CN104450621A - Regulating method of WDR63 gene in osteogenic differentiation and odontogenic differentiation processes of mesenchymal stem cell - Google Patents
Regulating method of WDR63 gene in osteogenic differentiation and odontogenic differentiation processes of mesenchymal stem cell Download PDFInfo
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Abstract
The invention discloses a regulating method of a WDR63 gene in osteogenic differentiation and odontogenicdifferentiation processes of a mesenchymal stem cell. The cell is replanted by virtue of cell culture, chromatin immunoprecipitation reaction and data analysis, plasmid construction and virus infection, western blotting analysis, cell reproductive capacity determination and alkaline phosphatase and alizarin red dyeing subcutaneously of a nude mouse, and the regulating effect of the WDR63 gene in osteogenic differentiation and odontogenic differentiation processes of the mesenchymal stem cell and the effect of promoting regeneration of tooth tissues are found. By virtue of the adjusting effect of gene activity and function of the mesenchymal stem cell by trimethylation of lysine on the fourth site of histone 3, the regulating effect of the WDR63 gene in osteogenic differentiation and odontogenicdifferentiation processes of the mesenchymal stem cell and the effect of the WDR63 gene in promoting regeneration of tooth tissues, the WDR63 gene obtained probably has a promoting effect on osteogenic differentiation of stem cells from apical papilla.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly relate to a kind of WDR63 gene mescenchymal stem cell bone to tooth to the regulate and control method in atomization.
Background technology
Odontopathy is one of modal disease of the mankind, and the disappearance of tooth have impact on the Health and Living quality of people greatly.Though existing dentures repai means are ripe, because it does not possess biological activity, are difficult to compare favourably with natural teeth, the vision that the mankind have " the 3rd secondary tooth " cannot be realized.For this reason, various countries attempt to utilize stem cell and tissue engineering technique to realize the reconstruction of tooth.How developing and a kind ofly have biologic activity and function and can rebuild the Dental Erosion missing tooth of periodontal relation in jawbone, is the key subjects that clinical odontologist and researcher are expected to solve jointly.Along with the progress of oral cavity developmental biology and tissue engineering technique, the work of regeneration of tooth research every aspect launches all, and the optimal strategy of full dental tissue engineering adopts autogenous cell, tooth and surrounding tissue (comprising tooth, periodontium, alveolar bone etc.) thereof are built simultaneously, then jaw defect district is implanted, to obtain the tooth with certain vigor.In tooth regeneration research field, stem cell is its important integral part, and stem cell is the undifferentiated cell of a class, has the of self-replication capacity and to many differentiation potentials.The Odontogenic cysts stem cell being mainly used in dental tissue engineering at present has 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 tissue of tooth root pointed orifice, there is multi-lineage potential, odontoblast, adipocyte and chondrocyte etc. can be induced to differentiate in vitro, dental pulp-dentine complex body spline structure can be formed in vivo, SCAP plays a significant role in root dentin forming process, it is a kind of dental tissue engineered seeds cell preferably, can be used for the regeneration of pulpodentinal and biology root of the tooth, there is good and wide potential applicability in clinical practice.There is dental pulp stem cell (DPSC) in dental pulp simultaneously, dental pulp stem cell can regenerate pulpodentinal sample complex body, and mineralized dentin matrix wherein arranges with dentinal tubule wherein and the dental pulp of fibrous tissue according to normal human-dentine complex body level of comprising blood vessel.And cell has remarkable self-renewal capacity and multi-direction differentiation capability, dystopy can form dentine in vivo, also can be divided into fat-like cell and neural-like cells.The Late Cambrian such as Miura in 2003 also report a kind of stem cell with many differentiation potentials be separated to from the deciduous teeth that come off of people.And by these stem cell called after deciduous teeth dental pulp stem cell (SHED).This kind of cell has the biological characteristicses such as high proliferative capacity, certain multi-lineage potential and self-renewal capacity equally, can to direction differentiation such as odontoblast, scleroblast, adipocyte, neurocyte.Periodontal ligament stem cell derives from pericemental adult stem cell, different types of mature cell with particular phenotype and function can be produced, the stable of periodontium function can be maintained, play the effect of physiological cell turnover and repair tissue damage, periodontal ligament stem cell can not only be divided into cementoblast like cell and osteoblast-like cells, forms dental cement sample and osteoid tissue; But also can fibroblast-like cells be divided into, form the reticular tissue of similar natural periodontium sample, energy formative tissue form, spatial disposition are similar to the structure of natural periodontium dental cement complex body, point out this to be a kind of autologous stem cells that can be used for effective osteanagenesis of tooth regenerative medicine.Odontotheca is the loose connective tissue held into around enamal organ, originates from outer embryo mesenchyme, plays an important role in tooth eruption process.Root of the tooth shaping age, periodontal tissue (as dental cement, periodontium and alveolar bone) is formed by odontotheca precursor cell, and odontotheca stem cell can be divided into scleroblast, cementoblast, adipocyte.After finding possible seed cell, the molecular mechanism of mescenchymal stem cell directed differentiation is still unclear, which also limits the potential application of mescenchymal stem cell.
