CN106868089A - A kind of method that utilization SF potassium-channel evaluates senile-resistant efficacy - Google Patents
A kind of method that utilization SF potassium-channel evaluates senile-resistant efficacy Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of method that senile-resistant efficacy to given the test agent is evaluated, the described method comprises the following steps：A) fibroblast is cultivated in cell culture fluid, is obtained comprising fibroblastic nutrient solution；B) fibroblastic potassium current is detected using patch clamp technique, obtains base current；C) by the nutrient solution of given the test agent addition step a)；D) fibroblastic potassium current is detected using patch clamp technique, obtains adding the electric current after given the test agent；E) testing result of step b) is compared with the testing result of step d), electric current is raised and then judges that the sample has senile-resistant efficacy compared with base current.The invention further relates to the device that the senile-resistant efficacy to given the test agent is evaluated.
Description
Technical field
The present invention relates to a kind of new appraisal procedure in anti-aging field, and in particular to one kind passes through patch-clamp
Technology for detection SF potassium-channel is come the method for evaluating product senile-resistant efficacy.Originally comment
The method of estimating can be used for including the cosmetics including skin preparations for extenal use, medicine, food senile-resistant efficacy
Evaluation areas.
Background technology
Anti-aging as the fields such as cosmetics, food, medicine and health products an important research class
Topic, has obtained extensive concern, and the research for anti-aging is also increasingly deep, relevant skin senescence
Mechanism theory also up to more than ten is planted.In recent years, the mechanism of most commonly seen skin senescence is free radical
Say, gene theory, mitochondrial DNA damage theory etc..
Skin senescence epidermis show as epidermal renewal slow down, barrier function weaken, keratinocyte
Vigor decline, have a superficial wound after repair ability weaken.Corium aging shows as corium to xenobiotics
Remove power to decline, dermis thickness is thinning, collagen and elastin laminin synthesis reduction, decomposition increase,
Catabolic enzyme increased activity.Dermis thickness is thinning, and collagen content is reduced, particularly soluble collagen content
Decline obvious；Hyaluronic acid and dermatan sulfate content are decreased obviously in extracellular interstitial, and glycosaminoglycan is total
Content declines.Additionally, collagenous fibres and elastomer arrangement are also gradually disorderly.As one of skin
Main cell type in important component and dermis of skin, fibroblast is in regulation skin physiology
Key effect is played with pathology aspect, because they can produce the extracellular matrix of dermal tissue.This
Outward, can not only be communicated with each other between fibroblast can also have with other kinds of cell in skin
Contact.And, fibroblast not only plays an active part in stimulates the propagation and apoptosis of cell, also assists in
In the inflammatory process of wound repair, immune response and corium, because they can be by autocrine and other point
The interaction secreted discharges various growth factors and cell factor.Therefore, fibroblastic increasing is adjusted
Grow closely bound up with anti aging effect.
In the evaluating in vitro method for anti-aging in currently available technology, relatively more widely used side
Method has：DPPH radicals scavengings detection, superoxide dismutase (SOD) Activity determination, DNA
The methods such as damage check, fibroblast proliferation.There is many lacking in the anti-ageing appraisal procedure of prior art
In point, such as DPPH radicals scavengings detection method, DPPH is both oxidant and free radical is indicated
Agent, when measured object and DPPH UV absorptions have overlapping, can influence measurement result.Additionally, DPPH
Although color it is main because single Electron Transfer Reactions are eliminated, but it is also possible to eliminated by hydrogen transfer reaction.By
Determine the tendency of reaction, therefore micromolecular compound due to possessing closer to free radical in steric hindrance
Of a relatively high oxidation resistance.It is many that the anti-oxidant of fast reaction occurs with peroxy radical in vivo
Agent may be difficult and DPPH radical reactions because of reasons such as steric hindrances.Additionally, most of anti-aging
Evaluation method, without the mechanism for deeply disclosing senile-resistant efficacy, easily causes false positive results, and need
Multiple test combinations, mutual authentication senile-resistant efficacy is operated more complicated, relatively costly.
