CN113667697A

CN113667697A - Evaluation method based on human TRPV1 receptor overexpression cell strain

Info

Publication number: CN113667697A
Application number: CN202010407275.2A
Authority: CN
Inventors: 殷庆飞; 陈媛祺; 陈�田
Original assignee: Shanghai Jahwa United Co Ltd
Current assignee: Shanghai Jahwa United Co Ltd
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2021-11-19
Also published as: WO2021227811A1

Abstract

The invention provides a virus overexpression vector and a cell containing the virus overexpression vector. The present invention also provides a method of assessing the pain causing risk of an agent or product, the method comprising the steps of: (a) constructing a virus over-expression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO: 1); (b) infecting SH-SY5Y cells with a virus over-expression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO:1) to construct a human TRPV1 receptor over-expression cell strain; (c) treating human TRPV1 receptor overexpression cells by using dihydrocapsaicin to establish a pain detection method; (d) and evaluating the pain-causing risk of the reagent or the product to be detected by adopting the established pain detection method.

Description

Evaluation method based on human TRPV1 receptor overexpression cell strain

Technical Field

The invention relates to the field of cosmetics and component safety evaluation, in particular to an eye irritation evaluation method, and specifically relates to a method for evaluating cosmetic raw materials and whether eye cosmetics are gout risks or not based on a human TRPV1 receptor overexpression cell line.

Background

Cosmetics are one of the chemicals commonly used by people, and are various, such as hair cosmetics, skin care cosmetics, color cosmetics, sun protection cosmetics, freckle removal cosmetics and the like. Some cosmetics may contact the eyes of a person during use and cause certain irritation and discomfort to the eyes, causing subjective sensations of lacrimation, conjunctival congestion, and/or itching, pain, etc. The toxicology experimental method in the technical Specification for cosmetic safety (2015 edition) in China is specified as follows: in general, before newly developed cosmetic products are put on the market, corresponding tests are performed according to the use and the category of the products to evaluate the safety of the products. The 'working specification for registration and record inspection of cosmetics' published in 2019 requires cosmetic toxicology test items: the hair products, skin care products and eye color cosmetics products which are easy to touch eyes in cosmetics with non-special purposes, and bath products are subjected to an acute eye irritation test; in cosmetics for special purposes, products for hair growth, hair dyeing and hair waving, spot removal and sun protection which are easy to touch eyes are subjected to acute eye irritation test.

Ocular irritancy refers to reversible inflammatory changes of the eye and its surrounding mucosa caused by direct exposure of the anterior segment of the eye to chemical substances. At present, various methods for evaluating acute eye irritation exist at home and abroad, and can be divided into an in vivo test method, an in vitro test method and a human body clinical test. The in vivo test method is the Draize rabbit eye test, is an international standard method for measuring acute eye irritation, is one of the most common acute eye irritation test methods, and is still the standard for eye irritation evaluation specified in cosmetic regulations in China at present.

With The Development of scientific technology and The continuous and intensive 3R (reduction, optimization, replacement) theory, many in vitro test Methods for replacing animal experiments appear, and The verification and recommendation of organizations such as The Organization for Economic Cooperation and Development (OECD), The inter-Organization coordination Committee for evaluation of The assessment of The Alternative Methods, ICCVAM, and The like are obtained. For example, Bovine Corneal Opacity and Permeability Test (Bovine Corneal Opacity and Permeability, BCOP, OECD document number TG 437), ex vivo corn Eye Test (ICE, OECD document number TG 438), cell culture type Fluorescein Leakage Test (fluoroescein leak, FL, OECD document number TG 460), in vitro Short-term Exposure Test (Short Time Exposure, STE, OECD document number TG 491), recombinant human Corneal epithelial model Test (Reconstructed human Corneal Cornea-like epitomeium, RhCE, OECD document number TG492), chick embryo chorionic Membrane Test (Hen's Egg Test-Chorioallogenic Membrane, HET-ps, file site number CAM/n. nie. cd. gorv/covar/gram/mouth. et al. Also, human clinical trials were conducted to evaluate acute eye irritation. These eye irritation test methods play an important role in cosmetic safety evaluation.

The in vivo test method and the in vitro test method have technical limitations in the aspect of evaluating acute eye irritation, and cannot reflect subjective feeling-pain, namely stabbing pain and burning pain, caused by substances entering eyes. For example, white rabbits in the in vivo test method Draize rabbit eye test do not complain of pain perception, and the test period is long, the cost is high, and the reliability of the test results from animals to humans is limited; the principle of the in vitro test method is focused on the barrier function damage of the corneal epithelium of the isolated eyeball, the matrix protein denaturation, the cytotoxicity, the cell barrier function, the stimulation reaction of blood vessels and the like, and the phenomena of in vivo defense mechanism, neuralgia and the like are difficult to react; although the human clinical test can reflect eye irritation and pain caused by the tested substances, the cost is high, the test period is long, certain ethical and safety risks exist, and the method is not suitable for large-scale preliminary screening of cosmetic formulas.

In the application process of the washing type cosmetics, for the infant group in the development and growth process, because the infant group has the advantages of small blinking frequency, long opening time, incomplete defensive eye closing, habitual eye rubbing and relatively small amount of tears, which make the eyes of the infant group very easily affected by external factors to generate eye irritation and pain, the eye irritation and pain generated by the cosmetics need to be further reduced in the development process of the cosmetics. Because the limitations of the in vivo and in vitro tests and the human clinical tests cannot meet the detection of the toxicological endpoint of pain, a new detection method needs to be established according to the pain generation mechanism, so as to evaluate whether cosmetic products and raw materials cause pain feeling, guide the development of milder products and meet the requirements of consumers.

The TRPV1 receptor (transient receptor potential vanilloid type 1, also known as capsaicin receptor) plays an important role in the generation and transmission of mammalian pain sensations. The TRPV1 receptor is distributed primarily in small to medium neurons in the dorsal root ganglia and trigeminal ganglia, particularly small diameter unmyelinated sensory nerve fibers. Wherein the sensory nerve fibers of the trigeminal nerve eye branch are distributed in the lacrimal gland, conjunctiva, iris, ciliary body and cornea, and the tail ends of the sensory nerve fibers on the cornea are very abundant, so the trigeminal nerve eye branch is very sensitive to external stimulation. The TRPV1 receptor is a nonselective cation channel composed of four subunits, each having 6 transmembrane helices and cytoplasmic N-and C-termini; the receptor can be activated by many factors, such as capsaicin, damaging high temperatures, low pH and endogenous substances, and is a multimodal receptor. After TRPV1 receptor is activated, monovalent and divalent cations (mainly calcium ions) enter the cell, trigger action potentials, and transmit into the higher central system, causing stinging and burning sensations.

The TRPV1 receptor protein sequences of human and rabbit are highly homologous in mammals, but differ in amino acid residues at the key binding site of the transmembrane helix TM3/4 capsaicin, which results in inconsistent response of TRPV1 receptors of different species to capsaicin. Therefore, in establishing a pain evaluation method, the species and practical application of the TRPV1 receptor need to be considered.

The invention starts from the practical problem that a human is a consumer user of cosmetics, simulates neurons related to corneal pain of a human eye based on the action mechanism of a human TRPV1 receptor in pain generation and transmission, constructs a nerve cell model over expressing the human TRPV1 receptor, and establishes a pain evaluation method based on the nerve cell model, so that the method can be used for evaluating whether the cosmetics and the components thereof have gout risk to the human eye. Meanwhile, as a comparative example, the invention also establishes a pain evaluation method based on the rabbit TRPV1 receptor, simulates the neurons related to the rabbit eye corneal pain in a Draize rabbit eye test, and compares the two pain evaluation methods based on the human and rabbit TRPV1 receptors respectively.

Compared with some methods for evaluating the pain based on the human TRPV1 receptor overexpression cell strain, the method for evaluating the pain based on the human TRPV1 receptor overexpression cell strain has the following advantages:

(1) more accords with the actual use feeling of people. The pain evaluation method of the invention is based on the human TRPV1 receptor, and can avoid the problems of false positive and false negative caused by species difference.

(2) The sensitivity is high. The pain test method of the invention detects the intracellular calcium ion concentration based on the green fluorescent calcium indicator Fluo-4, AM, the Fluo-4 emission intensity will depend on the level of bound calcium, i.e. the more calcium, the brighter the signal, and the indicator fluorescence optical density increases by at least 100-fold after binding with Ca2 +.

(3) The requirement on a fluorescence microplate reader is low. The detection method adopts a green fluorescent calcium indicator Fluo-4, AM, and only needs excitation light (494nm) with one wavelength; an automatic sample adding system is not needed for the fluorescence microplate reader.

(4) The detection method is simple, short in period, high in detection efficiency, low in cost, capable of being used for large-scale detection, and high in applicability and popularization.

The invention has the beneficial effects that: the pain evaluation method based on the human TRPV1 receptor overexpression cell strain is beneficial to evaluating whether cosmetic products and raw materials have eye pain risk, can guide the screening and development of mild cosmetic product formulas, and has high application value.

Disclosure of Invention

In one aspect, the invention provides a viral overexpression vector comprising the nucleotide sequence shown in SEQ ID No. 1 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID No. 1.

In another aspect, the invention provides a cell comprising a viral overexpression vector comprising the nucleotide sequence shown in SEQ ID No. 1 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID No. 1.

