CN117398448A - Ola1在治疗眼部血管异常增生性疾病中的应用 - Google Patents
Ola1在治疗眼部血管异常增生性疾病中的应用 Download PDFInfo
- Publication number
- CN117398448A CN117398448A CN202311227321.0A CN202311227321A CN117398448A CN 117398448 A CN117398448 A CN 117398448A CN 202311227321 A CN202311227321 A CN 202311227321A CN 117398448 A CN117398448 A CN 117398448A
- Authority
- CN
- China
- Prior art keywords
- vascular
- ola1
- oal1
- cornea
- diseases
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000002792 vascular Effects 0.000 title claims abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 35
- 101000597499 Homo sapiens Obg-like ATPase 1 Proteins 0.000 title claims abstract description 34
- 102100035415 Obg-like ATPase 1 Human genes 0.000 title claims abstract description 33
- 201000010099 disease Diseases 0.000 title claims description 26
- 230000002159 abnormal effect Effects 0.000 title claims description 16
- 230000035755 proliferation Effects 0.000 title claims description 10
- 210000004087 cornea Anatomy 0.000 claims abstract description 25
- 210000001525 retina Anatomy 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 13
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 206010058314 Dysplasia Diseases 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 14
- 238000012795 verification Methods 0.000 claims description 13
- 230000002062 proliferating effect Effects 0.000 claims description 12
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 11
- 241000699670 Mus sp. Species 0.000 claims description 11
- 239000011159 matrix material Substances 0.000 claims description 10
- 230000002207 retinal effect Effects 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 210000004204 blood vessel Anatomy 0.000 claims description 8
- 239000008188 pellet Substances 0.000 claims description 8
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 238000003125 immunofluorescent labeling Methods 0.000 claims description 6
- 230000033115 angiogenesis Effects 0.000 claims description 5
- 210000002889 endothelial cell Anatomy 0.000 claims description 5
- 238000010166 immunofluorescence Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 206010020718 hyperplasia Diseases 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims description 3
- 229920000669 heparin Polymers 0.000 claims description 3
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 3
- 229960001008 heparin sodium Drugs 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 230000006459 vascular development Effects 0.000 claims description 3
- 208000035475 disorder Diseases 0.000 claims 3
- 238000002360 preparation method Methods 0.000 claims 2
