37 DEG C.3-4 days half amounts change liquid, and cell fusion reaches 70%-
After 80%, under room temperature condition, 0.25% trypsinization 3 minutes, 1000rpm/min is centrifuged 5 minutes, reject supernatant, piping and druming
Precipitation, passes on 1:3 ratio after mixing, within every 3 days, changes liquid 1 time.The cell selecting for 3 generations is tested.
2) siRNA transfection.Experiment is divided into blank group (the normal cell culture fluid without transfection reagent), matched group (negative
Comparison buy from Santa Cruz, sc-37007), siRNA1-4 group (sequences of small interfering RNAs of synthesis).With 0.25% Trypsin
The dental pulp stem cell of enzymic digestion exponential phase makes single cell suspension (cell number 1 × 105), it is inoculated in 6 orifice plates, cultivates to carefully
Born of the same parents' degrees of fusion is more than 40%.With 250uL serum-free medium (Opti-MEM) dilution siRNA (transfectional cell final concentration of
33nM), pressure-vaccum mixing gently.5uL Lipofectamine TM2000, pressure-vaccum 3-5 time gently is diluted with 250uL Opti-MEM
Mixing, left at room temperature 10min.Mixing transfection reagent and siRNA diluent, gently pressure-vaccum mixing, left at room temperature 20min.
500uL transfection composite joins 24 porocyte plates (100uL/ hole) with 1.5mL serum-free DMEM after mixing, jog cell plates mix
Close uniformly.It is placed in 37 DEG C, 5%CO2Incubator.Changing culture fluid after transfection 24h, condition continues to cultivate routinely.
3) Western blot detects IRAK1 protein expression.After dental pulp stem cell transfection 24h, sucking culture medium, PBS is clear
Wash cell 1-2 time, in cell, add 100uL RIPA lysate, in collection cell to 1.5mL pipe, stand 30min on ice, centrifugal
12000rpm/min, 30min take supernatant, are prepared as protein sample for analyzing.The albumen of preparation is added sample-loading buffer RSB,
After boiling 5min with 100 DEG C, move into cooled on ice immediately, as the loading sample of electrophoresis.Glue and 10% separation gel is concentrated with 5%
Electrophoretic separation, applied sample amount is 20ug/ swimming lane, electrophoretic voltage, and concentration glue is 70V, and separation gel is 100V, electrophoresis time about 2.5h.
Internal reference is GD.
Result shows: only siRNA1 can significantly reduce the expression (see Fig. 1) of IRAK1 albumen.
Embodiment 3:Western blot detection siRNA dental pulp stem cell is become dentin differentiation capability impact (DPSS,
DMP-1 protein expression)
1) with the dental pulp stem cell of 0.25% trypsinization exponential phase make single cell suspension (cell number 1 ×
105), it is inoculated in 6 orifice plates, every hole 200uL.37 DEG C, 5%CO2After cultivating 24h in incubator, abandoning supernatant, every hole adds 400uL blood
Clear culture medium, transfection 4h (experiment is divided into blank group, matched group, siRNA group as previously mentioned).
2), after dental pulp stem cell transfection 24h, suck culture medium, PBS cell 1-2 time, in cell, add 100uL
RIPA lysate, in collection cell to 1.5mL pipe, stands 30min on ice, and centrifugal 12000rpm/min, 30min take supernatant, system
Standby one-tenth protein sample is for analyzing.The albumen of preparation is added sample-loading buffer RSB, after boiling 5min with 100 DEG C, moves into ice immediately
Upper cooling, as the loading sample of electrophoresis.Concentrating glue and 10% separation gel electrophoretic separation with 5%, applied sample amount is 20ug/ swimming lane,
Electrophoretic voltage, concentration glue is 70V, and separation gel is 100V, electrophoresis time about 2.5h.Internal reference is GD.
Result shows: siRNA can significantly improve the one-tenth dentin differentiation capability of dental pulp stem cell, particularly at 50ng/mL
Under TNF-α stimulates, the one-tenth dentin ability of dental pulp stem cell significantly improves, and the expression becoming dentin label DSPP, DMP1 is bright
Show and increase.Point out this siRNA to have and facilitate dentin differentiation capability (see Fig. 2).