The present invention is had been surprisingly found that based on following：Anti-aging effects and fibroblast potassium-channel
Opening is relevant.Potassium-channel is to allow K+ to flow into or out the ion channel egg of cell on cell membrane
In vain.It is also to have now been found that hypotype at most, the most complicated class ion channel of function.Potassium-channel
It is widely distributed with very strong generality.It is present in the various tissues and organ of body on a large scale
In, such as there is potassium-channel on heart, nerve, blood vessel, tracheae, skin and endocrine cell
Presence.Potassium-channel has stronger diversity, and different types of potassium-channel has
Respective specific blocking agent, such as：Apamin, Scyllatoxin and Curare can block SK；
And the calcium that Clotrimazole is then middle conductance activates the specific inhibition agent of potassium-channel.Potassium ion leads to
Road can not only participate in the foundation of neuron membrane resting potential, determine the granting frequency of its action potential
And amplitude, development, maturation also with cell etc. be relevant, potassium-channel as medicine target spot also just
Carrying out extensively.Therefore, research potassium-channel regulation and control and the effect in biological function and
Rule, clinical guidance value can be provided not only for research and development drug therapy potassium-channel disease, for
Effect of further investigation medicine (additive) is also significant, but for potassium-channel
Research does not relate to anti-aging field also.
The research of the ion channel expression on HSF is for the first time existed by Estacion
Annotated using patch clamp technique within 1991.Although HSF is listed in non-excitement
Property cell, but it can express the phenotype of a variety of ion channels, including valtage-gated extroversion is prolonged
Slow rectification potassium-channel (IK), calcium activates potassium-channel (IK (Ca)) and the instantaneous extroversion potassium of A types
Ion channel (IA).Recently, the research of Yun et al. shows that three class functional calciums activate potassium-channel,
Big conductance calcium-activated potassium-channel (BK), middle conductance calcium-activated potassium-channel (IK) and small electricity
Lead calcium and activate potassium-channel (SK), there is expression in HSF.However, on
Ion channel type that fibroblast possesses, function and the mechanism being conditioned are known little about it.
During potassium-channel on SF is studied, have been surprisingly found that, it is public in the prior art
The anti-senescence compounds resveratrol recognized can open fibroblast potassium ion lead to concentration dependent
Road.Meanwhile, the result of study of early stage shows, resveratrol can promote fibroblast proliferation, shows
Show its senile-resistant efficacy, in the specific blocking agent of addition potassium-channel, resveratrol no longer promotees
Enter fibroblastic propagation.It follows that resveratrol promote it is fibroblastic propagation with
The opening of potassium-channel is relevant, i.e., anti-aging effects have with the opening of fibroblast potassium-channel
Close.Inventor by detect SF potassium-channel open method, it was demonstrated that its with
The relation of senile-resistant efficacy.The senile-resistant efficacy that product is evaluated using the method for the present invention is quick, accurate
Really, it is sensitive, directly perceived.The method can provide one to the research of extensive senile-resistant efficacy raw material screening
Fast and effectively evaluation measures.
Therefore, present invention firstly provides SF potassium-channel opening and the cell
Propagation is related, so that for anti-aging study mechanism provides a new direction.
The content of the invention
The brief overview of one or more aspects given below is providing to the basic comprehension in terms of these.This is general
The extensive overview of the not all aspect for contemplating is stated, and is both not intended to identify the key of all aspects
Or decisive key element is also non-attempts to define the scope in terms of any or all.Its unique purpose is will be with simplification
Some concepts that form provides one or more aspects think the sequence of more detailed description given later.
The present invention verifies this method with the generally acknowledged anti-senescence compounds resveratrol of prior art as sample
Reliability, accuracy, repeatability.
Resveratrol (Resveratrol, Resveratrol) is polyphenol compound, be mainly derived from grape,
The plants such as giant knotweed, peanut.Resveratrol has become a generally acknowledged material with senile-resistant efficacy at present,
U.S. Ai Ermin Dare writings in 1998《Anti-aging canon》When, resveratrol is classified as " 100 kinds are most
One of popular effectively anti-aging material ".A large amount of evidences prove the anti-aging effects of resveratrol, and it not only may be used
Anti-lipid peroxidation, moreover it is possible to suppress the oxidation of human low density lipoprotein (LDL), reduces H2O2Generation,
Play a part of anti-oxidant, Green Tea Extract.Scientists also found that resveratrol can decline by adjusting control
The expression of old gene and imitate the path of calorie limitation (Caloric Restriction, CR) and resist aging very
To the extension life-span.Simultaneously external research shows that resveratrol can activate the table of anti-aging gene SIRTl
Reach.
