CN108498514A - Mdivi-1在制备治疗角膜碱烧伤药物中的应用 - Google Patents
Mdivi-1在制备治疗角膜碱烧伤药物中的应用 Download PDFInfo
- Publication number
- CN108498514A CN108498514A CN201810519514.6A CN201810519514A CN108498514A CN 108498514 A CN108498514 A CN 108498514A CN 201810519514 A CN201810519514 A CN 201810519514A CN 108498514 A CN108498514 A CN 108498514A
- Authority
- CN
- China
- Prior art keywords
- mdivi
- cornea
- drp1
- corneal alkali
- alkali burns
- 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.)
- Granted
Links
- NZJKEVWTYMOYOR-UHFFFAOYSA-N 3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanylidene-1h-quinazolin-4-one Chemical compound C1=C(Cl)C(OC)=CC(N2C(C3=CC=CC=C3NC2=S)=O)=C1Cl NZJKEVWTYMOYOR-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 229940079593 drug Drugs 0.000 title claims abstract description 7
- 239000003814 drug Substances 0.000 title claims abstract description 7
- 210000004087 cornea Anatomy 0.000 abstract description 51
- 230000014509 gene expression Effects 0.000 abstract description 14
- 230000002757 inflammatory effect Effects 0.000 abstract description 8
- 210000004204 blood vessel Anatomy 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 230000006735 deficit Effects 0.000 abstract description 5
- 101150062460 DNM1 gene Proteins 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 208000028006 Corneal injury Diseases 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 102100022692 Density-regulated protein Human genes 0.000 abstract 1
- 101001044612 Homo sapiens Density-regulated protein Proteins 0.000 abstract 1
- 101000909218 Homo sapiens Dynamin-1-like protein Proteins 0.000 abstract 1
- 101000841301 Homo sapiens Utrophin Proteins 0.000 abstract 1
- 101150006098 Dnm1l gene Proteins 0.000 description 33
- 239000003513 alkali Substances 0.000 description 31
- 210000001519 tissue Anatomy 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000003112 inhibitor Substances 0.000 description 14
- 241000700605 Viruses Species 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 102000007469 Actins Human genes 0.000 description 9
- 108010085238 Actins Proteins 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 102000004889 Interleukin-6 Human genes 0.000 description 8
- 108090001005 Interleukin-6 Proteins 0.000 description 8
- 210000005252 bulbus oculi Anatomy 0.000 description 8
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102100040247 Tumor necrosis factor Human genes 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 206010061218 Inflammation Diseases 0.000 description 6
