CN104450621B - WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization - Google Patents
WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization Download PDFInfo
- Publication number
- CN104450621B CN104450621B CN201410550443.8A CN201410550443A CN104450621B CN 104450621 B CN104450621 B CN 104450621B CN 201410550443 A CN201410550443 A CN 201410550443A CN 104450621 B CN104450621 B CN 104450621B
- Authority
- CN
- China
- Prior art keywords
- wdr63
- cell
- stem cell
- tooth
- tip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
It is measured to the regulation and control method with tooth into atomization by cell culture, chromatin imrnunoprecipitation reaction and data analysis, plasmid construction and virus transfection, western blot analysis, ability of cell proliferation, alkaline phosphatase and Alizarin red staining, nude mice by subcutaneous carry out cell Hui Zhi in mescenchymal stem cell bone the invention discloses a kind of WDR63 genes；It was found that WDR63 genes in mescenchymal stem cell bone to/regulating and controlling effect of the tooth into atomization, and promote dental tissue regeneration in effect.The present invention using the adjustment effect of the tri-methylated gene activation and function to mescenchymal stem cell for being related to the 4th lysine of Histone 3, WDR63 genes mescenchymal stem cell bone to regulating and controlling effect into atomization of/tooth, be related to effect of the WDR63 genes in promoting dental tissue regeneration, obtaining WDR63 may play a driving role in tip of a root dental papilla stem cell Osteoblast Differentiation.
Description
Technical field
The invention belongs to technical field of bioengineering more particularly to a kind of WDR63 genes mescenchymal stem cell bone to
Regulation and control method of the tooth into atomization.
Background technology
Odontopathy is one of most common disease of the mankind, and the missing strong influence of tooth people's health and life matter
Amount.Though existing dentures repai means are ripe, since it does not have bioactivity, it is difficult to it compares favourably with natural teeth, nothing
Method realizes that the mankind possess the vision of " third pair tooth ".For this purpose, various countries attempt to stem cell and tissue engineering technique to realize tooth
The reconstruction of tooth.How to develop it is a kind of have the function of biological activity with and the tooth of periodontal relationship can be rebuild in jawbone
Missing tooth is repaired, is that clinical dentist and researcher it is expected the key subjects solved jointly.With oral cavity development biology
It learns and the progress of tissue engineering technique, the work of regeneration of tooth research every aspect has been unfolded, and full dental tissue engineering
Optimal strategy is to use autogenous cell, and tooth and its surrounding tissue (including tooth, parodontium, alveolar bone etc.) are carried out at the same time structure
It builds, is then implanted into jaw defect area, to obtain the tooth with certain vigor.In tooth regenerates research field, stem cell is it
Important component part, stem cell are a kind of undifferentiated cells, have the of self-replication capacity and to more differentiation potentials.It is main at present
Be used for dental tissue engineering Odontogenic cysts stem cell have into type I collagen, deciduous teeth dental pulp stem cell, periodontal ligament stem cell,
Tip of a root nipple stem cell, Dental Follicle Cells.Wherein tip of a root dental papilla stem cell (SCAP) is present in the dental papilla group of tooth apical foramen of tooth
In knitting, there is multi-lineage potential, odontoblast, adipocyte and cartilage cell etc. can be induced to differentiate into vitro,
Dental pulp-dentine complex spline structure can be formed in vivo, SCAP plays a significant role in root dentin forming process,
It is a kind of preferable dental tissue engineered seeds cell, can be used for the regeneration of pulpodentinal and biology root of the tooth, has good
And wide potential applicability in clinical practice.There are dental pulp stem cell (DPSC), dental pulp stem cell can regenerate dental pulp tooth in dental pulp simultaneously
Essential sample complex, fibr tissue of the mineralized dentin matrix therein with dentinal tubule therein and comprising blood vessel is according to normal person
The dental pulp of body-dentine complex level arrangement.And cell has notable self-renewal capacity and multi-direction differentiation capability, energy
Dystopy forms dentine in vivo, can also differentiate into fatty like cell and neural-like cells.Miura in 2003 etc. has found simultaneously for the first time
Report a kind of stem cell with more differentiation potentials being separated to from the deciduous teeth that fall off of people.And the stem cell is named as breast
Tooth dental pulp stem cell (SHED).This kind of cell equally has high proliferative capacity, certain multi-lineage potential and self-renewing
The biological characteristics such as ability can break up to directions such as odontoblast, osteoblast, adipocyte, nerve cells.Periodontal
Film stem cell is derived from the adult stem cell of parodontium, and it is thin to generate different types of maturation with particular phenotype and function
Born of the same parents are able to maintain that the stabilization of parodontium function, play the effect of physiological cell turnover and repair tissue damage, and parodontium is dry thin
Born of the same parents can not only be divided into cementoblast like cell and osteoblast-like cells, form cementum sample and osteoid tissue；And
Fibroblast-like cells can be also divided into, the connective tissue of similar natural parodontium sample is formed, tissue morphology, space arrangement can be formed
The upper structure similar to natural parodontium cementum complex, it is a kind of effective osteanagenesis can be used for tooth regenerative medicine to prompt this
Autologous stem cells.Odontotheca is the loose connective tissue wrapped into around enamal organ, originates from outer embryo mesenchyma, in tooth eruption mistake
It plays an important role in journey.Root of the tooth shaping age, periodontium (such as cementum, parodontium and alveolar bone) is by odontotheca precursor
Cell is formed, and odontotheca stem cell can be divided into osteoblast, cementoblast, adipocyte.Finding possible seed
After cell, the molecular mechanism of mescenchymal stem cell directed differentiation is still unclear, and which also limits the potential of mescenchymal stem cell
Using.
