CN105879057A - Application of SFRP2 in promoting osteoblastic/odontoblastic differentiation of odontogenic mesenchymal stem cells - Google Patents
Application of SFRP2 in promoting osteoblastic/odontoblastic differentiation of odontogenic mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides application of SFRP2 in promoting osteoblastic/odontoblastic differentiation of odontogenic mesenchymal stem cells and also provides application of SFRP2 in the preparation of drugs for promoting regeneration of mediated tissues in odontogenic mesenchymal stem cells. By studying a mechanism to directionally differentiate mesenchymal stem cells under regulation with WNT signal regulation factor SFRP2, it is discovered that SFRP2 can function in promoting odontogenic-osteoblastic differentiation of stem cells; a possible mechanism of SFRP2 is described, thereby providing important leads for studying time-space specific molecular regulation mechanism for growth and development of teeth and bones and reconstructing biological full-tooth generated seed cells.
Description
This application claims and submitted Patent Office of the People's Republic of China, Application No. on 05 14th, 2015
201510246730.4, invention entitled " SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/one-tenth tooth
Application in differentiation " the priority of Chinese patent application, entire contents is hereby incorporated by the application
In.
Technical field
The present invention relates to stem cells technology field, be specifically related to WNT Signal Regulation factor S FRP2 and promote
Application in Odontogenic cysts mescenchymal stem cell skeletonization/one-tenth tooth directed differentiation.
Background technology
One of absence of tooth reparation and the paradenlal tissue regeneration research emphasis being always Oral Science.Mesenchyme
Stem cell plays an important role in tooth development and tissue regeneration processes, and development abnormalities of teeth includes number
Mesh, form, textural anomaly, these are all relevant to the gene expression during tooth development and dysfunction,
Thus cause the changes of function of cell particularly stem cells hyperplasia differentiation in growth course to cause sending out of disease
Raw.In recent years, the progress of mescenchymal stem cell and tissue engineering technique is to tooth or periodontal tissue again
Life brings hope.Mescenchymal stem cell, has multi-lineage potential and the ability freely updated, permissible
It is divided into corresponding tissue.Although the tooth of mescenchymal stem cell mediation and support tissue regeneration achieve
Achievement in research, but it is urgently to be resolved hurrily to there is also some key issues at present, and one of them is that the kind regenerated is careful
Born of the same parents-Odontogenic cysts source of human stem cell is limited, and the molecular regulation mechanism of the differentiation of Odontogenic cysts stem cell directional and propagation is still
Not the most clearly.Stem cell can carry out heteroplastic transplantation for treating, but due to the donor age and other one
The impact of a little factors, some stem cell is the oldest and the most feeble, and therapeutic effect is not good enough, and due to adult stem
Life cycle is of short duration causes its long-term treatment effects poor.If stem cell to be carried out heredity reconstruction so that it is
Reprogramming, activates or suppresses to regulate the gene of aging course, make stem cell the youngest, thus give and he
New life；Or it is used further to control after improving the qualitative differentiation capability of stem cell and extending its life cycle
Treat, thus improve the therapeutic effect of stem cell.Although a lot of researchs show mescenchymal stem cell heteroplastic transplantation
After there is immunosuppressive action, but all lack long-term follow-up report.Long-term heteroplastic transplantation is likely deposited
Rejection, ethics and the impact of some other factor, make patient be difficult to accept.With patient oneself
Stem cell cultivate in vitro during, carry out heredity reconstruction so that it is stem cell recovers normal
Function, especially for the patient of some heredopathia, discloses the pathogenesis of its disease, on the one hand helps
In disease early diagnosis, prevent and treat；On the other hand can pointedly their stem cell be adopted
Taking and activate abnormal quiet gene or the gene of quiet abnormal activation, so that reconstructing later, having
The stem cell of pluripotency has wide clinical value for autotransplantation.
SFRP2 is a member of the WNT Signal Regulation factor S FRPs gene family of a solubility.