In differentiation of stem cells process, set up special gene expression pattern detailedly can describe the expression of the lots of genes controlling its process with reticent.Covalent histone modifications plays an important role at adjustment chromatin power and function aspects.Methylate, a kind of modified types of histone, occurs in Methionin and arginine residues simultaneously.Such histone modification relates to various biological processes, and transcriptional regulatory.At present, the collection of illustrative plates of the histone modification be closely connected with gene expression pattern in differentiation of stem cells process, is not studied yet widely.At present, in a kind of body, the technological method of the genomic mapping of histone modification just grows up, and makes investigator can follow an extensive viewpoint about the distribution of histone modification.This method is " CHIP on chip ", and based on chromatin imrnunoprecipitation test, the probe utilizing interested genome area consistent is by the rich DNA fragmentation of gene chip hybridization identification.
Recently, investigator finds that tri-methylated H3K4 is relevant with function with the gene activation of mescenchymal stem cell, and especially bone is to differentiation.The object of research is by using the technique study mescenchymal stem cell bone of CHIP-on-chip to modify genomic change to the tri-methylated H3K4 of gene promoter region in atomization.Research in, by compare differentiation with do not break up the tri-methylated H3K4 collection of illustrative plates of tip of a root dental papilla stem cell (SCAP) gene promoter region, study tri-methylated H3K4 at tip of a root dental papilla stem cell bone to the function in differentiation potential.
Summary of the invention
The object of the embodiment of the present invention be to provide a kind of WDR63 gene mescenchymal stem cell bone to tooth to the regulate and control method in atomization, be intended to solve prior art and do not relate to WDR63 gene at mescenchymal stem cell bone to the problem regulated and controled in atomization with tooth.
The embodiment of the present invention be achieved in that a kind of WDR63 gene mescenchymal stem cell bone to tooth to the regulate and control method in atomization, this WDR63 gene comprises to the regulate and control method in atomization to tooth at mescenchymal stem cell bone:
Step one, cell cultures, wisdom tooth rinses with phosphate buffered saline (PBS) after with 75% alcohol disinfecting again, is separated and culture identification tip of a root dental papilla stem cell;
Step 2, chromatin imrnunoprecipitation reaction and data analysis, cell hatches 15 minutes in 1% formaldehyde solution, and 2 × 10
6the tri-methylated antibody of individual cell and anti-histone 3 the 4th Methionin is used for the reaction of this chromatin imrnunoprecipitation, and the precipitation DNA sample of all generations adopts real-time quantitative pcr to quantize, and data are expressed as the per-cent of DNA;
Step 3, plasmid construction and virus transfection, the structure of plasmid is carried out in standard method, and the SiRNA of design WDR63, is inserted on the shRNA carrier of slow virus, and order-checking qualification, is finally built into the plasmid of WDR63shRNA; The PCR primer of design WDR63 full length gene, the total length obtaining WDR63 by the method for PCR is connected on retroviral expression vector, and order-checking qualification, is finally built into plasmid; Then carry out virus packaging, collect, virus titer is identified, is kept at-80 degree refrigerators after packing; Virus transfection, carries out plating to tip of a root dental papilla stem cell and spends the night, and then retroviral infection or slow polybrene reach 6 hours, after 48 hours, with the cell that different antibiotic-screenings is transfected;
Step 4, western blot analysis, RIPA lysate dissolved cell, sample 10%SDS polyacrylamide gel is separated and utilizes half-dried transfer film device to transfer in polyvinylidene difluoride, film is smeared 5% evaporated milk and place 2h, then hatch a night by primary antibodie; Immunocomplex and rabbit or mouse immuning ball protein G antibody are together hatched and use chemical luminous substrate reagent to make it visual; Anti-WDR63 polyclonal antibody for WDR63;
Step 5, ability of cell proliferation measures, tip of a root dental papilla stem cell close 1.0 × 10
4individual cell density is plated on 60nm culture dish; And within 3,5,7 days, count in cell cultures, cell counting adopts automated cell calculating instrument and in cell suspension, adds trypan blue gets rid of dead cell; Carry out 3 independent experiments to average;
Step 6, alkaline phosphatase and Alizarin red staining, mineralising induced liquid induction tip of a root dental papilla stem cell, alkaline phosphatase activities detects to be analyzed according to the explanation of alkaline phosphatase activities detection kit, for detecting mineralization ability, after cell induction 2-3 week, fix with 70% ethanol, 2% Alizarin red staining; Quantitative assay calcium ion concn, the cetylpyridinium chloride(CPC) with 10% is dissolved in sodium phosphate at room temperature makes sodium alizarinsulfonate fade 30 minutes; Calcium ion concn is that the absorbancy by measuring 562nm determines, and converts with typical curve;
Step 7, nude mice by subcutaneous carries out cell Hui Zhi, by 4.0 × 10
6individual cell mixes with the hydroxyapatite/tricalcium phosphate ceramic particle of 40 milligrams, then be transplanted to 5 10 week age nude mice dorsal sc, each nude mice, the stem cell transplantation of blank tip of a root dental papilla under left dorsal surface skin, and WDR63 tip of a root dental papilla cells be transplanted to right side back surface subcutaneous; Carry out according to animal protocol approval specification; Transplant latter 8th week, obtain transplanted cells 10% formalin and fix, 10%EDTA decalcification, pH value 8.0, paraffin embedding, HE dyes, observational measurement tissue mineralization amount.