Special gene expression pattern is established in stem cell atomization detailed can describe to control a large amount of of its process
The expression of gene and silence.Covalent histone modifications play an important role in terms of adjusting chromatin power and function.It methylates, one
The modified types of kind histone, while being happened on lysine and arginine residues.Such histone modification is related to respectively
Kind biological processes and transcriptional regulatory.Currently, the histone being closely connected with gene expression pattern in stem cell atomization is repaiied
The collection of illustrative plates of decorations is not studied widely yet.Currently, a kind of technical method of the genomic mapping of internal histone modification just develops
Get up so that researcher can follow an extensive viewpoint about the distribution of histone modification.This method is " CHIP on
Chip " is tested based on chromatin imrnunoprecipitation, passes through genetic chip using the consistent probe of interested genome area
The rich DNA fragmentation of hybridization identification.
Recently, researcher has found that tri-methylated H3K4 is related with the gene activation of mescenchymal stem cell and function, especially
Bone is to differentiation.The purpose of research is the technique study mescenchymal stem cell bone by using CHIP-on-chip into atomization
The tri-methylated H3K4 of gene promoter region modifies the change to genome.Research in, by comparing differentiation with it is undifferentiated
The tri-methylated H3K4 collection of illustrative plates of tip of a root dental papilla stem cell (SCAP) gene promoter region, to study tri-methylated H3K4 in the tip of a root
Function of the dental papilla stem cell bone into differentiation potential.
Invention content
The embodiment of the present invention be designed to provide a kind of WDR63 genes mescenchymal stem cell bone to tooth to breaking up
Regulation and control method in journey, it is intended to solve the prior art without reference to WDR63 genes mescenchymal stem cell bone to tooth to differentiation
The problem of regulating and controlling in the process.
The embodiment of the present invention be achieved in that a kind of WDR63 genes mescenchymal stem cell bone to tooth to breaking up
Regulation and control method in journey, the WDR63 genes include to regulation and control method of the tooth into atomization in mescenchymal stem cell bone：
Step 1, cell culture, wisdom tooth are rinsed with phosphate buffered saline (PBS) again after with 75% alcohol disinfecting, are detached and are trained
Support identification tip of a root dental papilla stem cell；
Step 2, chromatin imrnunoprecipitation reaction and data analysis, cell are incubated 15 minutes in 1% formalin, 2 ×
106The tri-methylated antibody of the 4th lysine of a cell and anti-histone 3 is reacted for this chromatin imrnunoprecipitation, is owned
The precipitation DNA sample of generation is quantified using real-time quantitative pcr, and data are expressed as the percentage of DNA；
Step 3, plasmid construction and virus transfection, standard method carry out the structure of plasmid, design the SiRNA of WDR63, will
It is inserted on the shRNA carriers of slow virus, and sequencing identification is finally built into the plasmid of WDR63shRNA；It is complete to design WDR63 genes
Long PCR primer, the overall length that WDR63 is obtained with the method for PCR are connected on the expression vector of retrovirus, sequencing mirror
It is fixed, finally it is built into plasmid；Then it carries out viral packaging, collect, virus titer identification is stored in -80 degree refrigerators after packing；
Virus transfection to tip of a root dental papilla stem cell be electroplated overnight, and then retroviral infection or slow polybrene are small up to 6
When, after 48 hours, the cell that is transfected with different antibiotic-screenings；
Step 4, western blot analysis, RIPA lysates dissolve cell, sample 10%SDS polyacrylamide gels
It detaches and is transferred in polyvinylidene difluoride using half-dried transfer film device, 5% evaporated milk is smeared on film and places 2h, so
Afterwards a night is incubated with primary antibody；Immune complex is incubated together with rabbit or mouse immune globulin G antibody and uses chemiluminescent substrate
Reagent makes its visualization；It is anti-WDR63 polyclonal antibodies for WDR63；
Step 5, ability of cell proliferation measure, tip of a root dental papilla stem cell close 1.0 × 104A cell density is plated on
60nm culture dishes；And counted cell culture 3,5,7 days, cell count is using automated cell calculating instrument and in cell suspension
Middle addition trypan blue excludes dead cell；3 independent experiments are carried out to be averaged；
Step 6, alkaline phosphatase and Alizarin red staining, mineralising induce liquid to induce tip of a root dental papilla stem cell, alkaline phosphatase
Enzyme assay illustrates to be analyzed according to alkaline phosphatase activities detection kit, and to detect mineralization ability, cell induces 2-3
Zhou Hou is fixed with 70% ethyl alcohol, 2% Alizarin red staining；Calcium ion concentration is quantitative determined, it is molten with 10% cetylpyridinium chloride(CPC)
Alizarin red is set to fade at room temperature 30 minutes in sodium phosphate；Calcium ion concentration is determined by measuring the absorbance of 562nm, is used in combination
Standard curve converts；
Step 7, nude mice by subcutaneous carries out cell Hui Zhi, by 4.0 × 106A cell and 40 milligrams of hydroxyapatite/phosphoric acid
Tricalcium ceramic particle mixes, and 5 10 week old nude mice dorsal scs is then transplanted to, in each nude mice, blank tip of a root dental papilla
Under stem cell transplantation to left dorsal surface skin, and WDR63 tip of a root dental papilla cells be transplanted to right side back surface it is subcutaneous；According to animal
Agreement is ratified specification and is carried out；The 8th week after transplanting, obtains transplanted cells and fixed with 10% formalin, 10%EDTA decalcifications, pH
Value 8.0, paraffin embedding, HE dyeing, observational measurement tissue mineralization amount.
