CN108753701B - Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction - Google Patents
Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction Download PDFInfo
- Publication number
- CN108753701B CN108753701B CN201810418078.3A CN201810418078A CN108753701B CN 108753701 B CN108753701 B CN 108753701B CN 201810418078 A CN201810418078 A CN 201810418078A CN 108753701 B CN108753701 B CN 108753701B
- Authority
- CN
- China
- Prior art keywords
- culture
- ift80
- osteoblasts
- staining
- bone
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000019491 signal transduction Effects 0.000 title claims abstract description 23
- 210000002050 maxilla Anatomy 0.000 title claims abstract description 12
- 108010078791 Carrier Proteins Proteins 0.000 title claims abstract description 11
- 230000008844 regulatory mechanism Effects 0.000 title claims abstract description 11
- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract description 10
- 230000010189 intracellular transport Effects 0.000 title claims abstract description 6
- 210000000963 osteoblast Anatomy 0.000 claims abstract description 72
- 101001010727 Homo sapiens Intraflagellar transport protein 80 homolog Proteins 0.000 claims abstract description 68
- 102100030002 Intraflagellar transport protein 80 homolog Human genes 0.000 claims abstract description 51
- 210000004027 cell Anatomy 0.000 claims abstract description 46
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 38
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 31
- 210000004081 cilia Anatomy 0.000 claims abstract description 26
- 238000000338 in vitro Methods 0.000 claims abstract description 24
- 238000003501 co-culture Methods 0.000 claims abstract description 22
- 230000030279 gene silencing Effects 0.000 claims abstract description 19
- 230000035755 proliferation Effects 0.000 claims abstract description 18
- 238000002474 experimental method Methods 0.000 claims abstract description 16
- 238000012226 gene silencing method Methods 0.000 claims abstract description 16
- 230000033228 biological regulation Effects 0.000 claims abstract description 15
- 230000004069 differentiation Effects 0.000 claims abstract description 15
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 11
- 238000001727 in vivo Methods 0.000 claims abstract description 11
- 230000007246 mechanism Effects 0.000 claims abstract description 10
- 238000001890 transfection Methods 0.000 claims abstract description 8
- 238000010171 animal model Methods 0.000 claims abstract description 6
- 210000001519 tissue Anatomy 0.000 claims description 33
- 238000010186 staining Methods 0.000 claims description 30
- 238000001262 western blot Methods 0.000 claims description 27
- 238000003753 real-time PCR Methods 0.000 claims description 20
- 238000011532 immunohistochemical staining Methods 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 230000006870 function Effects 0.000 claims description 16
- 230000001744 histochemical effect Effects 0.000 claims description 16
- 238000003125 immunofluorescent labeling Methods 0.000 claims description 16
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 15
- 239000012188 paraffin wax Substances 0.000 claims description 15
- 102000014128 RANK Ligand Human genes 0.000 claims description 14
- 108010025832 RANK Ligand Proteins 0.000 claims description 14
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- 239000004055 small Interfering RNA Substances 0.000 claims description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 12
- 210000002449 bone cell Anatomy 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 230000003068 static effect Effects 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 claims description 10
- 101100072312 Mus musculus Ift80 gene Proteins 0.000 claims description 10
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 claims description 10
- 238000010603 microCT Methods 0.000 claims description 10
- 229960002378 oftasceine Drugs 0.000 claims description 10
- 102000015775 Core Binding Factor Alpha 1 Subunit Human genes 0.000 claims description 9
- 108010024682 Core Binding Factor Alpha 1 Subunit Proteins 0.000 claims description 9
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 9
- 108010088665 Zinc Finger Protein Gli2 Proteins 0.000 claims description 9
- 229920002866 paraformaldehyde Polymers 0.000 claims description 9
- 108060000903 Beta-catenin Proteins 0.000 claims description 8
- 102000015735 Beta-catenin Human genes 0.000 claims description 8
- 102000008186 Collagen Human genes 0.000 claims description 8
- 108010035532 Collagen Proteins 0.000 claims description 8
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 8
- 230000001886 ciliary effect Effects 0.000 claims description 8
- 229920001436 collagen Polymers 0.000 claims description 8
- 238000002965 ELISA Methods 0.000 claims description 7
- 230000000093 cytochemical effect Effects 0.000 claims description 7
- 241000699670 Mus sp. Species 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 210000001616 monocyte Anatomy 0.000 claims description 6
- 239000002243 precursor Substances 0.000 claims description 6
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 6
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 5
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 5
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 claims description 5
- 238000012755 real-time RT-PCR analysis Methods 0.000 claims description 5
- 230000037182 bone density Effects 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 4
- 230000001413 cellular effect Effects 0.000 claims description 4
- 238000012137 double-staining Methods 0.000 claims description 4
- 230000002055 immunohistochemical effect Effects 0.000 claims description 4
- 210000003625 skull Anatomy 0.000 claims description 4
- 230000004936 stimulating effect Effects 0.000 claims description 4
- -1 Col I Proteins 0.000 claims description 3
- 238000008157 ELISA kit Methods 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 claims description 3
- 229960002327 chloral hydrate Drugs 0.000 claims description 3
- 230000001605 fetal effect Effects 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000010348 incorporation Methods 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 210000004876 tela submucosa Anatomy 0.000 claims description 3
- 238000010361 transduction Methods 0.000 claims description 3
- 230000026683 transduction Effects 0.000 claims description 3
- 210000000689 upper leg Anatomy 0.000 claims description 3
- 238000004018 waxing Methods 0.000 claims description 3
- 101710189683 Alkaline protease 1 Proteins 0.000 claims 4
- 101710154562 Alkaline proteinase Proteins 0.000 claims 4
- 101710170876 Antileukoproteinase Proteins 0.000 claims 4
- 101710112538 C-C motif chemokine 27 Proteins 0.000 claims 4
- 101001086210 Homo sapiens Osteocalcin Proteins 0.000 claims 2
- 101000613820 Homo sapiens Osteopontin Proteins 0.000 claims 2
- 102100031475 Osteocalcin Human genes 0.000 claims 2
- 102100040557 Osteopontin Human genes 0.000 claims 2
- 102100021253 Antileukoproteinase Human genes 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 17
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 17
- WOVKYSAHUYNSMH-UHFFFAOYSA-N BROMODEOXYURIDINE Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-UHFFFAOYSA-N 0.000 description 8
- 229950004398 broxuridine Drugs 0.000 description 8
- 210000003254 palate Anatomy 0.000 description 8
- 102000004067 Osteocalcin Human genes 0.000 description 7
- 108090000573 Osteocalcin Proteins 0.000 description 7
