CN116676378A - Ap-2β基因在成骨细胞活性调控中的应用 - Google Patents
Ap-2β基因在成骨细胞活性调控中的应用 Download PDFInfo
- Publication number
- CN116676378A CN116676378A CN202310372291.6A CN202310372291A CN116676378A CN 116676378 A CN116676378 A CN 116676378A CN 202310372291 A CN202310372291 A CN 202310372291A CN 116676378 A CN116676378 A CN 116676378A
- Authority
- CN
- China
- Prior art keywords
- beta
- osteoblast activity
- gene
- osteoblast
- beta gene
- 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.)
- Pending
Links
- 210000000963 osteoblast Anatomy 0.000 title claims abstract description 106
- 230000000694 effects Effects 0.000 title claims abstract description 63
- 101100263837 Bovine ephemeral fever virus (strain BB7721) beta gene Proteins 0.000 title claims abstract description 40
- 101100316840 Enterobacteria phage P4 Beta gene Proteins 0.000 title claims abstract description 40
- 230000033228 biological regulation Effects 0.000 title claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 32
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 23
- 210000003625 skull Anatomy 0.000 claims abstract description 18
- 238000011161 development Methods 0.000 claims abstract description 12
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000001132 Osteoporosis Diseases 0.000 claims abstract description 7
- 230000014509 gene expression Effects 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 20
- 238000003753 real-time PCR Methods 0.000 claims description 19
- 239000003814 drug Substances 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 14
- 238000001262 western blot Methods 0.000 claims description 14
- 208000035475 disorder Diseases 0.000 claims description 12
- 239000012190 activator Substances 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 8
- 208000027418 Wounds and injury Diseases 0.000 claims description 6
- 230000006378 damage Effects 0.000 claims description 6
- 239000000975 dye Substances 0.000 claims description 6
- 208000014674 injury Diseases 0.000 claims description 6
- 230000008439 repair process Effects 0.000 claims description 6
- 238000002123 RNA extraction Methods 0.000 claims description 5
- 238000000751 protein extraction Methods 0.000 claims description 4
- 239000012807 PCR reagent Substances 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims 1
- 108060000903 Beta-catenin Proteins 0.000 abstract description 22
- 102000015735 Beta-catenin Human genes 0.000 abstract description 22
- 230000001105 regulatory effect Effects 0.000 abstract description 18
- 210000002454 frontal bone Anatomy 0.000 abstract description 12
- 230000019491 signal transduction Effects 0.000 abstract description 12
- 210000003455 parietal bone Anatomy 0.000 abstract description 10
- 230000004069 differentiation Effects 0.000 abstract description 8
- 230000002188 osteogenic effect Effects 0.000 abstract description 8
- 230000035755 proliferation Effects 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 208000020084 Bone disease Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 64