Further, the specific method of step 1：Tip of a root dental papilla tissue is gently removed, it is repeatedly clear with phosphate buffered saline (PBS)
It washes, shreds, be placed in the digestive juice of 3g/L containing collagenase type I and dispase 4g/L, digested 1 hour at 37 DEG C, cross 70 μm of cell sieves
Cell is collected, 1000rpm centrifuges 10min, single cell suspension is suspended into again with culture solution；By cell inoculation in 25cm2Cell
In culture bottle, contain 15% fetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml streptomysins in culture medium
In 37 DEG C, 5%CO2Culture, changes liquid 1 time in every 2~3 days；Daily cell growth condition is observed under inverted microscope；Work as cell
When growing to 80% converging state, 1: 2 had digestive transfer culture is pressed with 0.25% trypsase；After the stem cell in 2-4 generations is used for
Experiment.
Further, in step 2, signal must be standardized to carry out contrast experiment；LOWESS programs are used to weigh not
The changeability between array is reacted with chromatin imrnunoprecipitation, in order to determine the tri-methylated of the 4th lysine of Histone 3, no
Tip of a root dental papilla stem cell under the conditions of, which has differences, to methylate, therefore it is the 4th lysine of Histone 3 to define 2 times of variations
Tri-methylated significant changes threshold value；Equally, it is assumed that value threshold value 0.05 is also used for distinguishing gene alteration；Channel Image data
Than being standardized using LOWESS methods, and it is the expression analysis realized using identical method that assumed value, which calculates,.
Further, in step 3, the target sequence of shRNA is：
WDR63shRNA：5′-AAACCCAGGGCTGCCTTGGAAAAG-3′.
Further, the WDR63 genes mescenchymal stem cell bone to WDR63 in regulation and control method of the tooth into atomization
Enhance its alkaline phosphatase activities and mineralization ability, WDR63 can activate the expression of two key transcription factors OSX and RUNX2；
WDR63 is a crucial reinforcing agent of Osteoblast Differentiation；WDR63 promotes cell in vitro proliferative capacity that WDR63 is supported to have enhancing
The potential of regeneration；The tri-methylated tip of a root dental papilla that can enhance of the 4th lysine of Histone 3 of WDR63 promoters is done
The Osteoblast Differentiation potential of cell.
WDR63 genes provided in an embodiment of the present invention are in mescenchymal stem cell bone to the regulation and control side with tooth into atomization
Method, by a series of experiments find WDR63 genes in mescenchymal stem cell bone to/regulating and controlling effect of the tooth into atomization, with
And the effect in promoting dental tissue regeneration.The present invention, which uses, is related to the tri-methylated to mesenchyma of the 4th lysine of Histone 3
The gene activation of stem cell and the adjustment effect of function, WDR63 genes mescenchymal stem cell bone to/tooth into atomization
Regulating and controlling effect is related to effect of the WDR63 genes in promoting dental tissue regeneration, and obtaining WDR63 may be dry thin in tip of a root dental papilla
It plays a driving role in born of the same parents' Osteoblast Differentiation.
Description of the drawings
Fig. 1 is skeletonization culture provided in an embodiment of the present invention in WDR63, TREX1, FOXO24, these promoters of ARNT
Promote the tri-methylated of the 4th lysine of Histone 3, induces WDR63 and ARNTL expression；And in FOXP4, TMEM106B this
The 4th lysine of inhibition of histone 3 is tri-methylated in a little promoters, and FOXP4 is inhibited to express schematic diagram；
Fig. 2 is the bone of the overexpression enhancing tip of a root dental papilla stem cell of WDR63 provided in an embodiment of the present invention to differentiation potency
Power schematic diagram；
Fig. 3 is that the overexpression of WDR63 provided in an embodiment of the present invention increases internal mineralized tissue forming amount schematic diagram；
Fig. 4 be WDR63 provided in an embodiment of the present invention low expression can inhibit the stem cells hyperplasia of tip of a root dental papilla and bone to
Differentiation capability schematic diagram；
Fig. 5 be WDR63 genes provided in an embodiment of the present invention mescenchymal stem cell bone to tooth into atomization
Regulate and control method flow diagram.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
As shown in figure 5, the WDR63 genes of the embodiment of the present invention mescenchymal stem cell bone to tooth into atomization
Regulation and control method includes the following steps：
S501：Cell culture, wisdom tooth are rinsed with phosphate buffered saline (PBS) again after with 75% alcohol disinfecting, are detached and are cultivated
Identify tip of a root dental papilla stem cell；
S502：Chromatin imrnunoprecipitation reacts and data analysis, and cell is incubated 15 minutes in 1% formalin, 2 ×
106The tri-methylated antibody of the 4th lysine of a cell and anti-histone 3 is reacted for this chromatin imrnunoprecipitation, is owned
The precipitation DNA sample of generation is quantified using real-time quantitative pcr, and data are expressed as the percentage of DNA；
S503：The SiRNA for designing WDR63, is inserted on the shRNA carriers of slow virus, sequencing identification, final to build
At the plasmid of WDR63shRNA；The PCR primer for designing WDR63 full length genes, with the method for PCR obtain the overall length of WDR63 by its
It is connected on the expression vector of retrovirus, sequencing identification is finally built into plasmid；
S504：Then it carries out viral packaging, collect, virus titer identification is stored in -80 degree refrigerators after packing；Virus turns