Embodiment 4:ALP staining detection siRNA becomes the impact of dentin differentiation capability to dental pulp stem cell
1) with the dental pulp stem cell of 0.25% trypsinization exponential phase make single cell suspension (cell number 1 ×
105), it is inoculated in 6 orifice plates, every hole 200uL.37 DEG C, 5%CO2After cultivating 24h in incubator, abandoning supernatant, every hole adds 400uL blood
Clear culture medium, transfection 4h (experiment is divided into blank group, matched group, siRNA group as previously mentioned).
2) dyeing.Abandoning supernatant, every hole adds 200uL PBS cell 1-2 time, and under lucifuge, with fixing, A is liquid-solid determines cell
3min, is placed in 37 DEG C of baking ovens；Exhaustion fixative, with dyeing B liquid dye cell 15min under lucifuge, is placed in 37 DEG C of baking ovens；Exhaustion
B liquid, then with redying D liquid dye cell 5min, be placed in equally in 37 DEG C of baking ovens.Finally use PBS 3-5 time.Take pictures.
Result shows: compared with matched group, and siRNA can be obviously promoted the one-tenth dentin of dental pulp stem cell under height inflammation
Differentiation capability (see Fig. 3).
Embodiment 5: Alizarin red staining method detection siRNA becomes the impact of dentin differentiation capability to dental pulp stem cell
1) with the dental pulp stem cell of 0.25% trypsinization exponential phase make single cell suspension (cell number 1 ×
105), it is inoculated in 6 orifice plates, every hole 200uL.37 DEG C, 5%CO2After cultivating 24h in incubator, abandoning supernatant, every hole adds 400uL blood
Clear culture medium, transfection 4h (experiment is divided into blank group, matched group, siRNA group as previously mentioned).
2) dyeing.Abandoning supernatant, every hole adds 200uL PBS cell 1-2 time, alizarin red dye liquor dye cell 2h, is placed in
In 37 DEG C of baking ovens；Clean dye liquor, cetylpyridinium chloride(CPC) mineralising cell 2h under room temperature.Finally use PBS 3-5 time.Take pictures.
Result shows: compared with matched group, and siRNA can be obviously promoted the one-tenth dentin of dental pulp stem cell under height inflammation
Differentiation capability (see Fig. 4).
Below preferred embodiment to the invention is illustrated, but the invention is not limited to described
Embodiment, those of ordinary skill in the art it may also be made that all equivalents on the premise of the invention spirit
Modification or replacement, modification or the replacement of these equivalents are all contained in the application claim limited range.
Claims (7)
1. the target gene of the siRNA of a targeted human IRAK1 gene, it is characterised in that the core of described target gene
Nucleotide sequence is as shown in SEQ ID NO:1.
2. the siRNA of a targeted human IRAK1 gene, it is characterised in that described siRNA includes that positive-sense strand is with anti-
Justice chain, the nucleotide sequence of described positive-sense strand is as shown in SEQ ID NO:2；The nucleotide sequence of described antisense strand such as SEQ
Shown in ID NO:3.
The target gene of the siRNA of targeted human IRAK1 gene the most according to claim 1 is at preparation treatment dental caries medicine
Application in thing.
The siRNA of targeted human IRAK1 gene the most according to claim 2 answering in preparation treatment dental caries medicine
With.
The siRNA of targeted human IRAK1 gene the most according to claim 4 answering in preparation treatment dental caries medicine
With, it is characterised in that the siRNA specificity of described targeted human IRAK1 gene reduces the protein expression of IRAK1, suppression
The development of inflammation, promotes the one-tenth dentin differentiation of dental pulp stem cell.
6. the pharmaceutical composition treating dental caries, it is characterised in that described pharmaceutical composition includes described in claim 2
The siRNA of targeted human IRAK1 gene and pharmaceutically acceptable carrier.
The pharmaceutical composition for the treatment of dental caries the most according to claim 6, it is characterised in that described is pharmaceutically acceptable
Carrier is pharmaceutically acceptable excipient, suspending agent, filler and/or diluent.
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| CN101616910A (en) * | 2006-09-07 | 2009-12-30 | 比奥根艾迪克Ma公司 | Indazole derivatives as interleukin 1 receptor associated kinase conditioning agent |
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| CN101616910A (en) * | 2006-09-07 | 2009-12-30 | 比奥根艾迪克Ma公司 | Indazole derivatives as interleukin 1 receptor associated kinase conditioning agent |
| US20140303149A1 (en) * | 2010-07-13 | 2014-10-09 | Hoffmann-La Roche Inc. | PYRAZOLO[1,5a]PYRIMIDINE DERIVATIVES AS IRAK4 MODULATORS |
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