- 108090000623 proteins and genes Proteins 0.000 description 24
- 239000000243 solution Substances 0.000 description 23
- 238000002474 experimental method Methods 0.000 description 19
- 210000001519 tissue Anatomy 0.000 description 19
- 230000001936 parietal effect Effects 0.000 description 17
- 238000003197 gene knockdown Methods 0.000 description 16
- 239000012528 membrane Substances 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 102000013814 Wnt Human genes 0.000 description 15
- 108050003627 Wnt Proteins 0.000 description 15
- 230000002018 overexpression Effects 0.000 description 15
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 12
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 11
- 230000018109 developmental process Effects 0.000 description 10
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 10
- 230000003828 downregulation Effects 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 108010024682 Core Binding Factor Alpha 1 Subunit Proteins 0.000 description 7
- 102000015775 Core Binding Factor Alpha 1 Subunit Human genes 0.000 description 7
- 102000055102 bcl-2-Associated X Human genes 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 230000004072 osteoblast differentiation Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 6
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000002033 PVDF binder Substances 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 108700000707 bcl-2-Associated X Proteins 0.000 description 6
- 210000000988 bone and bone Anatomy 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000011164 ossification Effects 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 5
- 108050006400 Cyclin Proteins 0.000 description 5
- 241000713666 Lentivirus Species 0.000 description 5
- 108060001084 Luciferase Proteins 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 description 5
- 101150068698 Sp7 gene Proteins 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 239000003292 glue Substances 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 230000009456 molecular mechanism Effects 0.000 description 5
- 208000010392 Bone Fractures Diseases 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 206010017076 Fracture Diseases 0.000 description 4
- 108090001093 Lymphoid enhancer-binding factor 1 Proteins 0.000 description 4
- 102100022699 Lymphoid enhancer-binding factor 1 Human genes 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- 108091027967 Small hairpin RNA Proteins 0.000 description 4
- 108090001039 Transcription factor AP-2 Proteins 0.000 description 4
- 102000004893 Transcription factor AP-2 Human genes 0.000 description 4
- 230000004156 Wnt signaling pathway Effects 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000003531 protein hydrolysate Substances 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 230000002103 transcriptional effect Effects 0.000 description 4
- 108010058546 Cyclin D1 Proteins 0.000 description 3