Further, the concrete grammar of step one: peel off tip of a root dental papilla tissue gently, repeatedly clean with phosphate buffered saline (PBS), shred, be placed in the Digestive system containing type i collagen enzyme 3g/L and Dispase 4g/L, digest 1 hour at 37 DEG C, cross 70 μm of cell sieve collecting cells, the centrifugal 10min of 1000rpm, becomes single cell suspension with nutrient solution Eddy diffusion; Cell is inoculated in 25cm
2in Tissue Culture Flask, at substratum containing 37 DEG C, 5%CO in 15% foetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates
2cultivate, within every 2 ~ 3 days, change liquid 1 time; Every day is observation of cell upgrowth situation under inverted microscope; When Growth of Cells to 80% converging state, with 0.25% trypsinase by 1: 2 had digestive transfer culture; Experiment after the stem cell in 2-4 generation is used to.
Further, in step 2, signal must stdn to carry out contrast experiment; LOWESS program is used to weigh the mutability between coloured differently matter immunoprecipitation array, in order to determine the tri-methylated of H3 the 4th Methionin, tip of a root dental papilla stem cell under different condition there are differences and methylates, and therefore defines the threshold value that 2 times are changed to the tri-methylated noticeable change of H3 the 4th Methionin; Equally, assumed value threshold value 0.05 is also used for distinguishing gene alteration; Channel Image data are than using the standardization of LOWESS method, and assumed value calculating is the expression analysis using identical method to realize.
Further, in step 3, the target sequence of shRNA is:
WDR63shRNA：5′-AAACCCAGGGCTGCCTTGGAAAAG-3′。
Further, this WDR63 gene strengthens its alkaline phosphatase activities and mineralization ability to tooth to WDR63 in the regulate and control method in atomization at mescenchymal stem cell bone, and WDR63 can activate the expression of two key transcription factor OSX and RUNX2; WDR63 is the crucial toughener of of Osteoblast Differentiation; WDR63 promotes body outer cell proliferation ability to support, and WDR63 has the potential strengthening tissue regeneration; The tri-methylated Osteoblast Differentiation potential that can strengthen tip of a root dental papilla stem cell of H3 the 4th Methionin of WDR63 promotor.
The WDR63 gene that the embodiment of the present invention provides mescenchymal stem cell bone to tooth to the regulate and control method in atomization, find that WDR63 gene to the regulating and controlling effect in atomization, and is promoting the effect in dental tissue regeneration to/tooth at mescenchymal stem cell bone by series of experiments.The present invention adopt relate to the tri-methylated gene activation to mescenchymal stem cell of H3 the 4th Methionin and function regulating effect, WDR63 gene at mescenchymal stem cell bone to/tooth to the regulating and controlling effect in atomization, relate to WDR63 gene and promoting the effect in dental tissue regeneration, obtaining WDR63 may play a driving role in tip of a root dental papilla stem cell Osteoblast Differentiation.