- 102000004264 Osteopontin Human genes 0.000 description 7
- 108010081689 Osteopontin Proteins 0.000 description 7
- 230000011164 ossification Effects 0.000 description 7
- 230000009818 osteogenic differentiation Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 241000027355 Ferocactus setispinus Species 0.000 description 4
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 4
- 208000011580 syndromic disease Diseases 0.000 description 4
- 102000016921 Integrin-Binding Sialoprotein Human genes 0.000 description 3
- 108010028750 Integrin-Binding Sialoprotein Proteins 0.000 description 3
- 230000004156 Wnt signaling pathway Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 210000000412 mechanoreceptor Anatomy 0.000 description 3
- 108091008704 mechanoreceptors Proteins 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 208000030761 polycystic kidney disease Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 102100025802 Cystin-1 Human genes 0.000 description 2
- 101710106195 Cystin-1 Proteins 0.000 description 2
- 108090000031 Hedgehog Proteins Proteins 0.000 description 2
- 102000003693 Hedgehog Proteins Human genes 0.000 description 2
- 208000010043 Jeune syndrome Diseases 0.000 description 2
- 206010061274 Malocclusion Diseases 0.000 description 2
- 108010009711 Phalloidine Proteins 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000002308 calcification Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 102000037983 regulatory factors Human genes 0.000 description 2
- 108091008025 regulatory factors Proteins 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 208000026372 Congenital cystic kidney disease Diseases 0.000 description 1
- 108050006400 Cyclin Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 241000289669 Erinaceus europaeus Species 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 description 1
- 206010072610 Skeletal dysplasia Diseases 0.000 description 1
- 241000248384 Tetrahymena thermophila Species 0.000 description 1
- 102000013814 Wnt Human genes 0.000 description 1
- 108050003627 Wnt Proteins 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 208000031214 ciliopathy Diseases 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000002455 dental arch Anatomy 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000015155 detection of stimulus involved in sensory perception Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 208000003580 polydactyly Diseases 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000008410 smoothened signaling pathway Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108700033870 zebrafish IFT80 Proteins 0.000 description 1
Classifications
-
- 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
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Dispersion Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Virology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a method for researching a regulation mechanism of intracellular transport protein 80 in cilia of a cell in maxilla rapid enlargement machinery biological signal transduction, which comprises the following steps: a. constructing a mouse maxilla rapid expansion animal model; b. constructing IFT80 gene silencing osteoblast; c. constructing an in-vitro stretch culture osteoblast model; d. constructing a co-culture model of osteoblasts and osteoclasts; e. performing in vivo transfection experiments; f. determining the relationship between IFT80 space-time expression and reconstruction of maxillary rapid-expansion palatal suture bone; g. determining the regulation and control mechanism of IFT80 on the proliferation and differentiation of in vitro stretch culture osteoblasts, the formation of primary cilia and mechanical biological signal transduction; h. the effect of IFT80 gene silencing on the modulation of osteoclast function by osteoblasts in vitro stretch culture was determined. The research method has important guiding significance for treating the misfit deformity.
Description
Technical Field
The invention relates to the technical field of biological genes, in particular to a method for researching a regulation mechanism of cell ciliary internal transport protein 80 in maxilla rapid enlargement mechanical biological signal transduction.
Background
Insufficient development of maxillary width is a common malocclusion in orthodontics, and rapid enlargement of the upper jaw (RME) is the most common method for treating such malocclusions. The principle is that the maxillary is guided to move leftwards and rightwards separately by expanding the palatal suture, then the expanded palatal suture tissue proliferates, repairs and deposits bones, the top of the maxillary is widened, the width of the upper dental arch is increased, and finally the treatment purpose is achieved. However, after the palatal suture is rapidly enlarged, the mineralization and regeneration of new bone take more time, and the new bone which is not calcified at the edge of the palatal suture can not resist the resilience of the tissues, so that the recurrence is easy. Recurrence is the most major problem faced by this treatment technique. The research on the mechanism of reconstruction of maxillary rapid expansion palatal suture bone has important guiding significance for clinical reduction of relapse.
The maxilla is a Suture Distraction Osteogenesis (SDO) technology, and the proliferation and differentiation of bone suture tissue cells are stimulated by tension to promote the formation of new bone at the edge of a bone suture. Takahashi et al found that mechanical expansion forces promote differentiation of mesenchymal-like cells in the palate into osteoblasts. Hou et al found that mechanical expansion forces could induce new bone formation by promoting proliferation and differentiation of the suture periosteal cells in the palate. Rapid enlargement of the upper jaw involves complex mechanical bio-signal transduction: the tissue cells of the suture tissue sense the mechanical expansion stimulation and convert the mechanical expansion stimulation into biochemical signals to start signal conduction, stimulate effector cells to secrete cytokines to regulate and control the reconstruction of the suture bone tissue in the palate. Tang et al found that mechanical dilatory forces could promote the proliferation and differentiation of suture-like osteoblasts in the palate by activating the Wnt signaling pathway. Han et al found that in vitro mechanical stretch stimulation promoted osteoblast proliferation by activating the Indian Hedgehog (IHH) signaling pathway. The mechanical expansion force is found to increase the expression of related indexes of osteogenic differentiation of suture tissues in the palate, and the in vitro stretching stimulation can promote the osteogenic differentiation of osteoblasts. It was also found that the maxillary rapidly expanding palatine suture bone tissue remodelling was maintained by the intercoupling of osteoclast-mediated bone resorption and osteoblast-mediated bone formation, and that the palatine suture tissue osteoclasts were resistant to tartrate acid phosphatase (TRAP). And osteoblast alkaline phosphatase (ALP) histochemical staining positive rates both peaked after day seven of expansion. On day fourteen of the expansion, the TRAP positive rate decreased sharply, while the ALP positive rate decreased relatively slowly. However, the mechanism of how the mechanical stimulation signals are sensed and converted into biochemical signals by the suture tissue cells is not clear.