- 102000006311 Cyclin D1 Human genes 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 210000002449 bone cell Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 210000001652 frontal lobe Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 230000025308 nuclear transport Effects 0.000 description 3
- 230000009818 osteogenic differentiation Effects 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000007711 solidification Methods 0.000 description 3
- 230000008023 solidification Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 2
- 102100030091 Dickkopf-related protein 2 Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108090000331 Firefly luciferases Proteins 0.000 description 2
- 230000010190 G1 phase Effects 0.000 description 2
- 102000019058 Glycogen Synthase Kinase 3 beta Human genes 0.000 description 2
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 description 2
- 101000864647 Homo sapiens Dickkopf-related protein 2 Proteins 0.000 description 2
- 101000711796 Homo sapiens Sclerostin Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 230000018199 S phase Effects 0.000 description 2
- 102100034201 Sclerostin Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 101150025841 CCND1 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102100033601 Collagen alpha-1(I) chain Human genes 0.000 description 1
- 208000009283 Craniosynostoses Diseases 0.000 description 1
- 206010049889 Craniosynostosis Diseases 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 101150027068 DEGS1 gene Proteins 0.000 description 1
- 230000005971 DNA damage repair Effects 0.000 description 1
- 238000003718 Dual-Luciferase Reporter Assay System Methods 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 101150024147 bax gene Proteins 0.000 description 1
- 108700041737 bcl-2 Genes Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000009025 developmental regulation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000885 nephron Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 208000030761 polycystic kidney disease Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 210000004761 scalp Anatomy 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- -1 sp7 Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- C12Q2600/136—Screening for pharmacological compounds
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明涉及一种Ap‑2β基因在制备诊断和/或治疗成骨细胞活性调控相关病症的试剂中的应用。申请人研究证明,Ap‑2β基因是一种通过调控Wnt/β‑catenin信号通路来调节成骨细胞增殖和分化的新型调控因子,在调节额骨和顶骨成骨细胞的区域成骨潜能中发挥重要作用,因此在颅骨发育和相关骨类疾病如骨质疏松症中表现出一种新的治疗靶点。
Description
技术领域
本发明涉及生物医药领域,特别是涉及一种Ap-2β基因在成骨细胞活性调控中的应用。
背景技术
Ap-2β是一种存在于远端肾单位的转录因子,可以调节和控制细胞分裂,作为一个肿瘤抑制基因在发育的过程中能调控肿瘤细胞的生长、衰老和凋亡,是维系正常个体发育不可或缺的基因。
Ap-2β在胚胎发育过程中也具有重要作用,在小鼠出生后不久,小鼠体内的Ap-2β含量会显著下降,而缺少Ap-2β会出现多囊肾,并最终导致小鼠肾衰竭而死亡。研究发现,Ap-2β可以作为研究骨细胞增殖和凋亡的一个新的分子靶点。发明人之前的文章(CellPhysiol Biochem,2017)已经证明了顶骨、额骨成骨细胞具有基因表达模式和信号通路的差异,并发现转录因子Ap-2β在额骨组织中高度表达。现有技术证明Ap-2β的缺失会影响颅缝的发育,然而目前其在如何调控成骨发育相关方面还没未见报道。
发明内容
发明要解决的问题
本申请阐明了Ap-2β基因在调控成骨发育过程的分子机制。为颅骨发育调控的分子机制提供新的科学依据,为治疗颅骨发育相关疾病提供新的治疗靶点,也为临床再生医学提供新的理论基础。
用于解决问题的方案
发明人利用慢病毒介导稳转细胞系构建技术及shRNA基因敲低技术,在MC3T3-E1小鼠胚胎成骨细胞中过表达/敲低Ap-2β,获得Ap-2β过表达/敲低的稳转细胞系,研究Ap-2β基因对成骨发育的影响,并提取颅骨部分组织(顶骨、额骨)进行体外原代培养,深入阐明Ap-2β基因调控成骨发育的分子机制。
研究主要内容如下:
