CN117398404B - Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases - Google Patents
Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases Download PDFInfo
- Publication number
- CN117398404B CN117398404B CN202311724273.6A CN202311724273A CN117398404B CN 117398404 B CN117398404 B CN 117398404B CN 202311724273 A CN202311724273 A CN 202311724273A CN 117398404 B CN117398404 B CN 117398404B
- Authority
- CN
- China
- Prior art keywords
- mirna
- mir
- interleukin
- periodontitis
- chemokine receptor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 201000001245 periodontitis Diseases 0.000 title claims abstract description 47
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 22
- 239000003814 drug Substances 0.000 title claims abstract description 22
- 229940079593 drug Drugs 0.000 title abstract description 6
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 230000003278 mimic effect Effects 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 5
- 108091081021 Sense strand Proteins 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- 239000012454 non-polar solvent Substances 0.000 claims description 4
- 239000002798 polar solvent Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 239000011230 binding agent Substances 0.000 claims description 2
- 239000003086 colorant Substances 0.000 claims description 2
- 239000007884 disintegrant Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000008393 encapsulating agent Substances 0.000 claims description 2
- 239000010685 fatty oil Substances 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000000796 flavoring agent Substances 0.000 claims description 2
- 235000013355 food flavoring agent Nutrition 0.000 claims description 2
- 229940057995 liquid paraffin Drugs 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 230000000153 supplemental effect Effects 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 239000000080 wetting agent Substances 0.000 claims description 2
- 108091058688 miR-141 stem-loop Proteins 0.000 abstract description 73
- 102000004890 Interleukin-8 Human genes 0.000 abstract description 62
- 108090001007 Interleukin-8 Proteins 0.000 abstract description 62
- 108010018976 Interleukin-8A Receptors Proteins 0.000 abstract description 59
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 abstract description 59
- 229940096397 interleukin-8 Drugs 0.000 abstract description 59
- 102000001417 CXC chemokine receptor 1 Human genes 0.000 abstract description 57
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 abstract description 47
- 238000002474 experimental method Methods 0.000 abstract description 14
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- 239000005482 chemotactic factor Substances 0.000 abstract description 5
- 208000028169 periodontal disease Diseases 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000003334 potential effect Effects 0.000 abstract description 2
- 230000001276 controlling effect Effects 0.000 abstract 1
- 210000002950 fibroblast Anatomy 0.000 description 79
- 210000004027 cell Anatomy 0.000 description 54
- 108090000623 proteins and genes Proteins 0.000 description 42
- 108020004999 messenger RNA Proteins 0.000 description 41
- 102000004169 proteins and genes Human genes 0.000 description 38
- 239000002158 endotoxin Substances 0.000 description 35
- 229920006008 lipopolysaccharide Polymers 0.000 description 35
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 34
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 34
- 230000000638 stimulation Effects 0.000 description 34
- 210000004195 gingiva Anatomy 0.000 description 31
- 150000004032 porphyrins Chemical class 0.000 description 26
- 239000000243 solution Substances 0.000 description 26
- 230000008859 change Effects 0.000 description 21
- 239000000523 sample Substances 0.000 description 21
- 239000006228 supernatant Substances 0.000 description 21
- 238000001890 transfection Methods 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 230000003827 upregulation Effects 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 13
- 239000002609 medium Substances 0.000 description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 238000001514 detection method Methods 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 210000000988 bone and bone Anatomy 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 239000006180 TBST buffer Substances 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 230000003828 downregulation Effects 0.000 description 6
- 230000002757 inflammatory effect Effects 0.000 description 6
- 238000007747 plating Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 5
- 238000012286 ELISA Assay Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 208000027866 inflammatory disease Diseases 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 230000003239 periodontal effect Effects 0.000 description 4
- VCRBUDCZLSQJPZ-UHFFFAOYSA-N porphyrinogen Chemical compound C1C(N2)=CC=C2CC(N2)=CC=C2CC(N2)=CC=C2CC2=CC=C1N2 VCRBUDCZLSQJPZ-UHFFFAOYSA-N 0.000 description 4
- 208000006386 Bone Resorption Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102100036154 Platelet basic protein Human genes 0.000 description 3
- 101710195957 Platelet basic protein Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000037182 bone density Effects 0.000 description 3
- 230000024279 bone resorption Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000003399 chemotactic effect Effects 0.000 description 3
- 208000007565 gingivitis Diseases 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 210000002050 maxilla Anatomy 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000605862 Porphyromonas gingivalis Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 238000010603 microCT Methods 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000003254 palate Anatomy 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000008844 regulatory mechanism Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108091008928 CXC chemokine receptors Proteins 0.000 description 1
- 102000054900 CXCR Receptors Human genes 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000015617 Janus Kinases Human genes 0.000 description 1
- 108010024121 Janus Kinases Proteins 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091027977 Mir-200 Proteins 0.000 description 1
- -1 NF-KB Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009194 climbing Effects 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
- 238000001816 cooling Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008289 pathophysiological mechanism Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 201000011461 pre-eclampsia Diseases 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 210000004357 third molar Anatomy 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
Abstract
The application relates to the technical field of biological medicines, and particularly discloses application of miRNA-141-5p in preparation of a medicine for treating periodontitis diseases. The application proves that miRNA-141-5p can regulate and control the expression of periodontal disease related chemotactic factors CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein, and the mouse experiment also proves that the miR-141-5p mimic has a regulating and controlling effect on the mouse periodontitis disease. Therefore, miRNA-141-5p can be used as a potential action target point of a medicine related to periodontitis treatment, and the discovery of the new function provides a new thought for diagnosis and treatment of periodontitis diseases.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to an application of miRNA-141-5p in preparing a medicine for treating periodontitis diseases.
Background
Periodontitis is a chronic inflammatory disease occurring in periodontal supporting tissues, and is characterized in that plaque is used as an initiating factor, gingivitis gradually progresses along with accumulation of plaque biofilms, and complex interactions with host immune responses are carried out, and if inflammation is not effectively controlled, gingivitis can further progress to periodontitis, and meanwhile, the gingivitis is accompanied with a certain loss of periodontal supporting tissues.
miRNAs play an important role in the development of periodontal disease and in the host immune response. Current studies show that there is a significant difference in the miRNA profile of periodontitis tissue from that detected in healthy tissue. miR-141 is a member of the miR-200 family, and is directly involved in the regulation of various cell signaling pathways and key factors, including transforming growth factor-beta, NF-KB, janus kinase/signal transduction and transcriptional activator, MAPK and the like, and has been found to play an important role in various inflammatory diseases such as rheumatoid arthritis, sepsis, diabetes mellitus, periodontitis and the like.
miRNA-141-5p is a mature body generated by miR-141 after cleavage processing by Dicer ribonuclease in cytoplasm. Some prior researches report that miRNA-141-5p plays an important role in various diseases such as atherosclerosis, preeclampsia, endometriosis, cervical carcinoma and the like by targeting specific mRNAs, but a regulation mechanism based on miRNA-141-5p in periodontitis diseases is rarely reported.