In yet another aspect, the present invention provides a method of assessing the risk of pain development in an agent or product, the method comprising the steps of:

(a) constructing a virus over-expression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO: 1);

(b) infecting SH-SY5Y cells with a virus over-expression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO:1) to construct a human TRPV1 receptor over-expression cell strain;

(c) treating human TRPV1 receptor overexpression cells by using dihydrocapsaicin to establish a pain detection method;

(d) and evaluating the pain-causing risk of the reagent or the product to be detected by adopting the established pain detection method.

In a preferred embodiment, step (b) of the above-described method for evaluating the pain-causing risk of an agent or product comprises mixing the viral overexpression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO:1) with the packaging plasmids pLP/VSVG and pCMV-dR8.91 and transfecting HEK293FT cells to prepare viral particles containing the TRPV1 gene.

In a preferred embodiment, step (b) of the above method for evaluating pain-causing risk of an agent or product further comprises puromycin screening to obtain a stably expressed human TRPV1 receptor overexpressing cell strain.

In a preferred embodiment, step (c) of the above method for evaluating the pain-causing risk of an agent or product comprises measuring the intracellular calcium ion concentration based on a green fluorescent calcium indicator.

In a preferred embodiment, the green fluorescent calcium indicator in the above method of assessing the pain causing risk of an agent or product is Fluo-4, AM.

In a preferred embodiment, step (c) of the above method of assessing the pain causing risk of an agent or product further comprises the addition of a TRPV1 receptor inhibitor.

In a preferred embodiment, the TRPV1 receptor inhibitor in the above-described method for assessing the pain causing risk of an agent or product is ruthenium red.

In a preferred embodiment, the agent or product to be tested in step (d) of the above method of assessing the pain causing risk of an agent or product is selected from the group consisting of: cosmetics or cosmetic raw materials.

In a preferred embodiment, the cosmetic to be detected is a cosmetic that is easily touched to the eye, and the cosmetic raw material to be detected is an alkanolamide-based raw material. In a more preferred embodiment, the alkanolamide-based starting material to be detected is selected from: cocamide MEA, lauramide MEA, cocamide MIPA, palmitamide MEA, lactamide MEA, or any combination thereof.

Drawings

FIG. 1 shows that the RT-qPCR method detects the over-expression of TRPV1mRNA of a human TRPV1 receptor over-expression cell strain.

FIG. 2 shows fluorescence microscopy of activation of the human TRPV1 receptor by dihydrocapsaicin in an over-expressed cell line.

FIG. 3 shows a method for assessing pain based on the human TRPV1 receptor overexpressing cell line using different concentrations of dihydrocapsaicin.

Fig. 4 shows the detection of cocamide MEA based on a pain evaluation method of human TRPV1 receptor overexpressing cell line.

Fig. 5 shows the detection of a body wash product based on a pain evaluation method of a human TRPV1 receptor overexpression cell line.

FIG. 6 shows that RT-qPCR method detects TRPV1mRNA overexpression of rabbit TRPV1 receptor overexpression cell strain.

FIG. 7 shows fluorescence microscopy of the activation of the rabbit TRPV1 receptor by dihydrocapsaicin in an overexpressing cell line.

FIG. 8 shows a method for evaluating pain based on a rabbit TRPV1 receptor overexpressing cell line using different concentrations of dihydrocapsaicin.

FIG. 9 shows the detection of cocamide MEA based on a pain evaluation method of rabbit TRPV1 receptor overexpressing cell line.

Figure 10 shows the eye irritation of the chick embryo chorioallantoic membrane test with cocamide MEA.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill and research in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. For the purposes of the present invention, the following terms are defined.

According to the present invention, the term "cosmetic" refers to a chemical or fine chemical product that is applied by spreading, spraying or the like to any part of the surface of the human body, such as skin, hair, nails, lips, and teeth, for the purpose of cleaning, maintaining, beautifying, modifying and changing the appearance, or correcting the odor of the human body, maintaining a good condition.

According to the invention, the term "easy-to-touch eye cosmetic" refers to a cosmetic which can enter the front part of the eye during use, wherein the "front part of the eye" refers to the upper eyelid, the lower eyelid, the lacrimal gland, the bulbar conjunctiva, the cornea, the iris and other accessory tissues of the eye, and the "easy-to-touch eye cosmetic" refers to a hair product, a skin care product, an eye color cosmetic product, a bath product which are easy to touch the eye in cosmetics without special use, and a hair growing product, a hair dyeing product, a hair waving product, a spot removing product and a sun protection product in cosmetics with special use.

According to the present invention, the term "cosmetic raw material" refers to raw materials used in the production process of cosmetics, including cosmetic base raw materials such as oily raw materials, surfactants, solvents, powdery raw materials, high molecular polymers, and other additives; cosmetic auxiliary materials such as essence, colorant, pearling agent, antiseptic, bactericide, antioxidant and complexing agent; and special purpose raw materials, such as depilatory, hair dye, permanent wave agent, astringent, antiperspirant, deodorant, speckle removing agent, sunscreen agent, etc.

According to the present invention, the term "alkanolamide-based raw material" refers to alkanolamide compounds and alkanolamide derivatives which are useful in cosmetics. The alkanolamide is a non-ionic surfactant which is synthesized by taking alkyl alcohol amine and fatty acid or fatty acid ester as raw materials, is easy to biodegrade and is environment-friendly. The existence of amido bond in the molecular structure of the alkanolamide is beneficial to the formation of hydrogen bond, the critical micelle concentration is reduced, the hydrolysis resistance is improved, and the alkanolamide has the performances of viscosity increasing, foam stabilizing, dispersing, emulsifying, antistatic, rust prevention and the like, and is widely applied to the aspects of daily necessities, leather, metal cleaning, textile fiber, oil fields and the like. The alkanolamide derivative is a novel green surfactant which is derived and synthesized by taking alkanolamide as a raw material, and has more excellent performance than the alkanolamide.

The present invention is based on the following findings: human TRPV1 receptor is over-expressed in SH-SY5Y cells, a human corneal neuron pain perception mechanism is simulated, the condition of calcium ions entering cells is detected by a green fluorescent calcium indicator Fluo-4 and AM to judge the activation condition of the human TRPV1 receptor, and the activation condition is used as the basis of a pain evaluation method and can be used for predicting whether cosmetic raw materials and products have potential pain-causing risks.

The invention is briefly carried out as follows (examples 1 to 5): firstly, a human TRPV1 receptor overexpression cell strain is obtained by gene total synthesis, lentivirus packaging and SH-SY5Y cell infection methods and puromycin screening, and the overexpression of the human TRPV1 gene is detected by an RT-qPCR method. Secondly, human TRPV1 overexpression cells are treated by dihydrocapsaicin, the activation of human TRPV1 receptors is detected by a fluorescence microscope and a fluorescence microplate reader, and a pain detection method is established. Finally, the detection of cosmetic raw materials (taking cocamide MEA, lauramide MEA, cocamide MIPA, palmitamide MEA and lactamide MEA as examples) and products (taking bath cream A and bath cream B as examples) proves that the detection method can be used for detecting the cosmetic raw materials and products and evaluating whether the cosmetic raw materials and products have pain-causing risks.

Meanwhile, as comparative examples 6 to 9, a detection method based on the rabbit TRPV1 receptor was established. Firstly, a rabbit TRPV1 receptor overexpression cell strain is obtained by gene total synthesis, lentivirus packaging and SH-SY5Y cell infection methods and puromycin screening, and the overexpression of the rabbit TRPV1 gene is detected by an RT-qPCR method. Secondly, the rabbit TRPV1 overexpression cell is treated by dihydrocapsaicin, the activation of the rabbit TRPV1 receptor is detected by a fluorescence microscope and a fluorescence microplate reader, and a pain evaluation method based on the rabbit TRPV1 receptor is established. Finally, by examining cosmetic raw materials (taking cocamide MEA as an example) and comparing with example 4, it was demonstrated that the sensitivity of the pain evaluation method based on human TRPV1 is higher than that of the pain evaluation method based on rabbit TRPV1 receptor, and that the pain evaluation method based on human TRPV1 receptor is better than that of the pain evaluation method based on rabbit TRPV 1.

In addition, as comparative example 10, the presence or absence of eye irritation of cocamide MEA was examined by chick embryo chorioallantoic membrane test. The results show that the chick embryo chorioallantoic membrane test detects the non-ocular irritation of the cocamide MEA. By comparison with example 4, it is further demonstrated that the pain evaluation method based on the human TRPV1 receptor is superior to the chick chorioallantoic membrane test in evaluating whether a substance has nociceptive properties.

In conclusion, the invention provides a pain evaluation method which has application value and strong sensitivity and is based on the human TRPV1 receptor overexpression cell strain. The detection method can be used for detecting cosmetic raw materials and products, and evaluating whether the cosmetic raw materials and products have pain risk.

The present invention will be further illustrated below with reference to specific examples and comparative examples. However, these examples and comparative examples are only illustrative of the present invention and are not to be construed as limiting the scope of the present invention. The experimental procedures of the following examples and comparative examples, in which specific conditions are not specified, are generally conducted under conventional conditions or conditions recommended by the manufacturers. All percentages and parts are by weight unless otherwise indicated.

The reagents, consumables, instruments and vendor information used in the following examples and comparative examples are as follows:

1. cells, consumables and reagents

Human neuroblastoma cell SH-SY5Y was purchased from Shanghai Yangyi Bio-technology Ltd; t75 cell culture flasks, black-edged clear bottom 96-well plates were purchased from Corning, USA. Fetal bovine serum, cell culture medium high-glucose DMEM, antibiotic cocktail (PSN), 0.25% pancreatin-EDTA were purchased from thermo fisher, usa.