- 101150012171 OLA1 gene Proteins 0.000 claims 1
- 230000006496 vascular abnormality Effects 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 11
- 238000011269 treatment regimen Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 238000010276 construction Methods 0.000 description 6
- 238000003197 gene knockdown Methods 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 231100000216 vascular lesion Toxicity 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 206010029113 Neovascularisation Diseases 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000000159 corneal neovascularization Diseases 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000004263 retinal angiogenesis Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 101100406385 Caenorhabditis elegans ola-1 gene Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 206010010996 Corneal degeneration Diseases 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 206010015943 Eye inflammation Diseases 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000010899 TGS Human genes 0.000 description 1
- 108050001062 TGS Proteins 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000007960 cellular response to stress Effects 0.000 description 1
- 201000007717 corneal ulcer Diseases 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 201000011066 hemangioma Diseases 0.000 description 1
- 102000055243 human OLA1 Human genes 0.000 description 1
- 201000001371 inclusion conjunctivitis Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 206010023365 keratopathy Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000008646 thermal stress Effects 0.000 description 1
- 206010044325 trachoma Diseases 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/03—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; catalysing transmembrane movement of substances (3.6.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/05—Hydrolases acting on acid anhydrides (3.6) acting on GTP; involved in cellular and subcellular movement (3.6.5)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Urology & Nephrology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Heart & Thoracic Surgery (AREA)
- Animal Husbandry (AREA)
- Vascular Medicine (AREA)
- Ophthalmology & Optometry (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cardiology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Marine Sciences & Fisheries (AREA)
Abstract
本发明提供OLA1在治疗眼部血管异常增生性疾病中的应用，涉及生物医疗技术领域，其包括OLA1在制备治疗眼部血管异常增生疾病中药物中的应用，本申请中通过体内外实验验证了OLA1对视网膜和角膜中血管异常增生具有抑制作用，为临床上眼部血管异常增生性疾病提供治疗策略。
Description
技术领域
本发明涉及生物医疗技术领域，尤其涉及一种OLA1在治疗眼部血管异常增生性疾病中的应用。