Accompanying drawing explanation
Fig. 1 is that the skeletonization cultivation that the embodiment of the present invention provides promotes the tri-methylated of H3 the 4th Methionin in these promotors of WDR63, TREX1, FOXO24, ARNT, and induction WDR63 and ARNTL expresses; And inhibition of histone 3 the 4th Methionin is tri-methylated in these promotors of FOXP4, TMEM106B, FOXP4 is suppressed to express schematic diagram;
Fig. 2 is that the process LAN of the WDR63 that the embodiment of the present invention provides strengthens the bone of tip of a root dental papilla stem cell to differentiation capability schematic diagram;
Fig. 3 is that the process LAN of the WDR63 that the embodiment of the present invention provides increases mineralized tissue formation volume schematic diagram in body;
Fig. 4 is that the low expression of the WDR63 that the embodiment of the present invention provides can suppress the stem cells hyperplasia of tip of a root dental papilla and bone to differentiation capability schematic diagram;
Fig. 5 be the WDR63 gene that provides of the embodiment of the present invention mescenchymal stem cell bone to tooth to the regulate and control method schema in atomization.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
As shown in Figure 5, the WDR63 gene of the embodiment of the present invention comprises the following steps to the regulate and control method in atomization to tooth at mescenchymal stem cell bone:
S501: cell cultures, wisdom tooth rinses with phosphate buffered saline (PBS) after with 75% alcohol disinfecting again, is separated and culture identification tip of a root dental papilla stem cell;
S502: chromatin imrnunoprecipitation reaction and data analysis, cell hatches 15 minutes in 1% formaldehyde solution, and 2 × 10
6the tri-methylated antibody of individual cell and anti-histone 3 the 4th Methionin is used for the reaction of this chromatin imrnunoprecipitation, and the precipitation DNA sample of all generations adopts real-time quantitative pcr to quantize, and data are expressed as the per-cent of DNA;
S503: the SiRNA of design WDR63, is inserted on the shRNA carrier of slow virus, order-checking qualification, is finally built into the plasmid of WDR63shRNA; The PCR primer of design WDR63 full length gene, the total length obtaining WDR63 by the method for PCR is connected on retroviral expression vector, and order-checking qualification, is finally built into plasmid;
S504: then carry out virus packaging, collect, virus titer is identified, is kept at-80 degree refrigerators after packing; Virus transfection, carries out plating to tip of a root dental papilla stem cell and spends the night, and then retroviral infection or slow polybrene reach 6 hours, after 48 hours, with the cell that different antibiotic-screenings is transfected;
S505:RIPA lysate dissolved cell, sample 10%SDS polyacrylamide gel is separated and utilizes half-dried transfer film device to transfer in polyvinylidene difluoride, film is smeared 5% evaporated milk and places 2h, then hatch a night by primary antibodie; Immunocomplex and rabbit or mouse immuning ball protein G antibody are together hatched and use chemical luminous substrate reagent to make it visual;
S506: ability of cell proliferation measures, tip of a root dental papilla stem cell close 1.0 × 10
4individual cell density is plated on 60nm culture dish; And within 3,5,7 days, count in cell cultures, cell counting adopts automated cell calculating instrument and in cell suspension, adds trypan blue gets rid of dead cell; Carry out 3 independent experiments and get its mean value;
S507: mineralising induced liquid induction tip of a root dental papilla stem cell, alkaline phosphatase activities detects to be analyzed according to the explanation of alkaline phosphatase activities detection kit, after cell induction 2-3 week, fixes with 70% ethanol, 2% Alizarin red staining; Quantitative assay calcium ion concn, the cetylpyridinium chloride(CPC) with 10% is dissolved in sodium phosphate at room temperature makes sodium alizarinsulfonate fade 30 minutes; Calcium ion concn is that the absorbancy by measuring 562nm determines, and converts with typical curve;
S508: by 4.0 × 10
6individual cell mixes with the hydroxyapatite/tricalcium phosphate ceramic particle of 40 milligrams, then be transplanted to 5 10 week age nude mice dorsal sc, each nude mice, the stem cell transplantation of blank tip of a root dental papilla under left dorsal surface skin, and WDR63 tip of a root dental papilla cells be transplanted on the right side of back surface subcutaneous; These programs are carried out according to animal protocol approval specification; Transplant latter 8th week, obtain transplanted cells 10% formalin and fix, 10%EDTA decalcification (pH value 8.0), paraffin embedding, HE dyes, observational measurement tissue mineralization amount.
