CN107325062A - A kind of hydrogen peroxide activated fluorescence probe of detection and preparation and application - Google Patents
A kind of hydrogen peroxide activated fluorescence probe of detection and preparation and application Download PDFInfo
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- CN107325062A CN107325062A CN201710651377.7A CN201710651377A CN107325062A CN 107325062 A CN107325062 A CN 107325062A CN 201710651377 A CN201710651377 A CN 201710651377A CN 107325062 A CN107325062 A CN 107325062A
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- hydrogen peroxide
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 129
- 239000000523 sample Substances 0.000 title claims abstract description 89
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 39
- 230000006378 damage Effects 0.000 claims abstract description 14
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229960001138 acetylsalicylic acid Drugs 0.000 claims abstract description 11
- 230000001681 protective effect Effects 0.000 claims abstract description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 230000006837 decompression Effects 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 35
- 150000001875 compounds Chemical class 0.000 claims description 15
- 238000011282 treatment Methods 0.000 claims description 15
- 210000004556 brain Anatomy 0.000 claims description 13
- 239000000975 dye Substances 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000006907 apoptotic process Effects 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 7
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 5
- 210000001519 tissue Anatomy 0.000 claims description 5
- 239000005357 flat glass Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- 206010008089 Cerebral artery occlusion Diseases 0.000 claims description 3
- PQMOXTJVIYEOQL-UHFFFAOYSA-N Cumarin Natural products CC(C)=CCC1=C(O)C(C(=O)C(C)CC)=C(O)C2=C1OC(=O)C=C2CCC PQMOXTJVIYEOQL-UHFFFAOYSA-N 0.000 claims description 3
- FSOGIJPGPZWNGO-UHFFFAOYSA-N Meomammein Natural products CCC(C)C(=O)C1=C(O)C(CC=C(C)C)=C(O)C2=C1OC(=O)C=C2CCC FSOGIJPGPZWNGO-UHFFFAOYSA-N 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 210000005013 brain tissue Anatomy 0.000 claims description 3
- 238000007334 copolymerization reaction Methods 0.000 claims description 3
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 201000007309 middle cerebral artery infarction Diseases 0.000 claims description 3
- CNRNYORZJGVOSY-UHFFFAOYSA-N 2,5-diphenyl-1,3-oxazole Chemical class C=1N=C(C=2C=CC=CC=2)OC=1C1=CC=CC=C1 CNRNYORZJGVOSY-UHFFFAOYSA-N 0.000 claims description 2
- KYNSBQPICQTCGU-UHFFFAOYSA-N Benzopyrane Chemical compound C1=CC=C2C=CCOC2=C1 KYNSBQPICQTCGU-UHFFFAOYSA-N 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 108010040476 FITC-annexin A5 Proteins 0.000 claims description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 2
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 claims description 2
- 125000002252 acyl group Chemical group 0.000 claims description 2
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical class C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 238000004113 cell culture Methods 0.000 claims description 2
- 208000014674 injury Diseases 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 150000002790 naphthalenes Chemical class 0.000 claims description 2
- 150000003233 pyrroles Chemical class 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000000684 flow cytometry Methods 0.000 claims 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims 1
- 230000008733 trauma Effects 0.000 claims 1
- -1 ethyl (3 dimethylaminopropyl) Chemical group 0.000 abstract description 10
- 230000036542 oxidative stress Effects 0.000 abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000004440 column chromatography Methods 0.000 abstract description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 abstract 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 10
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 10
- 239000002994 raw material Substances 0.000 description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 102000006587 Glutathione peroxidase Human genes 0.000 description 2