Disclosure of Invention
The regulation and control mechanism of miR-141-5p in inflammatory diseases is explored, so that miRNA-141-5p can regulate and control chemokine CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein related to inflammatory diseases, and miRNA-141-5p can be used as a new target for diagnosis and treatment of inflammatory diseases. The application provides an application of miRNA-141-5p in preparing a medicament for treating periodontitis diseases.
In a first aspect, the application of miRNA-141-5p in preparing a medicament for treating periodontitis diseases adopts the following technical scheme:
the application of miRNA-141-5p in preparing a medicament for treating periodontitis diseases is provided, wherein the sequence of the miRNA-141-5p is shown as SEQ ID NO. 1.
The inventor of the application finds that miRNA-141-5p has a regulating effect on chemotactic factors CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein related to periodontitis diseases. The specific regulation mechanism is as follows: up-regulating miRNA-141-5p, can inhibit CXC chemokine receptor 1, interleukin-8 and the expression of protoplatelet basic protein, and plays a role in inhibiting periodontitis reaction; down-regulating miRNA-141-5p can up-regulate mRNA and protein levels of CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein, and plays a role similar to promoting periodontitis reaction. Therefore, the miRNA-141-5p can be used as a potential action target point of medicines related to periodontitis treatment, and the purpose of relieving or treating periodontitis diseases is realized to a certain extent by promoting the expression and the activity of the miRNA-141-5p.
In the application, miR-141-5p regulates the expression level of CXC chemokine receptor 1 through two ways, namely, inhibits protein translation by directly combining with mRNA targeting of CXC chemokine receptor 1 and regulates the expression level of CXC chemokine receptor 1 through NF- κB pathway. miR-141-5p is also capable of regulating the expression level of the chemokine interleukin-8 through the NF- κB pathway.
In a second aspect, the present application provides the use of an agent that expresses or enhances the function of miRNA-141-5p in the manufacture of a medicament for the treatment of periodontitis.
Optionally, the reagent comprises: a mimetic of miRNA-141-5p, a precursor of miRNA-141-5p, an agonist of miRNA-141-5p, and a vector carrying miRNA-141-5p.
Optionally, the mimetic of miRNA-141-5p comprises a sense strand and an antisense strand, wherein the sequence of the sense strand is shown as SEQ ID NO.1, and the sequence of the antisense strand is shown as SEQ ID NO. 2. The method comprises the following steps:
SEQ ID NO.1（sense）：5'-catcttccagtacagtgttgga-3’
SEQ ID NO.2（antisense）：5'-caacactgtactggaagatgtt-3’
in the application, miRNA-141-5p mimics are injected into mice in a local administration mode, and the administration amount of the miRNA-141-5p mimics is as follows: injections were given 1 time per day, 2 μl each. According to the method, a miRNA-141-5p mimic can be designed according to a miRNA-141-5p sequence, the miRNA-141-5p mimic is chemically synthesized small RNA, and experimental researches show that after the miRNA-141-5p mimic is transferred into a mouse body, the expression of the mouse miRNA-141-5p can be obviously up-regulated, so that the mRNA and protein levels of periodontal disease related chemotactic factors CXC chemokine receptor 1, interleukin-8 and protoplatelet alkaline protein are regulated and controlled, and the purpose of relieving or treating the periodontitis disease of the mouse is realized.
In the present application, the inventors first stimulated human gingival fibroblasts with inflammatory factor (tumor necrosis factor- α) and gingiva porphyrin monospora lipopolysaccharide to construct an inflammation model in which the relative expression amount of mRNA of CXC chemokine receptor 1 in human gingival fibroblasts is 1.5-2.0, the relative expression amount of mRNA of interleukin-8 is 1.8-2.3, and the relative expression amount of mRNA of protoplatelet basic protein is 1.5-2.5. Then, the cell transfection is carried out on the inflammation model for 12 hours by adopting the miRNA-141-5p mimic with the concentration of 20 mu M, and the relative expression amounts of CXC chemokine receptor 1, interleukin-8 and mRNA of protoplatelet basic protein in human gingival fibroblasts are obviously reduced; the relative expression level of mRNA of CXC chemokine receptor 1 is as low as 0.3-0.6, the relative expression level of mRNA of interleukin-8 is as low as 0.2-0.5, and the relative expression level of mRNA of protoplatelet basic protein is as low as 0.4-0.6. Therefore, the miRNA-141-5p mimics provided by the application can regulate and control mRNA and protein levels of periodontal disease related chemotactic factors CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein, and further can relieve or treat periodontitis diseases.
In a third aspect, the present application provides a medicament for treating periodontitis diseases, the medicament comprising an effective amount of miRNA-141-5p.
Optionally, the medicament further comprises a pharmaceutically acceptable carrier.
Optionally, the carrier is a polar solvent and a non-polar solvent; the polar solvent is selected from one or more of water, ethanol, glycerol, propylene glycol and DMSO; the nonpolar solvent is selected from one or two of fatty oil and liquid paraffin.
Optionally, the medicament further comprises a supplemental additive selected from one or more of diluents, buffers, encapsulating agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorption carriers.
Optionally, the medicament is in the form of suspension, emulsion, granule, spray, injection, transdermal absorbent, and is suitable for transfection, tablet, powder, granule or capsule.
In summary, the present application has the following beneficial effects:
the application reveals the close relation between miRNA-141-5p and the occurrence of periodontitis diseases for the first time, and can inhibit the expression of CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein by promoting the expression and activity of miRNA-141-5p so as to achieve the aim of inhibiting periodontitis reaction; in addition, the application also proves that the miR-141-5p mimic has a regulation and control effect on the periodontitis disease of the mice through a mouse experiment. Therefore, the research result of the application provides a pathophysiological mechanism for the occurrence of periodontitis, and miRNA-141-5p can be used as a potential acting target point of medicines related to periodontitis treatment.
Drawings
FIG. 1 shows the expression level of miR-141-5p in human gingival fibroblasts stimulated by different concentrations of inflammatory factors (tumor necrosis factor-alpha);
FIG. 2 shows the mRNA expression and protein expression levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein in human gingival fibroblasts stimulated by inflammatory factor (tumor necrosis factor-. Alpha.) at a concentration of 20 ng/mL;
FIG. 3 shows the expression level of miR-141-5p in human gingival fibroblasts stimulated by different concentrations of porphyromonas gingivalis lipopolysaccharide;
FIG. 4 shows the mRNA expression and protein expression levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet alkaline protein in human gingival fibroblasts stimulated by a 2.5 μg/mL concentration of gingiva porphyrin monospora lipopolysaccharide;
FIG. 5 is a graph of the cytofluorescent protein of miR-141-5p inhibitor after transfection of human gingival fibroblasts;
FIG. 6 shows miR-141-5p expression levels after various times of transfection of human gingival fibroblasts with miR-141-5p mimics;
FIG. 7 shows the mRNA expression levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein after transfection of human gingival fibroblasts with a miR-141-5p mimetic;
FIG. 8 shows the mRNA expression levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein following human gingival fibroblast cells under tumor necrosis factor- α stimulation following upregulation of miR-141-5 p;
FIG. 9 shows the mRNA expression levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein after human gingival fibroblasts are stimulated with the use of gingiva porphyrin monospora lipopolysaccharide after upregulation of miR-141-5 p.