- 102000003777 Interleukin-1 beta Human genes 0.000 description 6
- 108090000193 Interleukin-1 beta Proteins 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 5
- 229920002307 Dextran Polymers 0.000 description 5
- 108091030071 RNAI Proteins 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 5
- 210000001508 eye Anatomy 0.000 description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 5
- 230000009368 gene silencing by RNA Effects 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 108091027967 Small hairpin RNA Proteins 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229960002143 fluorescein Drugs 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- XYJODUBPWNZLML-UHFFFAOYSA-N 5-ethyl-6-phenyl-6h-phenanthridine-3,8-diamine Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2N(CC)C1C1=CC=CC=C1 XYJODUBPWNZLML-UHFFFAOYSA-N 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 3
- 102100037840 Dehydrogenase/reductase SDR family member 2, mitochondrial Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 101710188053 Protein D Proteins 0.000 description 3
- 101710132893 Resolvase Proteins 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 102000034356 gene-regulatory proteins Human genes 0.000 description 3
- 108091006104 gene-regulatory proteins Proteins 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001101998 Galium Species 0.000 description 2
- 235000014820 Galium aparine Nutrition 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 2
- 241000612118 Samolus valerandi Species 0.000 description 2
- 206010041047 Slow virus infection Diseases 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002651 drug therapy Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 210000005240 left ventricle Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 210000000115 thoracic cavity Anatomy 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 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 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108700021058 Dynamin Proteins 0.000 description 1
- 102000043859 Dynamin Human genes 0.000 description 1
- 108010036694 Dynamin I Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000002918 Fraxinus excelsior Nutrition 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- VAYOSLLFUXYJDT-RDTXWAMCSA-N Lysergic acid diethylamide Chemical compound C1=CC(C=2[C@H](N(C)C[C@@H](C=2)C(=O)N(CC)CC)C2)=C3C2=CNC3=C1 VAYOSLLFUXYJDT-RDTXWAMCSA-N 0.000 description 1
- 208000030768 Optic nerve injury Diseases 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 244000000231 Sesamum indicum Species 0.000 description 1
