CN103212161A - Photostimulation method and photostimulation device - Google Patents
Photostimulation method and photostimulation device Download PDFInfo
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Abstract
The invention relates to a photostimulation method and a photostimulation device. The photostimulation method comprises the following steps: providing a light-emitting diode light source which is one of clusters formed by a yellow light-emitting diode, a red light-emitting diode and a blue light-emitting diode, and irradiating the light source of the light-emitting diode to a main body to promote collagen protein synthesis, or to restrain growth of bacteria or to restrain melanogenesis, wherein the intensity of illumination of the red light-emitting diode is 1,000 lux to 3,500 lux, the intensity of illumination of the red light-emitting diode is 6,000 lux to 9,500 lux, and the intensity of illumination of the blue light-emitting diode is 3,000 lux to 7,000 lux.
Description
Technical field
The invention relates to a kind of photostimulation method and light stimulation device, refer to a kind of photostimulation method and light stimulation device that promotes collagen protein synthesis, strengthens bacteria growing inhibiting or the formation of inhibition melanin especially.
Background technology
In the department of dermatologry diagnosis, often with Drug therapy patient skin disease, as comedo etc., but for a long time, the result of Drug therapy is accompanied by side effect more, takes the burden that can cause on the body metabolism for a long time, and therapeutic effect is desirable not to the utmost, the anxiety that recurrence is often arranged can't effectively be improved patient's skin problem.
In recent years, the medical and beauty treatment industry is prosperous day by day, research is pointed out: wavelength can be used for the comedo treatment between the blue light of 400nm to 475nm, because of blue light can act on single-phase oxygen of toxigenicity and free radical with sclererythrin (coproporphyrin) in the light sensation in Propiobacterium (Propionibacterium acnes) or the histiocyte, and then destroy antibacterial and part sebaceous gland histiocyte, improve the inflamed of comedo.On the other hand, wavelength between the HONGGUANG of 600nm to 750nm, wavelength between the gold-tinted of 550nm to 600nm, and wavelength between the green glow of 500nm to 570nm, can stimulate the fibroblast of skin corium, and then promote collagen protein synthesis, prevent skin aging.
But, for reaching above-mentioned effect, industry is used laser or pulsed light mostly at present, and therefore the energy or the intensity of two kinds of light just are enough to reach above-mentioned effect, but cause cell injury easily.Recently just general light source of develop actively or LED source replace above-mentioned high-intensity light source, but at present LED source since energy a little less than, urgent need will find suitable illuminance just to be enough to be effective, otherwise light illumination is crossed and low can can't be brought into play curative effect; Otherwise, when light illumination is too high, except meeting cause cell impaired, device volume is promoted, can't develop the little and lightweight Portable phototherapy device of volume.
In view of the above, if can send out a kind of photostimulation method of output and light stimulation device, wherein utilize the light emitting diode of specific illumination range, reach to promote collagen protein synthesis, strengthen bacteria growing inhibiting or suppress melanin and form, and can save manpower and time cost, make the patient can have beautiful skin fast.
Summary of the invention
Main purpose of the present invention is that a kind of photostimulation method is being provided, and it can send the HONGGUANG or the gold-tinted of specific illumination range by light emitting diode, stimulates fibroblast, to increase collagen protein synthesis, while blood circulation promoting, the old useless cellular metabolism of quickening; Perhaps send the blue light of specific illumination range, suppress, kill Propiobacterium, or melanic synthetic in reduction and the inhibition melanocyte.
For reaching above-mentioned purpose, an aspect of the present invention provides a kind of photostimulation method, may further comprise the steps: a LED source is provided, and this LED source is to be selected from by wherein one of a Yellow light emitting diode, a red light-emitting diode and blue light-emitting diode institute cohort group; And this LED source shone in a main body, form to promote collagen protein synthesis, bacteria growing inhibiting or to suppress melanin, wherein, the illumination of this Yellow light emitting diode is 1,000 to 3,500 luxs (lux), the illumination of this red light-emitting diode are 6,000 to 9,500 luxs, the illumination of this blue light-emitting diode is 3,000 to 7,000 luxs.
Known technology uses different wavelength of laser light or pulsed light to reach comedo treatment usually, stimulates the fibroblast of skin corium to promote collagen protein synthesis, but because of laser light or pulsed light intensity is high and equipment is huge, ordinary consumer is difficult to have.Recently, though the light emitting diode of use is arranged as light source, expectation can reach above-mentioned treatment comedo and the effect that promotes collagen protein, but owing to knownly research is not arranged at the influence of the illumination of lumination of light emitting diode for cell or thalline, because of not setting under the prerequisite of specific illumination, it is unknown whether known method can reach the real genus of above-mentioned effect.Review, the method of the invention, utilization is sent the light emitting diode of blue light, gold-tinted or HONGGUANG as light source, and it is defined in different illumination ranges, it is hereby ensured to reach stimulates fibroblast to promote collagen protein synthesis, suppress or the poisoning Propiobacterium, melanic synthetic in reduction and the inhibition melanocyte.
