WO2018153037A1 - Barx1的用途 - Google Patents

Barx1的用途 Download PDF

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WO2018153037A1
WO2018153037A1 PCT/CN2017/098262 CN2017098262W WO2018153037A1 WO 2018153037 A1 WO2018153037 A1 WO 2018153037A1 CN 2017098262 W CN2017098262 W CN 2017098262W WO 2018153037 A1 WO2018153037 A1 WO 2018153037A1
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stem cells
barx1
mesenchymal stem
gene
expression
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PCT/CN2017/098262
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English (en)
French (fr)
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范志朋
林潇
王松灵
董蕊
李钧
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首都医科大学附属北京口腔医院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources

Definitions

  • Mesenchymal stem cells are multi-directional differentiation potential stem cells originally differentiated from bone marrow, and can differentiate into many different types of cells, including osteoblasts, chondrocytes, muscle cells, and fat cells. There is increasing evidence that mesenchymal stem cells can be present in non-myeloid tissues. Most adult mesenchymal stem cells can be used for cell-mediated tissue engineering. Mesenchymal stem cells derived from different tissues such as bone marrow, periosteum, and adipose tissue have similar surface marker characteristics, but there are significant differences in differentiation, proliferation, and migration of mesenchymal cells derived from different tissues.
  • the homeobox transcription factor Barx gene family participates in mesenchymal aggregation and formation through regulation of cell adhesion molecules and regulation of type II collagen.
  • the transcription factor BARX1 is expressed in the mesenchyme of the molar base, which not only participates in the regulation of tooth morphogenesis, but also plays an important role in the development of teeth and maxillofacial mesenchymal cells derived from neural crest.
  • Barx1 is expressed in mesenchymal agglutination sites, including the pharyngeal arch, limb buds, developing joints, molar nipples, and stomach wall.
  • the technical problem to be solved by the present invention is to provide the use of BARX1, and the present invention proves that the BARX1 gene can regulate the osteogenesis/dental orientation of mesenchymal stem cells by regulating the expression of genes related to osteogenic and odontogenic cells. Regulation of differentiation.
  • the present invention provides the use of BARX1 for the preparation of a preparation for regulating the expression of dentition-related genes in mesenchymal stem cells.
  • the dental related genes are DSPP, DMP1 and/or OSX.
  • the mesenchymal stem cells are apical papillary mesenchymal stem cells, umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.
  • the regulation is specifically: overexpressing the BARX1 gene, promoting the expression of the DSPP gene, the DMP1 gene and/or the OSX gene; knocking out the BARX1 gene, and inhibiting the expression of the DSPP gene, the DMP1 gene and/or the OSX gene.
  • the invention also provides the use of BARX1 in the preparation of a preparation for regulating osteogenic related genes in mesenchymal stem cells.
  • the osteogenic related genes are BSP and/or OSX.
  • the mesenchymal stem cells are apical papillary mesenchymal stem cells, umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.
  • the regulation is specifically: overexpressing the BARX1 gene, promoting the expression of the OSX gene, inhibiting the expression of the BSP gene; knocking out the BARX1 gene, inhibiting the expression of the OSX gene, and promoting the expression of the BSP gene.
  • the BARX1 of the present invention is a BARX1 protein, a BARX1 gene sequence, a substance capable of overexpressing the BARX1 gene in mesenchymal cells, or a substance capable of knocking out or knocking down the BARX1 gene in mesenchymal cells.
  • accession number of the nucleotide sequence of the BARX1 gene is GeneID: 56033, and the amino acid sequence of the BARX1 protein is translated from the nucleotide sequence of Accession No. GeneID:56033.
  • the substance capable of overexpressing the BARX1 gene in mesenchymal cells is an expression vector comprising the BARX1 gene; or a retrovirus transfected with an expression vector comprising the BARX1 gene.
  • the expression vector for the retrovirus is pQCXIP.
  • the substance capable of knocking out the BARX1 gene in mesenchymal cells is siRNA of BAXR1, and the sequence is TCCTGCTCAGTAAGCTGCTCG.
  • a shRNA plasmid of BAXR1 was constructed by inserting siRNA of BAXR1 into a shRNA vector of lentivirus.
  • the BARX1 gene in the umbilical cord mesenchymal stem cells is overexpressed, and the osteoblast-inducing culture medium induces stem cell differentiation after culture, and after 3 weeks, the osteogenesis-related and dental-related genes are detected at the mRNA level.
  • the results showed that overexpression of BARX1 inhibited the expression of BSP in umbilical cord mesenchymal stem cells, promoted the expression of DSPP and DMP1 in umbilical cord mesenchymal stem cells, and promoted the expression of OSX in umbilical cord mesenchymal stem cells. In the results, the promotion or inhibition was significantly different (p ⁇ 0.05 or p ⁇ 0.01) compared to the unexpressed normal umbilical cord mesenchymal stem cells.
  • the BARX1 gene in the root apical papilla mesenchymal stem cells is overexpressed, and the osteoblast-inducing culture medium induces stem cell differentiation after culture, and after 3 weeks, the osteogenesis-related, dental-related genes are detected at the mRNA level.
  • the results showed that overexpression of BARX1 inhibited the expression of BSP in root canal nipple mesenchymal stem cells, promoted the expression of DSPP and DMP1 in root canal nipple mesenchymal stem cells, and promoted the expression of OSX in root apical mesenchymal stem cells.
  • the promotion or inhibition was significantly different (p ⁇ 0.05 or p ⁇ 0.01) compared to the unexpressed normal root canine papilla mesenchymal stem cells.
  • the invention also provides the use of the BARX1 gene in the preparation of a preparation for regulating the mineralization ability of mesenchymal stem cells.
  • the mesenchymal stem cells are apical papillary mesenchymal stem cells or umbilical cord mesenchymal stem cells.
  • the mineralization refers to the formation of calcified nodules.
  • the BARX1 gene in the umbilical cord mesenchymal stem cells is overexpressed, and the stem cell differentiation is induced by the osteogenic induction culture medium, and the formation of calcium nodules is observed under the light microscope periodically.
  • the results indicate that overexpression of BARX1 inhibits the mineralization ability of umbilical cord mesenchymal stem cells in vitro.
  • the BARX1 gene in the root canal nipple mesenchymal stem cells is overexpressed, and the stem cell differentiation medium is induced to induce stem cell differentiation, and the formation of calcium nodules is observed under a light microscope periodically.
  • the results showed that overexpression of BARX1 inhibited the mineralization ability of root canal nipple mesenchymal stem cells in vitro.
  • the present invention also provides the use of BARX1 for the preparation of a preparation for regulating odontogenic and/or osteogenic differentiation of mesenchymal stem cells.
  • the mesenchymal stem cells are root tip nipple mesenchymal stem cells, umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.
  • the experiments of the present invention show that overexpression of the BARX1 gene can promote the expression of dentition-related genes in mesenchymal stem cells; and inhibit the expression of osteogenic related genes.
  • the final results indicate that overexpression of BARX1 gene can promote mesenchymal stem cell differentiation and inhibit osteogenic differentiation.
  • the present invention is implanted into a miniature pig dentin defect model by infecting root canine papilla stem cells (SCAPs) with a virus overexpressing the BARX1 vector.
  • SCAPs root canine papilla stem cells
  • the effect of expression of BARX1 on SCAPs differentiation in vivo was observed.
  • the ability of apical papilla stem cells to form dentin-like tissue was stronger than that of the control group.
  • the area of neonatal dentin tissue formation in the experimental group was increased compared with the control group.
  • the present inventors have shown that overexpression of BARX1 can promote the expression of osteoblast/osteogenesis-associated transcription factor OSX, while gene knockout of BARX1 inhibits the expression of OSX in mesenchymal stem cells.
  • the present invention shows that BARX1 directly binds to the OSX promoter sequence and positively regulates the transcription of OSX gene by OSX promoter analysis and chromatin immunoprecipitation (ChIP).
  • ChIP chromatin immunoprecipitation
  • co-immunoprecipitation (Co-IP) results indicate that BARX1 and OSX can form protein complexes in mesenchymal stem cells.
  • the OSX gene is used for regulating the expression of dentin-related genes and/or osteogenic related genes in mesenchymal stem cells; the osteogenic related gene is BSP; and the odontogene-related genes are DSPP and/or DMP1.
  • the present invention also provides a preparation for promoting the differentiation of mesenchymal stem cells into teeth, comprising a BARX1 protein or a substance capable of overexpressing the BARX1 gene.
  • the preparation for promoting the differentiation of mesenchymal stem cells into teeth comprises a substance capable of overexpressing the BARX1 gene.
  • a retrovirus transfected with an expression vector comprising the BARX1 gene e.g., pQCXIP.
  • the present invention also provides a preparation for promoting osteogenic differentiation of mesenchymal stem cells, comprising a BARX1 expression inhibitor or a substance capable of knocking out or knocking down the BARX1 gene.
  • the preparation for promoting osteogenic differentiation of mesenchymal stem cells includes a substance capable of knocking out or knocking down the BARX1 gene.
  • the substance capable of knocking out the BAXR1 gene was constructed by inserting a siRNA of BAXR1 into a shRNA vector of a lentivirus to construct a shRNA plasmid of BAXR1.