Sodium chloride, potassium chloride, magnesium sulfate heptahydrate, calcium chloride, potassium dihydrogen phosphate, glucose and DMSO are purchased from chemical reagents of national medicine group, Inc.; HEPES was purchased from Sigma, USA. The cosmetic raw materials, namely lauramide MEA is purchased from Shanghai Arlatin Biotechnology Co., Ltd, the palmitamide MEA and the cocoamide DEA are purchased from Shanghai Mecline Biotechnology Co., Ltd, and the cocoamide MEA and the cocoamide MIPA are the reserved raw materials of the company.

Dihydrocapsaicin (DHC), Fluo-4 as a green fluorescent calcium indicator, AM, and TRIzol as a total RNA extraction reagent were purchased from ThermoFisher, USA; calcium channel inhibitor Ruthenium Red (RR) was purchased from shanghai mclin biochemical technologies, ltd; reverse transcription Kit iScript cDNA Synthesis Kit and fluorescent real-time quantitative PCR reagent iTaq^TM Universal

Green Supermix was purchased from Bio-rad, USA.

KRH buffer (Krebs-Ringer HEPES buffer) formulation: 125mM NaCl, 5mM KCl, 1.2mM MgSO₄×7H₂O，2mM CaCl₂，1.2mM KH₂PO₄×2H₂O，25mM HEPES[free acid]6mM glucose; the pH was adjusted to 7.4 with 1M NaOH.

2. Instrument for measuring the position of a moving object

Ultramicro ultraviolet spectrophotometric NanoDrop ONE, Fluoroskan Ascent FL as a fluorescence microplate reader are purchased from Thermo corporation, a gene amplification instrument C1000 Touch and a real-time quantitative PCR instrument CFX Connect are purchased from Bio-rad corporation, a fluorescence microscope DMIL LED and a body type microscope M205C are purchased from Leica corporation, and an incubator Rcom MARUmax.

Example 1: construction of human TRPV1 overexpression cell strain and detection of overexpression

First, experiment principle

Synthesizing human TRPV1 gene by a whole gene synthesis method, and inserting the human TRPV1 gene into a lentivirus expression vector to obtain an over-expression vector pCDH-CMV-Puro-hTRPV 1; through virus packaging, virus infection of SH-SY5Y cells and puromycin screening, a human TRPV1 receptor overexpression cell strain is obtained, and the cell strain TRPV1mRNA overexpression condition is detected by using an RT-qPCR method.

Second, Experimental methods

1. Construction of viral overexpression vector pCDH-CMV-Puro-hTRPV1 (human)

1 (specific nucleic acid sequence is shown later) containing a Coding sequence (CDS) region of a protein of a human TRPV1 gene, a base of a restriction endonuclease XbaI site TCTAGA at the 5 'end of the CDS region, a base of GCCACC constituting a Kozak sequence, and a base of a restriction endonuclease NotI site GCGGCCGC at the 3' end of the CDS region. Wherein, the nucleic acid sequence information of the human TRPV1 gene is from a nucleic acid sequence database of the National Center for Biotechnology Information (NCBI), and the nucleic acid sequence number is NM-080704.3 (nucleic acid sequence website: https:// www.ncbi.nlm.nih.gov/nuccore/NM-080704.3). Then, SEQ ID NO:1 was inserted into the viral vector pCDH-CMV-MCS-Puro using restriction endonucleases XbaI and NotI to give the viral overexpression vector pCDH-CMV-Puro-hTRPV 1. GCCACC bases are added in front of the initiation codon ATG of the human TRPV1 gene to form a Kozak sequence, so that the protein translation efficiency can be improved, and the protein expression level can be improved.

2. Construction of human TRPV1 receptor overexpression cell line

Mixing a virus expression vector pCDH-CMV-Puro-hTRPV1 and packaging plasmids pLP/VSVG and pCMV-dR8.91 according to the mass ratio of 20:15:6, transfecting HEK293FT cells by using a calcium phosphate method, harvesting supernate, and performing ultracentrifugation to concentrate viruses to obtain virus particles containing TRPV1 genes. SH-SY5Y cells are infected by virus particles and screened by puromycin with the concentration of 3 mu g/ml, and finally, a human TRPV1 receptor overexpression cell strain with stable expression is obtained.

3. Detecting the overexpression condition of TRPV1mRNA of a human TRPV1 receptor overexpression cell strain

Collecting empty vector control cell strain and human TRPV1 receptor overexpression cell strain cells, extracting total RNA by TIRzol reagent, and carrying out reverse transcription by using a reverse transcription Kit iScript cDNA Synthesis Kit to obtain cDNA. Real-time quantitative PCR reagent iTaq using fluorescence^TM Universal

Green Supermix was subjected to fluorescent real-time quantitative (RT-qPCR) detection, and human TRPV1 receptor hyperrepresentation was analyzedExpression of TRPV1mRNA in cell lines. The primer sequences used in this example were designed as follows: the sequence of an upstream primer of the human TRPV1 gene is 5'-TCCCGGTGGATTGCCCTCAC-3', and the sequence of a downstream primer is 5'-TCTCGGTGCTGGCGGCGACA-3'; ACTIN gene (. beta. -ACTIN) was used as an internal control, the sequence of the upstream primer was 5'-CACCATTGGCAATGAGCGGTTC-3', and the sequence of the downstream primer was 5'-AGGTCTTTGCGGATGTCCACGT-3'.

Third, experimental results

The real-time fluorescent quantitative detection result shows that when the human TRPV1 receptor overexpression cell strain is successfully constructed, the expression of TRPV1mRNA in the cell strain is obviously improved.

As shown in fig. 1, TRPV1mRNA in the human TRPV1 receptor overexpressing cell line was 26-fold higher than that of the empty vector control cell line. The empty vector control cell line is denoted "pCDH-ctr" and the human TRPV1 receptor overexpressing cell line is denoted "hTRPV 1".

Example 2: fluorescent microscope observation of activation of human TRPV1 receptor by dihydrocapsaicin in over-expressed cell strain

First, experiment principle

Fluo-4, AM is an acetyl methyl ester derivative of Fluo-4, is a fluorescent dye capable of penetrating cell membranes, and is commonly used for detecting the concentration of intracellular calcium ions. Fluo-4, AM penetrates the cell membrane and enters the cell, and is cleaved by intracellular esterase to form Fluo-4, which is then retained in the cell. Fluo-4 is almost non-fluorescent when present as a free ligand, but produces strong fluorescence when bound to intracellular calcium ions with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516 nm. Changes in intracellular calcium ion concentration can be detected using fluorescence microscopy, fluorescent microplate readers, and the like. The TRPV1 receptor is a non-selective ion channel, when the TRPV1 receptor is activated by dihydrocapsaicin and the like, extracellular calcium ions enter cells through the TRPV1 receptor and are combined with Fluo-4 in the cells, and green fluorescence is generated under the excitation of excitation light. Therefore, the activation of TRPV1 receptor can be analyzed and judged by observing and recording the change of fluorescence intensity in cells. Ruthenium red is a TRPV1 receptor inhibitor and inhibits calcium ions from entering cells through TRPV1 receptors.

Second, Experimental methods

1. Seeding cells

The empty vector control cell line and the cells of the human TRPV1 receptor-overexpressing cell line were seeded in 96-well plates, 6 wells per cell line, 1X 10 wells per well⁵(ii) individual cells; culturing in cell culture box for about 48 hr (culture temperature 37 deg.C, 5% CO)₂) The cell fusion degree reaches more than 95%.

2. Green fluorescent calcium indicator loaded cells

The old medium was removed and 50. mu.L of DMEM-loaded medium containing 2. mu.M Fluo-4, AM was added to each well and incubated at 37 ℃ for 30 minutes to allow Fluo-4, AM to enter the cells and hydrolyze sufficiently.

3. Adding KRH buffer solution containing no inhibitor ruthenium red (KRH buffer solution preparation method, see "cell, consumable and reagent")

The loading medium was decanted and rinsed once with 100. mu.L of KRH buffer, 100. mu.L of KRH buffer was added to 3 wells of 6 wells per cell, and 100. mu.L of KRH buffer containing 10. mu.M of the inhibitor ruthenium red was added to the other 3 wells, followed by incubation at 37 ℃ for 15 minutes.

4. Fluorescent microscope observation and photographing

20 μ L of dihydrocapsaicin (final concentration: 10 μ M) with a concentration of 60 μ M was added to each of empty vector control cell wells and human TRPV1 receptor-overexpressing cell wells, and after mixing, the fluorescence of each well was immediately observed under a fluorescence microscope and analyzed by photography.

Third, experimental results

The TRPV1 receptor in the human TRPV1 receptor over-expression cell strain is activated by high concentration dihydrocapsaicin, and mediates the internal flow of calcium ions.

As shown in FIG. 2, no green fluorescence signal was observed in neither the ruthenium red group nor the ruthenium red group of the empty vector control cells; a clear green fluorescent signal was observed in the no ruthenium red group of human TRPV1 receptor overexpressing cells, while no green fluorescent signal was observed in the ruthenium red group. This indicates that human TRPV1 receptor in human TRPV1 receptor overexpressing cells mediates calcium influx under activation by the high concentration of dihydrocapsaicin and that the receptor mediated calcium influx can be inhibited by the inhibitor ruthenium red. The empty vector control cell line is denoted "pCDH-ctr" and the human TRPV1 receptor overexpressing cell line is denoted "hTRPV 1".