When using HONGGUANG or Yellow light emitting diode with suitable illuminance, when lasting appropriate time shines, can stimulate macrophage (macrophage) to discharge cytohormone (cytokine), promote the fibroblast division; (fibroblast growth factor FGF), and then increases collagen protein synthesis also can to stimulate simultaneously fibroblast synthetic DNA and secretion somatomedin.If this main body is cells in vivo, fibroblast or the macrophage in the skin corium for example, direct printing opacity then, irradiation skin reaches promotion wound healing and antidotal effect; Perhaps, if this main body is a cell in vitro, then can more treated cell be planted the object of bringing back to life and reach above-mentioned effect earlier through after the above-mentioned processing.Hence one can see that, and main body of the present invention is meant the object that is subjected to light stimulation.
In the above-mentioned photostimulation method of the present invention, when this LED source is during for this Yellow light emitting diode or this red light-emitting diode, this main body is preferably a fibroblast, a macrophage or its combination.In the present invention's one preferred embodiments, this main body is a fibroblast.In addition, the optical wavelength range of this Yellow light emitting diode can be between between the 570nm to 590nm, the optical wavelength range of this red light-emitting diode can be between 620nm to 750nm, and the irradiation time of the irradiation time of this red light-emitting diode or this Yellow light emitting diode does not have particular restriction, as long as can reach above-mentioned effect and can not damage to this main body, can adjust to some extent according to the predetermined luminous intensity of this red light-emitting diode and this Yellow light emitting diode emitted light, when illumination is higher, just can just reach same effect with short irradiation time; Otherwise when illumination was low, then available long irradiation time reached identical effect.
For example, send at red light-emitting diode under the illuminance of sending 1,000 to 3,500 lux of 6,000 to 9,500 luxs or Yellow light emitting diode, irradiation time can be between 5 minutes to 90 minutes.If exceed above-mentioned illumination range, when for example using red light-emitting diode to send the illuminance irradiation of 9890 luxs,, get off for a long time and can cause cell impaired because of illumination is too high though do not have too much influence in the short time; Otherwise, if when using red light-emitting diode to send the following illuminance irradiation of 6,000 luxs, then because of illumination is low excessively, even long-time use also is difficult to effectively be effective.
When using blue light-emitting diode with suitable illuminance, when lasting appropriate time shines, meeting stimulation melanin cell directly or indirectly influences tryrosinase (tyrosinase), and then reduces melanic synthesizing, and also can avoid melanin deposition certainly; On the other hand, also can influence the interior sclererythrin generation chemical action of sensing optical activity in the Propiobacterium, produce Cytotoxic single-phase oxygen and free radical, and then make Propiobacterium itself active impaired and dead, also can influence simultaneously part sebaceous gland histiocyte, reduce sebum secretion.If this main body is a skin, comedo or Propiobacterium for skin surface then can reach sterilization effects; Perhaps, then can suppress its synthesis of melanin, avoid skin colourity to increase and reach whitening effect for the melanocyte of skin surface.
In the above-mentioned photostimulation method of the present invention, when this LED source is during for this blue light-emitting diode, this main body then is a Propiobacterium, a melanocyte or its combination.In addition, the optical wavelength range of this blue light-emitting diode is between between the 450nm to 475nm, and the irradiation time of this blue light-emitting diode does not have particular restriction, as long as can reach above-mentioned effect and can not damage to this main body, can adjust to some extent according to the predetermined luminous intensity of this blue light-emitting diode emitted light, when illumination is higher, just can just reach same effect with short irradiation time; Otherwise when illumination was higher, then available long irradiation time reached identical effect.
For example, send at blue light-emitting diode under the illuminance of 3,000 to 7,000 luxs, irradiation time can be between 5 minutes to 90 minutes.If the illumination of using is higher than above-mentioned illumination range,, gets off for a long time and can cause cell impaired because of illumination is too high though do not have too much influence in the short time; Otherwise, if the illumination of using is lower than above-mentioned illumination range, then because of illumination is low excessively, even long-time use also is difficult to effectively be effective.In the present invention's one preferred embodiments, to send at blue light-emitting diode under the illuminance of 5,330 luxs, irradiation time surpasses 30 minutes and just can reduce the melanin generation; In the present invention's one preferred embodiments, to send at blue light-emitting diode under the illuminance of 5,710 luxs, irradiation time surpasses 10 minutes and just can reach the effect that suppresses Propiobacterium.
Another object of the present invention is that a kind of light stimulation device is being provided, wherein adopt red light-emitting diode or the Yellow light emitting diode that sends specific illumination range, in the hope of reaching the stimulation fibroblast, increase collagen protein synthesis, simultaneously blood circulation promoting, the old useless cellular metabolism of quickening; Perhaps send the blue light-emitting diode of specific illumination range,, or reduce and interior melanic synthesizing of inhibition melanocyte in the hope of inhibition, poisoning Propiobacterium.