Below in conjunction with embodiment, the present invention is described in further details;
One, cell cultures
All stem cells involved in the present invention are all in accordance with the Behavioral guidelines of hESC's research, and the utilization of tissue obtains 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 rinses with phosphate buffered saline (PBS) after with 75% alcohol disinfecting again, be separated and culture identification tip of a root dental papilla stem cell, be summarized as follows: peel off tip of a root dental papilla tissue gently, repeatedly clean with phosphate buffered saline (PBS), shred, be placed in the Digestive system containing type i collagen enzyme (3g/L) and Dispase (4g/L), digest 1 hour at 37 DEG C, cross 70 μm of cell sieve collecting cells, the centrifugal 10min of 1000rpm, becomes single cell suspension with nutrient solution Eddy diffusion; Cell is inoculated in 25cm
2in Tissue Culture Flask, 37 DEG C, 5%CO in substratum (containing 15% foetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates)
2cultivate, within every 2 ~ 3 days, change liquid 1 time; Every day is observation of cell upgrowth situation under inverted microscope; When Growth of Cells to 80% converging state, with 0.25% trypsinase by 1: 2 had digestive transfer culture; Experiment after the stem cell in 2-4 generation is used to;
Two, chromatin imrnunoprecipitation reaction and data analysis:
Chromatin imrnunoprecipitation reaction is carried out according to the description of product of manufacturers and uses human promoter 1.0R array analysis; Cell hatches 15 minutes in 1% formaldehyde solution, and 2 × 10
6the tri-methylated antibody of individual cell and anti-histone 3 the 4th Methionin is used for the reaction of this chromatin imrnunoprecipitation, and the precipitation DNA sample of all generations adopts real-time quantitative pcr to quantize, and data are expressed as the per-cent of DNA; Primer is as shown in supplementary table 1; Due to experiment and technique variation, signal must stdn to carry out suitable contrast experiment; LOWESS program is used to weigh the mutability between coloured differently matter immunoprecipitation array, in order to determine the tri-methylated of H3 the 4th Methionin, tip of a root dental papilla stem cell under different condition there are differences and methylates, and therefore defines the threshold value that 2 times are changed to the tri-methylated noticeable change of H3 the 4th Methionin; Equally, assumed value threshold value 0.05 is also used for distinguishing gene alteration; Simple, Channel Image data are than using the standardization of LOWESS method, and assumed value calculating is the expression analysis using identical method to realize;
Three, plasmid construction and virus transfection:
The structure of plasmid is carried out in standard method; All structures are all verified by suitable restriction digestion and/or order-checking; The SiRNA of design WDR63, is inserted on the shRNA carrier of slow virus, and order-checking qualification, is finally built into the plasmid of WDR63shRNA; The PCR primer of design WDR63 full length gene, the total length obtaining WDR63 by the method for PCR is connected on retroviral expression vector, and order-checking qualification, is finally built into plasmid; Then carry out virus packaging, collect, virus titer is identified, is kept at-80 degree refrigerators after packing; Virus transfection, carries out plating to tip of a root dental papilla stem cell and spends the night, and then retroviral infection or slow polybrene reach 6 hours, after 48 hours, with the cell that different antibiotic-screenings is transfected;
The target sequence of shRNA is:
WDR63shRNA：5′-AAACCCAGGGCTGCCTTGGAAAAG-3′；
Four, western blot analysis:
RIPA lysate dissolved cell, sample 10%SDS polyacrylamide gel is separated and utilizes half-dried transfer film device to transfer in polyvinylidene difluoride (PVDF), film is smeared 5% evaporated milk and places 2h, then hatch a night by primary antibodie; Immunocomplex and rabbit or mouse immuning ball protein G antibody are together hatched and use chemical luminous substrate reagent to make it visual; Anti-WDR63 polyclonal antibody mainly for WDR63;
Five, ability of cell proliferation measures:
Tip of a root dental papilla stem cell close 1.0 × 10
4individual cell density is plated on 60nm culture dish; And within 3,5,7 days, count in cell cultures, cell counting adopts automated cell calculating instrument and in cell suspension, adds trypan blue gets rid of dead cell; Carry out 3 independent experiments and get its mean value;
Six, alkaline phosphatase and Alizarin red staining:
Mineralising induced liquid induction tip of a root dental papilla stem cell, alkaline phosphatase activities detects to be analyzed according to the explanation of alkaline phosphatase activities detection kit, for detecting its mineralization ability, after cell induction 2-3 week, fixes with 70% ethanol, 2% Alizarin red staining; Quantitative assay calcium ion concn, the cetylpyridinium chloride(CPC) with 10% is dissolved in sodium phosphate at room temperature makes sodium alizarinsulfonate fade 30 minutes; Calcium ion concn is that the absorbancy by measuring 562nm determines, and converts with typical curve;
Seven, nude mice by subcutaneous carries out cell Hui Zhi:
The present invention is allowed by BJ Stomatological Hospital of Capital University of Medical Sciences Animal Care and the council of use; By about 4.0 × 10