- 235000003434 Sesamum indicum Nutrition 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000000320 anti-stroke effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002956 ash Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 210000003683 corneal stroma Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009483 enzymatic pathway Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 229940020947 fluorescein sodium Drugs 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- CMHHMUWAYWTMGS-UHFFFAOYSA-N oxybuprocaine Chemical compound CCCCOC1=CC(C(=O)OCCN(CC)CC)=CC=C1N CMHHMUWAYWTMGS-UHFFFAOYSA-N 0.000 description 1
- 229960003502 oxybuprocaine Drugs 0.000 description 1
- 230000004796 pathophysiological change Effects 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Ophthalmology & Optometry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Mdivi‑1在制备治疗角膜碱烧伤药物中的应用。通过动物实验证实了Mdivi‑1在治疗角膜碱烧伤的有效性。该物质通过抑制DRP1和Dnm1而抑制线粒体分裂，降低ROS产生、炎症因子IL‑1β和IL‑6以及TNF‑α表达水平，减少角膜新生血管的形成，从而改善了角膜碱烧伤引起的角膜损伤和视力损伤。
Description
技术领域
本发明涉及药物用途发明领域，涉及角膜碱烧伤药物中的应用。
背景技术
Mdivi-1(化学分子式：C15H10Cl2N2O2S，分子量：353.22，化学结构如下图)，为白色或卡其色固体，易溶于DMSO，不溶或难溶于H2O。
Mdivi-1是线粒体分裂dynamin相关GTP酶(DRP1)和线粒体分裂Dynamin I(Dnm1)的选择性细胞渗透抑制剂，在科学研究中，Mdivi-1的主要用途有两个方面：
在体外实验，用Mdivi-1处理细胞后抑制线粒体外膜透化导致细胞色素C释放减少，抑制了细胞凋亡。
在体外和体内实验，Mdivi-1被应用于抗中风和保护神经。
目前缺乏有效的治疗角膜碱烧伤的药物，现有的药物治疗主要有：局部或全身应用抗生素控制感染、局部或全身应用糖皮质激素抑制炎症和新生血管生成，补充维生素等。这些药物治疗仅能轻微缓解病情，对角膜碱烧伤后引起的炎症反应、视力损伤的疗效不佳。
发明内容
本发明的目的在于提供Mdivi-1的新用途，即Mdivi-1在制备治疗角膜碱烧伤药物中的应用。
Mdivi-1在治疗角膜碱烧伤的作用机理为：Mdivi-1通过抑制DRP1和Dnm1而抑制线粒体分裂，减少ROS、炎症因子IL-1β和IL-6的产生，减少角膜新生血管的形成，从而改善了角膜碱烧伤引起的角膜损伤和视力损伤。
本发明能有效减少角膜碱烧伤后角膜的ROS产生、炎症反应、新生血管形成。
附图说明
图1：A为Drp1代表性Westem-blot图。B为Drp1的灰度分析半定量检测结果。C为免疫荧光法检测角膜碱烧伤组织与正常小鼠组织中p-Drp1的表达。角膜碱烧伤第1天的时候p-Drp1的表达开始升高，第7天的时候降低，但是角膜碱烧伤组明显高于角膜正常对照组。数据表示为Mean+SD，n＝3，*P﹤0.05，**P﹤0.01，Control正常小鼠角膜组织VS碱烧伤小鼠角膜组织。图中，alkali burn：碱烧伤；Cont：正常对照组；1d、3d、7d、14d：碱烧伤后第、3、7、14天；The percentage of Drp1/β-actin：Drp1比上β-actin灰度值的百分比；AB-1、AB-3、AB-7、AB-14：分别为碱烧伤后第1、3、7、14天；merge：合并的图片。
图2：A为检测正常小鼠角膜组织中应用三种慢病毒后Drp1的敲低情况。B为Drp1灰度分析半定量检测结果。C、D分别为慢病毒感染4天与7天Drp1的Westem-blot图。E为Drp1抑制剂Mdivi-1与RNAi下调Drp1降低角膜碱烧伤引起的ROS含量的变化，显示在小鼠角膜碱烧伤的组织中，ROS的含量明显高于正常的角膜组织，Mdivi-1以及shRNA5-12明显降低了角膜碱烧伤组织中ROS的含量。数据表示为Mean+SD，n＝5,*p<0.05,**p<0.01，***P﹤0.001，正常小鼠角膜组织未处理VS正常小鼠角膜组织病毒处理。图中，shRNA:短发夹RNA；Cont：正常对照组；NC：阴性对照组；The percentage of Drp1/β-actin：Drp1比上β-actin灰度值的百分比；NaCl：氯化钠组。
图3：Drp1抑制剂Mdivi-1与RNAi沉默Drp1能显著减少炎症因子IL-1β、IL-6以及TNF-α的mRNA水平。角膜碱烧伤后IL-1β、IL-6以及TNF-α的mRNA水平显著升高，下调Drp1的表达可以显著地降低IL-1β、IL-6、TNF-α的mRNA水平。数据表示Mean+SD，n＝3,*p<0.05，**p<0.01，***p<0.001正常组VS Nacl组VS Mdivi-1VS shRNA5-1组。图中，IL-1βmRNA level：白介素-1βmRNA水平；IL-6mRNA level：白介素-6mRNA水平；TNF-αmRNAlevel：TNF-αmRNA水平；Cont：正常对照组；AB-7：碱烧伤第7天；sh RNA：短发夹RNA