Studied through the present inventor and found, resveratrol can promote fibroblastic propagation, and ability
Domain is it is well known that promote fibroblast proliferation related to anti-aging.It is an unexpected discovery of the invention that
Resveratrol promotes the effect of fibroblast proliferation to be produced by the potassium-channel of open cells film
Raw.
Therefore, the invention provides a kind of method that senile-resistant efficacy to given the test agent is evaluated, institute
The method of stating is comprised the following steps：
A) fibroblast is cultivated in cell culture fluid, is obtained comprising fibroblastic nutrient solution；
B) fibroblastic potassium current is detected using patch clamp technique, obtains base current；
C) by the nutrient solution of given the test agent addition step a)；
D) fibroblastic potassium current is detected using patch clamp technique, obtains adding test sample
Electric current after product；
E) testing result of step b) is compared with the testing result of step d), compared with base current
Electric current is raised and then judges that the sample has senile-resistant efficacy.
Base current of the present invention is in embodiment 2-3 the corresponding skin of negative control group into fiber finer
Born of the same parents' potassium current.
In one embodiment, the given the test agent can be any sample, including：Food, medicine and
/ or cosmetics.
In a specific embodiment, the electric current in the step e) is raised and included compared with base current
Electric current is raised at least above 100%.In a specific embodiment, the electric current liter in the step e)
Height includes that electric current raises at least 200% compared with base current.It is described in a specific embodiment
Electric current in step e) is raised includes that electric current raises at least 300% compared with base current.It is specific at one
In implementation method, the electric current in the step e) is raised includes that electric current raises at least 500 compared with base current
%.
On the other hand, present invention also offers the dress that a kind of senile-resistant efficacy to given the test agent is evaluated
Put, including：
Media to cell culture modules, cultivates fibroblast under cell culture condition；
Potassium current detection module, detects that fibroblastic potassium current and addition are received
Fibroblastic potassium current after test agent；
Calculating and comparing module, by fibroblastic potassium current and adds after given the test agent into fiber
The result of the potassium current of cell is calculated and compared, in this, as the anti-ageing of judgement given the test agent
The index of old effect.
In one embodiment, the given the test agent can be any sample, including：Food, medicine and
/ or cosmetics.
In a specific embodiment, the Judging index of the calculating and comparing module is to add given the test agent
The rising of fibroblastic potassium current afterwards.In a specific embodiment, the calculating
The Judging index of comparison module is that fibroblastic potassium current likens to fibre after adding given the test agent
The potassium current for tieing up cell is raised at least above 100%.In a specific embodiment, institute
The Judging index for stating calculating and comparing module is to add fibroblastic potassium current after given the test agent
At least 200% is raised than fibroblastic potassium current.In a specific embodiment,
The Judging index of the calculating and comparing module is to add fibroblastic potassium-channel electricity after given the test agent
Stream raises at least 300% than fibroblastic potassium current.In a specific embodiment,
The Judging index of the calculating and comparing module is to add fibroblastic potassium-channel electricity after given the test agent
Stream raises at least 500% than fibroblastic potassium current.
Judging index of the present invention is to add fibroblastic potassium current after given the test agent
Raise, judge whether given the test agent has senile-resistant efficacy, the Judging index of calculating and comparing module is to add to receive
Fibroblastic potassium current is raised than fibroblastic potassium current after test agent
At least above 100%.
The present invention provides a kind of new anti-aging study mechanism direction, by detect SF potassium from
The opening of subchannel is so as to judge the senile-resistant efficacy of additive.
Brief description of the drawings
Fig. 1 shows that under various concentrations resveratrol strengthens SF mitochondria activity
Result.
Fig. 2 shows that resveratrol promotes the microphoto (multiplication factor of SF propagation
200 times).
Fig. 3 shows that resveratrol promotes the elevated result of SF potassium channel current.
