Background of invention
Process skin, to avoid or to alleviate the effect of aging of aging and external environmental correclation of skin inherence, is the industry of a multi-million dollar, and what lay the first stone for this industry is the investment of fund even huger in the exploitation and checking of new technology.Electromagnetism (EM) energy is namely known before application to skin many decades by the application to skin surface, and is implemented in many different forms and by multiple delivery apparatus.In general, the skin processing method based on EM can be divided into separability with the process of non-peel-away, although the two all utilizes the Thermal release effect of EM energy apply.
Various product is had to make for improving skin and delaying and/or prevent typical aging sign for human consumer.This type of sign comprises such as microgroove, wrinkle, hyperpigmentation, sallow, sagging, black eye, swollen eye, uneven skin color, the pore of increase, the furfur of the epidermic cell metabolism of minimizing and exception or exfoliation.But for some human consumer, various obtainable product and the raising in technique in skin care still can not produce the result of expectation, and some human consumer feels that more invasive therapy sought help from by needs.
Separability process to be reinvented for the surface on general form or removing of skin is effective as separability laser has been proved to be, such as scar and removing in process of tatooing, and be proved to be for process and to improve aging be effective with the outward appearance of skin that is light injury.Although the mouth week of separability process fragility in the beauty treatment of skin of face and all regions of socket of the eye are effective for the outward appearance improving microgroove and wrinkle, its main shortcoming comprises long rehabilitation and restored cycle, this make human consumer undesirable over a long time in bear the aesthetic appearance be badly damaged.In addition, these the potential side effects irregular of the infection that may cause, cicatrization and pigementation are thought in beauty treatment it is unacceptable by human consumer, when especially relating to skin of face usually.
Therefore, recent research-and-development activity has focused on provides more cheap, more convenient and safer non-peel-away skin anti-aging and process of rejuvenating to human consumer.Relate to and apply heat energy to promote that the beauty treatment scheme of the skin appearance improved is well known in this area to skin.Electromagnetic energy delivery technique for the hand-held device form of human consumer house use has commercially been present in nearly ten years.The improvement of above-mentioned technology and improvement, to make to maximize the thermoinducible beneficial effect of darker target skin histology, make all to be minimised as center to target tissue with to the not damage desirably of peripheral and surface structure simultaneously.It is believed that optionally thermal treatment causes new collagen to be formed, and optionally thermal damage causes, particularly, dermal matrix is reinvented.But technology available is at present known causes debatable damage, this type of not the passive side effect continued desirably comprise overheated, burn, erythema and anochromasia.
In the past, effect of these devices and composition is determined only by externalist methodology.That is, if there is visible at the extexine of skin to improve, so energy delivery device must work.But, almost there is no the method for the Best Times determining these energy delivery device, temperature and composition.In addition, widely accepted, in the many visible change that skin surface occurs, be the result of the change occurred in the corium of the skin under surface and epidermal area.Not knowing that a kind of device is how cutaneous layer below, is to be difficult to know for sure the performance that how to control and maximize this device.
The selective photothermolysis of skin histology is the cosmetic treatments form of widespread use, particularly in the process of using comprising one-wavelength laser energy and wide spectrum intense pulsed light (IPL) energy.In these techniques, luminous energy is applied directly to the surface of skin, and infiltration depends on through the absorption in the transmission of epidermis and corium.Dark skin and supracutaneous dark color spots can hinder transmission and hinder effect of process by absorbing energy, and can cause the overheated of pigment regions, overheatedly cause blistering, burn and other cosmetically less desirable effects.
As the replacement scheme of the pyrolysis based on EM, radio frequency (RF) electric current of conductivity is also studied as beauty treatment skin processing mode.RF electric current and pulse radiation frequency (pRF) are known in the use of field of medicaments, but RF electric current is still relative novelty as the skin return of spring technology for the self-non-peel-away of sending of human consumer.At RF electric current in the application of skin, two electrodes directly applying to skin that transport through of bipolar RF electric current carry out.Therefore, electric current directly flows through skin corium, conducts to electrode from electrode, and this makes it be different from the application of EM energy, and EM energy is focused on by epidermis, and the restriction of those factors of the wavelength penetration degree of depth that is affected.
RF electric current use the significant skin treatment advantage seeming to provide the application being better than EM energy in theory.Such as, be different from electromagnetic energy, the RF energy of conductivity be do not rely on chromophoric, which avoid to pigment and complication that absorption difference between the skin of non-pigmented is relevant and efficacy problems, and cause have more endergonic melanocyte dark skin and can reflecting light light skin process in problem.In both cases, the consistence of result is all impaired, and the thermal control comprised in chromophoric skin is still problematic.
But, RF electric current with can provide the energy level of thermal treatment effect to skin use be limited to investigator can not parameters optimization to realize desired beneficial effect and don't to there is not skin injury desirably.RF electric current by the skin tissue delivery under skin surface, and is limited to the accessible surface of skin by the effect monitoring that temperature or humidity sensor carry out.RF current impedance is tissue composition and the function of various skin organizational attribution comprising Collagen density and integrity degree, water of hydration equality.Although the Distance geometry between electrode can be conditioned in the control theory of the parameter of such as pulse length and frequency, to make effect optimization and to avoid safety problem, but do not have certain equipment or other modes to monitor the benefit of the change of organization factors, this type of adjustment is almost impossible.Based in the sending of EM energy, the skin on surface reaches the highest treatment temp usually, makes the temperature monitoring on surface can avoid the hypodermic unexpected damage of his-and-hers watches.But with regard to RF electric current, in fact epidermis undertissue reaches the higher treatment temp of the skin of specific surface, makes damage may occur in darker tissue and not have measurable performance from the teeth outwards.Therefore, under the supervision of the medical worker through screwing up discipline and outside the clinical setting of the superior instrument of use, the independent device for human consumer individual use sent based on RF electric current and scheme are generally avoided, because when there is not monitoring mechanism under suitable epidermis, still there is safety issue at effective processing horizontal.There is provided RF electric current as unique processing form and for house human consumer use hand-held energy delivery device be in fact unknown.One claims device (STOP sotM, Ultragen Ltd) be positioned in Europe for human consumer individual, but the process tolerance level of this device is set so low in order to avoid not damage desirably, and the objective evidence of the clinical efficacy under control condition is not still existed.
Therefore, the effect of RF in skin treatment is limited in fact the additional or preparatory function as combining with other Thermal release processes.Such as, in 2002, Bitter and Mulholland(" Reportof a new technique for enhanced non-invasive skin rejuvenation using a dualmode pulsed light and radio frequency energy sources:selectiveradiothermolysis, " J.Cosmet Dermatol 2002; 1:142-145) propose the processing scheme of the combination based on RF electric current and IPL, and report the result of the face process based on 100 bit test experimenters, although above-mentioned author does not disclose concrete Treatment Design parameter.In this study, RF is increased the effect of IPL process by report.Side effect comprises cosmetically the report of not pigmentation effect desirably, and controls the pain perception of human consumer by the cooling on surface.