背景技术
在眼科疾病当中有一部分涉及到眼部血管病变，包括眼底血管病变：如早产儿视网膜病变、糖尿病视网膜病变、眼底血管瘤等；角膜血管病变：如角膜炎、角膜溃疡、角膜变性、沙眼性角膜病变等。这些眼部疾病的主要发病原因多为视网膜或角膜异常血管增生导致，过度的血管增生会造成眼部炎症、出血、视力障碍甚至失明。目前针对这类疾病的治疗手段较为局限，常采用手术治疗，大多数伴有眼内感染等并发症，给患者生活带来极大不变。因此寻找能够有效抑制眼部血管异常增生的相关基因将会为新药研发及应用提供可行方案，并将在眼科疾病治疗中提供新的策略。
人类Obg样ATPase-1(OLA1)属于GTPases大家族，属于TRAFAC(翻译因子)类和YchF亚家族。OLA1蛋白由中央胍结构域组成，两侧是盘绕的ATP酶结构域和C末端TGS。虽然它在细胞生物学中的功能大多未被表征，但OLA1参与DNA修复，肿瘤发生，细胞周期调节以及细胞应激对氧化和热应激的反应。OLA1在人体组织中分布广泛，包括血管内皮细胞，具有重要的病理生理学功能，然而其在眼部血管异常增生性疾病中是否发挥作用并提供可行治疗策略是本发明所要解决的问题。
发明内容
本发明的目的是为了解决现有技术中针对眼科疾病治疗手段较为局限，常采用手术治疗，大多数伴有眼内感染等并发症的技术问题。
为了实现上述目的，本发明采用了如下技术方案：
OLA1在制备治疗眼部血管异常增生疾病中药物中的应用。
优选的，所述药物用于抑制视网膜中血管新生。
优选的，所述药物用于抑制角膜中的新生血管。
本申请还提供了OLA1治疗眼部血管异常增生性疾病的验证方法，包括视网膜血管异常增生性疾病的验证方法及角膜血管增生性疾病的验证方法。
优选的，所述视网膜血管异常增生性疾病的验证方法包含以下步骤：
S1：建立过表达/敲除OAL1稳转细胞系
S2：在过表达/敲除OAL1稳转细胞系中利用成管实验检测OAL1对HRCEC管形成的影响；
S3：构建内皮细胞特性OAL1基因敲除小鼠，并通过免疫荧光观察出生后5天小鼠视网膜血管发育情况。
优选的，所述角膜血管增生性疾病的验证方法包含以下步骤：
S1：制备含肝素钠的基质胶微丸，并将基质胶微丸发分别植入到对照与敲除OAL1的小鼠角膜基质层中，1周后裂隙灯下观察小鼠角膜新生血管情况；
S2：将角膜进行COL IV免疫荧光染色，观察血管标志物表达情况。
与现有技术相比本申请所提供的OLA1在制备治疗眼部血管异常增生疾病中药物中的应用，通过体内外实验验证了OLA1对视网膜和角膜中血管异常增生具有抑制作用，为临床上眼部血管异常增生性疾病提供治疗策略。
附图说明
图1为本发明实施例1中构建HRCEC OLA1过表达稳转细胞系的Western结果显示图；
图2为本发明实施例1中HRCEC OLA1过表达稳转细胞系成管图，其中图A:HRCECOLA1过表达稳转细胞系8h成管图,4倍镜。图B:成管长度统计图。(*P<0.05，**P<0.01，***P<0.001，****P<0.0001)；
图3为本发明实施例2中构建HRCEC OLA1敲降稳转细胞系的Western结果显示图；
图4为本发明实施例2中HRCEC OLA1敲降稳转细胞系成管图，其中图A:HRCEC OLA1敲降稳转细胞系8h成管图，4倍镜，图B:成管长度统计图。
(*P<0.05，**P<0.01，***P<0.001，****P<0.0001)；
图5为本发明实施例3中小鼠视网膜免疫荧光显示OLA1抑制小鼠视网膜血管新生，其中图A:P5胎鼠COL IV染色，4倍镜，图B-C:血管半径，血管面积统计图，图D:P5胎鼠COL IV染色，10倍镜，图E-F:血管面积，分支点统计图(*P<0.05，**P<0.01，***P<0.001，****P<0.0001)；
图6为本发明实施例4中小鼠角膜微口袋以及免疫荧光染色显示OLA1抑制小鼠角膜血管新生，其中图A:小鼠角膜微口袋示意图；图B:术后第七天裂隙灯显微镜拍照以及免疫荧光染色；图C:血管面积统计图；图D:血管长度统计图(*P<0.05，**P<0.01，***P<0.001，****P<0.0001)。
具体实施方式
以下结合具体实施例，对本发明作进一步地详细说明。
OLA1在制备治疗眼部血管异常增生疾病中药物中的应用。
所述药物用于抑制视网膜中血管新生。
在另一实施方式中，所述药物用于抑制角膜中的新生血管。
本申请还提供了针对OLA1治疗眼部血管异常增生性疾病的验证实验，包括视网膜血管异常增生性疾病的验证及角膜血管增生性疾病的验证。
其中，所述视网膜血管异常增生性疾病的验证包含以下步骤：
S1：建立过表达/敲除OAL1稳转细胞系
S2：在过表达/敲除OAL1稳转细胞系中利用成管实验检测OAL1对HRCEC管形成的影响；
S3：构建内皮细胞特性OAL1基因敲除小鼠，并通过免疫荧光观察出生后5天小鼠视网膜血管发育情况。
所述角膜血管异常增生性疾病的验证包含以下步骤：
S1：制备含肝素钠的基质胶微丸，并将基质胶微丸发分别植入到对照与敲除OAL1的小鼠角膜基质层中，1周后裂隙灯下观察小鼠角膜新生血管情况；
S2：将角膜进行COL IV免疫荧光染色，观察血管标志物表达情况。
以下结合具体的实施例对本申请上述内容进行阐述：
本申请所使用的实验材料，如下所示：
1、Matrigel购于BD bioscience公司；
2、HRCEC购自上海青旗公司；
3、CCK8试剂购自南京诺唯赞生物科技股份有限公司；
4、DMEM购于HyClone公司；
5、Phosphate Bufffered Saline(1×)购于HyClone公司；
6、胎牛血清购于AllBio science公司。
7、lipo8000购自碧云天生物技术有限公司。
8、puromycin购自sigma公司。
9、flag抗体购自sigma公司。
10、opti-MEM购自gibco公司。
实施例1：过表达OAL1稳转细胞系的构建及成管实验：
A、过表达OAL1稳转细胞系的构建：
(1)把培养有HRCEC的六孔板每孔换成2ml新鲜DMEM培养液；
(2)取两个洁净无菌离心管，各加入125μl不含抗生素和血清的Opti-MEM，之后分别加2μg pCDH-Vector与pCDH-OLA1；并用枪轻轻吹打混匀；再加入4μl Lipo8000TM转染试剂，用枪轻轻吹打混匀；
(3)每孔125μl Lipo8000TM转染试剂-DNA混合物均匀加到孔内，轻轻混匀。
(4)48h后加puromycin(1μg/mL)进行筛选；
(5)筛选一周后，进行Westernblot实验检测转染效率；
请参阅图1，Western显示成功构建HRCEC OLA1过表达稳转细胞系，过表达效率达到95％。
B、成管实验：
C、(1)提前一天将matrigel从-80℃拿到4℃融化；
D、(2)于冰上将matrigel与DMEM 1：1混合，96孔板铺板，50μL/孔，放入培养箱30min；
E、(3)分别取每组细胞重悬于ECM培养基中，以3万/孔，每孔100μL种于96孔板中；
F、(3)4-8小时观察成管情况。
请参阅图2，结果显示过表达OLA1可抑制HRCEC的管形成能力。
实施例2：敲除OAL1稳转细胞系的构建及成管实验：
A、敲除OAL1稳转细胞系的构建
本步骤与实施例1中A步骤基本相同，其不同指出在于，本申请使用三个离心管，并且，三个离心管中分别加入的试剂为2μg sgVector、sgOLA1-H1、sgOLA1-H2和sgOLA1-H3质粒DNA。
结果如图3所示，Western显示成功构建HRCEC OLA1敲降稳转细胞系，敲降效率达到90％-95％。
B、成管实验
本步骤与实施例1中B步骤相同，结果如图4所示，结果显示敲降OLA1促进HRCEC成管。
实施例3：小鼠视网膜免疫荧光验证
(1)将8-10周龄的C57BL/6敲除及对照小鼠配种后持续观察，取同胞p5胎鼠的尾部尖端做基因型鉴定后，用眼科剪和镊子取出眼球后用PBS清洗并浸泡于4％PFA中常温固定2h。
(2)使用眼科剪和镊子于显微镜下仔细分离出视网膜，以防损坏。分离好的视网膜用PBS清洗一遍。
(3)将清洗好的视网膜置于70％乙醇中浸泡30min，之后用TBST(Triton)清洗3次，每次5min。
(4)将清洗好的视网膜置于1％BSA封闭液中室温封闭30min。
(5)将封闭好的视网膜浸没于一抗(COL IV)在4℃层析柜摇床摇晃孵育过夜，过夜后用TBST(Triton)清洗3次，每次5min。
(6)将清洗好的视网膜浸没于二抗(AlexaFluor-594Anti Rabbit)室温避光孵育2h，之后用TBST(Triton)清洗3次，每次5min。
(7)将清洗好的视网膜平展于准备好的载玻片上，将视网膜裁剪成4瓣，滴上防荧光淬灭剂，然后盖上盖玻片。
结果如图5所示，内皮细胞OLA1基因敲除小鼠出生5天后视网膜血管生成增加。
实施例4：小鼠角膜微口袋实验：
(1)实验前一天将基质胶微丸从-80℃冰箱取出，置于实验室4℃冰箱融解。
(2)取7-10周龄的雄性敲除与对照组小鼠，称重麻醉，如图6A所示，在其角膜缘做一开口，植入微丸，待其苏醒后放回笼中。
(3)实验7天后，于裂隙灯显微镜下观察小鼠角膜边缘血管生成情况并拍照，结果如图6B所示。随后取出角膜，做角膜血管免疫荧光染色，观察角膜血管生成情况，请参阅图6C、D。角膜微口袋及角膜免疫荧光染色显示，内皮细胞OLA1敲除小鼠角膜中新生血管增加。
综上，本发明通过体内外实验验证了OLA1对视网膜和角膜中血管异常增生具有抑制作用，为临床上眼部血管异常增生性疾病提供治疗策略。
Claims (6)
1.OLA1在制备治疗眼部血管异常增生疾病中药物中的应用。
2.根据权利要求1所述的OLA1在制备治疗眼部血管异常增生疾病中药物中的应用，其特征在于：所述药物用于抑制视网膜中血管新生。
3.根据权利要求1所述的OLA1在制备治疗眼部血管异常增生疾病中药物中的应用，其特征在于：所述药物用于抑制角膜中的新生血管。
4.OLA1治疗眼部血管异常增生性疾病的验证方法，其特征在于：包括视网膜血管异常增生性疾病的验证方法及角膜血管增生性疾病的验证方法。
5.根据权利要求4所述的OAL1治疗眼部血管异常增生性疾病的验证方法，其特征在于：所述视网膜血管异常增生性疾病的验证方法包含以下步骤：
S1：建立过表达/敲除OAL1稳转细胞系
S2：在过表达/敲除OAL1稳转细胞系中利用成管实验检测OAL1对HRCEC管形成的影响；
S3：构建内皮细胞特性OAL1基因敲除小鼠，并通过免疫荧光观察出生后5天小鼠视网膜血管发育情况。
6.根据权利要求4所述的OAL1治疗眼部血管异常增生性疾病的验证方法，其特征在于：所述角膜血管增生性疾病的验证方法包含以下步骤：
S1：制备含肝素钠的基质胶微丸，并将基质胶微丸发分别植入到对照与敲除OAL1的小鼠角膜基质层中，1周后裂隙灯下观察小鼠角膜新生血管情况；
S2：将角膜进行COLIV免疫荧光染色，观察血管标志物表达情况。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311227321.0A CN117398448A (zh) | 2023-09-22 | 2023-09-22 | Ola1在治疗眼部血管异常增生性疾病中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311227321.0A CN117398448A (zh) | 2023-09-22 | 2023-09-22 | Ola1在治疗眼部血管异常增生性疾病中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117398448A true CN117398448A (zh) | 2024-01-16 |
Family
ID=89486195
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311227321.0A Pending CN117398448A (zh) | 2023-09-22 | 2023-09-22 | Ola1在治疗眼部血管异常增生性疾病中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117398448A (zh) |
-
2023