Fig. 10 shows that the distance from the crest of the bone to the cementum boundary is significantly less in the administered group than in the periodontitis group, and the percentage of bone mass and bone density in the region of interest are significantly higher than in the periodontitis group.
The histological results of fig. 11 show that the administration group had lost adhesion, the alveolar bone resorption was less than that of the periodontitis control group, and the inflammatory cells infiltrated in the gingival lesion was less than that of the periodontitis control group.
Detailed Description
The embodiment of the application explores the influence relationship between miRNA-141-5p and periodontal disease related chemotactic factors CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein. It should be noted that the examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods referred to in the examples below are conventional in the art unless otherwise specified. The reagent consumables, the instruments, and the like, which are referred to in the examples below, are commercially available products unless otherwise specified.
The present application is described in further detail below with reference to examples, performance test and accompanying description.
Example 1
The level change of miR-141-5p and related molecules (CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein) in human gingival fibroblasts under the stimulation of inflammatory factors (tumor necrosis factor-alpha) is explored.
Primary culture of human gingival fibroblasts
(1) Primary culture: the gum is obtained from the patients in the oral hospital of Beijing university with the teeth Zhou Ke visit, and meets the requirements of whole body health, crown extension operation, periodontal tissue health of the operation part and the like. Fresh gingival tissue excised during surgery was immediately placed in alpha-MEM medium containing 5% diabody and immediately transferred to a biosafety cabinet. Tissue is organizedThe pieces were immersed in PBS containing 5% diabody for 5min, transferred to a 6cm diameter petri dish, 1mL of a-MEM medium containing 20% FBS and 1% diabody was previously added to the petri dish, the epithelial tissue was removed with a sterilized surgical instrument, and the remaining connective tissue was sheared to a size of about 1mm 3 Evenly spread on the bottom of a dish, medium was added to 2mL, incubated in a cell incubator (37 ℃,5% strength carbon dioxide), and changed every three days. Passage was performed when primary cells were observed to climb out of the tissue and grew 90% of the dish bottom.
(2) Identification of human gingival fibroblasts: when primary cells were cultured to the third generation, cell climbing sheets were prepared, and positive staining was characterized as human gingival fibroblasts according to cell morphology and anti-vimentin.
(3) Cell passaging and plating: passaging was performed when the cells were cultured in the petri dishes to a confluence of 90%. After the original medium was aspirated, the cells were washed three times with sterile PBS, 1mL of trypsin was added, and when the cells were observed under a microscope to be rounded but not dissociated, digestion was stopped by adding 1mL of complete medium (a-MEM medium containing 10% FBS and 1% diabody), gently swirled to cell suspension, and the cell suspension was collected in a 15mL conical tube, centrifuged at room temperature for 5min and at 1000rpm. After centrifugation, the supernatant is discarded, a complete culture medium is added into a conical tube to resuspend cell sediment, the cell sediment is gently and uniformly blown, cell counting is carried out under a microscope, and cell passage or plating is carried out according to a certain proportion as required. The number of human gingival fibroblasts used in the experiment is 3-6.
(4) Freezing and recovering: centrifuging the human gingival fibroblast suspension according to the method (3), pouring out the culture medium, adding 1mL of cell freezing solution to re-suspend cells, blowing and uniformly mixing, transferring the obtained solution to a 2mL cell freezing tube, marking cell names, algebra, freezing time and operators, immediately placing in a refrigerator at-80 ℃, and transferring to liquid nitrogen for freezing after 24 hours. When recovering cells, transferring the solution in the freezing tube to a 15mL conical tube, centrifuging at room temperature for 5min at the rotating speed of 1000rpm, discarding the freezing solution, adding 2mL of complete culture medium to resuspend the cells, taking a cell culture dish with the diameter of 10cm, adding 6mL of complete culture medium, transferring the cell suspension into the culture dish, blowing and mixing uniformly, and placing the cell culture dish in a cell culture box for culturing.
Culturing human gingival fibroblast with culture medium containing inflammatory factor (tumor necrosis factor-alpha)
Tumor necrosis factor-alpha is prepared into a storage solution with the concentration of 0.1 mg/mu L according to the reagent instruction, and frozen at the temperature of-80 ℃. Plating was performed the day before the experiment, cells were seeded in six well plates and the experiment was performed when cell confluence reached 70%. Preparing complete culture medium with tumor necrosis factor-alpha concentration of 0, 15, 20 and 25ng/mL, discarding original culture medium of six pore plates, washing with sterile PBS for three times, adding fresh culture medium containing tumor necrosis factor-alpha, placing in a cell incubator, culturing at constant temperature for 48h, and collecting cells and supernatant for subsequent experiment.
(III) Total DNA extraction from cells
(1) After collecting the supernatant from the six-well plates in Eppendorf tubes, washing three times with PBS, adding 500. Mu.L of TRIzol to each well plate, shaking quickly on a shaker for 10min, and collecting the solution in 1.5mL sterile Eppendorf tubes on ice.
(2) Obtaining an RNA aqueous phase: 200 mu L of chloroform is added into each Eppendorf tube, the tube cover is tightly covered, the mixture is uniformly mixed by shaking, and the mixture is kept stand at room temperature for 2-3min until obvious delamination is achieved, the temperature is 4 ℃, the centrifugation is carried out at 13000rpm for 10min, and the mixture is divided into three layers after the centrifugation: the lower layer is a red organic phenol chloroform layer, the middle layer is a white thin layer, the upper layer is a colorless water sample layer, and the water sample layer is sucked into a new Eppendorf tube.
(3) Obtaining RNA precipitation: adding equal volume of isopropanol, reversing upside down, shaking, mixing, standing at room temperature for 10min, centrifuging at 13000rpm for 15min at 4deg.C, discarding supernatant, and retaining white precipitate at bottom. The pellet was suspended by adding 75% alcohol (diluted with DEPC water) and gently inverting the pellet upside down, centrifuging at 13000rpm for 5min at 4℃and discarding the supernatant, taking care to preserve the bottom RNA pellet. Repeated 2 times.