In recent years, studies have shown that primary cilia may be mechanoreceptors for bone tissue, converting mechanoreceptor signals into biochemical signals. The primary cilia are a cell organ formed mainly by cell microtubules and protruding out of the cell surface, have sensory and signal transduction functions and exist in almost all eukaryotic cells. The primary ciliary surface binds a variety of signal receptors, ion channels and transporters, including: hedgehog (HH) and components of Wnt signaling pathway, polycystic proteins 1 (polycystic-1) and 2 (polycystic-2), etc., are regulators of various signaling pathways. The primary cilia are mechanoreceptors for renal epithelial cells and vascular endothelial cells. The fluid shear forces regulate proliferation and metabolism of renal epithelial cells by the primary cilia. Loss of primary ciliary function results in hyperproliferation of renal epithelial cells, eventually forming polycystic kidneys. Blood flow regulates the state of relaxation of blood vessels by stimulating primary cilia on the surface of vascular endothelial cells. The research proves that primary cilia structures exist in bone marrow mesenchymal cells, osteoblasts, osteocytes and chondrocytes. Primary cilia on the surface of osteoblasts were located using a fluorescence-labeled confocal microscope and a scanning electron microscope. Although the primary cilia structure of osteoclasts is not found, it has been reported that the primary cilia of osteoblasts can indirectly regulate the function of osteoclasts through the OPG/RANKL pathway. Abnormalities in primary ciliary structure or function cause a variety of ciliary disorders (ciliopathies), such as polycystic kidney disease, Bardet-Bied syndrome, and skeletal structural abnormalities.
Mutations in the human IFT80 gene can cause thoracic dysplasia syndrome (JATD) and short rib polydactyly type III Syndrome (SRPIII), both of which are accompanied by severe skeletal dysplasia. Knocking out zebrafish IFT80 gene to cause polycystic kidney; knocking out the tetrahymena thermophila IFT80 gene leads to shortening or deletion of cilia; the IFT80 gene of the knockout mouse has JATD and SRPIII syndrome-like phenotype accompanied with skull developmental defect, and a Hedgehog signal channel is inhibited; silencing the marrow mesenchymal cell IFT80 gene causes osteogenic differentiation disorder and primary cilium formation limitation, and the expression of Runx2 and Gli2 is obviously down-regulated; the above results all suggest that the primary ciliary IFT80 plays an important role in the reconstruction of the palatal suture bone for rapid maxillary enlargement.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art, and provide a research method for a regulation mechanism of cell cilia internal transport protein 80 in maxilla rapid-expansion mechanical signal transduction, which systematically explains a mechanism of IFT80 for regulating and controlling reconstruction of maxillary rapid-expansion palatal midjawbone from the overall, cell and molecular levels. Firstly, researching a space-time expression rule of IFT80 and a mechanical biological signal transduction related signal pathway regulation factor by utilizing a mouse maxilla rapid expansion animal model; secondly, silencing IFT80 gene of mouse osteoblast by using RNA interference technology, constructing an in vitro stretch-culture osteoblast model, and researching the regulation and control mechanism of IFT80 on proliferation and differentiation of the in vitro stretch-culture osteoblast, primary cilium formation and mechanical biological signal transduction; thirdly, researching the influence of IFT80 gene silencing on the regulation of osteoclast function by osteoblasts subjected to in vitro stretch culture by using an osteoblast and osteoclast co-culture model; finally, in vivo transfection experiments were performed to study the effect of in vivo IFT80 gene silencing on new bone formation by suture stretch in the palate. Provides basis for exploring ways of promoting the calcification of new bone formed by stretching the bone gap and reducing the absorption of the new bone and lays theoretical foundation for developing a method for effectively expanding the palatal suture.
In order to achieve the purpose, the invention adopts the technical scheme that: the method for researching the regulation mechanism of the intracellular transport protein 80 in the maxilla rapid-enlargement mechanical biological signal transduction comprises the following steps:
a. constructing a mouse upper jaw rapid enlargement animal model, wherein the first molars and the second molars of the mouse are already erupted when the mouse is not less than 6 weeks old;
b. constructing IFT80 gene silencing osteoblast; culturing osteoblasts from fetal rat skull bone, constructing negative shRNA interference sequences aiming at mouse IFT80 genes, packaging into lentiviral particles to infect the osteoblasts, and screening out shRNA interference sequences with the best inhibition efficiency;
c. constructing an in-vitro tension culture osteoblast model, wherein the mechanical tension stress parameters are as follows: 2% deformation, 0.5 Hz; the method comprises the following steps of dividing a static culture blank group, a static culture negative sequence interference group, a static culture IFT80 gene interference group, a stretch culture blank group, a stretch culture negative sequence interference group and a stretch culture IFT80 gene interference group;
d. constructing a co-culture model of osteoblasts and osteoclasts: culturing mouse femur bone marrow mesenchymal cells with 30ng/ml monocyte stimulating factor (M-CSF) for 6 days to induce monocyte differentiation into osteoclast precursor cells; then, osteoclast precursor cells are inoculated in a Transwell chamber, osteoblasts are inoculated in a Bioflex 6-hole stress culture plate, and a co-culture system is constructed; the method comprises the following steps of dividing a co-culture blank group, a co-culture negative sequence interference group, a co-culture IFT80 gene interference group and a co-culture chloral hydrate treatment group into a static state and a stretch culture state, and simultaneously observing the influence of bone cells on the function of osteoclasts;
e. performing in vivo transfection experiment, injecting the obtained lentiviral vector containing the interference sequence of the negative gene and the shRNA aiming at the mouse IFT80 gene under the submucosa of the maxillary rapidly-enlarged palatine suture region into the lentiviral vector containing the interference sequence of the shRNA aiming at the mouse IFT80 gene; the injection is injected once every other day and is continuously injected for 3 times;
f. determining the relationship between IFT80 space-time expression and reconstruction of maxillary rapid-expansion palatal suture bone;
g. determining the regulation and control mechanism of IFT80 on the proliferation and differentiation of in vitro stretch culture osteoblasts, the formation of primary cilia and mechanical biological signal transduction;
h. the effect of IFT80 gene silencing on the modulation of osteoclast function by osteoblasts in vitro stretch culture was determined.