For reaching above-mentioned purpose, another aspect of the present invention provides a kind of light stimulation device, comprising: a housing, and form an accommodation space and have an end face and a lateral margin, this end face is provided with a light-emitting window; One scattering sheet covers this light-emitting window of this housing; One first light source module, it is arranged in this accommodation space of this housing and has one first light emitting diode, this first light emitting diode is located at this scattering sheet below, and this first light emitting diode is to be selected from by red light-emitting diode, Yellow light emitting diode, and blue light-emitting diode institute cohort group wherein one, wherein, the light illumination that this Yellow light emitting diode sends through this scattering sheet is 1,000 to 3,500 luxs (lux), the light illumination that this red light-emitting diode sends through this scattering sheet is 6,000 to 9,500 luxs, and the light illumination that this blue light-emitting diode sends through this scattering sheet is 3,000 to 7,000 luxs; And a control module, it is to electrically connect this first light source module and a power module.
From the above, use in the light stimulation device of the present invention and send the light emitting diode of blue light, gold-tinted or HONGGUANG as light source, and the light of different colours all is defined in corresponding illumination range, therefore after light stimulation device irradiation of the present invention, can stimulate fibroblast, guarantee that collagen protein synthesis promotes, suppresses or kill Propiobacterium and reduction and suppresses melanic synthetic in the melanocyte.
In the above-mentioned light stimulation device of the present invention, this power module can be an external power source or is arranged in this accommodation space of this housing.If this power module is arranged in this accommodation space of this housing, it can comprise chargeable battery or can hold general aneroid battery or minicell, reaches the effect of supply power supply.On the other hand, if power module is an external power source or for being arranged at the chargeable battery in this accommodation space of this housing, this control module can have optionally and comprises: a charging hole electrically connects this control module for this power module.
In addition, in the above-mentioned light stimulation device of the present invention, this control module also can be had and optionally comprised: an on and off switch is arranged at this surface of shell, to control this power module supply power supply.In addition, this housing is preferably by the low material of light transmittance and is constituted, as reflexive height or the high material of density, to reduce the light leakage phenomena of light stimulation device.In addition, this area personage also can increase the integrally-built adaptation of light stimulation device by various structural designs, to reduce the light leakage phenomena of light stimulation device.
On the other hand, in the above-mentioned light stimulation device of the present invention, this lateral margin alternative of this housing is provided with a light hole.In the case, light stimulation device can also comprise: a light transmission piece covers this light hole; And a secondary light source module, it is corresponding light transmission piece setting and emits beam and pass this light transmission piece.In the case, this control module also can comprise again: a mode selector switch, all be arranged at this surface of shell, and to start this first light source module or this secondary light source module, in other words, promptly switch the start between first light source module and the secondary light source module.In addition, the light emitting diode that is adopted in this first light source module and this secondary light source module can be same color or different colours.
In the above-mentioned light stimulation device of the present invention, be located at this scattering sheet of this light-emitting window, can help even bright dipping, avoid diagnosis and treatment light direct irradiation user eyes, and the uniformity of raising device photostimulation effect, in other words be about to originally belong to the light emitting diode of point source, after the astigmatism effect, form area source at the light-emitting window place; In addition, be located at this light transmission piece of this light hole, then not necessarily be required to be scattering sheet, if scattering sheet then can reach above-mentioned effect, if not be scattering sheet, the light that then can directly transmit point source and provided.
In the above-mentioned light stimulation device of the present invention, this first light source module and this secondary light source module can be designed to replaceable, in other words use red light-emitting diode, Yellow light emitting diode or blue light-emitting diode to form this first light source module and this secondary light source module.If when needing red light irradiation, then replace to the light source module that constitutes by red light-emitting diode; And when needing blue light illumination, then replace to the light source module that constitutes by blue light-emitting diode.In addition, employed led designs in this first light source module and this secondary light source module can also be become replaceable, in other words, then the light emitting diode on the light source module be changed into red light-emitting diode if when needing red light irradiation.
In sum, photostimulation method of the present invention and light stimulation device, can be by adopting the light emitting diode of different colours, for example HONGGUANG, gold-tinted or blue light-emitting diode, carry out Photic Stimulation, therefore can reach and suppress or poisoning Propiobacterium and reduction or to suppress the melanin of melanocyte synthetic and promote collagen protein synthesis, and then reach and treat comedo and whiten or the aging-resistant effect.
Description of drawings
Below be by particular specific embodiment and description of drawings embodiments of the present invention, the personage who has the knack of this technology can understand other advantages of the present invention and effect easily by the content that this description disclosed.The present invention also can be implemented or be used by other different specific embodiments, and the every details in this description also can be carried out various modifications and change based on different viewpoints and application under not departing from spirit of the present invention, wherein:
Fig. 1 is the structural representation of the embodiment of the invention one light stimulation device
Fig. 2 is the side view of the embodiment of the invention one light stimulation device
Fig. 3 is the system block diagrams of the embodiment of the invention one light stimulation device.