(4) Dissolving: the RNA precipitate was dried at room temperature to be semitransparent, and 40-60 mu L RNase free water was added according to the amount of precipitate to sufficiently dissolve RNA.
(5) Concentration and purity determination: RNA concentration and purity were determined using NanoDrop, satisfying absorbance values at 260/280 ratios of 1.8-2.0 and 260/230 ratios greater than 1.8 were considered acceptable samples.
(IV) real-time quantitative fluorescence PCR
(1) Reverse transcription of mRNA into cDNA: reverse transcription was performed using ABScript II cDNA First Strand Synthesis Kit, 1. Mu.g of the required RNA volume was calculated from the measured RNA concentration, and each component was added to a 200. Mu.L sterile PCR tube as shown in Table 1 using Nuclease-free H 2 O was added to 20. Mu.L, mixed by shaking, centrifuged at 400rpm at room temperature for 1min, and reverse transcribed using a SimpliAmp PCR thermal cycler, the reaction procedure was: incubation at 37℃for 2min,55℃for 15min, heating at 85℃for 5min to inactivate the enzyme, and incubating at 4 ℃.
(2) Real-time quantitative fluorescent PCR: the PCR reaction system was prepared as shown in Table 2, and the primer sequences of the respective genes are shown in Table 3. Using 96-well plates or eight-row, 3 multiple wells were designed for each predicted gene for each sample, and the wells were centrifuged at 2500rpm for 1min at room temperature, and the 96-well plates were placed in a quantsudio 3 Real-time PCR instrument with the reaction program set to: pre-denaturation at 95℃for 10min, denaturation at 95℃for 15s, annealing at 60℃for 1min, and circulation 40 times.
(3) And (3) calculating and analyzing results: the relative expression level of the target gene in each sample was calculated by a relative quantification method (ΔΔct method).
TABLE 1 reverse transcription System Components and corresponding volumes
TABLE 2 Real-time PCR reaction System Components and corresponding volumes
TABLE 3 human Gene-specific primer sequences
Western Blot (Western Blot)
(1) Cell total protein extraction:
according to protease inhibitors (50×): RIPA lysate = 1:49, and preparing a cell lysate. After collecting the supernatant from the six-well plates in Eppendorf tubes, washing three times with PBS, adding 35-40. Mu.L of cell lysate to each well plate, shaking quickly on a shaker for 20min, and collecting the solution in 1.5mL sterile Eppendorf tubes using a cell spatula.
(2) Sample protein concentration determination
Diluting the BCA protein standard by adopting a gradient dilution method; then, 18 μl of PBS was added to 2 μl of the sample to be tested for dilution of the sample, and 20 μl of PBS, BCA standard diluent, and sample to be tested were sequentially added, each of which was provided with 3 wells. According to the reaction solution A: reaction liquid B was 50:1 preparing BCA working solution, adding 200 mu L of working solution into each hole, mixing uniformly, gently shaking, mixing uniformly, incubating at 37 ℃ for 30min, and measuring the absorbance (OD value) of each hole at 570nm by using an enzyme-labeled instrument. And calculating the final concentration of the sample protein according to the OD value and the standard curve.
(3) Western-blot experiment
According to the molecular weight of the target protein, adding 500mL of electrophoresis buffer (100 mL of 5 Xelectrophoresis solution+400 mL of distilled water), adding 40 mug of prepared protein samples into each sample hole, adding 5 mug of pre-dyeing markers into the front hole and the back hole, and keeping constant pressure of 120-125V until the samples reach the bottom of the separation gel. After electrophoresis, transferring the protein from the gel to the PVDF membrane by adopting a transfer solution at a constant flow of 300A in a cold room at 4 ℃ for 90min, then placing the PVDF membrane in a TBST solution, and sealing for 1.5 hours in a shaking table; PVDF membrane was trimmed, placed in hybridization bags, added with primary antibody (TBST dilution, concentration 1:1000), and incubated overnight at 4℃on a shaker. The PVDF membrane was then washed 3 times with TBST solution for 15min each. Goat anti-rabbit or goat anti-mouse peroxidase-labeled secondary antibody (TBST solution diluted at a concentration of 1:10000) was added, and the mixture was placed on a shaker and incubated at room temperature for 1h. The TBST solution was washed 3 times for 15min each. ECL hypersensitive chemiluminescence liquid is prepared, the ECL hypersensitive chemiluminescence liquid is dripped on a PVDF film, a gel imaging system is used for exposure and color development, and quantitative analysis is carried out on the gray value of a strip by Image J software.
TBST solution: taking 12.11g of Tris and 87.66g of NaCl in a measuring cylinder, and adding distilled water into the measuring cylinder to fix the volume to 1L to obtain TBS solution; then taking 100mL of the TBS solution, adding distilled water into a measuring cylinder to a volume of 1L, adding 1mL of TWEEN-20, and shaking and mixing uniformly to obtain the TBST solution;
electrophoresis liquid: taking 15.1g of Tris, 94g of Glycine and 5g of SDS in a measuring cylinder, adding distilled water into the measuring cylinder to fix the volume to 1L, and oscillating until the solution is fully dissolved to obtain the electrophoresis liquid.
Transfer film liquid: taking 30.2g of Tris and 144.13g of Glycine in a measuring cylinder, adding distilled water into the measuring cylinder to fix the volume to 1L, oscillating until the materials are fully dissolved, and preserving the materials at room temperature to obtain a transfer film liquid (10X); then taking 100mL of the film transfer liquid (10X) in a measuring cylinder, adding 200mL of anhydrous methanol and 700mL of distilled water into the measuring cylinder, and pre-cooling for 1h in a refrigerator at the temperature of minus 20 ℃ to obtain the film transfer liquid;
six ELISA assay (ELISA)
Cell supernatant collection: when the cells are collected, the supernatant is transferred to a 1.5mL sterile Eppendorf tube, centrifuged at 2000-3000rpm for 20min at 4 ℃, and then transferred to a new Eppendorf tube, and frozen in a refrigerator at-80 ℃.
ELISA standards were prepared as shown in Table 4; blank holes (without adding samples and enzyme-labeled reagents), standard holes and sample holes to be tested are respectively arranged, and 3 compound holes are respectively designed. And adding 50 mu L of corresponding standard substances into the standard substance holes on the enzyme-labeled coating plate, adding 40 mu L of sample diluent into the holes of the sample to be detected, then adding 10 mu L of the sample to be detected (the final dilution multiple is 5 times), and gently shaking and uniformly mixing. Placing the ELISA plate in a 37 ℃ incubator for incubation for 30min by using a sealing plate membrane sealing plate, and washing; then 50. Mu.L of the enzyme-labeled reagent was added to each well outside the blank wells. Incubating in a constant temperature box at 37 ℃ for 30min, and washing; after the completion, 50 mu L of the color-developing agent A and 50 mu L of the color-developing agent B are added into each hole, and the mixture is placed in a 37 ℃ incubator for light-shielding reaction for 10min after being gently vibrated and mixed. Then 50. Mu.L of stop solution was added to each well, at which point the solution was observed to change from blue to yellow. OD values were measured for each well at a wavelength of 450nm with a blank Kong Diaoling. This operation should be performed within 15 minutes after the addition of the stop solution.
