CN105879057B - SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application - Google Patents
SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application Download PDFInfo
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Abstract
The present invention provides SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application, additionally provide the application that SFRP2 promotes Odontogenic cysts mescenchymal stem cell mediating tissue regenerating medicine as preparation, the research that mechanism by regulating and controlling mescenchymal stem cell directed differentiation to WNT Signal Regulation factor S FRP2 carries out, promote stem cell tooth to-bone to the left and right of differentiation it was found that it has, and its possible mechanism is illustrated, to provide important clue for research tooth and the molecular regulation mechanism of bone growth and development Space-time speciality and the reconstruction of the biological regenerated seed cell of full tooth.
Description
This application claims in the submission of on May 14th, 2015 Patent Office of the People's Republic of China, application No. is 201510246730.4, invention
It is entitled " SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application " Chinese patent application it is excellent
It first weighs, entire contents are hereby incorporated by reference in the application.
Technical field
The present invention relates to stem cells technology fields, and in particular to WNT Signal Regulation factor S FRP2 promotes Odontogenic cysts mesenchyma
Stem cell skeletonization/at the application in tooth directed differentiation.
Background technique
Absence of tooth reparation and paradenlal tissue regeneration are always one of research emphasis of Oral Science.Mescenchymal stem cell exists
It playing an important role in tooth development and tissue regeneration processes, development abnormalities of teeth includes number, form, textural anomaly, this
A bit all to during tooth development gene expression and dysfunction it is related, so as to cause cell in growth course especially it is dry carefully
The changes of function of born of the same parents' Proliferation, Differentiation leads to the generation of disease.In recent years, the research of mescenchymal stem cell and tissue engineering technique into
It opens up and brings hope to tooth or paradenlal tissue regeneration.Mescenchymal stem cell, with multi-lineage potential and the energy freely updated
Power can be divided into corresponding tissue.Although tooth and support tissue regeneration that mescenchymal stem cell mediates achieve some researchs
Achievement, but at present there is also some critical issues are urgently to be resolved, one of them is that regenerated seed cell-Odontogenic cysts stem cell comes
Source is limited, and the molecular regulation mechanism of the differentiation of Odontogenic cysts stem cell directional and proliferation is completely clear not yet.Stem cell can carry out different
Body is transplanted for treating, but due to the influence at donor age and some other factor, the excessively aging of some stem cells, therapeutic effect
It is not good enough, and cause its long-term treatment effects poor since adult stem life cycle is of short duration.If lost to stem cell
Reconstruction is passed, its reprogramming is made, the gene for adjusting aging course is activated or inhibited, keeps stem cell again young, to give them
New life；Or improve the qualitative differentiation capability of stem cell and be used further to treat after extending its life cycle, to improve dry
The therapeutic effect of cell.Although many research shows that all being lacked with immunosuppressive action after mescenchymal stem cell heteroplastic transplantation
Weary long-term follow-up report.The influence of rejection, ethics and some other factor that long-term heteroplastic transplantation there may be,
Patient is set to be difficult to receive.During being cultivated in vitro with the stem cell of patient oneself, hereditary reconstruction is carried out, keeps it dry thin
Born of the same parents restore normal function and disclose the pathogenesis of its disease especially for the patient of certain hereditary diseases, on the one hand facilitate
Early diagnosis, prevention and the treatment of disease；On the other hand pointedly activation can be taken abnormal quiet their stem cell
Gene or quiet abnormal activation gene, to make to reconstruct later, the stem cell with pluripotency is used for autotransplantation
With wide clinical value.
SFRP2 is a member of a soluble WNT Signal Regulation factor S FRPs gene family.