(1)构建基因过表达及敲低模型:首先进行Ap-2β分子克隆构建重组质粒,后利用慢病毒介导稳转细胞系技术及shRNA基因敲低技术,在MC3T3-E1小鼠胚胎成骨细胞中过表达/敲低Ap-2β,获得Ap-2β过表达/敲低的稳转细胞系。
(2)确定Ap-2β对成骨细胞发育的影响:对Ap-2β稳转株分化各阶段的细胞进行RNA和蛋白的提取,通过ALP染色、qPCR(荧光定量PCR)、Western blotting(蛋白免疫印迹)对其表型进行观察,并分析各阶段成骨活性的变化。
(3)探究Ap-2β调控颅骨发育的机制:提取对照组与实验组成骨细胞的RNA,进行转录组测序。分析测序结果,利用qPCR、Western blotting、核质分离、TOPFlash荧光素酶报告基因等实验检测关键因子的转录水平和蛋白水平的变化。
(4)进一步探究Ap-2β调控成骨细胞的分子机制:提取颅骨组织进行体外原代培养,提取细胞蛋白和RNA,在原代成骨细胞上做成骨细胞活性调控干预实验,反向验证Ap-2β对于成骨活性的调控作用。
第一方面,本发明提供了一种Ap-2β基因在制备诊断和/或治疗成骨细胞活性调控相关病症的试剂中的应用。
优选地,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
优选地,所述应用选自:
(a)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的制备;
(b)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的筛选;或
(c)Ap-2β基因作为靶标应用于制备诊断成骨细胞活性调控相关病症的试剂。
第二方面,本发明还提供了一种用于辅助诊断成骨细胞活性调控相关病症的试剂盒,其特征在于,所述试剂盒包括Ap-2β基因的检测试剂。
优选地,所述检测试剂检测所述Ap-2β基因的表达量。
优选地,所述试剂盒采用的检测方法为Western Blot、荧光定量PCR。
优选地,所述试剂盒包括全蛋白提取试剂、PCR试剂、Western Blot检测试剂、RNA抽提试剂、荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针。
第三方面,本发明还提供了一种Ap-2β基因激活剂或调节剂在制备治疗成骨细胞活性调控相关病症中的应用,其特征在于,所述Ap-2β基因激活剂或调节剂为可调控Ap-2β基因表达水平的试剂。
优选地,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
第四方面,本发明还提供了一种Ap-2β基因激活剂或调节剂在制备治疗颅骨发育相关病症的药物中的应用。
发明的效果
本发明研究证明,Ap-2β基因是一种通过调控Wnt/β-catenin信号通路来调节成骨细胞增殖和分化的新型调控因子,在调节额骨和顶骨成骨细胞的区域成骨潜能中发挥重要作用,因此在颅骨发育中表现出一种新的治疗靶点。
附图说明
图1:Ap-2β在组织和成骨细胞中的表达。(A)Ap-2β蛋白在颅骨和肢体等多种组织中均有表达;(B)分化D0天Ap-2βmRNA在对照、过表达、敲低组的基因定量分析;(C)分化D0天Ap-2β蛋白在对照、过表达、敲低组的表达量。p***<0.001。
图2:Ap-2β对成骨前体细胞增殖和凋亡的影响。(A-B)用CCK-8法测定对照组、Ap-2β组和shAp-2β组MC3T3-E1细胞的生长曲线;(C-D)分化D0天用WB法测定Ap-2β、shAp-2β处理的成骨细胞中CyclinD1、PCNA、Bax和Bcl-2的蛋白变化;(E)Ap-2β、shAp-2β处理的成骨细胞EdU染色;(F)图E中EdU和DAPI之间的平均荧光强度;(G)shAp-2β处理的成骨细胞中检测Bax和Bcl-2的表达水平。p*<0.05,p**<0.01,p***<0.001。
图3:Ap-2β在MC3T3-E1细胞成骨分化中的作用。(A)分化D7天对照组、Ap-2β组和shAp-2β组MC3T3-E1细胞ALP染色的代表性图像;(B-C)WB及灰度分析显示Ap-2β在成骨细胞中的过表达和下调影响RUNX2和Sp7在分化第7天的活性;(D-G)RUNX2、Sp7、ALP、COL1A1mRNA相对水平在分化D7和D14时显著降低。p***<0.001。
图4:Ap-2β激活MC3T3-E1细胞Wnt/β-catenin信号通路。(A-B)Ap-2β过表达成骨细胞中活性β-catenin含量较高;(C)过表达Ap-2β可增强MC3T3-E1细胞中活性β-catenin的核转运;(D)Ap-2β过表达的成骨细胞在D7天时GSK3β和磷酸化的GSK3β没有改变;(E-F)Ap-2β敲低成骨细胞D7和D14时Wnt/β-catenin下游靶基因TCF、LEF-1、C-MYC和CD44显著降低;(G)在Ap-2β敲低的成骨细胞中,Wnt信号通路抑制剂DKK1、DKK2和SOST的相对mRNA水平在D7时显著升高;(H)当使用LiCl和未使用LiCl时,通过TOPFlash检测Wnt/β-catenin的转录活性。P*<0.05,p**<0.01,p***<0.001。
图5:Ap-2β对额骨和顶骨成骨细胞的区域成骨潜能的影响。(A)顶骨、额骨组织中Ap-2β的免疫荧光染色;(B-C)Western blot和Image J分析显示Ap-2β在额骨中表达高于顶骨;(D)慢病毒介导Ap-2β过表达和敲低后,顶骨、额骨成骨细胞中RUNX2、Sp7、Cnx-43、Ccnd1、TCF、LEF-1mRNA的相对表达水平;(E)ALP染色显示,在慢病毒介导的Ap-2β过表达和敲低方式下,顶骨、额骨成骨细胞有成骨活性差异;(F-G)Western blot和Image J显示,在慢病毒介导的Ap-2β过表达和敲低时,顶骨、额骨细胞蛋白量的变化。P*<0.05,p**<0.01,p***<0.001。
具体实施方式
为使本发明的技术方案和有益效果能够更加明显易懂,下面通过列举具体实施例的方式进行详细说明。其中,附图不一定是按比例绘制的,局部特征可以被放大或缩小,以更加清楚的显示局部特征的细节;除非另有定义,本文所使用的技术和科学术语与本申请所属的技术领域中的技术和科学术语的含义相同。
第一方面,本发明提供了一种Ap-2β基因在制备诊断和/或治疗成骨细胞活性调控相关病症的试剂中的应用。
在某些实施方案中,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
在某些实施方案中,所述应用选自:
(a)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的制备;
(b)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的筛选;或
(c)Ap-2β基因作为靶标应用于制备诊断成骨细胞活性调控相关病症的试剂。
第二方面,本发明还提供了一种用于辅助诊断成骨细胞活性调控相关病症的试剂盒,其特征在于,所述试剂盒包括Ap-2β基因的检测试剂。
在某些实施方案中,所述检测试剂检测所述Ap-2β基因的表达量。
在某些实施方案中,所述Ap-2β基因的检测试剂是本领域已知的任何可用于检测蛋白质表达水平的试剂。