  • the present invention also provides a method for promoting dentin differentiation of mesenchymal stem cells, overexpressing the BARX1 gene of mesenchymal stem cells, or inducing mesenchymal stem cells with an inducer containing BARX1.
  • the present invention also provides a method for promoting osteogenic differentiation of mesenchymal stem cells, which knocks out the BARX1 gene of mesenchymal stem cells, or induces mesenchymal stem cells with an inducing solution containing a BARX1 expression inhibitor.
  • Promoting the differentiation of mesenchymal stem cells into teeth can also be called promoting the regeneration of dental tissues; promoting osteogenic differentiation of mesenchymal stem cells can be called promoting bone regeneration.
  • the results of the present invention indicate that BARX1 can affect the differentiation of mesenchymal stem cells into osteogenesis and teeth by regulating the expression of osteogenic and odontogenic genes, thereby affecting the in vitro mineralization ability of mesenchymal stem cells.
  • the present invention provides technical support for preparing bone tissue or dental tissue regenerative drugs containing the BARX1 gene.
  • Figure 1 shows that overexpression of BARX1 inhibits osteogenic differentiation of umbilical cord mesenchymal stem cells and promotes dental differentiation;
  • Figure 1-a shows the expression of HA;
  • Figure 1-b shows alizarin red staining;
  • Figure 1-c shows quantitative analysis of calcium ions;
  • Figure 1-d shows qRT-PCR results showing OSX expression;
  • Figure 1-e shows qRT-PCR results showing BSP expression;
  • Figure 1-f shows qRT-PCR results showing DSPP expression;
  • Figure 1-g shows qRT-PCR The results show the expression of DMP1;
  • Figure 3 shows that overexpression of BARX1 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells and promotes dental differentiation;
  • Figure 3-a shows qRT-PCR results showing overexpression of BARX1;
  • Figure 3-b shows qRT-PCR results showing BSP expression;
  • 3-c shows that qRT-PCR results show the expression of DSPP;
  • Figure 4 shows that overexpression of BARX1 promotes dendritic differentiation of root apical mesenchymal stem cells in vivo;
  • Figure 4-a shows HE staining shows the results of simple ⁇ -TCP dentin defect replantation, and
  • Figure 4-a-1 shows 40 ⁇ mirror
  • Figure 4-a-2 is a 100 ⁇ microscopic image;
  • Figure 4-b shows HE staining showing the return of the dentin defect of the ⁇ -TCP composite empty vector root canine stem cells (SCAPs-pQCXIP+ ⁇ -TCP)
  • SCAPs-pQCXIP+ ⁇ -TCP SCAPs-pQCXIP+ ⁇ -TCP
  • FIG. 4-c shows a HE staining showing a ⁇ -TCP complex overexpressing BARX1 apical papilla stem cells ( SCAPs-HA-BARX1+ ⁇ -TCP) results of replantation of dentin defects
  • Figure 4-c-1 is a 40 ⁇ microscope image
  • Figure 4-c-2 is a 100 ⁇ microscope image
  • Figure 5 is a representation of BARX1 regulating OSX expression and protein complex formation with OSX;
  • Figure 5-a shows qRT-PCR results showing overexpression of OSX after BARX1;
  • Figure 5-b shows qRT-PCR results showing gene knockout BARX1;
  • Figure 5-c shows qRT-PCR results showing the expression of OSX after gene knockout of BARX1;
  • Figure 5-d shows that CHIP results show binding of BARX1 protein on OSX promoter;
  • Figure 5-e shows Co-IP results showing overexpression of BARX1 Formation of the BARX1-OSX protein complex;
  • Figure 5-f shows that the Co-IP results show the formation of the BARX1-OSX protein complex after gene knockout BARX1;
  • Figure 5-g shows that the polypeptide microarray results show that BARX1 binds to OSX Peptide
  • Figure 6 shows that knockout OSX inhibits osteogenic differentiation and dentate differentiation of root canal papilla stem cells;
  • Figure 6-a shows that Western blot results show gene knockout OSX;
  • Figure 6-b shows alkaline phosphatase staining;
  • Figure 6-c Alkaline phosphatase activity assay;
  • Figure 6-d shows alizarin red staining;
  • Figure 6-e shows calcium ion quantification;
  • Figure 6-f shows qRT-PCR results show BSP expression;
  • Figure 6-g shows qRT-PCR The results show the expression of DSPP;
  • Figure 6-h shows that qRT-PCR results show the expression of DMP1.
  • test materials used in the present invention are all commercially available products, all of which are commercially available.
  • Osteopontin plays a very important role in promoting bone formation and differentiation, chemotaxis and adhesion of osteoblasts.
  • Core transcription factor is a core transcription factor that regulates bone formation and tooth formation and plays a key role in cell osteogenic differentiation and dentition differentiation.
  • Dentin ⁇ protein DSPP is the only dentin-specific non-collagen protein synthesized and secreted by odontoblasts, secreted to the mineralization front, plays a role in initiating dentin mineralization and regulating the size and growth rate of hydroxyapatite crystals. effect.
  • DMP1 Dentin matrix acidic phosphorylated protein 1
  • DMP-1 is an acidic non-collagen protein essential for bone mineralization, bone, cartilage, enamel, cementum and dentin biomineralization.
  • DMP-1 whole gene sequence is a biomineralization initiation factor. .
  • the extracted teeth were immediately placed in a centrifuge tube filled with pre-cooled PBS, transferred into a cell chamber, and the root canine stem cells were isolated and cultured within 24 hours. Gently peel off the periodontal tissues around the teeth, take the apical papilla tissue, wash repeatedly with PBS, cut and place in a digestive solution containing type I collagenase (3g/L) and Dispase (4g/L), 37°C After digestion for 1 hour, the cells were collected through a 70 ⁇ m cell sieve, centrifuged at 1000 rpm for 10 min, and resuspended in a culture medium to form a single cell suspension.
  • type I collagenase 3g/L
  • Dispase 4g/L
  • the cells were seeded in a 25 cm 2 cell culture flask in ⁇ -MEM medium (containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin) at 37 ° C, 5%. CO 2 culture, change the solution once every 2 to 3 days. Cell growth was observed daily under an inverted microscope. When the cells were grown to 80% confluence, they were digested with 1:2 trypsin at 1:2. Mesenchymal stem cells were identified by detecting the multi-directional differentiation ability of CD44, CD90, CD146, STRO-1 and stem cells of mesenchymal stem cells and the ability of colony formation. The results showed that the obtained mesenchymal stem cells conformed to the characteristics of stem cells, and their differentiation ability and colony forming ability also met the requirements.
  • ⁇ -MEM medium containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml
  • the gene sequence of BAXR1 (GeneID:56033) was searched by NCBI database, and the siRNA of BAXR1 (such as SEQ ID NO: 19, the sequence is TCCTGCTCAGTAAGCTGCTCG) was designed by Whitehead, and inserted into the shRNA vector of lentivirus. Finally, a plasmid for the shRNA of BAXR1 was constructed.
  • the full-length PCR primers of BAXR1 gene were designed, and the full length of BAXR1 was obtained by PCR and the surface tag HA tag was added.
  • the recombinant plasmid was ligated into the retroviral expression vector (pQCXIP), sequenced and identified, and finally constructed into HA-BAXR1. Plasmid. Then, the virus is packaged, collected, and the virus titer is identified, and stored in a refrigerator at -80 ° C after being dispensed.
  • control plasmid and HA-BAXR1 virus were transfected into root canine papillary stem cells, umbilical cord stem cells and bone marrow stem cells. After transfection for 48 hours, BAXR1 overexpressing stably transfected stem cells were screened by drug, and HA expression was detected at mRNA and protein levels. The expression of exogenous BARX1 was identified.
  • Real-time RT-PCR The process of real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is:
  • Primer design bio software such as Primer 3 and oligo 6;
  • RNA/primer mix was prepared in a Microtube tube: template RNA: 1 ng to 5 ug; Oligo (dT) (500 ⁇ g/ml): 1 ⁇ l; 10 mM dNTP: 1 ⁇ l; sterilized distilled water was added to 12 ul.
  • the Real-time PCR reaction system was set up as follows: 5XSYBR Mix: 5 ⁇ l; PCR Primer (5 pmol): 0.5 ⁇ l; ddH 2 O: 5 ⁇ l; cDNA: 10.5 ⁇ l; Total 21 ⁇ l.
  • 2.1 BSA protein standard was diluted with double distilled water according to the instructions, so that the final concentration was 1000 ⁇ g/ml, 750 ⁇ g/ml, 500 ⁇ g/ml, 250 ⁇ g/ml, 125 ⁇ g/ml, 62.5 ⁇ g/ml, 0 ⁇ g/ml;
  • the filter was placed in a 5% skim milk powder diluted HRP-labeled secondary antibody and incubated for 1 hour at room temperature on a shaker;
  • Example 1 The umbilical cord stem cells overexpressing BARX1 constructed in Example 1 were seeded in a 6-well plate at a concentration of 2 ⁇ 10 3 /cm 2 , and the osteogenic induction medium was used after the cells were grown to 80% confluence (Invitrogen The stem cells were induced to differentiate into the osteogenesis/dentinogenesis in vitro, and the liquid was changed every 3 days, and the formation of calcium nodules was observed under a light microscope.