SFRPs gene family is positioned at chromosome 8p 12~11.1, including 5 members: SFRP1, SFRP2, SFRP3,
SFRP4 and SFRP5 belongs to secreting type frizzled GAP-associated protein GAP, as the soluble Wnt Signal Regulation factor, by with Wnt
The homologous Receptor Competition of signal path in conjunction with and inhibit Wnt signal path, to be formed in biological development, cell traffic, tumour
And it plays an important role in the life processes such as Apoptosis.Furthermore SFRP2 can also inhibit the activity of RANKL to inhibit broken
Bone cell activity, and being formed in conjunction with Tolloid metalloproteinases to adjust collagen, or with integrin and
Fibronectin composite bulk phase interaction regulating cell apoptosis.Current study show that SFRP2 is that mescenchymal stem cell itself is secreted
An important factor, play important regulative during the self-renewing of mescenchymal stem cell, and bone can be enhanced
Bone marrow-drived mesenchymal stem myocardial repair effect.But influence and mechanism of the SFRP2 to mescenchymal stem cell directed differentiation are unclear,
It is furtherd investigate.
Research by SFRP2 to mescenchymal stem cell function effect can be the procedural of research mescenchymal stem cell
Reactivation and Functional Remodeling carry out theoretical preparation, wish to find that corresponding drug or biological agent control are dry thin on this basis
The state of born of the same parents achievees the purpose that clinical application and treatment, solves the pain of patient, restores the physical and mental health of patient, and will generate
Preferable economic benefit and social benefit.
Summary of the invention
It is filled between a kind of WNT Signal Regulation factor S FRP2 regulation the purpose of the present invention is in view of the drawbacks of the prior art, providing
The application of matter stem cell directional differentiation, in order to achieve the object of the present invention, it is proposed to adopt the following technical solutions.
The present invention provides SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application.
The present invention also provides SFRP2 answering as preparation promotion Odontogenic cysts mescenchymal stem cell mediating tissue regenerating medicine
With.
Drug of the present invention needs to be prepared into any pharmaceutically acceptable dosage form according to processing, and configuration is logical
Often by well known to a person skilled in the art processing methods to prepare, i.e., active component is mixed with liquid solvent or solid carrier,
It is prepared adding at least one surfactant, wherein being more preferably tablet, granule, oral solution, injection or capsule
Agent.
Drug of the present invention can be used by user through dilution or directly before use.
Beneficial effects of the present invention are by regulating and controlling mescenchymal stem cell directed differentiation to WNT Signal Regulation factor S FRP2
The research that carries out of mechanism, it is found that it has and stem cell tooth promoted to explain to-bone to the left and right of differentiation, and to its possible mechanism
It states, thus for molecular regulation mechanism and the biological regenerated seed of full tooth of research tooth and bone growth and development Space-time speciality
The reconstruction of cell provides important clue.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows early stage Osteoblast Differentiation alkaline phosphatase index；A is that alkaline phosphatase activities refer to detect early stage Osteoblast Differentiation
Mark-alkaline phosphatase；B is to survey Alizarin red staining to detect late osteogenic index-cell mineralization ability；C is measurement calcium ion concentration
To detect late osteogenic index-cell mineralization ability；The different time points that d~i is 0d, 3d, 7d, 10d, 14d weeks harvest cell；
Fig. 2 shows that being overexpressed SFRP2 promotes SCAPs skeletonization/at dentine differentiation capability；
Fig. 3 shows that gene knockout SFRP2 inhibits SCAPs skeletonization/at dentine differentiation capability；
Fig. 4 shows the tip of a root dental papilla stem cell variation that SFRP2 is knocked out；
Fig. 5 shows that the method for alkaline phosphatase detection and Alizarin red staining observes the Osteoblast Differentiation ability of SCAPs；A is alkali
Acid phosphatase detection；B is Alizarin red staining.
Specific embodiment
The present invention will be further explained with reference to the examples below, as described below, is only to preferable implementation of the invention
Example, not limits the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents be changed to the equivalent embodiment changed on an equal basis.Without departing from the concept of the present invention, according to the present invention
Technical spirit any simple modification or equivalent variations that following embodiment is made, fall within the scope of protection of the present invention.