It is useful that RF is also suggested in the beauty treatment skin process be combined in the application of the luminous energy with orientation, and is studied.In general, design according to this processing scheme, RF is attached to luminous energy and is used, and is employed according to some parameters of luminous energy.Such as, at people (" Electro-opticalsynergy(ELOS such as HammestM) for nonablative skin rejuvenation:a preliminaryprospective study, " Journal of European Academy of Dermatology.andVenereology 2006, 20, 1070-1075), author focuses on the pulse-repetition coordinated between RF electric current and luminous energy, and propose to there is synergy between these form of energy, this can allow lower, the use of the luminous energy of less invasive level, and propose the application independent to RF relevant side effect and be lowered by the scheme of above-mentioned merging or avoid.In addition, the scheme of these devices is adopted to include in response to overheated with the method making physical skin lower the temperature or prevent overheated method.
In another example, U.S. Patent Application Publication 2008/0033516 A1 of Altshuler discloses " temperature controlled photo bio excites " of skin histology, and it relates to heated skin to the target degree of depth and the combination with electromagnetic radiation target area of irradiation territory.Altshuler mentions the prior art of the skin processing method stimulated based on low-level illumination, Low level laser, monochrome and quasi-monochromatic light, they are considered to improve the production of the production of ATP, cell proliferation and protein usually, and trigger growth response by the response of induction low grade inflammation, but also refer to the shortage of the report of inconsistent result and the clinical confirmation to effect.The application that Altshuler imagines heat energy can strengthen light stimulus response.The high heat that Altshuler teaches the skin of certain volume can be realized by any known source that the temperature of described volume can be made to be increased to preferably between 37 ° and 45 DEG C, and the heating that particular instantiation is undertaken by the EM radiation of any suitable wavelength within the scope of the use of warm air, AC or DC electric current, conductivity thermal source, ultrasonic wave or microwave radiation or 380-2700nm.But, in all embodiments of Altshuler, be rely on EM energy to realize desired treatment effect.Altshuler teaches the cooperative reinforcing that heat provides the desired effect of light stimulus, but it is also proposed when there is not EM, heat can cause not biostimulation desirably, such as slow down the reparation of radiation-induced DNA damage, the production of heat shock protein (it establishes the tolerance of the application to heat subsequently) and the modification to the regeneration and those zymetology process involved in repairing that are included in skin histology, and usually oppositely teach heat when there is not illumination as skin treatment mode.Altshuler does not propose how to overcome and the defect can not assessed or monitoring form subcutaneous skin situation is relevant.
In addition, for the device existed at present, consumer compliance always is a problem.Human consumer only has the limited amount time every day for their beauty treatment scheme.Although the manufacturers of device wants to recommend human consumer to use these devices longer time to guarantee that they obtain maximum beneficial effect, human consumer is unlikely to be deferred to.Therefore, using and admitting to only have using appts between there is a kind of balance limited amount free time at them one day of human consumer at suggestion extension fixture.
When design apparatus, composition and scheme, the experience of human consumer is also important.Such as, audiogram is the method that generally uses and provides huge medical beneficial effect.But the gel used in audiogram process dense thick and be difficult to removing, result in the discomfort of human consumer.In addition, the skin of a lot of energy delivery device heating surface is too fast or too hot, result in and makes us unhappy consumer experience.
Therefore, need can substantially improve skin with the method avoiding needing more invasive therapy He avoid associated risk always.Further, there is the demand of better method to the effect measuring energy delivery device, these methods can be used to set up the experience that human consumer can accept more, the result of the skin appearance of the improvement desired by simultaneously realizing.
The demand to safety and effective non-peel-away skin treatment and the individual scheme used of return of spring device, methods for the treatment of and applicable human consumer is also there is in this area.Particularly, exist by promoting collage synthesis and corium to reinvent processing list subcutaneous skin and don't the demand of the not injury pattern desirably to handled target tissue or tissue around can being caused.The effect to assessing and monitor the process performed based on the human consumer of RF electric current is there is in this area, to optimize the demand of the method for the application for the RF electric current providing desired beneficial effect, and the demand existed the methods for the treatment of based on RF electric current optimized, described methods for the treatment of avoids the problem relevant to the methods for the treatment of based on EM and does not rely on and process the Physical temperature-lowering combined.
Summary of drawings
Although this specification sheets is by particularly point out and clearly claimed claims of the present invention is drawn a conclusion, should believes by following explanation and the present invention can be understood better by reference to the accompanying drawings, wherein:
Fig. 1 is the schematic diagram of naturally occurring injuring and repairing circulation in human skin;
Fig. 2 A, 2B show the characteristic of human collagen;
Fig. 3 is the schematic diagram of the biological model of RF electric current efficacy;
Fig. 4 A, 4B are 24 hourly average multiple changes of compiling gene;
Fig. 5 A, 5B are 24 hourly average multiple changes of Exemplary gene;
Fig. 6 A, 6B are 1 monthly average multiple changes of compiling gene;
Fig. 7 A, 7B are 1 monthly average multiple changes of Exemplary gene;
Fig. 8 A, 8B are two width figure of RF simple heat transfer;
Fig. 9 is the schematic diagram of RF simple heat transfer;
The schematic diagram of the crows feet area of Figure 10 human consumer's around eyes;
Figure 11 is the region A of consumer skin;
Figure 12 is the region B of consumer skin;
Figure 13 is the region C of consumer skin;
Figure 14 is the region D of consumer skin;
Figure 15 is the region E of consumer skin; And
Figure 16 is the region F of consumer skin.
Detailed Description Of The Invention
Except as otherwise noted, the Science and Technology term used relatively with invention as herein described should have the implication that those of ordinary skill in the art understands usually.In addition, unless the context otherwise requires, singular references should comprise plural number, and plural term should comprise odd number.In general, cell and tissue culture as herein described, molecular biology and protein and few or polynucleotide are chemical and the technology of hybridization and with the name that these use relatively, be well known in the art and generally use those.Such as, the standard technique for the purifying of genetic material (nucleic acid) and preparation, chemical analysis and oligonucleotide synthesis is employed.Enzyme reaction and purification technique are according to the ordinary method in the specification sheets of manufacturer or this area or as described hereinly carry out.Technology described herein and program generally according to ordinary method known in the art, and are implemented as described in the various routines quoting in this specification sheets and discuss and more concrete reference.Laboratory protocols as herein described and technology and the name used relatively be well known in the art and generally use those.
For the object explaining and illustrate many aspects of the present invention, a large amount of detail is listed in hereafter.But those skilled in the relevant art will be understood that, do not have these detail embodiment of the present invention can be implemented yet.In other instances, for the present invention being described effectively and avoiding the object of fuzzy creative aspect, show more in general manner and discuss known structure and device.It should be pointed out that many different and substituting structure, device, software and the technology that have that invention disclosed and their embodiment of illustration can be suitable for.Multiple embodiments and these full breadth of inventing self are not limited to specifically described embodiment.
All when providing the scope of value, be to be understood that, each value between two parties, until 1/10th of the unit of lower limit (unless the context clearly determines otherwise), any other statement between the upper and lower bound of this scope and in the scope of this statement or value between two parties, include within the scope of the invention.Be subject to the constraint of any concrete removing property restriction in stated scope, in scope less described in the upper and lower bound of these less scopes can be included in independently, and be also included within the scope of the invention.When all stated scope comprises one or all two in its limit, the scope eliminating one of these limits be included or whole two is also included within the present invention.Except as otherwise noted, represent whole numerals of the amount of the reactant, reaction conditions etc. that use in this specification sheets and claim, all should be understood to be in all situations all by the modification of term " about ".