6individual cell mixes with the hydroxyapatite/tricalcium phosphate ceramic particle of 40 milligrams, then by previously described be transplanted to 5 10 week age nude mice dorsal sc, each nude mice, the stem cell transplantation of blank tip of a root dental papilla under left dorsal surface skin, and WDR63 tip of a root dental papilla cells be transplanted on the right side of back surface subcutaneous; These programs are carried out according to animal protocol approval specification; Transplant latter 8th week, obtain transplanted cells 10% formalin and fix, 10%EDTA decalcification (pH value 8.0), paraffin embedding, HE dyes, observational measurement tissue mineralization amount;
Eight, experimental result:
Chromatin imrnunoprecipitation reaction is used to generate gene promoter region histone modification gene mapping:
The tri-methylated full-length genome gene of H3 the 4th Methionin of promoter region distribution when attempting to detect tip of a root dental papilla stem cell Osteoblast Differentiation; Cultivate tip of a root dental papilla stem cell 7 days with osteogenic and ordinary culture medium respectively, the concentrated chip cohybridization using the tri-methylated antibody of anti-histone 3 the 4th Methionin and difference to mark carries out chromatin imrnunoprecipitation reaction; Chromatin imrnunoprecipitation response data shows, and after osteogenic induction, gene promoter is rich in tri-methylated H3 the 4th Methionin; In addition, do not breaking up and breaking up the 4th the Methionin gene mapping discovery of the tri-methylated H3 of tip of a root dental papilla stem cell gene promoter region, 119 gene promoters show the growth being greater than twice at tri-methylated H3 the 4th Methionin, and 21 gene promoters show the reduction being greater than twice at tri-methylated H3 the 4th Methionin; In order to confirm chromatin imrnunoprecipitation reaction experiment data, these gene proofing chips of WDR63, TREX1, FOXO24, ARNTL, FOXP4, TMEM106B are analyzed; Result shows relative to ordinary culture medium, at WDR63 during osteogenic induction, TREX1, FOXO24, H3 tri-methylated in these promotors of ARNTL the 4th Methionin significantly increases, and H3 the 4th Methionin tri-methylated in these promotors of FOXP4, TMEM106B significantly reduces (picture 1a-f); In addition, real-time quantitative PCR result is presented in tip of a root dental papilla differentiation of stem cells process, and WDR63, ARNTL present the high expression level being greater than twice, and FOXP4 then expresses (picture 1g-i) with being; But TREX1, FOXO24, TMEM106B are at differentiation and the expression no significant difference do not broken up in tip of a root dental papilla stem cell; Generally speaking, these data allow and infer that WDR63 may play a driving role in tip of a root dental papilla stem cell Osteoblast Differentiation;
WDR63 process LAN strengthens the Osteoblast Differentiation potential of tip of a root dental papilla stem cell
The function of further confirmation WDR63 in tip of a root dental papilla stem cell, inserts a retroviral vector by WDR63 sequence; Overexpression WDR63 when this structure is attached to tip of a root dental papilla stem cell by retroviral infection; Also by the overexpression (picture 2a) of Western blot experiment checking WDR63; Next, in conjunction with after tip of a root dental papilla stem cell carry out osteogenic induction to detect its Osteoblast Differentiation potential, result shows, the overexpression of WDR63 adds alkaline phosphatase activities (picture 2b); Therefore, by Alizarin red staining and calcium ion quantitative measurment, the tip of a root dental papilla stem cell of overexpression WDR63 strengthens (picture 2c-d) relative to its mineralization ability of tip of a root dental papilla stem cell of empty vectors; Real-time quantitative PCR result also shows, and at the 7th day and the 14th day that the tip of a root dental papilla stem cell of overexpression WDR63 is induced, the expression of resorption lacunae was significantly increased (picture 2e); Next, have detected the expression of the key transcription factor regulating Osteoblast Differentiation, comprise RUNX2 and OSX; The rna level of RUNX2 and OSX significantly increases the tip of a root dental papilla stem cell (picture 2f-g) relative to empty vectors in the tip of a root dental papilla stem cell of overexpression WDR63; Next, examine whether to add WDR63 and can affect the osteogenesis in vivo of tip of a root dental papilla stem cell; SCA unloaded tip of a root dental papilla stem cell and year WDR63 tip of a root dental papilla stem cell transplantation are to nude mice by subcutaneous; The 8th week after the transfer, HE dyeed display, had more bone sample mineralized tissue to compare blank group (picture 3a) carrying in WDR63 tip of a root dental papilla stem cell transplants of obtaining; Observational measurement mineralized tissue display bone sample mineralized tissue amount is higher than blank group (picture 3b) in year WDR63 tip of a root dental papilla stem cell transplants; Therefore, in body, transplantation experiments shows that a year WDR63 tip of a root dental papilla stem cell produces more bone sample mineralized tissue and compares unloaded tip of a root dental papilla stem cell; In sum, these results show, WDR63 expresses the tip of a root dental papilla stem cell Osteoblast Differentiation greatly triggered;
The minimizing of WDR63 suppresses the bone of tip of a root dental papilla stem cell to differentiation
In order to illustrate the function of WDR63 in tip of a root dental papilla stem cell further, devising a ShorthairpinRNA target and be to suppress WDR63 expression formula and it is introduced tip of a root dental papilla stem cell by slow-virus transfection; After selection, detect the usefulness (picture 4a) lowered with immunoblotting assay; Next, the osteogenic ability whether WDR63 have impact on tip of a root dental papilla stem cell is in itself detected; Tip of a root dental papilla is cultivated by stem cell in osteogenic, finds to suppress its alkaline phosphatase activities of tip of a root dental papilla stem cell of WDR63 significantly lower than blank tip of a root dental papilla stem cell (picture 4b); By Alizarin red staining and calcium ion quantitative measurment after osteogenic induction, the tip of a root dental papilla stem cell of WDR63 is suppressed obviously to reduce (picture 4c-d) relative to blank its mineralization ability of tip of a root dental papilla stem cell; In addition, carry out ability of cell proliferation detection display in cell cultures after 7 days, suppress the tip of a root dental papilla stem cell of WDR63 obviously to reduce (picture 4e) relative to blank its multiplication capacity of tip of a root dental papilla stem cell;
Nine, conclusion
WDR63 is a kind of WD repetitive proteins of unknown function, the function of research WDR63 in tip of a root dental papilla differentiation of stem cells process, find inside and outside tip of a root dental papilla stem cell body in atomization, WDR63 strengthens its alkaline phosphatase activities and mineralization ability, and this shows that WDR63 may be the key factor controlling mescenchymal stem cell Osteoblast Differentiation potential; Mescenchymal stem cell to the differentiation of scleroblast system require other clone break up between coordination and suppression, the activation of multiple transcription factor is relevant with Derived from Mesenchymal Stem Cells, two crucial transcription factors, RUNX2 and OSX is that Osteoblast Differentiation is necessary; Result of study shows, WDR63 can activate the expression of two key transcription factor OSX and RUNX2; Next, have detected the mRNA level in-site of resorption lacunae, it is the major structural protein of ground substance of bone; Result of study shows, and WDR63 induces the expression of resorption lacunae gene; These results show that WDR63 is the crucial toughener of of Osteoblast Differentiation; In addition, cell growth curve shows to suppress WDR63 can suppress the propagation of tip of a root dental papilla stem cell; The vitro detection of the growth of mescenchymal stem cell, propagation and viability can mescenchymal stem cell function in Accurate Prediction body; These find significantly to show to strengthen the growth of mescenchymal stem cell, propagation, and viability may improve their blood vessel and tissue regeneration potentiality; In current research, for WDR63, result of study promotes that body outer cell proliferation ability support WDR63 has the potential strengthening tissue regeneration;
In a word, result represents the modification of international viewpoint and tri-methylated H3 the 4th Methionin and tip of a root dental papilla stem cell bone to functionally contacting of breaking up, and shows by changing tri-methylated H3 the 4th Methionin controlling gene and activate and reticent most important to the Osteoblast Differentiation of mescenchymal stem cell; Also find a crucial Osteoblast Differentiation toughener-WDR63, the tri-methylated Osteoblast Differentiation potential that can strengthen tip of a root dental papilla stem cell of H3 the 4th Methionin of WDR63 promotor.
Supplement chart 1: the primer carrying out chromatin imrnunoprecipitation reaction real-time quantitative PCR
Supplement chart 2: the primer carrying out real-time quantitative PCR test
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. WDR63 gene mescenchymal stem cell bone to tooth to the regulate and control method in atomization, it is characterized in that, this WDR63 gene comprises to the regulate and control method in atomization to tooth at mescenchymal stem cell bone:
Step one, cell cultures, wisdom tooth rinses with phosphate buffered saline (PBS) after with 75% alcohol disinfecting again, is separated and culture identification tip of a root dental papilla stem cell;
Step 2, chromatin imrnunoprecipitation reaction and data analysis, cell hatches 15 minutes in 1% formaldehyde solution, and 2 × 10
6the tri-methylated antibody of individual cell and anti-histone 3 the 4th Methionin is used for the reaction of this chromatin imrnunoprecipitation, and the precipitation DNA sample of all generations adopts real-time quantitative pcr to quantize, and data are expressed as the per-cent of DNA;
Step 3, plasmid construction and virus transfection, the structure of plasmid is carried out in standard method, and the SiRNA of design WDR63, is inserted on the shRNA carrier of slow virus, and order-checking qualification, is finally built into the plasmid of WDR63shRNA; The PCR primer of design WDR63 full length gene, the total length obtaining WDR63 by the method for PCR is connected on retroviral expression vector, and order-checking qualification, is finally built into plasmid; Then carry out virus packaging, collect, virus titer is identified, is kept at-80 degree refrigerators after packing; Virus transfection, carries out plating to tip of a root dental papilla stem cell and spends the night, and then retroviral infection or slow polybrene reach 6 hours, after 48 hours, with the cell that different antibiotic-screenings is transfected;