Dye be electroplated overnight tip of a root dental papilla stem cell, and then retroviral infection or slow polybrene are up to 6 hours, and 48
After hour, the cell that is transfected with different antibiotic-screenings；
S505：RIPA lysates dissolve cell, and sample is detached with 10%SDS polyacrylamide gels and utilizes half-dried transfer
Film device is transferred in polyvinylidene difluoride, and 5% evaporated milk is smeared on film and places 2h, is then incubated a night with primary antibody；
Immune complex is incubated together with rabbit or mouse immune globulin G antibody and makes its visualization with chemiluminescent substrate reagent；
S506：Ability of cell proliferation measures, tip of a root dental papilla stem cell close 1.0 × 104A cell density is plated on 60nm
Culture dish；And counted cell culture 3,5,7 days, cell count is added using automated cell calculating instrument and in cell suspension
Enter trypan blue and excludes dead cell；It carries out 3 independent experiments and takes its average value；
S507：Mineralising induces liquid that tip of a root dental papilla stem cell, alkaline phosphatase activities is induced to detect according to alkaline phosphatase
Activity detection kit illustrates to be analyzed, and after cell induces 2-3 weeks, is fixed with 70% ethyl alcohol, 2% Alizarin red staining；It is quantitative
Calcium ion concentration is measured, being dissolved in sodium phosphate at room temperature with 10% cetylpyridinium chloride(CPC) makes alizarin red fade 30 minutes；Calcium
Ion concentration is determined by measuring the absorbance of 562nm, and standard curve is used in combination to convert；
S508：By 4.0 × 106A cell is mixed with 40 milligrams of hydroxyapatite/tricalcium phosphate ceramic particle, then will
It is transplanted to 5 10 week old nude mice dorsal scs, in each nude mice, blank tip of a root dental papilla stem cell transplantation to left dorsal table
Under musculus cutaneus, and WDR63 tip of a root dental papilla cells be transplanted to right side back surface it is subcutaneous；These programs ratify specification according to animal protocol
It carries out；The 8th week after transplanting, obtains transplanted cells and fixed with 10% formalin, 10%EDTA decalcifications (pH value 8.0), paraffin packet
It buries, HE dyeing, observational measurement tissue mineralization amount.
The present invention is described in further details with reference to embodiment；
One, cell culture
All stem cells according to the present invention abide by the Behavioral guidelines of hESC's research, tissue
Using obtaining the approval of Ethics Committee of the Capital University of Medical Sciences, the preoperative signing informed consent form of the equal informed consent of volunteer；Wisdom tooth
It is rinsed again with phosphate buffered saline (PBS) after with 75% alcohol disinfecting, separation and culture identification tip of a root dental papilla stem cell, summary
It is as follows：Tip of a root dental papilla tissue is gently removed, is cleaned, is shredded repeatedly with phosphate buffered saline (PBS), is placed in containing collagenase type I (3g/
L) and the digestive juice of dispase (4g/L), digest 1 hour at 37 DEG C, cross 70 μm of cell sieves and collect cells, 1000rpm centrifugations
10min suspends into single cell suspension again with culture solution；By cell inoculation in 25cm2In Tissue Culture Flask, (contain in culture medium
15% fetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml streptomysins) in 37 DEG C, 5%CO2Culture,
Change liquid 1 time within every 2~3 days；Daily cell growth condition is observed under inverted microscope；When cell growth to 80% converging state
When, press 1: 2 had digestive transfer culture with 0.25% trypsase；The stem cell in 2-4 generations be used for after experiment；
Two, chromatin imrnunoprecipitation reaction and data analysis：
Chromatin imrnunoprecipitation reaction carries out according to the description of product of manufacturer and uses human promoter's 1.0R arrays point
Analysis；Cell is incubated 15 minutes in 1% formalin, and 2 × 106The trimethyl of the 4th lysine of a cell and anti-histone 3
To change antibody to react for this chromatin imrnunoprecipitation, the precipitation DNA sample of all generations is quantified using real-time quantitative pcr,
Data are expressed as the percentage of DNA；Primer is as shown in supplementary table 1；Due to experiment and technique variation, signal must standardize with into
Row contrast experiment appropriate；LOWESS programs are used to weigh the changeability between different chromatin imrnunoprecipitation reaction arrays,
In order to determine the tri-methylated of the 4th lysine of Histone 3, the tip of a root dental papilla stem cell under different condition has differences methyl
Change, therefore defines the threshold value for the tri-methylated significant changes that 2 times of variations are the 4th lysine of Histone 3；Equally, it is assumed that value threshold
Value 0.05 is also used for distinguishing gene alteration；Simple, Channel Image data ratio is standardized using LOWESS methods, and assumed value
Calculating is the expression analysis realized using identical method；
Three, plasmid construction and virus transfection：
Standard method carries out the structure of plasmid；All structures, which are all digested and/or are sequenced by limitation appropriate, to be verified；
The SiRNA for designing WDR63, is inserted on the shRNA carriers of slow virus, and sequencing identification is finally built into WDR63shRNA's
Plasmid；The PCR primer of WDR63 full length genes is designed, the overall length that WDR63 is obtained with the method for PCR is connected to reverse transcription disease
On the expression vector of poison, sequencing identification is finally built into plasmid；Then it carries out viral packaging, collect, virus titer identification, point