图4：Drp1抑制剂Mdivi-1抑制角膜碱烧伤引起的新生血管形成。荧光素钠角膜铺片代表性图片，在小鼠角膜碱烧伤的组织中，角膜新生血管的面积明显增加，Mdivi-1可明显降低角膜新生血管的形成。图中，alkali burn(14d)：碱烧伤第14天；Cont：正常对照组；NaCl：氯化钠组。
具体实施方式
下面结合实施例并对照附图对本发明作进一步详细说明。
为了更好地理解本发明的实质，下面以动物实验和结果来证明Mdivi-1的用途。
一.材料与方法
1.动物
本实验选用健康6-8周C57BL/6实验用小鼠，雌雄随机，购买自湖南长沙斯莱克景达有限责任公司，经过中科院上海实验动物中心审核，饲养于南昌大学眼科研究所专用动物房。所有实验动物的喂养及实验程序均严格遵照实验动物保护条款。动物房内环境安静，室温25℃左右，12h/天光照(7：00～19：00)。
2.主要试剂
荧光素FITC-dextran(sigma)；WesternBright ECL发光试剂(K-12045-D10)(advansta)；qPCR试剂盒(AQ131-01)(Trans Gen)；逆转录试剂盒(AE301-02)荧光定量PCR试剂盒(Bio-rad)；Trizol试剂(Invitrogen)；线粒体分裂蛋白Drp1抑制剂mdivi-1(sigma)；Dihydroethidium(D7008)(sigma)。
3.抗体：兔单克隆抗体Drp1，兔单克隆抗体Phospho-Drp1(Ser579)(美国CST公司)；Beta actin antibody(β-actin)(美国Affinity公司)；FITC标记兔抗兔IgG二抗(美国Proteintech公司)；HRP标记山羊抗兔IgG二抗(CW0103T)(北京康为世纪公司)。
4.主要实验仪器
IX71倒置荧光显微镜(日本Olympus公司)；超声粉碎机(宁波新芝)；冰冻切片机(CM1950，德国Lecia公司)；垂直电泳系统，凝胶成像分析系统(美国Bio-Rad公司)；荧光定量PCR仪(CFX connect,美国BIO-RAD公司)。
5.实验方法
(1)模型建立
挑选6-8周健康实验小鼠(C57BL/6)，经腹腔注射10％水合氯醛(0.3ml/100g)全身麻醉，0.4％的奥布卡因进行眼球表面麻醉，将直径为2mm大小的滤纸浸入装有1mol/L的氢氧化钠的EP管内，镊子取出滤纸，吸除表面多余液体后贴于小鼠右眼的角膜中央，计时40s，立即用10ml 0.9％的生理盐水冲洗眼表及眼睑。将造好模的小鼠分为2组：生理盐水(AB+0.9％NaCl组)、5μM Mdivi-1溶液(AB+Mdivi-1组)，左眼作为正常对照组。给药方式为滴眼，每天4次，每次20ul。
(2)Drp1抑制剂的治疗
将造好模的小鼠分为2组：生理盐水(AB+0.9％NaCl组)、5μM Mdivi-1溶液(AB+Mdivi-1组)，左眼作为正常对照组。给药方式为滴眼，每天4次，每次20ul。
(3)蛋白质免疫印迹
取出各组眼球，解剖分离角膜，置于EP管中，在低温液氮下将角膜碾碎成粉末，加入提前配好的RIPA裂解液120ul，吹打混匀后于冰上裂解40min，每10min用涡旋混合器涡旋10s，超声破碎仪超声破碎、离心，取上清，加入蛋白上样缓冲液，吹打均匀后100摄氏度金属浴10min，于-20摄氏度保存。取适量蛋白样品进行上样、跑胶、转膜、孵育一抗和二抗，最后显影。用Image J软件分析条带灰度值，并与β-actin比较计算结果，每组标本重复实验至少3次。
(4)免疫荧光染色实验
角膜碱烧伤7天后取各组小鼠眼球各一只，4％的多聚甲醛固定30min，然后依次用15％、30％蔗糖溶液进行梯度脱水。将眼球进行液氮冰冻包埋，制成7μm的冰冻切片用于免疫荧光。切片放入实验台自然晾干，PBS洗涤3次。5％BSA封闭1h(封闭液含5％BSA以及0.05％的TritonX-100)。用滤纸吸去封闭液，加入抗体p-Drp1(1：200)，玻片放于湿盒，4℃孵育过夜。PBS洗3次，滤纸吸干多余液体，加入Alexa Fluor488标记的抗兔二抗(1：150)，放入避光湿盒内，37℃孵育1h。PBS洗涤3次，滤纸吸干多余液体，Mounting medium(含DAPI)封片。倒置荧光显微镜下观察，拍照。
(5)qPCR实验
1)RNA提取
取各实验组及对照组小鼠眼球，分离出角膜。每个无核酶EP管放入1个角膜，在液氮超低温的情况下研碎角膜组织。每管加入0.5ml Trizol，液氮反复冻融三次，把组织吹散，室温孵育5min，按说明书以每ml Trizol体积加入0.2ml的氯仿，震荡孵育后，以10000×g4℃离心15min，取上层水相(RNA主要在水相层)于一新的无核酶EP管。每个EP管中加入0.25mL的异丙醇，轻轻颠倒混匀30次，室温放置10min，以10000×g4℃离心10min。管侧壁与管底形成胶状沉淀，弃上清。用DEPC水配制75％乙醇，每个EP加入1ml，涡旋混匀，4℃7500×g离心5min。弃上清，室温自然晾干后，加入20ul的DEPC水，吹打、溶解，简短离心。测RNA浓度。保存于-80℃冰箱以备使用。
2)cDNA的合成
轻轻混匀，放入PCR仪，42℃孵育15min；85℃加热5秒钟失活EasyScript○R RT/RI。逆转录后所得cDNA用作qPCR反应模板。
3)qPCR
上机(qPCR仪)。数据分析。q PCR反应条件：94℃预变性30s；94℃变性5s，60℃延伸30s；共40个循环。实验重复6次。引物序列:
TNF-a的上游引物：5’-ATGGCCTCCCTCTCATCAGT-3’
TNF-a的下游引物：5’-TGGTTTGCTACGACGTGGG-3’