And drawing a standard curve by using Excel software with the concentration of the standard substance as an abscissa and the OD value as an ordinate, wherein the linear correlation coefficient R value of the standard curve is more than 0.99, substituting the OD value measured by the sample into the standard curve to calculate the corresponding concentration, and the actual concentration of the sample is the measured concentration multiplied by the dilution factor 5. The final results were plotted and analyzed using GraphPad Prism 9 software.
Table 4 ELISA standard preparation
Detection result
(1) Results of detection of changes in levels of miR-141-5 p: the change in levels of miR-141-5p in human gingival fibroblasts stimulated by tumor necrosis factor-alpha at concentrations of 0, 15, 20, 25ng/mL, respectively, is shown in FIG. 1.
The results in FIG. 1 show that downregulation of miR-141-5p was observed at tumor necrosis factor-alpha concentrations of 20ng/mL and 25ng/mL, and that the differences were statistically significant.
(2) Detection of changes in CXC chemokine receptor 1, interleukin-8, and levels of the original platelet basic protein: the change in CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein levels at tumor necrosis factor-alpha stimulation at 20ng/mL, respectively, is shown in FIG. 2.
In FIG. 2, (a) mRNA expression level of CXC chemokine receptor 1 was changed after stimulation of human gingival fibroblasts with tumor necrosis factor- α of 20ng/mL for 48 hours; (b) mRNA expression level of interleukin-8 was changed 48h after stimulation of human gingival fibroblast with tumor necrosis factor-alpha at 20 ng/mL; (c) mRNA expression level of the basic protein of the original platelet after stimulating human gingival fibroblasts for 48 hours by using the tumor necrosis factor-alpha of 20 ng/mL; (d) Gray scale analysis result of CXC chemokine receptor 1 after tumor necrosis factor-alpha of 20ng/mL stimulates human gingival fibroblast for 48 hours; (e) Protein level change of interleukin-8 after tumor necrosis factor-alpha of 20ng/mL stimulated human gingival fibroblast for 48 hours; (f) Protein level change of interleukin-8 after tumor necrosis factor-alpha of 20ng/mL stimulated human gingival fibroblast for 48 hours; (g) Tumor necrosis factor-alpha at 20ng/mL stimulated changes in the levels of the basic protein of the platelet in human gingival fibroblasts after 48 h.
The results in FIG. 2 show that CXC chemokine receptor 1, interleukin-8 and the mRNA of the platelet-derived basic protein and the protein of CXC chemokine receptor 1 are significantly upregulated in human gingival fibroblasts under 20ng/mL tumor necrosis factor- α stimulation, and interleukin-8 and the platelet-derived basic protein in the cell supernatant are also significantly upregulated.
In conclusion, 20ng/mL tumor necrosis factor-alpha stimulation can induce the down regulation of miR-141-5p expression in human gingival fibroblasts, and simultaneously, the levels of mRNA and protein of chemokine receptor CXC chemokine receptor 1, chemokine interleukin-8 and protoplatelet alkaline protein are increased, and miR-141-5p and the chemotactic related molecules show completely opposite reaction trends.
Example 2
The level change of miR-141-5p and chemotactic related molecules in human gingival fibroblasts under the stimulation of gingival porphyrin monospora lipopolysaccharide is explored.
Example 2 was performed as in example 1, except that: step two; procedure of example 2
(II) is specifically as follows
(II) culture medium containing gingiva porphyrin monospore lipopolysaccharide for culturing human gingiva fibroblast
The porphyromonas gingivalis lipopolysaccharide is prepared into a storage solution with the concentration of 1mg/mL according to the instruction book of the reagent, and the storage solution is frozen at the temperature of minus 80 ℃. Plating was performed the day before the experiment, cells were seeded in six well plates and the experiment was performed when cell confluence reached 70%. Preparing complete culture medium with the concentration of 0, 0.1, 1, 2.5 and 5 mug/mL of the gingivalis lipopolysaccharide, discarding the original culture medium of a six-hole plate, washing three times by using sterile PBS, adding fresh culture medium containing the gingivalis lipopolysaccharide, placing the culture medium in a cell culture box, culturing at constant temperature for 48 hours, and collecting cells and supernatant for subsequent experiments.
Detection result
(1) Results of detection of changes in levels of miR-141-5 p: the change in levels of miR-141-5p in human gingival fibroblasts stimulated by the gum porphyrins monospora lipopolysaccharide at concentrations of 0, 0.1, 1, 2.5, 5 μg/mL, respectively, is shown in FIG. 3.
The results of FIG. 3 show that significant downregulation of miR-141-5p was observed at gum porphyrinogen lipopolysaccharide concentrations of 1. Mu.g/mL, 2.5. Mu.g/mL, and 5. Mu.g/mL.
(2) Detection of changes in CXC chemokine receptor 1, interleukin-8, and levels of the original platelet basic protein: the change in CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein levels at concentrations of 0, 0.1, 1, 2.5, 5 μg/mL, respectively, stimulated by the gingiva porphyrin monospora lipopolysaccharide is shown in FIG. 4.
In FIG. 4, (a) mRNA expression level of CXC chemokine receptor 1 was changed after stimulation of human gingival fibroblasts with 2.5 μg/mL of gingiva porphyrin monospora lipopolysaccharide for 48 hours; (b) mRNA expression level of interleukin-8 was changed after stimulation of human gingival fibroblasts with 2.5. Mu.g/mL of gingiva porphyrin monospora lipopolysaccharide for 48 hours; (c) mRNA expression level change of the basic protein of the protoplatelet after 2.5 mug/mL gingiva porphyrin monospora lipopolysaccharide stimulates human gingiva fibroblast for 48 hours; (d) Gray scale analysis result of CXC chemokine receptor 1 after 2.5 mug/mL of gingiva porphyrin monospora lipopolysaccharide stimulates human gingiva fibroblast for 48 hours; (e) Protein level change of interleukin-8 after stimulation of human gingival fibroblasts with 2.5 μg/mL gingiva porphyrin monospora lipopolysaccharide for 48 h; (f) Protein level change of interleukin-8 after stimulation of human gingival fibroblasts with 2.5 μg/mL gingiva porphyrin monospora lipopolysaccharide for 48 h; (g) 2.5 μg/mL of gingiva porphyrin monospora lipopolysaccharide stimulated changes in the protein level of the platelet alkaline protein after 48h of human gingiva fibroblasts.