- 108700016172 Glutathione peroxidases Proteins 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 229940049706 benzodiazepine Drugs 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000006213 oxygenation reaction Methods 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- YNMGRZLDRLHRTN-UHFFFAOYSA-N 1,2,3,3-tetramethyl-2h-indole Chemical class C1=CC=C2C(C)(C)C(C)N(C)C2=C1 YNMGRZLDRLHRTN-UHFFFAOYSA-N 0.000 description 1
- HOSGXJWQVBHGLT-UHFFFAOYSA-N 6-hydroxy-3,4-dihydro-1h-quinolin-2-one Chemical group N1C(=O)CCC2=CC(O)=CC=C21 HOSGXJWQVBHGLT-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010048554 Endothelial dysfunction Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 102000002933 Thioredoxin Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007323 disproportionation reaction Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000008694 endothelial dysfunction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- AWJUIBRHMBBTKR-UHFFFAOYSA-N isoquinoline Chemical compound C1=NC=CC2=CC=CC=C21 AWJUIBRHMBBTKR-UHFFFAOYSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- FNEZBBILNYNQGC-UHFFFAOYSA-N methyl 2-(3,6-diamino-9h-xanthen-9-yl)benzoate Chemical compound COC(=O)C1=CC=CC=C1C1C2=CC=C(N)C=C2OC2=CC(N)=CC=C21 FNEZBBILNYNQGC-UHFFFAOYSA-N 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 238000012831 peritoneal equilibrium test Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 238000012636 positron electron tomography Methods 0.000 description 1
- 238000012877 positron emission topography Methods 0.000 description 1
- 230000009993 protective function Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- SMQUZDBALVYZAC-UHFFFAOYSA-N salicylaldehyde Chemical compound OC1=CC=CC=C1C=O SMQUZDBALVYZAC-UHFFFAOYSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 125000000437 thiazol-2-yl group Chemical group [H]C1=C([H])N=C(*)S1 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
- C07C69/84—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring
- C07C69/90—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring with esterified hydroxyl and carboxyl groups
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/12—Radicals substituted by oxygen atoms
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1018—Heterocyclic compounds
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Materials Engineering (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The present invention provides a kind of detection hydrogen peroxide activated fluorescence probe, by the way that aspirin is dissolved in dichloromethane, add 1 hydroxybenzotriazole, 1 ethyl (3 dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, the fluorogen containing hydroxyl is added, is spin-dried for through extracting decompression, column chromatography is obtained.The detection method for the fluorescence probe that the present invention is provided is easy, and probe is added in detection architecture and is incubated 10 minutes survey fluorescence intensities, you can result is provided, without other reagents auxiliary；Detection hydrogen peroxide activated in living cells level tissue can be realized；While being detected to hydrogen peroxide, certain protective effect can be played to oxidativestress damage.The fluorescence probe of the present invention can be in detection solution in concentration of hydrogen peroxide application, also can it is hydrogen peroxide activated in detection cell or tissue in application and the application in detection living cells to the protective effect of oxidativestress damage.The structural formula of the fluorescence probe：
Description
Technical field
The invention belongs to field of biological detection, it is related to a class and detects hydrogen peroxide activated fluorescence probe, and preparation method thereof
With the application in hydrogen peroxide activated in detecting biological specimen.
Background technology
Generation development of the endothelial dysfunction to cardiovascular and cerebrovascular disease plays important facilitation.Although the two it
Between potential molecular events do not have clear and definite final conclusion yet, still, the generation of oxidative stress is necessarily accompanied with pathogenesis.
Therefore, in neural blood vessel and angiocardiopathy develop, active oxygen radical (ROS) plays the effect of key.Peroxide
Key members in Hua Qingshi active oxygen radicals family, have participation in the pathomechanism of major disease such as including apoplexy.
Hydrogen peroxide level in endothelial cell it is unbalance, cellular redox can be caused disorderly, endothelial cell normal function is destroyed
Performance, to cell cause damage.As enzymatic oxidation molecule, hydrogen peroxide is mainly spontaneous or with superoxides by superoxides
Disproportionation generation.Hydrogen peroxide can be by glutathione peroxidase (GPx), catalase, thioredoxin or peroxide
Change the enzymes such as reductase (Prx) to remove.Hydrogen peroxide can aoxidize some large biological molecules, such as lipid, protein and DNA, so that
Cause cytotoxic damage.Studies have reported that showing that hydrogen peroxide is not limited only to the one side of performance damage, hydrogen peroxide may be used also
So that as second messenger, important regulating and controlling effect is played in physiology course.Therefore, the quality of hydrogen peroxide performance effect is main
Depending on position, local concentration and the accumulation situation of its generation.Carrying out detection to hydrogen peroxide level in biological tissue has weight
The medical diagnosis on disease meaning wanted.
For the detection of hydrogen peroxide, it there is now various ways, including it is isotope of redox-sensitive fluorescin, nanotube, super
Polarization, ultrasound, mass spectrum, PET, chemiluminescence, and hydroperoxidation small-molecule fluorescent probe.It is worth noting that, small
Fluorescence probe is due to high sensitivity, to characteristics such as tissue not damageds, increasingly causing the concern of researcher.However,
Existing commercially available fluorescence probe, such as dichlorodifluoro fluorescein, dihydro rhodamine, lack specificity to ROS.To solve this
Problem, Chang laboratories take the lead in using can carry out the aryl boric acid esters of biocompatibility reaction generation phenols with hydrogen peroxide
Probe.This method provides important method for the biological study of hydrogen peroxide, however, aryl boric acid esters probe is to mistake
Aoxidize nitroso anion (ONOO-) reactivity be higher than hydrogen peroxide, reaction of missing the target in vivo is serious, this shortcoming
Limit its extensive use.Meanwhile, the biological effect of the accessory substance boric acid after such probe reaction is unknown.Therefore, we are right
Its structure is designed transformation.Design has synthesized the novel probe to hydrogen peroxide with high degree of specificity, meanwhile, after it reacts
Product certain protectiveness effect can be played to the oxidativestress damage that hydrogen peroxide is caused.
The content of the invention
Detect hydrogen peroxide activated fluorescence probe it is an object of the invention to provide a kind of, be one kind can quickly in vitro and
Vivo detection is hydrogen peroxide activated and the fluorescence probe of obvious protective function is played to cellular oxidation stress damage.With Formulas I institute
The structure shown：
R is fluorogen such as benzothiazoles, cumarin, fluorescein, 1,8- benzene-naphthalene diimides class, naphthalene series dyes, Luo Dan
Bright, flower cyanines prime system row, TCF (2- dicyano methylene -3- cyano group -4,5,5- trimethyl -2,5- dihydrofuran), NBD (7- nitre
Base-benzo -2- oxa- -1,3- diazole) dyestuff, benzo pyran dyestuff, 2,5- diphenyloxazoles class, the pyrroles of dyestuff fluorine boron two
(BODIPY) etc..
It is real by following preparation method it is a further object to provide the preparation method of the compound shown in Formulas I
It is existing：Aspirin is dissolved in dichloromethane, I-hydroxybenzotriazole, 1- ethyls-(3- dimethylaminopropyls) carbon is added
Acyl diimmonium salt hydrochlorate, adds the fluorogen containing hydroxyl, room temperature reaction, through extraction, depressurizes and is spin-dried for solvent, column chromatography, i.e.,
Compound of formula I can be obtained.
It is also another object of the present invention to provide the answering in concentration of hydrogen peroxide in detecting solution of the compound shown in Formulas I
With being realized by following steps：By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, plus
Enter in system to be detected, make final concentration of 10 micromole of probe, be incubated at room temperature, system is determined on XRF after one hour
Fluorescence intensity, and then determine the concentration of hydrogen peroxide in solution to be detected.
It is hydrogen peroxide activated in detection cell or tissue that a further object of the present invention is to provide the compound shown in Formulas I
In application, realized by following steps：
(1) in cell external source concentration of hydrogen peroxide detection：In vitro culture EA.hy926 cells, are inoculated on sheet glass,
It is used for subsequent experimental after cell density reaches 70-80%.Cell is incubated 5 μM, 10 μM, 20 μM of probe observation probe respectively
Effective working concentration；Give 50 μM, 100 μM, 200 μM, hydrogen peroxide treatment, detection probe minimum detection limit；
(2) in cell endogenous concentration of hydrogen peroxide detection：In vitro culture EA.hy926 cells, are inoculated on sheet glass,
It is used for subsequent experimental after cell density reaches 70-80%.15 minutes incubation probes, cell culture is replaced with sugar-free HBSS in advance
Liquid, is placed in hermetically sealed can, and scarce sugared oxygen deprivation stress damage (OGD) processing of different time is carried out to cell.By copolymerization, Jiao is right
The fluorescence of probe is imaged, and observes the response of probe under the different OGD times；
(3) after mouse brain middle cerebral artery occlusion (MCAO, Middle Cerebral Artery Occlusion) damage
The response of probe：Mouse gives MCAO model treatments, and 3% chloraldurate is given after 24 hours, brain is taken.In cerebrospinal fluid, oxygenation
Under the conditions of to brain tissue carry out slicing treatment, 200 micron thickness.After section, under the conditions of 34 C water baths, cerebrospinal fluid is placed on
Middle incubation probe 15 minutes, using Nikon A1R Laser Scanning Confocal Microscopes to brain piece picture mosaic, observes the fluorescence of full brain middle probe.
The 5th purpose of the present invention is to provide guarantor of the compound shown in Formulas I to oxidativestress damage in detection living cells
The application of shield effect.Realized by following steps：In vitro culture EA.hy926 cells, are inoculated in six orifice plates, treat cell density
Reaching is used for subsequent experimental after 70-80%, give cell 200 μM of hydrogen peroxide, and different time is handled respectively, observes Apoptosis
Rate, and choose right times progress subsequent experimental；It is as follows that experiment sets group：Control group, hydrogen peroxide treatment group, be incubated probe groups,
Probe is incubated in advance and gives hydrogen peroxide treatment group；The double dye method streams of Annexin V-FITC/PI are utilized to above-mentioned packet cell
Formula cell art detects Apoptosis, and the protective effect to probe is estimated.
The fluorescence probe that the present invention is provided unstressed configuration in aqueous, but can occur specific reaction with hydrogen peroxide in itself,
Product of the generation with hyperfluorescence, while the aspirin with antioxidant effect is discharged, so as to realize the special of hydrogen peroxide
Sensitivity is detected.The fluorescence probe and detection method that the present invention is provided have the advantages that：(1) detection method is easy, inspection
Probe is added in survey system and is incubated 10 minutes survey fluorescence intensities, you can result is provided, without other reagents auxiliary；(2) it can realize
Hydrogen peroxide activated detection in living cells level tissue；(3), can be to oxidative stress while being detected to hydrogen peroxide
Certain protective effect is played in damage.
Brief description of the drawings
Fig. 1 is probe molecule Ia with changing before and after hydroperoxidation.
Fig. 2 is probe molecule Ib with changing before and after hydroperoxidation.
Fig. 3 is probe molecule Ic with changing before and after hydroperoxidation.
Fig. 4 is probe molecule Id with changing before and after hydroperoxidation.
Fig. 5 is probe molecule Ie with changing before and after hydroperoxidation.
Fig. 6 is to give various concentrations hydrogen peroxide, and the fluorescence intensity of probe gradually strengthens.
Fig. 7 is with OGD time lengthenings, fluorescence probe intensity enhancing.
Fig. 8 is that after MCAO is damaged, fluorescence is shown after being incubated probe.
Fig. 9 is the Apoptosis protective effect that probe is caused to hydrogen peroxide.
Embodiment
The following examples are that the present invention is further illustrated, rather than limitation the scope of the present invention.
Embodiment 1：Probe molecule Formulas I a synthesis
Raw material aspirin (386mg, 2.14 mMs) is dissolved in 20 milliliters of dichloromethane, HOBT is added under ice bath
(261mg, 1.93 mMs), EDC hydrochlorides (370mg, 1.93 mMs), is stirred at room temperature 20 minutes, adds 2- (benzos [d]
Thiazol-2-yl) -4- fluorophenols (262mg, 1.07 mMs), DIPEA (44 μ L, 2.68 mMs) is stirred at room temperature, TLC detections
Raw material reaction is finished, and is added water and is quenched, dichloromethane extraction, then is washed with saturated sodium-chloride, and anhydrous sodium sulfate drying was spin-dried for post.
Probe I a is obtained, yield is 36.7%.1H NMR(500MHz,CDCl3,δ):8.43 (dd, J=7.9,1.6Hz, 1H), 8.15 (dd,
J=9.2,2.9Hz, 1H), 7.99 (d, J=8.1Hz, 1H), 7.85 (d, J=8.0Hz, 1H), 7.73 (td, J=8.0,
1.6Hz, 1H), 7.48 (tt, J=7.5,1.0Hz, 2H), 7.38 (t, J=7.5Hz, 1H), 7.31-7.23 (m, 3H), 2.27
(s,3H)。
Embodiment 2：Probe molecule Formulas I b synthesis
Raw material aspirin (386mg, 2.14 mMs) is dissolved in 20 milliliters of dichloromethane, HOBT is added under ice bath
(261mg, 1.93 mMs), EDC hydrochlorides (370mg, 1.93 mMs), is stirred at room temperature 20 minutes, adds 1- (6- hydroxyls
Naphthalene -2- bases) second -1- ketone (200mg, 1.07 mMs), DIPEA (44 μ L, 2.68 mMs) is stirred at room temperature, TLC detection raw materials
Reaction is finished, and is added water and is quenched, dichloromethane extraction, then is washed with saturated sodium-chloride, and anhydrous sodium sulfate drying was spin-dried for post.It must visit
Pin Ib, yield is 87.4%.1H NMR(500MHz,CDCl3,δ):8.43 (dd, J=7.9,1.6Hz, 1H), 8.15 (dd, J=
9.2,2.9Hz, 1H), 7.99 (d, J=8.1Hz, 1H), 7.85 (d, J=8.0Hz, 1H), 7.73 (td, J=8.0,1.6Hz,
1H), 7.48 (tt, J=7.5,1.0Hz, 2H), 7.38 (t, J=7.5Hz, 1H), 7.31-7.23 (m, 3H), 2.27 (s, 3H).
Embodiment 3：Probe molecule Formulas I c synthesis
Raw material aspirin (1.110g, 6.16 mMs) is dissolved 20 milliliters in dichloromethane, added under ice bath
HOBT (749mg, 5.54 mMs), EDC hydrochlorides (1.062g, 5.54 mMs), is stirred at room temperature 20 minutes, adds 7- hydroxyls
Cumarin (500mg, 3.08 mMs), DIPEA (1.367mL, 7.7 mMs), is stirred at room temperature, and TLC detection raw materials have reacted
Finish, add water and be quenched, dichloromethane extraction, then washed with saturated sodium-chloride, anhydrous sodium sulfate drying was spin-dried for post.Obtain probe I c, production
Rate is 78.6%.1H NMR(500MHz,CDCl3,δ):8.49 (s, 1H), 8.29 (d, J=8.0,1H), 8.08 (d, J=
8.5Hz, 1H), 8.04 (d, J=9.0Hz, 1H), 7.89 (d, J=8.5Hz, 1H), 7.70 (s, 1H), 7.68 (d, J=8.0Hz,
1H), 7.44-7.40 (m, 2H), 7.22 (d, J=8.0Hz, 1H), 2.73 (d, J=1.0Hz, 3H), 2.32 (d, J=1.5Hz,
3H)。
Embodiment 4：Probe molecule Formulas I d synthesis
Raw material aspirin (317mg, 1.76 mMs) is dissolved in 20 milliliters of dichloromethane, HOBT is added under ice bath
(213mg, 1.58 mMs), EDC hydrochlorides (303mg, 1.58 mMs), is stirred at room temperature 20 minutes, adds 6- hydroxyl -2- first
Base -1H- benzos [de] isoquinolin -1,3 (2H)-diketone (200mg, 0.88 mM), DIPEA (363 μ L, 2.2 mMs), room
Temperature stirring, TLC detection raw material reactions are finished, and are added water and are quenched, dichloromethane extraction, then are washed with saturated sodium-chloride, anhydrous sodium sulfate
Dry, be spin-dried for post.Probe-type Id is obtained, yield is 43.9%.1H NMR(500MHz,CDCl3,δ):8.67-8.64(m,2H),
8.39 (dd, J=8.0,1.5Hz, 1H), 8.31 (dd, J=8.5,1.0Hz, 1H), 7.80-7.77 (m, 1H), 7.76 (td, J=
7.8,1.5Hz, 1H), 7.64 (d, J=8.0Hz, 1H), 7.50 (dt, J=7.5,1.0Hz, 1H), 7.27 (dd, J=8.0,
1.0Hz,1H),3.58(s,3H),2.26(s,3H)。
Embodiment 5：Probe molecule Formulas I e synthesis
Raw material aspirin (2.955g, 16.4 mMs) is dissolved in 70 milliliters of dichloromethane, HOBT is added under ice bath
(1.994g, 14.76 mMs), EDC hydrochlorides (2.829g, 14.76 mMs), is stirred at room temperature 20 minutes, adds to hydroxyl
Benzaldehyde (1g, 8.2 mMs), DIPEA (3.388mL, 20.5 mMs), is stirred at room temperature, and TLC detection raw material reactions are finished,
Add water and be quenched, dichloromethane extraction 10ml, then wash with saturated sodium-chloride 10 milliliters, anhydrous sodium sulfate drying was spin-dried for post.In obtaining
Mesosome Aspirin 4- formoxyl phenyl esters, yield is 85.8%.
By Aspirin 4- formoxyls phenyl ester (324mg, 1.14 mMs) and 1,2,3,3- tetramethyl -3H-
Indoles -1- (100mg, 0.57 mM) is dissolved in 15 milliliters of toluene, adds piperidines (30 μ L, 0.33 mM), heating
80 C overnights react, and suction filtration washes solid with toluene twice.Vacuum drying, obtains probe I e, yield is 63.7%.1H NMR
(500MHz,CDCl3,δ):8.26 (s, 1H), 8.25 (d, J=15Hz, 2H), 8.21 (dd, J=7.5,1.5Hz, 1H), 7.84
(d, J=16Hz, 1H), 7.69-7.64 (m, 2H), 7.60-7.55 (m, 3H), 7.43 (dt, J=7.8,1.0Hz, 1H), 7.36
(d, J=8.5Hz, 2H), 7.20 (dd, J=8.0,0.5Hz, 1H), 4.47 (s, 3H), 2.31 (s, 3H), 1.87 (s, 6H),
1.63(s,3H)。
Embodiment 6：H in compound test PBS solution shown in Formulas I a2O2The application of concentration
By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, adds and contains H2O2PBS
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree.With maximum excitation wavelength, 375 nanometers are to excite, and collect 475 nanometers of maximum emission wavelength, and then determine the mistake in PBS solution
Hydrogen peroxide concentration, as a result referring to Fig. 1.
Embodiment 7：H in compound test PBS solution shown in Formulas I b2O2The application of concentration
By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, adds and contains H2O2PBS
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree.With maximum excitation wavelength, 324 nanometers are to excite, and collect 497 nanometers of maximum emission wavelength, and then determine the mistake in PBS solution
Hydrogen peroxide concentration, as a result referring to Fig. 2.
Embodiment 8：H in compound test PBS solution shown in Formulas I c2O2The application of concentration
By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, adds and contains H2O2PBS
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree.With maximum excitation wavelength, 324 nanometers are to excite, and collect 453 nanometers of maximum emission wavelength, and then determine the mistake in PBS solution
Hydrogen peroxide concentration, as a result referring to Fig. 3.
Embodiment 9：H in compound test PBS solution shown in Formulas I d2O2The application of concentration
By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, adds and contains H2O2PBS
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree.With maximum excitation wavelength, 374 nanometers are to excite, and collect 545 nanometers of maximum emission wavelength, and then determine the mistake in PBS solution
Hydrogen peroxide concentration, as a result referring to Fig. 4.
Embodiment 10：H in compound test PBS solution shown in Formulas I d2O2The application of concentration
By a small amount of dmso solution of probe molecule, storing solution is prepared；A small amount of storing solution is taken, adds and contains H2O2PBS
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree.With maximum excitation wavelength, 517 nanometers are to excite, and collect 552 nanometers of maximum emission wavelength, and then determine the mistake in PBS solution
Hydrogen peroxide concentration, as a result referring to Fig. 5.
Embodiment 11：It is hydrogen peroxide activated in probe molecule detection EA.hy926 cells
EA.hy926 cells shift to an earlier date after 15 minutes incubation probes, and the hydrogen peroxide of various concentrations is given respectively, passes through copolymerization
Focusing microscope is observed, and can substantially find the raising with concentration of hydrogen peroxide, the fluorescence intensity of probe substantially increases.
EA.hy926 cells shift to an earlier date after 15 minutes incubation probes, and the hydrogen peroxide for giving same concentrations is handled.Pass through
Confocal microscopy, it has been found that with the extension of processing time, the fluorescence intensity of probe gradually increases.
EA.hy926 cells shift to an earlier date after 15 minutes incubation probes, give OGD processing.By confocal microscopy, I
Find, with the extension of OGD processing times, the fluorescence intensity of probe gradually increases.Concrete outcome is referring to Fig. 6 and Fig. 7.
Similar phenomenon, we can be repeated in HUVEC cell lines.
Embodiment 12：It is hydrogen peroxide activated in probe molecule detection mouse brain slices
Mouse gives MCAO model treatments, after modeling 24 hours, injects 3% chloraldurate, takes brain.In cerebrospinal fluid, oxygenation
Under the conditions of to brain tissue carry out slicing treatment, 200 micron thickness.After section, under the conditions of 34 C water baths, cerebrospinal fluid is placed on
Middle incubation probe 15 minutes, using Nikon A1R Laser Scanning Confocal Microscopes to brain piece picture mosaic, observes the fluorescence of full brain middle probe.It is logical
Experimental result is crossed it was found that after modeling, the fluorescence intensity of the probe in brain piece substantially increases, and concrete outcome is referring to Fig. 8.
Embodiment 13：Protective effect of the probe molecule to Hydroperoxide injury
In vitro culture EA.hy926 cells, are inoculated in six orifice plates, are used for after cell density reaches 70-80% follow-up real
Test.Give cell 200 μM of hydrogen peroxide, different time is handled respectively, observe apoptosis rate, and choose after right times progress
Continuous experiment.It is as follows that experiment sets group：Control group, hydrogen peroxide treatment group, probe groups are incubated, probe is incubated in advance and peroxidating is given
Hydrogen treatment group.FCM analysis is carried out to above-mentioned packet cell, passes through experimental result, it has been found that hydrogen peroxide treatment group
The apoptosis rate of middle cell substantially increases, and in probe incubation group, apoptosis rate is decreased obviously.Specific experiment result is referring to Fig. 9.
Claims (8)
1. a kind of detect hydrogen peroxide activated fluorescence probe, it is characterised in that with the structure shown in Formulas I：
R is fluorogen such as benzothiazoles, cumarin, fluorescein, 1,8- benzene-naphthalene diimides class, naphthalene series dyes, rhodamine, flower
Cyanines prime system row, 2- dicyano methylene -3- cyano group -4,5,5- trimethyl -2,5- dihydrofuran dyestuff, 7- nitros-benzo -2- oxygen
The miscellaneous thiiazole dyes of -1,3- two, benzo pyran dyestuff, 2,5- diphenyloxazoles class, the pyrroles of dyestuff fluorine boron two.
2. the preparation method of the compound of formula I described in claim 1, it is characterised in that realized by following steps：
Aspirin is dissolved in dichloromethane, I-hydroxybenzotriazole, 1- ethyls-(3- dimethylaminopropyls) carbon is added
Acyl diimmonium salt hydrochlorate, adds the fluorogen containing hydroxyl afterwards, room temperature reaction, through extraction, and decompression is spin-dried for solvent, post layer
Analysis, produces compound of formula I.
3. application of the compound of formula I according to claim 1 in detection solution in concentration of hydrogen peroxide, its feature exists
In being realized by following steps：By probe molecule dmso solution, storing solution is prepared；Storing solution is taken, is added to be detected
In system, make final concentration of 10 micromole of probe, be incubated at room temperature, measure system fluorescence is strong on XRF after one hour
Degree, and then determine the concentration of hydrogen peroxide in solution to be detected.
The application during 4. compound of formula I according to claim 1 is hydrogen peroxide activated in detection cell or tissue.
5. application according to claim 4, it is characterised in that realized by following steps：
(1) in cell external source concentration of hydrogen peroxide detection：In vitro culture EA.hy926 cells, are inoculated on sheet glass, treat thin
Born of the same parents' density, which reaches, is used for subsequent experimental after 70-80%, cell is incubated the effective of 5 μM, 10 μM, 20 μM of probe observation probe respectively
Working concentration；Give 50 μM, 100 μM, 200 μM, hydrogen peroxide treatment, detection probe minimum detection limit；
(2) in cell endogenous hydrogen peroxide concentration detection：In vitro culture EA.hy926 cells, are inoculated on sheet glass, treat
Cell density, which reaches, is used for subsequent experimental after 70-80%, in advance 15 minutes incubation probes, and cell culture is replaced with sugar-free HBSS
Liquid, is placed in hermetically sealed can, and the scarce sugared oxygen deprivation stress damage that different time is carried out to cell is handled, by copolymerization Jiao to probe
Fluorescence is imaged, the different responses for lacking probe under sugared oxygen deprivation stress trauma time of observation.
(3) response of probe after mouse brain middle cerebral artery occlusion is damaged：Mouse gives MCAO model treatments, is given after 24 hours
3% chloraldurate is given, brain is taken, slicing treatment, 200 micron thickness, section is carried out to brain tissue under cerebrospinal fluid, aerobic condition
Afterwards, under the conditions of 34 C water baths, it is placed in cerebrospinal fluid and is incubated probe 15 minutes, utilize Nikon A1R Laser Scanning Confocal Microscopes
To brain piece picture mosaic, the fluorescence of full brain middle probe is observed.
6. compound of formula I according to claim 1 is made in detection living cells to the protectiveness of hydrogen peroxide stress damage
Application in.
7. application according to claim 6, it is characterised in that realized by following steps：In vitro culture EA.hy926 is thin
Born of the same parents, are inoculated in six orifice plates, and subsequent experimental is used for after cell density reaches 70-80%, gives cell 200 μM of hydrogen peroxide,
Different time is handled respectively, apoptosis rate is observed, and chooses right times progress subsequent experimental, and it is as follows that experiment sets group：Control
Group, hydrogen peroxide treatment group, probe groups are incubated, is incubated probe and gives hydrogen peroxide treatment group in advance, to above-mentioned packet cell
FCM analysis is carried out, the protective effect to probe is estimated.
8. application according to claim 7, it is characterised in that Annexin V-FITC/PI are utilized to above-mentioned packet cell
Double dye method Apoptosis by Flow Cytometries.
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