Example 3: method for evaluating pain based on human TRPV1 receptor overexpression cell line by using different concentrations of dihydrocapsaicin

First, experiment principle

Second, Experimental methods

1. Seeding cells

The empty vector control cell line and the human TRPV1 receptor overexpressing cell line cells were seeded in 96-well plates with a clear bottom in black. For each test substance (dihydrocapsaicin), 1 96-well plate was inoculated per cell, 60 wells per plate (well plate numbers B2-B11, C2-C11, …, G2-G11), and 1X 10 wells per plate⁵For each cell, 100. mu.L of 1 XPBS buffer was added to wells around a 96-well plate to reduce the effect of liquid evaporation on cell proliferation. Culturing in cell culture box for about 48 hr (culture condition 37 deg.C, 5% CO)₂) The cell fusion degree is more than 95%.

2. Preparing dihydrocapsaicinoid-like substance

The dihydrocapsaicin mother liquor is prepared by DMSO solvent, and the concentration is 60 mM. 10 times of equal dilution solution is prepared by KRH buffer solution, and 8 concentration gradients are respectively 60pM, 600pM, 6nM, 60nM, 600nM, 6. mu.M, 60. mu.M and 600. mu.M from low to high. KRH buffer containing 10. mu.M of ruthenium red inhibitor was prepared.

3. Green fluorescent calcium indicator loaded cells

The old medium was removed and 50. mu.L of DMEM-loaded medium containing 2. mu.M Fluo-4, AM was added to each well and incubated at 37 ℃ for 25-30 minutes.

4. KRH buffer solution added with inhibitor-free/inhibitor-containing ruthenium red

The loading medium was decanted, rinsed once with 100. mu.L of KRH buffer, then 100. mu.L of KRH buffer was added to each of wells B2-B11, C2-C11 and D2-D11, and 100. mu.L of KRH buffer containing 10. mu.M inhibitor red ruthenium was added to each of wells E2-E11, F2-F11 and G2-G11, and incubated at 37 ℃ for 15 minutes in a cell incubator.

5. Data collected by fluorescent microplate reader

The 96-well plate was removed from the incubator, the lid was removed, and the plate was placed in a fluorescence microplate reader to measure the basal fluorescence value. mu.L of KRH buffer was then added immediately to the blank control wells (6 wells in column 2) using a multi-channel pipette, and 20. mu.L of different concentrations of dihydrocapsaicin (8 gradients from low to high at final concentrations of 10pM, 100pM, 1nM, 10nM, 100nM, 1. mu.M, 10. mu.M, 100. mu.M) were added sequentially to the assay wells (column 3-column 10, and 8 columns of 48 wells). After mixing, the fluorescence value of each well was measured immediately with a fluorescence microplate reader.

6. Data analysis

And (3) importing data collected by a fluorescence microplate reader into an Excel table, firstly calculating fluorescence difference values (namely reaction fluorescence value-basic fluorescence value) of each hole before and after adding a detection substance, then deducting blank control hole difference values from each hole of a detection group, taking the value of 10 mu M dihydrocapsaicin as 100%, and comparing the values of the holes with different concentrations of the detection group to obtain relative percentage. Nonlinear regression analysis was performed using GraphPad Prism 7 software, according to the quantity-effect equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) HillSlope)) (setting parameters Bottom 0 and Top 100) to obtainDose-response curve and EC₅₀The value is obtained. Linear regression analysis was performed on data that could not be fitted with a non-linear curve. The difference between the values of the test group (without inhibitor) and the test group (with inhibitor) at the same concentration was subjected to t-test, and each group was analyzed independently.

Third, experimental results

The pain evaluation method based on the human TRPV1 receptor overexpression cell line proves that the dihydrocapsaicin activates the human TRPV1 receptor in a concentration-dependent manner and has a dose-effect relationship, EC₅₀The value was 1.35 nM.

As shown in FIG. 3, panel A shows that no fluorescence signal was generated in the non-inhibitor ruthenium red group (pCDH-ctr) and the inhibitor ruthenium red group (pCDH-ctr + RR) after treatment of empty vector control cells with different concentrations of dihydrocapsaicin. Panel B shows that in the non-inhibitor ruthenium red group (hTRPV1), low concentrations (e.g., 10pM) of dihydrocapsaicin did not produce a fluorescent signal when treating human TRPV1 receptor overexpressing cells, and the human TRPV1 receptor was not activated; when human TRPV1 receptor-overexpressing cells were treated with higher concentrations (e.g., 10nM) of dihydrocapsaicin, a fluorescent signal was collected, indicating that human TRPV1 receptor was activated by dihydrocapsaicin, calcium influx from the extracellular space, producing a fluorescent signal, and had a concentration-dependent profile. No fluorescence was observed in the inhibitor added ruthenium red group (hTRPV1+ RR), indicating that human TRPV1 receptor was inhibited and calcium ions did not enter the cell. According to the analysis of GraphPad Prism 7 software, the corresponding quantity-effect relation curve and EC are obtained₅₀The value is obtained. Dihydrocapsaicin activates EC of human TRPV1 in human TRPV1 receptor overexpressing cell line₅₀The value was 1.35 nM. p value<0.05 indicates that the difference between the two groups of values is statistically significant, indicated by "+"; p value<0.01 indicates that the difference between the two sets of values is of high statistical significance, indicated by ". x".

Example 4: method for detecting cosmetic raw material based on pain evaluation method of human TRPV1 receptor overexpression cell strain

First, experiment principle

Second, Experimental methods

1. Seeding cells

Cells of the human TRPV1 receptor overexpressing cell line were seeded in 96-well plates with a black-edged clear bottom. For each test substance (5 cosmetic raw materials: Cocamide MEA, lauramide MEA, Cocamide MIPA, palmitamide MEA, and lactamide MEA), 1 96-well plate was inoculated, and 60-well plates (well plate numbers B2-B11, C2-C11, …, G2-G11) were inoculated per well, 1X 10⁵For each cell, 100. mu.L of 1 XPBS buffer was added to wells around a 96-well plate to reduce the effect of liquid evaporation on cell proliferation. Culturing in cell culture box for about 48 hr (culture condition 37 deg.C, 5% CO)₂) The cell fusion degree is more than 95%.

2. Preparing a sample to be tested

Preparing mother liquor of cocamide MEA, lauramide MEA, cocamide MIPA, palmitamide MEA and lactamide MEA by KRH buffer solution, wherein the concentration is 6mM, and the substances which are not easy to dissolve are accelerated to dissolve by ultrasound. Then, 3.16-fold equal dilution of the solution was prepared with KRH buffer, and 8 concentration gradients were set from low to high, respectively, at 1.9. mu.M, 6. mu.M, 19. mu.M, 60. mu.M, 190. mu.M, 600. mu.M, 1.9mM, and 6 mM. A positive control sample was prepared with 60. mu.M dihydrocapsaicin (KRH buffer), and KRH buffer containing 10. mu.M of ruthenium red inhibitor.

3. Green fluorescent calcium indicator loaded cells

The old medium was removed and 50. mu.L of DMEM-loaded medium containing 2. mu.M Fluo-4, AM was added to each well and incubated at 37 ℃ for 30 minutes.

5. Data collected by fluorescent microplate reader

The 96-well plate was removed from the incubator, the lid was removed, and the plate was placed in a fluorescence microplate reader to measure the basal fluorescence value. Then 20. mu.L of KRH buffer was immediately added to the blank control wells (6 wells in column 2) using a multi-channel pipette, and 20. mu.L of 60. mu.M dihydrocapsaicin (final concentration of 10. mu.M) was added to the positive control wells (6 wells in column 11). mu.L of 3.16 fold equal dilution samples (8 concentration gradients from low to high with final concentrations of 316nM, 1. mu.M, 3.16. mu.M, 10. mu.M, 31.6. mu.M, 100. mu.M, 316. mu.M, 1mM) were added sequentially to the assay wells (column 3-column 10, total 8 columns of 48 wells). After mixing, the fluorescence value of each well was measured immediately with a fluorescence microplate reader.

6. Data analysis

And (3) importing data collected by a fluorescence microplate reader into an Excel table, firstly calculating fluorescence difference values (namely reaction fluorescence value-basic fluorescence value) of each hole before and after adding a detection substance, then deducting blank control hole difference values from each hole of a detection group, taking the value of 10 mu M dihydrocapsaicin as 100%, and comparing the values of the holes with different concentrations of the detection group to obtain relative percentage. Nonlinear regression analysis was performed using GraphPad Prism 7 software, according to the quantity-effect equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) HillSlope)) (setting parameters Bottom 0 and Top 100) to obtain the quantity-effect curves and EC₅₀The value is obtained. Linear regression analysis was performed on data that could not be fitted with a non-linear curve. The number of the detection group (without inhibitor) and the number of the detection group (with inhibitor) at the same concentration are comparedValue differences were subjected to t-test and each group was analyzed independently.

Third, experimental results

Detecting cocamide MEA, lauramide MEA, cocamide MIPA, palmitamide MEA and lactamide MEA which are cosmetic raw materials based on a pain evaluation method of a human TRPV1 receptor overexpression cell strain, and finding that the activities of the cocamide MEA, the lauramide MEA and the cocamide MIPA and a human TRPV1 activator exist to cause gout risk; whereas palmitamide MEA and lactamide MEA do not produce a fluorescent signal and there is no pain-causing risk.