The results of FIG. 4 show that, upon stimulation with 2.5. Mu.g/mL of the gingiva porphyrin monospora lipopolysaccharide, the levels of CXC chemokine receptor 1, interleukin-8, and the mRNA and CXC chemokine receptor 1 proteins of the protoplatelet basic protein in human gingiva fibroblasts were significantly increased, and that interleukin-8 and the protoplatelet basic protein in the cell culture supernatant were also significantly upregulated compared to those before stimulation.
In conclusion, 2.5 mug/mL of gingiva porphyrin monospora lipopolysaccharide stimulation can induce the down regulation of miR-141-5p expression in human gingiva fibroblasts, and simultaneously, the levels of mRNA and protein of chemokine receptor CXC chemokine receptor 1, chemokine interleukin-8 and protoplatelet alkaline protein are increased, and miR-141-5p and the chemotactic related molecules show completely opposite reaction trends.
Example 3
Investigation of the Regulation and control effects of miR-141-5p on CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein expression levels in human gingival fibroblasts
Primary culture of human gingival fibroblasts
The primary culture of human gingival fibroblasts was performed as in example 1, except that: in step (3), plating was performed using six well plates when cell confluency reached 70%.
(II) transfection of cells
The next day after plating, the original medium in the six well plates was discarded, PBS was washed twice, 1.25mL of freshly prepared α -MEM medium containing 10% FBS was added to each well plate, the transfection mixture was added dropwise to the cell well plates uniformly, 250. Mu.L of each well plate was added, and mixed with gentle shaking. And placing the six-hole plate in a cell incubator, and culturing for a corresponding period of time according to experimental requirements, and then carrying out subsequent experiments.
Preparation of transfection mixture: mu.L of 20. Mu.M RNA oligo (RNA oligo from Gimar Gene Co., ltd., sequences shown in Table 5) was added to 125. Mu.L of the serum-reduced medium in a 1.5mL sterile Eppendorf tube, wherein the experimental group was transfected with miR-141-5p mimetic, the control group was transfected with mimetic control, and 5. Mu. L HighGene plus Transfection reagent was added to 125. Mu.L of the serum-reduced medium in another Eppendorf tube, and the mixture was gently stirred and mixed, and after incubation at room temperature for 5min, the two were mixed, stirred and mixed, and incubated at room temperature for 20min.
TABLE 5 RNA oligo sequence
(III) total DNA extraction of cells and real-time quantitative fluorescence PCR
The "total DNA extraction of cells and real-time quantitative fluorescent PCR" was performed as described in example 1.
(IV) Western Blot (Western Blot)
The "Western Blot" of example 1 was followed.
(V) ELISA assay (ELISA assay)
The "enzyme-linked immunosorbent assay (ELISA) test" of example 1 was followed.
Detection result
(1) Transfection efficiency evaluation of miR-141-5p inhibitor: cell fluorescent protein expression was observed and recorded using an inverted fluorescent microscope by transfecting human gingival fibroblasts with Carboxyfluorescein (FAM) -labeled miR-141-5p inhibitor for 24 h. The results are shown in FIG. 5.
As can be seen from FIG. 5, more than 75% of human gingival fibroblasts were observed to express fluorescent protein 24 hours after transfection, indicating successful transfection and higher transfection efficiency.
(2) Transfection efficiency evaluation of miR-141-5p mimics
Human gingival fibroblasts 6h, 12h, 24h and 36h were transfected with miR-141-5p mimics, respectively, and the level change of miR-141-5p in the human gingival fibroblasts was detected, and the results are shown in FIG. 6.
As can be seen from FIG. 6, a significant increase in miR-141-5p levels in human gingival fibroblasts was observed after 6h, 12h, 24h and 36h of transfection.
(3) Upregulation of miR-141-5p effects on human gingival fibroblast expression CXC chemokine receptor 1, interleukin-8, and protoplatelet basic protein:
human gingival fibroblasts were transfected with miR-141-5p mimics for 12h and 36h, respectively, and mRNA level changes of CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein in the human gingival fibroblasts were detected. Human gingival fibroblasts were then transfected with the miR-141-5p mimetic for 48h, and protein level changes of CXC chemokine receptor 1 in human gingival fibroblasts and interleukin-8 and protoplatelet basic proteins in cell supernatants were detected. The results are shown in FIG. 7.
As can be seen from FIG. 7, significant downregulation of the mRNA levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein was observed after upregulation of miR-141-5p 12h and 36 h. After up-regulation of miR-141-5p 48h, significant down-regulation of CXC chemokine receptor 1 in human gingival fibroblasts and interleukin-8 and protoplatelet basic protein levels in cell supernatants was observed.
In conclusion, the experiment proves that miR-141-5p has a regulation and control effect on CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein expression in human gingival fibroblasts, and inhibiting miR-141-5p can up-regulate mRNA and protein levels of CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein and plays a role similar to pro-inflammatory factors; up-regulating miR-141-5p has the function of inhibiting human gingival fibroblast from expressing CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein.
Example 4
Exploring the effect of inhibiting or upregulating miR-141-5p on inflammatory factor (tumor necrosis factor-alpha) to induce human gingival fibroblast to express CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein
Primary culture of human gingival fibroblasts
The primary culture of human gingival fibroblasts was performed as in example 1.
(II) transfection of cells
Reference is made to the procedure for cell transfection in example 3; human gingival fibroblasts were transfected with the miR-141-5p mimetic for 12h, and with the mimetic NC as a control, for 12h. The original medium was then aspirated, washed three times with sterile PBS, and human gingival fibroblasts were stimulated with tumor necrosis factor- α at a concentration of 20ng/mL for 48h, and cells and supernatant were collected.
(III) total DNA extraction of cells and real-time quantitative fluorescence PCR
mRNA level changes of CXC chemokine receptor 1, interleukin-8, and protoplatelet basic protein in human gingival fibroblasts were detected by "total cell DNA extraction and real-time quantitative fluorescent PCR" according to example 1.
(IV) Western Blot (Western Blot)
Protein level changes of CXC chemokine receptor 1 in human gingival fibroblasts were detected according to the "Western Blot" (Western Blot) of example 1.
(V) ELISA assay (ELISA assay)
The protein level changes of interleukin-8, protoplatelet basic protein in the cell supernatant were detected according to the "enzyme-linked immunosorbent assay (ELISA experiment)" of example 1.
Detection result
mRNA and protein level changes of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein were detected by stimulating human gingival fibroblasts with 20ng/mL tumor necrosis factor-alpha for 48h after upregulation of miR-141-5 p. The results are shown in FIG. 8.
