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Evaluation method of overexpression cell strain based on human TRPV1 receptor

A technology of overexpression and overexpression vector, applied in the direction of receptors/cell surface antigens/cell surface determinants, chemical instruments and methods, botany equipment and methods, etc., can solve problems such as inconsistent capsaicin responses

Pending Publication Date: 2021-11-19
SHANGHAI JAHWA UNITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The human and rabbit TRPV1 receptor protein sequences in mammals are highly homologous, but the amino acid residues in the key binding site of capsaicin in the transmembrane helix TM3 / 4 differ, which leads to the response of different species of TRPV1 receptors to capsaicin inconsistent

Method used

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  • Evaluation method of overexpression cell strain based on human TRPV1 receptor
  • Evaluation method of overexpression cell strain based on human TRPV1 receptor
  • Evaluation method of overexpression cell strain based on human TRPV1 receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Construction of human TRPV1 overexpression cell line and detection of overexpression

[0061] 1. Experimental principle

[0062]The human TRPV1 gene was synthesized by the whole gene synthesis method and inserted into the lentiviral expression vector to obtain the overexpression vector pCDH-CMV-Puro-hTRPV1; the human TRPV1 was obtained by virus packaging, virus infection of SH-SY5Y cells and puromycin selection TRPV1 mRNA overexpression in the cell line was detected by RT-qPCR method.

[0063] 2. Experimental method

[0064] 1. Construction of virus overexpression vector pCDH-CMV-Puro-hTRPV1 (human)

[0065] Synthesize SEQ ID NO:1 (see below for specific nucleic acid sequence) by whole gene synthesis method, this sequence contains human TRPV1 gene protein coding sequence (Coding sequence, CDS) region, CDS region 5' endonuclease XbaI site TCTAGA base base and GCCACC base constituting the Kozak sequence, and the 3' endonuclease NotI site base GCGGCCGC in the...

Embodiment 2

[0073] Example 2: Fluorescent microscope observation of human TRPV1 receptor activation by dihydrocapsaicin in overexpressed cell lines

[0074] 1. Experimental principle

[0075] Fluo-4, AM is an acetyl methyl ester derivative of Fluo-4, a fluorescent dye that can penetrate cell membranes, and is often used to detect intracellular calcium ion concentration. After Fluo-4, AM penetrates the cell membrane and enters the cell, it is cleaved by intracellular esterase to form Fluo-4, which is retained in the cell. Fluo-4 is almost non-fluorescent in the form of free ligand, but it can produce strong fluorescence when combined with intracellular calcium ions, with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516nm. Fluorescence microscope, fluorescence microplate reader, etc. can be used to detect the change of intracellular calcium ion concentration. The TRPV1 receptor is a non-selective ion channel. When the TRPV1 receptor is activated by substan...

Embodiment 3

[0088] Example 3: Using different concentrations of dihydrocapsaicin to evaluate the pain evaluation method based on human TRPV1 receptor overexpression cell line

[0089] 1. Experimental principle

[0090] Fluo-4, AM is an acetyl methyl ester derivative of Fluo-4, a fluorescent dye that can penetrate cell membranes, and is often used to detect intracellular calcium ion concentration. After Fluo-4, AM penetrates the cell membrane and enters the cell, it is cleaved by intracellular esterase to form Fluo-4, which is retained in the cell. Fluo-4 is almost non-fluorescent in the form of free ligand, but it can produce strong fluorescence when combined with intracellular calcium ions, with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516nm. Fluorescence microscope, fluorescence microplate reader, etc. can be used to detect the change of intracellular calcium ion concentration. The TRPV1 receptor is a non-selective ion channel. When the TRPV1 recep...

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Abstract

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

Description

technical field [0001] The present invention relates to the field of safety evaluation of cosmetics and their ingredients, and is actually an eye irritation evaluation method, specifically a method for evaluating whether there is a risk of pain in cosmetic raw materials and eye-touching cosmetics based on human TRPV1 receptor overexpression cell lines method. Background technique [0002] Cosmetics are one of the chemicals commonly used by people, and there are many types, such as hair care, skin care, make-up, sunscreen, and freckle removal. Some cosmetics may contact people's eyes during use, and cause certain irritation and discomfort to the eyes, causing tears, conjunctival hyperemia, and / or subjective feelings such as itching and pain. "Toxicological Experimental Methods" in my country's "Safety and Technical Specifications for Cosmetics" (2015 Edition) stipulates that under normal circumstances, before a newly developed cosmetic product is put on the market, correspon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/12C12N5/10C12Q1/02C12Q1/6851G01N21/64
CPCC12N15/86C07K14/705G01N33/5014G01N33/5058G01N33/5044G01N21/6428C12Q1/6851C12Q2531/113C12Q2521/107C12Q2563/107C07K14/435C12N5/10C12N15/867C12Q1/02G01N21/64
Inventor 殷庆飞陈媛祺陈田
Owner SHANGHAI JAHWA UNITED
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