The step f comprises the following substeps:
1) material taking: taking palatine suture tissue, fixing the palatine suture tissue by using PL (2% paraformaldehyde, 75mM lysine and 10mM sodium periodate) fixing liquid, and scanning by using Micro-CT; then decalcifying, dehydrating, waxing, paraffin embedding, slicing, and then using for HE, histochemical and immunohistochemical staining, or not decalcifying, embedding hard tissue, slicing, and directly observing calcein double-label under a fluorescence microscope; taking fresh palatine suture tissue, and extracting RNA for Real-time RT-PCR detection; extracting protein for Western blot analysis;
2) Micro-CT scanning and three-dimensional reconstruction: carrying out Micro-CT scanning and three-dimensional reconstruction on palatal suture tissue, and observing palatal suture form and bone density change;
3) calcein double-label experiment: injecting calcein (10mg/g body weight) into abdominal cavity twice 1 day and 6 days before killing the mice, and observing with a fluorescence microscope;
4) histochemical staining: taking palatine suture paraffin sections, and carrying out histochemical staining on HE, total collagen, ALP, TRAP and the like;
5) immunohistochemical staining: taking palatal suture paraffin sections for immunohistochemical staining and immunohistochemical double staining;
6) real-time RT-PCR: taking total RNA extracted from palatine suture tissue for Real-time RT-PCR analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, ALP, Col I, Gli1, Gli2, beta-catenin, etc.;
7) western blot: taking protein extracted from palatal suture tissue for Western blot analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, Gli1, Gli2, beta-catenin and the like.
The step g comprises the following substeps:
1) confocal microscope of fine cell immunofluorescence labeling: after the culture is finished, fixing cells by 4% paraformaldehyde, and observing by an immunofluorescence labeling confocal microscope;
2) scanning electron microscope: removing the culture solution from osteoblasts, fixing, freeze-drying, and observing primary cilia structure on the cell surface by a scanning electron microscope;
3) BrdU incorporation experiments: 5mM BrdU is added 6 hours before the cell culture is terminated, after the cell culture is terminated, the cell is washed by PBS and digested by pancreatin, and the cell is collected to be analyzed by a flow cytometer or subjected to BrdU immunofluorescence staining to detect the proliferation capacity of osteoblasts;
4) and (3) cytochemical staining: removing the culture solution of osteoblasts, fixing, and staining with ALP and alizarin red respectively;
5) real-time RT-PCR: extracting total RNA of each bone cell composition for Real-time RT-PCR analysis;
6) western blot: and extracting proteins of each bone cell component, and performing Western blot analysis.
The step f comprises the following substeps:
1) cell immunofluorescence labeling confocal microscope: fixing osteoclast with 4% paraformaldehyde, and observing with immunofluorescence labeling confocal microscope;
2) and (3) cytochemical staining: osteoclasts were fixed after discarding the culture medium and TRAP stained using a TRAP staining kit (Sigma);
3) real-time RT-PCR: extracting total RNA of each component bone cell, and detecting the expression of OPG and RANKL by Real-time RT-PCR;
4) western blot: extracting proteins of each bone osteoblast, and detecting the expression of OPG and RANKL by Western blot;
5) enzyme-linked immunosorbent assay (ELISA): collecting cell culture solution, and detecting the content of OPG and RANKL by using an ELISA kit.
The step g comprises the following substeps:
1) material taking: killing the mice in the laboratory and the control group on the seventh day, and taking palatal suture tissues;
2) histochemical staining: taking palatine suture paraffin sections, and carrying out histochemical staining on HE, total collagen, ALP, TRAP and the like;
3) immunohistochemical staining: paraffin sections of the palatine suture were taken and subjected to IFT80 immunohistochemical staining, while the fluorescence intensity of GFP was observed with a confocal laser microscope.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
starting from the overall, cellular and molecular levels respectively, the system explains the mechanism of IFT80 for regulating and controlling the reconstruction of maxillary rapid expansion palatal suture bone.