Following experimental method is conventional technical means in the art in the case where not carrying out specified otherwise.
Separation, culture and the identification of stem cell:
The utilization of tissue obtains the approval of Ethics Committee, the Capital University of Medical Sciences, the preoperative label of the equal informed consent of volunteer
Order informed consent form.People's permanent teeth periodontium and permanent teeth inflammatory periodontium are obtained, pulp tissue or correction needs are pulled out
Permanent teeth (correction subtrahend extraction or young wisdom tooth) tip of a root dental papilla tissue, according to previous literature report method separation and
PDLSCs, DPSCs, SCAPs are cultivated, is summarized as follows: the tooth pulled out is immediately placed in the sterile centrifuge tube equipped with pre-cooling PBS,
It transfers into Cytology Lab, is separately cultured periodontal ligament stem cell, pulp cells, tip of a root dental papilla cells interior for 24 hours.Gently remove tooth
The periodontium of surrounding takes the periodontium in middle section, and mill opens pulp cavity, takes out dental pulp, or directly cuts tip of a root dental papilla position
Tissue, is cleaned repeatedly with PBS, is shredded, be placed in the digestive juice of enzyme containing Type I collagen (3g/L) and Dispase (4g/L), disappear at 37 DEG C
Change 1 hour, crosses 70 μm of cell sieves and collect cell, 1000rpm is centrifuged 10min, suspends into single cell suspension again with culture solution.It will
Cell inoculation in 25cm2 Tissue Culture Flask, α-MEM culture medium (containing 15% fetal calf serum, 2mmol/L glutamine,
100U/ml penicillin and 100 μ g/ml streptomysins) in 37 DEG C, 5%CO2 culture, change within every 2~3 days liquid 1 time.It is aobvious in inversion daily
Micro- microscopic observation cell growth condition.When cell grows to 80% converging state, passed with 0.25% trypsase by 1:2 digestion
Generation.By detecting surface marker CD44, CD90, CD146, STRO-1 of mescenchymal stem cell and the Multidirectional Differentiation energy of stem cell
The methods of power, clonality identify mescenchymal stem cell.
It constructs virus particle: using the SiRNA of the programming SFRP2 of Whitehead research institute, being inserted into slow virus carrier
On be built into the plasmid of SFRP2shRNA.
Virus packaging and collection: slow virus plasmid and corresponding packaging plasmid (VSVG and dv-8.2) transfect 293T cell,
48 hours after transfection, supernatant is collected, virus titer identification is carried out, is saved after packing.
The foundation of stably transfected cell line: slow-virus transfection SCAPs stem cell uses drug 48 hours after transfection
Puromycin is screened 3 days, and screening obtains the stably transfected cell line of Scramsh, SFRP2sh and SFRP2, SFRP2Vector.
1 stem cell skeletonization of embodiment/at the in vitro study of tooth differentiation biology performance influence
By detecting early stage Osteoblast Differentiation index-alkaline phosphatase, late osteogenic index-calcium tubercle forms and in mRNA water
Flat detection division guideline (RUNX2, COL1, OPN, BSP, OCN etc.) observation is in stem cell in vitro to skeletonization/at dentine direction
The ability of differentiation.
Take the tip of a root dental papilla stem cell for being overexpressed SFRP2 and control group with 2 × 103/cm2Concentration be inoculated in 6 orifice plates
In, after cell grows to 80% fusion, with osteogenic induction culture solution by umbilical cord stem cells in vitro to skeletonization/at dentine side
To induction, 1 not good liquor is changed within every 2 days, in light microscopic observation calcium tubercle formational situation.