SFRPs gene family is positioned chromosome 8p 12～11.1, including 5 member: SFRP1, SFRP2,
SFRP3, SFRP4 and SFRP5, belong to secreting type frizzled associated protein, as the Wnt of solubility
The Signal Regulation factor, suppresses Wnt signal to lead to by being combined with the Receptor Competition of Wnt signal path homology
Road, thus play weight in the life processes such as biological development, cell traffic, tumor formation and apoptosis
The effect wanted.In addition SFRP2 can also suppress the activity of RANKL thus suppress osteoclast activity,
And be combined with Tolloid metalloproteases and to regulate collagen and formed, or with integrin and fibronectin
Composite bulk phase interaction regulating cell apoptosis.Current study show that SFRP2 is this status of mescenchymal stem cell
The important factor secreted, plays important regulative during the self renewal of mescenchymal stem cell,
And mesenchymal stem cells MSCs myocardial repair effect can be strengthened.But SFRP2 is fixed to mescenchymal stem cell
Unclear to impact and the mechanism of differentiation, need to further investigate.
By the SFRP2 research to mescenchymal stem cell function effect, it can be research mescenchymal stem cell
Procedural reactivation and Functional Remodeling carry out theoretical preparation, wish on this basis find corresponding medicine
The state of thing or biological preparation control stem cell reaches the purpose of clinical practice and treatment, solves patient's
Misery, recovers the physical and mental health of patient, and will produce preferable economic benefit and social benefit.
Summary of the invention
It is an object of the invention to the defect for prior art, it is provided that a kind of WNT Signal Regulation factor
The application of SFRP2 regulation and control mescenchymal stem cell directed differentiation, in order to realize the purpose of the present invention, intends using
Following technical scheme.
The invention provides SFRP2 answering in promoting Odontogenic cysts mescenchymal stem cell skeletonization/one-tenth tooth differentiation
With.
Present invention also offers SFRP2 and promote the regeneration of Odontogenic cysts mescenchymal stem cell mediating tissue as preparation
The application of medicine.
Medicine of the present invention needs to can be prepared as any pharmaceutically acceptable dosage form according to processing,
Its configuration generally by well known to a person skilled in the art prepared by processing method, will active component molten with liquid
Agent or solid carrier mixing, be prepared from adding at least one surfactant, be the most more preferably
Tablet, granule, oral liquid, injection or capsule.
Medicine of the present invention or directly can be used through dilution by user before use.
Beneficial effects of the present invention is by fixed to WNT Signal Regulation factor S FRP2 regulation and control mescenchymal stem cell
To the research that carries out of mechanism of differentiation, find that it has and promote that stem cell tooth is to the left and right of differentiation and right to-bone
Its possible mechanism is illustrated, thus for studying tooth and the molecule of bone growth and development Space-time speciality
The reconstruction of the seed cell of regulatory mechanism and the regeneration of biological full tooth provides important clue.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to reality
Execute the required accompanying drawing used in example or description of the prior art to be briefly described.
Fig. 1 shows Osteoblast Differentiation alkali phosphatase index in early days；A is that alkaline phosphatase activities detects to become in early days
Bone division guideline-alkali phosphatase；B is for surveying Alizarin red staining detection late osteogenic index-cell mineralization ability；
C detects late osteogenic index-cell mineralization ability for measuring calcium ion concentration；D～i is 0d, 3d, 7d,
The different time points harvesting in 10d, 14d week；
Fig. 2 shows that process LAN SFRP2 promotes SCAPs skeletonization/one-tenth dentin differentiation capability；
Fig. 3 shows that gene knockout SFRP2 suppresses SCAPs skeletonization/one-tenth dentin differentiation capability；
Fig. 4 shows the tip of a root dental papilla stem cell change that SFRP2 knocks out；
Fig. 5 shows that the method for alkali phosphatase detection and Alizarin red staining observes the Osteoblast Differentiation energy of SCAPs
Power；A is alkali phosphatase detection；B is Alizarin red staining.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further, the following stated, only to the present invention
Preferred embodiment, not does the restriction of other forms, any technology being familiar with this specialty to the present invention
Personnel are changed to the Equivalent embodiments changed on an equal basis possibly also with the technology contents of the disclosure above.Every
Without departing from the present invention program content, any letter following example done according to the technical spirit of the present invention
Single amendment or equivalent variations, all fall within protection scope of the present invention.Following experimental technique is not carrying out spy
It is this area routine techniques means in the case of different explanation.