IL-1b的上游引物：5’-GCCACCTTTTGACAGTGATGAG-3’
IL-1b的下游引物：5’-ATCAGGACAGCCCAGGTCAA-3’
IL-6的上游引物：5’-AAAGCCAGAGTCCTTCAGAGAG-3’
IL-6的下游引物：5’-TGGAAATTGGGGTAGGAAGGAC-3’
(6)ROS含量检测
角膜碱烧伤7天后取各组小鼠眼球各一只，4％的多聚甲醛固定30min，然后依次用15％、30％蔗糖溶液进行梯度脱水。OCT冷冻包埋(注意不能有泡)，制成7μm的冰冻切片。切片自然晾干，PBS洗涤3次，0.5％Triton-X作用10min。加入终浓度为10μM的Dihydroethidium探针，放入湿盒37℃孵育30分钟。PBS洗涤3次，甘油封片，于倒置荧光显微镜下，利用535/610nm波长的发射光进行ROS荧光信号的采集和拍照。用Image J对ROS的荧光数据进行分析。
(7)角膜新生血管检测
实验分为三组：正常对照组、角膜碱烧伤组+0.9％NaCl组、角膜碱烧伤组+Mdivi-1组。配置荧光素FITC-dextran溶液，5g/L，6-8周小鼠大概用量为1.5ml。于造模后14天，全身麻醉，固定四肢。手术剪剪开横隔处皮肤，沿胸骨向上剪开胸腔皮肤及肋骨，暴露胸腔，剪开心包膜、暴露心脏。将2ml的注射器针头磨平，左手持血管钳固定心脏，右手持注射器进针向左心室灌注荧光素FITC-dextran，边注射边在显微镜下观察角膜，大约6min左右(最好不要超过9min)，发现小鼠的鼻尖、口唇、四肢末梢、肝脏等变黄，迅速取出小鼠眼球。将眼球用4％多聚甲醛固定30min。在解剖显微镜下分离出角膜，剪成四叶草形状，进行角膜铺片。将玻片放于倒置荧光显微镜下485/520nm波长激发光激发FITC-dextran的荧光观察采集图像，并用Cellses软件对新生血管面积进行定量，实验至少重复三次。
二.结果
1.线粒体形态调节蛋白Drp1在碱烧伤角膜组织中的转录表达及磷酸化修饰水平上调。
已经有文献报道显示，线粒体动态变化参与了视神经损伤过程，为了进一步验证线粒体动态相关蛋白Drp1是否参与角膜碱烧伤的发生发展过程及其机制,我们通过Western blot的方法检测了小鼠角膜碱烧伤1、3、7天中Drp1的表达水平。制备好角膜碱烧伤模型，在显微镜下取角膜细胞，研磨、裂解和总蛋白提取；BCA法测定蛋白质浓度。蛋白样本经10％SDS-PAGE电泳后进行半干转，转膜后分别用Drp 1和β-actin一抗及相应的HRP-二抗孵育，并利用化学发光显像检测角膜内线粒体形态调节蛋白Drp 1，的表达水平，β-actin为内参。实验结果如图1所示，和正常角膜相比，角膜碱烧伤组线粒体调节线粒体分裂的蛋白Drp 1的水平在第1天起开始上调，第3天显著上调，第14天降低。。这一结果提示我们角膜碱烧伤使线粒体动态相关蛋白Drp1发生变化，并且Drp1磷酸化所介导的线粒体分裂有可能参与碱烧伤所致角膜组织损伤过程(见图1)。
2.Drp1抑制剂Mdivi-1与RNAi下调Drp1表达显著抑制了角膜碱烧伤后角膜组织中的氧化应激水平。
有研究发现，碱烧伤能刺激角膜组织中ROS的产生。ROS主要是通过酶途径和线粒体途经产生，为了进一步确定Drp1介导的线粒体分裂是否能通过调节线粒体来源ROS水平，以此方式参与碱烧伤所致角膜组织损伤过程，为此，我们构建了表达靶向Drp1的shRNA的三种慢病毒载体[Dnm1l-RNAi(42333-1)组、Dnm1l-RNAi(42334-1)组、Dnm1l-RNAi(42335-12)组]和阴性对照慢病毒载体,看是否影响ROS的产生。Western blot的方法检测结果如下图所示：A图：慢病毒感染复数MOI＝5条件下，三种慢病毒中Dnm1l-RNAi(42335-12)(以下简称shRNA5-12)的敲低效果最明显，故选择shRNA5-12，为了摸索慢病毒在体内表达的时间高峰，分别感染4天与7天后取角膜Western blot结果显示7天时敲低最明显，又考虑到角膜碱烧伤模型为7天模型，为了能使角膜碱烧伤7天时Drp1的敲低效果更为明显，故选择角膜碱烧伤模型制备前4天进行病毒感染。实验分为四组：正常对照组、角膜碱烧伤+Nacl组、角膜碱烧伤+Drp1抑制剂Mdivi-1组与角膜碱烧伤+慢病毒shRNA5-12组，Dihydroethidium试剂用于检测角膜组织中的ROS的含量。结果显示：碱烧伤后角膜中ROS的含量明显高于正常组，抑制剂组与慢病毒组ROS的含量明显低于角膜碱烧伤NaCl组(见图2)。
3.Drp1抑制剂Mdivi-1与RNAi下调Drp1表达显著抑制了角膜碱烧伤后角膜的炎症因子表达水平。
角膜碱烧伤后主要的病理生理改变为角膜基质层炎症因子的浸润。上述实验结果显示角膜碱烧伤ROS水平明显高于正常组，细胞内ROS的大量积累，可以诱导组织炎症反应和细胞死亡。而下调Drp1的表达后碱烧伤角膜组织的ROS水平下调。为了验证Mdivi-1和RNAi降低碱烧伤角膜氧化应激水平，从而抑制角膜组织的炎症反应，我们用qPCR检测角膜炎症因子IL-1β、IL-6以及TNF-α的mRNA的表达量。实验分为四组：正常对照组、角膜碱烧伤+Nacl组、角膜碱烧伤+Drp1抑制剂Mdivi-1组与角膜碱烧伤+慢病毒shRNA5-12组，结果显示碱烧伤后角膜中炎症因子的含量明显高于正常组，抑制剂组与慢病毒组炎症因子的含量明显低于角膜碱烧伤NaCl组(见图3)。
4.Drp1抑制剂Mdivi-1显著降低了碱烧伤后角膜新生血管的形成。
角膜病理性新生血管是角膜碱烧伤最常见的并发症之一也是最棘手的问题之一，常导致严重的视力损伤甚至失明。在手术显微镜下观察碱烧伤角膜模型，发现新生血管从第3天开始出现，7天时新生血管面积达到整个角膜的一半，在14天时达到高峰。为了观察解新生血管的情况，用荧光素FITC-dextran左心室灌注，然后取角膜剪四叶草进行角膜铺片，用cellsens软件计算新生血管面积。结果显示角膜碱烧伤后有大量病理性新生血管产生，应用Drp1抑制剂Mdivi-1后新生血管明显减少(见图4)。