Firstly, researching a space-time expression rule of IFT80 and a mechanical biological signal transduction related signal pathway regulation factor by utilizing a mouse maxilla rapid expansion animal model;
secondly, silencing IFT80 gene of mouse osteoblast by using RNA interference technology, constructing an in vitro stretch-culture osteoblast model, and researching the regulation and control mechanism of IFT80 on proliferation and differentiation of the in vitro stretch-culture osteoblast, primary cilium formation and mechanical biological signal transduction;
thirdly, researching the influence of IFT80 gene silencing on the regulation of osteoclast function by osteoblasts subjected to in vitro stretch culture by using an osteoblast and osteoclast co-culture model;
finally, in vivo transfection experiments were performed to study the effect of in vivo IFT80 gene silencing on new bone formation by suture stretch in the palate. Provides basis for exploring ways of promoting the calcification of new bone formed by stretching the bone gap and reducing the absorption of the new bone and lays theoretical foundation for developing a method for effectively expanding the palatal suture.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example one
The method for researching the regulation mechanism of the intracellular transport protein 80 in the maxilla rapid-enlargement mechanical biological signal transduction comprises the following steps:
a. constructing a mouse upper jaw rapid enlargement animal model, wherein the first molars and the second molars of the mouse are already erupted when the mouse is not less than 6 weeks old;
b. constructing IFT80 gene silencing osteoblast; culturing osteoblasts from fetal rat skull bone, constructing negative shRNA interference sequences aiming at mouse IFT80 genes, packaging into lentiviral particles to infect the osteoblasts, and screening out shRNA interference sequences with the best inhibition efficiency;
c. constructing an in-vitro tension culture osteoblast model, wherein the mechanical tension stress parameters are as follows: 2% deformation, 0.5 Hz; the method comprises the following steps of dividing a static culture blank group, a static culture negative sequence interference group, a static culture IFT80 gene interference group, a stretch culture blank group, a stretch culture negative sequence interference group and a stretch culture IFT80 gene interference group;
d. constructing a co-culture model of osteoblasts and osteoclasts: culturing mouse femur bone marrow mesenchymal cells with 30ng/ml monocyte stimulating factor (M-CSF) for 6 days to induce monocyte differentiation into osteoclast precursor cells; then, osteoclast precursor cells are inoculated in a Transwell chamber, osteoblasts are inoculated in a Bioflex 6-hole stress culture plate, and a co-culture system is constructed; the method comprises the following steps of dividing a co-culture blank group, a co-culture negative sequence interference group, a co-culture IFT80 gene interference group and a co-culture chloral hydrate treatment group into a static state and a stretch culture state, and simultaneously observing the influence of bone cells on the function of osteoclasts;
e. performing in vivo transfection experiment, injecting the obtained lentiviral vector containing the interference sequence of the negative gene and the shRNA aiming at the mouse IFT80 gene under the submucosa of the maxillary rapidly-enlarged palatine suture region into the lentiviral vector containing the interference sequence of the shRNA aiming at the mouse IFT80 gene; the injection is injected once every other day and is continuously injected for 3 times;
f. determining the relationship between IFT80 space-time expression and reconstruction of maxillary rapid-expansion palatal suture bone;
g. determining the regulation and control mechanism of IFT80 on the proliferation and differentiation of in vitro stretch culture osteoblasts, the formation of primary cilia and mechanical biological signal transduction;
h. the effect of IFT80 gene silencing on the modulation of osteoclast function by osteoblasts in vitro stretch culture was determined.
The step f comprises the following substeps:
1) material taking: taking palatine suture tissue, fixing the palatine suture tissue by using PL (2% paraformaldehyde, 75mM lysine and 10mM sodium periodate) fixing liquid, and scanning by using Micro-CT; then decalcifying, dehydrating, waxing, paraffin embedding, slicing, and then using for HE, histochemical and immunohistochemical staining, or not decalcifying, embedding hard tissue, slicing, and directly observing calcein double-label under a fluorescence microscope; taking fresh palatine suture tissue, and extracting RNA for Real-time RT-PCR detection; extracting protein for Western blot analysis;
2) Micro-CT scanning and three-dimensional reconstruction: carrying out Micro-CT scanning and three-dimensional reconstruction on palatal suture tissue, and observing palatal suture form and bone density change;
3) calcein double-label experiment: injecting calcein (10mg/g body weight) into abdominal cavity twice 1 day and 6 days before killing the mice, and observing with a fluorescence microscope;
4) histochemical staining: taking palatine suture paraffin sections, and carrying out histochemical staining on HE, total collagen, ALP, TRAP and the like;
5) immunohistochemical staining: taking palatal suture paraffin sections for immunohistochemical staining and immunohistochemical double staining;
6) real-time RT-PCR: taking total RNA extracted from palatine suture tissue for Real-time RT-PCR analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, ALP, Col I, Gli1, Gli2, beta-catenin, etc.;
7) western blot: taking protein extracted from palatal suture tissue for Western blot analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, Gli1, Gli2, beta-catenin and the like.
The step g comprises the following substeps:
1) confocal microscope of fine cell immunofluorescence labeling: after the culture is finished, fixing cells by 4% paraformaldehyde, and observing by an immunofluorescence labeling confocal microscope;
2) scanning electron microscope: removing the culture solution from osteoblasts, fixing, freeze-drying, and observing primary cilia structure on the cell surface by a scanning electron microscope;
3) BrdU incorporation experiments: 5mM BrdU is added 6 hours before the cell culture is terminated, after the cell culture is terminated, the cell is washed by PBS and digested by pancreatin, and the cell is collected to be analyzed by a flow cytometer or subjected to BrdU immunofluorescence staining to detect the proliferation capacity of osteoblasts;
4) and (3) cytochemical staining: removing the culture solution of osteoblasts, fixing, and staining with ALP and alizarin red respectively;
5) real-time RT-PCR: extracting total RNA of each bone cell composition for Real-time RT-PCR analysis;
6) western blot: and extracting proteins of each bone cell component, and performing Western blot analysis.
The step f comprises the following substeps:
1) cell immunofluorescence labeling confocal microscope: fixing osteoclast with 4% paraformaldehyde, and observing with immunofluorescence labeling confocal microscope;
2) and (3) cytochemical staining: osteoclasts were fixed after discarding the culture medium and TRAP stained using a TRAP staining kit (Sigma);
3) real-time RT-PCR: extracting total RNA of each component bone cell, and detecting the expression of OPG and RANKL by Real-time RT-PCR;
4) western blot: extracting proteins of each bone osteoblast, and detecting the expression of OPG and RANKL by Western blot;
5) enzyme-linked immunosorbent assay (ELISA): collecting cell culture solution, and detecting the content of OPG and RANKL by using an ELISA kit.