Alkaline phosphatase activities are measured as shown in Fig. 1 (a), after 3 days to detect early stage Osteoblast Differentiation index-alkaline phosphatase
Enzyme detects Alizarin red staining and measurement calcium ion concentration as shown in Fig. 1 (b, c), after 2 weeks, 3 weeks to detect late osteogenic index-
Cell mineralization ability.Cell is harvested such as Fig. 1 (d~i) and in the different time points of induction 0d, 3d, 7d, 10d, 14d weeks, in mRNA
Level detection skeletonization/and at tooth division guideline: BSP, OPN, COL1A2, DSPP, DMP1, OSX.
The result shows that overexpression SFRP2 promotion tip of a root dental papilla stem cell in vitro skeletonization/break up at tooth.
Specific experiment method:
1, determination of alkaline phosphatase activity:
Prepare buffer: lysate
Stock substrate Sol.1 capsule 5ml pure water (4 DEG C of storages).
Operating procedure:
1) standard curve is made with standard items；
2) culture medium is removed, PBS is washed 2 times；
3) add 300 μ l lysates, 37 DEG C of shaking tables, 15min；
4) cell is gently scraped, moves to 1.5ml EP pipe, 4 DEG C of centrifugation 5min；
5) supernatant is taken, another EP pipe is moved to；
6) capsule is taken, 5ml pure water dissolution (Stock substrate Sol.) is added；
7) it takes 50 μ l ALP buffers that 50 μ l Stock substrate Sol. are added, mixes well；
8) it is placed in 96 orifice plates, 100 μ l (7) liquid, 10 μ l supernatants, 37 DEG C of incubation 15min is added；
9) 110 μ l 0.5N NaOH are added and terminate reaction；
10) 405nm wavelength absorbance value (OD value) is read in microplate reader；
2, Alizarin red staining:
1) culture medium is discarded, PBS is washed 2 times；
2) 70% ethyl alcohol is fixed, and 4 DEG C, 1h；
3) distilled water is washed 2 times；
4) 40mM alizarin red aqueous solution (pH 4.2) room temperature dyes 1-10min, visually observes coloring case；
5) distilled water is washed 5 times, is gently blown and beaten；
6) scanner takes the photograph type collection image thoroughly.
3, Ca2+ Concentration Testing:
1) after Alizarin red staining, 10%w/v CPC is added, is placed at room temperature for 30min (AR-S is dissolved in CPC)；
2) with 1:10 dilute solution, its absorbance value (OD) is measured with 562nm wavelength in microplate reader
3) Ca is calculated with AR-S standard curve2+Relative concentration.
4, RT-PCR primer sequence:
5, RNA is extracted
1) culture dish cell abandons supernatant, and PBS is rinsed 2 times, adds 700ul QIAZOL, and piping and druming mixes, and closes at ep pipe, and room temperature is incubated
5min is educated, 140ul chloroform is added, strength concussion mixes 15s, is incubated at room temperature 3min, and 4 DEG C of 12000g are centrifuged 15min, receives supernatant in new
EP pipe；
2) take 700ul sample to RNeasy Mini column, 4 DEG C of 8000g centrifugation 15s, abandoning lower liquid；
3) plus 700ul Buffer RWT to RNeasy Mini column, 4 DEG C of 8000g are centrifuged 15s, abandon lower liquid；
4) plus 500ul Buffer RPE to RNeasy Mini column, 4 DEG C of 8000g are centrifuged 15s, abandon lower liquid；
5) step (4) are repeated；
6) the new 2ml collection tube of RNeasy Mini column to one is shifted, 4 DEG C of 1000g are centrifuged 1min, abandon
Lower liquid；
7) the new 2ml collection tube of RNeasy Mini column to one is shifted, 30-50ul RNase- is added
Free water, 4 DEG C of 8000g are centrifuged 15s, collect lower liquid and manage in new EP, survey RNA concentration, -80 DEG C of preservations.