The separation of stem cell, cultivate and identify:
The utilization of tissue obtains the approval of Ethics Committee of the Capital University of Medical Sciences, and volunteer all knows the inside story same
Anticipate preoperative signing Informed Consent Form.Obtain people permanent teeth periodontal tissue and permanent teeth inflammatory periodontal tissue, dental pulp
Tissue, or permanent teeth (exodontia of correction subtrahend or the young wisdom teeth) tip of a root dental papilla that correction needs are pulled out
Tissue, separates and cultivates PDLSCs, DPSCs, SCAPs, summary according to the method for previous literature report
It is as follows: the tooth pulled out to be immediately placed in the aseptic centrifuge tube equipped with pre-cooling PBS, transfers into Cytology Lab,
Separation and Culture periodontal ligament stem cell, pulp cells, tip of a root dental papilla cells in 24h.Peel off tooth gently
The periodontal tissue that tooth rim encloses, takes the periodontal tissue in stage casing, and mill leaves pulp cavity, takes out dental pulp, or directly cuts
Take tip of a root dental papilla site tissue, repeatedly clean with PBS, shred, be placed in containing NTx enzyme (3g/L)
With the Digestive system of Dispase (4g/L), digest 1 hour at 37 DEG C, cross 70 μm cell sieves and collect cell,
1000rpm is centrifuged 10min, becomes single cell suspension with culture fluid Eddy diffusion.Cell is inoculated in 25cm2
In Tissue Culture Flask, α-MEM culture medium (containing 15% hyclone, 2mmol/L glutamine, 100
U/ml penicillin and 100 μ g/ml streptomycins) in 37 DEG C, 5%CO2 cultivate, within every 2～3 days, change liquid 1
Secondary.Every day is observation of cell upgrowth situation under inverted microscope.When cell grows to 80% converging state,
1:2 had digestive transfer culture is pressed with 0.25% trypsin.By detecting the surface marker of mescenchymal stem cell
The Multidirectional Differentiation ability of CD44, CD90, CD146, STRO-1 and stem cell, clonality etc.
Method identifies mescenchymal stem cell.
Build virus particle: apply the SiRNA of the programming SFRP2 of Whitehead institute, insert
Enter to be built on slow virus carrier the plasmid of SFRP2shRNA.
Virus packaging and collection: slow virus plasmid and corresponding packaging plasmid (VSVG and dv-8.2) transfection
293T cell, transfects latter 48 hours, collects supernatant, carry out virus titer qualification, preserves after subpackage.
The foundation of stably transfected cell line: slow-virus transfection SCAPs stem cell, transfects latter 48 hours, uses
Medicine puromycin screens 3 days, and screening obtains Scramsh, SFRP2sh and SFRP2, SFRP2Vector
Stably transfected cell line.
The in vitro study of embodiment 1 stem cell skeletonization/one-tenth tooth differentiation biology performance impact
By detection in early days Osteoblast Differentiation index-alkali phosphatase, late osteogenic index-calcium tuberosity formed and
MRNA level in-site detection division guideline (RUNX2, COL1, OPN, BSP, OCN etc.) is observed
Stem cell is in vitro to the ability of skeletonization/one-tenth dentin direction differentiation.