Fig. 4 shows the human fibroblast survival rate of the embodiment of the invention two.
Fig. 5 shows the human fibroblast survival rate of the embodiment of the invention three.
Fig. 6 shows the collagen protein synthesis rate of the embodiment of the invention four.
Fig. 7 shows the human fibroblast survival rate of the embodiment of the invention five.
Fig. 8 shows the human fibroblast survival rate and the collagen protein synthesis rate of the embodiment of the invention six.
Fig. 9 shows the human melanocyte survival rate of the embodiment of the invention seven.
Figure 10 shows the melanin synthetic ratio of the embodiment of the invention eight.
Figure 11 shows the Propiobacterium survival rate of the embodiment of the invention nine.
Figure 12 shows the human fibroblast survival rate of comparative example of the present invention.
The specific embodiment
Accompanying drawing described in the embodiments of the invention is the sketch map of simplification.Only described accompanying drawing only shows the element relevant with the present invention, and the aspect that its shown element is non-when be actual enforcement, component number, the shape equal proportion during its actual enforcement are one optionally to design, and its component placement kenel may be more complicated.
Embodiment one
Referring to figs. 1 to Fig. 3, wherein Fig. 1 is the structural representation of light stimulation device of the present invention, and Fig. 2 is the side view of light stimulation device of the present invention, and Fig. 3 is the system block diagrams of light stimulation device of the present invention.
As shown in Figure 1 to Figure 3, light stimulation device of the present invention comprises: a housing 10, a scattering sheet 12, a light transmission piece 13, one first light source module 40, a secondary light source module 50 and a control module 30.
This housing 10 forms an accommodation space, can hold each module.In addition, this housing 10 has an end face 11 and a lateral margin 12, and this end face 11 is provided with a light-emitting window 111, and this lateral margin 12 is provided with a light hole 121.
Be positioned at these light-emitting window 111 places of this end face 11, use this scattering sheet 12 to cover; Be positioned at this light hole 121 of this lateral margin 12, use this light transmission piece 13 to cover.In addition, these secondary light source module 50 corresponding light transmission piece 13 are arranged at this accommodation space of this housing 10, and emit beam and pass this light transmission piece 13, and have at least one second light emitting diode 51.In this, if only being used for printing opacity merely, this light transmission piece 13 is used for astigmatism, then 50 of secondary light source modules become point source.
This first light source module 40 is arranged in this accommodation space of this housing 10 and a plurality of first light emitting diodes 41 with arrayed, this first light emitting diode 41 is located at this scattering sheet 12 belows, and this first light emitting diode 41 is to be selected from by red light-emitting diode, Yellow light emitting diode, and blue light-emitting diode institute cohort group wherein one, wherein, the light illumination that this Yellow light emitting diode sends through this scattering sheet is 1,000 to 3,500 luxs (lux), the light illumination that this red light-emitting diode sends through this scattering sheet is 6,000 to 9,500 luxs, and the light illumination that this blue light-emitting diode sends through this scattering sheet is 3,000 to 7,000 lux.
This control module 30 electrically connects this first light source module 40 and a power module 20, and this control module 30 comprises: a charging hole 33 electrically connects this control module 30 for this power module 20; One on and off switch 31 is arranged at this housing 10 surfaces, to control this power module 20 supply power supplys; And a mode selector switch 32, all be arranged at this housing 10 surfaces, to start this first light source module 40 or this secondary light source module 50.
This power module 20 can be an external power source or is arranged in this accommodation space of this housing 10.In this power module 20 was arranged at this accommodation space of this housing 10, this power module 20 can contain chargeable battery or can hold general aneroid battery or minicell, reached the effect of supply power supply.
Therefore, above-mentioned light stimulation device adopts red light-emitting diode or the Yellow light emitting diode that sends specific illumination range, just can reach the stimulation fibroblast, increases collagen protein synthesis, simultaneously blood circulation promoting, the old useless cellular metabolism of quickening; If adopt the blue light-emitting diode that sends specific illumination range, just can suppress, kill Propiobacterium, or melanic synthetic in reduction and the inhibition melanocyte.
Embodiment two
Utilize the light stimulation device of the foregoing description one to shine human fibroblast, study its influence for cell survival rate.In present embodiment, the employed light emitting diode of light source module is that to send illumination be 9 in this light stimulation device, the HONGGUANG of 250lux.
At first, will contain the DMEM cell culture fluid of human fibroblast, and add in the 48 hole culture plates, the cell number of being inoculated is 2 * 10
4Individual/hole, the common 0.5ml of the cumulative volume of culture fluid (containing cell) in each hole places CO in 48 holes
2Incubator was cultivated 24 hours.Afterwards, take out whole culture fluid, add PBS buffer 0.5ml again, and (Lux 9 to use the HONGGUANG stimulating apparatus of embodiment one, 250) irradiation is after 5,10,15,30,45,60,90 minutes, and whole PBS buffer adds the culture fluid of 0.5ml again in the taking-up hole, cultivates 24 hours again.
Then, the MTT reagent of culture medium 0.5ml that more renews and adding 0.125ml is put to 37 ℃, 5%CO
2Cell culture incubator internal reaction after 4 hours, whole culture medium are taken out, add the DMSO dissolving first of 0.5ml
(formazan), get and utilize ELISA trace dish analyser (ELISA Reader SpectraMax M2) in 0.2ml to 96 hole, measure it at OD
570Light absorption value during nm.Being calculated as of cell survival rate: cell survival rate (%)=(OD behind the irradiation
570/ control group OD
570) * 100%, wherein control group are meant the cell that does not use the light stimulation device irradiation, and experimental result is with reference to figure 4.
As shown in Figure 4, in illumination is 9, the cell survival rate of 250lux red light irradiation after 5 minutes is 116%, the cell survival rate that shines after 10 minutes is 116%, and the cell survival rate that shines after 15 minutes is 111%, and the cell survival rate that shines after 30 minutes is 110%, the cell survival rate that shines after 45 minutes is 109%, the cell survival rate that shines after 60 minutes is 108%, and the cell survival rate that shines after 90 minutes is 103%, and the result is all three multiple mean values of independence of experiment.Under the prerequisite of manual operation error amount positive and negative 10%, light application time is that 5 minutes to 30 minutes cell survival rate has and exceeds this value.Hence one can see that, using illumination is 9, the red light-emitting diode irradiation of 250lux is after 5 minutes to 30 minutes, have and promote the effect that human fibroblast survival rate promotes slightly, and learn that by the result HONGGUANG can't reduce cell survival rate, therefore using illumination is 9, and the red light-emitting diode irradiation of 250lux conforms with the safety of the course of treatment.
Embodiment three
Learn that by above embodiment two results using illumination is 9, after the red light-emitting diode of 250lux is radiated at 5 minutes to 30 minutes, have and promote the phenomenon that human fibroblast survival rate promotes, and all irradiation times conforms with the safety of the course of treatment.In present embodiment, further with the irradiation number of times by once changing twice into, and the illumination of red light-emitting diode is weakened to 7 again, 800lux carries out the cell survival rate test again.
At first, will contain the DMEM cell culture fluid of human fibroblast, and add in the 48 hole culture plates, the cell number of being inoculated is 2 * 10
4Individual/hole, the common 0.5ml of the cumulative volume of culture fluid (containing cell) in each hole places CO in 48 holes
2Incubator was cultivated 24 hours.Afterwards, take out whole culture fluid, add PBS buffer 0.5ml again, and (Lux 7 to use the HONGGUANG stimulating apparatus of embodiment one, 800) irradiation is after 5,10,15,30,45,60,90 minutes, whole PBS buffer adds the culture fluid of 0.5ml again in the taking-up hole, cultivates 24 hours again, and repeats once above-mentioned photostimulation step.
Then, the culture medium 0.5ml that more renews and add the MTT reagent of 0.125ml is put to 37 ℃, the cell culture incubator internal reaction of 5%CO2 after 4 hours, whole culture medium is taken out, and adds the DMSO dissolving first of 0.5ml
(formazan), get and utilize ELISA trace dish analyser in 0.2ml to 96 hole, measure it at OD
570Light absorption value during nm.Being calculated as of cell survival rate: cell survival rate (%)=(OD behind the irradiation
570/ control group OD
570) * 100%, wherein control group are meant the cell that does not use the light stimulation device irradiation, and experimental result is with reference to figure 5.
As shown in Figure 5, in illumination is 7, the cell survival rate of 800lux red light irradiation after 5 minutes is 122%, the cell survival rate that shines after 10 minutes is 132%, the cell survival rate that shines after 15 minutes is 121%, and the cell survival rate that shines after 30 minutes is 119%, and the cell survival rate that shines after 45 minutes is 121%, the cell survival rate that shines after 60 minutes is 116%, and the cell survival rate that shines after 90 minutes is 107%.
The above results result is all three multiple mean values of independence of experiment, and under the prerequisite of manual operation error amount positive and negative 10%, can learn that light application time is that 5 minutes to 60 minutes cell survival rate all exceeds this value.Can get conclusion thus, using illumination is 7, and the red light-emitting diode irradiation of 800lux has the effect of promoting human fibroblast survival rate after 5 minutes to 60 minutes, wherein comparatively obvious with the effect of shining after 5 to 45 minutes.
Embodiment four
Utilize the light stimulation device of the foregoing description one to shine human fibroblast, study its influence for human fibroblast secretion collagen protein.In present embodiment, the employed light emitting diode of light source module is that to send illumination be 7 in this light stimulation device, the HONGGUANG of 800lux.
At first, will contain the DMEM cell culture fluid of human fibroblast, and add in the 48 hole culture plates, the cell number of being inoculated is 2 * 10
4Individual/hole, the common 0.5ml of the cumulative volume of culture fluid (containing cell) in each hole places CO in 48 holes
2Incubator was cultivated 24 hours.Afterwards, take out whole culture fluid, add PBS buffer 0.5ml again, and the HONGGUANG stimulating apparatus (Lux7 of use embodiment one, 800) irradiation is after 5,10,15,30,45 minutes, whole PBS buffer adds the culture fluid of 0.5ml again in the taking-up hole, cultivates 24 hours again, and repeats once above-mentioned photostimulation step.
Then, culture medium in the hole is all taken out, and put into the 1.5ml centrifuge tube, the hole of cultivating cell afterwards distinctly adds the aqueous acetic acid of 0.5ml 0.5M (4 ℃), after placing dissolvings in 20 minutes collagen protein wherein, take out aqueous solutions whole in the hole and put into the 1.5ml centrifuge tube, centrifuge tube successively adds 50 μ l acid neutralizing agent (acid neutralizing reagent more respectively, Biocolor), the separation concentrating agents of 4 ℃ of 100 μ l (Isolation ﹠ Concentration Reagent, and in 4 ℃ of refrigerators, place and spend the night Biocolor).Afterwards, centrifuge tube taken out and centrifugal 10 minutes with 12000rpm, remove supernatant, in centrifuge tube, add 1ml photoghraphic coupler (Sircol Dye Reagent again, Biocolor) add in the centrifuge tube and shook 30 minutes, again with 12000rpm after centrifugal 10 minutes, remove supernatant, add again 4 ℃ of 750 μ l the hydrochlorate abluent (Acid-Salt Wash Reagent, Biocolor), again with 12000rpm after centrifugal 10 minutes, remove supernatant, centrifuge tube add again 250 μ l alkaline agents (Alkali Reagent, Biocolor), take out 200 μ l in last every pipe and add in 96 porose discs, measure the light absorption value of 555nm.At collagen protein production rate (%)=(the collagen protein growing amount behind the irradiation/control group collagen protein growing amount) * 100%, wherein control group is meant the cell that does not use the light stimulation device irradiation, experimental result is with reference to figure 6, the also survival rate of display fibers blast cell wherein, this cell survival rate are that the method according to the foregoing description two records.
As shown in Figure 6, using illumination is 7, the collagen protein production rate of 800lux red light-emitting diode irradiation after 30 minutes is 123%, illumination is 7, after the red light-emitting diode irradiation of 800lux, the effect of promoting human fibroblast secretion collagen protein is arranged, wherein obvious with the effect of shining after 30 minutes.
Embodiment five
Utilize the light stimulation device of the foregoing description one to shine human fibroblast, study its influence for the fibroblast survival rate, and in present embodiment, the employed light emitting diode of light source module is to send the gold-tinted that illumination is 2290lux in this light stimulation device, and analyze with reference to embodiment three described methods, and the rayed time is 5,10,15,30,45 minutes, and experimental result is with reference to figure 7.
As shown in Figure 7, using the cell survival rate of Yellow light emitting diode irradiation after 15 minutes of 2290lux is 115%, and this represents that it has the effect of promoting human fibroblast survival rate, wherein comparatively obvious with the effect of shining after 10 to 45 minutes.
Embodiment six
Utilize the light stimulation device of the foregoing description one to shine human fibroblast, study its influence for fibroblast secretion collagen protein, and in present embodiment, the employed light emitting diode of light source module is to send the gold-tinted that illumination is 2290lux in this light stimulation device, and analyze with reference to embodiment four described methods, experimental result is with reference to figure 8, the also survival rate of display fibers blast cell wherein, and this cell survival rate is that the method according to the foregoing description five records.
As shown in Figure 8, using the collagen protein production rate of Yellow light emitting diode irradiation after 15 minutes of 2290lux is 125%, and unmanned and cell poisoning phenomenon, this represents that it has and promote the effect that people's fibrid mother stock is secreted collagen protein, and is wherein comparatively obvious with the effect of shining after 10 to 45 minutes.
Embodiment seven
Utilize the light stimulation device of the foregoing description one to shine human melanocyte, study its influence for human melanocyte survival rate, and in present embodiment, the employed light emitting diode of light source module is that to send illumination be 5 in this light stimulation device, the blue light of 330lux.
At first, be seeded in the 24 hole culture plates being incubated at the human melanocyte that contains α-MSH culture medium, cell number is 7 * 10
4Individual/hole.Then, every hole adds the culture medium that contains 10%FBS (Hyclone), and celliferous cumulative volume is 0.5ml altogether, places CO
2Incubator was cultivated 24 hours.Afterwards, take out whole culture medium, add PBS buffer 0.5ml, use the blue light stimulating apparatus (5 of embodiment one, 330lux, and increase fan heat radiation holding temperature) irradiation was taken out the culture medium that whole PBS buffer add 0.5ml again after 5,10,15,30,45,60,90 minutes, cultivated 24 hours.
Then, the MTT reagent of culture medium 0.5ml that more renews and adding 0.125ml is put to 37 ℃, 5%CO
2Cell culture incubator internal reaction after 4 hours, whole culture medium are taken out, add the DMSO dissolving first of 0.5ml
(formazan), get and utilize ELISA trace dish analyser in 0.2ml to 96 hole, measure it at OD
570Light absorption value during nm.Being calculated as of cell survival rate: cell survival rate (%)=(OD behind the irradiation
570/ control group OD
570) * 100%, wherein control group are meant the cell that does not use the light stimulation device irradiation, and experimental result is with reference to figure 9.
As shown in Figure 9, any cell poisoning phenomenon is not arranged, all be worth after relatively positive and negatively 10% in personal error with the control group, this represents that the blue light under this illumination still belongs to safety range.
Embodiment eight
Utilize the light stimulation device of the foregoing description one to shine human melanocyte, study it for the synthetic influence of melanin, and in present embodiment, the employed light emitting diode of light source module is that to send illumination be 5 in this light stimulation device, the blue light of 330lux.
At first, be seeded in the 24 hole culture plates being incubated at the human melanocyte that contains α-MSH culture medium, cell number is 1x105/hole, adds the culture medium and the Cell sap that contain 10%FBS, and cumulative volume is 0.5ml altogether, places CO
2Incubator was cultivated 24 hours.Then, take out whole culture medium, add PBS buffer 0.5ml, use the blue light stimulating apparatus (5 of embodiment one, 330lux, and increase fan heat radiation holding temperature) irradiation was taken out the culture medium that whole PBS buffer add 0.5ml again after 5,10,15,30,45,60,90 minutes, cultivated 24 hours.Afterwards, take out whole culture medium, cell is washed the back with 1 with Trypsin-EDTA solution (1X), centrifugal 10 minutes of 000rpm removes supernatant, adds the 1M NaOH of 200 μ l and places boiling water bath 10 minutes, the interior melanin of cell is dissolved among the NaOH, and the reuse spectrophotometer is with OD
490Nm measures its melanin content, and the result is with reference to Figure 10.
As shown in figure 10, use 5, the irradiation of the blue light-emitting diode of 330lux after 5 minutes the melanin generation rate be 105%, shine that the melanin generation rate is 101% after 10 minutes, shine that the melanin generation rate is 105% after 15 minutes, shine that the melanin generation rate is 108% behind 30 clocks, shine melanin generation rate 96% after 45 minutes, shine that the melanin generation rate is 98% after 60 minutes, shine melanin generation 91% after 90 minutes, above-mentioned experimental result is all three multiple mean values of independence of experiment, and hence one can see that, and melanocyte can make the melanin amount descend about about 10% through blue light illumination after 90 minutes.
Embodiment nine
Utilize the light stimulation device irradiation Propiobacterium of the foregoing description one, study its influence for the Propiobacterium survival rate, and in present embodiment, the employed light emitting diode of light source module is that to send illumination be 5 in this light stimulation device, the blue light of 710lux.
At first, freezing preservation bacterium liquid is taken out, done three rides and cultivate, select single bacterium colony, bacterium colony is provoked with the aseptic inoculation ring, coat on the plating medium, treat that scraping hypothallus from plating medium after 48 hours is dissolved in the sterilized water, transfer OD value (OD with sterilized water
600=0.1) after, with sterilized water dilution twice, just can obtain the bacterium number is 10 again
6Bacterium liquid.
Then, bacterium liquid is inserted in the culture dish of 6cm, totally nine coil, every cup fungi liquid measure is respectively 5ml, and the blue light stimulating apparatus of use embodiment one (5,710lux) shone 5,10,15,20,30,45,60,90 minutes.Then, with the bacterium liquid after the illumination, with continuous 10 times of dilutions, concentration is 10
-3, 10
-4, 10
-5, each concentration is got the 0.1ml culture fluid and is applied on RCM (BD biosciences) culture dish, respectively is divided into three dishes, cultivates 48 hours in 37 ℃ of anaerobic environments.Afterwards, take out culture dish and calculate the bacterium number, be effective clump count with 30 to 300 clump counts/coil.
On the other hand,, respectively get 0.1ml and be incubated at the liquid RCM culture medium of 5ml, cultivate in 37 ℃ of anaerobic environments after 48 hours, with OD with remaining bacterium liquid after the illumination
600The growth change of Propiobacterium after the observation illumination, the result is with reference to Figure 11.As shown in figure 11, shine after 45 minutes, the efficient that suppresses Propiobacterium promptly reaches 95%, and this expression fungistatic effect is fairly obvious.
Comparative example
Utilize light stimulation device to shine human fibroblast, study its influence for human fibroblast survival rate, and in this comparative example, the employed light emitting diode of light source module is that to send illumination be 9, the HONGGUANG of 890lux, and analyze with reference to embodiment two described methods, experimental result is with reference to Figure 12.
As shown in figure 12, use 9, the irradiation of the red light-emitting diode of 890lux after 5 minutes cell survival rate be 111%, the cell survival rate that shines after 10 minutes is 105%, and the cell survival rate that shines after 15 minutes is 108%, and the cell survival rate that shines after 30 minutes is 91%, the cell survival rate that shines after 45 minutes is 82%, the cell survival rate that shines after 60 minutes is 75%, and the cell survival rate that shines after 90 minutes is 85%, and the result is all three multiple mean values of independence of experiment.
Under the prerequisite of manual operation error amount positive and negative 10%, can learn that light application time is that 5 minutes cell survival rate exceeds this value; Yet light application time is that 45 minutes to 90 minutes cell survival rate is lower than this value.Can get conclusion thus, using 9, the red light-emitting diode irradiation of 890lux is after 5 minutes, though the effect of promoting human fibroblast survival rate is slightly arranged, but along with the irradiation time is elongated, then human fibroblast survival rate begins to descend, and the survival rate than the control group was low until beginning in 30 minutes, surpassed more than 45 minutes, the phenomenon of Jian Shaoing then, so use 9, the red light-emitting diode irradiating cell of 890lux has the doubt in the safety.
The foregoing description only is to give an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that the claim scope is described certainly, but not only limits to the foregoing description.
Claims (16)
1. photostimulation method may further comprise the steps:
One LED source is provided, and this LED source is selected from by wherein one of a Yellow light emitting diode, a red light-emitting diode and blue light-emitting diode institute cohort group; And
This LED source is shone in a main body, forms to promote collagen protein synthesis, bacteria growing inhibiting or to suppress melanin,
Wherein, the illumination of this Yellow light emitting diode is 1,000 to 3,500 lux, and the illumination of this red light-emitting diode is 6,000 to 9,500 luxs, and the illumination of this blue light-emitting diode is 3,000 to 7,000 luxs.
2. photostimulation method as claimed in claim 1, wherein, this LED source is this Yellow light emitting diode or this red light-emitting diode.
3. photostimulation method as claimed in claim 2, wherein, this main body is a fibroblast, a macrophage or its combination.
4. photostimulation method as claimed in claim 3, wherein, the optical wavelength range of this Yellow light emitting diode is between between the 570nm to 590nm, and the optical wavelength range of this red light-emitting diode is between 620nm to 750nm.
5. photostimulation method as claimed in claim 3, wherein, the irradiation time of this red light-emitting diode or the irradiation time of this Yellow light emitting diode were between 5 minutes to 90 minutes.
6. photostimulation method as claimed in claim 1, wherein, this LED source is this blue light-emitting diode.
7. photostimulation method as claimed in claim 6, wherein, this main body is a Propiobacterium, a melanocyte or its combination.
8. photostimulation method as claimed in claim 7, wherein, the optical wavelength range of this blue light-emitting diode is between between the 450nm to 475nm.
9. photostimulation method as claimed in claim 7, wherein, the irradiation time of this blue light-emitting diode was between 5 minutes to 90 minutes.
10. light stimulation device comprises:
One housing forms an accommodation space and has an end face and a lateral margin, and this end face is provided with a light-emitting window;
One scattering sheet covers this light-emitting window of this housing;
One first light source module, it is arranged in this accommodation space of this housing and has one first light emitting diode, this first light emitting diode is located at this scattering sheet below, and this first light emitting diode is selected from by red light-emitting diode, Yellow light emitting diode, and blue light-emitting diode institute cohort group wherein one, wherein, the light illumination that this Yellow light emitting diode sends through this scattering sheet is 1,000 to 3,500 luxs, the light illumination that this red light-emitting diode sends through this scattering sheet is 6,000 to 9,500 luxs, and the light illumination that this blue light-emitting diode sends through this scattering sheet is 3,000 to 7,000 luxs; And
One control module, it electrically connects this first light source module and a power module.
11. light stimulation device as claimed in claim 10, wherein, this power module is in an external power source or this accommodation space that is arranged at this housing.
12. light stimulation device as claimed in claim 11, wherein, this control module comprises: a charging hole electrically connects this control module for this power module.
13. light stimulation device as claimed in claim 10, wherein, this control module comprises: an on and off switch is arranged at this surface of shell, to control this power module supply power supply.
14. light stimulation device as claimed in claim 10, wherein, this lateral margin of this housing is provided with a light hole.
15. light stimulation device as claimed in claim 14 also comprises: a light transmission piece covers this light hole; And a secondary light source module, it is corresponding light transmission piece setting and emits beam and pass this light transmission piece.
16. light stimulation device as claimed in claim 15, wherein, this control module comprises: a mode selector switch all is arranged at this surface of shell, to start this first light source module or this secondary light source module.
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