As shown in fig. 4A, in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR), a lower concentration (e.g., 1 μ M) of cocamide MEA treated human TRPV1 receptor overexpressing cells did not produce a fluorescent signal, and human TRPV1 receptor was not activated; when the human TRPV1 receptor overexpression cells are treated by the cocamide MEA with higher concentration (such as 100 mu M), the group without inhibitor ruthenium red (hTRPV1) generates fluorescence signals, and the group with inhibitor ruthenium red (hTRPV1+ RR) generates no fluorescence signals, which indicates that the human TRPV1 receptor is activated by the cocamide MEA, calcium ion influx is mediated, and the activation has concentration dependence. The quantity-effect relationship curve and EC of the cocamide MEA activated human TRPV1 receptor are obtained according to the analysis of GraphPad Prism 7 software₅₀The value was 34.37. mu.M.

As shown in fig. 4B, in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR), no fluorescence signal was generated and the human TRPV1 receptor was not activated when human TRPV1 receptor overexpressing cells were treated with lower concentrations (e.g., 1 μ M) of lauramide MEA; when the human TRPV1 receptor overexpression cells are treated by the lauramide MEA with higher concentration (such as 1000 mu M), the inhibitor-free ruthenium red group (hTRPV1) generates fluorescence signals, and the inhibitor-added ruthenium red group (hTRPV1+ RR) generates no fluorescence signals, which indicates that the human TRPV1 receptor is activated by the lauramide MEA, calcium ion influx is mediated, and the activation has concentration dependence. According to the analysis of GraphPad Prism 7 software, the quantity-effect relation curve and EC of the lauramide MEA for activating the human TRPV1 receptor are obtained₅₀The value 364. mu.M.

As shown in FIG. 4C, lower concentrations were found in the non-inhibitor ruthenium Red group (hTRPV1) and the inhibitor ruthenium Red group (hTRPV1+ RR)None of the cocamide MIPA (e.g., 1 μ M) produced a fluorescent signal when human TRPV1 receptor overexpressing cells, and the human TRPV1 receptor was not activated; when human TRPV1 receptor overexpression cells are treated by a higher concentration (such as 1000 mu M) of cocamide MIPA, the group without inhibitor ruthenium red (hTRPV1) generates a fluorescence signal, and the group with inhibitor ruthenium red (hTRPV1+ RR) generates no fluorescence signal, which indicates that the human TRPV1 receptor is activated by the cocamide MIPA, mediates calcium ion influx, and the activation has concentration dependence. According to the analysis of GraphPad Prism 7 software, the quantity-effect relation curve and EC of activating human TRPV1 receptor by cocamide MIPA are obtained₅₀The value was 145.3. mu.M.

As shown in FIG. 4D, no fluorescence signal was generated when different concentrations of palmitoyl-MEA treated human TRPV1 receptor overexpressing cells in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR). This indicates that the palmitoamide MEA does not have human TRPV1 activator activity, with no pain-causing risk.

As shown in fig. 4E, no fluorescence signal was generated when different concentrations of lactamide MEA treated human TRPV1 receptor overexpressing cells in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR). This indicates that lactamide MEA does not have human TRPV1 activator activity and there is no pain-causing risk.

In fig. 4, a p value <0.05 indicates that the difference between the two sets of values is statistically significant, indicated by "+"; a p value <0.01 indicates that the difference between the two sets of values is highly statistical and is indicated by an ".

Example 5: method for detecting shower gel product based on pain evaluation method of human TRPV1 receptor overexpression cell strain

First, experiment principle

Second, Experimental methods

1. Seeding cells

2. Preparing a sample to be tested

Preparation of shower gel A

Preparation of shower gel B

The KRH buffer solution is used for preparing the test mother liquor of the shower cream A and the shower cream B, and the concentration is 6 percent (mass to volume ratio). Then, KRH buffer solution is used to prepare 3.16 times of solution diluted in equal ratio, and 8 concentration gradients are respectively 0.0019%, 0.006%, 0.019%, 0.06%, 0.19%, 0.6%, 1.9% and 6% from low to high. A positive control sample was prepared with 60. mu.M dihydrocapsaicin (KRH buffer), and KRH buffer containing 10. mu.M of ruthenium red inhibitor.

3. Green fluorescent calcium indicator loaded cells

The loading medium was decanted, rinsed once with 100. mu.L of KRH buffer, then 100. mu.L of KRH buffer was added to each of wells B2-B11, C2-C11 and D2-D11, and 100. mu.L of KRH buffer containing 10. mu.M inhibitor red ruthenium was added to each of wells E2-E11, F2-F11 and G2-D11, and incubated at 37 ℃ for 15 minutes in a cell incubator.

5. Data collected by fluorescent microplate reader

The 96-well plate was removed from the incubator, the lid was removed, and the plate was placed in a fluorescence microplate reader to measure the basal fluorescence value. Then 20. mu.L of KRH buffer was immediately added to the blank control wells (6 wells in column 2) using a multi-channel pipette, and 20. mu.L of 60. mu.M dihydrocapsaicin (final concentration of 10M) was added to the positive control wells (6 wells in column 11). mu.L of 3.16 fold equal dilution samples (8 gradients from low to high with a final concentration of 0.000316%, 0.001%, 0.00316%, 0.01%, 0.0316%, 0.1%, 0.316%, 1%) were added sequentially to the assay wells (column 3-column 10, 8 columns of 48 wells). After mixing, the fluorescence value of each well was measured immediately with a fluorescence microplate reader.

6. Data analysis

And (3) importing data collected by a fluorescence microplate reader into an Excel table, firstly calculating fluorescence difference values (namely reaction fluorescence value-basic fluorescence value) of each hole before and after adding a detection substance, then deducting blank control hole difference values from each hole of a detection group, taking the value of 10 mu M dihydrocapsaicin as 100%, and comparing the values of the holes with different concentrations of the detection group to obtain relative percentage. Nonlinear regression analysis was performed using GraphPad Prism 7 software, according to the quantity-effect equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) HillSlope)) (setting parameters Bottom 0 and Top 100) to obtain the quantity-effect relationshipCurves and EC₅₀The value is obtained. Linear regression analysis was performed on data that could not be fitted with a non-linear curve. The difference between the values of the test group (without inhibitor) and the test group (with inhibitor) at the same concentration was subjected to t-test, and each group was analyzed independently.

Third, experimental results

The pain evaluation method based on the human TRPV1 receptor overexpression cell strain predicts that the shower gel A sample has no pain risk and the shower gel B sample has gout risk.

As shown in fig. 5A, in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR), the low concentration (e.g., 1 μ M) of body wash a sample treated human TRPV1 receptor overexpressing cells did not produce a fluorescent signal, and human TRPV1 receptor was not activated; when the human TRPV1 receptor overexpression cells are treated by a bath lotion A sample with higher concentration (such as 0.1% -1%), fluorescence signals are collected in the inhibitor-free ruthenium red group (hTRPV1) and the inhibitor-added ruthenium red group (hTRPV1+ RR), but no TRPV1 receptor specific fluorescence signals are generated. The pain evaluation method predicts that the shower gel a sample is not at risk of causing pain.

As shown in fig. 5B, in both the non-inhibitor ruthenium red group (hTRPV1) and the inhibitor ruthenium red group (hTRPV1+ RR), the low concentration (e.g., 1 μ M) of the shower gel B sample treated human TRPV1 receptor overexpressing cells did not produce a fluorescent signal, and the human TRPV1 receptor was not activated; when the human TRPV1 receptor overexpression cells are treated by a bath lotion B sample with higher concentration (such as 0.0316% -1%), fluorescence signals are collected from the inhibitor-free ruthenium red group (hTRPV1) and the inhibitor-added ruthenium red group (hTRPV1+ RR), and a fluorescence signal specific to the human TRPV1 receptor is generated in the inhibitor-free ruthenium red group (hTRPV 1). The pain evaluation method predicts the gout risk of the shower gel B sample.

In fig. 5, a p value <0.05 indicates that the difference between the two sets of values is statistically significant, indicated by "+"; a p value <0.01 indicates that the difference between the two sets of values is highly statistical and is indicated by an ".

Comparative example 6: construction of rabbit TRPV1 overexpression cell strain and detection of overexpression

First, experiment principle

Synthesizing a rabbit TRPV1 gene by a whole-gene synthesis method, and inserting the rabbit TRPV1 gene into a lentivirus expression vector to obtain an over-expression vector pCDH-CMV-Puro-oTRPV 1; the rabbit TRPV1 receptor overexpression cell strain is obtained by virus packaging, virus infection of SH-SY5Y cells and puromycin screening, and the cell strain TRPV1mRNA overexpression condition is detected by an RT-qPCR method.

Second, Experimental methods

1. Construction of viral overexpression vector pCDH-CMV-Puro-oTRPV1 (Rabbit)

2 (specific nucleic acid sequence shown below) containing the CDS region of rabbit TRPV1 gene, the base of restriction endonuclease XbaI site TCTAGA at 5 'end of CDS region and the base of GCCACC constituting Kozak sequence, and the base of restriction endonuclease NotI site GCGGCCGC at 3' end of CDS region. Among them, the nucleic acid sequence information of rabbit TRPV1 gene is from nucleic acid sequence database of the National Center for Biotechnology Information (NCBI) with nucleic acid sequence number NM-001082166 (nucleic acid sequence website: https:// www.ncbi.nlm.nih.gov/nuccore/NM-001082166). Then, the viral overexpression vector pCDH-CMV-Puro-oTRPV1 was obtained by inserting SEQ ID NO. 2 into the viral vector pCDH-CMV-MCS-Puro using restriction endonucleases XbaI and NotI. GCCACC bases are added in front of the ATG of the start codon of the rabbit TRPV1 gene to form a Kozak sequence, so that the protein translation efficiency can be improved, and the protein expression level can be improved.