Fig. 4 shows the potassium-channel that potassium-channel blocker IBTX blocking resveratrols cause
The elevated result of electric current.
Fig. 5 shows that blocking agent IBTX suppresses the result of the fibroblast proliferation that resveratrol causes.
Specific embodiment
Inventor is by research it was unexpectedly observed that the activating agent with senile-resistant efficacy such as resveratrol promotes into
The effect of the propagation of fibrocyte is produced by the potassium-channel of open cells film.That is,
Present inventors have unexpectedly found that, generally acknowledged anti-senescence compounds resveratrol being capable of concentration dependent ground in the prior art
Open fibroblast potassium-channel, is achieved in the purpose of anti-aging.
Therefore, whether have the invention provides a kind of quick, accurate, sensitive, visual evaluation product anti-ageing
The method of old effect.
The present invention is expanded on further with reference to specific embodiment.It should be appreciated, however, that these realities
Example is applied to be merely to illustrate the present invention and be not meant to limit the scope of the invention.Do not noted in the following example
The test method of bright actual conditions, generally according to normal condition, or according to the bar proposed by manufacturer
Part.Unless otherwise indicated, all of percentage and number be by weight.
The XTT used in following examples is purchased from sigma companies；Resveratrol is purchased from sigma companies；
Fibroblast obtains from the Shanghai hospitals of traditional Chinese and western medicine；Blocking agent IBTX is purchased from sigma companies；
TTX is purchased from sigma companies；The model MK3 of ELIASA, it is public purchased from Thermo Labsystems
Department；Microscopical model OLYMPUS IX50, purchased from Olympus Corp；Whole-cell patch-clamp
The model Axopatch 200B amplifier of record amplifier, purchased from Axopatch companies；Manually
The model MSC-200 of converter, purchased from Bio-Logic-Science Instruments companies.
Embodiment 1：Resveratrol promotes SF propagation
First, experimental principle
(4- methoxyl group -6- nitros) benzene sulfonic acid sodium salts (XTT) of 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles]-two
It is a kind of tetrazolium azepine derivatives of new synthesis, can be reduced into by living cells mitochondria dehydrogenase water miscible
Brown formazan product, when XTT and electron pair mixture are used in conjunction with, growing amount and the cell of formazan
Propagation degree is proportionate.XTT methods can extrapolate living cells quantity by colorimetric estimation, with enzyme-linked
Immune detector determines its optical density, and the proliferation rate that can obtain experimental group is compared with control group, so as to sentence
The cell cultivation effect of disconnected given the test agent.
2nd, experimental technique
1st, Fibroblast cell-culture and drug-treated
Dermal fibroblast (fibroblast) is inoculated in the blake bottle containing DMEM nutrient solutions,
It is placed in 37 DEG C, CO2(CO2Volume fraction is 5%) to cultivate 5 days in constant incubator.By cell
Blake bottle takes out, and with Trypsin Induced, cell then is inoculated in into 96 orifice plates, is incubated 24 hours,
Control group adds 200 μ L cell culture fluids per hole, and dosing group adds 200 μ L resveratrols, white lamb's-quarters per hole
The concentration of reed alcohol uses 2,10,20 μM of three different concentration gradients, is cultivated in incubator after dosing.
Second sample-adding is carried out after 48 hours, 200 μ L solution are added per hole, method ibid, is incubated 72h
It is used as cell afterwards and breeds detection.
2nd, XTT methods detection
After agent-feeding treatment terminates, it is the 0.2gL-1 XTT aqueous solution and 5mmolL-1 to prepare mass concentration
The PMS aqueous solution, the PMS aqueous solution and the XTT aqueous solution are with volume ratio 1:1000 mixing, by 96
Cell culture fluid in orifice plate is removed, and the mixed liquor of 125 μ L XTT and PMS is added per hole, in 37
DEG C cell culture incubator is incubated 2h；Using ELIASA at 450nm mensuration absorbance, absorbance is higher,
Show that cell mitochondrial activity is higher.
3rd, experimental result
1st, resveratrol enhancing SF mitochondria activity
As shown in figure 1, XTT testing results show, resveratrol is 10 μM and 20 μM in concentration
When can promote the mitochondria activity of SF, that is, promote fibroblastic propagation.It is real
Test result to show, resveratrol can promote fibroblastic propagation (compared with the control, its result
With significant difference, * P<0.05).
