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Construction method of barrier function weakening model

A technology for constructing a method and a model, which is applied in the field of constructing a barrier function weakening model, can solve the problems of increased degradation rate of Loricrin protein and difficult to detect, and achieves the effect of shortening model differentiation time, improving proliferation and differentiation, and promoting proliferation and differentiation.

Active Publication Date: 2020-01-14
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, studies have also found that Loricrin-derived polypeptides can be detected in diabetic or aging human and animal skin, but it is difficult to detect in healthy young people, indicating that the degradation rate of Loricrin protein increases with age. will also increase

Method used

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  • Construction method of barrier function weakening model
  • Construction method of barrier function weakening model
  • Construction method of barrier function weakening model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] In order to provide a method for testing the safety and efficacy of baby skin care products and cosmetics with skin barrier repairing effects; the present invention provides a method for constructing a model of barrier function weakening, comprising the following method steps:

[0033] Step 1. Cell preparation

[0034] The original epidermal cells were taken, recovered and expanded, and the P4-P20 generation cells were used, digested with trypsin to make a single-cell suspension, and the cell density was adjusted to 5.0×10 5 pcs / mL~5.0×10 6 cells / mL, inoculated in a culture bottle, added FAD culture solution, gently shook the culture bottle to make the cells evenly dispersed, and placed at 37°C, 5% CO 2 cultivated under conditions;

[0035] The FAD culture solution is prepared by mixing F12 and culture solution DMEM at a volume ratio of 1:3.

[0036] Step 2. Submerged inoculation and culture of the weakened barrier function model

[0037] Take cells in the logarithm...

Embodiment 2

[0056] This example focuses on the in vitro construction method steps of the barrier function weakening model constructed in vitro using primary epidermal cells:

[0057] Step 1. Cell preparation

[0058] Take the original epidermal cells, recover and expand, use the P7 generation cells, digest with trypsin to make a single cell suspension, and adjust the cell density to 5.0×10 5 cells / mL, inoculated in a culture bottle, added FAD culture solution, gently shook the culture bottle to make the cells evenly dispersed, and placed at 37°C, 5% CO 2 cultivated under conditions;

[0059] The FAD culture solution is prepared by mixing F12 and culture solution DMEM at a volume ratio of 1:3.

[0060] Step 2. Submerged inoculation and culture of the weakened barrier function model

[0061] Take cells in the logarithmic growth phase, make a single cell suspension with culture medium I, and adjust the cell density to 5×10 5 cells / mL, with a volume of 200 μL / chamber, inoculated in the sm...

Embodiment 3

[0071] This example focuses on the use of the weakened barrier function model constructed by primary epidermal cells in vitro for ET 50 Detection steps:

[0072] 1) Put the model into a 6-well plate, and add 0.9 mL of culture medium into the well plate.

[0073] 2) Aspirate 80 μL of 1.0% Triton X-100, and slowly drop it on the surface of the tissue to ensure that the reagent can cover the surface of the tissue as much as possible.

[0074] 3) The administration time is 0, 1, 2 hours respectively.

[0075] 4) After the last piece of tissue is administered, transfer all 6-well plates to a constant temperature incubator (37±1°C, 5±1% CO 2 , 95% relative humidity).

[0076] 5) 30 minutes before the end of tissue incubation, prepare 1 mg / mL MTT solution, and add 300 μL of MTT solution to each well of the 24-well plate.

[0077] 6) After the drug administration, start the washing procedure, use a washing bottle containing sterile DPBS to wash the tissue, after washing 10 times, ...

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Abstract

The invention relates to a construction method of a barrier function weakening model. The construction method comprises the following steps: step 1, preparation of cells; step 2, inoculation and culture of the barrier function weakening model under liquid; step 3, gas-liquid surface proliferation and differentiation culture of the barrier function weakening model; and step 4, gas-liquid surface differentiation inhibition culture of the barrier function weakening model. The barrier function weakening model can well replace an animal model, becomes a core tool for in-vitro testing, and can be applied to in-vitro testing of safety and efficacy of products such as infant skin care products, cosmetics with human skin barrier repairing efficacy and the like.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering biomaterials, and in particular relates to a method for constructing a barrier function weakening model. Background technique [0002] The skin barrier plays a major role in the skin's functions of resisting the external environment, maintaining the homeostasis of the internal environment, and preventing skin moisture loss. The structural basis of the skin barrier is the "brick-and-gray" structure of the stratum corneum. The "brick" structure is mainly composed of stratum corneum cells and intracellular protein envelopes, the "gray" structure is mainly composed of lipid encapsulation, and the essence of the "brick-gray" structure is the intersection of cuticle protein envelopes and lipid envelopes. It is the basis for the barrier function of the stratum corneum. Its functional barrier is mainly composed of three parts: ① transmembrane proteins and intramembrane proteins of keratinocyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0625G01N33/5008G01N33/5044C12N2500/84C12N2501/11C12N2500/40C12N2500/24C12N2501/117C12N2501/39C12N2500/38C12N2501/999C12N2500/14C12N2501/727C12N2503/00
Inventor 何欣胡成虎卢永波刘文佳张勇杰
Owner GUANGDONG BOXI BIO TECH CO LTD
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