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Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte

A technology of melanocytes and whitening agent, applied in the field of toxicology and efficacy experiments, can solve the problems of high cost, immaturity, and unstable melanin expression.

Inactive Publication Date: 2009-09-16
程树军 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing in vitro methods have their shortcomings: usually, the expression of melanin is not stable enough, and there are certain differences in morphology and genes between tumor or animal cells and normal human cells
[0005] The development of tissue-engineered skin containing melanocytes is another way to evaluate the toxicity and efficacy of whitening. For example, in 2003, Yoon T.J. et al. added isolated and cultured primary melanocytes to tissue-engineered skin to construct artificial skin containing melanocytes. Skin (Yoon T.J., Lei T.C., Yamaguchi Y., Batzer J., Wolber R., Hearing V.J., Anal.Biochem., 318, 260-269 (2003)), but this engineered skin containing melanocytes has not yet Mature, expensive and difficult to cultivate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] I. Culture and Identification of Human Skin Primary Melanocytes

[0062] (1) Configuration of human skin melanocyte culture medium

[0063] 1). Preparation of human melanocyte culture medium:

[0064]A. Complete culture solution: Mix keratinocyte serum-free medium K-SFM (Keratinocyte Serum-Free Medium) with fetal calf serum at a volume ratio of 9:1, and add the following cofactors: Hydrocortisone (Hydrocortisone), and its final The concentration is 0.4ug / ML; bovine insulin (insuilin), its final concentration is 10ug / ML; L-glutamine (L-glutamine), its final concentration is 6mmol; 12-o-tetradecanoyl phorbol acetate -13 (abbreviated as TPA, English name 12-o-tetradecanoylphorbol-13-acetate), its final concentration is 81.06nmol / L; 3-isobutyl-1-methylxanthine (abbreviated as IBMX, namely 3-isobutyl- l-methylxanthine), its final concentration is 0.1nmol / L; transferrin (transferrin), its final concentration is 10ug / ML; cholera toxin (cholera toxin, CT), its final concentra...

Embodiment 2

[0093] I. Culture and Identification of Human Skin Primary Melanocytes

[0094] (1) Configuration of human skin melanocyte culture medium

[0095] 1) Preparation of human melanocyte culture medium:

[0096] A. Complete culture solution: Mix keratinocyte serum-free medium K-SFM (Keratinocyte Serum-Free Medium) with fetal bovine serum at a volume ratio of 9:1, and add the following cofactors: hydrocortisone (Hydrocortisone), and its final Concentration is 0.4ug / ML; Bovine insulin (insuilin), its final concentration is 10ug / ML; L-glutamine (L-glutamine), its final concentration is 6mmol; 12-o-tetradecanoyl phorbol acetate -13 (abbreviated as TPA, English name 12-o-tetradecanoylphorbol-13-acetate), its final concentration is 81.06nmol / L; 3-isobutyl-1-methylxanthine (abbreviated as IBMX, namely 3-isobutyl- l-methylxanthine), its final concentration is 0.1nmol / L; transferrin (transferrin), its final concentration is 10ug / ML; cholera toxin (choleratoxin, CT), its final concentratio...

Embodiment 3

[0125] I. Culture and Identification of Human Skin Primary Melanocytes

[0126] (1) Configuration of human skin melanocyte culture medium

[0127] 1) Preparation of human melanocyte culture medium:

[0128] A. Complete culture solution: Mix keratinocyte serum-free medium K-SFM (Keratinocyte Serum-Free Medium) with fetal bovine serum at a volume ratio of 9:1, and add the following cofactors: hydrocortisone (Hydrocortisone), and its final The concentration is 0.4ug / ML; bovine insulin (insuilin), its final concentration is 10ug / ML; L-glutamine (L-glutamine), its final concentration is 6mmol; 12-o-tetradecanoyl phorbol acetate -13 (abbreviated as TPA, English name 12-o-tetradecanoylphorbol-13-acetate), its final concentration is 81.06nmol / L; 3-isobutyl-1-methylxanthine (abbreviated as IBMX, namely 3-isobutyl- l-methylxanthine), its final concentration is 0.1nmol / L; transferrin (transferrin), its final concentration is 10ug / ML; cholera toxin (choleratoxin, CT), its final concentr...

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PUM

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Abstract

The invention relates to an experimental method for analyzing the toxicity and the effect of a skin whitening agent through a primary culture human being skin melanocyte. The method comprises the following steps: 1, culturing and identifying the primary human being skin melanocyte; 2, carrying out the analysis of the toxicity of skin whitening substances through the primary human being skin melanocyte; and 3, carrying out the analysis of the effect of the skin whitening substances through the primary human being skin melanocyte, namely carrying out the analysis on two aspects of the melanin content and the tyrosinase activity. The method has the following advantages that: the human being melanocyte cultured in vitro has a high division and multiplication capacity, a high standardization degree, little difference between batches, and the same function of producing melanin granules with the melanocyte in vivo; the result obtained through the healthy human being melanocyte is more reliable than the result obtained through animal or human-derived melanoma cells; and the toxic action and the whitening effect of the substances to be tested are evaluated from the two aspects of characterization and quantification. The method can replace the living body animal and human being skin, and can be directly applied to toxicity and effect experiments of the whitening substances in products such as chemicals, cosmetics, medicaments, and the like.

Description

technical field [0001] The invention relates to a method of using primary cultured human skin melanocytes for toxicity analysis and efficacy analysis of whitening agents, which can replace living animal (or human) skin and neoplastic melanocytes, and can be applied to whitening chemical raw materials or containing whitening agents Experimental field of toxicology and efficacy of cosmetic formulations. Background technique [0002] The skin is the largest organ of the human body, and it is the main organ for the toxic effects of exogenous chemical factors (chemicals, drugs, cosmetics, etc.) or physical and mechanical factors (ultraviolet rays, microwaves, etc.). Skin toxicity testing and efficacy testing using animal or human skin are routine testing items for health and safety evaluation of cosmetics, pharmaceuticals, food additives and raw materials, pesticides, biological products, and chemicals. Traditional skin safety evaluation experiments not only require a certain nu...

Claims

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Application Information

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IPC IPC(8): G01N21/31C12Q1/18
Inventor 程树军
Owner 程树军
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