2nd, resveratrol promotes fibroblast proliferation
As shown in Fig. 2 resveratrol can promote fibroblast proliferation.
Experimental result shows, after agent-feeding treatment 72h, 96 orifice plates is directly clapped under inverted microscope
According to microscope magnification is 200 times.From the graph, it is apparent that resveratrol is in concentration
Fibroblastic propagation can be remarkably promoted at 10 μM, compared with negative control group, cell quantity is bright
It is aobvious to increase.That is resveratrol can promote the propagation of SF, and fibroblast proliferation
It is able to demonstrate that the senile-resistant efficacy of resveratrol.
Embodiment 2：SF potassium-channel is detected
First, patch clamp technique principle
Patch clamp technique is developed on the basis of voltage clamp, and it is one kind to record by ion
The gas current of passage reflects the molecule activity of single (or multiple) ion channel on cell membrane, Ke Yijian
The temporal resolution of the current sensitivity, 1 μm of spatial resolution and 10 μ s of 1pA is measured, is from thin
Extracellular molecule level research biomembrane information provides direct approach.Full cell pattern patch-clamp is thrown through heat
The micro-pipe electrode of light is placed in cleaning, and smooth surface of cell membrane forms high resistance seals, in surface of cell membrane
A small pieces film is suctioned out, is continued with negative-pressure ward, make electrode tube inner cell film rupture, intracellular fluid and electrode
Interior liquid is directly communicated and obtained.This mode not only can record membrance current but also recordable film potential.Full cell note
The electric current of record is the summation of all ion channel electrical activities on whole cell membrane.This recording mode it is excellent
Point is the integrality that can keep cell and its reactivity.Whole-cell recording technique can be seen on a cellular level
Examine the general reaction feature of Cellular electrical activity after the effect by medicine.
2nd, experimental technique
1st, Fibroblast cell-culture
With embodiment 1
2nd, Whole-cell recording
Cultured HSF is taken out, is recorded with patch clamp technique whole-cell recording technique pattern
Fibroblast potassium current.It is soft that rheometer employs Axopatch 200B amplifier and pClamp
Part, the record of potassium current, culture medium is replaced by extracellular fluid.
1) extracellular fluid is prepared：Containing 140mM NaCl, 2.5mM KCl, 10mM HEPES, 1
mM MgCl2,1mM CaCl2And 0.001mM TTX (TTX, tetraodotoxin, can prevent appoint
What sodium current)
2) extracellular fluid pH value：PH value to 7.4 is adjusted with NaOH
3) intracellular fluid is prepared：Interior liquid is filled with glass electrode, wherein containing 135mM glucose
Sour potassium, 10mM KCl, 10mM HEPES, 1mM CaCl2And 2mM MgCl2.In electrode injection
The resistance of liquid is 5~7M Ω.
4) intracellular fluid pH value：PH is adjusted to 7.3 with KOH
5) resveratrol solution is prepared：Resveratrol is made into concentration respectively for 1,5,10,50 and
100 μM of solution
6) nutrient solution is replaced by extracellular fluid by cell before recording
7) resveratrol perfusate continuous perfusion is used as needed
8) fill and rush in potassium current record after liquid, electrode resistance is 5~8M Ω
9) extracellular use manual converter (MSC-200Manual Solution Changer) gravity administration,
Duration is 60~90 seconds, and flow velocity is 50 μ l/ minutes
All of record is all to carry out at room temperature (23~25 DEG C).
3rd, data collection and analysis
Computer is connected to sound pick-up outfit and is used with digidata1440a A/D interfaces.The electricity of digital sample
Flow not 100 μ s (10kHz).Current signal is filtered by a pole Bessel filter of 3-kHz tri-.Electricity
Stream corrects online leakage and residual capacitance transient state and phosphorus/4 agreement.Data acquisition and issuance is to use pClamp
10.2 softwares (Axon Instruments, Foster City, CA, USA) and (Microcal of Origin 8.0
analysis software,Northampton,MA,USA).Statistical analysis using t- inspections not into
Pair or paired correlation compare.The mode of test value mean value ± S.E.M is expressed, and is represented with n and detected
Cell quantity.P<0.05 is used for representing between statistic mass there is significant difference.Multigroup is compared and adopts
Detected with One-way ANOVA one-way analysis of variance (ONE-WAY ANOVA).
3rd, experimental result
1st, resveratrol can promote SF calcium to activate potassium current rising
As shown in figure 3, resveratrol can promote SF potassium current.
Experimental result shows that administration concentration substantially increases from 1 to 100 μM of resveratrol, current amplitude
Appear in 10 μM.When resveratrol concentration be 10,50 and 100 μM with negative control compared with have show
Write sex differernce (* P<0.05).That is, resveratrol and base current (potassium corresponding to negative control from
Electron current) compare, can conspicuousness cause the rising of SF potassium current
(*p<0.05)。
Influence detection of the potassium-channel blocker of embodiment 3 to SF potassium-channel
First, patch clamp technique principle
With embodiment 2
2nd, experimental technique
1st, Fibroblast cell-culture
With embodiment 1
2nd, Whole-cell recording
Cultured HSF is taken out, is recorded with patch clamp technique whole-cell recording technique pattern
Fibroblast potassium current.The record of potassium current, culture medium is replaced by extracellular fluid.
1) extracellular fluid is prepared：Containing 140mM NaCl, 2.5mM KCl, 10mM HEPES, 1
mM MgCl2,1mM CaCl2And 0.001mM TTX (TTX, tetraodotoxin, can prevent appoint
What sodium current)
2) extracellular fluid pH value：PH value to 7.4 is adjusted with NaOH
3) intracellular fluid is prepared：Interior liquid is filled with glass electrode, wherein containing 135mM glucose
Sour potassium, 10mM KCl, 10mM HEPES, 1mM CaCl2And 2mM MgCl2.In electrode injection
The resistance of liquid is 5~7M Ω.
4) intracellular fluid pH value：PH is adjusted to 7.3 with KOH
5) blocking agent IBTX is prepared：100nM IBTX
6) nutrient solution is replaced by extracellular fluid by cell before recording
7) resveratrol and IBTX perfusate continuous perfusions are used as needed
8) in perfusion potassium current record after liquid, electrode resistance is 5~8M Ω
9) extracellular use manual converter (MSC-200Manual Solution Changer) gravity administration,
Duration is 60~90 seconds, and flow velocity is 50 μ l/ minutes
All of record is all to carry out at room temperature (23~25 DEG C).
2nd, data collection and analysis
With embodiment 2
3rd, experimental result
As shown in figure 4, being entered to fibroblast by the specific inhibition agent IBTX of potassium-channel
Row is intervened, it is found that the blocking agent can block the opening of the cell potassium-channel that resveratrol causes,
So as to judge the potassium-channel on the passage as fibroblast.
Experimental result shows that Fig. 4 A show, starts first to add potassium-channel special in extracellular fluid
Property blocking agent IBTX, detect potassium current, (1 is marked in Fig. 4 A without significantly rising
Part), add resveratrol and detect that potassium current finds that compare negative control simultaneously
Electric current also no longer raises (part of Fig. 4 A marks 2), afterwards removes resveratrol, also no longer right
Potassium current size produces any influence (part of Fig. 4 A marks 3).Fig. 4 B are Nogata
Figure, is after results of weak current statistics, to add blocking agent IBTX that cell potassium current can not be caused to raise,
After the result illustrates that blocking agent IBTX blocks potassium-channel, resveratrol can not cause passage
It is open, the passage is further demonstrated for potassium-channel.Add potassium-channel specific inhibition
The elevated electric current that resveratrol causes after agent is potassium current.
Embodiment 4：The detection that potassium-channel blocker influences on fibroblast proliferation
First, experimental principle
With embodiment 1
2nd, experimental technique
1st, cell culture and drug-treated
Dermal fibroblast (fibroblast) is inoculated in the blake bottle containing DMEM nutrient solutions,
It is placed in 37 DEG C, CO2(CO2Volume fraction is 5%) to cultivate 5 days in constant incubator.By cell
Blake bottle takes out, and with Trypsin Induced, cell then is inoculated in into 96 orifice plates, is incubated 24 hours,
Control group adds 200 μ L cell culture fluids per hole, and dosing group adds 200 μ L different quality concentration white per hole
Veratryl alcohol, and resveratrol+potassium-channel blocker IBTX, cultivate in incubator.48 is small
When after carry out second sample-adding, per hole add 200 μ L solution, method ibid, be incubated 72h after use
Make cell propagation detection.
2nd, XTT detections
With embodiment 1
3rd, experimental result
1st, potassium-channel blocker IBTX suppresses the fibroblast proliferation that resveratrol causes
As shown in figure 5, when only 10 μM resveratrols are added, fibroblastic propagation can be caused,
And potassium-channel specific inhibition agent IBTX is added simultaneously, and concentration is when reaching 20nM, into fiber
Cell does not have cultivation effect.
Experimental result shown, fibroblast proliferation can be promoted during resveratrol individualism (with the moon
Property control compare, its result has significant difference, * P<0.05.) potassium-channel is added in cell
Blocking agent IBTX is incubated simultaneously, and resveratrol is not then after IBTX reaches finite concentration (20nM)
Fibroblastic propagation (P can be caused>0.05), this explanation potassium-channel blocker IBTX can
Suppress the fibroblast proliferation that resveratrol causes, point out resveratrol to promote fibroblast proliferation
Effect possibly through it cause potassium-channel open effect.Add potassium-channel
After specific inhibition agent, resveratrol can not promote fibroblast proliferation.
Therefore, resveratrol can cause the propagation of SF, verify its anti-ageing effect, and
And senile-resistant efficacy is that (* p are improved by conspicuousness<0.05) fibroblast potassium current
What opening caused.Fibroblastic potassium current is than fibroblastic potassium after adding given the test agent
Ion channel current is raised at least above 100%.
Claims (7)
1. a kind of method for carrying out senile-resistant efficacy evaluation to given the test agent, methods described includes following step
Suddenly：
A) fibroblast is cultivated in cell culture fluid, fibroblast is obtained；
B) fibroblastic potassium current is detected using patch clamp technique, obtains base current；
C) by the fibroblast of given the test agent addition step a)；
D) fibroblastic potassium current is detected using patch clamp technique, obtains adding test sample
Electric current after product；
E) testing result of step b) is compared with the testing result of step d), compared with base current
Electric current is raised and then judges that the sample has senile-resistant efficacy.
2. the method for claim 1, it is characterised in that the given the test agent includes：Food,
Medicine and/or cosmetics.
3. the method for claim 1, it is characterised in that the electric current in the step e) raises bag
The electric current compared with base current is included to raise at least above 100%.
4. the device that a kind of senile-resistant efficacy to given the test agent is evaluated, including：
Media to cell culture modules, cultivates fibroblast under cell culture condition；
Potassium current detection module, detects that fibroblastic potassium current and addition are received
Fibroblastic potassium current after test agent；
Calculating and comparing module, by fibroblastic potassium current and adds after given the test agent into fiber
The result of the potassium current of cell is calculated and compared, in this, as the anti-ageing of judgement given the test agent
The index of old effect.
5. device as claimed in claim 4, it is characterised in that the given the test agent includes：Food,
Medicine and/or cosmetics.
6. device as claimed in claim 4, it is characterised in that the judgement of the calculating and comparing module refers to
Mark is the rising of fibroblastic potassium current after addition given the test agent.
7. device as claimed in claim 4, it is characterised in that the judgement of the calculating and comparing module refers to
Mark is that fibroblastic potassium current is more logical than fibroblastic potassium ion after adding given the test agent
Road electric current is raised at least above 100%.
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Cited By (2)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114292894A (en) * | 2022-01-14 | 2022-04-08 | 广东悠质检测技术有限公司 | Method for evaluating tightening effects of cosmetics and raw materials by using in-vitro cells |
| CN114292894B (en) * | 2022-01-14 | 2023-08-11 | 广东悠质检测技术有限公司 | Method for evaluating tightening effect of cosmetics and raw materials by using in vitro cells |
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| CN106868089B (en) | 2020-02-28 |
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