综上所述，本发明经动物实验证实：线粒体形态调节蛋白Drp1的抑制剂Mdivi-1能显著抑制碱烧伤后角膜组织的氧化应激水平，显著降低碱烧伤后角膜组织的炎症因子表达水平以及角膜新生血管的形成，有效改善了碱烧伤后角膜的损伤和视力损伤。
实施例。
用本技术领域公知的方法，以DMSO为溶媒制成Mdivi-1母液，生理盐水稀释后制成5μM的Mdivi-1点眼液。
Claims (1)
1.Mdivi-1在制备治疗角膜碱烧伤药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810519514.6A CN108498514B (zh) | 2018-05-28 | 2018-05-28 | Mdivi-1在制备治疗角膜碱烧伤药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810519514.6A CN108498514B (zh) | 2018-05-28 | 2018-05-28 | Mdivi-1在制备治疗角膜碱烧伤药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108498514A true CN108498514A (zh) | 2018-09-07 |
| CN108498514B CN108498514B (zh) | 2020-01-17 |
Family
ID=63401825
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810519514.6A Expired - Fee Related CN108498514B (zh) | 2018-05-28 | 2018-05-28 | Mdivi-1在制备治疗角膜碱烧伤药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108498514B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055216A (zh) * | 2019-05-09 | 2019-07-26 | 张秀明 | 一种改善间质干细胞生物学功能的方法 |
| CN111281965A (zh) * | 2019-08-05 | 2020-06-16 | 无锡市人民医院 | Tir/bb环拟似物as-1在制备治疗角膜新生血管性疾病的药物中的应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (zh) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | 一种羊膜提取液的制备方法及用途 |
| CN101658491A (zh) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | 一种治疗角膜碱烧伤的羊膜滴眼液 |
| CN102488674A (zh) * | 2011-11-26 | 2012-06-13 | 山东省眼科研究所 | 一种异甘草素滴眼液及其制备方法 |
| CN106265682A (zh) * | 2016-09-06 | 2017-01-04 | 河南省眼科研究所 | 雷公藤红素在制备抑制碱烧伤角膜新生血管及促进角膜碱烧伤愈合滴眼液制剂中的应用 |
-
2018
- 2018-05-28 CN CN201810519514.6A patent/CN108498514B/zh not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (zh) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | 一种羊膜提取液的制备方法及用途 |
| CN101658491A (zh) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | 一种治疗角膜碱烧伤的羊膜滴眼液 |
| CN102488674A (zh) * | 2011-11-26 | 2012-06-13 | 山东省眼科研究所 | 一种异甘草素滴眼液及其制备方法 |
| CN106265682A (zh) * | 2016-09-06 | 2017-01-04 | 河南省眼科研究所 | 雷公藤红素在制备抑制碱烧伤角膜新生血管及促进角膜碱烧伤愈合滴眼液制剂中的应用 |
Non-Patent Citations (5)
| Title |
|---|
| JI-HONG WU ET AL.: "RNAi screening identifies GSK3β as a regulator of DRP1 and the neuroprotection of lithium chloride against elevated pressure involved in down regulation of DRP1", 《NEUROSCIENCE LETTERS》 * |
| XIANGJUN SHE等: "Inhibition of mitochondrial fission preserves 1 photoreceptors after retinal detachment", 《PATHOLOGY》 * |
| 刘文凤等: "线粒体功能障碍与角膜疾病研究进展", 《眼科新进展》 * |
| 王健等: "角膜碱性烧伤药物治疗的现状与研究进展", 《中华眼科医学杂志》 * |
| 白军强: "托珠单抗对小鼠碱烧伤视网膜作用的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055216A (zh) * | 2019-05-09 | 2019-07-26 | 张秀明 | 一种改善间质干细胞生物学功能的方法 |
| CN111281965A (zh) * | 2019-08-05 | 2020-06-16 | 无锡市人民医院 | Tir/bb环拟似物as-1在制备治疗角膜新生血管性疾病的药物中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108498514B (zh) | 2020-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mazzocca et al. | Inhibition of transforming growth factor β receptor I kinase blocks hepatocellular carcinoma growth through neo‐angiogenesis regulation | |
| Upreti et al. | Tumor-endothelial cell three-dimensional spheroids: new aspects to enhance radiation and drug therapeutics | |
| Li et al. | Celastrol nanomicelles attenuate cytokine secretion in macrophages and inhibit macrophage-induced corneal neovascularization in rats | |
| Li et al. | Riboflavin and ultraviolet A irradiation for the prevention of progressive myopia in a guinea pig model | |
| CN108498514A (zh) | Mdivi-1在制备治疗角膜碱烧伤药物中的应用 | |
| Hu et al. | High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene | |
| WO2023240958A1 (zh) | Jwa多肽在制备抗新生血管性眼病药物方面的应用 | |
| Duncan et al. | Lens cell survival after exposure to stress in the closed capsular bag | |
| Nemet et al. | Protective effect of free-radical scavengers on corneal endothelial damage in phacoemulsification | |
| CN104640559B (zh) | 含c‑肽的用于预防或治疗糖尿病性血管渗漏诱导的疾病的组合物 | |
| Shi et al. | Effects of trimetazidine on mitochondrial respiratory function, biosynthesis, and fission/fusion in rats with acute myocardial ischemia | |
| Yamagishi-Kimura et al. | Interaction between pilocarpine and ripasudil on intraocular pressure, pupil diameter, and the aqueous-outflow pathway | |
| Yan et al. | Acquired adenoma of the nonpigmented ciliary epithelium: analysis of five cases | |
| Loy et al. | Ultrastructural evidence of a secretory role for melatonin in the human parotid gland | |
| Liu et al. | CF101 alleviates OA progression and inhibits the inflammatory process via the AMP/ATP/AMPK/mTOR axis | |
| CN104981696B (zh) | 诊断和治疗前列腺癌的方法 | |
| Meng et al. | Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas | |
| Muñoz et al. | Testicular structure and description of the seminal pathway in Leporinus macrocephalus (Anostomidae, Teleostei) | |
| He et al. | Regulatory role of TIGAR on endothelial metabolism and angiogenesis | |
| CN113577066B (zh) | 芳基胍化合物或其药学上可接受的盐的用途 | |
| CN111214660A (zh) | Pax4基因表达抑制剂在制备抑制纤维化的药物中的应用 | |
| Zhang et al. | Aquaporin 5 in the eye: expression, function, and roles in ocular diseases | |
| JP6722201B2 (ja) | 内眼手術時の酸化ストレス障害に対する水素分子含有予防又は治療薬 | |
| CN107029237A (zh) | 产热增强化合物增强去甲肾上腺素类化合物诱导褐色脂肪细胞产热的应用 | |
| CN109975499A (zh) | 一种提升利托那韦药物抗癌有效性的检测方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200117 |