6, reverse transcription PCR
1) following template ribonucleic acid/primer mixed liquor is prepared in Microtube pipe；
2) 65 DEG C of heat preservations rapid freezing on ice 1 minute or more after five minutes；
3) the centrifugation several seconds makes template ribonucleic acid/primer denaturing soln be gathered in Microtube bottom of the tube；
4) it is prepared in above-mentioned Microtube pipe and following inverse transcription reaction liquid is added；
5) mix each component, 37 DEG C heat preservation 2 minutes after cooled on ice；
6) 1ul M-MLV reverse transcriptase is added, gently piping and druming mixes；
7) 2 minutes are kept the temperature for 37 DEG C, 70 DEG C keep the temperature 15 minutes, and 4 DEG C of heat preservations, sample closes at -20 DEG C.
7、Real-time PCR
It is as follows to configure Real-time PCR reaction system:
Real-time PCR reaction system is as follows:
The In vivo study that 2 stem cell biology performance of embodiment influences
It will be overexpressed and the tip of a root dental papilla stem cell of knockout group mix with hydroxyapatite and calcium triphosphate (HA/TCP)
After be transplanted to nude mice by subcutaneous, observe histological variation to study skeletonization in stem cell body/at tooth differentiation capability.As shown in Fig. 2,
It is overexpressed SFRP2 as the result is shown and promotes SCAPs skeletonization/at dentine differentiation capability.As shown in figure 3, gene knockout as the result is shown
SFRP2 inhibition SCAPs skeletonization/at dentine differentiation capability.
The tip of a root dental papilla stem cell for taking SFRP2 to knock out is with 2 × 103/cm2Concentration be inoculated in 6 orifice plates, culture discovery
Cell gradually apoptosis.It detects transcription factor discovery OSX expression relevant to skeletonization and is decreased obviously (Fig. 4).
Internal transplantation experiments method:
1) the good 4th generation above-mentioned different grouping cell of growth conditions is taken respectively, is inoculated with the density of 1 × 105/ware
10cm culture dish abandons culture medium when cell length to 80% convergence degree, and (to guarantee cell state, cell confluency degree is or not PBS rinsing
It is preferably excessive).
2) every group of cell adds II Collagenase Type of 2.5ml (TLC containing 100ug/ml), 37 DEG C of incubation 5min, by cell from culture
It elutes on ware, then cell suspension is transferred in the centrifuge tube of 50ml.Be added in advance in centrifuge tube 5-10ml containing 20%FBS,
α-MEM the culture medium of 10-8M dexamethasone and L-Glutamine.
2) 37 DEG C of incubation 2-5min of 2.5ml pancreatin are added into each centrifuge tube.
3) 1000rpm, 4 DEG C of centrifugation 5min.
4) cell, meter is resuspended containing 20%FBS, 10-8M dexamethasone and the α-MEM culture medium of L-Glutamine with above-mentioned
Number, take 1ml cell suspension into the 2ml round bottom EP pipe for containing 40mg hydroxyapatite (HA) (to guarantee that every group of cell concentration is consistent,
About 2-3 × 106 cell/group).
5) 37 DEG C, 1.5h is incubated under the conditions of 5%CO2.To guarantee that cell is sufficiently combined with HA, should be incubated in the case where shake
It educates.
6) it is centrifuged in short-term, HA is made to be sunken to tube bottom, suct clearly, cell and HA mixture are stand-by.
7) nude mice is weighed, 1% yellow Jackets intraperitoneal injection of anesthesia.75% alcohol disinfecting skin of back, in dorsal midline
Cut the notch of a 2-3cm in place.
8) cell and HA mixture are implanted into nude mice by subcutaneous skin suture by blunt separation subcutaneous connective tissue.