2. Construction of rabbit TRPV1 receptor overexpression cell line

Mixing a virus expression vector pCDH-CMV-Puro-hTRPV1 and packaging plasmids pLP/VSVG and pCMV-dR8.91 according to the mass ratio of 20:15:6, transfecting HEK293FT cells by using a calcium phosphate method, harvesting supernate, and performing ultracentrifugation to concentrate viruses to obtain virus particles containing TRPV1 genes. SH-SY5Y cells are infected by virus particles, puromycin (puromycin) with the concentration of 3 mug/ml is used for screening, and finally, the rabbit TRPV1 receptor overexpression cell strain with stable expression is obtained.

3. Detecting the over-expression condition of TRPV1mRNA of a rabbit TRPV1 receptor over-expression cell strain

Collecting empty vector control cell line and rabbit TRPV1 receptor overexpression cell line cells, extracting total RNA by TIRzol reagent, and performing reverse transcriptionThe Kit iScript cDNA Synthesis Kit is reverse transcribed to obtain cDNA. Real-time quantitative PCR reagent iTaq using fluorescence^TM Universal

Green Supermix is used for carrying out fluorescence real-time quantitative (RT-qPCR) detection, and the overexpression condition of TRPV1mRNA in the rabbit TRPV1 receptor overexpression cell strain is analyzed. The primer sequences used in this comparative example were designed as follows: the sequence of the upstream primer of the rabbit TRPV1 gene is 5'-TCCCGGTGGATTGCCCTCAC-3', and the sequence of the downstream primer is 5'-TCTCGGTGCTGGCGGCGACA-3'; ACTIN gene (. beta. -ACTIN) was used as an internal control, the sequence of the upstream primer was 5'-CACCATTGGCAATGAGCGGTTC-3', and the sequence of the downstream primer was 5'-AGGTCTTTGCGGATGTCCACGT-3'.

Third, experimental results

The real-time fluorescent quantitative detection result shows that the rabbit TRPV1 receptor overexpression cell strain is successfully constructed, and the expression of TRPV1mRNA in the cell strain is obviously increased.

As shown in figure 1, TRPV1mRNA in the rabbit TRPV1 receptor overexpressing cell line was 77-fold higher than the empty vector control cell line. The empty vector control cell line is denoted "pCDH-ctr" and the rabbit TRPV1 receptor overexpressing cell line is denoted "oTRPV 1".

Comparative example 7: fluorescent microscope observation of rabbit TRPV1 receptor activated by dihydrocapsaicin in over-expressed cell strain

First, experiment principle

Second, Experimental methods

1. Seeding cells

Cells of the rabbit TRPV1 receptor-overexpressing cell line were seeded in 96-well plates at 1X 10 wells per well, 6 wells per cell line⁵(ii) individual cells; culturing in cell culture box for about 48 hr (culture temperature 37 deg.C, 5% CO)₂) The cell fusion degree reaches more than 95%.

2. Green fluorescent calcium indicator loaded cells

3. KRH buffer solution added with inhibitor-free/inhibitor-containing ruthenium red

4. Fluorescent microscope observation and photographing

20 μ L of dihydrocapsaicin (final concentration 10 μ M) with a concentration of 60 μ M was added to each of the empty vector control cell wells and the rabbit TRPV1 receptor-overexpressing cell wells, and after mixing, the fluorescence of each well was immediately observed under a fluorescence microscope and analyzed by photography.

Third, experimental results

The TRPV1 receptor in the rabbit TRPV1 receptor over-expression cell strain is activated by high-concentration dihydrocapsaicin, and mediates the inflow of calcium ions.

As shown in FIG. 2, no green fluorescence signal was observed in neither the ruthenium red group nor the ruthenium red group of the empty vector control cells; a clear green fluorescence signal was observed in the no ruthenium red group of rabbit TRPV1 receptor overexpressing cells, whereas no green fluorescence signal was observed in the ruthenium red group. This indicates that rabbit TRPV1 receptor in rabbit TRPV1 receptor overexpressing cells mediates calcium influx under activation by high concentrations of dihydrocapsaicin and that the receptor mediated calcium influx can be inhibited by the inhibitor ruthenium red. The empty vector control cell line is denoted "pCDH-ctr" and the rabbit TRPV1 receptor overexpressing cell line is denoted "oTRPV 1".

Comparative example 8: method for evaluating pain based on rabbit TRPV1 receptor overexpression cell line by using different concentrations of dihydrocapsaicin

First, experiment principle

Second, Experimental methods

1. Seeding cells

Cells of the rabbit TRPV1 receptor overexpressing cell line were seeded in 96-well plates with a black-edged clear bottom. For each test substance (dihydrocapsaicin), 1 96-well plate was inoculated per cell, 60 wells per plate (well plate numbers B2-B11, C2-C11, …, G2-G11), and 1X 10 wells per plate⁵Adding 100 μ L of 1 XPBS buffer solution into peripheral wells of 96-well plate, and reducing liquid volatilization to increase cell volumeThe effects of colonization. Culturing in cell culture box for about 48 hr (culture condition 37 deg.C, 5% CO)₂) The cell fusion degree is more than 95%.

2. Preparing dihydrocapsaicinoid-like substance

The dihydrocapsaicin mother liquor is prepared by DMSO solvent, and the concentration is 60 mM. The 10-fold equal dilution solution was prepared with KRH buffer, and the concentration gradient of 8 solutions was 190nM, 600nM, 1.9. mu.M, 6. mu.M, 19. mu.M, 60. mu.M, 190. mu.M, and 600. mu.M, respectively, from low to high. KRH buffer containing 10. mu.M of ruthenium red inhibitor was prepared.

3. Green fluorescent calcium indicator loaded cells

5. Data collected by fluorescent microplate reader

The 96-well plate was removed from the incubator, the lid was removed, and the plate was placed in a fluorescence microplate reader to measure the basal fluorescence value. mu.L of KRH buffer was then added immediately to the blank control wells (6 wells in column 2) using a multi-channel pipette, and 20. mu.L of different concentrations of dihydrocapsaicin (8 concentration gradients ranging from low to high at 31.6nM, 100nM, 316nM, 1. mu.M, 3.16. mu.M, 10. mu.M, 31.6. mu.M, 100. mu.M) were added sequentially to the assay wells (column 3-column 10, and 8 columns of 48 wells). After mixing, the fluorescence value of each well was measured immediately with a fluorescence microplate reader.

6. Data analysis

Introducing data collected by a fluorescence microplate reader into an Excel table, firstly calculating fluorescence difference values (namely reaction fluorescence value-basic fluorescence value) of each hole before and after adding a detection substance, then deducting blank control hole difference values from each hole of a detection group, taking the value of 10 mu M dihydrocapsaicin as 100%, and detecting the values of different concentration holes of the detection group and the values of different concentration holes of the detection groupThe relative percentages are obtained. Nonlinear regression analysis was performed using GraphPad Prism 7 software, according to the quantity-effect equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) HillSlope)) (setting parameters Bottom 0 and Top 100) to obtain the quantity-effect curves and EC₅₀The value is obtained. Linear regression analysis was performed on data that could not be fitted with a non-linear curve. The difference between the values of the test group (without inhibitor) and the test group (with inhibitor) at the same concentration was subjected to t-test, and each group was analyzed independently.

Third, experimental results

The pain evaluation method based on the rabbit TRPV1 receptor overexpression cell line proves that the dihydrocapsaicin activates the rabbit TRPV1 receptor in a concentration-dependent manner and has a dose-effect relationship, EC₅₀The value was 354.7 nM.

As shown in FIG. 8, it was revealed that fluorescence signals were collected when rabbit TRPV1 receptor overexpressing cells were treated with dihydrocapsaicin at a higher concentration (e.g., 1. mu.M) in the ruthenium red group without inhibitor (hTRPV1), indicating that the rabbit TRPV1 receptor was activated by dihydrocapsaicin and calcium ion influx in the extracellular space, generating fluorescence signals. No fluorescence was observed in the inhibitor added ruthenium red group (hTRPV1+ RR), indicating that the rabbit TRPV1 receptor was inhibited and calcium ions did not enter the cell. According to the analysis of GraphPad Prism 7 software, the corresponding quantity-effect relation curve and EC are obtained₅₀The value is obtained. Dihydrocapsaicin activates EC of rabbit TRPV1 in rabbit TRPV1 receptor overexpression cell line₅₀The value was 354.7 nM. p value<0.05 indicates that the difference between the two groups of values is statistically significant, indicated by "+"; p value<0.01 indicates that the difference between the two sets of values is of high statistical significance, indicated by ". x".

Compared with example 3, the sensitivity of the human TRPV1 overexpression receptor cell line to dihydrocapsaicin is higher than that of the rabbit TRPV1 overexpression receptor cell line, and the method is more suitable for a pain evaluation method.