Take the tip of a root dental papilla stem cell of process LAN SFRP2 and matched group with 2 × 103/cm2Concentration inoculation
In 6 orifice plates, after cell grows to 80% fusion, with osteogenic induction culture fluid, umbilical cord stem cells is existed
External within every 2 days, change 1 not good liquor to skeletonization/one-tenth dentin direction induction, at light Microscopic observation calcium tuberosity
Formational situation.
As shown in Fig. 1 (a), measure after 3 days alkaline phosphatase activities detect in early days Osteoblast Differentiation index-
Alkali phosphatase, as shown in Fig. 1 (b, c), 2 weeks, after 3 weeks detect Alizarin red staining and measure calcium from
Sub-concentration detects late osteogenic index-cell mineralization ability.As Fig. 1 (d～i) and induction 0d, 3d,
The different time points harvesting in 7d, 10d, 14d week, detects skeletonization/one-tenth tooth differentiation in mRNA level in-site
Index: BSP, OPN, COL1A2, DSPP, DMP1, OSX.
Result shows that process LAN SFRP2 promotes tip of a root dental papilla stem cell in vitro skeletonization/one-tenth tooth differentiation.
Specific experiment method:
1, determination of alkaline phosphatase activity:
Preparation buffer: lysate
Stock substrate Sol.1 capsule 5ml pure water (4 DEG C of storages).
Operating procedure:
1) standard curve is made with standard substance；
2) going culture medium, PBS washes 2 times；
3) 300 μ l lysates, 37 DEG C of shaking tables, 15min are added；
4) cell is scraped gently, move to 1.5ml EP pipe, 4 DEG C of centrifugal 5min；
5) take supernatant, move to another EP pipe；
6) take capsule, add 5ml pure water and dissolve (Stock substrate Sol.)；
7) take 50 μ l ALP buffer and add 50 μ l Stock substrate Sol., fully mix；
8) it is placed in 96 orifice plates, adds 100 μ l (7) liquid, 10 μ l supernatant, hatch 15min for 37 DEG C；
9) add 110 μ l 0.5N NaOH and terminate reaction；
10) 405nm wavelength absorbance value (OD value) is read in microplate reader；
2, Alizarin red staining:
1) discarding culture medium, PBS washes 2 times；
2) 70% ethanol is fixed, 4 DEG C, 1h；
3) distilled water washes 2 times；
4) 40mM alizarin red aqueous solution (pH 4.2) room temperature dyeing 1-10min, perusal coloring case；
5) distilled water is washed 5 times, blows and beats gently；
6) scanner takes the photograph type collection image thoroughly.
3, Ca2+ Concentration Testing:
1) after Alizarin red staining, adding 10%w/v CPC, room temperature places 30min, and (AR-S is dissolved to
In CPC)；
2) with 1:10 dilute solution, in microplate reader, its absorbance (OD) is measured with 562nm wavelength
3) Ca is calculated with AR-S standard curve2+Relative concentration.
4, RT-PCR primer sequence:
5, RNA extracts
1) culture dish cell abandons supernatant, and PBS rinses 2 times, adds 700ul QIAZOL, and piping and druming mixes,
Closing at ep pipe, incubated at room 5min, add 140ul chloroform, strength is shaken and is mixed 15s, incubated at room 3min,
4 DEG C of 12000g are centrifuged 15min, receive supernatant and manage in new EP；
2) take 700ul sample to be centrifuged 15s to RNeasy Mini column, 4 DEG C of 8000g, abandon subnatant
Body；
3) add 700ul Buffer RWT to RNeasy Mini column, 4 DEG C of 8000g and be centrifuged 15s,
Abandon lower floor's liquid；
4) add 500ul Buffer RPE to RNeasy Mini column, 4 DEG C of 8000g to be centrifuged 15s, abandon
Lower floor's liquid；
5) step (4) is repeated；
6) transfer RNeasy Mini column to new 2ml collection tube, 4 DEG C of 1000g is centrifuged 1
Min, abandons lower floor's liquid；
7) the new 2ml collection tube of transfer RNeasy Mini column to, adds 30-50ul
RNase-free water, 4 DEG C of 8000g are centrifuged 15s, collect lower floor's liquid and manage in new EP, survey RNA dense
Degree ,-80 DEG C of preservations.