9) the 8th week after transplanting, transplanted cells is obtained and are fixed with 10% formalin, 10%EDTA decalcification (pH value 8.0),
Paraffin embedding, HE dyeing, observational measurement tissue mineralization amount；
The research of 3 regulatory mechanism of embodiment:
By transfect shRNA method respectively realizes knock out KDM2A and BCOR, by RT-PCR detection knockout as a result,
Using H3K36me2 the and H3K4me3 first in the ChIP method detection site SFRP2 transcription site+666~+583bp of upstream promoter
The case where base, the results showed that knocking out KDM2A or BCOR can be improved+666~+583bp of SFRP2 transcription site upstream promoter
The level of H3K36me2 and the H3K4me3 methylation in site.It knocks out KDM2A or BCOR and passes through the group egg of raising SFRP2 promoter
White methylation is to improve expression of the SFRP2 in tip of a root dental papilla stem cell.
Experimental method:
(1) culture medium in tissue culture plate is sopped up, is added 1% formaldehyde of 10ml (10% formaldehyde of 9ml PBS+1ml), 37
DEG C 10 minutes, supernatant is abandoned, is washed 2 times using the PBS that protease is added.
(2) supernatant is abandoned, cell is moved into 1.5ml EP and is managed, 4 DEG C, 2500rpmx4min
(3) 200ul SDS endochylema lysate is added according to 1 × 106 cell, is incubated for 10 minutes on ice,
(4) DNA is processed into 200~1000 base size segments on ice
(5) 4 DEG C, 13000rpm is centrifuged 10 minutes, and supernatant is taken to be transferred to new EP pipe.
(6) supernatant is diluted according to the ChIP buffer that protease inhibitors is added in 10 times of applications
(7) 75ulSalmon Sperm DNA/Protein agar syrup is added in 2ml dilution to stir 30 minutes, centrifugation
Supernatant is put into new EP and managed by removal agar syrup.
(8) primary antibody is added the pretreated supernatant of 2ml, 4 DEG C of rotations be incubated for 4 hours~it is overnight.
(9) it is anti-to collect that 4 DEG C of syrup of 60ul Salmon Sperm DNA/Protein agar rotation processing 1 hour is added
Body histone complexes
(10) low-speed centrifugal (1000G 1 minute) removes agar syrup, and careful removal is comprising being not associated with chromatinic supernatant
(11) sediment composite successively is cleaned with following solutions.The step of cleaning: being added solution, runs at 4 degree and turns 10min, and 4
Degree stands 10min precipitating, and 700rpm is centrifuged 1min, removes supernatant.
(12) after cleaning, start to elute.Every pipe is added 250ul and elutes buffer, and top turns 15min at room temperature, stands
After centrifugation, supernatant is collected.Repeated washing is primary.Final eluent is every pipe 500ul.
(13) 20ul 5M NaCl (the final concentration of 0.2M of NaCl) solution crosslinking: is added in every pipe.It mixes, 65 degree of solution crosslinkings 4
Hour.10ul0.5M EDTA is added, 20ul1M Tris-Hcl, PH 6.5 and 45 DEG C of 2ul 10mg/ml Proteinase K are incubated for 1
Hour.
(14) recycling of DNA sample, chloroform recovery and alcohol precipitation, 70% alcohol wash drying after precipitating, are dissolved in water preservation.
(15) PCR carries out DNA detection
Embodiment 4SFRP2 by inhibit WNT access come realize skeletonization/at tooth differentiation direction regulate and control
Phosphorylation beta-catenin can be raised by being overexpressed SFRP2, reduce the content of beta-catenin in core, on the contrary
Phosphorylation beta-catenin can be lowered by knocking out SFRP2, increase the content of beta-catenin in core.By WNT1A using inverse
Retroviral is observed after being knocked out, as shown in figure 5, observing using the method for alkaline phosphatase detection and calcium ion quantitative analysis
Osteoblast Differentiation ability to SCAPs obviously weakens, and design can be bright after being saved using transwell experimental applications SFRP2
The aobvious reduction for improving SCAPs osteogenic ability.