Should understand, unless otherwise indicated, the step of any method enumerated in claims additional herein can any order be carried out.Such as, in the claim to a method book of enumerated steps (a) and (b) and (c), step (c) can step (a) and (b) before or between carry out.In addition, although be enumerated as different steps, independent step can between the cycle partly overlap to carry out or fully overlap is carried out.
" adjustment keratinous tissue conditions " refers to and improves skin and/or preventative regulation of skin situation herein, and comprises and such as protect described tissue to exempt from ultraviolet radiation, and regulation of skin aging sign." improve mammiferous skin " herein and refer to and cause visually and/or appreciable positive change in sense of touch on the look and feel of described tissue.The situation that can be conditioned and/or improve includes but not limited to one or more following situations: the appearance reducing wrinkle and coarse dark line, microgroove, crackle, lump and enlarged pores; The thickening of skin (such as, the epidermis of structure skin and/or corium and/or subcutaneous layer, and the stratum corneum of finger/toenail and hair shaft (if applicable) is to alleviate skin, hair or finger/toenail atrophy); Increase the spiral structure (being also called as relations with network) at dermal-epidermal edge; Prevent from causing skin or hair loss of elasticity due to the forfeiture of such as skin function elastin, damage and/or inactivation, cause such as elastosis, sagging, forfeiture skin or these situations of hair deformation-recovery function; Reduce liparitosis; Change the painted of skin, hair or finger/toenail, such as black eye, rash spot (the red spotty staining as caused due to such as acne erythematosa), sallow, the variable color caused due to telangiectasis or spider shape blood vessel, drying, embrittlement and hair graying.
" signs of skin aging " used herein includes but not limited to, perceptible performance in the apparent visual caused due to skin aging and sense of touch, and any macroscopic view or microphenomenon.These signs are produced by following effect, and described effect includes but not limited to the development of texture discontinuity, as wrinkle and coarse dark line, microgroove, skin striped, crackle, lump, enlarged pores, unevenness or anchor; Furfur; Dry; Lose skin elasticity; Variable color (comprising black eye); Rash spot; Shoal; Cutaneous pigmentation region, as senile plaque and freckle; Keratinization; Abnormal differentiation; Parakeratosis of skin; Elastosis; Decomposition of collagen, and stratum corneum, corium, epidermis, vascular system (as telangiectasis or spider shape blood vessel) and especially near skin those lower-hierarchies (as fat and/or muscle) in other histological change.
As used herein, " hyperpigmentation " refers to that wherein pigmentation is greater than the Pigmented skin area of skin adjacent domain (as eyespot, senile plaque etc.).
In this article, " personal care composition " refers to the composition being suitable for being applied topically on mammal skin.Personal care composition as herein described can comprise one or more skin care actives.As used herein, " skin care actives " or " active substance " refers to the compound such as contributing to regulation of skin and other mammiferous skin by providing beneficial effect or improve described skin.
As used herein, " energy delivery device " refers to for any device to mammal skin and/or hair delivery of energy.In this article, " delivery of energy " instigates skin surface and/or layer to be exposed to the energy launched by energy delivery device, and wherein it can penetrate into required skin layer, comprises hair shaft and/or hair follicle.
As used herein, " constant level " refers between device start time and device down time, and the energy that device is sent or energy output remain in level constant in fact.
As used herein, " pulse " refers between device start time and device down time.Energy output, with predictable variation, is characterized in that higher output period (pulse) exports the period alternately occur with lower.The beginning of pulse can be unexpected or progressive." predictable " refers to that the timed interval between pulse peak intensity, pulse shape, pulse duration and pulse is in fact identical.Pulse duration and time between the pulses can be changed.
As used herein, " hand-held " is that finger device has the comfortable weight held of applicable general grownup and size.
Human skin can be divided into two main structural sheets: the corium of epidermis and below the epidermis.Epidermis has the stratum corneum as the biological containment to environment.At the stratum basale of epidermis, exist and be called as melanocytic chromatogenous cell, they are main determining factors of skin color.
Corium below provides the main structural support of skin.It is primarily of the extracellular protein composition being called as collagen.Collagen by fibroblasts produces, and is synthesized by three polypeptide chains as triple helices, and these three polypeptide chains are connected by heat-labile and heat-staple chemical bond.When the tissue comprising collagen is heated, the physical property of these albumen substrates changes in characteristic temperature.Collagen contraction collagen stroma occurring along with heating and the structural transformation reinvented.
In skin, produce the repairing activity of some amounts, to promote the generation that collagen continues.In weathering process, the speed that damage occurs can raise faster than repairing activity, or damage can continue to occur with less constant speed, but repairing activity slows down.Result is all the minimizing of collagen and the impaired outward appearance as evidence for senescence appearance in both cases, comprises the outward appearance of surface imperfection as microgroove, wrinkle and Hyperpigmented spot.Normal repairing activity circulation and along with being shown in Fig. 1 and 3 impact that outward appearance produces time lapse.
In FIG, box indicating causes the regular element of collagen production.Due to many reasons, collagen increase in skin, and its speed of repairing and replacing contributes to skin and looks fuller, more healthy and more attractive.The propagation continued and corium are reinvented and are occurred natively, but regrettably, and these processes slow down along with our ageing.When skin sustains damage, such as, in inflammation or after being subject to as the radiation from the sun infringement, also collagen can be produced.Circle in Fig. 1 illustrates more collagenogenic mechanism and how can follow the trail of it and generates.MMP and cytokine activity are only contribute to following the trail of the reparation of collagen and two measurable amounts of replenishment cycles.
Fig. 3 is the schematic diagram of the dual-use function biological model of Elure effect 20.Skin 21 has been divided into three layers, stratum corneum 30, epidermis 32 and corium 34.Reinventing of collagen betides in skin corium with the production of new collagen.The normal infringement 22 of skin 21 is occurred constantly, comprise normal aging, UV infringement, pH change, chemical attack and other.Normal infringement 22 causes impaired collagen 24.Similarly, normal low-level inflammation 26 cause cytokine, HSP and HSF active.Finally, low-level heat energy repeatedly causes the MMP activity 36 raised, and result is the production of collagen fragment.The increase of heat makes the rise 38 causing the formation of collagen and the cytoactive of reparation.Mechanism 36 and 38 all causes the formation of healthy collagen 28 and reinvents.
Although collagen has the melting temperature (Tm) of paramount 50 DEG C through measuring, repairing circulation can be changed by the interpolation of the heat of lower level and be strengthened.The thermo-cracking that intramolecular hydrogen bond is cross-linked is produced by the balance between cracking event and relaxation events (formation again of hydrogen bond).The generation of this process does not need external force.Therefore, molecule internal stress is produced by the thermo-cracking of intramolecular hydrogen bond.The contraction structure of the tertiary structure of crosslinked molecule creates initial intermolecular contraction vector.RF model heating curve is shown in Fig. 8.
Dermis forms primarily of collagen 1.Collagen is expressed by inoblast with the precollagenous form of the protein of strand.Precollagen is truncated to collagen 1 when expressing, and is folded into the triple helices structure being called as " tropocollagen ".RF model heating curve is shown in Fig. 8 A, Fig. 8 B and Fig. 9.