6, reverse transcriptional PCR
1) Microtube pipe is prepared following template ribonucleic acid/primer mixed liquor；
2) rapidly at freezing more than 1 minute on ice after 65 DEG C of insulations 5 minutes；
3) the centrifugal several seconds makes the denaturing soln of template ribonucleic acid/primer be gathered in bottom Microtube pipe；
4) in above-mentioned Microtube pipe, preparation adds following inverse transcription reaction liquid；
5) mix each component, 37 DEG C insulation 2 minutes after cooled on ice；
6) add 1ul M-MLV reverse transcriptase, blow and beat mixing gently；
7) 37 DEG C are incubated 2 minutes, and 70 DEG C are incubated 15 minutes, and 4 DEG C of insulations, sample closes at-20 DEG C.
7、Real-time PCR
Configuration Real-time PCR reaction system is as follows:
Real-time PCR reaction system is as follows:
The In vivo study of embodiment 2 stem cell biology performance impact
The tip of a root dental papilla stem cell by process LAN and knocking out group and hydroxyapatite and calcium triphosphate
(HA/TCP) being transplanted to nude mice by subcutaneous after mixing, the change of tissues observed becomes in studying stem cell body
Bone/one-tenth tooth differentiation capability.As in figure 2 it is shown, result display process LAN SFRP2 promotes SCAPs skeletonization/one-tenth
Dentin differentiation capability.As it is shown on figure 3, result display gene knockout SFRP2 suppression SCAPs skeletonization/
Become dentin differentiation capability.
Take tip of a root dental papilla stem cell that SFRP2 knocks out with 2 × 103/cm2Concentration be inoculated in 6 orifice plates,
Cultivate and find cell gradually apoptosis.Detect the transcription factor relevant to skeletonization and find that OSX expression is obvious
Decline (Fig. 4).
Internal transplantation experiments method:
1) the 4th generation above-mentioned different grouping cell that growth conditions is good is taken respectively, close with 1 × 105/ware
Degree inoculation 10cm culture dish, abandons culture medium when cell length to 80% degree of converging, and PBS rinsing is (for ensureing
Cell state, cell confluency degree is unsuitable excessive).
2) often group cell adds 2.5ml II Collagenase Type (containing 100ug/ml TLC) 37 DEG C and hatches 5min, will
Cell is eluting from culture dish, is then transferred in the centrifuge tube of 50ml by cell suspension.Centrifuge tube carries
Front addition 5-10ml contains 20%FBS, 10-8M dexamethasone and the α-MEM culture medium of L-glutaminate.
2) in each centrifuge tube, add 2.5ml pancreatin 37 DEG C and hatch 2-5min.
3) 1000rpm, 4 DEG C of centrifugal 5min.
4) by the above-mentioned α-MEM culture medium containing 20%FBS, 10-8M dexamethasone and L-glutaminate
Re-suspended cell, counting, take the 1ml cell suspension 2ml extremely containing 40mg hydroxyapatite (HA)
Round bottom EP pipe (to ensure often to organize cell concentration consistent, about 2-3 × 106 cell/group).
5) 37 DEG C, 1.5h under the conditions of 5%CO2, is hatched.For ensureing that cell is fully combined with HA, Ying
Hatch in the case of shake.
6) being centrifuged in short-term, make HA be sunken at the bottom of pipe, suct clear, cell is stand-by with HA mixture.
7) nude mice is weighed, 1% pentobarbital sodium intraperitoneal injection of anesthesia.75% alcohol disinfecting skin of back,
The otch of a 2-3cm is cut at dorsal midline.
8) blunt separation subcutaneous connective tissue, implants nude mice by subcutaneous skin suture by cell with HA mixture.