Comparative example 9: method for detecting cocamide MEA (Mea-membrane electrode assembly) based on rabbit TRPV1 receptor overexpression cell strain pain evaluation method

First, experiment principle

Second, Experimental methods

1. Seeding cells

Cells of the rabbit TRPV1 receptor overexpressing cell line were seeded in 96-well plates with a black-edged clear bottom. For the test substance, cocamide MEA, 1 96-well plate was inoculated, 60 wells per well plate (well plate numbers B2-B11, C2-C11, …, G2-G11), 1X 10 wells per well⁵For each cell, 100. mu.L of 1 XPBS buffer was added to wells around a 96-well plate to reduce the effect of liquid evaporation on cell proliferation. Culturing in cell culture box for about 48 hr (culture condition 37 deg.C, 5% CO)₂) The cell fusion degree is more than 95%.

2. Preparing a sample to be tested

The mother liquor of cocamide MEA was prepared with KRH buffer solution at 6mM, and then 3.16-fold equal dilution was prepared with KRH buffer solution at 8 concentration gradients from low to high of 1.9. mu.M, 6. mu.M, 19. mu.M, 60. mu.M, 190. mu.M, 600. mu.M, 1.9mM, and 6mM, respectively. A positive control sample was prepared with 60. mu.M dihydrocapsaicin (KRH buffer), and KRH buffer containing 10. mu.M of ruthenium red inhibitor.

3. Green fluorescent calcium indicator loaded cells

5. Data collected by fluorescent microplate reader

6. Data analysis

And (3) importing data collected by a fluorescence microplate reader into an Excel table, firstly calculating fluorescence difference values (namely reaction fluorescence value-basic fluorescence value) of each hole before and after adding a detection substance, then deducting blank control hole difference values from each hole of a detection group, taking the value of 10 mu M dihydrocapsaicin as 100%, and comparing the values of the holes with different concentrations of the detection group to obtain relative percentage. Nonlinear regression analysis was performed using GraphPad Prism 7 software, according to the quantity-effect equation Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) HillSlope)) (setting parameters Bottom 0 and Top 100) to obtain the quantity-effect curves and EC₅₀The value is obtained. Linear regression analysis was performed on data that could not be fitted with a non-linear curve. The difference between the values of the test group (without inhibitor) and the test group (with inhibitor) at the same concentration was subjected to t-test, and each group was analyzed independently.

Third, experimental results

The method for evaluating pain based on rabbit TRPV1 receptor over-expression cell strain detects cocamide MEA as cosmetic raw material, and finds that the cocamide MEA has rabbit TRPV1 activator activity and EC₅₀Value 2251 μ M, there is a risk of gout.

As shown in FIG. 9, in both the non-inhibitor ruthenium red group (oTRPV1) and the inhibitor ruthenium red group (oTRPV1+ RR), the lower concentration (e.g., 1 μ M) of cocamide MEA treated rabbit TRPV1 receptor overexpressing cells did not produce a fluorescent signal and rabbit TRPV1 receptor was not activated; when rabbit TRPV1 receptor overexpression cells are treated by a higher concentration (such as 1000 muM) of cocamide MEA, the group without inhibitor ruthenium red (hTRPV1) generates a fluorescence signal, and the group with inhibitor ruthenium red (hTRPV1+ RR) generates no fluorescence signal, which indicates that the rabbit TRPV1 receptor is activated by the cocamide MEA, calcium ion influx is mediated, and the activation has concentration dependence. According to the analysis of GraphPad Prism 7 software, the quantity-effect relation curve and EC of the cocamide MEA activated rabbit TRPV1 receptor are obtained₅₀Value 2251 μ M.

Compared with example 4, the sensitivity of human TRPV1 overexpression receptor cell strain to cocamide MEA is higher than that of rabbit TRPV1 overexpression receptor cell strain, and the method is more suitable for pain evaluation method.

Comparative example 10: chick embryo chorioallantoic membrane test for testing eye irritation of Cocamide MEA

First, experiment principle

The chick embryo Chorioallantoic Membrane Test (Hen's Egg Test on the Chorioallonic Membrane, HET-CAM) is one method used to perform in vitro assessment of ocular irritability. The chorioallantoic membrane vascular system in the middle stage of chick embryo incubation is similar to the structure of eye conjunctiva tissue, and has the characteristics of completeness, clearness and transparency. In the test, a test subject is contacted directly with the chick embryo chorioallantoic membrane for a period of time, and the changes in chorioallantoic membrane toxicity effect indicators (e.g., bleeding, clotting, and vascular thawing) are observed and combined to produce a score that is used to assess ocular irritation of the test subject. The end-point method was used for the test.

Second, Experimental methods

1. Chick embryo incubation

After the white Lai Hangzhou chicken fertilized SPF (specific pathogen free) chick embryo is purchased, the chick embryo is placed in an incubator with the air chamber end facing upwards, the chick embryo is incubated under the conditions that the temperature is 37.5 +/-0.5 ℃ and the relative humidity is 55-70%, the chick embryo is automatically turned over for 3-6 times when the chick embryo is horizontally inclined for 45 degrees every hour, the chick embryo is automatically ventilated, and the chick embryo is used for testing when the chick embryo is incubated to the 9 th day.

2. Preparing a sample to be tested

0.9% physiological saline was prepared as a negative control sample. A1% solution of a mixture of sodium salts of fatty alcohol ether sulfates (Texapon ASV) was prepared in 0.9% physiological saline as a positive control sample. A stock solution of 2% cocamide MEA was prepared with KRH buffer, and then 0.06% cocamide MEA was prepared with 0.9% physiological saline as a sample to be tested. Then, the mixture was incubated in a 37 ℃ water bath for 15 minutes or more.

3. Preparation of chorioallantoic membrane

And (4) examining the 9-day-old chick embryo by photographing eggs, marking the positions of the air chambers on the surface of the eggshells, and stripping the positions of the marks of the eggshells by using tweezers to expose white egg membranes. The egg membranes were wetted with 1-2 ml of 0.9% saline by pipette and aspirated, and then carefully removed with forceps to ensure the integrity of the chorioallantoic membrane.

4. Test procedure

0.3 ml of the sample was dropped onto the CAM, ensuring that at least 50% of the CAM face was covered by the sample. After 3 minutes of action, the sample on the chorioallantoic membrane was gently rinsed with 0.9% physiological saline and observed after the completion of the rinsing within 30 seconds. At least 3 chick embryos per group were tested.

5. Observation of results

The extent of the change in hemorrhagic, thrombogenic and vasolytic toxic effects was observed and recorded. And taking a picture and storing the test picture.

Third, experimental results

Chick embryo chorioallantoic membrane testing showed no eye irritation at 0.06% of cocamide MEA.

As shown in FIG. 10, panel A shows no significant change in the chorioallantoic membrane before and after treatment with 0.9% saline, and no bleeding, clotting, and vasolytic changes in the chorioallantoic membrane were observed; panel B shows moderate bleeding of the chorioallantoic membrane following 1% ASV treatment; panel C shows no significant change in the chorioallantoic membrane after 0.06% cocamide MEA treatment, and no bleeding, clotting, and vasolysis of the chorioallantoic membrane were observed.

EC of Cocamide MEA in pain assessment method compared to example 4₅₀The concentration of the amount of material at 34.37 μ M, which is much lower than 0.06% of cocamide MEA in the chick embryo chorioallantoic membrane test at 2465.3 μ M, i.e. the concentration of cocamide MEA where no eye irritation was detected in the chick embryo chorioallantoic membrane test, resulted in a significant TRPV1 specific signal in the pain assessment method, predicting the risk of pain. This indicates that the pain assessment method based on the human TRPV1 overexpression receptor cell line has a clear advantage in predicting pain, whereas the chick chorioallantoic membrane test has a limitation in assessing pain. Therefore, the pain assessment method based on the human TRPV1 overexpression receptor cell line has higher application value in the aspect of predicting whether the substance has gout risk evaluation, and can be used for screening cosmetic formulations on a large scale and guiding milder cosmetic development.

Sequence listing

<110> Shanghai's family Joint sharps Ltd

<120> evaluation method of overexpression cell line based on human TRPV1 receptor

