Human nasal mucosa epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide as well as preparation method and application

A technology of epithelial cells and inflammatory models, applied in the direction of artificial cell constructs, epidermal cells/skin cells, animal cells, etc., can solve the problems of unstable inflammatory models and high reducibility of nasal inflammatory responses, and achieve high reproducibility. The effect of excellent mold effect

Active Publication Date: 2021-06-29
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The model described in [0008] has high reducibility to nasal inflammation, and can more perfectly and truly evaluate the anti-inflammatory effect of nasal products and the treatment or symptom relief of chronic rhinitis Active substance screening experiment and mechanism research, and solved the instability problem of single lipopolysaccharide-induced inflammation model

Method used

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  • Human nasal mucosa epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide as well as preparation method and application
  • Human nasal mucosa epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide as well as preparation method and application
  • Human nasal mucosa epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide as well as preparation method and application

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Experimental program
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preparation example Construction

[0062] The present invention provides a kind of preparation method of human nasal mucosal epithelial cell inflammation model, which comprises the following steps:

[0063] Inoculating human nasal mucosal epithelial cells in culture medium to obtain adherent human nasal mucosal epithelial cells;

[0064] The culture medium was replaced, and Oncostatin M was added to cultivate the human nasal mucosal epithelial cells adhered to the wall, and then lipopolysaccharide was added for cultivation to obtain a human nasal mucosal epithelial cell inflammation model.

[0065] After obtaining the human nasal mucosal epithelial cell inflammation model, the mRNA expression level of claudin-4 of the HNEpC cells and the contents of the pro-inflammatory factors IL-β and TNF-α in the cell supernatant were determined.

[0066] The TNF-α is tumor necrosis factor-α, which is produced by many different cell types, such as monocytes, macrophages, T cells and B cells. TNF-α has many functions, includ...

Embodiment 1

[0085] Example 1 Effects of Different Concentrations of OSM Treatment on Cell Viability and Claudin-4 Expression

[0086] Set up blank control group and experimental group. Set 5 concentrations for concentration exploration. OSM was respectively set at 1ng / ml, 5ng / ml, 10ng / ml, 15ng / ml, and 30ng / ml. Adjust the concentration of human nasal epithelial cells to 1×10 with MEM medium containing 10% FBS 5 cells / mL, and seeded in 96-well plate, 100 μL per well, 37°C, 5% CO 2 Cultivate overnight in an incubator;

[0087] Add 100 μL of different concentrations of OSM diluted with serum-free MEM medium to each well of the experimental group, add the same amount of serum-free MEM medium to the blank control group, and store at 37°C, 5% CO 2 Continue to cultivate in the incubator for 6h, 12h, 24h.

[0088] (1) Detection of cell viability by WST-1 method: Discard the old culture medium, add 100 μL of serum-free medium and 10 μL of WST-1 (KG Biotech, product number: KGA316) into each we...

Embodiment 2

[0111] Example 2 10ng / ml OSM, different concentrations of lipopolysaccharide, 10ng / ml OSM combined with different concentrations of lipopolysaccharide, and 20ng / ml OSM combined with 80 μg / mL lipopolysaccharide have effects on cell viability, claudin-4 expression and cell neutralization Effect of TNF-α, IL-1β levels in cell culture supernatant

[0112] (1) For the effects of 10ng / ml OSM, different concentrations of lipopolysaccharide, 10ng / ml OSM combined with different concentrations of lipopolysaccharide, and 20ng / ml OSM combined with 80μg / mL lipopolysaccharide on the cell survival rate and the expression of claudin-4, specifically Operation method is with reference to embodiment 1, and the result of 24h is respectively as Figure 3-1 to Figure 3-2 shown.

[0113] (2) For 10ng / ml OSM, different concentrations of LPS, 10ng / ml OSM combined with different concentrations of LPS and 20ng / ml OSM combined with 80 μg / mL LPS to treat TNF-α in cells and cell culture supernatant, The ...

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Abstract

The invention discloses a human nasal mucosa epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide as well as a preparation method and application. The model is prepared by a method comprising the following steps: inoculating human nasal mucosa epithelial cells into a culture medium for culturing to obtain human nasal mucosa epithelial cells growing in an adherent manner; replacing the culture medium, adding oncostatin M to culture the human nasal mucosa epithelial cells growing in an adherent manner, and then adding lipopolysaccharide to culture to obtain the model. According to the invention, OSM and LPS are combined to serve as irritants to treat human nasal mucosa epithelial cells HNEpC for the first time, OSM serving as a cell factor can destroy a barrier function of the epithelial cells, so that LPS binds to an intracellular receptor of the LPS to initiate an inflammatory reaction, as an induction condition, to construct a more effective and stable in-vitro nasal cavity inflammation model; and the model can be used to more truly, comprehensively and accurately reflect screening of nasal cavity medicine raw materials and products and evaluation of anti-inflammatory effects.

Description

technical field [0001] The invention relates to the technical field of safety and efficacy detection of nasal cavity care products, in particular to a human nasal epithelial cell inflammation model induced by oncostatin M combined with lipopolysaccharide, a preparation method and an application. Background technique [0002] Chronic rhinitis is chronic inflammation of nasal mucosa and submucosa. Its main feature is that the inflammation lasts for more than three months or recurs repeatedly, protractedly, and cannot return to normal during the intermittent period, and there is no clear pathogenic microorganism, accompanied by varying degrees of nasal congestion, increased secretions, and swelling or thickening of the nasal mucosa and other obstacles. According to the degree of pathology and dysfunction of chronic rhinitis, it can be divided into chronic simple rhinitis and chronic hypertrophic rhinitis. Even chronic nasal inflammation characterized by bone limitations or di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0625G01N33/5008C12N2501/90C12N2501/2306
Inventor 谢文娟张晓鸥毛华李芬郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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