  • the cells were harvested at different time points of induction 0, 3, 7, 14 and 21 days, and the osteogenic differentiation index-BSP was detected at the mRNA level (Fig. 2-c).
  • the results showed that overexpression of BARX1 inhibited apical papillary mesenchyme Stem cell BSP expression.
  • the bone marrow mesenchymal stem cells were seeded in a 6-well plate at a concentration of 2 ⁇ 10 3 /cm 2 . After the cells were grown to 80% confluence, the osteoblast-inducing medium was used to direct the stem cells to the osteogenesis/dentation direction in vitro. Differentiation was induced, and the liquid was changed every 3 days, and the formation of calcium nodules was observed under a light microscope.
  • the cells were harvested at different time points of 0, 3, 7 and 10 days, and the osteogenic differentiation index-bone ⁇ protein (BSP, Figure 3-b) was detected at the mRNA level.
  • BSP osteogenic differentiation index-bone ⁇ protein
  • the procedure is the same as in the first embodiment.
  • the RT-PCR primer sequence is:
  • OSX-F CCTCCTCAGCTCACCTTCTC (SEQ ID NO: 1);
  • OSX-R GTTGGGAGCCCAAATAGAAA (SEQ ID NO: 2);
  • BSP-F CAGGCCACGATATTATCTTTACA (SEQ ID NO: 3);
  • BSP-R CTCCTCTTCTTCCTCCTCCTC (SEQ ID NO: 4);
  • DSPP-F CGACATAGGTCACAATGAGGATGTCG (SEQ ID NO: 5);
  • DSPP-R TTGCTTCCAGCTACTTGAGGTC (SEQ ID NO: 6);
  • DMP1-F GACAGCCTCTCACTGGATTCGCTGTC (SEQ ID NO: 7);
  • DMP1-R CTCGCACACACTCTCCCACTC (SEQ ID NO: 8);
  • GAPDH-F CGGACCAATACGACCAAATCCG (SEQ ID NO: 9);
  • GAPDH-R AGCCACATCGCTCAGACACC (SEQ ID NO: 10).
  • SCAPs were mixed with HA/TCP and implanted into a miniature pig dentin defect model to observe the effect of overexpression of BARX1 on SCAPs differentiation in vivo. .
  • the fourth generation cells with good growth state were inoculated into a 10 cm culture dish at a density of 1 ⁇ 10 5 cells/dish. When the cells were grown to 80% confluence, the medium was discarded and rinsed with PBS (to ensure cell status). , cell confluence should not be too large).
  • ⁇ -MEM medium containing 15% FBS, 1% double antibody and 1% L-glutamine, count, and then take 5 ⁇ 10 5 cells in a 12-well plate with a diameter of 3-3.5.
  • Mm, 1 mm thick ⁇ -tricalcium phosphate ( ⁇ -TCP) incubated at 37 ° C, 5% CO 2 for 2 h, each 2 ml containing 15% FBS, 1% double antibody and 1% L-glutamine
  • the ⁇ -MEM medium was further incubated at 37 ° C under 5% CO 2 for 3 days until use.
  • the cell-free ⁇ -TCP was also cultured for 3 days in 2 ml of the same medium under the same conditions.
  • the first and second premolars were opened (diameter 3-4 mm open medullary holes), and 3 teeth on different small pigs were randomly selected and placed into the cell-free ⁇ -TCP as described in 1.5.
  • Six teeth were placed in 1.5 and the control cells (SCAP-pQCXIP) were incubated with ⁇ -TCP (SCAP-pQCXIP+ ⁇ -TCP), and 6 teeth were placed in 1.5.
  • the experimental group cells were incubated (SCAPs- HA-BARX1) ⁇ -TCP (SCAPs-HA-BARX1+ ⁇ -TCP), a total of 15 premolars, glass ionomer cement temporarily sealed.
  • the mini-pigs were sacrificed, and the tissue samples were taken out and fixed in 4% paraformaldehyde, then transferred to 10% EDTA with a pH of 7 (7.4-7.6). After complete decalcification. The running water was rinsed for 24 hours, and then the tissue was dehydrated and embedded.
  • Dehydration step 75% ethanol I: 10 min; 75% ethanol II: 10 min; 85% ethanol I: 10 min; 85% ethanol II: 10 min; 95% ethanol I: 10 min; 95% ethanol II: 10 min; 30 min; absolute ethanol II: 30 min; xylene I: 30 min; xylene II: 30 min; dip wax I: 30 min; dip wax II: overnight.
  • Embedding After the dipping wax is finished, transfer the tissue to the preheated embedding frame, quickly pour the molten wax, adjust the position of the tissue block with warm tweezers, place on the refrigerator, and solidify the molten wax.
  • Sectioning After trimming the wax block, 5 ⁇ m thick serial sections were cut in a rotary microtome, and the sections were placed on a polylysine-treated slide and dried overnight at 37 °C.
  • Example 4 BARX1 positively regulates gene expression of OSX in mesenchymal stem cells and forms a protein complex with OSX.
  • BARX1 lentivirus-mediated shRNA
  • BARX1sh1, BARX1sh2 lentivirus-mediated shRNA
  • Co-IP co-immunoprecipitation
  • BARX1 not only directly regulates the expression of OSX in mesenchymal stem cells, but also forms a protein complex with OSX to regulate the differentiation function of mesenchymal stem cells.
  • the procedure is the same as in the first embodiment.
  • the RT-PCR primer sequence is:
  • BARX1-F CGCTTCGAGAAGCAGAAGTA (SEQ ID NO: 11)
  • BARX1-R CTTCATCCTCCGATTCTGGT (SEQ ID NO: 12)
  • the final concentration is 1%, 37 ° C, 15 min;
  • the procedure is the same as in the first embodiment.
  • the RT-PCR primer sequence is:
  • OSX-1-F GGTATGTATTAGGGCATGTGTCA (SEQ ID NO: 13)
  • OSX-1-R GGTATGTATTAGGGCATGTGTCA (SEQ ID NO: 14)
  • OSX-2-F AGGGAGGAAGGGAGTGTTGA (SEQ ID NO: 15)
  • OSX-2-R GAAAGGGGAGCCAAGAACCA (SEQ ID NO: 16)
  • OSX-NC-F GAAAGGGGAGCCAAGAACCA (SEQ ID NO: 17)
  • OSX-NC-R GGCAGGGACCTAAAAGACCA (SEQ ID NO: 18)
  • Example 3 3.5 according to the concentration of the protein sample the sample is prepared into the same concentration of the same volume of the sample, take 25ug protein sample as Input, protein denaturation, the same steps as in Example 1;
  • the remaining samples were equally divided into two groups.
  • the target protein group was added with 2 ug of immunoprecipitated antibody, and the negative control group was added with IgG 2 ug, and incubated for 1 h at 4 ° C on a rotary shaker;
  • OSX protein synthesis peptide chip 431 amino acids, Overlapping design, interval 3 amino acids, polypeptide length 15 amino acids, a chip 140 peptides;
  • the chromogenic reagent is HRP-labeled streptavidin, diluted with blocking solution (1:10000), shaken at room temperature for 2 h, and washed;
  • Chip scanning and color point data analysis The color developing chip was scanned and imaged by Bio-Rad ChemiDoc XRS+ chemiluminescence imaging system at 425 nm, and the color development time was 300 s. The image was analyzed using TotalLab image analysis software to analyze the color point density values. Using the “Spot Edge Average” algorithm in the software, the optical density values of each color point were calculated with reference to the background value of each color point.
  • OSXsh Mesenchymal stem cells were infected with lentiviral-mediated shRNA (OSXsh). After screening with 2 ⁇ g/mL puromycin, the expression of OSX was detected at the protein level. The results showed that OSX can be effectively knocked by OSXsh in apical mesenchymal stem cells. Except ( Figure 6-a).
  • the root nipple mesenchymal stem cells were seeded in a 6-well plate at a concentration of 2 ⁇ 10 3 /cm 2 . After the cells were grown to 80% confluence, the stem cells were osteogenesis/in vitro by osteogenic induction medium. In the direction of dentin differentiation, the liquid was changed every 3 days, and the formation of calcium nodules was observed under a light microscope.
  • the cells were harvested at different time points of induction 0, 3, 7, 10 and 14 days, and the osteogenic differentiation index-BSP was detected at the mRNA level (Fig. 6-f). The results showed that the gene knockout OSX inhibited the apical papilla between the apex. Expression of BSP in mesenchymal stem cells.

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Abstract

一种BARX1基因的用途，研究表明BARX1能够通过调控成骨、成牙基因的表达，从而影响间充质干细胞体外矿化能力，进而影响间充质干细胞的向成骨、成牙的分化。该用途为制备含有BARX1基因的骨组织或牙组织再生药物提供技术支持。

Description

BARX1的用途
本申请要求于2017年02月24日提交中国专利局、申请号为201710104434.X、发明名称为“BARX1的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医疗技术领域，尤其涉及BARX1的用途。
背景技术
间充质干细胞是最初从骨髓中分化而来的多向分化潜能干细胞，可分化为多种不同种类的细胞，包括成骨细胞、软骨细胞、肌细胞以及脂肪细胞等。越来越多的证据证明间充质干细胞可存在于非骨髓组织中。多数成人的间充质干细胞可用于细胞介导的组织工程。源于骨髓、骨膜、以及脂肪组织等不同组织的间充质干细胞拥有相似的表面标记特征，但是不同组织来源的间充质细胞在分化、增殖以及迁移等方面存在着显著差异。在过去的几十年里，基于其干细胞的特性，一类新的干细胞从口腔颌面组织中分离出来，包括源于牙周膜、牙髓以及根尖乳头等组织的干细胞。这些细胞具有多向分化潜能、成骨/成牙本质分化及自我更新能力。将其移植到小鼠或小型猪体内，可以生成骨样/牙本质样矿化组织并具有修复牙体组织缺损的能力。相比较源于骨髓的间充质干细胞，这类干细胞更易获得，而且与牙体组织有着更紧密的联系。因此牙源性间充质干细胞为牙齿组织的再生提供了可靠的细胞来源，但是其成牙定向分化的分子机制尚未明确，限制了其潜在的应用。
同源盒转录因子Barx基因族通过对细胞粘附分子调控及对II型胶原蛋白的调节参与间充质凝聚及形成。其中，转录因子BARX1在磨牙始基的间充质中表达，不仅参与牙齿形态发生的调控，也在牙齿及源于神经嵴的颌面部间充质细胞的发育中发挥着重要作用。在小鼠体内，Barx1表达于间充质凝聚位点，包括咽弓、肢芽、发育中的关节、磨牙乳头以及胃壁。异位表达Barx1基因会导致小鼠下颌培养中的切牙转变为异常的磨牙样的 形态。在人类，罕见的Barx1基因重复或缺失会导致颌面部及关节的畸形。这些研究结果表明Barx1调节细胞粘附分子的表达并参与牙齿发育及形态的控制。然而，BARX1在牙源性间充质干细胞中的作用及作用机制尚不明确。
发明内容
有鉴于此，本发明要解决的技术问题在于提供BARX1的用途，本发明证明，BARX1基因能够调控通过调控与成骨、成牙相关基因的表达，实现对间充质干细胞成骨/成牙定向分化的调控。
本发明提供了BARX1在制备调控间充质干细胞成牙相关基因表达的制剂中的应用。本发明实施例中，成牙相关基因为DSPP、DMP1和/或OSX。
所述间充质干细胞为根尖牙乳头间充质干细胞、脐带间充质干细胞或骨髓间充质干细胞。
所述调控具体为：过表达BARX1基因，促进DSPP基因、DMP1基因和/或OSX基因的表达；敲除BARX1基因，抑制DSPP基因、DMP1基因和/或OSX基因的表达。
本发明还提供了BARX1在制备调控间充质干细胞内成骨相关基因的制剂中的应用。本发明实施例中，成骨相关基因为BSP和/或OSX。
所述间充质干细胞为根尖牙乳头间充质干细胞、脐带间充质干细胞或骨髓间充质干细胞。
所述调控具体为：过表达BARX1基因，促进OSX基因的表达，抑制BSP基因的表达；敲除BARX1基因，抑制OSX基因的表达，促进BSP基因的表达。
本发明所述BARX1为BARX1蛋白、BARX1基因序列、能够过表达间充质细胞中BARX1基因的物质，或者能够敲除或敲低间充质细胞中BARX1基因的物质。
所述BARX1基因核苷酸序列的登录号为GeneID:56033，所述BARX1蛋白氨基酸序列有登录号为GeneID:56033的核苷酸序列翻译而来。
所述能够过表达间充质细胞中BARX1基因的物质为包含BARX1基因的表达载体；或者由包含BARX1基因的表达载体转染的逆转录病毒。所述逆转录病毒的表达载体为pQCXIP。
所述能够敲除间充质细胞中BARX1基因的物质为BAXR1的siRNA，序列为TCCTGCTCAGTAAGCTGCTCG。或者为将BAXR1的siRNA插入慢病毒的shRNA载体上构建而成BAXR1的shRNA质粒。
本发明实施例中，过表达脐带间充质干细胞中的BARX1基因，培养后以成骨诱导培养液诱导干细胞分化，3周后，在mRNA水平检测与成骨相关、成牙相关基因。结果表明，过表达BARX1抑制脐带间充质干细胞BSP的表达；促进脐带间充质干细胞DSPP和DMP1的表达；促进脐带间充质干细胞OSX的表达。结果中，所述促进或抑制与未经过表达的正常脐带间充质干细胞相比，具有显著性差异(p<0.05或p<0.01)。
本发明实施例中，过表达根尖牙乳头间充质干细胞中的BARX1基因，培养后以成骨诱导培养液诱导干细胞分化，3周后，在mRNA水平检测与成骨相关、成牙相关基因。结果表明，过表达BARX1抑制根尖牙乳头间充质干细胞BSP的表达；促进根尖牙乳头间充质干细胞DSPP和DMP1的表达；促进根尖牙乳头间充质干细胞OSX的表达。结果中，所述促进或抑制与未经过表达的正常根尖牙乳头间充质干细胞相比，具有显著性差异(p<0.05或p<0.01)。
本发明实施例中，过表达骨髓间充质干细胞中的BARX1基因，培养后以成骨诱导培养液诱导干细胞分化，3周后，在mRNA水平检测与成骨相关、成牙相关基因。结果表明，过表达BARX1抑制骨髓间充质干细胞BSP的表达；促进骨髓间充质干细胞DSPP的表达。结果中，所述促进或抑制与未经过表达的正常骨髓间充质干细胞相比，具有显著性差异(p<0.05或p<0.01)。
本发明还提供了BARX1基因在制备调控间充质干细胞矿化能力的制剂中的应用。
所述调控具体为过表达BARX1基因抑制间充质干细胞的体外矿化能力。
所述间充质干细胞为根尖牙乳头间充质干细胞或脐带间充质干细胞。
所述矿化指钙化结节的形成。
本发明实施例中，过表达脐带间充质干细胞中的BARX1基因，培养后以成骨诱导培养液诱导干细胞分化，定期在光镜下观察钙结节形成情况。结果表明过表达BARX1抑制脐带间充质干细胞体外矿化能力。
本发明实施例中，过表达根尖牙乳头间充质干细胞中的BARX1基因，培养后以成骨诱导培养液诱导干细胞分化，定期在光镜下观察钙结节形成情况。结果表明过表达BARX1抑制根尖牙乳头间充质干细胞体外矿化能力。
本发明还提供了BARX1在制备调控间充质干细胞成牙和/或成骨分化的制剂中的应用。
本发明实施例中，间充质干细胞为根尖牙乳头间充质干细胞、脐带间充质干细胞或骨髓间充质干细胞。
如前所述，本发明实验表明，过表达BARX1基因能够促进间充质干细胞内成牙相关基因的表达；抑制成骨相关基因的表达。最终结果表明，过表达BARX1基因能够促进间充质干细胞成牙分化，抑制成骨分化。
并且，为了验证过表达BARX1对根尖牙乳头干细胞牙向/骨向分化的影响，本发明通过携带过表达BARX1载体的病毒感染根尖牙乳头干细胞(SCAPs)，植入小型猪牙本质缺损模型，观察过表达BARX1后在体内环境中对SCAPs分化的影响。根尖牙乳头干细胞成牙本质样组织能力强于对照组，实验组新生牙本质组织形成面积较对照组增加。
本发明研究表明，过表达BARX1能够促进干细胞成骨/成牙相关转录因子OSX的表达，而基因敲除BARX1则会抑制OSX在间充质干细胞的表达。本发明通过OSX启动子分析及染色质免疫共沉淀(ChIP)表明，BARX1直接结合OSX启动序列，正向调控OSX的基因转录。此外，免疫共沉淀(Co-IP)结果表明BARX1和OSX在间充质干细胞中可以形成蛋白复合体。上述结果表明在间充质干细胞中BARX1不但可以直接调控OSX的表达，而且可以和OSX形成蛋白复合体，共同调节间充质干细胞的分化功能。
OSX基因在调控间充质干细胞内成牙本质相关基因和/或成骨相关基因表达中的应用；所述成骨相关基因为BSP；所述成牙本质相关基因为DSPP和/或DMP1。
本发明研究结果表明，基因敲除OSX抑制根尖牙乳头间充质干细胞BSP、DSPP和DMP1的表达。进而能够抑制根尖牙乳头间充质干细胞早期体外矿化能力。基因敲除OSX抑制根尖牙乳头间充质干细胞体外矿化能力。
本发明还提供了一种促进间充质干细胞成牙分化的制剂，包括BARX1蛋白或能够过表达BARX1基因的物质。
本发明实施例中，促进间充质干细胞成牙分化的制剂包括能够过表达BARX1基因的物质。具体的为：由包含BARX1基因的表达载体转染的逆转录病毒。所述逆转录病毒的表达载体为pQCXIP。
本发明还提供了一种促进间充质干细胞成骨分化的制剂，包括BARX1表达抑制剂或能够敲除或敲低BARX1基因的物质。
本发明实施例中，促进间充质干细胞成骨分化的制剂包括能够敲除或敲低BARX1基因的物质。能够敲除BAXR1基因的物质为BAXR1的siRNA插入慢病毒的shRNA载体上构建而成BAXR1的shRNA质粒。
本发明还提供了一种促进间充质干细胞成牙分化的方法，过表达间充质干细胞的BARX1基因，或以含有BARX1的诱导液对间充质干细胞进行诱导。
本发明还提供了一种促进间充质干细胞成骨分化的方法，敲除间充质干细胞的BARX1基因，或以含有BARX1表达抑制剂的诱导液对间充质干细胞进行诱导。
促进间充质干细胞成牙分化亦可称为促进牙组织再生；促进间充质干细胞成骨分化可称为促进骨组织再生。
本发明研究结果表明，BARX1能够通过调控成骨、成牙基因的表达，影响间充质干细胞向成骨、成牙的分化，从而影响间充质干细胞体外内矿化能力。本发明为制备含有BARX1基因的骨组织或牙组织再生药物提供技术支持。
附图说明
图1是过表达BARX1抑制脐带间充质干细胞成骨分化、促进成牙分化；图1-a示HA的表达；图1-b示茜素红染色；图1-c示钙离子定量分析；图1-d示qRT-PCR结果显示OSX的表达；图1-e示qRT-PCR结果显示BSP的表达；图1-f示qRT-PCR结果显示DSPP的表达；图1-g示qRT-PCR结果显示DMP1的表达；
图2是过表达BARX1抑制根尖牙乳头间充质干细胞成骨分化、促进成牙分化；图2-a示BARX1的表达；图2-b示BARX1的表达；图2-c示茜素红染色；图2-d示钙离子定量分析；图2-e示qRT-PCR结果显示BSP的表达；图2-f示qRT-PCR结果显示DSPP的表达；图2-g示qRT-PCR结果显示DMP1的表达；图2-h示qRT-PCR结果显示BARX1的表达；
图3是过表达BARX1抑制骨髓间充质干细胞成骨分化、促进成牙分化；图3-a示qRT-PCR结果显示过表达BARX1；图3-b示qRT-PCR结果显示BSP的表达；图3-c示qRT-PCR结果显示DSPP的表达；
图4是过表达BARX1促进根尖牙乳头间充质干细胞体内成牙分化；图4-a示HE染色显示单纯β-TCP牙本质缺损处回植结果，图4-a-1为40×镜下图像，图4-a-2为100×镜下图像；图4-b示HE染色显示β-TCP复合空载体根尖牙乳头干细胞(SCAPs-pQCXIP+β-TCP)牙本质缺损处回植结果，图4-b-1为40×镜下图像，图4-b-2为100×镜下图像；图4-c示HE染色显示β-TCP复合过表达BARX1的根尖牙乳头干细胞(SCAPs-HA-BARX1+β-TCP)牙本质缺损处回植结果，图4-c-1为40×镜下图像，图4-c-2为100×镜下图像；
图5是BARX1调控OSX的表达，并与OSX形成蛋白复合体；图5-a示qRT-PCR结果显示过表达BARX1后OSX的表达；图5-b示qRT-PCR结果显示基因敲除BARX1；图5-c示qRT-PCR结果显示基因敲除BARX1后OSX的表达；图5-d示CHIP结果显示OSX启动子上BARX1蛋白的结合；图5-e示Co-IP结果显示过表达BARX1后BARX1-OSX蛋白复合体的形成；图5-f示Co-IP结果显示基因敲除BARX1后BARX1-OSX蛋白复合体的形成；图5-g示多肽微阵列结果显示BARX1与OSX结合的 肽段；
图6是基因敲除OSX抑制根尖牙乳头干细胞成骨分化及成牙分化；图6-a示Western blot结果显示基因敲除OSX；图6-b示碱性磷酸酶染色；图6-c示碱性磷酸酶活性测定；图6-d示茜素红染色；图6-e示钙离子定量分析；图6-f示qRT-PCR结果显示BSP的表达；图6-g示qRT-PCR结果显示DSPP的表达；图6-h示qRT-PCR结果显示DMP1的表达。
具体实施方式
本发明提供了BARX1的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
骨涎蛋白(BSP)具有促进骨的形成及分化、成骨细胞的趋化、黏附等功能,在骨转换方面起着非常重要的作用。
核心转录因子(OSX)是调控骨形成和牙形成的核心转录因子,在细胞成骨分化、成牙分化过程中发挥着关键作用
牙本质涎蛋白DSPP是唯一由成牙本质细胞合成和分泌的牙本质特异性非胶原蛋白，分泌至矿化前沿，起着启动牙本质矿化及调节羟基磷灰石晶体的大小和生长速度的作用。
牙本质基质酸性磷酸化蛋白1(DMP1)是骨、软骨、釉质、牙骨质和牙本质生物矿化所必需的一种酸性非胶原蛋白，DMP-1全基因序列是生物矿化的启动因子。
下面结合实施例，进一步阐述本发明：
实施例1
1)干细胞的获得
1.1根尖牙乳头间充质干细胞的分离、培养与鉴定
人体组织的利用得到首都医科大学伦理委员会的批准，志愿者均知情同意术前签订知情同意书。获取了人恒牙根尖牙乳头组织，按照以往文献报道的方法分离和培养根尖牙乳头干细胞。
将拔除的牙齿立即放入无菌装有预冷PBS的离心管，移送进细胞室，在24h内分离培养根尖牙乳头干细胞。轻轻剥离牙齿周围的牙周组织，取根尖牙乳头组织，用PBS反复清洗，剪碎，置于含Ⅰ型胶原酶(3g/L)和Dispase(4g/L)的消化液，37℃下消化1小时，过70μm细胞筛收集细胞，1000rpm离心10min，用培养液重新悬浮成单细胞悬液。将细胞接种于25cm2细胞培养瓶中，在α-MEM培养基(含15％胎牛血清、2mmol/L谷氨酰胺、100U/ml青霉素和100μg/ml链霉素)中37℃、5％CO2培养，每2～3天换液1次。每天在倒置显微镜下观察细胞生长状况。当细胞生长至80％汇合状态时，用0.25％胰蛋白酶按1:2消化传代。通过检测间充质干细胞的表面标志物CD44、CD90、CD146、STRO-1和干细胞的多向分化能力、克隆形成能力等方法鉴定间充质干细胞。结果表明：所获得的间充质干细胞符合干细胞特性，其分化能力和克隆形成能力也符合要求。
1.2购买脐带间充质干细胞和脊髓间充质干细胞。
2)构建病毒质粒
通过NCBI数据库查询BAXR1的基因序列(GeneID:56033)，应用Whitehead提供的程序设计BAXR1的siRNA(如SEQ ID NO:19所示，序列为TCCTGCTCAGTAAGCTGCTCG)，将其插入慢病毒的shRNA载体上，测序鉴定，最终构建成BAXR1的shRNA的质粒。
设计BAXR1基因全长的PCR引物，用PCR的方法得到BAXR1的全长并加表面标记HA Tag，将其连接到逆转录病毒的表达载体(pQCXIP)上，测序鉴定，最终构建成HA-BAXR1的质粒。然后进行病毒包装、收集，病毒滴度鉴定，分装后保存在-80℃冰箱。
3)稳定转染细胞系的建立
对照Scramble的shRNA，BAXR1的shRNA转染根尖牙乳头干细胞、 脐带干细胞、骨髓间充质干细胞，转染48小时后，用puromycin筛选7天后得到对照Scramble和BAXR1基因敲除的稳定转染干细胞，提取细胞的mRNA和蛋白，在mRNA水平和蛋白水平检测shRNA的敲除效果。
对照质粒及HA-BAXR1的病毒转染根尖牙乳头干细胞、脐带干细胞、骨髓干细胞，转染48小时后，用药物筛选得到BAXR1过表达的稳定转染干细胞，在mRNA和蛋白水平检测HA的表达鉴定外源性BARX1的表达。
结果表明：本发明成功构建了BAXR1基因敲除及过表达的根尖牙乳头干细胞、脐带干细胞、骨髓干细胞。
本实施例中所采用的具体方法：
1)实时反转录聚合酶链式反应(Real-time RT-PCR)的过程为：
1.引物设计，Primer 3及oligo 6等生物软件；
2.RNA提取：
2.1培养皿细胞弃上清，PBS冲洗2遍，加700ul QIAZOL，吹打混匀，收于ep管，室温孵育5min，加140ul氯仿，强力震荡混匀15s，室温孵育3min，4℃12000g离心15min，收上清于新EP管；
2.2取700ul样本至RNeasy Mini column，4℃8000g离心15s，弃下层液体；
2.3加700ul Buffer RWT至RNeasy Mini column，4℃8000g离心15s，弃下层液体；
2.4加500ul Buffer RPE至RNeasy Mini column，4℃8000g离心15s，弃下层液体；
2.5重复步骤2.4；
2.6转移RNeasy Mini column至一新2ml collection tube，4℃1000g离心1min，弃下层液体；
2.7转移RNeasy Mini column至一新2ml collection tube，加30-50ul RNase-free water，4℃8000g离心15s，收集下层液体于新EP管，测RNA浓度，-80℃保存。
3.反转录PCR
3.1 Microtube管中配制下列模板RNA/引物混合液：模板RNA：1ng～5ug；Oligo(dT)(500μg/ml)：1μl；10mM dNTP：1μl；加入灭菌蒸馏水至12ul。
3.2 65℃保温5分钟后迅速在冰上冷冻1分钟以上。
3.3离心数秒使模板RNA/引物的变性溶液聚集于Microtube管底部。
3.4在上述Microtube管中配制加入下列反转录反应液：5×first strand buffer：4ul；0.1M DTT：2ul；RNase OUT：1ul。
3.5混匀各组分，37℃保温2分钟后冰上冷却。
3.6加入1ul M-MLV逆转录酶，轻轻吹打混匀。
3.7 37℃保温2分钟，70℃保温15分钟，4℃保温，样本收于-20℃。
4.实时定量荧光PCR
配置Real-time PCR反应体系如下：5XSYBR Mix：5μl；PCR Primer(5pmol)：0.5μl；ddH2O：5μl；cDNA：10.5μl；Total 21μl。
反应体系如表1：
表1 反应体系
Figure PCTCN2017098262-appb-000001
2)Western Blot的过程为：
1.细胞总蛋白的提取
1.1弃培养基，用4℃预冷的5ml PBS漂洗细胞两次，加入5ml PBS后，用细胞刮刮下培养皿中的细胞，收于15ml离心管，1100rpm离心6min；弃上清，加入1ml PBS重悬细胞，收于EP管，7200rpm离心2min；
1.2弃上清，以1:5(细胞：裂解液)体积比加裂解液(100ul RIPA+1ul  PMSF+1ul PIC)，冰上15min，每2-3min混悬一次；
1.3 4℃，14000rpm离心15min，收集上清液于新EP管中，-80℃保存。
2. Bradford法测定蛋白浓度
2.1 BSA蛋白标准品按说明依次用双蒸水稀释，使终浓度分别为1000μg/ml，750μg/ml，500μg/ml，250μg/ml，125μg/ml，62.5μg/ml，0μg/ml；
2.2根据样品和标准品数量，按每孔加200ul 1X考马斯亮蓝液，取1ul样品和标准品加入96孔板中，室温孵育5min，设副孔和空白孔；
2.3测定595nm波长的吸光度。根据标准曲线计算蛋白浓度；
2.4根据蛋白浓度计算上样体积，每组蛋白的上样量为25μg。
3.聚丙烯酰胺凝胶电泳
3.1准备预成胶；
3.2变性：每孔25μg蛋白量上样,用蒸馏水将待测样本体积稀释至20ul，每样本加入5ul loading buffer(溴酚蓝)，95℃加热10min；
3.3上样，以80V电压电泳至溴酚蓝电泳至分离胶，将电压调到100V，直至溴酚蓝到达分离胶底部，终止电泳。
4.转膜
4.1准备预成膜；
4.2将电泳胶转移至预成膜中，使用BioRad半干转进行转膜；
4.3取出PVDF膜，TBST漂洗5min。
5. Western blot滤膜杂交
5.1将转印后的PVDF膜用5％脱脂奶粉室温摇床封闭1小时；
5.2 TBST漂洗10min×3次；
5.3将滤膜取出，放入5％脱脂奶粉稀释的一抗稀释液中，4℃摇床过夜；
5.4 TBST洗膜，10min×3次；
5.5将滤膜放入5％脱脂奶粉稀释的HRP标记的二抗中，室温摇床孵育1小时；
5.6 TBST洗膜，10min×3次。
6.显色
将PVDF膜胶面朝上放在保鲜膜上，加入1:1混合的显影液，使其均匀覆盖膜，暗室曝光，扫描。
实施例2 BARX1对间充质干细胞成骨/成牙分化功能影响的体外研究
1)取实施例1构建的过表达BARX1的脐带干细胞，以2×103/cm2的浓度接种于6孔板中，待细胞生长至80％融合后，用成骨诱导培养液(Invitrogen公司)将干细胞在体外向成骨/成牙本质方向分化诱导，每3天换1次液，在光镜下观察钙结节形成情况。
3周后检测茜素红染色及测定钙离子浓度来检测晚期成骨指标-细胞矿化能力，结果表明过表达BARX1抑制脐带间充质干细胞体外矿化能力(图1-a,图1-b)。并在诱导0，3，7，14和21天的不同时间点收获细胞，在mRNA水平检测成骨分化指标-骨涎蛋白(BSP，图1-c)，结果表明过表达BARX1抑制脐带间充质干细胞BSP的表达。
在mRNA水平检测成牙本质分化指标-牙本质涎蛋白(DSPP，图1-d)、牙本质基质酸性磷酸化蛋白1(DMP1，图1-e)，结果表明过表达BARX1促进脐带间充质干细胞DSPP和DMP1的表达。
在mRNA水平检测相关核心转录因子OSX，结果表明过表达BARX1促进脐带间充质干细胞OSX的表达(图1-f)。
上述结果表明过表达BARX1抑制脐带间充质干细胞成骨分化，促进成牙分化。
2)取取实施例1构建的过表达BARX1的根尖牙乳头间充质干细胞以2×103/cm2的浓度接种于6孔板中，待细胞生长至80％融合后，用成骨诱导培养液将干细胞在体外向成骨/成牙本质方向分化诱导，每3天换1次液，在光镜下观察钙结节形成情况。
2、3周后检测茜素红染色及测定钙离子浓度来检测晚期成骨指标-细胞矿化能力，结果表明过表达BARX1抑制根尖牙乳头间充质干细胞体外矿化能力(图2-a,图2-b)。
并在诱导0，3，7，14和21天的不同时间点收获细胞，在mRNA水平检测成骨分化指标-BSP(图2-c)，结果表明过表达BARX1抑制根尖牙乳头间充质干细胞BSP的表达。
在mRNA水平检测成牙本质分化指标-DSPP(图2-d)、DMP1(图2-e)，结果表明过表达BARX1促进根尖牙乳头间充质干细胞DSPP和DMP1的表达。
在mRNA水平检测相关核心转录因子OSX，结果表明过表达BARX1促进根尖牙乳头间充质干细胞OSX的表达(图2-f)。
上述结果表明过表达BARX1抑制根尖牙乳头间充质干细胞成骨分化，促进成牙分化。
3)取实施例1构建的过表达BARX1的骨髓间充质干细胞被使用表达有野生型BARX1的慢病毒感染，2μg/mL嘌呤霉素筛选后，实时定量RT-PCR结果显示，BARX1在骨髓间充质干细胞异位表达(图3-a)。
取骨髓间充质干细胞以2×103/cm2的浓度接种于6孔板中，待细胞生长至80％融合后，用成骨诱导培养液将干细胞在体外向成骨/成牙本质方向分化诱导，每3天换1次液，在光镜下观察钙结节形成情况。
诱导0，3，7和10天的不同时间点收获细胞，在mRNA水平检测成骨分化指标-骨涎蛋白(BSP，图3-b)，结果表明过表达BARX1抑制骨髓间充质干细胞BSP的表达。
在mRNA水平检测成牙本质分化指标-牙本质涎蛋白(DSPP，图3-c)结果表明过表达BARX1促进骨髓间充质干细胞DSPP的表达。
上述结果表明过表达BARX1抑制骨髓间充质干细胞成骨分化，促进成牙分化。
具体过程为：
1.茜素红染色
1.1弃掉培养基，PBS洗2次；
1.2 70％乙醇固定，4℃，1h；
1.3双蒸水洗2次；
1.4 40mM茜素红溶液(pH 4.2)室温染色1-10min，肉眼观察着色 情况；
1.5双蒸水洗5次，轻轻吹打；
1.6扫描仪透摄模式采集图像。
2. Ca2+浓度检测
2.1茜素红染色后，加入10％w/v CPC，室温放置30min(AR-S被溶解至CPC中)；
2.2以1:10稀释溶液，在酶标仪中以562nm波长测定其吸光度值(OD)；
2.3以AR-S标准曲线计算Ca2+的相对浓度。
3. Real-time RT-PCR
步骤同实施例1。RT-PCR引物序列为：
OSX-F：CCTCCTCAGCTCACCTTCTC(SEQ ID NO:1)；
OSX-R：GTTGGGAGCCCAAATAGAAA(SEQ ID NO:2)；
BSP-F：CAGGCCACGATATTATCTTTACA(SEQ ID NO:3)；
BSP-R：CTCCTCTTCTTCCTCCTCCTC(SEQ ID NO:4)；
DSPP-F：CGACATAGGTCACAATGAGGATGTCG(SEQ ID NO:5)；
DSPP-R：TTGCTTCCAGCTACTTGAGGTC(SEQ ID NO:6)；
DMP1-F：GACAGCCTCTCACTGGATTCGCTGTC(SEQ ID NO:7)；
DMP1-R：CTCGCACACACTCTCCCACTC(SEQ ID NO:8)；
GAPDH-F：CGGACCAATACGACCAAATCCG(SEQ ID NO:9)；
GAPDH-R：AGCCACATCGCTCAGACACC(SEQ ID NO:10)。
实施例3 BARX1对间充质干细胞成骨/成牙分化功能影响的体内研究
为了验证过表达BARX1对根尖牙乳头干细胞牙向/骨向分化的影响，将SCAPs与HA/TCP混合，植入小型猪牙本质缺损模型，观察过表达BARX1后在体内环境中对SCAPs分化的影响。
取4只小型猪，在其下颌第一、二前磨牙上开髓，随机植入SCAPs与β-TCP混合物或单植入β-TCP，共15个位点，其中空白对照组(β-TCP)3个(图3-a)，对照组(SCAPs-pQCXIP+β-TCP)6个(图3-b)， 实验组(SCAPs-HA-BARX1+β-TCP)6个(图3-c)。植入8周后，取出组织标本进行切片染色分析，结果显示，实验组根尖牙乳头干细胞成牙本质样组织能力强于对照组，实验组新生牙本质组织形成面积较对照组增加(图4-a～图4-c)。
具体过程为：
1.细胞移植
1.1分别取生长状态良好的第4代细胞，以1×105个/皿的密度接种于10cm培养皿，待细胞长至80％汇合度时弃掉培养基，用PBS漂洗(为保证细胞状态，细胞汇合度不宜过大)。
1.2每皿细胞加3ml不含EDTA的胰酶，于37℃孵育2min，将细胞从培养皿上洗脱，加入4ml含15％FBS、1％双抗和1％L-谷氨酰胺的α-MEM培养基中和胰酶作用，之后将细胞悬液转移至50ml的离心管中。
1.3于1100rpm，离心6min。
1.5用上述含15％FBS、1％双抗和1％L-谷氨酰胺的α-MEM培养基重悬细胞，计数，然后取5×105个细胞置于12孔板中直径3-3.5mm、厚1mm的β-磷酸三钙(β-TCP)片上，37℃，5％CO2条件下孵育2h，每加入2ml的含15％FBS、1％双抗和1％L-谷氨酰胺的α-MEM培养基，继续于37℃，5％CO2条件下孵育3天待用。不含细胞的β-TCP也于2ml相同培养基、相同条件下培养3天。
1.6小型猪称重，麻醉。
1.7于小型猪下颌第一、二前磨牙上开髓(直径3-4mm开髓孔)，随机选取不同小型猪个体上的3颗牙放入1.5中所述的不含细胞的β-TCP，6颗牙放入1.5中所述孵育有对照组细胞(SCAP-pQCXIP)的β-TCP(SCAPs-pQCXIP+β-TCP)，6颗牙放入1.5中所述孵育有实验组细胞(SCAPs-HA-BARX1)的β-TCP(SCAPs-HA-BARX1+β-TCP)，共15颗前磨牙，玻璃离子水门汀暂封。
2.标本固定，脱钙，脱水包埋，制作石蜡切片
在体内移植10周后处死小型猪，取出组织标本，置于4％多聚甲醛中固定，之后转至pH值等于7(7.4～7.6)的10％EDTA内，完全脱钙后 流水冲洗24小时，随后将组织脱水包埋。
脱水步骤：75％乙醇Ⅰ：10min；75％乙醇Ⅱ：10min；85％乙醇Ⅰ：10min；85％乙醇Ⅱ：10min；95％乙醇Ⅰ：10min；95％乙醇Ⅱ：10min；无水乙醇Ⅰ：30min；无水乙醇Ⅱ：30min；二甲苯Ⅰ：30min；二甲苯Ⅱ：30min；浸蜡Ⅰ：30min；浸蜡Ⅱ：过夜。
包埋：浸蜡结束后，将组织转到预热的包埋框中，迅速倾入熔蜡，用温热的镊子调整组织块的位置，制冷机上放置，使熔蜡凝固。
切片：修整蜡块后在轮转切片机切5μm厚连续切片，将切片裱于多聚赖氨酸处理的玻片上，37℃干燥过夜。
3.石蜡切片HE染色步骤：二甲苯Ⅰ：10min；二甲苯Ⅱ：10min；无水乙醇Ⅰ：3min；无水乙醇Ⅱ：3min；95％乙醇Ⅰ：3min；95％乙醇Ⅱ：3min；85％乙醇：3min；75％乙醇：3min；流水冲洗：3min；蒸馏水洗：1min；苏木精液染色：2min；流水洗去苏木精液：30s；1％盐酸-乙醇：3s；流水冲洗返蓝：10min；蒸馏水洗：1min；0.5％伊红液染色：1min；蒸馏水稍洗：2s；80％乙醇：10s；95％乙醇Ⅰ：10s；95％乙醇Ⅱ：10s；无水乙醇Ⅰ：10s；无水乙醇Ⅱ：10s；二甲苯Ⅰ：2min；二甲苯Ⅱ：2min；中性树胶封固。
实施例4 在间充质干细胞中BARX1正向调控OSX的基因表达，且与OSX形成蛋白复合体。
为了揭示BARX1促进间充质干细胞成牙分化、抑制成骨分化的分子机制，通过候选基因筛查发现，过表达BARX1促进干细胞成骨/成牙相关转录因子OSX的表达(图5-a)。为了进一步证实BARX1对OSX的调控，间充质干细胞被使用慢病毒介导shRNA(BARX1sh1、BARX1sh2)感染，2μg/mL嘌呤霉素筛选后，实时定量RT-PCR检测结果表明，BARX1可以被BARX1sh1、BARX1sh2有效的敲除(图5-b)；实时定量RT-PCR检测结果表明，基因敲除BARX1抑制OSX在间充质干细胞的表达(图5-c)。OSX启动子分析及染色质免疫共沉淀(ChIP)结果表明，BARX1直接结合OSX启动序列，正向调控OSX的基因转录(图5-d)。
此外，免疫共沉淀(Co-IP)结果表明BARX1和OSX在间充质干细胞中可以形成蛋白复合体(图5-e，图5-f)；多肽微阵列实验结果显示了BARX1蛋白结合OSX的多肽序列(图5-g)。
上述结果表明在间充质干细胞中BARX1不但可以直接调控OSX的表达，而且可以和OSX形成蛋白复合体，共同调节间充质干细胞的分化功能。
具体过程为：
1. Real-time RT-PCR
步骤同实施例1。RT-PCR引物序列为：
BARX1-F：CGCTTCGAGAAGCAGAAGTA(SEQ ID NO:11)
BARX1-R：CTTCATCCTCCGATTCTGGT(SEQ ID NO:12)
2. CHIP
2.1培养皿细胞中加入甲醛，终浓度为1％，37℃，15min；
2.2弃含甲醛的培养基，用4℃预冷的5ml PBS漂洗细胞两次，加入5ml PBS后，用细胞刮刮下培养皿中的细胞，收于50ml离心管，4℃2000rpm离心4min；
2.3弃上清，每样本加入含蛋白酶抑制剂的室温SDS裂解液200ul(100ul SDS裂解液+1ul PMSF+1ul PIC)，冰上10min；
2.4超声破碎，功率75％，3次，每次7-8s，间隔90s；
2.5 4℃，13000rpm离心10min，收集上清液于新EP管中；
2.6测DNA浓度，将各样本制成同浓度、同体积样本；
2.7用CHIP稀释缓冲液(含蛋白酶抑制剂)将上述样本稀释10倍；取1％稀释后的样本作为Input，加入20ul 5M NaCl，65℃，4h，-20℃保存待用；
2.8将剩余样本分组，目的蛋白组中加入免疫沉淀抗体2ug，阴性对照组中加入IgG 2ug，4℃旋转摇床孵育1h；
2.9加入60ul蛋白A琼脂糖珠子，4℃旋转摇床孵育过夜；
2.10轻柔离心，小心去除上清液，得到琼脂糖凝胶/抗体/组蛋白复合体，分别加入1ml下列缓冲液清洗上述复合体：
1)低盐免疫沉淀复合体清洗缓冲液，1次，1min/次，4℃5000rpm离心；
2)高盐免疫沉淀复合体清洗缓冲液，1次，3min/次，4℃5000rpm离心；
3)氯化锂免疫沉淀复合体清洗缓冲液，1次，1min/次，4℃5000rpm离心；
4)TE缓冲液，2次，1min/次，4℃5000rpm离心；
2.11在清洗后的复合体中加入250ul洗脱液(1％SDS，0.1M NaHCO3)，旋转摇床室温孵育15min，4℃5000rpm离心，将上清液转入新的EP管内，再重复一遍，共得到500ul洗脱后的样本；
2.12在洗脱样本中加入20ul 5M NaCl 65℃4h；
2.13DNA纯化：
2.13.1在Input和洗脱样本中加入10ul 0.5M EDTA，20ul 1M Tris-HCl(pH6.5)和1ul 20mg/ml蛋白酶K，45℃，1h；
2.13.2加入500ul酚/氯仿，涡旋混匀，静置5min，4℃14000rpm 5min离心；
2.13.3收集上清液至新EP管，加入1ml乙醇+1‰糖原，-20℃30min-1h，4℃14000rpm 10min离心；
2.13.4弃上清液，室温晾干，加入100-200ul双蒸水重悬。
2.14 Real-time PCR:
步骤同实施例1。RT-PCR引物序列为：
OSX-1-F：GGTATGTATTAGGGCATGTGTCA(SEQ ID NO:13)
OSX-1-R：GGTATGTATTAGGGCATGTGTCA(SEQ ID NO:14)
OSX-2-F：AGGGAGGAAGGGAGTGTTGA(SEQ ID NO:15)
OSX-2-R：GAAAGGGGAGCCAAGAACCA(SEQ ID NO:16)
OSX-NC-F：GAAAGGGGAGCCAAGAACCA(SEQ ID NO:17)
OSX-NC-R：GGCAGGGACCTAAAAGACCA(SEQ ID NO:18)
3. Co-IP
3.1弃培养基，用4℃预冷的5ml PBS漂洗细胞两次，加入5ml PBS 后，用细胞刮刮下培养皿中的细胞，收于50ml离心管，1100rpm离心6min；弃上清，加入1ml PBS重悬细胞，收于EP管，7200rpm离心2min；
3.2弃上清，加入1ml IP裂解液，冰上15min，每2-3min混悬一次；
3.3 4℃，14000rpm离心15min，收集上清液于新EP管中；
3.4 Bradford法测定蛋白浓度，步骤同实施例1；
3.5根据蛋白样本的浓度将样本配制成同浓度同体积样本，取25ug蛋白样本作为Input，蛋白变性，步骤同实施例1；
3.6将剩余样本平均分为两组，目的蛋白组中加入免疫沉淀抗体2ug，阴性对照组中加入IgG 2ug，4℃旋转摇床孵育1h；
3.7加入40ul蛋白A琼脂糖珠子，4℃旋转摇床孵育过夜；
3.8轻柔离心，小心去除上清液，加入1ml 20％免疫共沉淀洗脱液清洗，4℃5000rpm离心30s，去除上清液，重复3-4次；
3.9加入30ul 2×loading buffer(溴酚蓝)，95℃加热5min变性；
3.10 Input与免疫共沉淀样本一起进行Western Blot，步骤同实施例1。
4.多肽微阵列
4.1合成BARX1重组蛋白；
4.2根据根据OSX蛋白的序列合成多肽芯片，431个氨基酸，Overlapping设计，间隔3个氨基酸，多肽长度15个氨基酸，一个芯片140条多肽；
4.3多肽阵列合成：活化的膜放置于全自动合成仪上，根据程序自动转移Fmoc-氨基酸溶液到活化膜上的特定位置与膜进行反应。膜按序浸入封闭液I中和封闭液II中，进行侧链封闭，DMF洗膜。膜放入去保护溶液中，用于移除氨基端的Fmoc保护基团，去保护之后，用DMF洗膜，而后再用乙醇干燥。重复以上步骤，直至多肽阵列全部合成完毕。全部合成后,用溶液去除侧链保护基团，再用CH2Cl2洗膜，而后用乙醇干燥,立即使用或-20℃保存用于下一步实验；
4.4多肽阵列与目标蛋白结合反应
4.4.1目标蛋白生物素标记：4℃操作，取1mlBARX1蛋白溶液，用生物素标记试剂0.266umol，约13.3ul与蛋白溶液混匀，室温孵育60min； 将孵育完成的蛋白溶液通过过滤柱，1000xg离心2min收集蛋白溶液即为生物素标记的BARX1蛋白溶液；
4.4.2封闭：阵列膜活化后加入封闭液，室温震荡封闭4h，洗膜；
4.4.3蛋白样品孵育：封闭液稀释的标记BARX1蛋白样品(终浓度0.1ug/ml)与阵列膜4℃孵育过夜；
4.4.4显色试剂孵育：显色试剂为HRP标记的链霉亲和素，封闭液稀释(1:10000)后，室温震荡2h，洗膜；
4.4.5显色：加入ECL发光试剂，数字成像；
4.5芯片扫描及显色点数据分析：显色芯片使用Bio-Rad ChemiDoc XRS+化学发光成像系统425nm扫描成像，显色时长300s。成像图片使用TotalLab图像分析软件分析显色点光密度值，使用软件中“Spot Edge Average”算法，以每个显色点周边背景值为参照计算每个显色点的光密度值。
实施例5 OSX对根尖牙乳头间充质干细胞成骨/成牙分化功能影响
间充质干细胞被使用慢病毒介导shRNA(OSXsh)感染，2μg/mL嘌呤霉素筛选后，在蛋白水平检测OSX的表达，结果表明根尖牙乳头间充质干细胞中OSX可以被OSXsh有效敲除(图6-a)。
取根尖牙乳头间充质干细胞以2×103/cm2的浓度接种于6孔板中，待细胞生长至80％融合后，用成骨诱导培养液将干细胞在体外向成骨/成牙本质方向分化诱导，每3天换1次液，在光镜下观察钙结节形成情况。
5天后后检测碱性磷酸酶染色及测定碱性磷酸酶活性检测早期矿化指标-碱性磷酸酶，结果表明基因敲除OSX抑制根尖牙乳头间充质干细胞早期体外矿化能力(图6-b,图6-c)。
3周后检测茜素红染色及测定钙离子浓度来检测晚期成骨指标-细胞矿化能力，结果表明基因敲除OSX抑制根尖牙乳头间充质干细胞体外矿化能力(图6-d,图6-e)。
并在诱导0，3，7，10和14天的不同时间点收获细胞，在mRNA水平检测成骨分化指标-BSP(图6-f)，结果表明过基因敲除OSX抑制根尖牙乳头间充质干细胞BSP的表达。
在mRNA水平检测成牙本质分化指标-DSPP(图6-g)、DMP1(图6-h)，结果表明基因敲除OSX抑制根尖牙乳头间充质干细胞DSPP和DMP1的表达。
上述结果表明基因敲除OSX抑制根尖牙乳头间充质干细胞成骨分化及成牙分化。
具体过程为：
1. Western Blot
步骤同实施例1。
2.碱性磷酸酶(ALP)染色
2.1弃掉培养基，PBS洗2次；
2.2柠檬酸-丙酮-甲醛固定液(25：65：8)固定，室温，0.5h；
2.3双蒸水洗2次；
2.4配置染色液：1ml Sodium Nitrite溶液与1ml FRV-Alkaline溶液轻柔混匀，静置2min，将其加入45ml去离子水中，最后加入1ml Naphthol AS-BI Alkaline溶液，充分混匀；
2.5上述染色液室温染色15min，肉眼观察着色情况；
2.6双蒸水洗5次，轻轻吹打；
2.7扫描仪透摄模式采集图像。
3.碱性磷酸酶(ALP)活性测定
3.1弃掉培养基，PBS洗2次；
3.2加入500ul裂解液，37℃，15min；
3.3刮刀刮净细胞，收集细胞及裂解液于EP管中，涡旋，4℃，14000rpm离心10min，收集上清液于新EP管中，冰上放置待用；
3.4于96孔板中每孔加入50ul碱性磷酸酶Buffer溶液、50ul Stock Substrate溶液和10ul上述样本，37℃，15min；
3.5加和110ul 0.5M NaOH中止反应；在酶标仪中以405nm波长测定其吸光度值(OD)；
3.6用Bradford法测定样本蛋白浓度，步骤同实施例1；
3.7以标准曲线及样本蛋白量计算ALP活性。
4.茜素红染色
步骤同实施例2。
5. Ca2+浓度检测
步骤同实施例2。
6. Real-time RT-PCR
步骤及引物同实施例2。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2017098262-appb-000002
Figure PCTCN2017098262-appb-000003
Figure PCTCN2017098262-appb-000004
Figure PCTCN2017098262-appb-000005
Figure PCTCN2017098262-appb-000006

Claims (11)

  1. BARX1在制备调控间充质干细胞成牙相关基因表达的制剂中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述成牙相关基因为DSPP、DMP1和/或OSX。
  3. BARX1在制备调控间充质干细胞内成骨相关基因的制剂中的应用。
  4. 根据权利要求1所述的应用，其特征在于，所述成骨相关基因为BSP和/或OSX。
  5. BARX1在制备调控间充质干细胞成牙和/或成骨分化的制剂中的应用。
  6. 根据权利要求1～5任一项所述的应用，其特征在于，所述间充质干细胞为根尖牙乳头间充质干细胞、脐带间充质干细胞或骨髓间充质干细胞。
  7. OSX在制备调控间充质干细胞内成牙本质相关基因和/或成骨相关基因表达的制剂中的应用；所述成骨相关基因为BSP；所述成牙本质相关基因为DSPP和/或DMP1。
  8. 一种促进间充质干细胞成牙分化的制剂，其特征在于，包括BARX1蛋白或能够过表达BARX1基因的物质。
  9. 一种促进间充质干细胞成骨分化的制剂，其特征在于，包括BARX1表达抑制剂或能够敲除或敲低BARX1基因的物质。
  10. 一种促进间充质干细胞成牙分化的方法，其特征在于，过表达间充质干细胞的BARX1基因，或以含有BARX1的诱导液对间充质干细胞进行诱导。
  11. 一种促进间充质干细胞成骨分化的方法，其特征在于，敲除或敲低间充质干细胞的BARX1基因，或以含有BARX1表达抑制剂的诱导液对间充质干细胞进行诱导。
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