- 2023-09-22 CN CN202311227321.0A patent/CN117398448A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Landsend et al. | Congenital aniridia–A comprehensive review of clinical features and therapeutic approaches | |
| Ho et al. | Experience with a subretinal cell-based therapy in patients with geographic atrophy secondary to age-related macular degeneration | |
| Mora et al. | Comparative Analysis of the Safety and Functional Outcomes of Anterior versus Retropupillary Iris‐Claw IOL Fixation | |
| Lang | Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege | |
| CN102048756B (zh) | 人脂肪来源的间充质干细胞在肾脏、眼底疾病中的用途 | |
| Lois et al. | A new model of posterior capsule opacification in rodents | |
| WO2020083007A1 (zh) | Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用 | |
| JP2020511166A (ja) | 融合タンパク質とその製造方法及び使用 | |
| Zhao et al. | IL-10 inhibits retinal pigment epithelium cell proliferation and migration through regulation of VEGF in rhegmatogenous retinal detachment | |
| Zhang et al. | Experimental models and examination methods of retinal detachment | |
| Liu et al. | The differential expression of cytokines and growth factors after SMILE compared with FS-LASIK in rabbits | |
| Shihan et al. | αVβ8 integrin targeting to prevent posterior capsular opacification | |
| CN105418769B (zh) | 一种具有抗肿瘤、抗炎症和治疗眼科疾病功能的融合蛋白及其制备方法和应用 | |
| Fries et al. | A cross-sectional analysis of 556 eyes entering the homburg aniridia centre | |
| CN102205110B (zh) | 整合素阻断剂在制备治疗新生血管性眼病药物中的应用 | |
| CN117398448A (zh) | Ola1在治疗眼部血管异常增生性疾病中的应用 | |
| Dong et al. | RNA interference reveals the coregulatory effects of cylindromatosis on apoptosis and necroptosis of photoreceptor cells in experimental retinal detachment | |
| Khurana et al. | Surgical outcomes of cataract surgery in anterior and combined persistent fetal vasculature using a novel surgical technique: a single center, prospective study | |
| Gour et al. | New horizons in aniridia management: Clinical insights and therapeutic advances | |
| CN113577066B (zh) | 芳基胍化合物或其药学上可接受的盐的用途 | |
| CN108721315B (zh) | 小分子核酸miR-21在治疗青光眼中的应用 | |
| Caporossi et al. | DSAEK‐derived glider technique to introduce human amniotic membrane patch through small‐gauge trocar for retinal pathologies. | |
| Alsalloum et al. | Culture of human retinal explants as an ex vivo model for retinal gene therapy | |
| Wang et al. | Clinical Observation of Phacoemulsification Combined with Intravitreal Injection of Conbercept in Cataract Patients with Diabetic Macular Edema | |
| WO2023078099A1 (zh) | 一种用于治疗神经损伤疾病的基因药物 |
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 |