在某些实施方案中,所述试剂盒采用的检测方法为Western Blot、荧光定量PCR。
在某些实施方案中,所述试剂盒包括全蛋白提取试剂、PCR试剂、Western Blot检测试剂、RNA抽提试剂、荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针。
在某些实施方案中,所述试剂盒还包括样品处理剂、如样品裂解试剂、样品纯化试剂及核酸提取试剂等。
第三方面,本发明还提供了一种Ap-2β基因激活剂或调节剂在制备治疗成骨细胞活性调控相关病症中的应用,其特征在于,所述Ap-2β基因激活剂或调节剂为可调控Ap-2β基因表达水平的试剂。
在某些实施方案中,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
第四方面,本发明还提供了一种Ap-2β基因激活剂或调节剂在制备治疗颅骨发育相关病症的药物中的应用。
实验1:CCK-8检测细胞增殖
为了解Ap-2β转录因子在细胞增殖中的作用,采用CCK-8法评估Ap-2β对细胞增殖的影响。
实验步骤:
实验前一天在96孔板中按照5×104细胞密度铺板,保证第二天实验细胞密度达到60%-70%。实验当天待细胞密度达到所需量时,从培养箱中拿出细胞,在每孔中加入10μLCCK-8溶液。可以用加了相应量细胞培养液和CCK-8溶液但没有加入细胞的孔作为空白对照。由于96孔板较易蒸发,故采取弃用周围一圈的办法,改加相同量的PBS以缓解蒸发。在CO2培养箱内继续孵育1h后在450nm测定吸光度。记录数据利用GraphPad绘制折线图。
实验2:细胞ALP染色
探究体外成骨分化中Ap-2β在细胞分化的作用时,碱性磷酸酶(ALP)的活性是评价分化成骨细胞活性的关键因素。
实验步骤:
从培养箱中取出分化D7天的成骨细胞系,用移液枪轻轻吸去培养基,用常温PBS清洗残留培养基3次,随后加入适量4%多聚甲醛固定30min。固定后完成用PBS洗3次。
ALP工作液配制:每5mL ddH2O中加入96μL Vector Reagent 1,96μL VectorReagent 2,54μL Vector Reagent 3。现用现配,未用完的弃掉。将配置好的工作液沿壁适量加入细胞孔中,使其完全覆盖细胞。将细胞板放入暗室避光孵育30min,在中途检查是否挥发过多,若挥发则及时补充ALP染液。染色完成后弃掉染液,用ddH2O清洗3遍。倒扣沥干水分,在显微镜下观察并拍照记录。
实验3:稳转株细胞EdU染色
EdU是胸苷的核苷类似物,可以在S期细胞周期合并到DNA中。荧光染料可以与EdU发生“Click”化学反应(点击法),从而将增殖的细胞标记上荧光。可用于细胞成像、流式、细胞示踪、DNA损伤修复检测、线粒体活性检测以及病毒增殖活性检测等。
实验步骤:
在6孔板中培养适当数量的细胞,保证第二天细胞密度达到70%-80%。细胞培养过夜并且恢复到正常状态后,进行所需的药物处理。将37℃预热的2×EdU工作液等体积加入6孔板中,使EdU在培养液中的终浓度为10μM。如果培养基体积过大,可以先吸除适量的培养液,更换所有的培养液可能会对细胞的增殖有影响。将细胞放入CO2培养箱继续孵育2h。该孵育时间的长短取决于细胞生长速率,通常宜继续孵育细胞周期10%左右的时间。EdU标记细胞完成后,去除培养液,加入1mL 4%多聚甲醛室温固定15min。去除固定液,每孔用1mL含3% BSA的PBS洗涤细胞3次,每次5min。随后用含0.3% TritonX-100的PBS通透液孵育15min。配置Click反应液,每孔加入0.5mL,轻轻摇晃培养板以确保反应混合物可以均匀覆盖样品,室温避光孵育30min。吸除反应液,用洗涤液洗3次,每次5min。后用DAPI染液染细胞核30min。弃DAPI染液,用PBS洗3次每次5min,最后用ddH2O洗1次5min。沥干水分,在室温下晾干。在荧光显微镜下利用紫外和绿光激发DAPI和EdU,观察实验结果并记录。
实验4:蛋白免疫印迹
4.1细胞全蛋白提取
实验步骤:
从培养箱中取出分化D0天和D7天的成骨细胞,用预冷的PBS洗3次。配置总蛋白裂解液:取适量RIPA裂解液与1.5mL离心管中,并加入磷酸酶抑制剂和蛋白酶抑制剂,最后加入PMSF使其终浓度为1mM。用1mL枪头尽量吸去细胞板中的PBS,10cm dish中加入200μL的蛋白裂解液,用细胞刮轻刮下细胞,转移到1.5mL离心管中,在冰上裂解30min。在低温高速离心机中12000rpm,4℃,15min离心,取上清即为细胞总蛋白,注意不要吸到底部沉淀。将样品保存在-80℃。
4.2蛋白免疫印迹
实验步骤:
配置聚丙烯酰胺凝胶:加入少量洗涤剂清洗制胶用的玻璃板,将薄板和厚板底部对齐卡到制胶架子中,用ddH2O捡漏。用配胶试剂盒在15mL离心管中配置分离胶,用移液器将分离胶加到玻璃板至顶端1.5cm-2cm处,用ddH2O封胶,静置大约30min后分离胶凝固。用滤纸将分离胶上面的ddH2O吸干,再用配胶试剂盒配置浓缩胶,均匀加到玻璃板中,插上梳子避免产生气泡,室温放置待中间出现明显界限即可,将玻璃板放入电泳槽中,直立将梳子拔出,并加入1×SDS电泳缓冲液。
蛋白电泳:按照一定顺序上等量的蛋白样品,并在第一个孔里加5μL蛋白Marker,先用80V恒压把样品在浓缩胶压成一条直线,待Marker到分离胶时改为120V恒压。根据Marker的位置来确定蛋白分离的程度,以最大化的分离目标蛋白。
转膜:提前将1×转膜液预冷,并将PVDF膜在甲醇中浸泡10min活化,然后在转膜液中平衡膜直到没有气泡。将转膜用海绵、厚滤纸在转膜液中充分浸湿。电泳结束后,取出凝胶割掉上层浓缩胶,按海绵垫、滤纸、蛋白凝胶、PVDF膜、滤纸、海绵垫的顺序依次放在转膜夹板上,用滚轮将PVDF膜和蛋白凝胶之间的气泡推离,夹紧转膜板并扣住,注意正负极放入转膜槽中,倒入1×转膜液使夹板浸没。将整个转膜槽放置于大冰盒中,周围用冰填充,120V,恒压转膜2h。
封闭:转膜完成后小心取出PVDF膜。在TBST中清洗3次每次5min,用TBST配置5%的脱脂奶粉溶液即为封闭液,将PVDF膜在封闭液中室温封闭1.5h。弃封闭液,将膜放在摇床上用TBST洗3次每次5min,用一抗稀释液配置所需的一抗,比例为1:1000,并加入终浓度为0.02%的NaN3,在4℃摇床中轻微摇晃孵育过夜。第二天回收一抗,用TBST洗3次每次10min。用二抗稀释液配置与一抗种属匹配的二抗,比例1:5000,在室温下孵育1h。弃二抗,用TBST洗3次每次5min。
显影:将等量的ECL发光液A和B混匀,缓慢滴加在PVDF膜上,用超灵敏化学发光成像仪曝光,曝光时间根据蛋白调整,直到扫出清晰的条带,并保留带有Marker的白光图,将条带和Marker合并。最后根据结果分析蛋白表达情况,使用Image J进行灰度分析。
实验5:荧光定量PCR
5.1细胞总RNA抽提
实验步骤:
在超净台中使用核酸酶清除试剂喷洒后灭菌30min,准备好总RNA抽提时所需的RNase free环境。吸净细胞上清培养液,用预冷的PBS沿壁轻缓冲洗细胞3遍。在10cm dish中加入1mL TRIzol并反复吹散细胞,室温放置5min使细胞充分裂解释放RNA。随后将其转移至RNase free的1.5mL离心管中。在管内加入200μL氯仿,用力盖紧并在漩涡振荡器上剧烈振荡混匀15s,室温静置3min。放入4℃预冷的离心机中12000rpm离心15min。离心完成后取出离心管放入冰盒中,小心吸取上层清澈液约400μL到新的离心管中,注意枪头不能吸到液体中间层及下层沉淀物。在管中加入等量400μL的异丙醇,盖紧后上下颠倒混匀10次,在冰上静置15min。接着于4℃离心机中12000rpm离心15min后取出。弃置上清,可看到管底有微量白色胶状沉淀附着。加入1mL DEPC水配置的75%乙醇,用枪头冲打洗涤沉淀。重复此步骤漂洗一次。12000rpm,4℃,15min离心。弃上清,倒扣沥干水分并放置通风处干燥直至离心管中无水分。加入30μL DEPC水反复吹打促进溶解沉淀。用紫外分光光度计测定RNA纯度与浓度,将数值记录在管壁于-80℃保存。
5.2荧光定量PCR
荧光定量PCR是指在PCR扩增反应体系中加入荧光基团,通过对扩增反应中每一个循环产物荧光信号的实时检测,最后通过标准曲线对未知模板进行定量分析的方法。
实验步骤:
根据逆转录试剂盒说明,配置第一步混合液,轻轻吹打混匀,在微量离心机中离心后放于PCR仪42℃孵育2min,以去除残留基因组DNA。在第一步的反应管中直接加入5μL4×ⅢSuperMix plus,轻轻吹打混匀,放入PCR仪进行逆转录程序,产物即为cDNA。用cDNA、各基因所需引物和荧光定量PCR试剂盒,在冰盒中配置反应体系,配置完取20μL混合液加入到荧光定量PCR 96孔板中。加样时注意避免产生气泡,加完后瞬离。进入ABI 7500荧光定量PCR仪,采用两步法扩增目的基因。设置好反应程序,将反应结束后获取的Ct值根据2-ΔΔCt计算得出mRNA的相对含量,通过GraphPad绘制数据结果图。
实验6:核质分离实验
实验步骤:
从培养箱中取出分化7天的稳转细胞,倒弃培养液,用预冷的PBS洗涤3次,加入500μLBuffer 1裂解液。立即刮下细胞,转移至1.5mL离心管中。用1mL针头反复抽打20次,然后置于冰上裂解20min 3000rpm,4℃离心10min,上清是粗制的胞浆蛋白组分,沉淀为核粗提物。
胞浆组分制备:将上清转入另一预冷的离心管,8000rpm,4℃离心10min,弃去沉淀,取上清即为胞质蛋白组分。
核粗提物处理:取步骤3中核粗提物沉淀,加入250μL Buffer 2裂解液,用1mL针头反复抽打20次,冰浴1min,用离心机3000rpm,4℃离心10min。
核蛋白制备:弃置步骤5中上清液,向离心沉淀物中加入250μL Buffer 3裂解液,混匀后冰浴超声破碎5次,每次3s。用离心机12000rpm,4℃离心10min。取上清,即得到细胞核蛋白样品。
实验7:TOPFlash报告基因实验
实验步骤:
第一天下午四点左右铺板:以六孔板为例,按照每孔5×105细胞数铺板,使第二天细胞密度达到70%-80%左右(Lipo 8000转染时细胞密度70%-80%为佳)。同时设置阳性和阴性对照。
第二天上午九点左右瞬转:对于每孔细胞,在离心管中依次配置125μL Opti-MEM、2.5μg质粒DNA、4μL Lipo 8000转染混液。混匀并加入每孔细胞。培养6h后换成α-MEM全培液(可选:6h后换成无血清培养基饥饿12h)。利用翊圣双荧光素酶报告基因检测试剂盒检测荧光表达量。对于贴壁细胞,先吸去上清培养液,再用预冷的PBS冲洗3遍,最后加入500μL裂解液。冰上孵育5min,充分裂解细胞。裂解完成后用高速离心机12000rpm离心1min,收集上清,即为蛋白裂解液。配置萤火虫荧光素酶底物,即按照所需检测量,将50×的萤火虫荧光素酶底物用底物稀释液稀释至1×。取100μL稀释后的底物加入到黑色酶标板中,同时设置3-6个重复。取20μL蛋白裂解液加入每个复孔中,室温孵育5min后即可使用紫外分光光度仪检测荧光表达量(检测需在30min内完成)。
实验8:细胞爬片免疫荧光
实验步骤:
第一天:在培养板中将已爬好细胞的玻片用PBS浸洗3次,每次5min。用4%多聚甲醛固定爬片15min,PBS浸洗玻片3次,每次5min。用0.3% Triton X-100(PBS配制)室温通透20min。PBS浸洗玻片3次,每次5min,吸水纸吸干PBS,在玻片上滴加正常山羊血清,室温封闭30min。吸水纸吸掉封闭液,不洗,每张玻片滴加足够量1:1000稀释好的一抗并放入湿盒,4℃孵育过夜。
第二天:加荧光二抗,PBST浸洗爬片3次,每次5min,吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗,湿盒中室温孵育1h,PBST浸洗切片3次,每次5min(注意:从加荧光二抗起,后面所有操作步骤都尽量在较暗处进行)。
复染核:滴加DAPI避光孵育5min,对标本进行染核,PBST 5min×4次洗去多余的DAPI。用吸水纸吸干爬片上的液体,用含抗荧光淬灭剂封片液封片,然后在荧光显微镜下观察采集图像。
实验9::头颅组织冰冻切片
实验步骤:
将固定完储存在甲醇中的组织取出,用自来水冲洗。将组织浸入用PBS配置的30%蔗糖中脱水,在4℃中过夜,直至组织沉底。组织按需求以横切面或纵切面方式放在模具中,并加入OCT包埋剂使组织浸没,平放进-80℃冰箱迅速冷却凝固。凝固后取出进入冰冻切片机,在样本托上涂适量OCT包埋剂,迅速放上冰冻组织,在速冻架上静置10min。
切片:样本固定在样品托上后,固定到标本台,调整好切片角度。安装预冷刀片,调整刀台位置和角度,开始匀速切片。
收片:冰冻切片分成A、B、C和D 4个平行组,按顺序依次收片,每片载玻片上收6-8次,标记。放入-20℃冰箱长期保存。
实验10:小鼠顶骨、额骨原代细胞培养
实验步骤:
将孕期为18.5天的母鼠断颈处死。高温高压灭菌手术器械,用剪刀小心剖开孕肚,在体视镜下取出E18.5天胚胎小鼠,随后用PBS将血液冲洗干净,用精细镊子剥开头皮,用手术剪刀沿着冠状缝和矢状缝边缘剪下两块顶骨和额骨,并轻轻剥去颅骨内外两侧的骨膜和结缔组织,放于无血清的α-1640培养液中,标记好Pb与Fb,放置于冰上,全部剔除完后立即转移至超净台。在超净台中取灭菌过的1.5mL离心管,加入1mLα-1640培养液,用镊子夹取小鼠头盖骨上下反复清洗5次,每次都要换新的离心管和α-1640培养液,转入装有200μL酶解液的离心管底部,保证充分消化,然后在37℃,CO2培养箱中消化20min。20min后取出,用1mLα-1640培养液清洗一次,再转到另一新的有200μL酶解液的1.5mL离心管中,在37℃,CO2培养箱中消化30min(前两次酶解出来的细胞舍弃)。30min后取出,用1mLα-1640培养液再清洗一次,转到装有200μL酶解液的1.5mL离心管底部,用钝头剪将小鼠颅骨剪碎至无大块骨片,在37℃,CO2培养箱中消化90min。90min后取出,在离心管中加入1mLα-MEM全培液,吹打3s,静置60s后,将上层悬浮液转移到新的15mL离心管中。用离心机4℃,1000rpm离心10min。弃上清,加2mL新鲜α-MEM全培液,上下轻轻吹打细胞底部使其完全悬浮,并吸取10μL细胞悬液到血球计数板中,在显微镜下进行细胞计数。按照实验需要将一定数量的细胞铺于6孔/12孔板中,并在37℃,CO2培养箱中培养。过夜后,全部活细胞都将贴壁,随后可以进行常规细胞培养操作。