The step g comprises the following substeps:
1) material taking: killing the mice in the laboratory and the control group on the seventh day, and taking palatal suture tissues;
2) histochemical staining: taking palatine suture paraffin sections, and carrying out histochemical staining on HE, total collagen, ALP, TRAP and the like;
3) immunohistochemical staining: paraffin sections of the palatine suture were taken and subjected to IFT80 immunohistochemical staining, while the fluorescence intensity of GFP was observed with a confocal laser microscope.
Example two
Determining the reconstruction process of the maxillary rapid expansion palatal suture bone, and observing the palatal suture form and the change of bone density by using Micro-CT at continuous time points; HE. Staining total collagen, ALP, TRAP and the like to observe the formation and absorption of new bones of tissues in the middle gap of the palate, and evaluating the expression change of indexes related to osteogenic differentiation such as Bone Sialoprotein (BSP), Osteopontin (OPN), Osteocalcin (OCN), ALP and type I collagen (Col I) by Real-time RT-PCR and Western-blot; calcein double-label experiment was performed to evaluate the deposition rate of new bone in the palatal suture 14 days after arch expansion.
The proliferation of cells in palatal suture tissue of maxillary rapid enlargement is confirmed, and immunohistochemical staining is carried out on cell nucleus proliferation antigens PCNA and Ki 67.
Defining a space-time expression rule of IFT80 in a palatal suture stretch bone forming process, and detecting the distribution and expression of IFT80 by continuous time point immunohistochemical staining; meanwhile, immunohistochemical staining detects the distribution and expression of cilia-associated proteins Tg737 and Kif3 a.
The relation between IFT80 spatiotemporal expression and palatine suture stretch bone formation is clarified, and IFT80 and osteoblast specific transcription factor Runx2 as well as IFT80 and Col I immunohistochemical double staining experiments are carried out.
And (3) determining whether a Hedgehog and Wnt signaling pathway is involved in palatine suture stretch bone reconstruction, and detecting expression changes of Gli1, Gli2 and beta-catenin by using a Real-timeRT-PCR and Western-blot method.
The regulation and control effects of IFT80 on the proliferation and differentiation of stretch-cultured osteoblasts are determined, osteoblasts with IFT80 gene silencing are subjected to stretch-culture in vitro, and the proliferation capacity of the cells is detected by 5-bromodeoxyuridine (BrdU) immunofluorescence staining and flow cytometry analysis; detecting the differentiation capacity of the cells by using ALP activity detection, ALP and alizarin erythrocyte chemical staining; and detecting the expression change of osteogenic differentiation related indexes such as BSP, OPN, OCN, ALP and Col I by adopting Real-time RT-PCR and Western blot.
To confirm the regulation effect of IFT80 on primary cilia formation of stretch-cultured osteoblasts, we performed IFT80 and Acetylated alpha-tubulin (Acetylated alpha-tubulin) cell immunofluorescence dual-labeling experiments on osteoblasts with silenced IFT80 gene in vitro stretch-cultured; simultaneously detecting the change of the length of the primary cilia on the cell surface by using a fluorescence labeling confocal microscope and a scanning electron microscope; and detecting the expression changes of cilia-associated proteins Tg737 and Kif3a by using Western-blot.
In order to determine a regulation mechanism of IFT80 on osteoblast mechanical biological signal transduction, IFT80 gene-silenced osteoblasts are subjected to in vitro stretch culture, and the expression changes of key regulatory factors (Gli1, Gli2 and beta-catenin) of Hedgehog and Wnt signal pathways and Runx2 are detected by adopting methods such as Real-time RT-PCR, Western blot, cell immunofluorescence and the like; cells were then treated with the corresponding agonist (or antagonist) and observed for changes in expression of regulatory factors as well as Runx 2.
Although the primary cilia structure of the osteoclast is not found, researches report that the primary cilia of the osteoblast can indirectly regulate the function of the osteoclast through an OPG/RANKL pathway to construct a co-culture model of the osteoblast and the osteoclast, and the influence of IFT80 gene silencing on the regulation of the function of the osteoclast by the osteoblast in vitro stretch culture is observed.
In order to determine the influence of IFT80 gene silencing on the regulation of osteoclast function by stretch-cultured osteoblasts, the change of osteoclast skeleton formation is detected by Phalloidin (Phalloidin) cellular immunofluorescence; TRAP activity assay and TRAP cytochemical staining assay changes in osteoclast function.
In order to determine whether the OPG/RANKL pathway participates in the regulation process, Real-time RT-PCR and Western blot are used for detecting the expression levels of OPG and RANKL of osteoblasts; enzyme-linked immunosorbent assay (ELISA) detects the expression level of OPG and RANKL in the culture medium.
To clarify the effect of in vivo IFT80 gene silencing on palatopalatine suture stretch bone formation, we performed in vivo transfection experiments by injecting a lentiviral vector containing an shRNA interference sequence against mouse IFT80 gene under the submucosal region of the maxillo-rapidly enlarged palatal suture region to locally silence the IFT80 gene.
To confirm the transfection efficiency in vivo, GFP fluorescence intensity detection and IFT80 immunohistochemical staining were performed.
To clarify the reconstruction of the palatal suture bone, staining was performed with HE, total collagen, ALP, TRAP, and the like.
The above is only a specific application example of the present invention, and the protection scope of the present invention is not limited in any way. All the technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.
