CN108693183B - Method for detecting sensitization of cosmetic components through ultra-weak biophoton imaging system - Google Patents
Method for detecting sensitization of cosmetic components through ultra-weak biophoton imaging system Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The invention discloses a method for detecting sensitization of cosmetic components through an ultra-weak biophoton imaging system, which comprises the following steps: s1, preprocessing materials; s2, culturing tissue blocks; s3, setting an experimental group; s4, preparing an experiment and preheating an imaging system; s5, positioning and photographing; s6, experimental imaging; and S7, processing the sequence images and analyzing the data. The invention uses ultra-weak biological photon imaging technology and immunohistochemical staining method to detect the change of biological photon radiation intensity after DNCB treatment of common cosmetic sensitizers on cultured skin tissues donated by dead remains, so as to quickly detect skin sensitizers in cosmetics.
Description
Technical Field
The invention relates to cosmetic safety detection, which utilizes an ultra-weak biophoton imaging system to detect the change of the biophoton intensity of spontaneous radiation of human skin treated by cosmetics so as to determine whether the cosmetics contain chemical components capable of causing skin allergic damage. Belongs to the technical field of biomedical detection, and the technology can also be applied to the fields of life science research, medical clinical examination, environmental protection and the like.
Background
Most cosmetics have complicated components, and common sensitizers include perfume, antiseptic, metal elements (lead, mercury, nickel, arsenic), surfactant, emulsifier, hormone (corticosteroid hormone, estrogen, etc.), humectant, 1-chloro-2, 4-Dinitrobenzene (DNCB), natural plant components, etc. The diversification and high use rate of cosmetics has led to an increasing incidence of cosmetic contact dermatitis. After skin contacts with cosmetics, cosmetic contact dermatitis can be induced by sensitizers in the cosmetics, and the skin is divided into two main categories according to sensitization mechanisms: one is allergic contact dermatitis and the other is irritant contact dermatitis. Skin-contact inflammation may be caused by allergic reactions, such as primary stimulation of the body by antigens, antigen delivery by langerhans cells in the skin, and antigen-specific T lymphocytes by lymph nodes; irritant contact dermatitis does not produce specific antibodies, and skin inflammation can be caused by one-time smearing or multiple times of irritation.
At present, the method for evaluating the safety of cosmetics generally adopts a human body patch test, namely, a standard patch device is used for carrying out the human body skin patch test on a subject, and the results of the patch part are subjectively evaluated and statistically analyzed to judge the allergen and the sensitization degree. However, the detection process of the human body patch test takes a long time and requires a large number of volunteers to participate, and the individual differences among the volunteers are large. However, currently popular in vitro substitution animal experimental methods such as human cell line activation tests still have the defects of long time consumption, high cost and the like in the research of predicting the sensitization of chemicals.
In view of the above, it is urgently needed to provide a method for simply, rapidly and accurately exploring cosmetic sensitization detection through an ultra-weak biophotonic imaging system to determine whether cosmetics are sensitized or not.
Disclosure of Invention
In order to solve the technical problems, the technical scheme adopted by the invention is to provide a method for detecting the allergenicity of cosmetics by an ultra-weak biophoton imaging system, which comprises the following steps:
s1, preprocessing materials; s2, culturing tissue blocks; s3, setting experimental groups, including setting 4 DNCB solution research groups with concentration, wherein each experimental group is provided with a Williams E culture solution blank control group and an AOO solvent control group; s4, preparing an experiment and preheating an imaging system; s5, positioning and photographing; s6, experimental imaging; and S7, processing the sequence images and analyzing the data.
In the above method, the step S1 includes:
the donated remains provided by the red cross are taken back to the skin on the back of the lower arm, and subcutaneous fat is removed by a scalpel and a sharp blade in a large culture dish containing a large amount of PBS buffer solution, so that only the dermis layer and the epidermis layer are left as far as possible;
after fat is removed, the tissue is cut into a plurality of tissue blocks, the tissue blocks are put into anti-freezing liquid and infiltrated in a refrigerator at the temperature of minus 4 ℃, and the infiltrated tissue blocks are subpackaged into a vertical external rotation round bottom freezing storage tube and stored in liquid nitrogen at the temperature of minus 80 ℃.
In the above method, the step S2 includes the steps of:
taking out the required number of frozen tissue blocks from the liquid nitrogen, respectively placing the frozen tissue blocks in a vertical external rotation round bottom freezing tube containing a PBS solution, and rewarming for 10min through a water bath at 37 ℃;
the workbench was sterilized by turning on the uv lamp half an hour in advance, and in the superclean workbench, the tissue mass was rinsed 2 times with 1% double antibody in PBS solution and cultured for 2 days with the gas-liquid interface method.
In the above method, the step S3 includes the following steps:
4 concentration DNCB solution research groups are set, namely 0.5%, 1%, 5% and 7% DNCB treatment experiment groups, and each experiment group is provided with a Williams E culture solution blank control group and an AOO solvent control group, and the specific experiment setting groups are as follows:
experiment group one: detecting the change of the biological photon radiation intensity of the skin tissue after DNCB stimulation of different concentrations;
experimental group 1: 0.5% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 2: 1% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 3: 5% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 4: 7% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes.
In the above method, the step S4 includes the steps of:
experiment group two: the intensity of biophoton radiation of human skin tissue changes after DNCB stimulation under different culture times;
the experiment included culturing for 1 day, 2 days and 3 days for any of groups 1-4, and observing the change in the intensity of the biophoton radiation of the tissue mass after culturing for 1 day, 2 days and 3 days, respectively.
In the above method, the step S4 includes:
and (3) stabilizing the room temperature, keeping the room temperature at 24-25 ℃ during the experiment, starting a cooling liquid circulating pump to refrigerate the photon imaging device, enabling the EMCCD to stably work at the temperature of about-92 ℃, waiting for cooling, starting HCimage imaging control software, starting a shooting program of the photon imaging device according to the setting during imaging before formal imaging, and preheating the shooting program to stabilize the background baseline of the photon imaging device.
In the above method, the step S5 includes the steps of:
respectively taking out the culture dishes in the first experimental group and the second experimental group from the culture box according to the sequence of the experimental groups, taking off the culture dish covers, putting the culture dishes in the dark box, placing the culture dishes in the perfusion tank, introducing perfusion liquid flowing at a constant temperature and a constant speed of 37 +/-0.5 ℃, ensuring that the perfusion liquid completely covers the tissue blocks, closing the dark box after the liquid level of the perfusion liquid is stable, enabling the surface skin of the tissue blocks to be opposite to the lens, closing indoor lighting, taking off the lens cover, starting shooting a positioning photo in a CCD (charge coupled device) mode when the HCImage shows that the temperature of an EMCCD (electron-coupled device) is-92 ℃, finishing shooting the positioning photo when the image is clear and storing the positioning photo in a working directory; exposure time 0.04s, photon mode 0.
In the above method, the step S7 includes the steps of:
using image processing software HCIamge and Matlab7.0 software matched with EMCCD to process an original image, extracting a sample gray value and continuing statistical analysis, and specifically comprising the following steps: converting the sequence image obtained by the EMCCD into a TIF format; introducing the sequence image into Matlab7.0, and performing speckle removing treatment on the image in the target folder by using a watershed program to obtain a treated sequence image; then, the sequence image processed by Matlab7.0 is imported into HCIamge again, a corresponding target area is selected from the processed image by referring to the position of the sample in the positioning picture, the average gray value of the tissue target area of interest and the non-tissue background area is extracted, and the average gray value of the non-sample area is also extracted as the average background gray value of the image; finally, subtracting the background gray value from the sample gray value to obtain a relative gray value of the region; finally, statistical analysis was performed using Microsoft Excel and Two-tailed Student's t-test and mapping was performed using Graphpad.
In the above method, further comprising the steps of:
immunohistochemical staining:
s81, taking out tissue blocks in culture dishes of the experiment group I and the experiment group II, placing the tissue blocks on a glass slide, fixing the tissue blocks by using 4% paraformaldehyde, respectively placing the tissue blocks in culture dishes of sucrose TBS solutions with the concentration of 30% and placing the culture dishes in a refrigerator at 4 ℃ overnight for freezing protection until the tissue blocks are precipitated to the bottom of the culture dishes;
s82, taking the experimental group as a unit, cutting the tissue blocks in the experimental group into sections with the thickness of 10 μm by using a constant temperature freezing microtome, and respectively attaching the sections to the glass slides coated with gelatin for microscopic examination.
In the above method, the step S82 specifically includes:
circling the section on the glass slide by using a grouping pen, waiting for the mark of the grouping pen to be dried, and putting the section into TBS solution to wash for 2 times, wherein each washing time is 15 min;
determining the dilution ratio of the antibody stock solution, specifically Langerhans cell specific antibody S-100, the dilution ratio is 1:500, respectively adding 200 μ l of diluted antibody drops on the slices in the pen ring, covering the whole slices, standing at room temperature for 2h, and transferring to a 4 ℃ refrigerator for 12 h; taking out all the sections from a refrigerator, respectively washing the glass slide with TBS solution, each time for 15min, preparing biotin-labeled anti-rabbit secondary antibody 1:400, respectively dripping 200 mul of diluted anti-rabbit secondary antibody solution into a grouping pen ring, standing at room temperature for 2h, and respectively washing the anti-rabbit secondary antibody with TBS solution; then preparing ABC compound, diluting to 1:80, respectively dripping the ABC compound on the slices, standing at room temperature for 1.5h, and then washing with TBS solution; finally, respectively dripping the diaminobenzidine color developing solution on the slices for dyeing for 5-10min, washing the slices with double distilled water for several times, and drying the slices at room temperature;
the next day, adopting an alcohol gradient dehydration method, sequentially soaking the slices in 50%, 70%, 80%, 95% I, 95% II, 100% I and 100% II alcohol for 15min, then respectively and transparently soaking the slices in xylene I and xylene II for 15min, and finally, sealing the slices with neutral gum and performing microscopic examination.
The invention uses ultra-weak biological photon imaging technology and immunohistochemical staining method to detect the change of biological photon radiation intensity after DNCB treatment of common cosmetic sensitizers on cultured skin tissues donated by dead remains, so as to rapidly detect skin sensitizers in cosmetics.
Drawings
FIG. 1 is a flow chart of a system for detecting freshness of beef provided by the present invention;
FIG. 2 is a bar graph of the intensity of biophoton radiation in human skin tissue of the experimental groups 1-4 of the present invention at different concentrations of DNCB during incubation; wherein,
a is an experimental histogram of the experimental group 1, B is an experimental histogram of the experimental group 2, C is an experimental histogram of the experimental group 3, and D is an experimental histogram of the experimental group 4;
FIG. 3 is a graph showing the biophoton intensity profiles of human skin tissues in the DNCB treatment test groups of different concentrations according to the present invention;
FIG. 4 is a graph showing the trend of the content of volatile basic nitrogen in beef of the experimental group 5 provided by the present invention over time;
FIG. 5 is a graph of Langerhans cell activation in human skin tissue under different treatment conditions of the present invention.
Detailed Description
Skin, as an organic tissue itself, radiates biophoton outwardly, and skin, as the outermost layer of the body, provides convenience for in vivo biophoton detection. The method detects the sensitization of cosmetics by researching the change of the biophoton intensity of skin tissue radiation after common cosmetics sensitizers 1-chloro-2, 4-Dinitrobenzene (DNCB) act on skin, the DNCB contained in the cosmetics is a positive sensitizer, contact dermatitis can be generated after the skin is contacted, and the research shows that the biophoton intensity of the skin tissue radiation which is inflamed can be changed. Therefore, the method for detecting skin-sensitizing substances in cosmetics by using the ultra-weak biophoton imaging system is a technology for rapidly evaluating the safety of the cosmetics.
The invention is described in detail below with reference to specific embodiments and the accompanying drawings.
As shown in fig. 1-5, the present invention provides a method for detecting cosmetic sensitization by using an ultra-weak biophoton imaging system, wherein the imaging device is a currently commonly used electron multiplying ccd (emccd), as specifically disclosed in patent CN 103536277A.
Example (b): method for detecting cosmetic sensitization by using ultra-weak biophoton imaging system
Firstly, experimental materials:
human skin tissue from Chinese brain pool center (CBBC)
Second, the experimental procedure
S1, pretreatment of the material, comprising the following steps:
s11, the skin of the back of the lower arm was retrieved from the donated body provided by the red cross, and the subcutaneous fat was removed with a scalpel and a sharp blade in a large dish containing a large amount of PBS buffer, leaving as far as possible only the dermis and epidermis.
S12, removing fat, cutting the tissue into multiple tissue blocks of 0.5 × 0.5cm, putting into anti-freezing liquid, infiltrating in a refrigerator at-4 deg.C, subpackaging the infiltrated tissue blocks into 2.0ml round bottom freezing tubes capable of rotating outwards, and storing in liquid nitrogen at-80 deg.C.
S2, tissue block culture, comprising the following steps:
s21, taking out the required number of frozen tissue blocks from the liquid nitrogen in the step S12, respectively placing the frozen tissue blocks in 2.0ml of standable external rotation round bottom freezing tubes containing PBS solution, and rewarming for 10min through 37 ℃ water bath.
And S22, turning on an ultraviolet sterilization lamp half an hour in advance to sterilize the worktable, rinsing the tissue block obtained in the step S21 with a PBS solution containing 1% double antibody for 2 times in a super-clean worktable, culturing the tissue block for 2 days by using a gas-liquid interface method, namely placing the tissue block in a 35X 10mm culture dish with the surface facing upwards and the dermis facing downwards, adding 1ml of Williams E culture solution into the culture dish, completely soaking the dermis part of the tissue block in the culture solution, and contacting the epidermis with air.
S3, setting an experimental group, and specifically comprising the following steps:
to investigate whether DNCB can be the most reliable positive control compound for skin sensitization for the evaluation of ultra-weak biophotonic imaging, we investigated the change in intensity of biophotonic radiation after stimulation of skin tissue with DNCB solutions of different concentrations. 4 concentration DNCB solution study groups are set, and 0.5%, 1%, 5% and 7% DNCB treatment experiment groups are respectively set, and each experiment group is provided with a Williams E culture solution blank control group and an AOO solvent control group. The skin tissue culture time was 2 days, and the culture time was 48 hours from 24 hours after the AOO solvent control group and the DNCB study group. The specific experimental setup groups were as follows:
experiment group one: the intensity change of the biological photon radiation of the skin tissue after the DNCB stimulation of different concentrations is detected.
DNCB solution research group: in 4 dishes obtained in step S22, 5. mu.l of each of 0.5% DNCB solution, 1% DNCB solution, 5% DNCB solution and 7% DNCB solution was dropped onto the surface of the skin in the 4 dishes.
Solvent control group: corresponding to the DNCB solution test group, 4 dishes of step S22 were taken, and 5. mu.l of the AOO solution was added dropwise to the surface of the skin in each dish.
Blank control group: the 4 dishes obtained in step S22 were taken and were not processed.
All the plates were placed at 37 ℃ in 5% CO2Culturing in an incubator, and changing the culture solution every other day. The experimental group one is as follows:
experimental group 1: 0.5% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 2: 1% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 3: 5% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes;
experimental group 4: 7% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes.
Experiment group two: the intensity of biophoton radiation of human skin tissue after DNCB stimulation varied at different incubation times.
The experiment included culturing for 1 day, 2 days, and 3 days for any of the above experimental groups, and observing the change in the intensity of the biophoton radiation from the tissue mass after culturing for 1 day, 2 days, and 3 days, respectively. The experiment takes the tissue block after the stimulation of 0.5 percent DNCB solution as an example to observe the change of the biological photon radiation intensity of the skin tissue.
Experimental group 5: 0.5% DNCB Petri dishes, AOO Petri dishes and Williams E Petri dishes.
S4, experimental preparation and imaging system preheating: comprises that
And (3) stabilizing the room temperature, keeping the room temperature at 24-25 ℃ during the experiment, starting a cooling liquid circulating pump to refrigerate the photon imaging device, enabling the EMCCD to stably work at the temperature of about-92 ℃, waiting for cooling, starting HCimage imaging control software, starting a shooting program of the photon imaging device according to the setting during imaging before formal imaging, and preheating the shooting program to stabilize the background baseline of the photon imaging device.
S5, positioning and photographing: respectively taking out the culture dishes in the first experimental group and the second experimental group from the culture box according to the sequence of the experimental groups, taking off the culture dish covers, putting the culture dishes in the dark box, placing the culture dishes in the perfusion tank, introducing perfusion liquid flowing at a constant temperature and a constant speed of 37 +/-0.5 ℃, ensuring that the perfusion liquid completely covers the tissue blocks, closing the dark box after the liquid level of the perfusion liquid is stable, enabling the surface skin of the tissue blocks to be opposite to the lens, closing indoor lighting, taking off the lens cover, starting shooting a positioning photo in a CCD (charge coupled device) mode when the HCImage shows that the temperature of an EMCCD (electron-coupled device) is-92 ℃, finishing shooting the positioning photo when the image is clear and storing the positioning photo in a working directory; exposure time 0.04s, photon mode 0.
S6, experimental imaging: after the positioning photograph shooting is finished, the mode is switched to an 'EMCCD' mode, the gain is switched to the maximum x 1200, the photon mode is 3, the exposure time of each frame is 60s, the shooting is started, and 90 frames of signal images are obtained in total.
S7, sequence image processing and data analysis: using image processing software HCIamge and Matlab7.0 software matched with EMCCD to process an original image, extracting a sample gray value and continuing statistical analysis, and specifically comprising the following steps: converting the sequence image obtained by the EMCCD into a TIF format; introducing the sequence image into Matlab7.0, and performing speckle removing treatment on the image in the target folder by using a watershed program to obtain a treated sequence image; and then, the sequence image processed by Matlab7.0 is imported into HCIamge again, a corresponding target area is selected from the processed image by referring to the position of the sample in the positioning picture, the Average Gray Value (AGVs) is extracted for the tissue target area of interest and the non-tissue Background area, and simultaneously, the Average gray value of the non-sample area is also extracted as the Average Background Gray Value (BGVs) of the image. Finally, subtracting the background gray value from the sample gray value to obtain a Relative gray value (Relative gray values, RGVs) of the region; finally, statistical analysis was performed using Microsoft Excel and Two-tailed Student's t-test and mapping was performed using Graphpad.
S8, immunohistochemical staining, comprising the following steps:
s81, taking out tissue blocks in culture dishes of the experiment group I and the experiment group II, placing the tissue blocks on a glass slide, fixing the tissue blocks by using 4% paraformaldehyde, respectively placing the tissue blocks in culture dishes of sucrose TBS solutions with the concentration of 30% and placing the culture dishes in a refrigerator at 4 ℃ overnight for freezing protection until the tissue blocks are precipitated to the bottom of the culture dishes;
s82, taking an experimental group as a unit, respectively cutting the tissue blocks in the experimental group I and the experimental group II into sections with the thickness of 10 mu m by using a constant-temperature freezing microtome, and respectively attaching the sections to glass slides coated with gelatin for microscopic examination, wherein the method specifically comprises the following steps:
circling the section on the glass slide by using a grouping pen, waiting for the mark of the grouping pen to be dried, and putting the section into TBS solution to wash for 2 times, wherein each washing time is 15 min;
determining the dilution ratio of the antibody stock solution, specifically Langerhans cell specific antibody S-100, the dilution ratio is 1:500, respectively adding 200 μ l of diluted antibody drops on the slices in the pen ring, covering the whole slices, standing at room temperature for 2h, and transferring to a 4 ℃ refrigerator for 12 h; taking out all the sections from a refrigerator, respectively washing the glass slide with TBS solution, each time for 15min, preparing biotin-labeled anti-rabbit secondary antibody 1:400, respectively dripping 200 mul of diluted anti-rabbit secondary antibody solution into a grouping pen ring, standing at room temperature for 2h, and respectively washing the anti-rabbit secondary antibody with TBS solution; then preparing ABC compound, diluting to 1:80, respectively dripping the ABC compound on the slices, standing at room temperature for 1.5h, and then washing with TBS solution; and finally, respectively dripping the Diaminobenzidine (DAB) color development solution on the slices for dyeing for 5-10min, washing the slices for several times by double-distilled water, and drying the slices at room temperature.
The next day, adopting an alcohol gradient dehydration method, sequentially soaking the slices in 50%, 70%, 80%, 95% I, 95% II, 100% I and 100% II alcohol for 15min, then respectively and transparently soaking the slices in xylene I and xylene II for 15min, and finally, sealing the slices with neutral gum and performing microscopic examination.
Thirdly, detecting results:
as shown in FIG. 2, the A, B, C, D plots are the biophoton radiation intensity versus gray scale value for human skin tissue from the 0.5%, 1%, 5%, and 7% DNCB solution treatment experimental groups, respectively.
FIG. 3 is a graph showing the variation trend of the biophoton intensity of human skin tissue in the experimental groups 1-4 treated with DNCB solutions of different concentrations, as can be seen from FIGS. 2 and 3, the intensity of the biophoton radiation of human skin tissue treated with DNCB solution of 0.5% is weakest, the intensity of the biophoton radiation of 1% is obviously increased compared with that of 0.5%, but is not obviously changed when the intensity of the biophoton radiation is increased to 5%, while the intensity of the biophoton radiation of 7% is obviously decreased compared with that of 1%, and no obvious difference is found in statistical analysis. The Williams E blank culture group and the AOO solvent control group have basically the same trend, and the biophoton radiation intensity of the two groups is higher than that of the DNCB group, which shows that the DNCB with high difficulty can inhibit the biophoton radiation phenomenon of skin tissues. From the results of treatment of human skin by the Williams E culture solution group, the AOO solvent group and the DNCB experimental group, the AOO solvent has no obvious effect on the sensitization of human skin. The DNCB solution group had an inhibitory effect on biophotonic radiation of human skin compared to the other two groups of treatments.
As shown in FIG. 5, A, B in the figure and the top panel C in the figure are 100-fold micrographs of 10 μm sections of human skin tissue after Williams E blank treatment, 0.5% DNCB treatment and AOO solution treatment, respectively, of panel 1, and the panels under D, E and F are 200-fold micrographs of the area indicated by the arrows; a plurality of S-100 staining positive Langerhans cells are scattered under the epidermis after being treated by 0.5% DNCB and AOO solution, positive Langerhans cells are not found in blank control, AOO and DNCB dripped on the surface of the skin activate antigen presenting cells in the skin, namely Langerhans cells, which shows that the skin tissue cultured in vitro is activated under the treatment condition of the AOO and the DNCB, an immune response reaction is generated, the generation of the immune response reaction shows that the biophoton radiation activity of the skin epidermal cells is enhanced, the skin surface change image obtained by the immunohistochemistry technology is visible to naked eyes, and the enhancement of the skin biophoton activity of a DNCB treatment group detected by an ultra-weak biophoton imaging system is proved from the tissue level.
The invention provides a method for detecting cosmetic sensitization through an ultra-weak biophoton imaging system, which has the following characteristics:
(1) the skin tissue donated by the dead human body is cultured in vitro by a gas-liquid interface method, and the obtained model can be used for evaluating the sensitization of the cosmetic components on the skin.
(2) The ultra-weak biological photon imaging system taking EMCCD as the core can detect the biological photon radiation intensity of a skin tissue in-vitro culture model donated by a dead human body, and has the potential to be developed into a novel cosmetic safety detection technology.
(3) And (3) rapid detection: the ultra-weak biophoton imaging technology can be used for rapidly detecting whether cosmetics contain chemical components causing skin allergic damage or not, is simple and convenient to operate, is safe and non-toxic, and consumes less time than the traditional human cell line activation test method and the like.
(4) The functions are rich: different experimental parameters can be adjusted according to detection conditions, and the method can be widely applied to the fields of cosmetic safety detection, life science research, environmental protection and the like.
The present invention is not limited to the above-mentioned preferred embodiments, and any structural changes made under the teaching of the present invention shall fall within the protection scope of the present invention, which has the same or similar technical solutions as the present invention.
Claims (3)
1. The method for detecting the allergenicity of the cosmetic ingredients through the ultra-weak biophoton imaging system is characterized by comprising the following steps of:
s1, preprocessing materials, namely, taking back the skin of the back of the lower arm from the donated corpses provided by the red cross, and removing subcutaneous fat by using a scalpel and a sharp blade in a large culture dish containing a large amount of PBS buffer solution to leave only a dermis layer and an epidermis layer as far as possible;
after fat is removed, cutting the tissue into a plurality of tissue blocks, putting the tissue blocks into anti-freezing liquid and permeating the tissue blocks in a refrigerator at the temperature of-4 ℃, subpackaging the permeated tissue blocks into a vertical external rotation round bottom freezing storage tube, and storing the frozen tissue blocks in liquid nitrogen at the temperature of-80 ℃;
s2, tissue block culture, namely taking out the required number of frozen tissue blocks from liquid nitrogen, respectively placing the frozen tissue blocks in a vertical external rotation round bottom freezing tube containing a PBS solution, and rewarming for 10min through a 37 ℃ water bath;
turning on an ultraviolet sterilizing lamp half an hour in advance to sterilize the workbench, rinsing the tissue block for 2 times by using 1% double-antibody-containing PBS (phosphate buffer solution) in a super-clean workbench, and culturing the tissue block for 2 days by using a gas-liquid interface method;
s3, setting experimental groups, including setting 4 DNCB solution research groups with concentration, wherein each experimental group is provided with a WilliamsE culture solution blank control group and an AOO solvent control group;
the specific experimental setup groups were as follows:
experiment group one: detecting the change of the biological photon radiation intensity of the skin tissue after DNCB stimulation of different concentrations;
experimental group 1: 0.5% DNCB Petri dishes, AOO Petri dishes and WilliamsE Petri dishes;
experimental group 2: 1% DNCB petri dishes, AOO petri dishes, and WilliamsE petri dishes;
experimental group 3: 5% DNCB Petri dishes, AOO Petri dishes and WilliamsE Petri dishes;
experimental group 4: 7% DNCB Petri dishes, AOO Petri dishes and WilliamsE Petri dishes;
s4, preparing an experiment and preheating an imaging system;
experiment group two: the intensity of biophoton radiation of human skin tissue changes after DNCB stimulation under different culture times;
the experiment comprises culturing any group of 1-4 experimental groups for 1 day, 2 days and 3 days, and observing the change of the radiation intensity of the biological photons of the tissue block after culturing for 1 day, 2 days and 3 days;
stabilizing the room temperature, keeping the room temperature at 24-25 ℃ during the experiment, starting a cooling liquid circulating pump to refrigerate the photon imaging device, enabling the EMCCD to stably work at about-92 ℃, waiting for cooling, starting HCimage imaging control software, starting a shooting program of the photon imaging device according to the setting during imaging before formal imaging, and preheating the shooting program to stabilize the background baseline of the photon imaging device;
s5, positioning and photographing;
s6, experimental imaging;
s7, processing sequence images and analyzing data;
s8, immunohistochemical staining, comprising the following steps:
s81, taking out tissue blocks in culture dishes of the experiment group I and the experiment group II, placing the tissue blocks on a glass slide, fixing the tissue blocks by using 4% paraformaldehyde, respectively placing the tissue blocks in culture dishes of sucrose TBS solutions with the concentration of 30% and placing the culture dishes in a refrigerator at 4 ℃ overnight for freezing protection until the tissue blocks are precipitated to the bottom of the culture dishes;
s82, taking the experimental group as a unit, respectively cutting the tissue blocks in the experimental group into sections with the thickness of 10 mu m by using a constant temperature freezing microtome, and respectively attaching the sections to glass slides coated with gelatin for microscopic examination, wherein the method specifically comprises the following steps:
circling the section on the glass slide by using a grouping pen, waiting for the mark of the grouping pen to be dried, and putting the section into TBS solution to wash for 2 times, wherein each washing time is 15 min;
determining the dilution ratio of the antibody stock solution, specifically Langerhans cell specific antibody S-100, the dilution ratio is 1:500, respectively adding 200 μ l of diluted antibody drops on the slices in the pen ring, covering the whole slices, standing at room temperature for 2h, and transferring to a 4 ℃ refrigerator for 12 h; taking out all the sections from a refrigerator, respectively washing the glass slide with TBS solution, each time for 15min, preparing biotin-labeled anti-rabbit secondary antibody 1:400, respectively dripping 200 mul of diluted anti-rabbit secondary antibody solution into a grouping pen ring, standing at room temperature for 2h, and respectively washing the anti-rabbit secondary antibody with TBS solution; then preparing ABC compound, diluting to 1:80, respectively dripping the ABC compound on the slices, standing at room temperature for 1.5h, and then washing with TBS solution; finally, respectively dripping the diaminobenzidine color developing solution on the slices for dyeing for 5-10min, washing the slices with double distilled water for several times, and drying the slices at room temperature;
the next day, adopting an alcohol gradient dehydration method, sequentially soaking the slices in 50%, 70%, 80%, 95% I, 95% II, 100% I and 100% II alcohol for 15min, then respectively and transparently soaking the slices in xylene I and xylene II for 15min, and finally, sealing the slices with neutral gum and performing microscopic examination.
2. The method of claim 1, wherein the step S5 includes the steps of:
respectively taking out the culture dishes in the first experimental group and the second experimental group from the culture box according to the sequence of the experimental groups, taking off the culture dish covers, putting the culture dishes in the dark box, placing the culture dishes in the perfusion tank, introducing perfusion liquid flowing at a constant temperature and a constant speed of 37 +/-0.5 ℃, ensuring that the perfusion liquid completely covers the tissue blocks, closing the dark box after the liquid level of the perfusion liquid is stable, enabling the surface skin of the tissue blocks to be opposite to the lens, closing indoor lighting, taking off the lens cover, starting shooting a positioning photo in a CCD (charge coupled device) mode when the HCImage shows that the temperature of an EMCCD (electron-coupled device) is-92 ℃, finishing shooting the positioning photo when the image is clear and storing the positioning photo in a working directory; exposure time 0.04s, photon mode 0.
3. The method of claim 2, wherein the step S7 includes the steps of:
processing an original image by using image processing software HCIamge and Matlab7.0 software matched with EMCCD, extracting a sample gray value and continuing statistical analysis, wherein the method comprises the following specific steps: converting the sequence image obtained by the EMCCD into a TIF format; introducing the sequence image into Madlab7.0, and performing quantitative speckle removal processing on the image in the target folder by using a watershed program to obtain a processed sequence image; then, the sequence image processed by Matlab7.0 is imported into HCIamge again, a corresponding target area is selected from the processed image by referring to the position of the sample in the positioning picture, the average gray value of the tissue target area of interest and the non-tissue background area is extracted, and the average gray value of the non-sample area is also extracted as the average background gray value of the image; finally, subtracting the background gray value from the sample gray value to obtain a relative gray value of the region; finally statistical analysis was performed using Microsoft excel and Two-tailed student' st-test and mapping was performed using Graphpad.
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