In FIG. 8, (a) mRNA expression level of CXC chemokine receptor 1 in human gingival fibroblasts after upregulation of miR-141-5p under stimulation with tumor necrosis factor- α at 20 ng/mL; (b) Up-regulating mRNA expression level change of interleukin-8 in human gingival fibroblast after miR-141-5p under the stimulation of tumor necrosis factor-alpha of 20 ng/mL; (c) Up-regulating mRNA expression level change of protoplatelet basic protein in human gingival fibroblast after miR-141-5p under the stimulation of tumor necrosis factor-alpha of 20 ng/mL; (d) Up-regulating the gray level analysis result of CXC chemokine receptor 1 in human gingival fibroblast after miR-141-5p under the stimulation of tumor necrosis factor-alpha of 20 ng/mL; (e) Up-regulating protein level change of interleukin-8 in supernatant of miR-141-5p under stimulation of tumor necrosis factor-alpha of 20 ng/mL; (f) Up-regulating protein level change of interleukin-8 in supernatant of miR-141-5p under stimulation of tumor necrosis factor-alpha of 20 ng/mL; (g) Up-regulating the level of original platelet basic protein in supernatant of miR-141-5p under the stimulation of tumor necrosis factor-alpha of 20 ng/mL.
As can be seen from FIG. 8, the upregulation of mRNA and protein levels of CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein in human gingival fibroblasts under tumor necrosis factor-alpha stimulation can be attenuated and even prevented by upregulation of miR-141-5p, and the differences are statistically significant.
In conclusion, inhibiting miR-141-5p increases CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein expression caused by tumor necrosis factor-alpha stimulation; upregulation of miR-141-5p has the effect of attenuating or even preventing an increase in CXC chemokine receptor 1, interleukin-8, and tropoplatelet basic protein expression due to tumor necrosis factor-alpha stimulation.
Example 5
Exploring the influence of up-regulating miR-141-5p on gingiva porphyrin monospora lipopolysaccharide to induce human gingiva fibroblast to express CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein
Example 5 was performed as in example 4, except that: the tumor necrosis factor-alpha in the step (II) is replaced by gingiva porphyrin monospore lipopolysaccharide, and the method concretely comprises the following steps:
(II) transfection of cells
Reference is made to the procedure for cell transfection in example 5; human gingival fibroblasts were transfected with miR-141-5p mimetic for 12h, and with mimetic control as control, the same transfection was performed for 12h. The crude medium was then aspirated, washed three times with sterile PBS, human gingival fibroblasts were stimulated with 2.5 μg/mL of gum porphyrinogen lipopolysaccharide for 48h, and cells and supernatant were collected.
Detection result
Human gingival fibroblasts were stimulated with 2.5ng/mL of gingiva porphyrin monospora lipopolysaccharide for 48h after up-regulation of miR-141-5p, and changes in CXC chemokine receptor 1, interleukin-8, and the mRNA and protein levels of the protoplatelet alkaline proteins were detected. The results are shown in FIG. 9
In FIG. 9, (a) mRNA expression level of CXC chemokine receptor 1 in human gingival fibroblasts after upregulation of miR-141-5p under stimulation with 2.5 μg/mL of gingiva porphyrin monospora lipopolysaccharide; (b) Up-regulating mRNA expression level change of interleukin-8 in human gingival fibroblast after miR-141-5p under the stimulation of 2.5 mug/mL gingiva porphyrin monospora lipopolysaccharide; (c) Up-regulating mRNA expression level change of protoplatelet alkaline protein in human gingival fibroblast after miR-141-5p under the stimulation of gingiva porphyrin monospora lipopolysaccharide of 22.5 mug/mL; (d) 1. Mu.g/mL of gray level analysis result of CXC chemokine receptor in human gingival fibroblast after up-regulating miR-141-5p under the stimulation of gingiva porphyrin monospora lipopolysaccharide; (e) Up-regulating protein level change of interleukin-8 in cell supernatant after miR-141-5p under the stimulation of 2.5 mug/mL gingivalis porphyrinogen lipopolysaccharide; (f) Up-regulating protein level change of interleukin-8 in cell supernatant after miR-141-5p under the stimulation of 2.5 mug/mL gingivalis porphyrinogen lipopolysaccharide; (g) 2.5 mug/mL of the gingiva porphyrin monospora lipopolysaccharide, and up-regulating the level of the protoplatelet basic protein in the supernatant of the cells after miR-141-5 p.
As can be seen from FIG. 9, up-regulation of miR-141-5p can attenuate or even prevent up-regulation of CXC chemokine receptor 1, interleukin-8, and protoplatelet basic protein expression in human gingival fibroblasts under stimulation of gingiva porphyrin monospora lipopolysaccharide, and the difference is statistically significant.
In conclusion, inhibiting miR-141-5p enables CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein expression caused by stimulation of gingivalis lipopolysaccharide to be up-regulated to be further increased; upregulation of miR-141-5p has the effect of attenuating or even preventing an increase in CXC chemokine receptor 1, interleukin-8 and protoplatelet basic protein expression due to stimulation by the gingiva porphyrin monospora lipopolysaccharide.
Example 6
Exploring the effect of miR-141-5p mimic in mouse periodontitis model
1. Selection and grouping of mice: selecting 12 male wild C57BL/6 mice with the age of 6-8 weeks, and dividing the mice into 3 groups, wherein 1 group is used as a healthy control group without any treatment; and constructing a mouse periodontitis model in the other 2 groups.
2. Constructing a mouse periodontitis model: injecting the sodium pentobarbital injection solution with concentration of 1% into the abdominal cavity of the mouse to anesthetize the mouse; then, separating the inter-dental space of the first molar and the second molar of the upper jaw of the mouse by using a dental probe of the mouse; ligating the neck of the second molar of the upper jaw of the mouse by using a ligature wire of 5-0, clamping the ligature wire by using a needle holder to enter from the far middle palate side of the first molar of the upper jaw of the mouse, ligating and fixing the ligature wire at the position close to the middle palate side after encircling the second molar of the upper jaw for one week; ligating for 1 week to obtain a mouse periodontitis model; wherein, the periodontitis group, the administration group and the healthy control group are respectively 8 sample volumes.
3. And (3) testing: the healthy control group did not perform any treatment; ligating the periodontitis group for 1 week; the administration group was locally injected with miR-141-5p mimic at the ligation site 1 time per day, 2. Mu.L each time, for 1 week. After 1 week, all mice were sacrificed and maxillary tissues were taken for analysis.
4. Detection method
(1) Micro-CT scans and reconstructions were performed using the Inveon MM system. The method comprises the steps of taking the maxilla of a mouse to scan bone mass and microstructure, respectively carrying out three-dimensional reconstruction on the maxilla of each group, selecting an interested region (the alveolar bone around the second molar of the maxilla of the mouse, the mesial-distal range from the distal side of the distal root of the first molar to the mesial side of the proximal root of the third molar, and the direction range of the coronal root from bifurcation of the second molar to the root tip), carrying out data analysis, and obtaining the vertical distance from the crest of the alveolar bone to the cementum boundary, the bone mass percentage and the periodontal bone density, and taking an average value of each group of mice.