Step 4, western blot analysis, RIPA lysate dissolved cell, sample 10%SDS polyacrylamide gel is separated and utilizes half-dried transfer film device to transfer in polyvinylidene difluoride, film is smeared 5% evaporated milk and place 2h, then hatch a night by primary antibodie; Immunocomplex and rabbit or mouse immuning ball protein G antibody are together hatched and use chemical luminous substrate reagent to make it visual; Anti-WDR63 polyclonal antibody for WDR63;
Step 5, ability of cell proliferation measures, tip of a root dental papilla stem cell close 1.0 × 10
4individual cell density is plated on 60nm culture dish; And within 3,5,7 days, count in cell cultures, cell counting adopts automated cell calculating instrument and in cell suspension, adds trypan blue gets rid of dead cell; Carry out 3 independent experiments to average;
Step 6, alkaline phosphatase and Alizarin red staining, mineralising induced liquid induction tip of a root dental papilla stem cell, alkaline phosphatase activities detects to be analyzed according to the explanation of alkaline phosphatase activities detection kit, for detecting mineralization ability, after cell induction 2-3 week, fix with 70% ethanol, 2% Alizarin red staining; Quantitative assay calcium ion concn, the cetylpyridinium chloride(CPC) with 10% is dissolved in sodium phosphate at room temperature makes sodium alizarinsulfonate fade 30 minutes; Calcium ion concn is that the absorbancy by measuring 562nm determines, and converts with typical curve;
Step 7, nude mice by subcutaneous carries out cell Hui Zhi, by 4.0 × 10
6individual cell mixes with the hydroxyapatite/tricalcium phosphate ceramic particle of 40 milligrams, then be transplanted to 5 10 week age nude mice dorsal sc, each nude mice, the stem cell transplantation of blank tip of a root dental papilla under left dorsal surface skin, and WDR63 tip of a root dental papilla cells be transplanted to right side back surface subcutaneous; Carry out according to animal protocol approval specification; Transplant latter 8th week, obtain transplanted cells 10% formalin and fix, 10%EDTA decalcification, pH value 8.0, paraffin embedding, HE dyes, observational measurement tissue mineralization amount.
2. WDR63 gene as claimed in claim 1 mescenchymal stem cell bone to tooth to the regulate and control method in atomization, it is characterized in that, the concrete grammar of step one: peel off tip of a root dental papilla tissue gently, repeatedly clean with phosphate buffered saline (PBS), shred, be placed in the Digestive system containing type i collagen enzyme 3g/L and Dispase 4g/L, digest 1 hour at 37 DEG C, cross 70 μm of cell sieve collecting cells, the centrifugal 10min of 1000rpm, becomes single cell suspension with nutrient solution Eddy diffusion; Cell is inoculated in 25cm
2in Tissue Culture Flask, at substratum containing 37 DEG C, 5%CO in 15% foetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates
2cultivate, within every 2 ~ 3 days, change liquid 1 time; Every day is observation of cell upgrowth situation under inverted microscope; When Growth of Cells to 80% converging state, with 0.25% trypsinase by 1: 2 had digestive transfer culture; Experiment after the stem cell in 2-4 generation is used to.
3. WDR63 gene as claimed in claim 1 mescenchymal stem cell bone to tooth to the regulate and control method in atomization, it is characterized in that, in step 2, signal must stdn to carry out contrast experiment; LOWESS program is used to weigh the mutability between coloured differently matter immunoprecipitation array, in order to determine the tri-methylated of H3 the 4th Methionin, tip of a root dental papilla stem cell under different condition there are differences and methylates, and therefore defines the threshold value that 2 times are changed to the tri-methylated noticeable change of H3 the 4th Methionin; Equally, assumed value threshold value 0.05 is also used for distinguishing gene alteration; Channel Image data are than using the standardization of LOWESS method, and assumed value calculating is the expression analysis using identical method to realize.
4. WDR63 gene as claimed in claim 1 mescenchymal stem cell bone to tooth to the regulate and control method in atomization, it is characterized in that, in step 3, the target sequence of shRNA is:
WDR63shRNA：5′-AAACCCAGGGCTGCCTTGGAAAAG-3′。
5. WDR63 gene as claimed in claim 1 mescenchymal stem cell bone to tooth to the regulate and control method in atomization, it is characterized in that, this WDR63 gene strengthens its alkaline phosphatase activities and mineralization ability to tooth to WDR63 in the regulate and control method in atomization at mescenchymal stem cell bone, and WDR63 can activate the expression of two key transcription factor OSX and RUNX2; WDR63 is the crucial toughener of of Osteoblast Differentiation; WDR63 promotes body outer cell proliferation ability to support, and WDR63 has the potential strengthening tissue regeneration; The tri-methylated Osteoblast Differentiation potential that can strengthen tip of a root dental papilla stem cell of H3 the 4th Methionin of WDR63 promotor.
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