Western Blot:
1) extraction of total protein of cell
1. culture medium is abandoned, twice with the 4 DEG C of 5ml PBS being pre-chilled rinsing cells, after 5ml PBS is added, with training under cell scraper
The cell in ware is supported, 15ml centrifuge tube is closed at, 1100rpm is centrifuged 6min；Supernatant is abandoned, 1ml PBS is added, cell is resuspended, close at EP
Pipe, 7200rpm are centrifuged 2min；
2. abandoning supernatant, lysate (100ul RIPA+1ul PMSF+1ul is added with 1:5 (cell: lysate) volume ratio
PIC), 15min, every 2-3min are suspended primary on ice；
3. 4 DEG C, 14,000rpm centrifugation 15min collect supernatant in new EP pipe, -80 DEG C of preservations.
2) Bradford method measures protein concentration
1. BSA protein standard substance is illustratively successively diluted with distilled water, making final concentration is respectively 1000 μ g/ml, 750 μ g/
Ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 0 μ g/ml；
2. adding 200ul 1X Coomassie brilliant blue liquid according to sample and standard items quantity by every hole, taking 1ul sample and standard items
It is added in 96 orifice plates, 5min is incubated at room temperature, if secondary orifices and blank well；
3. measuring the absorbance of 595nm wavelength.Protein concentration is calculated according to standard curve；
4. calculating loading volume according to protein concentration, the applied sample amount of every histone is 25 μ g.
3) polyacrylamide gel electrophoresis
1. preparing pre- plastic；
2. denaturation: every 25 μ g protein content loading of hole, with distilled water by sample to be tested volume dilution to 20ul, every sample is added
5ul loading buffer (bromophenol blue), 95 DEG C of heating 10min；
3. voltage is transferred to 100V with 80V electrophoresis to bromophenol blue electrophoresis to separation gel by loading, until bromophenol blue arrives
Up to separation gel bottom, electrophoresis is terminated.
4) transferring film
1. preparing membranae praeformativa；
2. running gel is transferred in membranae praeformativa, row transferring film is rotated into using BioRad is half-dried；
3. taking out pvdf membrane, TBST rinses 5min.
5) Western blot filter hybridization
1. 5% skimmed milk power room temperature shaker of the pvdf membrane after transfer is closed 1 hour；
2. TBST rinses 10min × 3 time；
3. filter membrane is taken out, it is put into the diluted primary antibody dilution of 5% skimmed milk power, 4 DEG C of shaking tables are stayed overnight；
4. TBST washes film, 10min × 3 time；
5. filter membrane is put into the secondary antibody of the diluted HRP label of 5% skimmed milk power, room temperature shaker is incubated for 1 hour；
6. TBST washes film, 10min × 3 time.
6) it develops the color
Pvdf membrane glue surface is placed on preservative film upward, the developer solution of 1:1 mixing is added, makes its uniform fold film, darkroom
Exposure, scanning.
Determination of alkaline phosphatase activity and Alizarin red staining method are the same.
In conclusion SFRP2 can promote the tooth of SCAP cell to/bone to differentiation, have an effect may by with tune
It is related to relevant important transcription factor OSX is broken up to control bone, BCOR and KDM2A complex can be by starting sub-portion to SFRP2
The change of position methylation is to regulate and control the transcriptional expression of SFRP2.It to tooth to differentiation is to pass through that it, which promotes dental papilla stem cell bone,
The WNT access of classics is inhibited to realize.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (3)
1. having and only SFRP2 being in the drug or preparation preparation promotion Odontogenic cysts mescenchymal stem cell skeletonization and/or broken up at tooth
In application.
2. having and only SFRP2 in preparation promoting Odontogenic cysts mescenchymal stem cell to mediate application in the regenerated drug of full tooth.
3. application according to claim 2, it is characterised in that: the drug is tablet, granule, oral solution, injection
Liquid or capsule.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510246730.4A CN105561338A (en) | 2015-05-14 | 2015-05-14 | Application of SFRP2 to promotion of odontogenic mesenchymal stem cell osteogenic/odontoblastic differentiation |
| CN2015102467304 | 2015-05-14 |
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| CN105879057A CN105879057A (en) | 2016-08-24 |
| CN105879057B true CN105879057B (en) | 2019-06-07 |
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| CN201610262569.4A Active CN105879057B (en) | 2015-05-14 | 2016-04-25 | SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application |
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| CN110499283A (en) * | 2018-05-17 | 2019-11-26 | 西安组织工程与再生医学研究所 | Wnt signal path activator improves the application in low alkalinity phosphate disease stem cell Osteoblast Differentiation ability abnormal products in preparation |
| CN111979184B (en) * | 2020-06-30 | 2023-02-17 | 首都医科大学附属北京口腔医院 | Application of KDM3B gene in differentiation, proliferation and migration chemotaxis of mesenchymal stem cells in bone direction/tooth direction |
| CN114107184A (en) * | 2021-11-26 | 2022-03-01 | 山东中医药大学第二附属医院 | Application of siRNA in osteogenic differentiation of dental pulp stem cells |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449448A (en) * | 2000-08-19 | 2003-10-15 | 爱克斯澳迪亚有限公司 | Stem cell differentiation |
| EP1639097A2 (en) * | 2003-06-25 | 2006-03-29 | Ottawa Health Research Institute | Methods and compositions for modulating stem cell growth and differentiation |
| CN1833033A (en) * | 2003-06-06 | 2006-09-13 | 惠氏公司 | Methods and materials for identifying agents which modulate bone remodeling and agents identified thereby |
| EP1940445A2 (en) * | 2005-08-19 | 2008-07-09 | Duke University | Stem cell derived factors for treating pathologic conditions |
| CN104450621A (en) * | 2014-09-30 | 2015-03-25 | 首都医科大学附属北京口腔医院 | Regulating method of WDR63 gene in osteogenic differentiation and odontogenic differentiation processes of mesenchymal stem cell |
-
2015
- 2015-05-14 CN CN201510246730.4A patent/CN105561338A/en active Pending
-
2016
- 2016-04-25 CN CN201610262569.4A patent/CN105879057B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449448A (en) * | 2000-08-19 | 2003-10-15 | 爱克斯澳迪亚有限公司 | Stem cell differentiation |
| CN1833033A (en) * | 2003-06-06 | 2006-09-13 | 惠氏公司 | Methods and materials for identifying agents which modulate bone remodeling and agents identified thereby |
| EP1639097A2 (en) * | 2003-06-25 | 2006-03-29 | Ottawa Health Research Institute | Methods and compositions for modulating stem cell growth and differentiation |
| EP1940445A2 (en) * | 2005-08-19 | 2008-07-09 | Duke University | Stem cell derived factors for treating pathologic conditions |
| CN104450621A (en) * | 2014-09-30 | 2015-03-25 | 首都医科大学附属北京口腔医院 | Regulating method of WDR63 gene in osteogenic differentiation and odontogenic differentiation processes of mesenchymal stem cell |
Non-Patent Citations (3)
| Title |
|---|
| sFrp2和sFrp3在小鼠磨牙发育中的作用;韩萍萍;《中国优秀硕士学位论文全文数据库基础科学辑》;20120228;摘要、第1.2.2节 * |
| 间充质干细胞向成骨细胞分化中的Wnt信号通路;潘京华等;《中国组织工程研究》;20131024;摘要、第7147页左栏最后一段 * |
| 韩萍萍.sFrp2和sFrp3在小鼠磨牙发育中的作用.《中国优秀硕士学位论文全文数据库基础科学辑》.2012,摘要、第1.2.2节. * |
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Inventor after: Fan Zhipeng Inventor after: Wang Songling Inventor after: Yu Guoxia Inventor after: Wang Jinsong Inventor after: Yang Haoqing Inventor after: Li Guoqing Inventor before: Fan Zhipeng Inventor before: Wang Songling Inventor before: Yu Guoxia Inventor before: Wang Jinsong |