<130> 203078

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2540

<212> DNA

<213> Artificial sequence

<400> 1

tctagagcca ccatgaagaa atggagcagc acagacttgg gggcagctgc ggacccactc 60

caaaaggaca cctgcccaga ccccctggat ggagacccta actccaggcc acctccagcc 120

aagccccagc tctccacggc caagagccgc acccggctct ttgggaaggg tgactcggag 180

gaggctttcc cggtggattg ccctcacgag gaaggtgagc tggactcctg cccgaccatc 240

acagtcagcc ctgttatcac catccagagg ccaggagacg gccccaccgg tgccaggctg 300

ctgtcccagg actctgtcgc cgccagcacc gagaagaccc tcaggctcta tgatcgcagg 360

agtatctttg aagccgttgc tcagaataac tgccaggatc tggagagcct gctgctcttc 420

ctgcagaaga gcaagaagca cctcacagac aacgagttca aagaccctga gacagggaag 480

acctgtctgc tgaaagccat gctcaacctg cacgacggac agaacaccac catccccctg 540

ctcctggaga tcgcgcggca aacggacagc ctgaaggagc ttgtcaacgc cagctacacg 600

gacagctact acaagggcca gacagcactg cacatcgcca tcgagagacg caacatggcc 660

ctggtgaccc tcctggtgga gaacggagca gacgtccagg ctgcggccca tggggacttc 720

tttaagaaaa ccaaagggcg gcctggattc tacttcggtg aactgcccct gtccctggcc 780

gcgtgcacca accagctggg catcgtgaag ttcctgctgc agaactcctg gcagacggcc 840

gacatcagcg ccagggactc ggtgggcaac acggtgctgc acgccctggt ggaggtggcc 900

gacaacacgg ccgacaacac gaagtttgtg acgagcatgt acaatgagat tctgatgctg 960

ggggccaaac tgcacccgac gctgaagctg gaggagctca ccaacaagaa gggaatgacg 1020

ccgctggctc tggcagctgg gaccgggaag atcggggtct tggcctatat tctccagcgg 1080

gagatccagg agcccgagtg caggcacctg tccaggaagt tcaccgagtg ggcctacggg 1140

cccgtgcact cctcgctgta cgacctgtcc tgcatcgaca cctgcgagaa gaactcggtg 1200

ctggaggtga tcgcctacag cagcagcgag acccctaatc gccacgacat gctcttggtg 1260

gagccgctga accgactcct gcaggacaag tgggacagat tcgtcaagcg catcttctac 1320

ttcaacttcc tggtctactg cctgtacatg atcatcttca ccatggctgc ctactacagg 1380

cccgtggatg gcttgcctcc ctttaagatg gaaaaaactg gagactattt ccgagttact 1440

ggagagatcc tgtctgtgtt aggaggagtc tacttctttt tccgagggat tcagtatttc 1500

ctgcagaggc ggccgtcgat gaagaccctg tttgtggaca gctacagtga gatgcttttc 1560

tttctgcagt cactgttcat gctggccacc gtggtgctgt acttcagcca cctcaaggag 1620

tatgtggctt ccatggtatt ctccctggcc ttgggctgga ccaacatgct ctactacacc 1680

cgcggtttcc agcagatggg catctatgcc gtcatgatag agaagatgat cctgagagac 1740

ctgtgccgtt tcatgtttgt ctacatcgtc ttcttgttcg ggttttccac agcggtggtg 1800

acgctgattg aagacgggaa gaatgactcc ctgccgtctg agtccacgtc gcacaggtgg 1860

cgggggcctg cctgcaggcc ccccgatagc tcctacaaca gcctgtactc cacctgcctg 1920

gagctgttca agttcaccat cggcatgggc gacctggagt tcactgagaa ctatgacttc 1980

aaggctgtct tcatcatcct gctgctggcc tatgtaattc tcacctacat cctcctgctc 2040

aacatgctca tcgccctcat gggtgagact gtcaacaaga tcgcacagga gagcaagaac 2100

atctggaagc tgcagagagc catcaccatc ctggacacgg agaagagctt ccttaagtgc 2160

atgaggaagg ccttccgctc aggcaagctg ctgcaggtgg ggtacacacc tgatggcaag 2220

gacgactacc ggtggtgctt cagggtggac gaggtgaact ggaccacctg gaacaccaac 2280

gtgggcatca tcaacgaaga cccgggcaac tgtgagggcg tcaagcgcac cctgagcttc 2340

tccctgcggt caagcagagt ttcaggcaga cactggaaga actttgccct ggtccccctt 2400

ttaagagagg caagtgctcg agataggcag tctgctcagc ccgaggaagt ttatctgcga 2460

cagttttcag ggtctctgaa gccagaggac gctgaggtct tcaagagtcc tgccgcttcc 2520

ggggagaagt gagcggccgc 2540

<210> 2

<211> 2549

<212> DNA

<213> Artificial sequence

<400> 2

tctagagcca ccatgaagag atgggtgagc ttggactcgg gggaatctga ggacccgctt 60

ccagaagaca cctgcccaga cctcctggat ggagattcca acgccaagcc acctccagcc 120

aagccccaca tcttctccac ggccaagagc cgcagccggc tctttgggaa aggcgactca 180

gaggagacgt cccccatgga ttgctcttat gaggaaggcg aactggcccc ctgcccggcc 240

attacggtca gctctgtgat cattgtccag aggtctgggg atggccccac ctgtgccagg 300

cagctgtcgc aggactccgt ggcagccgcc ggcgccgaga agcccctgaa actctacgat 360

cgccggcgga tcttcgaggc cgtggcccag aacaactgcc aggagctgga gagcctgctg 420

tgcttcctgc agaggagcaa gaagcgcctg acggacagcg agttcaaaga ccctgagaca 480

gggaagacct gtctgctcaa agccatgctc aacctgcaca gcgggcaaaa cgacaccatc 540

ccgctgctcc tggagatcgc gcggcagacg gacagcctga aggagttcgt caacgccagc 600

tataccgaca gttactacaa gggccagaca gctctgcaca ttgccatcga gaggcggaac 660

atggcgctgg tgaccctcct ggtggagaac ggagcggatg tccaggctgc ggccaacggg 720

gacttcttta agaaaaccaa ggggcggcct ggcttctact ttggtgagct gcccctgtcc 780

ctggctgcgt gcaccaacca gctggccatc gtgaagttcc tgctgcagaa ctcctggcag 840

ccggcggaca tcagcgccag ggactcggtg ggcaacacgg tgctgcacgc cctggtggag 900

gtggccgaca acacccccga caacaccaag ttcgtgacga gcatgtacaa cgagatcctg 960

atcctggggg ccaaactcca ccccacgctg aagctggagg agctcatcaa taagaaaggg 1020

ctgacgccgc tggccctggc tgccggcagt gggaagattg gggtgctggc ctacattctg 1080

cagcgggaga tcctggagcc cgagtgccgg cacctgtccc ggaagttcac cgagtgggcc 1140

tacgggcctg tgcactcctc gctctacgac ctgtcctgca tcgacacctg cgagaggaac 1200

tccgtgctgg aggtgatcgc ctacagcagc agtgagaccc ctaatcgcca cgacatgctc 1260

ttggtggagc cactgaaccg actgctgcag gacaagtggg acagagttgt caagcgcatc 1320

ttctacttca acttcttcgt ctactgcctg tacatgatca tcttcaccac cgccgcctac 1380

tacaggcccg tggatggcct gcctccttat aagctgagaa acctccctgg agactatttc 1440

cgagtcactg gagagattct gtccgtagca gggggcgtct actttttttt ccgagggatc 1500

cagtatttcc tgcagaggcg gccgtccatg aaggccctgt ttgtggacag ctacagtgag 1560

atgctcttct tcgtgcaggc cctgttcatg ctggcgacgg tggtgctgta cttcagccac 1620

tgcaaggagt atgtggcgac catggtgttc tccctggcct tgggctggat caacatgctc 1680

tactacaccc gtgggttcca gcagatgggc atctacgccg tcatgatcga gaagatgatc 1740

ctgagggacc tgtgtcgttt catgtttgtc tacctggtgt tcttgttcgg cttttccaca 1800

gcggtggtga ctctgattga ggacgggaag aacagctcga cgtctgccga gtccacgtcg 1860

cacaggtggc gggggtttgg ctgtcggtcg tccgatagct cctacaacag cctgtactcc 1920

acatgcctgg agctgttcaa gttcaccatt ggcatgggcg acctggagtt caccgagaac 1980

tacgacttca aagccgtctt catcatcctg ctgctggcct acgtgattct cacctacatc 2040

ctcctgctca acatgctcat cgcgctcatg ggcgagacgg tcaacaagat cgcacaggag 2100

agcaagagca tctggaagct gcagagagct atcaccatcc tggacacaga gaagggcttc 2160

ctgaagtgca tgaggaaggc cttccgctcc ggcaagctgc tgcaggtggg ctacaccccc 2220

gacggcaagg acgactgcag gtggtgcttc agggtggacg aggtgaactg gaccacctgg 2280

aacaccaacg tgggcatcat caacgaggac ccgggcaact gtgagggcgt caagcgcacc 2340

ctgagcttct ccctgaggtc gggcagagtt tcagggagaa actggaagaa cttcgccttg 2400

gttccccttt taagggatgc cagtacccga gaccggcacc ccgcccctcc cgaggacgtc 2460

cacctgaggc cctttgtggg ctccctgaag ccgggggacg ccgagctctt caaggactct 2520

gtggccgcgg cagagaagtg agcggccgc 2549

Claims

1. A viral overexpression vector comprising the nucleotide sequence shown in SEQ ID No. 1 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID No. 1.

2. A cell comprising the viral overexpression vector of claim 1.

3. A method of assessing the pain causing risk of an agent or product, the method comprising the steps of:

4. The method of claim 3, wherein step (b) comprises mixing the viral overexpression vector pCDH-CMV-Puro-hTRPV1(SEQ ID NO:1) with the packaging plasmids pLP/VSVG and pCMV-dR8.91 and transfecting HEK293FT cells to produce viral particles containing the TRPV1 gene.

5. The method according to claim 3, wherein the step (b) further comprises puromycin screening to obtain a stably expressed human TRPV1 receptor overexpressing cell strain.

6. The method of claim 3, wherein step (c) comprises detecting intracellular calcium ion concentration based on a green fluorescent calcium indicator.

7. The method of claim 6, wherein the green fluorescent calcium indicator is Fluo-4, AM.

8. The method of claim 3, wherein said step (c) further comprises adding a TRPV1 receptor inhibitor.

9. The method of claim 8, wherein said TRPV1 receptor inhibitor is ruthenium red.

10. A method according to claim 3, wherein the reagent or product to be detected in step (d) is selected from: cosmetics or cosmetic raw materials.

11. The method of claim 10 wherein the cosmetic is a readily accessible to the eye and the cosmetic material is an alkanolamide based material.

12. The method of claim 11, wherein the alkanolamide-based starting material is cocamide MEA, lauramide MEA, cocamide MIPA, palmitamide MEA, lactamide MEA, or any combination thereof.