9) transplant after the 8th week, acquisition transplanted cells fix with 10% formalin, 10%EDTA decalcification
(pH value 8.0), paraffin embedding, HE dyes, observation measurements tissue mineralization amount；
Embodiment 3 regulatory mechanism is studied:
Realize respectively knocking out KDM2A and BCOR by the method for transfection shRNA, pass through RT-PCR
The result that detection knocks out, application ChIP method detection SFRP2 transcription site upstream promoter+666～+583
The methylated situation of H3K36me2 and H3K4me3 in bp site, result show to knock out KDM2A or
BCOR can improve the H3K36me2 in SFRP2 transcription site upstream promoter+666～+583bp site
And the methylated level of H3K4me3.Knock out KDM2A or BCOR and start by improving SFRP2
The histone methylated of son improves SFRP2 expression in tip of a root dental papilla stem cell.
Experimental technique:
(1) sop up the culture medium in Tissue Culture Plate, add 10ml 1% formaldehyde (9ml
PBS+1ml 10% formaldehyde), 37 DEG C 10 minutes, abandon supernatant, application adds the PBS of protease
Wash 2 times.
(2) abandon supernatant, cell is moved into 1.5ml EP and manages, 4 DEG C, 2500rpmx4min
(3) add 200ul SDS endochylema lysate according to 1 × 106 cell, hatch on ice
10 minutes,
(4) on ice, DNA is processed into 200～1000 base size fragments
(5) 4 DEG C, 13000rpm is centrifuged 10 minutes, takes supernatant and proceeds to new EP pipe.
(6) supernatant is added according to 10 times of application the ChIP buffer dilution of protease inhibitor
(7) 2ml diluent is added 75ulSalmon Sperm DNA/Protein agar syrup
Stirring 30 minutes, supernatant is put into new EP pipe by centrifugal segregation agar syrup.
(8) by the supernatant of an anti-addition 2ml pretreatment, 4 DEG C of rotations are hatched 4 hours～overnight.
(9) 60ul Salmon Sperm 4 DEG C of rotation processing of DNA/Protein agar syrup are added
1 hour to collect antibody histone complexes
(10) low-speed centrifugal (1000G 1 minute) removes agar syrup, and careful removal comprises
Uncombined chromatinic supernatant
(11) sediment composite is cleaned with following solutions successively.The step cleaned: add solution, 4
Degree top turns 10min, and 4 degree stand 10min precipitation, and 700rpm is centrifuged 1min, removes supernatant.
(12), after cleaning, eluting is started.Often pipe adds 250ul eluting buffer, and under room temperature, top turns
15min, stands after being centrifuged, collects supernatant.Repeated washing is once.Final eluent is every pipe 500ul.
(13) crosslinking is solved: pipe often adds 20ul 5M NaCl (the final concentration of 0.2M of NaCl).Mixed
Even, 65 degree solve crosslinking 4 hours.Add 10ul0.5M EDTA, 20ul1M Tris-Hcl, PH 6.5 with
And 2ul 10mg/ml E.C. 3.4.21.64 45 DEG C hatches 1 hour.
(14) recovery of DNA sample, chloroform extraction and alcohol precipitation, 70% ethanol is dried after washing precipitation,
It is dissolved in water to preserve.
(15) PCR carries out DNA detection
Embodiment 4SFRP2 realizes skeletonization/one-tenth tooth differentiation direction by suppression WNT path and regulates and controls
Process LAN SFRP2 can raise phosphorylation beta-catenin, the content of beta-catenin in minimizing core,
Otherwise knock out SFRP2 and can lower phosphorylation beta-catenin, the content of beta-catenin in increase core.
Observe after WNT1A application retrovirus is knocked out, as it is shown in figure 5, application alkali phosphatase
The method of detection and calcium ion quantitative analysis observes that the Osteoblast Differentiation ability of SCAPs substantially weakens, design
Application transwell experimental applications SFRP2 can be obviously improved the fall of SCAPs osteogenic ability after saving
Low.