Claims (3)
1. The method for researching the regulation mechanism of the intracellular transport protein 80 in the maxilla rapid-enlargement mechanical biological signal transduction is characterized by comprising the following steps:
a. constructing a mouse upper jaw rapid expansion animal model, wherein the mouse is not less than 6 weeks old, and ensuring that the first molars and the second molars sprout;
b. constructing IFT80 gene silencing osteoblast; culturing osteoblasts from fetal rat skull bone, constructing negative shRNA interference sequences aiming at mouse IFT80 genes, packaging into lentiviral particles to infect the osteoblasts, and screening out shRNA interference sequences with the best inhibition efficiency;
c. constructing an in-vitro tension culture osteoblast model, wherein the mechanical tension stress parameters are as follows: 2% deformation, 0.5 Hz; the method comprises the following steps of dividing a static culture blank group, a static culture negative sequence interference group, a static culture IFT80 gene interference group, a stretch culture blank group, a stretch culture negative sequence interference group and a stretch culture IFT80 gene interference group;
d. constructing a co-culture model of osteoblasts and osteoclasts: culturing mouse femur bone marrow mesenchymal cells with 30ng/ml monocyte stimulating factor (M-CSF) for 6 days to induce monocyte differentiation into osteoclast precursor cells; then, osteoclast precursor cells are inoculated in a Transwell chamber, osteoblasts are inoculated in a Bioflex 6-hole stress culture plate, and a co-culture system is constructed; the method comprises the following steps of dividing a co-culture blank group, a co-culture negative sequence interference group, a co-culture IFT80 gene interference group and a co-culture chloral hydrate treatment group into a static state and a stretch culture state, and simultaneously observing the influence of bone cells on the function of osteoclasts;
e. performing in vivo transfection experiment, injecting the obtained lentiviral vector containing the interference sequence of the negative gene and the shRNA aiming at the mouse IFT80 gene under the submucosa of the maxillary rapidly-enlarged palatine suture region into the lentiviral vector containing the interference sequence of the shRNA aiming at the mouse IFT80 gene; the injection is injected once every other day and is continuously injected for 3 times;
f. determining the relationship between IFT80 space-time expression and reconstruction of maxillary rapid-expansion palatal suture bone; 1) material taking: fixing palatine suture tissue by using PL fixing solution containing 2% paraformaldehyde, 75mM lysine and 10mM sodium periodate, and scanning by Micro-CT; then decalcifying, dehydrating, waxing, paraffin embedding, slicing, and then using for HE, histochemical and immunohistochemical staining, or not decalcifying, embedding hard tissue, slicing, and directly observing calcein double-label under a fluorescence microscope; taking fresh palatine suture tissue, and extracting RNA for Real-time RT-PCR detection; extracting protein for Western blot analysis;
2) Micro-CT scanning and three-dimensional reconstruction: carrying out Micro-CT scanning and three-dimensional reconstruction on palatal suture tissue, and observing palatal suture form and bone density change;
3) calcein double-label experiment: injecting 10mg/g of calcein into abdominal cavity twice 1 day and 6 days before killing the mice, and observing with a fluorescence microscope;
4) histochemical staining: taking palatine suture paraffin sections, and carrying out histochemical staining on HE, total collagen, ALP, TRAP and the like;
5) immunohistochemical staining: taking palatal suture paraffin sections for immunohistochemical staining and immunohistochemical double staining;
6) real-time RT-PCR: taking total RNA extracted from palatine suture tissue for Real-time RT-PCR analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, ALP, Col I, Gli1, Gli2 and beta-catenin;
7) western blot: taking protein extracted from palatal suture tissue for Western blot analysis, wherein the detection indexes are as follows: IFT80, Runx2, OPN, OCN, Gli1, Gli2 and beta-catenin;
g. determining the regulation and control mechanism of IFT80 on the proliferation and differentiation of in vitro stretch culture osteoblasts, the formation of primary cilia and mechanical biological signal transduction; 1) confocal microscope of fine cell immunofluorescence labeling: after the culture is finished, fixing cells by 4% paraformaldehyde, and observing by an immunofluorescence labeling confocal microscope;
2) scanning electron microscope: removing the culture solution from osteoblasts, fixing, freeze-drying, and observing primary cilia structure on the cell surface by a scanning electron microscope;
3) BrdU incorporation experiments: 5mM BrdU was added 6 hours before the termination of the cell culture, and after the termination of the culture,
washing with PBS, digesting with pancreatin, collecting cells, analyzing with flow cytometer or BrdU immunofluorescence staining
Detecting the proliferation capacity of osteoblasts;
4) and (3) cytochemical staining: removing the culture solution of osteoblasts, fixing, and staining with ALP and alizarin red respectively;
5) real-time RT-PCR: total RNA of each component bone cell was extracted and used as Real-time RT-PCR fraction
Separating out;
6) western blot: extracting proteins of each component bone cell, and carrying out Western blot analysis;
h. the effect of IFT80 gene silencing on the modulation of osteoclast function by osteoblasts in vitro stretch culture was determined.
2. The method for studying the regulatory mechanism of cellular ciliary transport protein 80 in maxillomandibular rapid-expansion mechanical biosignal transduction according to claim 1, wherein said step f comprises the following substeps:
1) cell immunofluorescence labeling confocal microscope: fixing osteoclast with 4% paraformaldehyde, and observing with immunofluorescence labeling confocal microscope;
2) and (3) cytochemical staining: fixing the osteoclast after removing the culture solution, and performing TRAP staining by using a TRAP staining kit;
3) real-time RT-PCR: extracting total RNA of each component bone cell, and detecting the expression of OPG and RANKL by Real-time RT-PCR;
4) western blot: extracting proteins of each bone osteoblast, and detecting the expression of OPG and RANKL by Western blot;
5) enzyme-linked immunosorbent assay: collecting cell culture solution, and detecting the content of OPG and RANKL by using an ELISA kit.