Dermis is primarily of collagen 1, and 50 form, Fig. 2 A and Fig. 2 B.Collagen is expressed by inoblast with the form of the protein precollagen 52 of strand.Precollagen 52 is truncated to collagen 150 when expressing, and is folded into the triple helices structure 54 being called as " tropocollagen ".This process graphical is in Fig. 2 A and 2B.
The crosslinked of collagen can be intramolecular (covalent linkage or hydrogen bond) or intermolecular (covalent linkage or ionic linkage).Cause that collagen comprises heat energy infringement, machinery infringement according to the reason of age sex change, pH is on the impact of collagenase and MMP speed, hydrated state and can " pull on " or the generality of natural balance of collagen microfibril of " untiing " is destroyed, described destruction causes them easily to be digested by MMP.Although these represent multiple infringement type, it is temperature controlled to be all that speed is subject to.In addition, the normal collagen replacement cycle can be regulated and controled in the temperature range of <37 DEG C-43 DEG C.
In the cracking that lower temperature also can send out key collagenogenic, but be with lower speed.Be associated with relaxation phenomenon in a lot of situation of low-level thermo-cracking, in relaxation phenomenon, key is formed again when not having the net change of molecular length.
It is the biophysical phenomenon occurred at cell and molecular level that corium is reinvented.The molecular contraction of collagen or partially denaturing relate to the effect of energy source, and it makes the longitudinal axis of molecule unstable by the heat-labile key of cracking triple helices.Therefore, the stress of the inter-molecular linkage breaking matrix is created.This is in fact extracellular process, and the contraction of cell needs one period of lag-phase for migration of fibroblast cells and the region of breeding to damaged.Callus response relates generally to initial inflammatory process, and it comprises the white cell of process cell debris or leukocytic infiltration.Following by the propagation of inoblast in injury site and/or the increase of renewal, is finally can for the increase of the collagen of deposition.Inoblast is divided into contractile myofibroblast, and the latter is the source of cellular soft tissue contraction.After cellular contraction, collagen is deposited in the soft tissue structure of tension as static supported matrix.With regard to object attractive in appearance, the deposition of the scar matrix at this initial stage and reinventing to provide and change the denseness of soft tissue and the approach of geometrical shape subsequently.
Heat application causes the cascade reaction of injuring and repairing, finally to realize the improvement of its surface appearance, be as known in the art, but technology available is at present associated with known shortcoming.Such as, laser delivery apparatus uses the light of specific wavelength, the light transdermal of described specific wavelength, to be combined and to remove multiple pigment and pigment by the process being called as selective light heat effect from skin with specific chromophoric group.Laser apparatus is large-scale, expensive capital equipment, only processes particular problem or color in skin, is easy to that laser burn occurs, stays scar, can cause superpigmentation and/or very few and may cause user and patient that ocular injury occurs.Strong wide band light is according to the light of system generation multi-wavelength, and the same variable color by selective photothermolysis improved effect skin, percutaneous heating non-specifically improves texture.Above-mentioned system is relatively large equally and expensive, and the improvement of texture and wrinkle is very little, and there is skin burn, hypopigmentation or problem that is too much and scar equally.Generally speaking, the application of electromagnetic energy is realized by epidermis, and penetration degree is limited to the pigmentation factor on surface and the composition of skin corium.
Radio-frequency technique is also known in skin treatment field.RF technology uses electric current to heat corium, and stimulates the production of collagen and elastin fiber that skin firm is compacted.But, due to can not optimization process parameter, in the Status of development that this field is current, there is shortcoming in essence.The application of RF electric current produces the thermal gradient contrary with other heat energy delivery technique in skin.The application of RF electric current is undertaken by the electrode at a distance of some distances be placed on skin.Electric current conducts in-between the electrodes, by corium, the temperature of corium is raised quickly than the temperature of skin surface.Because most of skin parameters measuring apparatus is designed the measurement for skin surface, the superheated of corium may just be fully realized for various reasons after occurring.
Therefore, avoided in the art do not exist be designed to go out for the hot-quenching controlling inner heating, the process by RF electric current in Physical temperature-lowering or other technologies situation.
But, utilize the new development of the biotechnology of genomics and proteomics, inventor developed for the method for assessment RF electric current in the effect used of corium.Particularly, the present inventor has screened one group and is accredited as and relates to dermal collagen matrix, dermal inflammation and reinvent and the potential gene of epidermal differentiation.By analysis and verifying when standing specific RF current processing parameter, condition and scheme, the difference regulation and control of above-mentioned potential gene, determine the gene label based on obtained gene expression profile and gene chip component.
The energy being delivered to skin and/or skin layer can be the form of RF energy, comprises such as rf wave and microwave.Exemplary RF energy device is disclosed in following U.S. Patent Publication: 6,889,090; 6,702,808; 6,662,054; 5,569,242; 5,755,753; 6,241,753; 6,430,446; 6,350,276; 5,919,219; 5,660,836; 6,413,255; 6,228,078; 5,366,443; With 6,766,202.
The effect for the RF process desired by optimizing can be designed by the application of the RF of corium and processing scheme first time of application of comprising RF electric current.When analysis and verifying gene expression data, have been surprisingly found that, potential gene data energy collecting is enough reduced to three subsets, in optimization process scheme not exist under more debatable inflammatory cytokine damage response, in providing the corium of the stress-inducing of the increase of dermal collagen and desired poisonous substance excitement to reinvent, above-mentioned three subsets have unique effect.With regard to known to the present inventor, this represent genomics and be applied to for the first time using in the process of RF current technique.Above-mentioned express spectra display, controlled causes coercing of positive poisonous substance excitement with optimized application, the corium desired by causing and maintaining of coercing of described positive poisonous substance excitement is reinvented, and avoids the traditional damage be associated with not biological effect desirably simultaneously.The optimizable scheme that the invention provides novel gene chip, gene label, screening method and find based on these.
Gene (FBN1, FBLN1, TNXB, the FN1 relevant to the integrity of dermal matrix is comprised as the gene that potential gene is studied, LOXL2, COL3A1, COL1A1, ELN and LOXL1), cause to dermal inflammation reinvent relevant gene (TIMP2, IL1A, TIMP1, TNF, MMP1, MMP9, MMP3, SOD2 and IL1B) and gene (KRT2, the KRT6A relevant to the barrier function of epidermis, CLDN1, LOR, FLG, IVL, DRT10, AQP3 and KRT14).The subset deriving from the analysis of the expression data to these genes is as table 1(corium mark), table 2(matrix remodeling mark, namely, positive poisonous substance excitement coerce releaser) and the mark of table 3(inflammatory cytokine response, that is, repair the damage outside response) in listed by.
Gene expression profile provides the information about the cell response to one group of situation.Gene comprises the instruction for manufacturing messenger RNA(mRNA) (mRNA).But at any given time point, each cell only manufactures mRNA from a part for its gene carried.If gene is used to produce mRNA, being then referred to as is " opening ", otherwise to be just referred to as be " closedown ".Term " regulation and control " refers to the transcriptional state causing the contrast state being different from gene.Such as, " rise " can comprise and only opening, or can refer to improve transcription rate to higher than the baseline rate deriving from contrast or reference conditions.
In expression pattern analysis, the relative quantity of the mRNA expressed in two or more experiment conditions is measured.The change of mRNA level in-site hint to the demand of the protein of being encoded by this mRNA changed.Such as, corresponding to the rising of substrate level in cellular environment of enzyme, transcribing of the rising of enzyme catalyst or cofactor is observed.
In general, gene expression profile shows those genes of significant difference under being included in the experiment condition of change.The subset of this normally some data set (it can comprise whole genome).For the cell of a type, the expression pattern of its combination is one group of gene of the specific characteristic of specified criteria, constitutes the gene label of this condition.Gene label can be used to, and such as, select the patient that can benefit from particular procedure, or design is in order to make the maximized processing scheme of desired feature.
The gene expression profile of potential gene data collection can use microarray to measure.The exemplary commercially available acquisition of cDNA microarray, and can purchased from such as Agilent Technologies, Affymetrix Inc.(Santa Clara, Calif.), Nanogen(San Diego, Calif.) and Protogene Laboratories(Palo Alto, Calif.) company.The specific hybridization techniques that can be used to produce the express spectra adopted in method of the present invention is equally described in U.S. Patent Publication 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800, in 992; Above-mentionedly openly to be incorporated herein by reference; And WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203; With in EP 785 280.Generally speaking, in these methods, for each, it expresses the array that the detected decisive gene of phenotype includes " probe " nucleic acid of a probe, contacts with such as listed above target nucleic acid.Contact as carried out under stringent hybridization condition, then removes unconjugated nucleic acid in hybridization conditions.The pattern of the nucleic acid of the hybridization obtained provides the information of the expression about often kind of detected gene, wherein said expressing information be whether be expressed about gene and, normally, which kind of horizontal expression (to work as expression data with, i.e. express spectra, when can be both qualitative and quantitative) aspect.Alternatively, described express spectra can measure by quantitative PCR or for other quantivative approachs detecting mRNA.
One embodiment of the invention provide gene panel, described gene panel comprises in response to the generation of the radio-frequency current in the tissue volume of mammal skin that, by the gene regulated and controled in described mammal skin, described tissue volume is enough heated to treatment temp by described radio-frequency current.At least one gene is selected from table 1 or table 2, and at least one gene is selected from table 3.In particular embodiments, at least one gene is selected from each of these three tables.All gene panels according to the present invention are all expected and are comprised the gene that at least one is selected from table 3, because these genetic expressions do not change show do not have " bad " to damage response.Probe can be designed to often kind of gene for composition gene chip of the present invention, to build very specific microarray, for design, screening, adapt to or monitor the processing scheme for desired effect, or verifying described processing scheme in the experimenter accepting process.Contemplate microarray, described microarray comprises one group of fixing nucleic acid probe, and described fixing nucleic acid probe can detect this gene with composition according to the gene recombination of gene panel of the present invention.
As used herein, " probe " refers to oligonucleotide, polynucleotide or DNA molecular, is no matter naturally occurring or synthesis produces, its can specifically with there is the nucleic acid hybridization with the sequence of this probes complementary.Probe of the present invention specifically refers to the oligonucleotide be connected with solid support in DNA microarray substrate.Probe can be strand or double-strand.Probe comprises the Nucleotide of 15-25 or more usually, although it can comprise less Nucleotide.Probe is herein selected as complementary from the different chains of specific target nucleic acid sequence, and therefore must be fully complementary, can hybridize specifically with their respective target chains under one group of predetermined condition.Therefore, probe sequence need not reflect the sequence of the exact complementarity of target.Such as, non-complementary nucleotide fragments can be connected to 5' or the 3' end of probe, and the rest part of described probe sequence and the complementation of target chain.Alternatively, non-complementary base or longer sequence can be dispersed in probe, as long as have the complementarity being enough to anneal specifically accordingly between described probe sequence and the sequence of target nucleic acid.
Another embodiment of the invention comprises the method providing beneficial effect to mammal skin.Described beneficial effect comprises, when the inflammatory cytokine response that there is not injured skin, in the skin corium of mammal skin, induce the formation of collagen and/or corium to reinvent, and described method is included in the tissue volume of the inherent described mammal skin for the treatment of cycle and produces radio-frequency current, described tissue volume is enough heated to treatment temp by described radio-frequency current, avoids the rise of the expression of listed gene in table 3 simultaneously.In a treatment cycle, RF electric current can produce repeatedly.
As used herein, term " processes " and " process " etc. generally refers to beauty treatment desired by acquisition or aesthetic, and described effect is confirmed by the biological answer-reply of target.As used herein, " process " covers the process in the Mammals especially mankind, and comprise: (a) prevents or avoid the development of cosmetically not skin desirably, such as microgroove, wrinkle, Hyperpigmented spot and due to the aging of aging or environmental correclation or other skin abnormalities of causing the impact of skin, (b) suppresses, alleviate or postpone the appearance of cosmetically not skin desirably; C () reverses cosmetically not skin desirably or cause it to restore.
In one section of toeatment period, one or more treatment in accordance with the present invention cycle can be carried out.Treatment cycle in order to realize desired response can be short as much as possible in the time length.In particular embodiments, treatment cycle is about one minute or less, and in other specific embodiments, treatment cycle is for being greater than about one minute.In other specific embodiments, treatment cycle continued between about 1 and about 6 minute, and continued between about 2 and about 6 minutes in other embodiments.Treatment in accordance with the present invention comprises one or more treatment cycle period, and is at least one week in particular embodiments and comprises at least one treatment cycle.In other specific embodiments, described toeatment period is between one week and 12 weeks.In a more particular embodiment, described treatment cycle is between 3 weeks and 8 weeks.In certain embodiments, each week in toeatment period comprises the treatment cycle between and six, although should understand in the art, this can change along with the characteristic accepting the individuality processed.
Generally speaking, although using of RF electric current result in than in the higher temperature of skin surface in the dermis, the temperature of skin on surface by most conveniently and non-invasively measuring.Therefore, in certain embodiments, treatment temp (being defined as the temperature of the tissue volume that RF electric current flows through herein) achieves the skin surface temperature on described tissue volume being less than about 45 DEG C.In a more particular embodiment, described treatment temp achieves the skin surface temperature on described tissue volume between about 37 DEG C and about 43 DEG C.
Can pass through from the sample extraction mRNA deriving from the tissue volume that RF electric current flows through according to desired beneficial effect of the present invention; Be selected from by least one the express spectra that the gene of table 1 and/or table 2 and at least one be selected from the gene panel of the genomic constitution of table 3 assess with measuring.The rise that express spectra reflects the gene being selected from table 1 and/or 2 and the genetic expression being selected from table 3 do not change in fact, namely show to provide beneficial effect.
Generally speaking, be reflected among expression multiple delta data according to the regulation and control of gene of the present invention.Be known in the art and there is many experiments for analyzing based on microarray with qualification method of difference expression gene between different condition, and the known selection on method can affect the set of the gene of qualification.Multiple changes the result seeming to provide most repeatability.Multiple change can be defined as the ratio (or difference of contrast average log value and treatment group data average log value) of the mean value of contrast mean value and treatment group observed value.Therefore, multiple is changed to 1 expression and does not change relative to contrast observed value.Positive multiple change represents the rising expressed through for some time, is called rise herein, and negative multiple change represents the reduction expressed through for some time, is called downward herein.The significance of multiple change can be determined by common statistical method.
According to some aspect of the present invention, beneficial effect can be optimized by adjustment treatment temp and/or other processing parameters, described adjustment maximizes in response to the rise of the gene being selected from table 1 and/or table 2 wherein, and the expression being simultaneously selected from the gene of table 3 maintains express spectra constant in fact.Processing scheme of the present invention can comprise safeguards process, and wherein beneficial effect is safeguarded that process is maintained exceeded described toeatment period by one or more, and each safeguards that process comprises at least one maintenance intervals.
The present invention creative skin treatment scheme imagination by the RF electric current heat application via handheld RF current delivery device conductivity.According to preferred RF delivery apparatus of the present invention, send RF with the current forms of conductivity between the electrode being placed in the some distances apart on skin.The delivery apparatus realizing beneficial effect of the present invention conducts radio-frequency current by tissue volume, and there is not electromagnetic radiation in the visible ray of electromagnetic spectrum or infrared frequency, there is not the monochrome of supplementing or polychromatic source of pointing to described tissue volume and there is not Physical temperature-lowering.
Certain embodiments of the present invention provide assessment any energy delivery device or energy delivery technique the ability provided according to the effect in beneficial effect of the present invention is provided.Therefore, assessment is designed to provide the concrete grammar of process effect of the energy delivery device of beneficial effect to comprise by heated skin to described skin: by applying skin described in described energy delivery device process; MRNA is extracted from the sample of the skin after described process; And produce express spectra for gene panel as described in the present invention.As shown in embodiment 3 hereafter, there is RF current delivery device, it comprises can by the parameter specification dividing value counting to produce according to express spectra of the present invention.A kind of applicable device is manufactured by Syneron.The example being not designed to realize the device of genomics of the present invention response object is the RF delivery apparatus that a kind of hand-held individual manufactured by Ultragen Ltd uses, its in Europe with STOPtMbrand is issued.This device is also shown in figure 3 for comparing, and it fails the rise of the gene caused in table 1 and 2.The specification of this device is designed to be applicable to the scheme provided based on the heat energy less be in fact applied on larger skin area, and can not reach the level needed for the effect realizing the positive poisonous substance excitement realized by device according to the present invention and scheme.
According to another embodiment of the invention, provide the method for screening skin of face processing scheme.Described method is screened for stimulating the effect in the inflammatory cytokine response of injured skin providing collagen and/or corium to reinvent beneficial effect to mammal skin.Described method generally comprises according to processing scheme process facial skin; MRNA is extracted from the sample of the skin of face after described process; Gene expression profile is produced for gene panel as described in the present invention; Described gene expression profile and reference spectrum are compared; And when described express spectra reflects the rise of the gene being selected from table 1 and/or table 2 and is selected from the shortage in fact of regulation and control of gene of table 3, determine that described facial processing scheme is effective.In particular embodiments, described skin of face processing scheme is included in treatment cycle the pulse radiation frequency electric current using the generation of radio-frequency current generation device by the first tissue volume of described skin of face.Described processing scheme can be included in described treatment cycle, and mobile described radio-frequency current generation device also produces the pulse radiation frequency electric current by the minor microstructure volume of described skin of face.
In screening method, have selected the region of skin for the consistence with the target treatment zone especially characteristic of face.The region of the beauty treatment aspect fragility of skin of face comprises the region in socket of the eye week and mouth week.Therefore, the skin of face around the ear being called as ear week skin, close with process targeting regions in structure, but be not easy to be seen with regard to the infringement that may be caused by micro-biopsy procedure.Therefore, the present invention need to carry out bioptic in, required sample by after process ear week skin examination of living tissue obtain, and with reference to by before treatment or untreated ear week skin examination of living tissue obtain.Generally speaking, the described before treatment or all skin comprising all skin of ear after contiguous described process substantially with reference to skin of untreated ear.In unusual specific embodiment, the express spectra being selected from the gene of table 1 and/or table 2 shows the multiple change being statistically greater than described reference one times.
It should be noted that, although because skin of face causes maximum cosmetic treatments challenge (because it the most easily shows aging and the aging of environmental correclation and the effect of damage, and it initial thinner and than skin more fragile in other regions of health) and in illustrative embodiment, highlight skin of face, but the present invention can also provide beneficial effect to the skin outside skin of face.In addition, for the human consumer of cosmetic treatments technology, skin of face is the region with topmost importance attractive in appearance.
In certain embodiments, by the generation with pulse radiation frequency electric current synergistically to skin of face application beautifying active substance or composition, the beneficial effect provided by the application of RF electric current can extended or enhancing.Described active substance or composition can have strengthening to gene expression profile according to the present invention or synergistic effect.The limiting examples of the beautifying active substance be applicable to comprises retinyl propionate and derivative, caffeine, hyaluronic acid and general plant milk extract.
With regard to treatment cycle, toeatment period, treatment temp and delivery apparatus, screening method according to the present invention comprises the parameter identical in fact with treatment in accordance with the present invention method, and described parameter is adjusted with regard to desired screening dividing value.
According to other embodiments of the present invention, provide the gene label being applicable to the difference expression gene identifying cosmetic skin beneficial effect.Described beneficial effect comprises, when the inflammatory cytokine response that there is not injured skin, in the skin corium of mammal skin, induce the formation of collagen and/or corium to reinvent, described gene label comprises at least one gene in each being selected from table 1, table 2 and table 3.Generally speaking, gene label is the subset of the gene obtained from the data set of the gene relevant to particular characteristics, proterties or biological function.Gene label can obtain from all or part of of gene data collection, and can comprise from least about two kinds of genes or the information of any amount of gene of quantity forming total data set at the most according to label of the present invention.When the subset of described data set is used, described subset can comprise the gene of rise, downward gene, in fact not by the gene that regulates and controls or their combination.
Method of the present invention comprises to mammal skin region coating first man care composition and the step optionally applying the second personal care composition.Described first and second personal care compositions can be various ways, include but not limited to lotion, white cream, slurries, foam, gel, spray, salve, facial mask, club, wetting Agent for Printing Inks, patch, powder and/or cleaning piece.In one embodiment, described first man care composition before delivery of energy and/or period coating.In an alternative embodiment, described second personal care composition applies after described first composition of coating and delivery of energy.Optionally, method of the present invention can comprise the step to cutaneous application the 3rd personal care composition, and wherein said 3rd composition comprises amendment.In one embodiment, described 3rd personal care composition applied before the described first man care composition of coating.Preferably, described 3rd composition coating before delivery of energy at least 24 hours.In an alternative embodiment, described first man care composition applies twice every day, and energy is sent once every day, or once in a week, or monthly.In one embodiment, twice daily described first man care composition is coated on skin, and weekly to dermal delivery energy.
Described first, second, and third personal care composition can comprise Multiple components, and its limiting examples can be present in The CTFA International Cosmetic Ingredient Dictionary andHandbook, the tenth edition (2004).
Composition of the present invention can comprise the dermatology acceptable carrier of 50% to 99.9%.Carrier of the present invention is emulsion form.Herein, " emulsion " generally includes containing aqueous phase and oil phase.Oil derived from animal, plant or oil, can be able to be natural or synthesis, and can comprise silicone oil.Emulsion carriers includes but not limited to oil-in-water, water-in-oil, water-in-oil-in-water and water-in-silicone bag fat liquor.In one embodiment, dermatology acceptable carrier comprises O/w emulsion, or water-covered siloxane.Described emulsion can also comprise wetting agent, such as, and glycerine and emulsifying agent that is non-ionic, cationic and/or negatively charged ion.Suitable emulsifying agent is disclosed in, and such as, authorizes the United States Patent (USP) 3 of the people such as Dickert, 755,560, authorize the United States Patent (USP) 4 of the people such as Dixon, 421,769 and McCutcheon ' s Detergentsand Emulsifiers, in North America version 317-324 page (1986).
Can within the scope of larger number change, use composition of the present invention to improve skin.The amount being coated to the personal care composition on skin can change according to the beneficial effect of the position of health and expectation.Exemplary amount comprises 0.1mg/cm2to 40mg/cm2.An available amount of application is 0.5mg/cm2to 10mg/cm2.
In skin or being administered in the composition on skin surface, temperature variation can be produced simultaneously.This temperature variation is the change except any temperature variation produced by delivery of energy self.Such as, before energy is sent, skin can be heated, or before, during and/or after energy is sent, skin be cooled.
Use the gene panel after process and gene label, and human consumer's investigation widely, establish the region of scheme, make to develop the size of optimized treatment zone and optimized treatment time.Find contrary with the instruction of this area, using appts is not best treatment process over a large area.When excessive region is processed, the part of beginning can human consumer again inswept above it before cooling and replying.If the region of process is too little, human consumer can emit the risk of excess processes and unnecessary damage.In addition, process itself is the work of high strength for human consumer, process is divided into little block to make human consumer more easily fully observe scheme.Equally, if the personal care composition that the personal care composition used together with device can be applied to less region, process this region, then removes any remnants by human consumer, so described composition will to human consumer's more attractive many.Personal care composition is applied on the face whole, or or even one side of something on the face, can cause process experience make us unhappy.This has negative impact for the compliance of human consumer then.
See now lower Figure 10, wherein marked the crows feet area 60 of human consumer 62 by dotted circle, it to occur in above and below eye 64 and contiguous every tail of the eye at a glance.As everyone knows, along with the ageing of people, crows feet area 60 is easy to form wrinkle and microgroove, and is easy to suffer environmental injury.Figure 11 shows region A 70, and the arrow wherein shown in the A of region illustrates the exemplary process approach of described device (not shown).Above has been given the treatment time.Figure 10 shows region B 72, and it is positioned at the top of the same eyes of human consumer 62, and overlapping with crows feet area 60.Show exemplary arrow, to show possible process approach.By localized area A 70 and region B 73 by this way, crows feet area 60 has been processed the length of non-crows feet area twice of region A and B.This is an important discovery, because need on face to be processed human consumer, most microgroove and wrinkle are positioned at crows feet area.
Figure 13 and 14 shows region C 74 on the another eyes 64 of human consumer 62 and D 76.Can expect as people, these treatment zones and region A and B similar in fact and be processed in an identical manner.
A kind of alternative processing scheme is shown in Figure 15 and 16, wherein on human consumer 62, shows region E 80 and F 82.These " C " type treatment zones each time through the crows feet area 60 of out-of-date process Figure 10 once, but longer " C " type treatment zone allow each region of processed skin return the process of carrying out another at device before cooling a little.So, the comfort level of human consumer adds, but the crows feet area 60 of Figure 10 is only processed in this treatment cycle.
Second personal care composition optionally uses together with aforesaid method.Described second personal care composition can use between the described first man care composition of use and the continuous treatment cycle of thermal.Described second personal care composition preferably comprises at least one and is not present in skin care actives in described first man care composition.
Following embodiment is provided some feature and advantage for display multiple embodiments of the present invention, and should not be understood to limit its scope.
Embodiment
Embodiment 1:processing scheme
This example illustrates the process to all regions of the ear of skin of face, the object of process carries out genomics and histological assessment.Therefore, be in control object, whole process is all performed by technician under clinical setting.In process range four region is processed, process total 16 minutes, 4 minutes, each region, secondaryly on every Wendesdays carried out six weeks.This code is according to the face/neck subregion research adopting the design of experimenter own control, and process is only applied to the ear week region of side, and adds that false process that the equipment be not energized form is applied to all regions of ear of the correspondence of opposite side by gel.With Thermal Imaging Camera monitoring process, to guarantee that target skin surface temperature reached 40-43 DEG C in first minute, and keep in the process of each 4 minutes but be no more than.
Treatment site is positioned at below ear and hits exactly.About 1.5 inches wide of this site, about 4 inches long.If process the region too dry of skin or overheat, extra gel can be used.The face of experimenter and neck are clean, and do not have astringent, perfume etc.Hair is cut short by from treatment zone.
device process:
1. the amount (about 1.4g gel) of squeezing is administered to treatment site, and coating evenly, avoids friction.If skin overheats, represented by experimenter, the gel of the amount can squeezed additional if desired during process is applied to the cheek above described treatment zone, cools to make skin.
2. opening unit set the timer to 4 minutes.
3. the function head of device is applied on skin, and adopts and seesaw and slightly touch and move along treatment site.The head of operating device is across remaining connective while treatment zone.Shown by the lens on application device with suitable contact of skin, it should continue to dodge ruddiness to indicate contact.
4. thermal camera image is monitored during processing.Within 30-60 second, the skin in treatment zone reaches the ideal temperature of 40-43 DEG C.Color mode on heat picture illustrates temperature.Generally speaking, along with device heating, application device is moved across skin rapidly, causes discomfort to avoid the temperature because raising.Along with process proceeds, move and slowed down to maintain desirable temperature.Experimenter is alerted, and device will make us sensible heat, but can not be burnt.
If 5. heat picture shows solid red area, then temperature has exceeded 43 DEG C.By following manner control temperature: improve the processing speed seesawed; Slightly increase the size for the treatment of zone;
Hocket and seesaw and 8-shaped motor pattern; Or by making function head contact the extra gel for this accidental situation be deposited on cheek, and add more gel.
If 6. treatment zone could not reach desirable temperature, or it has cooled off, should make seesaws slows down, until the major part of described treatment zone is in target temperature.
Embodiment 2:examination of living tissue code
The present embodiment has demonstrated for collecting the relative gentle examination of living tissue code of the assessment according to some aspect of the present invention with monitoring object specimen in use, and supported data disclosed herein.
In 30 adult female experimenters, use RF current delivery device to carry out clinical biopsy studies.Process is provided in ear week (around the ear) region of skin of face.Ear week skin as socket of the eye week skin suitable substitute be known at beauty treatment fields.Process clinically use SyneronV8 device (see embodiment 3) carry out, four minutes add Thermogram as target spectrogram.Experimenter accepts weekly three process, amounts to and carries out six weeks.Carry out examination of living tissue, with the change of the histology of detection of skin according to process, and produce genome spectrum.
examination of living tissue flow process:
The living tissue sample of 4mm is obtained from the left side of neck and ear week region (this region is positioned at immediately below ear) on right side.Use the aseptic syringe needle of 30 wire gauges, by containing the adrenergic fix comprising 2% Xylocitin under the skin treating biopsy.Once experimenter represents that this region is numb, what namely adopt the aseptic technique of standard to collect suitable size drills through living tissue sample, then passes through sewn closed.If desired, the aluminum chloride aqueous solution of 20% is used to homeostasis.After examination of living tissue, the normal healing of monitoring suture, and remove suture line after 7 days.
sample preparation after examination of living tissue:
Bioptic tissue is divided into two points of other samples, and one is detected for genetic expression, and another is for Histological assessment.Sample is divided into equal two parts to be parallel to the lines drawn to the line of corium from stratum corneum.The process of sample is carried out according to industry standard.
Embodiment 3:mRNA extracts and analyzes
This example illustrates and support to comprise gene chip, gene label and instruct RF to send qualification and the analysis of the RNA biomarker of certain embodiments of the present invention of the discovery of the biological model of the design of scheme.
As described above from the both sides of the face/neck immediately below ear, obtain the living tissue sample 2 times of 4mm according to embodiment 2, once after final process 24 hours, once after the treatment 4 weeks.
Biopsy samples be transferred to comprise 1.5ml tissue storage reagent (solution, invitrogen, Life Technologies, Carlsbad, CA) 2ml centrifuge tube in.Centrifuge tube is at 2-8 DEG C of refrigerated overnight.Remove storage reagent, and by sample as in-80 DEG C of refrigerators until process.Before facing process, from refrigerator, shift out sample, to each pipe add 1.5ml phenol and guanidinium isothiocyanate (reagent, Invitrogen, Life Technologies, Carlsbad, CA) a single phase soln and a 3mM wolfram varbide globule (3mM Tungsten Carbide beads, Qiagen, catalog number (Cat.No.) 69997).Homogenize immediately sample in mixed grinding device (Quiagen Inc.), carries out the process of four times 3 minutes with 30/ second, overturns joint after each.With 12,000rpm Centrifuge A sample 10 minutes, to remove impurity.Then, supernatant liquid is transferred to the PhaseLock gel rotated through in advance and heavily manages (Phase Lock gel heavy tubes, Eppendorf, New York, NY, catalog number (Cat.No.) 0032-005-152) in, add 300 μ L chloroforms (Sigma), concuss, avoids excessive vortex.With 12,000rpm Centrifuge A sample 10 minutes, afterwards supernatant liquid is transferred in new 2ml centrifuge tube.
By add 800 μ L 70% ethanol regulate conjugation conditions, vortex pipe with mixing, then of short duration rotation.830ml sample is transferred in the RNeasy centrifugal mini column chromatography in vacuum manifold, and applies vacuum.Remaining sample (about 830mil) to be transferred on identical RNeasy post and to apply vacuum.By damping fluid RW1 to the RNeasy post that pipettes 700 μ l and 500 μ l successively applies vacuum after each wash, eliminate pollutent.
Above-mentioned RNeasy centrifugal column is transferred in new 2mil collection tube, and with the centrifugal pipe of 14,000rpm 2 minutes, by posts transfer in new 1.5ml collection tube.The ethanol that ridge suction is remaining in each pillar.30 μ l preheatings directly do not moved liquid on RNeasy film containing the water of RNA enzyme, hatch film 5 minutes, then with 14,000rpm centrifugal 2 minutes.Collect elutriant, obtain the RNA of water-soluble instant.
After separation, use can purchased from RNA 6000 Nano of Agilent Technologies, Inc.of Santa Clara, CAkit#5065-4476 measures RNA output.RNA is assessed by One step RT-PCR.With regard to RT-PCR biomarker analysis, RNA is diluted to the final concentration in 5ng/ hole.
Use RT-PCR kit (QScripttMone-Step Fast MGB qRT-PCR test kit, can purchased from Quanta BioScience, Inc., Gaithersburg, MD) RNA of purifying is converted into cDNA.Then by RNA and the QScript of 500 nanograms enzyme/damping fluid premixed liquid mixing, and run on thermal cycler according to the specification sheets of test kit.Then mixed by cDNA and the Quanta the obtained Perfecta Master Mix of a μ l, uniformly distributing is to custom arrays flat board (the Custom RT Profiler comprising the primer for following gene verified in advancetMpCR Array, can purchased from SABiosciences, Corp., Federick, MD): KRT2, KRT6A, CLDN1, LOR, FLG, IVL, KRT10, AQP3, KRT14, FBN1, FBNL1, TNXB, FN1, LOXL2, COL3A1, COL1A1, ELN, LOXL1, TIMP2, IL1A, TIMP1, TNF, MMP1, MMP9, MMP3, SOD2 and IL1B.Then above-mentioned Array Plate is sealed, and at thermal cycler (StepOnePlustMreal-Time PCR System Upgrade, from AppliedBiosystems, Inc.Foster City, CA) upper operation.
Data analysis uses by SABiosciences of Frederick, and the data analysis software that MD provides is carried out.
27 listed above compiling genes at the expression multiple delta data of the target spot time of 24 hours and 4 weeks respectively as shown in Fig. 4 A, 4B and Fig. 6 A, 6B above.The gene relevant to dermal matrix is grouped into 90 and 91, and reinvent relevant gene to dermal inflammation and be grouped into 92 and 93, the gene relevant to epidermal differentiation is grouped into 94 and 95.The multiple delta data of Exemplary gene is as shown in Fig. 5 A collagen 1A1,5B elastin and Fig. 7 A collagen 1A1 and 7B elastin.Please note the common regulation and control between many collagen e1A1 genes and elastin gene.
Dimension disclosed herein and value are not appreciated that and are strictly limited to quoted exact value.On the contrary, except as otherwise noted, each such dimension is all intended to both represent quoted value, also represents the functionally equivalent scope around this value.Such as, the dimension being disclosed as " 40mm " is intended to represent " about 40mm ".
Unless expressly excluded, or in other words limit to some extent, each document quoted herein, comprise any cross reference or Patents or patent application, be incorporated in full herein with way of reference all accordingly.Quoting of any document is not to its prior art as disclosed herein or claimed any invention; or its individually or with any combination of other reference any, or with reference to, propose, suggestion or disclose any accreditation that this type of is invented.In addition, if any implication of term or definition and any implication of same term in the document be incorporated to way of reference or define is conflicted mutually in this document, be as the criterion with the implication or definition of giving that term in this document.
Although illustrate and describe particular of the present invention, it should be apparent to those skilled in the art that can to make when not departing from the spirit and scope of the invention many other change and modification.Therefore, all these being intended to contain in the scope of the invention of claims change and modification.
Multiple delta data is checked and analyzes, its grouping is found surprisingly, when showing the separated (IL1A of gene of few adjustment in response to described process, IL1B and TNF) time, they comprise inflammatory cytokine response gene, and reinvent relevant gene to the synthesis of collagen and renewal and corium and raised under processing conditions.According to multiple delta data select gene correspondingly list in table 1 hereafter, 2 and 3.
In addition, also find surprisingly, 24 months time, processing scheme is on the positive impact of genetic expression, after treatment 1 month time maintain weaken hardly.For maintenance genomics effect to exceeding the above-mentioned time, safeguard that process may be needs.
Expection gene expression profile reflects the rise in translation product middle and lower reaches.Although viewed multiple change may be relatively low, the chronic up-regulation of the synthesis of some protein and enzyme in the long term, is considered to impart substantial lasting Beauty benefits to skin.
Embodiment 4:personal care composition
Following is for the Exemplary personal care's composition with device use of the present invention.
table 1
corium marks
table 2
matrix remodeling marks
table 3
inflammatory cytokine respond flag