(2) Fixing the scanned sample for 36h by using a fixing solution, then placing the sample in 10% EDTA decalcification solution, decalcification for 2 weeks at room temperature, and changing the decalcification solution 1 time every 2 days; then dehydrated, embedded, sectioned, and HE stained to evaluate alveolar bone resorption and local inflammation.
5. Detection result:
the data obtained from the scan of Micro-CT is shown in FIG. 10, and it can be seen in FIG. 10 that the distance from the crest of the bone to the cementum boundary of the administration group is significantly less than that of the periodontitis group, and that the percentage of bone mass and bone density in the region of interest of the administration group are significantly higher than those of the periodontitis group.
The sample staining results are shown in fig. 11, and it can be seen from fig. 11 that the administration group had lost adhesion, less alveolar bone resorption than the periodontitis group, and less inflammatory cells infiltrated in the gingival lesion area than the periodontitis group.
In conclusion, the miR-141-5p mimic provided by the application has a regulatory effect on the periodontitis disease of mice; therefore, miR-141-5p can be used as a potential target for treating periodontitis diseases.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1. The application of miRNA-141-5p in preparing a medicament for treating periodontitis diseases is provided, wherein the sequence of the miRNA-141-5p is shown as SEQ ID NO. 1.
2. Use of an agent that promotes expression of miRNA-141-5p in the manufacture of a medicament for the treatment of periodontitis diseases;
the reagent is a mimic of miRNA-141-5 p;
the miRNA-141-5p mimic comprises a sense strand and an antisense strand, wherein the sequence of the sense strand is shown as SEQ ID NO.1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.
3. The use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier.
4. The use according to claim 3, wherein the carrier is a polar solvent and a non-polar solvent;
the polar solvent is selected from one or more of water, ethanol, glycerol and propylene glycol, and the nonpolar solvent is selected from one or two of fatty oil and liquid paraffin.
5. The use according to claim 1 or 2, wherein the medicament further comprises a supplemental additive selected from one or more of diluents, buffers, encapsulating agents, excipients, fillers, binders, wetting agents, disintegrants, surfactants, colorants and flavoring agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311724273.6A CN117398404B (en) | 2023-12-15 | 2023-12-15 | Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311724273.6A CN117398404B (en) | 2023-12-15 | 2023-12-15 | Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117398404A CN117398404A (en) | 2024-01-16 |
| CN117398404B true CN117398404B (en) | 2024-03-08 |
Family
ID=89492983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311724273.6A Active CN117398404B (en) | 2023-12-15 | 2023-12-15 | Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117398404B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109689027A (en) * | 2016-06-29 | 2019-04-26 | 奥德纳米有限公司 | Triglycerides aural preparations and application thereof |
| CN116251126A (en) * | 2023-02-14 | 2023-06-13 | 重庆医科大学 | Application of periodontal ligament stem cell-derived exosome in relieving cell aging and treating periodontitis |
| CN116333994A (en) * | 2021-12-07 | 2023-06-27 | 上海鲸奇生物科技有限公司 | Treatment of non-coding RNA mediated neurological diseases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6830976B2 (en) * | 2019-03-08 | 2021-02-17 | キユーピー株式会社 | MicroRNA expression enhancer and food and drink for microRNA expression enhancer |
-
2023
- 2023-12-15 CN CN202311724273.6A patent/CN117398404B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109689027A (en) * | 2016-06-29 | 2019-04-26 | 奥德纳米有限公司 | Triglycerides aural preparations and application thereof |
| CN116333994A (en) * | 2021-12-07 | 2023-06-27 | 上海鲸奇生物科技有限公司 | Treatment of non-coding RNA mediated neurological diseases |
| CN116251126A (en) * | 2023-02-14 | 2023-06-13 | 重庆医科大学 | Application of periodontal ligament stem cell-derived exosome in relieving cell aging and treating periodontitis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117398404A (en) | 2024-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Goto et al. | Hyperocclusion stimulates osteoclastogenesis via CCL2 expression | |
| JP2021183629A (en) | Applications of hexokinase 2-specific inhibitor in acute central nervous system injury disease | |
| Hu et al. | Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis. | |
| US11453915B2 (en) | MiRNA marker and kit associated with postmenopausal osteoporosis | |
| WO2008141438A1 (en) | Gabaergic modulators for treating airway conditions | |
| CN110596406B (en) | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament | |
| Dai et al. | Evaluation of interleukin-10 producing CD19+ B cells in human gingival tissue | |
| Tian et al. | HIF‐1α regulates osteoclast activation and mediates osteogenesis during mandibular bone repair via CT‐1 | |
| Zhang et al. | ULK1 suppresses osteoclast differentiation and bone resorption via inhibiting Syk-JNK through DOK3 | |
| Menini et al. | MicroRNAs in Peri-implant Crevicular Fluid Can Predict Peri-implant Bone Resorption: Clinical Trial with a 5-Year Follow-up. | |
| CN117398404B (en) | Application of miRNA-141-5p in preparation of medicines for treating periodontitis diseases | |
| Manfrin et al. | Expression of OPG, RANK, and RANKL proteins in tooth repair processes after immediate and delayed tooth | |
| Sun et al. | Rebalancing liver-infiltrating CCR3+ and CD206+ monocytes improves diet-induced NAFLD | |
| CN109512833A (en) | The function and purposes of E2F6 inhibitor | |
| Zhang et al. | Extracellular vesicles secreted by human periodontal ligament induced osteoclast differentiation by transporting miR-28 to osteoclast precursor cells and further promoted orthodontic tooth movement | |
| CN113679735A (en) | Application of SLC7A11 gene in hepatocellular carcinoma interventional embolization operation | |
| CN101712960A (en) | Mac-2BP tumor antigen gene, protein, antibody and application thereof | |
| WO2006104136A1 (en) | Therapeutic agent for non-alcoholic fatty liver disease, and screening method for drug candidate compound for treatment or prevention of non-alcoholic fatty liver disease | |
| US20210038688A1 (en) | Icam-1 marker and application thereof | |
| KR20100096010A (en) | Novel use of drg2 gene | |
| Tang et al. | Hypoxia‐induced factor 1α promotes osteotomy‐induced regional acceleratory phenomenon via DC‐STAMP mediated membrane fusion | |
| CN107641653A (en) | MACC1 AS1 probes are preparing the application in being used to predict the diagnostic reagent of stomach cancer clinical prognosis | |
| CN116240176B (en) | Secretory neutrophil and application thereof | |
| CN108034713A (en) | Postmenopausal Osteoporosis diagnosis and treatment target spot and its application | |
| CN115120727B (en) | Application of S100A9 inhibitor in preparation of medicine for preventing and treating clostridium perfringens infection diarrhea |
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 |