3. The method for studying the regulatory mechanism of cellular ciliary transport protein 80 in maxillomandibular rapid-expansion mechanical biosignal transduction according to claim 1, wherein said step g comprises the substeps of:
1) material taking: killing the mice in the laboratory and the control group on the seventh day, and taking palatal suture tissues;
2) histochemical staining: collecting Paraffin section of palatine suture, and performing HE, total collagen, ALP and TRAP
Histochemical staining;
3) immunohistochemical staining: paraffin sections of the palatine suture were taken and subjected to IFT80 immunohistochemical staining, while the fluorescence intensity of GFP was observed with a confocal laser microscope.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810418078.3A CN108753701B (en) | 2018-05-04 | 2018-05-04 | Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810418078.3A CN108753701B (en) | 2018-05-04 | 2018-05-04 | Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108753701A CN108753701A (en) | 2018-11-06 |
| CN108753701B true CN108753701B (en) | 2021-10-29 |
Family
ID=64009787
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810418078.3A Expired - Fee Related CN108753701B (en) | 2018-05-04 | 2018-05-04 | Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108753701B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173247A (en) * | 2007-08-08 | 2008-05-07 | 中国人民解放军军事医学科学院基础医学研究所 | Method for culturing osteoclast with mesenchyma stem cell combined with cell factor |
| CN104651302A (en) * | 2015-02-09 | 2015-05-27 | 苏州大学附属第一医院 | Method for extracting myelomonocyte and differentiating to osteoclast |
-
2018
- 2018-05-04 CN CN201810418078.3A patent/CN108753701B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173247A (en) * | 2007-08-08 | 2008-05-07 | 中国人民解放军军事医学科学院基础医学研究所 | Method for culturing osteoclast with mesenchyma stem cell combined with cell factor |
| CN104651302A (en) * | 2015-02-09 | 2015-05-27 | 苏州大学附属第一医院 | Method for extracting myelomonocyte and differentiating to osteoclast |
Non-Patent Citations (9)
| Title |
|---|
| Hedgehog信号通路调控骨形成及BMSCs成骨分化的研究进展;迟博婧 等;《中国修复重建外科杂志》;20161201;摘要，第1548页右栏第3.4节 * |
| Indian Hedgehog在牵张力促进成骨细胞增殖中的作用研究;韩磊 等;《华西口腔医学杂志》;20120601;摘要，第234-244页第一段，第235页右栏1.2方法及2.1节 * |
| The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis;Yang S et al;《Bone》;20120704;407-417页 * |
| 上颌快速扩大兔腭中缝组织改建过程中血管内皮生长因子的表达和意义;张卫兵;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20051015;E074-52页 * |
| 初级纤毛对人牙周膜细胞成骨相关基因表达的影响;蒋思琪 等;《口腔医学研究》;20180124;10-13页 * |
| 多囊蛋白1在上颌快速扩大机械生物信号转导中的调控机制;王华;《中国博士学位论文全文数据库医药卫生科技辑》;20180615;E074-10页 * |
| 小鼠成骨细胞和破骨细胞共培养模型的建立;鲁秀敏;《中国骨质疏松杂志》;20080320;第160页第1.4-1.7节 * |
| 机械扩张力作用下小鼠腭中缝成骨与破骨的研究;苗毅等;《口腔医学》;20090428(第04期);摘要，第170页第1.2-1.6节，第170-171页讨论部分 * |
| 纤毛转运蛋白对骨发育影响的研究进展;李功臣等;《口腔颌面外科杂志》;20170828(第04期);285-289页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108753701A (en) | 2018-11-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roberts et al. | The combined bone forming capacity of human periosteal derived cells and calcium phosphates | |
| Li et al. | Platelet‐rich fibrin promotes periodontal regeneration and enhances alveolar bone augmentation | |
| Klees et al. | Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway | |
| Harunaga et al. | Dynamics of salivary gland morphogenesis | |
| Yang et al. | Cyclic tensile stretch modulates osteogenic differentiation of adipose-derived stem cells via the BMP-2 pathway | |
| Kim et al. | Dynamic mechanical and nanofibrous topological combinatory cues designed for periodontal ligament engineering | |
| Wan et al. | Synergistic effect of matrix stiffness and inflammatory factors on osteogenic differentiation of MSC | |
| WO2008053892A1 (en) | Pharmaceutical for promoting functional regeneration of damaged tissue | |
| JP6613380B2 (en) | Novel use as a pharmaceutical composition for the prevention and treatment of fibrosis using the inhibitory activity of chromone derivatives on epithelial-mesenchymal transition | |
| US20130029292A1 (en) | Method of implanting mesenchymal stem cells for natural tooth regeneration in surgically prepared extraction socket and compositions thereof | |
| CN110616190B (en) | Method for regulating and controlling periodontal ligament stem cell osteogenic differentiation based on extracellular matrix | |
| Lu et al. | Rejuvenation of tendon stem/progenitor cells for functional tendon regeneration through platelet-derived exosomes loaded with recombinant Yap1 | |
| Wu et al. | SCAPs regulate differentiation of DFSCs during tooth root development in swine | |
| CN108753701B (en) | Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction | |
| CN105255824A (en) | Periodontal ligament stem cell osteogenic differentiation inducing liquid and method | |
| CN103387959A (en) | Stem cell from deciduous teeth, and preparation method and application thereof | |
| Huang et al. | Calcitonin gene-related peptide-induced calcium alginate gel combined with adipose-derived stem cells differentiating to osteoblasts | |
| Zhu et al. | Aging induced p53/p21 in genioglossus muscle stem cells and enhanced upper airway injury | |
| Tian et al. | The effects of Sema3A overexpression on the proliferation and differentiation of rat gingival mesenchymal stem cells in the LPS-induced inflammatory environment | |
| Fujisaki et al. | Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells | |
| US20210180018A1 (en) | Composition for promoting stem cell differentiation, comprising progenitor cell culture solution and multilayer graphene film, and use thereof | |
| JP2009256350A (en) | Bone differentiation inducing agent | |
| Staszyk et al. | Primary culture of fibroblasts and cementoblasts of the equine periodontium | |
| CN104127862A (en) | Application of Semaphorin 4D in preventing and treating bone metabolic diseases | |
| Santos | Mechanical regulation of cardiac fibroblasts. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20211029 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |