CN108004329A

CN108004329A - A kind of skin care item method for customizing for improving allergy skin quality

Info

Publication number: CN108004329A
Application number: CN201711320052.7A
Authority: CN
Inventors: 杜立波
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-12-12
Filing date: 2017-12-12
Publication date: 2018-05-08

Abstract

The invention discloses a kind of skin care item method for customizing for improving allergy skin quality, comprise the steps of：Step 1, user's cell sample is gathered；Step 2, the mRNA in cell sample is extracted；Step 3, mRNA reverse transcriptions are synthesized into cDNA；Step 4, the expression quantity of the polymorphism of allergy related gene and corresponding cDNA in cDNA are detected, allergy related gene includes at least one of FLG, DDB2, NLRP3；Step 5, judge susceptibility according to gene pleiomorphism and corresponding cDNA expression quantity, and improve the skin care item component and dosage of allergy skin quality for susceptibility selection, prepare the suitable skin care item for improving allergy skin quality.This method obtains the susceptibility of different user by measuring the expression quantity of allergy related gene, and the skin care item of suitable improvement allergy skin quality are prepared according to the specific susceptibility of user.

Description

Method for customizing skin care product for improving allergic skin

Technical Field

The invention relates to the technical field of skin care products, in particular to a skin care product customization method for improving allergic skin.

Background

With the development of scientific technology, the whole gene spectrogram of human can be obtained by sequencing. Numerous studies currently indicate that the state of human skin is closely related to genes. The genotype of a specific site (or single nucleotide polymorphism of gene, SNPs, gene polymorphism for short) in these allergy-associated genes can largely affect the state of the skin, and different genotypes are key factors causing skin problems. People can find or predict the problems of the skin by detecting different genotypes, and the problems are used as guidance to select a suitable skin care product for improving the allergic skin. Further studies have found that, in addition to the genotype, the expressed protein due to the genotype is the key factor causing skin problems.

There are a large number of skin care and beauty products with different functions available on the market today. But each individual's skin has its uniqueness, e.g., varying levels of anti-allergy and sensitivity, among different human skins. The selection of skin care products suitable for self improvement of allergic skin is an important problem. The current method of choice for consumers is to verify whether a skin care product for improving allergic skin is suitable for themselves in a selection-purchase-try manner, in which process the consumer not only wastes a great deal of time and money, but also may cause skin discomfort. Therefore, how to select a skin care product which is suitable for individual consumers and can improve allergic skin becomes an important problem to be solved urgently.

Disclosure of Invention

The invention provides a skin care product customization method for improving allergic skin, which obtains sensitivities of different users by measuring the polymorphism of allergy related genes and the expression quantity of corresponding cDNA (complementary deoxyribonucleic acid), and prepares a skin care product suitable for improving the allergic skin according to the specific sensitivities of the users.

The customization method of the skin care product for improving the allergic skin quality comprises the following steps:

step 1, collecting a user cell sample;

step 2, extracting mRNA in the cell sample;

step 3, reverse transcription of mRNA to synthesize cDNA;

step 4, detecting the polymorphism of allergy-related genes in the cDNA and the expression level of the corresponding cDNA, wherein the allergy-related genes comprise at least one of FLG, DDB2 and NLRP 3;

and 5, judging the sensitivity according to the gene polymorphism and the expression quantity of the corresponding cDNA, and selecting components and dosage according to the sensitivity to prepare a skin care product for improving the allergic skin.

Further, the step 4 comprises:

step 4a), searching the mRNA sequence of the allergy-related gene on NCBI, and designing a corresponding primer;

and 4b), adopting a delta-deltaCt method, taking the gene GAPDH as an internal reference, and detecting the polymorphism of the allergy-related gene and the relative expression amount of the corresponding cDNA through the primer.

Further, the primer comprises:

GAPDH Forword：5’-CGGGAAACTGTGGCGTGATG-3’

Reverse：5’-ATGACCTTGCCCACAGCCTT-3’

FLG Forword：5’-GACAGGGATCCTACCACGAG-3’

Reverse：5’-TCTCTGCTTGCACTTCTGGA-3’

DDB2Forword：5’-GCGTGGCACCCAACTCAC-3’

Reverse：5’-GGTGGGTTTGTCCTTGATGCC-3’

NLRP3Forword：5’-GAATGCCCGTCTGGGTGAGA-3’

Reverse：5’-GGGCTCTCACACGTCTTGGT-3’。

furthermore, the ingredients of the skin care product for improving the allergic skin comprise an anti-allergic agent, polypeptide and an auxiliary agent.

Further, the anti-allergic agent comprises one or more of dipotassium glycyrrhizinate, tetrandrine, chamomile extract and centella asiatica extract.

Further, the polypeptide comprises alanine/histidine/lysine polypeptide copper HCl, soy bean (GlycinEMAX) polypeptide, dipeptide-1, dipeptide-2, dipeptide-4, oligopeptide-1, oligopeptide-2, oligopeptide-3, oligopeptide-4, oligopeptide-5, oligopeptide-6, oligopeptide-29, oligopeptide-34, carnosine, yeast polypeptide, arginine/lysine polypeptide, nonapeptide-1, poly (tripeptide-6), ascorbic acid polypeptide, sodium surfactin, hexapeptide-1, 2, 3, 5, 9, 11, heptapeptide-6, myristoyl hexapeptide-5, myristoyl pentapeptide-4, tripeptide-1, 2, 3, 10, 32, tripeptide-1 copper, decapeptide-4, tetrapeptide-1, 3, 4, pentapeptide-1, 3, 34, oat peptide, wild soybean peptide, acetyl octapeptide-3, acetyl dipeptide-1 cetyl esters, acetyl hexapeptide 1, 7, 8, acetyl heptapeptide-4, acetyl tetrapeptide 2, 3, 5, 9, 11, palmitoyl dipeptide-7, palmitoyl hexapeptide-12, 14, 15, palmitoyl tripeptide-1, 5, 8, palmitoyl tetrapeptide-5, 7, 10, palmitoyl pentapeptide-4, 5.

Further, the adjuvant comprises a first group of moisturizers, a second group of moisturizers, grease, an organic solvent, a preservative, a solubilizer, a thickener, a skin conditioner, a chelating agent, a fragrance, an antioxidant, and water.

Further, the step 5 comprises:

step 5a) selecting a suitable mass ratio of said first group of moisturizers, said second group of moisturizers, said oil, said organic solvent, said preservative, said solubilizer, said thickener, said skin conditioner, said chelator, said antioxidant, said polypeptide, said fragrance, said anti-sensitivity agent and water for the skin condition of the user;

step 5b) stirring and mixing the solubilizer, the antioxidant, the grease, the organic solvent and the aromatic agent at high speed in an emulsifying machine to prepare a mixture A;

step 5c), putting the first group of humectants into a stirring pot, stirring and dissolving to prepare a mixture B;

step 5d), adding the second group of the moisturizing agents, the chelating agents and the thickening agents into a stirring pot, heating to 85-95 ℃ while stirring, fully dissolving the components, and keeping the temperature for 15-20 minutes to prepare a mixture C;

step 5e) cooling the mixture C to 40-45 ℃, sequentially adding the skin conditioner, the anti-allergy agent and the polypeptide, stirring and dissolving to prepare a mixture D;

and 5f) adding the preservative into the mixture D under the condition that the temperature is kept to be 40-45 ℃, fully stirring the mixture A and the mixture B, cooling to room temperature, discharging and standing.

Further, the skin care product for improving the allergic skin quality comprises the following components in percentage by mass: 3.5-7.5 parts of a first group of moisturizers, 10-12 parts of a second group of moisturizers, 6-10 parts of grease, 4-4.5 parts of organic solvent, 0.2-3 parts of preservative, 3.5-8 parts of solubilizer, 1-2 parts of thickener, 5-8 parts of skin conditioner, 0.2-0.4 part of chelating agent, 1.5-8.5 parts of antioxidant, 0.5-2 parts of polypeptide, 0.03-0.04 part of aromatic, 1.6-4 parts of anti-allergic agent and 40-60 parts of water.

Further, the skin care product for improving the allergic skin quality comprises the following components in percentage by mass: 6 parts of a first group of moisturizing agents, 12 parts of a second group of moisturizing agents, 7 parts of grease, 4.4 parts of organic solvents, 0.24 part of preservatives, 5.4 parts of solubilizing agents, 2 parts of thickening agents, 7.2 parts of skin conditioning agents, 0.2 part of chelating agents, 6 parts of antioxidants, 1.8 parts of polypeptides, 0.03 part of aromatic agents, 3.2 parts of anti-allergic agents and 45 parts of water.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) according to the customization method, one-person-one formula is realized, and the skin care product with the personal special formula and capable of improving the allergic skin is prepared according to the specific sensitivities of different users.

(2) The sensitivity can be more accurately characterized by taking the allergy-related gene polymorphism and the expression quantity of the cDNA corresponding to the allergy-related gene polymorphism as the root cause for the skin to present different skin sensitivity states.

(3) According to the customization method, oral mucosa cells or facial skin exfoliative cells are selected as samples to detect the allergy related gene polymorphism and the expression quantity of the cDNA corresponding to the gene polymorphism, so that the skin can be prevented from being stimulated by laser or medicaments, and the customization method is safe and reliable.

(4) The skin care product for improving the allergic skin quality prepared by the customization method of the invention mostly adopts natural animal and plant extracts and polypeptide as raw materials, is mild and non-irritant to the skin, can be used together with skin care products with other efficacies, and has good skin care effect.

(5) The skin care product prepared by the customized method aims at improving the anti-allergic capability of a user and can fundamentally solve the problem of skin sensitivity of the user.

(6) The invention realizes the new application of FLG, DDB2, NLRP3 and other genes in the field of skin care products.

Drawings

The embodiments illustrated in the drawings are meant to be illustrative of the invention. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. In the drawings:

fig. 1 is a schematic view of an embodiment of a method for customizing a skin care product for improving allergic skin according to the present invention.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have their meanings unless specifically defined otherwise.

Gene expression (genexpression) "means that a cell transcribes and translates genetic information stored in a DNA sequence during its life into a biologically active protein molecule. The similar proteins of different gene products from different tissues can be generated by the same gene in different tissues, and can be generated by the same gene code, and the phenomenon is firstly that the same gene can generate different transcripts and post-transcriptional processing effects in different tissues because the enhancer in the gene has tissue specificity and can be combined with tissue specific factors in different tissues.

"Gene polymorphism" refers to the phenomenon of two or more kinds of variation occurring at a certain genetic locus (gene sequence or non-gene sequence) in a population at an appropriate frequency, and can be determined by direct analysis of DNA or gene products. Essentially, polymorphisms arise as variations at the gene level, typically in regions of the gene sequence that do not encode proteins and regions that do not have important regulatory functions.

"polymerase chain reaction (RT-PCR)" is a technique that combines reverse transcription (RT-PCR) of RNA with polymerase chain amplification (PCR) of cDNA. First, cDNA is synthesized from RNA by the action of reverse transcriptase, and then the target fragment is amplified and synthesized by using the cDNA as a template. The RT-PCR technology is sensitive and has wide application, and can be used for detecting the gene expression level in cells, the content of RNA viruses in the cells and directly cloning cDNA sequences of specific genes. The RNA used as a template may be total RNA, mRNA or an in vitro transcribed RNA product. Whatever RNA is used, it is critical to ensure that the RNA is free of RNase and genomic DNA contamination. The method utilizes the related technology to extract total RNA in tissues or cells, takes mRNA in the total RNA as a template, and utilizes oligo (dT) or random primers to carry out reverse transcription by utilizing reverse transcriptase to obtain cDNA. Then using cDNA as template to make PCR amplification so as to obtain target gene or detect gene expression. Real-time PCR (real-time PCR), which is one of quantitative PCR (Q-PCR), performs quantitative analysis of DNA based on the amplification of DNA over a certain period of time.

As shown in fig. 1, the method for customizing a skin care product for improving allergic skin quality according to the present invention mainly comprises the following steps:

step 1, collecting a user cell sample.The cell sample can be oral mucosa epithelial cells, facial skin exfoliative cells or other cells capable of extracting user genes.

In the embodiment of the invention, the specific steps of collecting the oral mucosa epithelial cells comprise (1) preparation before collection: when gargling with clear water, attention needs to be paid to the fact that in order to ensure that cell samples are not polluted, drinks containing caffeine, carbonated drinks or fruit juice and the like are not drunk as much as possible half an hour before oral mucosa cells are collected; (2) collecting oral mucosa epithelial cells: the mouth swab package is opened, swallowing is performed, then the mouth swab is put into the mouth, the mouth swab is scraped back and forth for 20 times close to the inner side of the cheek, the mouth swab is taken out, the mouth swab is marked, and the genomic DNA is immediately extracted or stored in a 1.5ml centrifuge tube which is added with 0.5ml mouth swab genomic DNA storage solution (Baiolaibo, Cat: BTN 80802).

In another embodiment of the present invention, the specific steps of collecting exfoliated cells from facial skin comprise (1) preparing before collection: washing the face with clear water, wherein in order to ensure that the cell sample is not polluted, the tested person removes all used cosmetic residues with the cleansing oil; (2) collecting facial exfoliated skin cells: after the face is dried, the skin cells of the face are repeatedly adhered to the face by using a cell adhering device (if the collected cells are too few, the cells can be collected by using a plurality of adhering devices). The adhesive is properly preserved after being completely taken, so that pollution is avoided. Note that the labeling is done to prevent confusing the samples.

And 2, extracting mRNA in the cell sample.The embodiment of the invention adopts RNeasy Plus Microkit (cat # 74034) of QIAGEN company to extract total RNA, and comprises the following steps:

a) aiming at an oral mucosa epithelial cell sample, putting an oral swab into a 1.5ml centrifuge tube of RNase-free, cutting off the handle part of the oral swab, and leaving a cotton head of the medicine in the centrifuge tube; for the face skin exfoliative cell samples, the adhesive film with skin cells adhered on the cell adhesive device is torn off by tweezers and put into a 1.5ml centrifuge tube of RNase-free (note: one tweezers is used for each sample to avoid contamination between samples). Cells were lysed by adding 350. mu.l of RLT buffer and vortexed for 1 min.

b) The cell lysate from the previous step was added to the gDNA-depleted spin column provided in the kit and centrifuged at >10,000 rpm for 30 s. Discarding the centrifugal column and retaining the filtrate.

c) An equal volume (typically 350. mu.l) of 70% ethanol was added to the filtrate and mixed.

d) The sample from the previous step (including the formed pellet) was transferred to an RNeasyMinElute spin column provided in the kit, centrifuged at >10,000 rpm for 15s, and the filtrate was discarded.

e) To RNeasy MinElute spin column was added 700. mu.l buffer RW1, >10, 000rpm centrifugation for 15s and the filtrate was discarded.

f) Add 500. mu.l buffer RPE to RNeasy MinElute spin column, centrifuge at >10,000 rpm for 15s, discard filtrate.

g) Add 500. mu.l 80% ethanol to RNeasy MinElute spin column, centrifuge for 2min at >10,000 rpm, discard filtrate.

h) RNeasy MinElute spin columns were transferred to new 2ml collection tubes provided with the kit and centrifuged 5min at maximum speed with the lid opened. The collecting pipe and the filtrate therein are discarded.

i) Transferring the RNeasy MinElute centrifugal column into a new 1.5ml centrifuge tube provided by the kit, adding 14 μ l of RNase-freewater into the center of the centrifugal column, standing for 2min, and centrifuging at the highest rotation speed for 1min to obtain the extracted RNA in the centrifuge tube.

j) The concentration and quality of RNA (A260/A280) were determined using a NanoDrop (thermo) spectrophotometer, and 1. mu.g of the RNA was immediately reverse transcribed or the extracted RNA was stored at-80 ℃.

And 3, reverse transcription of mRNA to synthesize cDNA.In the examples of the present invention, cDNA synthesis was carried out using ReverTraAceQPCRTMasterMixWithDNAremover (cat. No.: FSQ-301) from TOYOBO. The specific operation steps are as follows:

a)4 mixing of xDNMasterMix with gdnraver: to the whole tube of 4 xDMastermix (440. mu.l) was added 8.8. mu.l (1/50 amount) of gDNAROMOVER, and the mixture was mixed by inversion. The 4 xDMastermix added with the gDNARemover can keep stable for at least 3 months at the temperature of-20 ℃.

b) Denaturation of RNA: after heat denaturation of 1. mu.g RNA at 65 ℃ for 5 minutes, the cells were immediately cooled on ice.

c) Removal of genomic DNA reaction (DNase reaction): the following reaction solution was prepared on ice.

After the reaction solution was gently stirred to homogeneity, the reaction solution was incubated at 37 ℃ for 5 minutes.

d) Reverse transcription reaction: the following reaction solution was prepared on ice.

After the reaction solution was gently stirred to be uniform, the reaction was carried out at the following temperature.

After the reaction is finished, the product is stored at the temperature of minus 20 ℃. Real-timeRTPCR was diluted 10-fold as a template and added.

And 4, detecting the polymorphism of the allergy-related gene in the cDNA and the expression level of the corresponding cDNA.

In an embodiment of the present invention, the allergy-associated gene may comprise one or a combination of FLG, DDB2, NLRP3, and the like.

FLG (filaggrin) is an inflammatory allergy-associated gene, is located at 1q21.3 position of chromosome 1, has a full length of 23.03kb, and has 3 exons in total. FLG mRNA was 12747nt in length and encoded a protein consisting of 4061 amino acid residues. The gene functions are as follows: FLG is abundant in the epidermis and plays an important role in the barrier function of the epidermis. The reduction or lack of the expression of FLG leads to the damage of the barrier function of the epidermis, so that allergens, irritants and the like in the environment can easily enter the epidermis, and then various pathophysiological changes are caused, namely, the human skin barrier of the genotype is strong, and the higher the expression amount of the gene is, the lower the risk of skin allergy is.

DDB2(DNA damage binding protein 2) is an allergy-associated gene, which is located on 11p11.2 of chromosome 11, has a total length of 24.78kb and has 10 exons. DDB2 has a full mRNA length of 1870nt and encodes a protein consisting of 427 amino acid residues. The gene functions are as follows: DDB2 is a product of E gene of xeroderma pigmentosum group and is one of important genes of nucleotide excision repair pathway, which can influence the generation and development of acne by participating in androgen metabolism, inflammation process and scar formation of acne, i.e. people of the genotype are at risk of forming acne, and the higher the expression level of the gene, the higher the skin sensitivity is.

NLRP3 (inflammasome 3) is an allergy-associated gene located on chromosome 1q44, 32.95kb in total length, 9 exons, NLRP3 mRNA 4299nt in total length, which encodes a protein consisting of 1036 amino acid residues.the inflammasome is a complex composed of multiple proteins, which can regulate the activation of caspase-1 (caspase-1) and promote the cleavage maturation of cytokine precursors pro-IL-1 β and pro-IL-18 during natural immune defense, and can also regulate caspase-1 dependent apoptosis (pyroptosis), inducing cell death under inflammatory and stress pathological conditions, i.e., the genotype of a human has low ability to resist inflammation, and the higher the expression level of the gene, the higher the risk of developing skin allergy.

The embodiment of the invention adopts real-timeRTPCR to detect the quantity of related genes, and comprises the following specific steps:

a) the mRNA sequence of the related gene is searched on NCBI, and a corresponding primer is designed.

In the embodiment of the invention, the gene GAPDH is used as an internal reference, namely, the expression level of the GAPDH gene is set to be 1, and the relative quantitative analysis of the skin-related gene is carried out by adopting a delta-deltaCt method. The primer sequences of GAPDH and skin-associated genes are shown in table 1:

TABLE 1

The amplified fragment sequences of the respective genes shown in Table 1 are shown in Table 2:

TABLE 2

b) Adopted is SYBRGreenRealtimePCRMasterMix (cat No.: QPK-201) by RocheAnd (6) performing detection on the machine at 96.

The reaction solution was prepared according to the following scheme shown in Table 3:

TABLE 3

Cycling conditions for PCR are shown in table 4:

TABLE 4

c) And (3) data analysis: the analysis of the dissolution curve is a single peak, which shows that the specificity of the primer is good, and the primer can be used for subsequent experiments; if the solubility curve is non-unimodal, the specificity of the primer is not good, and the primer needs to be redesigned; each pair of primers of each sample is repeated for three times, so that the error in the operation process is reduced; GAPDH was used as an internal reference, that is, the expression level of GAPDH gene was set to 1, and relative quantitative analysis was performed by delta-deltaCt method.

Step 5, judging the sensitivity according to the expression quantity of the allergy related gene, and selecting and improving the allergy aiming at the user sensitivity The skin care product for improving the allergic skin is prepared by the components and the dosage of the skin care product for improving the allergic skin.

The active ingredients with specific content in the skin care product for improving allergic skin can regulate and control the gene expression quantity of different types, thereby realizing the purpose of improving skin. For example, the anti-allergic agent for controlling the expression level of allergy-related genes such as SLC24a5, DDB2, NLRP3 comprises one or more of dipotassium glycyrrhizinate, tetrandrine, chamomile extract, and centella asiatica extract. The polypeptide can realize the purpose of regulating and controlling the expression of protein by regulating a signal path related to the expression of the allergic gene in the skin.

Based on the principle, the skin care product for improving allergic skin generally comprises an anti-allergic agent, polypeptide and an auxiliary agent. Wherein the adjuvant may comprise a first group of moisturizers, a second group of moisturizers, a lipid, an organic solvent, a preservative, a solubilizer, a thickener, a skin conditioner, a chelating agent, a fragrance, an antioxidant, and water. Specifically, the skin care product for improving allergic skin, which is prepared aiming at different users, comprises 3.5-7.5 parts by weight of a first group of moisturizers, 10-12 parts by weight of a second group of moisturizers, 6-10 parts by weight of grease, 4-4.5 parts by weight of an organic solvent, 0.2-3 parts by weight of a preservative, 3.5-8 parts by weight of a solubilizer, 1-2 parts by weight of a thickener, 5-8 parts by weight of a skin conditioner, 0.2-0.4 part by weight of a chelating agent, 1.5-8.5 parts by weight of an antioxidant, 0.5-2 parts by weight of a polypeptide, 0.03-0.04 part by weight of an aromatic, 1.6-4 parts by weight of an anti-allergic agent and 40-60 parts by weight of water. Wherein,

the humectant comprises one or more of sodium hyaluronate, polyglutamic acid, tremella polysaccharide, ginseng polysaccharide, ganoderan, propylene glycol, dipropylene glycol, butanediol and glycerol, wherein the humectant is divided into a first group of humectants and a second group of humectants according to different adding sequences in the preparation process;

the oil comprises one or more of olive oil, white oil, vaseline, paraffin oil, almond oil, squalane, nut oil, jojoba oil, and shea butter;

the organic solvent comprises one or more of dodecanol, hexadecanol, octadecanol, docosanol and cetyl alcohol;

the preservative comprises one or more of propyl hydroxybenzoate, iodopropynyl butylcarbamate, 2-phenoxyethanol and methyl hydroxybenzoate;

the solubilizer comprises one or more of stearic acid, PEG20 hydrogenated castor oil, PEG20 glyceryl stearate, PEG60 hydrogenated castor oil, PEG60 glyceryl isostearate, polyglycerol-10 stearate, and PEG/PPG-17/6 copolymer;

the thickening agent comprises one or more of hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl guar gum and a polymeric acrylate thickening agent;

the skin conditioner comprises one or more of herba Centellae extract, lycopene, ceramide, lecithin, propolis extract, chamomile extract, Ginseng radix extract, yeast extract, and herba Houttuyniae extract;

the chelating agent comprises disodium EDTA;

the antioxidant comprises one or more of brown algae extract, Ginseng radix extract, ginsenoside, Galla chinensis extract, tea polyphenols, ganoderan, algal polysaccharides, Rubi fructus extract, resveratrol, avocado oil, grapeseed oil, astaxanthin, ferulic acid, coenzyme Q10, N-acetylcysteine, curcumin, tanshinone IIA, glabridin, and thioctic acid;

the antiallergic agent comprises one or more of dipotassium glycyrrhizinate, tetrandrine, flos Matricariae Chamomillae extract, and herba Centellae extract;

the aromatic comprises one or more of water extract of flos Magnoliae, water extract of Aloe, and water extract of flos Rosae Rugosae;

polypeptide, which can realize the purpose of regulating protein expression by regulating signal pathways related to gene expression in skin and comprises alanine/histidine/lysine polypeptide copper HCl, soybean (Glycinemax) polypeptide, dipeptide-1, dipeptide-2, dipeptide-4, oligopeptide-1, oligopeptide-2, oligopeptide-3, oligopeptide-4, oligopeptide-5, oligopeptide-6, oligopeptide-29, oligopeptide-34, carnosine, saccharomycete polypeptide, arginine/lysine polypeptide, nonapeptide-1, poly (tripeptide-6), ascorbic acid polypeptide, sodium bacitracin, hexapeptide-1, 2, 3, 5, 9, 11, heptapeptide-6, myristoyl hexapeptide-5, myristoyl pentapeptide-4, tripeptide-1, 2, 3, 10, 32, tripeptide-1 copper, oligopeptide-6, and polypeptide-6, One or more of decapeptide-4, tetrapeptide-1, 3, 4, pentapeptide-1, 3, 34, oat peptide, wild soybean peptide, acetyl octapeptide-3, acetyl dipeptide-1 cetyl esters, acetyl hexapeptide 1, 7, 8, acetyl heptapeptide-4, acetyl tetrapeptide 2, 3, 5, 9, 11, palmitoyl dipeptide-7, palmitoyl hexapeptide-12, 14, 15, palmitoyl tripeptide-1, 5, 8, palmitoyl tetrapeptide-5, 7, 10 and palmitoyl pentapeptide-4, 5.

The preparation method of the skin care product for improving the allergic skin generally comprises the following steps:

step 5a) selecting a humectant, oil, an organic solvent, a preservative, a solubilizer, a thickener, a skin conditioner, a chelating agent, an antioxidant, a polypeptide, an aromatic, an anti-allergy agent and water in a proper mass ratio of the skin care product for improving the allergic skin types according to the user sensitivity;

step 5b) stirring and mixing the solubilizer, the antioxidant, the grease, the organic solvent and the aromatic agent in an emulsifying machine at an overspeed to prepare a mixture A;

step 5c), placing the first group of humectants into a stirring pot, stirring and dissolving to prepare a mixture B;

step 5d) adding the second group of humectant, chelating agent and thickener into a stirring pot, heating to 85-95 ℃ while stirring, fully dissolving, and keeping the temperature for 15-20 minutes to prepare a mixture C;

step 5e) cooling the mixture C to 40-45 ℃, sequentially adding a skin conditioner, an anti-allergy agent and polypeptide, and stirring for dissolving to prepare a mixture D;

and step 5f), adding a preservative into the mixture D under the condition that the temperature is kept at 40-45 ℃, fully stirring the mixture A and the mixture B, cooling to room temperature, discharging and standing.

Example 1

Example 1 of the present invention was carried out on a 55-year-old volunteer 1, and the genes tested included: allergy-related genes FLG, DDB2 and NLRP 3. Preparing the following skin care product according to the detection result: wherein the total mass of the skin care product is 50Kg,

the volunteers 1 performed the gene examination again after using the skin care product for improving allergic skin property of the above formulation for 100 days, and the relative expression amounts of the genes before and after using the skin care product for improving allergic skin property are shown in table 5:

TABLE 5

Name of gene	FLG	DDB2	NLRP3	GAPDH
					Before use	3.514534	1.60956	3.482202	1
After use	5.62326	1.223476	2.12687	1
					Reference example	2.971445	1.65306	2.976469	1

Among them, the reference examples of examples 1 to 4 are the average values of the relative expression amounts of the respective genes in 100 volunteers aged 20 to 30 years.

The expression level of the gene shown in Table 5 indicates that the expression level of gene NLRP3 is high and the skin is sensitive before the skin care product for improving allergic skin is used in volunteers 1. After the skin care product for improving allergic skin with the formula is used, the expression level of the gene NLRP3 is obviously reduced, the expression levels of the genes FLG and DDB24 are also well regulated, and correspondingly, the anti-allergic capability is improved.

Example 2

The example 2 of the present invention performed gene detection on volunteer 2 aged 45, and the detected genes included: allergy-related genes FLG, DDB2 and NLRP 3. Preparing the following skin care product according to the detection result: wherein the total mass of the skin care product is 50Kg,

first group of humectants: 1Kg of polyglutamic acid, 1Kg of tremella polysaccharide and 1Kg of ganoderma lucidum polysaccharide

Second group of humectants: butanediol 0.2Kg, dipropylene glycol 0.8Kg, glycerin 5Kg

Grease: almond oil 1Kg, squalane 0.5Kg, nut oil 0.5Kg, jojoba oil 1.5Kg

Organic solvent: 1Kg of docosanol and 1.2Kg of cetyl alcohol

Preservative: iodoproparganol butyl carbamate 0.12Kg

Solubilizer: PEG20 hydrogenated castor oil 0.8Kg, PEG20 glyceryl stearate 1.1Kg, PEG60 hydrogenated castor oil 0.8Kg

Thickening agent: hydroxymethyl cellulose 1Kg

Skin conditioner: 1.5Kg of ginseng root extract, 2Kg of yeast extract and 0.1Kg of houttuynia cordata extract

Chelating agent: EDTA disodium 0.1Kg

Antioxidant: ginseng extract 0.2Kg, tea polyphenol 0.5Kg, brown algae extract 0.5Kg, curcumin 0.2Kg, gallnut extract 0.4Kg, ferulic acid 0.1Kg, coenzyme Q100.1Kg, N-acetylcysteine 1Kg

Polypeptide: tripeptide-1 copper 0.6Kg, decapeptide-40.1 Kg, tetrapeptide-10.1 Kg, pentapeptide-340.1 Kg

Anti-allergic agent: dipotassium glycyrrhizinate 0.7Kg, centella asiatica extract 0.9Kg

Aromatic agent: 0.015Kg of rose aqueous extract

And others: balance of water

The relative expression levels of genes before and after the skin care product for improving allergic skin properties was used in volunteers 2 for 100 days, which were obtained by performing gene testing again, are shown in table 6:

TABLE 6

Name of gene	FLG	DDB2	NLRP3	GAPDH
					Before use	0.538369	0.874583	0.239539	1
After use	0.93326	0.733476	0.21874	1
					Reference example	2.971445	1.65306	2.976469	1

The expression levels of the genes shown in table 6 indicate that the expression level of gene FLG was low and the skin was sensitive before the skin care product for improving allergic skin was applied to volunteer 2. After the skin care product for improving allergic skin with the formula is used, the expression level of the gene FLG is obviously improved, the expression levels of the genes NLRP3 and DDB24 are also well adjusted, and correspondingly, the anti-sensitivity capability is improved.

Example 3

The embodiment 3 of the invention aims at carrying out gene detection on a 44-year-old volunteer 3, and the detected genes comprise: allergy-related genes FLG, DDB2 and NLRP 3. Preparing the following skin care product according to the detection result: wherein the total mass of the skin care product is 50Kg,

first group of humectants: 0.75Kg of sodium hyaluronate, 0.75Kg of tremella polysaccharide and 1Kg of ganoderma lucidum polysaccharide

Second group of humectants: dipropylene glycol 2Kg and glycerin 4Kg

Grease: almond oil 1Kg, squalane 1Kg, jojoba oil 1Kg

Organic solvent: 1Kg of docosanol and 1Kg of cetyl alcohol

Preservative: 0.2Kg of 2-phenoxyethanol and 0.2Kg of propylparaben

Solubilizer: PEG60 glyceryl isostearate 0.85Kg, polyglycerol-10 stearate 0.85Kg

Thickening agent: carboxymethyl cellulose 0.5Kg

Skin conditioner: propolis extract 1Kg, Ginseng radix extract 1Kg, and yeast extract 0.5Kg

Chelating agent: EDTA disodium 0.2Kg

Antioxidant: raspberry extract 0.35Kg, resveratrol 0.25Kg, grape seed oil 0.25Kg, coenzyme Q100.1Kg, N-acetylcysteine 0.8Kg

Polypeptide: 0.1Kg of amino acid/lysine polypeptide, 10.05 Kg of nonapeptide, 0.01Kg of ascorbic acid polypeptide, 0.08Kg of surfactin sodium, 120.1 Kg of palmitoyl hexapeptide, and 60.05 Kg of heptapeptide

Anti-allergic agent: dipotassium glycyrrhizinate 0.8Kg

Aromatic agent: aloe water extract 0.02Kg

And others: balance of water

The relative expression levels of genes before and after applying the skin care product for improving allergic skin properties of the above formulation to the volunteers 3 for 100 days were shown in table 7:

TABLE 7

The expression levels of the genes shown in table 7 indicate that volunteer 3 had a lower expression level of FLG gene and had more sensitive skin before using the skin care product for improving allergic skin conditions. After the skin care product for improving allergic skin with the formula is used, the expression level of the gene FLG is obviously improved, the expression levels of the genes NLRP3 and DDB24 are also well adjusted, and correspondingly, the anti-sensitivity capability is improved.

Example 4

Example 4 of the present invention was carried out on a 43-year-old volunteer 4, and the genes detected included: allergy-related genes FLG, DDB2 and NLRP 3. Preparing the following skin care product according to the detection result: wherein the total mass of the skin care product is 50Kg,

first group of humectants: 1Kg of polyglutamic acid, 0.5Kg of tremella polysaccharide and 0.3Kg of ginseng polysaccharide

Second group of humectants: dipropylene glycol 2Kg, butanediol 1Kg and glycerin 3Kg

Grease: 2Kg of vaseline, 1Kg of paraffin oil, 1Kg of nut oil and 1Kg of shea butter oil

Organic solvent: octadecanol 1Kg and docosanol 1.2Kg

Preservative: iodoproparganol butyl carbamate 0.2Kg

Solubilizer: PEG20 hydrogenated castor oil 1Kg, PEG60 glyceryl isostearate 1Kg, polyglycerol-10 stearate 0.6Kg, PEG/PPG-17/6 copolymer 1.2Kg

Thickening agent: hydroxypropyl guar gum 1Kg

Skin conditioner: lecithin 0.9Kg, propolis extract 0.9Kg, chamomile extract 1Kg, ginseng root extract 0.2Kg, yeast extract 0.9Kg

Chelating agent: EDTA disodium 0.15Kg

Antioxidant: ginsenoside 0.3Kg, tea polyphenol 0.2Kg, brown algae extract 0.2Kg, and Galla chinensis extract 1.2Kg

Polypeptide: carnosine 0.1Kg, saccharomycete polypeptide 0.09Kg, surfactin sodium 0.02Kg, hexapeptide-10.03 Kg, palmitoyl dipeptide-70.05 Kg

Anti-allergic agent: dipotassium glycyrrhizinate 1Kg and chamomile extract 1Kg

Aromatic agent: 0.015Kg of rose aqueous extract

And others: balance of water

The relative expression levels of genes before and after the skin care product for improving allergic skin properties was used in volunteers 4 for 100 days, which were obtained by performing gene testing again, are shown in table 8:

TABLE 8

Name of gene	FLG	DDB2	NLRP3	GAPDH
					Before use	0.239816	0.203063	0.017824	1
After use	0.42226	0.16476	0.01874	1
					Reference example	2.971445	1.65306	2.976469	1

The expression levels of the genes shown in Table 8 indicate that the expression level of FLG gene was low and the skin was sensitive before the skin care product for improving allergic skin was applied to volunteers 4. After the skin care product for improving allergic skin with the formula is used, the expression level of the gene FLG is obviously improved, the expression levels of the genes NLRP3 and DDB24 are also well adjusted, and correspondingly, the anti-sensitivity capability is improved.

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> Du Libo

<120> customization method of skin care product for improving allergic skin

<130>1

<160>12

<170>PatentIn version 3.5

<210>1

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> GAPDH primer 1

<400>1

cgggaaactg tggcgtgatg 20

<210>2

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> GAPDH primer 2

<400>2

atgaccttgc ccacagcctt 20

<210>3

<211>0

<212>DNA

<213>Artificial Sequence

<220>

<223> FLG primer 1

<400>3

gacagggatc ctaccacgag 20

<210>4

<211>0

<212>DNA

<213>Artificial Sequence

<220>

<223> FLG primer 2

<400>4

tctctgcttg cacttctgga 20

<210>5

<211>18

<212>DNA

<213>Artificial Sequence

<220>

<223> primer 1 DDB2

<400>5

gcgtggcacc caactcac 18

<210>6

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<223> primer 2 DDB2

<400>6

ggtgggtttg tccttgatgc c 21

<210>7

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> NLRP3 primer 1

<400>7

gaatgcccgt ctgggtgaga 20

<210>8

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> NLRP3 primer 2

<400>8

gggctctcac acgtcttggt 20

<210>9

<211>87

<212>DNA

<213>Artificial Sequence

<220>

<223> GAPDH amplified fragment

<400>9

cgggaaactg tggcgtgatg gccgcggggc tctccagaac atcatccctg cctctactgg 60

cgctgccaag gctgtgggca aggtcat 87

<210>10

<211>130

<212>DNA

<213>Artificial Sequence

<220>

<223> FLG amplification fragment

<400>10

gacagggatc ctaccacgag caatcggtag ataggtctgg acactcaggg tcccatcaca 60

gccacaccac atcccaggga aggtctgatg cctcccatgg gcagtcagga tccagaagtg 120

caagcagaga 130

<210>11

<211>93

<212>DNA

<213>Artificial Sequence

<220>

<223> DDB2 amplified fragment

<400>11

gcgtggcacc caactcaccc cagcaccgtg gctgtgggtt ccaaaggggg agatatcatg 60

ctctggaatt ttggcatcaa ggacaaaccc acc 93

<210>12

<211>135

<212>DNA

<213>Artificial Sequence

<220>

<223> NLRP3 amplified fragment

<400>12

gaatgcccgt ctgggtgaga gtgtgagcct caacaaacgc tacacacgac tgcgtctcat 60

caaggagcac cggagccagc aggagaggga gcaggagctt ctggccatcg gcaagaccaa 120

gacgtgtgag agccc 135

Claims

1. A method for customizing a skin care product for improving allergic skin is characterized by comprising the following steps:

step 1, collecting a user cell sample;

step 2, extracting mRNA in the cell sample;

step 3, reverse transcription of mRNA to synthesize cDNA;

2. The method of customizing a skin care product for improving allergic skin according to claim 1, wherein the step 4 comprises:

3. The method of customizing a skin care product for improving allergic skin according to claim 2, wherein the primer comprises:

GAPDH Forword：5’-CGGGAAACTGTGGCGTGATG-3’

Reverse：5’-ATGACCTTGCCCACAGCCTT-3’

FLG Forword：5’-GACAGGGATCCTACCACGAG-3’

Reverse：5’-TCTCTGCTTGCACTTCTGGA-3’

DDB2 Forword：5’-GCGTGGCACCCAACTCAC-3’

Reverse：5’-GGTGGGTTTGTCCTTGATGCC-3’

NLRP3 Forword：5’-GAATGCCCGTCTGGGTGAGA-3’

Reverse：5’-GGGCTCTCACACGTCTTGGT-3’。

4. the method for customizing skin care products for improving allergic skin according to claim 1, wherein the ingredients of the skin care products for improving allergic skin comprise an anti-sensitization agent, a polypeptide and an auxiliary agent.

5. The method of claim 4, wherein the anti-sensitization agent comprises one or more of dipotassium glycyrrhizinate, tetrandrine, chamomile extract, and centella asiatica extract.

6. The method of claim 4, wherein the polypeptide comprises alanine/histidine/lysine polypeptide copper HCl, soy (Glycinemax) polypeptide, dipeptide-1, dipeptide-2, dipeptide-4, oligopeptide-1, oligopeptide-2, oligopeptide-3, oligopeptide-4, oligopeptide-5, oligopeptide-6, oligopeptide-29, oligopeptide-34, carnosine, saccharomycete polypeptide, arginine/lysine polypeptide, nonapeptide-1, poly (tripeptide-6), ascorbic acid polypeptide, sodium surfactin, hexapeptide-1, 2, 3, 5, 9, 11, heptapeptide-6, myristoyl hexapeptide-5, myristoyl pentapeptide-4, tripeptide-1, 2, 3, 10, 32, myristoyl pentapeptide-4, and the like, Tripeptide-1 copper, decapeptide-4, tetrapeptide-1, 3, 4, pentapeptide-1, 3, 34, oat peptide, soybean peptide, acetyl octapeptide-3, acetyl dipeptide-1 cetyl esters, acetyl hexapeptide 1, 7, 8, acetyl heptapeptide-4, acetyl tetrapeptide 2, 3, 5, 9, 11, palmitoyl dipeptide-7, palmitoyl hexapeptide-12, 14, 15, palmitoyl tripeptide-1, 5, 8, palmitoyl tetrapeptide-5, 7, 10, palmitoyl pentapeptide-4, 5.

7. The method of customizing skin care products for improving allergic skin conditions according to claim 5, wherein the adjunct comprises a first group of moisturizers, a second group of moisturizers, a lipid, an organic solvent, a preservative, a solubilizer, a thickener, a skin conditioner, a chelating agent, a fragrance, an antioxidant, and water.

8. The method of customizing a skin care product for improving allergic skin conditions according to claim 7, wherein said step 5 comprises:

9. The method for customizing the skin care product for improving the allergic skin according to claim 8, wherein the skin care product for improving the allergic skin comprises the following components in percentage by mass: 3.5-7.5 parts of a first group of moisturizers, 10-12 parts of a second group of moisturizers, 6-10 parts of grease, 4-4.5 parts of organic solvent, 0.2-3 parts of preservative, 3.5-8 parts of solubilizer, 1-2 parts of thickener, 5-8 parts of skin conditioner, 0.2-0.4 part of chelating agent, 1.5-8.5 parts of antioxidant, 0.5-2 parts of polypeptide, 0.03-0.04 part of aromatic, 1.6-4 parts of anti-allergic agent and 40-60 parts of water.

10. The method for customizing the skin care product for improving the allergic skin according to claim 9, wherein the skin care product for improving the allergic skin comprises the following components in percentage by mass: 6 parts of a first group of moisturizing agents, 12 parts of a second group of moisturizing agents, 7 parts of grease, 4.4 parts of organic solvents, 0.24 part of preservatives, 5.4 parts of solubilizing agents, 2 parts of thickening agents, 7.2 parts of skin conditioning agents, 0.2 part of chelating agents, 6 parts of antioxidants, 1.8 parts of polypeptides, 0.03 part of aromatic agents, 3.2 parts of anti-allergic agents and 45 parts of water.