实施例1:Ap-2β过表达和下调在成骨细胞中的验证结果
蛋白免疫印迹的结果证实了Ap-2β蛋白在包括颅骨和肢体在内的骨骼组织中高度表达(图1A),这表明Ap-2β在调节颅骨发育中具有潜在的作用,但Ap-2β在颅骨中的具体功能尚不清楚。为了表征Ap-2β在成骨细胞中的功能,本申请使用慢病毒载体系统在成骨祖细胞MC3T3-E1细胞中高效地过表达Ap-2β(Ap-2β组)和用shRNA转导敲除Ap-2β(shAp-2β组)。用shRNA control慢病毒颗粒转导的成骨细胞被指定为阴性对照(Scramble组)。通过qPCR分析(图1B)和Western blot分析(图1C)检测MC3T3-E1细胞中Ap-2β在基因和蛋白上的相对表达水平,数据显示Ap-2βmRNA充分过表达超过2倍,下调量超过80%。蛋白表达量在Ap-2β组中具有上升趋势,在shAp-2β组中明显下降。
实施例2:Ap-2β对成骨细胞增殖和凋亡的影响
CCK-8检测数据显示,下调的Ap-2β在处理48h后显著抑制了成骨细胞的细胞数量(图2A),而过表达Ap-2β在处理48h后显著促进了成骨细胞的细胞数量(图2B)。在细胞增殖过程中,Cyclin D1被证明是细胞周期G1期的调节因子。增殖细胞核抗原(PCNA)是DNA“夹子”,是DNA复制的正向因子。数据显示,在Ap-2β过表达的成骨细胞中,Cyclin D1和PCNA显著上调(图2C-D),而Ap-2β在成骨细胞中的下调导致Cyclin D1和PCNA的表达显著降低。EdU检测显示,EdU+细胞在过表达Ap-2β的成骨细胞中显著分布,而在成骨细胞中下调Ap-2β会导致EdU+成骨细胞减少(图2E-F),这些数据表明Ap-2β是成骨细胞增殖过程中S期、G1期和DNA复制的正向调节因子。
为了探究增殖减少是否由于凋亡的增加,本申请评估了凋亡相关蛋白,如凋亡调节因子Bax,它是促凋亡的因子,而Bcl-2具有抗凋亡的特征。数据显示,Ap-2β下调成骨细胞中Bax升高,Bcl-2降低,而Ap-2β过表达成骨细胞中Bax活性降低,Bcl-2活性升高(图2C-D、G),Bax和Bcl-2基因表达水平及其比值也与蛋白活性一致。这些数据表明Ap-2β在一定程度上通过调节Bax和Bcl-2相互作用的活性来调控细胞凋亡。
实施例3:Ap-2β对成骨细胞分化的影响
通过ALP细胞染色实验用于观察成骨细胞分化活性,数据显示,shAp-2β成骨细胞的成骨分化受损,过表达Ap-2β显示成骨细胞分化活性增加(图3A)。在转录水平上,RUNX2和Sp7被报道为驱动成骨细胞分化的主要调节因子。研究发现,Ap-2β可显著提高分化阶段RUNX2和Sp7的水平(图3B-C),而Ap-2β的下调可导致RUNX2和Sp7活性的显著降低。成骨细胞特异性标志物如ALP、COL1A1、RUNX2和Sp7在Ap-2β敲低组中并分化D7和D14天时,可以观察到它们的含量显著降低(图3D-G)。以上数据表明,Ap-2β能够促进成骨分化。
实施例4:Ap-2β通过刺激Wnt信号通路调控成骨细胞分化
为了了解Ap-2β调控成骨细胞分化的分子机制,本申请测试了几种由DEGs聚类的信号通路。结果表明,过表达Ap-2β的总activeβ-catenin在D7时被强烈激活,且差异显著(图4A-B)。
Ap-2β的过表达增强了成骨细胞中activeβ-catenin的核转运,而Ap-2β的下调导致成骨细胞中activeβ-catenin的核转运减少(图4C),这表明成骨细胞中Ap-2β可以激活Wnt/β-catenin信号通路。但在D7过表达Ap-2β的成骨细胞中,Wnt/β-catenin信号GSK-3β的下游成分和磷酸化的GSK-3β没有改变(图4D)。这提示我们Ap-2β可能不是通过Wnt信号通路降解途径实现调控的。
此外,Ap-2β敲除的成骨细胞在D7和D14时Wnt/β-catenin靶基因TCF、C-MYC、CD44和LEF-1的表达也显著降低(图4E-F)。Ap-2β敲低的模型下,Wnt信号通路下游抑制因子SOST、DKK1、DKK2等表达明显升高,说明Wnt信号通路的激活部分是通过调控其抑制因子(图4G)。
为了检测MC3T3-E1成骨细胞中Wnt/β-catenin信号通路的转录水平是否依赖于Ap-2β,本申请使用TOPFlash双荧光素酶报告检测系统评估TCF/LEF的转录活性。结果显示,过表达Ap-2β可显著提高成骨细胞系中β-catenin/TCF/LEF报告基因荧光素酶活性约1.9倍。同时,Ap-2β敲低成骨细胞中的荧光素酶活性明显受到抑制(图4H)。使用LiCl(20mM)对过表达Ap-2β的成骨细胞进行Wnt信号通路的药物激活,荧光素酶活性与未LiCl处理的Ap-2β过表达成骨细胞相比显著增加,但β-catenin/TCF/LEF报告细胞的荧光素酶活性在Ap-2β下调的成骨细胞中无论是否使用LiCl处理都有显著差异。这些数据表明,Ap-2β通过激活Wnt/β-catenin信号通路刺激成骨分化。
实施例5:Ap-2β对额骨和顶骨成骨细胞的区域成骨潜能的影响
在额骨和顶骨中,Ap-2β+免疫信号在额骨组织中大量分布,但在顶骨中Ap-2β+免疫信号相对较少(图5A),说明Ap-2β在额骨组织中具有较高的表达量。额骨、顶骨组织的WB分析也证实了它们相同的表达模式(图5B-C)。
Ap-2β在额骨和顶骨成骨细胞中具有调节潜能,其存在不同信号通路共调控。通过培养额骨成骨细胞和顶骨成骨细胞,利用慢病毒介导Ap-2β在额骨和顶骨成骨细胞中过表达和下调,以及分析Wnt/β-catenin信号通路靶基因的表达,结果表明,与顶骨成骨细胞相比,Ap-2β在额骨成骨细胞中表达较高,而在额骨成骨细胞中下调Ap-2β会导致其表达降低。Wnt/β-catenin信号通路下游靶基因TCF、LEF-1、Cnx-43、CCND-1在Ap-2β过表达的顶骨成骨细胞中高表达,而在Ap-2β敲低的额骨成骨细胞中,TCF、LEF-1、Cnx-43、CCND-1显著降低(图5D),提示Ap-2β可诱导原代成骨细胞中Wnt/β-catenin靶基因表达。
在正常额叶成骨细胞中,ALP细胞染色显示额叶成骨细胞比顶骨成骨细胞具有更强的成骨潜能,且额叶成骨细胞中慢病毒介导的Ap-2β下调导致成骨细胞活性降低,而顶骨成骨细胞中慢病毒介导的Ap-2β过表达导致顶骨成骨细胞活性增加(图5E),提示Ap-2β具有可以调节额叶和顶骨成骨细胞的区域成骨潜能。此外,本申请还证实了β-catenin、Runx2、Sp7和Ap-2β在额骨和顶骨成骨细胞中呈Ap-2β依赖性表达(图5F-G),这再次表明Ap-2β通过调控Wnt/β-catenin信号通路调控额骨和顶骨区域成骨活性。
应当理解,以上实施例均为示例性的,不用于包含权利要求所包含的所有可能的实施方式。在不脱离本公开的范围的情况下,还可以在以上实施例的基础上做出各种变形和改变。同样的,也可以对以上实施例的各个技术特征进行任意组合,以形成可能没有被明确描述的本发明的另外的实施例。因此,上述实施例仅表达了本发明的几种实施方式,不对本发明专利的保护范围进行限制。
Claims (10)
1.一种Ap-2β基因在制备诊断和/或治疗成骨细胞活性调控相关病症的试剂中的应用。
2.根据权利要求1所述的应用,其特征在于,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
3.根据权利要求1所述的应用,其特征在于,所述应用选自:
(a)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的制备;
(b)Ap-2β基因作为靶标应用于治疗成骨细胞活性调控相关病症的药物的筛选;或
(c)Ap-2β基因作为靶标应用于制备诊断成骨细胞活性调控相关病症的试剂。
4.一种用于辅助诊断成骨细胞活性调控相关病症的试剂盒,其特征在于,所述试剂盒包括Ap-2β基因的检测试剂。
5.根据权利要求4所述的试剂盒,其特征在于,所述检测试剂检测所述Ap-2β基因的表达量。
6.根据权利要求5所述的试剂盒,其特征在于,所述试剂盒采用的检测方法为WesternBlot、荧光定量PCR。
7.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒包括全蛋白提取试剂、PCR试剂、Western Blot检测试剂、RNA抽提试剂、荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针。
8.一种Ap-2β基因激活剂或调节剂在制备治疗成骨细胞活性调控相关病症中的应用,其特征在于,所述Ap-2β基因激活剂或调节剂为可调控Ap-2β基因表达水平的试剂。
9.根据权利要求8所述的应用,其特征在于,所述成骨细胞活性调控相关病症包括由于成骨细胞活性降低导致的骨质疏松或骨折修复损伤等。
10.一种Ap-2β基因激活剂或调节剂在制备治疗颅骨发育相关病症的药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310372291.6A CN116676378A (zh) | 2023-04-10 | 2023-04-10 | Ap-2β基因在成骨细胞活性调控中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310372291.6A CN116676378A (zh) | 2023-04-10 | 2023-04-10 | Ap-2β基因在成骨细胞活性调控中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116676378A true CN116676378A (zh) | 2023-09-01 |
Family
ID=87784311
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310372291.6A Pending CN116676378A (zh) | 2023-04-10 | 2023-04-10 | Ap-2β基因在成骨细胞活性调控中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116676378A (zh) |
-
2023
- 2023-04-10 CN CN202310372291.6A patent/CN116676378A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109136174B (zh) | 一种延缓衰老的干细胞来源外泌体制剂 | |
Parreno et al. | Tropomyosin 3.1 association with actin stress fibers is required for lens epithelial to mesenchymal transition | |
Yu et al. | ERBB2 gene expression silencing involved in ovarian cancer cell migration and invasion through mediating MAPK1/MAPK3 signaling pathway. | |
CN108570453A (zh) | 一种慢病毒过表达载体介导的e6-ap促进结肠癌细胞生长和转移的实验方法 | |
CN107217060B (zh) | Kdm1a的用途 | |
CN116676378A (zh) | Ap-2β基因在成骨细胞活性调控中的应用 | |
CN101705227B (zh) | 抑制人AP-2alpha基因表达的siRNA在抗宫颈癌药物制备中的应用 | |
CN113209312B (zh) | 一种抑制转录因子mef2c表达的试剂在制备治疗瘢痕疙瘩的药物中的应用 | |
CN116115759A (zh) | 联合抑制nat10/kif23的物质在制备防治结直肠癌药物中的应用 | |
CN114134145A (zh) | Hoxc10在胃癌发病机制中的作用 | |
CN113925972A (zh) | Otub1蛋白用于治疗骨质疏松症的应用 | |
CN112877425A (zh) | 检测snf5基因表达的试剂在制备动脉粥样硬化诊断产品中的应用 | |
CN114134147B (zh) | 一种调控fzd9的非编码rna及其应用 | |
CN112656805A (zh) | 抑制ythdf1活性的物质在制备预防或治疗胃癌产品中的应用 | |
CN114574583B (zh) | Tmc5在乳腺癌特异性骨转移中的诊断、治疗的应用 | |
CN107881240A (zh) | 骨肉瘤的诊治标志物 | |
CN116426469B (zh) | LAP2α在间充质干细胞成脂向分化中的应用 | |
Cao et al. | Astragaloside-IV induces the differentiation of bone marrow mesenchymal stem cells into osteoblasts through NMUR2-mediated Wnt/β-catenin pathway | |
CN110201145B (zh) | Epha7在制备促排卵剂中的应用 | |
CN117987526A (zh) | Nf2基因在缺损颅骨修复再生中的用途 | |
CN114134146A (zh) | 一种长链非编码rna及其应用 | |
CN115873804A (zh) | Bhlhe40基因在调控骨髓间充质干细胞增殖、分化和衰老中的应用 | |
CN117618570A (zh) | eIF6基因及其蛋白在制备调控皮肤组织创面愈合药物中的应用 | |
CN104083775B (zh) | 磷酸化dcbld2y750在诊断和治疗神经胶质瘤中的应用 | |
CN116430048A (zh) | BTF3L4基因及其RNAi干扰系统的应用 |
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 |