Western Blot:
1) extraction of total protein of cell
1. abandon culture medium, rinse cell twice with the 5ml PBS of 4 DEG C of pre-coolings, after adding 5ml PBS, use
Cell scrapes the cell in culture dish, closes at 15ml centrifuge tube, and 1100rpm is centrifuged 6min；Abandon supernatant,
Adding 1ml PBS re-suspended cell, close at EP pipe, 7200rpm is centrifuged 2min；
2. abandon supernatant, add lysate (100ul RIPA+1ul with 1:5 (cell: lysate) volume ratio
PMSF+1ul PIC), 15min on ice, every 2-3min suspendible is once；
3. 4 DEG C, 14,000rpm are centrifuged 15min, collect supernatant in new EP pipe ,-80 DEG C of preservations.
2) Bradford method measures protein concentration
1. BSA protein standard substance dilutes with distilled water the most successively, makes final concentration be 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. according to sample and standard substance quantity, add 200ul 1X Coomassie brilliant blue liquid by every hole, take 1ul sample
Product and standard substance add in 96 orifice plates, incubated at room 5min, if secondary orifices and blank well；
3. the absorbance of 595nm wavelength is measured.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. pre-plastic is prepared；
2. degeneration: every hole 25 μ g protein content loading, with distilled water by sample to be tested volume dilution to 20ul,
Every sample adds 5ul loading buffer (bromophenol blue), 95 DEG C of heating 10min；
3. loading, with 80V electrophoresis to bromophenol blue electrophoresis to separation gel, is transferred to 100V by voltage, directly
Arrive bottom separation gel to bromophenol blue, terminate electrophoresis.
4) transferring film
1. membranae praeformativa is prepared；
2. running gel is transferred in membranae praeformativa, uses that BioRad is half-dried rotates into row transferring film；
3. taking out pvdf membrane, TBST rinses 5min.
5) Western blot filter hybridization
1. the pvdf membrane after transfer is closed 1 hour with 5% defatted milk powder room temperature shaker；
2. TBST rinses 10min × 3 time；
3. filter membrane is taken out, put in an anti-diluent of 5% defatted milk powder dilution, 4 DEG C of incubator overnight；
4. TBST washes film, 10min × 3 time；
5. filter membrane is put into 5% defatted milk powder dilution HRP labelling two anti-in, room temperature shaker hatches 1
Hour；
6. TBST washes film, 10min × 3 time.
6) colour developing
Pvdf membrane glue surface is placed on preservative film upward, adds the developer solution of 1:1 mixing so that it is uniformly cover
Epiphragma, darkroom exposes, scanning.
Determination of alkaline phosphatase activity and Alizarin red staining method are the same.
In sum, SFRP2 can promote the tooth of SCAP cell to/bone to differentiation, and it is had an effect and may lead to
Cross relevant to important transcription factor OSX that differentiation is relevant with regulation and control bone, BCOR Yu KDM2A complex
Can by change methylated to SFRP2 promoter site thus regulate and control the transcriptional expression of SFRP2.Its
Promote that dental papilla stem cell bone is to be realized by the WNT path that suppression is classical to tooth to differentiation.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (3)
1.SFRP2 promotes Odontogenic cysts mescenchymal stem cell skeletonization and/or the medicine becoming tooth differentiation or system in preparation
Application in agent.
2.SFRP2 answering in preparation promotes the medicine of Odontogenic cysts mescenchymal stem cell mediating tissue regeneration
With.
Application the most according to claim 2, it is characterised in that: described medicine is tablet, granule
Agent, oral liquid, injection 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 true CN105879057A (en) | 2016-08-24 |
| CN105879057B 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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| CN111979184A (en) * | 2020-06-30 | 2020-11-24 | 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 cell osseous/odontogenic |
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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 |
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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 |