- 80 degree refrigerators are stored in after dress；Virus transfection to tip of a root dental papilla stem cell be electroplated overnight, then infects reverse transcription disease
Malicious or slow polybrene is up to 6 hours, after 48 hours, the cell that is transfected with different antibiotic-screenings；
The target sequence of shRNA is：
WDR63shRNA：5′-AAACCCAGGGCTGCCTTGGAAAAG-3′；
Four, western blot analysis：
RIPA lysates dissolve cell, and sample is detached with 10%SDS polyacrylamide gels and half-dried transfer membrane is utilized to fill
It sets and is transferred in polyvinylidene difluoride (PVDF), 5% evaporated milk is smeared on film and places 2h, is then incubated one with primary antibody
Night；Immune complex is incubated together with rabbit or mouse immune globulin G antibody and makes its visualization with chemiluminescent substrate reagent；
It is anti-WDR63 polyclonal antibodies mainly for WDR63；
Five, ability of cell proliferation measures：
Tip of a root dental papilla stem cell close 1.0 × 104A cell density is plated on 60nm culture dishes；And cell culture 3,5,
It is counted within 7 days, cell count is added trypan blue using automated cell calculating instrument and in cell suspension and excludes dead cell；It carries out
3 independent experiments take its average value；
Six, alkaline phosphatase and Alizarin red staining：
Mineralising induces liquid that tip of a root dental papilla stem cell, alkaline phosphatase activities detection is induced to be examined according to alkaline phosphatase activities
Test agent box illustrates to be analyzed, and is fixed with 70% ethyl alcohol, 2% alizarin after cell induces 2-3 weeks to detect its mineralization ability
Red colouring；Calcium ion concentration is quantitative determined, be dissolved in sodium phosphate with 10% cetylpyridinium chloride(CPC) makes alizarin red move back at room temperature
Color 30 minutes；Calcium ion concentration is determined by measuring the absorbance of 562nm, and standard curve is used in combination to convert；
Seven, nude mice by subcutaneous carries out cell Hui Zhi：
The present invention is allowed by the animal care of BJ Stomatological Hospital of the Capital University of Medical Sciences and using the committee；Will about 4.0 ×
106A cell is mixed with 40 milligrams of hydroxyapatite/tricalcium phosphate ceramic particle, is then transplanted to 5 by previously described
Only 10 week old nude mice dorsal scs, under each nude mice, blank tip of a root dental papilla stem cell transplantation to left dorsal surface skin,
And WDR63 tip of a root dental papilla cells be transplanted to right side back surface it is subcutaneous；These programs are carried out according to animal protocol approval specification；It moves
The 8th week after plant, obtains transplanted cells and fixed with 10% formalin, 10%EDTA decalcifications (pH value 8.0), paraffin embedding, HE dyes
Color, observational measurement tissue mineralization amount；
Eight, experimental result：
It is reacted using chromatin imrnunoprecipitation and generates gene promoter region histone modification genome：
The 4th lysine of Histone 3 of promoter region distribution when attempting to detect tip of a root dental papilla stem cell Osteoblast Differentiation
Tri-methylated full-length genome gene；Osteogenic and ordinary culture medium culture tip of a root dental papilla stem cell 7 days are used respectively,
The concentration chip cohybridization marked with difference using the tri-methylated antibody of the 4th lysine of anti-histone 3 is carried out chromatin and exempted from
Epidemic disease precipitation reaction；Chromatin imrnunoprecipitation response data shows that gene promoter is rich in tri-methylated group after osteogenic induction
The 4th lysine of albumen 3；In addition, in more undifferentiated and differentiation tip of a root dental papilla stem cell gene promoter region trimethyl
The 4th lysine genome of Histone 3 of change find, 119 gene promoters are shown in tri-methylated Histone 3 the
4 lysines are more than twice of growth, and 21 gene promoters show big in the 4th lysine of tri-methylated Histone 3
In twice of reduction；In order to confirm chromatin imrnunoprecipitation reaction experiment data, WDR63, TREX1, FOXO24, ARNTL,
FOXP4, TMEM106B these gene proofing chips analysis；The result shows that relative to ordinary culture medium, when osteogenic induction
Tri-methylated the 4th lysine of Histone 3 dramatically increases in these promoters of WDR63, TREX1, FOXO24, ARNTL, and
Tri-methylated the 4th lysine of Histone 3 significantly reduces (picture 1a-f) in these promoters of FOXP4, TMEM106B；Separately
Outside, real-time quantitative PCR result is shown in tip of a root dental papilla stem cell atomization, and WDR63, ARNTL are presented more than twice
Height expression, and FOXP4 is then ground expression (picture 1g-i)；However TREX1, FOXO24, TMEM106B are in differentiation and undifferentiated
Expression no significant difference in canine tooth nipple stem cell；Generally speaking, these data, which allow, speculates that WDR63 may be in tip of a root dental papilla
It plays a driving role in stem cell Osteoblast Differentiation；
WDR63 is overexpressed the Osteoblast Differentiation potential of enhancing tip of a root dental papilla stem cell
It further confirms functions of the WDR63 in tip of a root dental papilla stem cell, WDR63 sequences is inserted into a reverse transcription
Viral vectors；Overexpression WDR63 when this construction is attached to tip of a root dental papilla stem cell by retroviral infection；Also lead to
Cross the overexpression (picture 2a) of Western blot experiment verification WDR63；Next, tip of a root dental papilla stem cell in conjunction with after into
Row osteogenic induction is to detect its Osteoblast Differentiation potential, the results showed that, the overexpression of WDR63 increases alkaline phosphatase activities
(picture 2b)；Therefore, pass through Alizarin red staining and calcium ion quantitative measurment, the tip of a root dental papilla stem cell of overexpression WDR63
Relative to the tip of a root dental papilla stem cell of empty vectors, its mineralization ability enhances (picture 2c-d)；Real-time quantitative PCR result is also shown
Show, the 7th day and the 14th day induced in the tip of a root dental papilla stem cell of overexpression WDR63, the expression of resorption lacunae significantly increases
High (picture 2e)；Next, having detected the expression for the key transcription factor for adjusting Osteoblast Differentiation, including RUNX2 and OSX；RUNX2
Significantly increase the tip of a root in the tip of a root dental papilla stem cell of overexpression WDR63 relative to empty vectors with the rna level of OSX
Dental papilla stem cell (picture 2f-g)；Next, tip of a root dental papilla stem cell can be influenced in vivo by examining whether addition WDR63
Ostosis；SCA zero loads tip of a root dental papilla stem cell and load WDR63 tip of a root dental papilla stem cell transplantations to nude mice by subcutaneous；It is transplanting
The 8th week afterwards, HE dyeing displays had more bone sample mineralising groups in the load WDR63 tip of a root dental papilla stem cell transplants of acquisition
It knits and compares blank group (picture 3a)；Observational measurement mineralized tissue shows that bone sample mineralized tissue amount is dry in load WDR63 tip of a root dental papilla
It is higher than blank group (picture 3b) in cell transplantation body；Therefore, internal transplantation experiments show to carry WDR63 tip of a root dental papilla stem cells
It generates more bone sample mineralized tissues and compares unloaded tip of a root dental papilla stem cell；In conclusion these results indicate that WDR63 tables
Up to the tip of a root dental papilla stem cell Osteoblast Differentiation triggered significantly；
The reduction of WDR63 inhibits the bone of tip of a root dental papilla stem cell to differentiation
In order to further elucidate functions of the WDR63 in tip of a root dental papilla stem cell, a ShorthairpinRNA target is devised
It is to inhibit WDR63 expression formulas and it is introduced into tip of a root dental papilla stem cell by slow-virus transfection；After selection, immunoblotting is used
The efficiency (picture 4a) that analysis detection is lowered；Next, detecting whether that WDR63 affects tip of a root dental papilla stem cell in itself
Osteogenic ability；Tip of a root dental papilla stem cell is cultivated in osteogenic, finds the tip of a root dental papilla stem cell for inhibiting WDR63
Its alkaline phosphatase activities is substantially less than blank tip of a root dental papilla stem cell (picture 4b)；Pass through Alizarin red staining after osteogenic induction
With calcium ion quantitative measurment, inhibit the tip of a root dental papilla stem cell of WDR63 relative to its mineralising of blank tip of a root dental papilla stem cell
Ability is substantially reduced (picture 4c-d)；In addition, carrying out ability of cell proliferation detection display after cell culture 7 days, inhibit WDR63
Tip of a root dental papilla stem cell relative to blank tip of a root dental papilla stem cell, its proliferative capacity is substantially reduced (picture 4e)；
Nine, conclusion
WDR63 is a kind of WD repetitive proteins of unknown function, research WDR63 in tip of a root dental papilla stem cell atomization
Function, find in atomization inside and outside tip of a root dental papilla stem cell body, WDR63 enhances its alkaline phosphatase activities and mineralising
Ability, this shows that WDR63 may be a key factor for controlling mescenchymal stem cell Osteoblast Differentiation potential；Mescenchymal stem cell
Require the coordination and inhibition between the differentiation of other cell lines, the activation of multiple transcription factors and mesenchyma dry to the differentiation of osteoblast system
Cell differentiation is related, and two crucial transcription factors, RUNX2 and OSX are necessary to Osteoblast Differentiation；Result of study shows
WDR63 can activate the expression of two key transcription factors OSX and RUNX2；Next, having detected the mRNA water of resorption lacunae
It is flat, it is the major structural protein of bone matrix；Result of study shows that WDR63 induces the expression of resorption lacunae gene；These results
Show that WDR63 is a crucial reinforcing agent of Osteoblast Differentiation；In addition, cell growth curve shows to inhibit WDR63 that can inhibit root
The proliferation of canine tooth nipple stem cell；The vitro detection of the growth of mescenchymal stem cell, proliferation and survival ability being capable of Accurate Prediction
Internal mescenchymal stem cell function；These find the growth for significantly showing to enhance mescenchymal stem cell, and proliferation, survival ability may
Improve their blood vessel and regeneration potentiality；In current research, result of study promotes cell in vitro proliferation for WDR63
Ability supports WDR63 to have the potential of enhancing regeneration；
In short, as a result represent modification and the tip of a root tooth of the 4th lysine of the i.e. tri-methylated Histone 3 of international viewpoint
Nipple stem cell bone is contacted to the functionality of differentiation, and is shown by changing the 4th lysine control of tri-methylated Histone 3
Gene activation and silence are most important to the Osteoblast Differentiation of mescenchymal stem cell；A crucial Osteoblast Differentiation enhancing is also found
The tri-methylated of the 4th lysine of Histone 3 of agent-WDR63, WDR63 promoter can enhance tip of a root dental papilla stem cell
Osteoblast Differentiation potential.
Supplement chart 1：Carry out the primer of chromatin imrnunoprecipitation reaction real-time quantitative PCR
Supplement chart 2：Carry out the primer of real-time quantitative PCR experiment
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Claims (5)
1. a kind of WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization, which is characterized in that should
WDR63 genes include to regulation and control method of the tooth into atomization in mescenchymal stem cell bone：
Step 1, cell culture, wisdom tooth are rinsed with phosphate buffered saline (PBS) again after with 75% alcohol disinfecting, detach and cultivate mirror
Determine tip of a root dental papilla stem cell；
Step 2, chromatin imrnunoprecipitation reaction and data analysis, cell are incubated 15 minutes in 1% formalin, and 2 × 106It is a
The tri-methylated antibody of the 4th lysine of cell and anti-histone 3 is reacted for this chromatin imrnunoprecipitation, all generations
Precipitation DNA sample is quantified using real-time quantitative pcr, and data are expressed as the percentage of DNA；
Step 3, plasmid construction and virus transfection, standard method carry out the structure of plasmid, design the SiRNA of WDR63, inserted
Enter on the shRNA carriers of slow virus, sequencing identification is finally built into the plasmid of WDR63shRNA；Design WDR63 full length genes
PCR primer, the overall length that WDR63 is obtained with the method for PCR are connected on the expression vector of retrovirus, sequencing identification,
Finally it is built into plasmid；Then it carries out viral packaging, collect, virus titer identification is stored in -80 degree refrigerators after packing；Virus
Transfection to tip of a root dental papilla stem cell be electroplated overnight, and then retroviral infection is up to 6 hours, after 48 hours, with not
The cell that same antibiotic-screening is transfected；
Step 4, western blot analysis, RIPA lysates dissolve cell, and sample is detached with 10% sds page
And be transferred in polyvinylidene difluoride using half-dried transfer film device, 5% skim milk is smeared on film and places 2 h, is then used
Primary antibody is incubated a night；Immune complex is incubated together with rabbit or mouse immune globulin G antibody and with chemiluminescent substrate reagent
Make its visualization；Primary antibody for WDR63 is anti-WDR63 polyclonal antibodies；
Step 5, ability of cell proliferation measure, tip of a root dental papilla stem cell 1.0 × 104A cell density is plated on 60nm cultures
Ware；And counted cell culture 3,5,7 days, cell count is using automated cell calculating instrument and platform is added in cell suspension
Expect blue exclusion dead cell；3 independent experiments are carried out to be averaged；
Step 6, alkaline phosphatase and Alizarin red staining, mineralising induce liquid to induce tip of a root dental papilla stem cell, alkaline phosphatase enzyme activity
Property detection illustrate to be analyzed according to alkaline phosphatase activities detection kit, for detect mineralization ability, cell induction 2-3 week
Afterwards, it is fixed with 70% ethyl alcohol, 2% Alizarin red staining；Calcium ion concentration is quantitative determined, is dissolved in phosphorus with 10% cetylpyridinium chloride(CPC)
Sour sodium makes alizarin red fade 30 minutes at room temperature；Calcium ion concentration is determined by measuring the absorbance of 562nm, and standard is used in combination
Curve converts；
The mescenchymal stem cell is tip of a root dental papilla stem cell.
2. WDR63 genes as described in claim 1 in mescenchymal stem cell bone to the regulation and control method with tooth into atomization,
It is characterized in that, the specific method of step 1：Tip of a root dental papilla tissue is gently removed, is cleaned repeatedly with phosphate buffered saline (PBS),
It shreds, is placed in the digestive juice of 3 g/L and 4 g/L of dispase of enzyme containing Type I collagen, is digested 1 hour at 37 DEG C, cross 70 μm of cells
Sieve collects cell, and 1000 rpm centrifuge 10 min, single cell suspension is suspended into again with culture solution；By cell inoculation in 25 cm2
In Tissue Culture Flask, containing 15% fetal calf serum, 2 mmol/L glutamine, 100 U/ ml penicillin and 100 μ g/ ml chains
37 DEG C, 5% CO in the culture medium of mycin2Culture, changes liquid 1 time in every 2 ~ 3 days；Daily cell growth is observed under inverted microscope
Situation；When cell growth to 80% converging state, 1 is pressed with 0.25% trypsase:2 had digestive transfer cultures；The stem cell in 2-4 generations by with
In experiment later.
3. WDR63 genes as described in claim 1 in mescenchymal stem cell bone to the regulation and control method with tooth into atomization,
It is characterized in that, in step 2, signal must be standardized to carry out contrast experiment；LOWESS programs are used to weigh different dyes
Changeability between chromaticness immune precipitation array, in order to determine tri-methylated, the different items of the 4th lysine of Histone 3
Tip of a root dental papilla stem cell under part, which has differences, to methylate, therefore it is the three of the 4th lysine of Histone 3 to define 2 times of variations
The threshold value for the significant changes that methylate；Equally, it is assumed that value threshold value 0.05 is also used for distinguishing gene alteration；Channel Image data ratio makes
Standardized with LOWESS methods, and it is the expression analysis realized using identical method that assumed value, which calculates,.
4. WDR63 genes as described in claim 1 in mescenchymal stem cell bone to the regulation and control method with tooth into atomization,
It is characterized in that, in step 3, the target sequence of shRNA is：
WDR63shRNA: 5'-AAACCCAGGGCTGCCTTGGAAAAG-3'。
5. WDR63 genes as described in claim 1 in mescenchymal stem cell bone to the regulation and control method with tooth into atomization,
It is characterized in that, the WDR63 genes enhance in mescenchymal stem cell bone to WDR63 in regulation and control method of the tooth into atomization
Its alkaline phosphatase activities and mineralization ability, WDR63 can activate the expression of two key transcription factors OSX and RUNX2；
WDR63 is a crucial reinforcing agent of Osteoblast Differentiation；WDR63 promotes cell in vitro proliferative capacity that WDR63 is supported to have enhancing
The potential of regeneration；The tri-methylated tip of a root dental papilla that can enhance of the 4th lysine of Histone 3 of WDR63 promoters is done
The Osteoblast Differentiation potential of cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550443.8A CN104450621B (en) | 2014-09-30 | 2014-09-30 | WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550443.8A CN104450621B (en) | 2014-09-30 | 2014-09-30 | WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450621A CN104450621A (en) | 2015-03-25 |
| CN104450621B true CN104450621B (en) | 2018-10-19 |
Family
ID=52897380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410550443.8A Active CN104450621B (en) | 2014-09-30 | 2014-09-30 | WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450621B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105561338A (en) * | 2015-05-14 | 2016-05-11 | 首都医科大学附属北京口腔医院 | Application of SFRP2 to promotion of odontogenic mesenchymal stem cell osteogenic/odontoblastic differentiation |
| KR20200108315A (en) * | 2018-02-09 | 2020-09-17 | 제넨테크, 인크. | Oligonucleotide to regulate the expression of TMEM106B |
| CN115184239A (en) * | 2018-04-17 | 2022-10-14 | 中国矿业大学 | Visual test device and method for fractured coal rock mass permeation grouting |
| CN109504710B (en) * | 2018-12-04 | 2021-03-26 | 首都医科大学附属北京口腔医院 | Application of KDM4D |
| CN110938669B (en) * | 2019-12-20 | 2023-03-28 | 南昌大学第二附属医院 | Verification method for stimulating osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN111849993B (en) * | 2020-03-18 | 2022-01-14 | 商丘华原生物科技发展有限公司 | Gene inhibitor for promoting osteogenic differentiation of mesenchymal stem cells |
| CN114058591B (en) * | 2021-11-17 | 2022-10-14 | 中国人民解放军空军军医大学 | Recombinant mesenchymal stem cell and application thereof |
| CN114522233B (en) * | 2021-11-30 | 2023-07-14 | 首都医科大学附属北京口腔医院 | Polypeptide sequence of KDM6B and regulation and control application of mesenchymal stem cell function |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101842477A (en) * | 2007-01-22 | 2010-09-22 | 株式会社器官再生工学 | Method for production of mesenchymal cell, method for production of tooth, and mesenchymal cell for formation of tooth |
| CN102335457A (en) * | 2010-07-28 | 2012-02-01 | 郭山辉 | Composition for tooth regeneration |
| CN104000737A (en) * | 2014-06-10 | 2014-08-27 | 上海交通大学医学院附属第九人民医院 | Tissue-engineered dental pulp-dentin complex |
-
2014
- 2014-09-30 CN CN201410550443.8A patent/CN104450621B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101842477A (en) * | 2007-01-22 | 2010-09-22 | 株式会社器官再生工学 | Method for production of mesenchymal cell, method for production of tooth, and mesenchymal cell for formation of tooth |
| CN102335457A (en) * | 2010-07-28 | 2012-02-01 | 郭山辉 | Composition for tooth regeneration |
| CN104000737A (en) * | 2014-06-10 | 2014-08-27 | 上海交通大学医学院附属第九人民医院 | Tissue-engineered dental pulp-dentin complex |
Non-Patent Citations (1)
| Title |
|---|
| 牙乳头细胞多向分化的观察;庄姮等;《北京大学学报（医学版）》;20090408;第41卷(第1期);第62-65页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450621A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104450621B (en) | WDR63 genes are in mescenchymal stem cell bone to the regulation and control method with tooth into atomization | |
| Jung et al. | Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells | |
| KR101343460B1 (en) | Composition for hard tissue formation and, dentin or pulp regeneration containing ameloblast, apical bud cell or its culture fluid as an active ingredient | |
| US11578308B2 (en) | Method of preparing an artificial tooth primordium in vitro and artificial tooth primordium derived therefrom | |
| Tabatabaei et al. | Effects of equiaxial strain on the differentiation of dental pulp stem cells without using biochemical reagents | |
| CN110577931A (en) | Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues | |
| CN109504710B (en) | Application of KDM4D | |
| CN106456673A (en) | Enamel products and methods of use | |
| ITNA20060017A1 (en) | TECHNIQUE OF COLLECTION AND SELECTION OF AN EMBRYONIC-TYPE POPLATION OF STEM CELLS FROM PERIDENT FOLLICULAR FABRICS OF ADULT MEN. | |
| Huang et al. | Proliferation and odontogenic differentiation of human umbilical cord mesenchymal stem cells and human dental pulp cells co-cultured in hydrogel | |
| CN102008360B (en) | A kind of structure of the artificial neural network sample catheter for repairing spinal cord injury | |
| Kunwong et al. | Optimization of poly (lactic-co-glycolic acid)-bioactive glass composite scaffold for bone tissue engineering using stem cells from human exfoliated deciduous teeth | |
| CN110499285A (en) | Application of the ALPL gene in the product of preparation prevention and/or treatment low alkalinity phosphatase disease | |
| CN104306958B (en) | Application of the insulin-like growth factor binding protein 5 in promoting paradenlal tissue regeneration | |
| CN105879057B (en) | SFRP2 promote Odontogenic cysts mescenchymal stem cell skeletonization/at tooth break up in application | |
| CN112755052A (en) | Application of human deciduous tooth pulp stem cell exosome | |
| Bi et al. | Hertwig's epithelial root sheath cells show potential for periodontal complex regeneration | |
| CN104755610B (en) | Fat tissue cell | |
| CN104174064B (en) | Tissue-engineered bone of genetic modification and its preparation method and application | |
| WO2021012718A1 (en) | Pluripotent stem cells mdpscs for regeneration of dental pulp, separation culture method therefor, and use thereof | |
| REZENDE et al. | Dental Pulp Stem Cells from Natal Teeth: Isolation and Morphological Study. | |
| CN106497871A (en) | A kind of method for improving human amnion mesenchymal stem cell Osteoblast Differentiation efficiency and application | |
| Bai et al. | Substrate stiffness promotes dentinogenesis via LAMB1–FAK–MEK1/2 signaling axis | |
| RU2716485C1 (en) | Method of growing enamel in experiment | |
| Song et al. | The Effects of Transforming Growth Factor‐β1 on the Differentiation of Cell Organoids Composed of Gingiva‐Derived Stem Cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |