CN107496432A - A kind of method by suppressing caveolin delaying cell aging - Google Patents
A kind of method by suppressing caveolin delaying cell aging Download PDFInfo
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- A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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Abstract
The invention belongs to biotechnology, medicine, health products and cosmetic field, and in particular to a kind of method by suppressing caveolin delaying cell aging and its application in medicine, health products and cosmetic field.The inventive method is by suppressing caveolin, reduce the combination of p47 regulation subunits and cell membrane in nicotinamide-adenine dinucleotide phosphate oxidizing ferment NOX2 hypotypes, hinder the performance of nicotinamide-adenine dinucleotide phosphate oxidizing ferment NOX2 hypotypes effect, and then reduce the generation of reactive oxygen species, suppress the generation of factor M MP2/9 downstream, the degree of Cellular Oxidation is reduced, so as to realize the purpose of delaying cell aging process.Using the inventive method prepare anti-aging product is widely applicable, safe to use, Small side effects and can fundamentally intervene aging course, be easy to be easily accepted by the patient.
Description
Technical field
The invention belongs to biotechnology, medicine, health products and cosmetic field, and in particular to one kind is by suppressing alveole egg
The method of white delaying cell aging and its application in medicine, health products and cosmetic field.
Background technology
Skin aging is the skin senescence phenomenon as caused by natural cause or non-natural factor, and its essence is Skin Cell
Aging.Skin aging is mainly manifested in skin histology decline and low two aspects of physiological function.Many factors can all cause skin
Skin aging, such as age growth, disease, mental element, malnutrition, bad life habits, environmental factor, endocrine disturbance, skin
Skin, which maintains improper, inappropriate medication etc., can cause and accelerate skin aging.
The method for the treatment of skin aging includes non-operative treatment and the major class of operation method two.Non-operative treatment is treated including medicine
Method, Chemical peeling method, microwave therapy, cosmetics, ferment etc., wherein, medicinal treatment mainly carries out bioactivity to Skin Cell
Regulate and control to improve skin-nourishing situation；Chemical peeling method mainly by removing the horny layer of epidermis of aging, promotes basal cell to increase
It is raw, aging collagenous fibres are repaired, improve skin tension and elasticity；Microwave therapy using the microwave action of different wave length in skin and
Subcutaneous each level, promote to recover skin elasticity vigor, stimulate collagen fiber hyperplasia reparation；Cosmetics are by removing free radical, pre-
Antiultraviolet, promote skin cell metabolism, replenishing collagen and elastin laminin, moisturizing and repair skin barrier function etc.
Play a role；Ferment can eliminate bodily waste, balance intestinal bacterium, strengthen metabolism.Operation method includes wiping skin, skin
Lower filling and face crinkle-removing etc..
However, above-mentioned therapy still respectively has weak point, for example, medicine, Chemical peeling agent and microwave can produce different journeys
The side effect of degree, pigmentation and superficial scar are such as caused, drug dependence is produced, causes microwave radiation damage；Cosmetics
Reach skin effect by keeping the skin wet, increasing the means such as collagen with ferment etc., but can not fundamentally intervene aging
Journey, it can only play a part of " cover and modify "；And operation method can cause surgical injury, be also easy to produce postoperative complications, risk
It is larger and costly, therefore applicable surface is narrower.
Caveolin (Caveolin-1) be in a kind of main membrane in caveolae of cell surface (caveolae)
Albumen, played a role in caveolae integrality, the transport of utricle, the conduction of signal is kept.Caveolin is being permitted
Important adjustment effect has occurred for high expression in many cells, the infiltration of transhipment, endothelium to material and tumour, in addition, alveole
Albumen is probably a kind of tumor suppressor gene, and its mutation is had detected that in human tumor.
However, up to the present, there is not yet by suppressing caveolin come the relevant report of delaying cell aging, do not have yet
There is the anti-aging product prepared using the method to emerge.
The content of the invention
The main cause that human inner cell's vigor lowers is cell ageing caused by Cellular Oxidation, active oxygen
(ROS) fundamental role is played in oxidizing process in the cell.The generation of ROS materials makes cell enter accelerated ageing, under it
Factor M MP2/9 generation is swum, is the immediate cause of skin senescence.In ROS various sources, NOX2 hypotype nicotinoyl amine glands are fast
Nicotinamide adenine dinucleotide phosphoric acid oxidizing ferment (NADPH oxidase) approach is representative one.
Nadph oxidase NOX2 hypotypes are the complexs of a multiple proteins, one of regulation subunit p47 and cell
The combination of film, drive whole oxidizing process.Our research finds that the combination of p47 regulation subunits and cell membrane is because its envelope
On caveolin attracted.The combination of p47 regulation subunits and cell membrane can be reduced by suppressing caveolin, hinder NADPH oxygen
Change the performance of enzyme effect, and then reduce the generation of ROS materials, it is suppressed that MMP2/9 generation is thin so as to greatly slow down
Born of the same parents' aging course, it is significant to antagonism skin aging.
Specifically, nadph oxidase NOX2 can be activated by p47 phosphorylations, activation signals, then endochylema subunit moves
Functioning cell pigment b558 is formed to film.In resting cell, an automatic holddown, its dual SH3 domain is presented
Comprising C-terminal sequence, excessive stimulation causes its phosphorylation, phosphorylation site on some residues generally to reflect different stimulated
Property, and fine setting consequence can be produced.However, in a general activation method, P47 phosphorylation causes film aggregation and ROS
Generation.
Atherosclerosis is inflammation and the result of cell ageing, living during the occurrence and development of atherosclerosis
The generation of property oxygen (ROS) is considered as a crucial promoting factor, and nadph oxidase NOX2 is then ROS main source.
In addition, membrane structure is deposited on vascular endothelial cell with OxLDL ELISA, and caveolin is how to participate in oxLDL mediations
Active oxygen generation process at present it is still unclear.It was found that the protein expression exposed to OxLDL ELISA raises
Macrophage, and and then raise that P47 in nadph oxidase NOX2 is horizontal, and this synergy causes the increase of cell ageing, demonstrate,prove
Real oxLDL processing causes P47 from cytoplasm indexing to Plasma membranes.In addition, immunoprecipitation experiment confirm, caveolin with
P47 has the relevance of height.The above results show that caveolin-1 stimulates ROS possibly as to P47 films target and for oxLDL
Caused switch.Our result of study discloses what molecular events unknown before one mediated in OxLDL ELISA
Effect in cell senescence, and a new target may be provided for the clinical intervention of atherosclerosis.
As a kind of membrane structure for participating in atherosclerosis, alveole is that 50-100 nanofilmstructures combine to form with Lipid Rafts
's.After being upset, they may form flask shape and cave in into cell and interior body.It is few different from traditional end body
Evidence shows that alveole internalization forms to have with interior body/lysosome and associated.On atherosclerosis, caveolin system has transport
LDL steps up the effect of chrotoplast and vascular wall cholesterol.However, increasing evidence shows that caveolin simultaneously participates in
Signal transduction.The expression that the formation of caveolae structures is to rely on cFLIP forms the support for supporting ampuliform mould trace.In addition,
Caveolin can be associated with substantial amounts of protein and lipid material.Caveolin has an internal sequence, be embedded in N and
In the free cytoplasm of C-terminal.One caveolin domain (CSD) is bound with multiple protein, such as Src family kinases, MAPKs and NO
Synthase, although in some cases, this association is the negativity regulation and control of related protein function.
Since have been acknowledged that nadph oxidase and caveolin-1 take part in cell ageing process, it is therefore necessary to enter
How one step research P47 and caveolin interact and cause the aging course of cell, especially for atherosclerosis
Meaning.In our current research, we show the aging of OxLDL ELISA mediation.We first confirm that oxLDL mediates table
Up to Caveolin-1 and P47 chain event, the missing of caveolin causes P47 protein expressions to reduce, Caveolin-1 expression
With increase oxidation reaction and the relation of cell ageing；Secondly, it has been found that stimulated in increase OxLDL ELISA
Caveolin-1 and P47 and ROS, which is produced, to be seemed to be only limited to caveolin.These discoveries help that machine occurs to atherosclerosis
Reason is furtherd investigate.
Based on the studies above, the invention provides a kind of method by suppressing caveolin delaying cell aging, pass through
Suppress caveolin, reduce p47 regulation subunits and cell membrane in nicotinamide-adenine dinucleotide phosphate oxidizing ferment NOX2 hypotypes
Combination, hinder the performance of nicotinamide-adenine dinucleotide phosphate oxidizing ferment NOX2 hypotypes effect, and then reduce active oxygen thing
The generation of matter, suppress the generation of factor M MP2/9 downstream, the degree of Cellular Oxidation is reduced, so as to realize delaying cell aging
The purpose of process.
Preferably, suppress caveolin in the inventive method to complete using caveolin inhibitor.
It is further preferred that the caveolin inhibitor used in the inventive method includes incadronate
(incadronate), filipin rhzomorph (filipin) and Genistein (genistein).
Moreover, it relates to a kind of declined preparing anti-skin by suppressing the method for caveolin delaying cell aging
Application in old medicine.
Meanwhile the invention further relates to a kind of method by suppressing caveolin delaying cell aging in preparation treatment artery
Application in anti-atherosclerotic agent.
Meanwhile declined the invention further relates to a kind of by suppressing the method for caveolin delaying cell aging preparing anti-skin
Application in old health products.
Meanwhile declined the invention further relates to a kind of by suppressing the method for caveolin delaying cell aging preparing anti-skin
Application in aging cosmetic.
The present invention is delaying cell aging, especially slows down skin aging and provides new, more preferable treatment method.Pass through
The method for the suppression caveolin delaying cell aging that the present invention discloses, can be further prepared into system by caveolin inhibitor
Or external application anti aging effect medicine, health products or cosmetics, and then develop the monomer production comprising all kinds of caveolin inhibitor
Product or compound preparation, for delay skin aging.Using the inventive method prepare anti-aging product it is widely applicable, use peace
Entirely, it Small side effects and can fundamentally intervene aging course, be easy to be easily accepted by the patient, it is contemplated that will have huge application
Prospect.
Brief description of the drawings
During Fig. 1 is driven caused by MMP2/9 using the OxLDL ELISA of various concentrations, pass through RNAi technology
MMP2/9 generation can dramatically be reduced by suppressing caveolin.
During Fig. 2 is driven caused by MMP2/9 using the OxLDL ELISA of various concentrations, pass through RNAi technology
Suppressing caveolin can be such that the generation of cell ageing index beta galactosidase (β-galactosidase) also drastically declines.
Embodiment
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
Explicitly point out in addition, singulative "one", " one " and " this " include plural form.
Unless otherwise defined, all technologies and scientific terminology that are used in the present invention and those skilled in the art of the present technique are usual
The meaning of understanding is identical.Except used in embodiment specific method, equipment, in addition to material, according to those skilled in the art
The record of grasp and the present invention to prior art, can also be used and the method described in the embodiment of the present invention, equipment, material
Any method, equipment and the material of similar or equivalent prior art realizes the present invention.
The experimental method of unreceipted actual conditions in following embodiments, generally use conventional laboratory conditions or experiment equipment system
Make the condition proposed by manufacturer.
Embodiment 1
Material and method
Cell culture
Raw264.7 cells are placed in RPMI-1640 nutrient solutions (hyclone) and cultivated, and 10% is added into nutrient solution
FBS (Gibco), 100U/ml penicillin and 100mg/ml streptomysin, and 10mM hydroxyethyl piperazine second thiosulfonic acid and 50 μM
Beta -mercaptoethanol.HEK293T cells are cultivated in the DMEM nutrient solutions containing 10% hyclone and above-mentioned antibiotic.
Reagent and antibody
Beta galactosidase staining kit is purchased from Beyotime Biotechnology, is analyzed for co-immunoprecipitation
(Co-immunoprecipitation assay) and immunofluorescence dyeing (immune fluorescent staining)
Anti-p47 antibody and anti-Caveolin antibody are purchased from Santa Cruz and Cell Signaling Technology respectively.
Gene knockout
Five kinds of Cav1 targeting shRNA clones are purchased from Sigma companies, and sequence is as follows：
F11：CCGGCCAGTTAGATTTAGGGATTTACTCGAGTAAATCCCTAAATCTAACTGGTTTTTG；
F12：CCGGCCGCTTGTTGTCTACGATCTTCTCGAGAAGATCGTAGACAACAAGCGGTTTTTG；
G1：CCGGCGACGTGGTCAAGATTGACTTCTCGAGAAGTCAATCTTGACCACGTCGTTTTTG；
G2：CCGGTGAAGCTATTGGCAAGATATTCTCGAGAATATCTTGCCAATAGCTTCATTTTTG；
G3：CCGGGCTTCCTGATTGAGATTCAGTCTCGAGACTGAATCTCAATCAGGAAGCTTTTTG.
Lentiviral particle is packed using the reagent of liposome 2000 (Invitrogen) in 293FT.By shRNA matter
Grain is transfected on packaging plasmid pCMV and envelope plasmid pHCMV-G.Scramble shRNA are as control.After 48 hours, it will contain
The supernatant 2000rpm for having virion is centrifuged 5 minutes, 4 DEG C.Raw264.7 cells press 5 × 105/ hole is seeded on 6 orifice plates
(about 70% fusion).After 12 hours, the 0.5ml culture supernatants for containing slow virus are added to connecing containing 8 μ g/ml polybrenes
In kind hole.Then inoculation plate is continued to cultivate at 32 DEG C after 2500rpm is centrifuged 1 hour.After 24 hours, 3 μ g/ml purine will be contained
The fresh medium of mycin is added in inoculation plate and screened, and antibiotic nutrient solution every other day changes once, screens within the 7th day
Stable shRNA expression cells system.
Cell ageing detects
In cell ageing detection, the Raw264.7 cellular portions through ctrl shRNA or caveolin-1shRNA transfection
With oxLDL processing, after 24 hours, sample is fixed according to the operation instruction of manufacturer and beta galactosidase dyes.Then
Sample is observed under the microscope, counts all cells and positive cell number.
Gelatin histopathological examinations
In gelatin histopathological examinations, cell supernatant is put into 10%SDS-PAGE gel electrophoresises and adds 1% gelatin.
After electrophoresis, gel is dyed with 30% methanol containing 0.05% coomassie brilliant blue R250 and 10% acetum under the conditions of 37 DEG C
3 hours, then gel handled with decolouring buffer solution until there is clearly band, finally, with digital gel imaging system to solidifying
Glue is scanned.
Co-immunoprecipitation is analyzed
In co-immunoprecipitation analysis, oxLDL processing is carried out to part Raw264.7 cells, after 4 hours, cell dissolved
In NP-40 lysis buffers (50mM HEPES, pH7.4,150mM NaCl, 1%NP-40,1mM EDTA, 1mM PMSF, 1mM
NaF, 1mM NaVO3With the mixed liquor of protease inhibitors) in, cytolysate mixes with antibody-pearl mixture under the conditions of 4 DEG C
One night of culture is closed, antibody pearl is then collected by low-speed centrifugation (1000rpm centrifuges 5min) and (added with IP lysis buffers
Add 300mM NaCl) rinse 4 times.After flushing, globule is suspended in 70 μ l 1X SDS sample loading buffers and boiled 5 minutes,
Detected for western blot.
IF is dyed and the micro- detection of fluorescence imaging
In immunofluorescence dyeing, cell is immersed in 4% paraformaldehyde 20 minutes at room temperature, and with containing 10% tire ox
The PBS of serum is handled 30 minutes.Then sample is dyed with anti-p47 and the antibody of anti-caveolin 1, washed with PBS
Wash 3 times, be then incubated and washed again with two kinds of antibody.Finally, in fluorescence microscopy Microscopic observation sample.
Result of the test
Horizontal to Raw264.7 cell the matrix metalloproteinases MMP2 and MMP9 after oxLDL is handled by us
Research, the results showed that：Under conditions of 60 and 80 μ g/ml, oxLDL promotes dramatically increasing for MMP9 and MMP2, shows inflammation
Position effect (Fig. 1).However, Caveolin-1shRNA can not prevent MMP9 generation completely, but its aggregate level is in proportion
Reduction.On the other hand, MMP2 is then wholly absent.In order to support caveolin-1 to take part in the judgement of aging course, when
After caveolin-1 is knocked, the generation of beta galactosidase is also substantially obstructed in cell after oxLDL is handled, so as to confirm
The ability of oxLDL Induction of Cellular senescence needs caveolin-1 participation (Fig. 2).
The preferred embodiments of the disclosure and embodiment are explained in detail above, but the present invention is not limited to
The above-described embodiment and examples, in those skilled in the art's possessed knowledge, the present invention can also not departed from
Various changes can be made on the premise of design.
Claims (7)
- A kind of 1. method by suppressing caveolin delaying cell aging, it is characterised in that：By suppressing caveolin, reduce P47 adjusts the combination of subunit and cell membrane in nicotinamide-adenine dinucleotide phosphate oxidizing ferment NOX2 hypotypes, hinders niacinamide The performance of adenine-dinucleotide phosphoric acid oxidizing ferment NOX2 hypotypes effect, and then the generation of reactive oxygen species is reduced, suppress under it Factor M MP2/9 generation is swum, the degree of Cellular Oxidation is reduced, so as to realize the purpose of delaying cell aging process.
- 2. according to claim 1 by suppressing the method for caveolin delaying cell aging, it is characterised in that：The suppression Caveolin is completed using caveolin inhibitor.
- 3. according to claim 2 by suppressing the method for caveolin delaying cell aging, it is characterised in that：The alveole Protein inhibitor includes incadronate, filipin rhzomorph and Genistein.
- 4. the method by suppressing caveolin delaying cell aging according to claim any one of 1-3, its feature exist In：Application of the methods described in anti aging effect medicine is prepared.
- 5. the method by suppressing caveolin delaying cell aging according to claim any one of 1-3, its feature exist In：Methods described is preparing the application in treating atherosclerosis medicine.
- 6. the method by suppressing caveolin delaying cell aging according to claim any one of 1-3, its feature exist In：Application of the methods described in anti aging effect health products are prepared.
- 7. the method by suppressing caveolin delaying cell aging according to claim any one of 1-3, its feature exist In：Application of the methods described in anti aging effect cosmetics are prepared.
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| CN1460181A (en) * | 2000-09-08 | 2003-12-03 | 汉城国立大学工业基金会 | Nucleic acid sequences and protein involved in cellular senescence |
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| CN1460181A (en) * | 2000-09-08 | 2003-12-03 | 汉城国立大学工业基金会 | Nucleic acid sequences and protein involved in cellular senescence |
Non-Patent Citations (1)
| Title |
|---|
| 宋琳 等: "小窝蛋白-1在细胞衰老及衰老相关疾病中的作用", 《中国生物化学与分子生物学报》 * |
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Application publication date: 20171222 |
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