Application of Hedgehog signal channel gene and regulator in regulating physiological functions of airway epithelial cells

A technology of airway epithelial cells and signaling pathways, applied in the identification of gene function and small molecule function, in the field of biomedicine

Pending Publication Date: 2022-05-27
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genes involved in the Hh signaling pathway, and the role of inhibitors or activators of this signaling pathway in airway epithelial regeneration and mucus secretion are unknown.

Method used

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  • Application of Hedgehog signal channel gene and regulator in regulating physiological functions of airway epithelial cells
  • Application of Hedgehog signal channel gene and regulator in regulating physiological functions of airway epithelial cells
  • Application of Hedgehog signal channel gene and regulator in regulating physiological functions of airway epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] This example intends to explore the role of the Hedgehog signaling pathway ligand gene Shh in regulating the differentiation of mouse airway epithelial cells. The specific test methods are as follows:

[0026] Nkx2.1Cre; Shhflox / + mice (referred to as E18.5 control) and airway epithelium-specific knockout Shh mice (Nkx2.1Cre; Shhflox / flox, referred to as ShhCKO) were used to stain the frozen sections of mouse airway tissues. Methods as below:

[0027] Trachea and lungs of E18.5 control and ShhCKO mice were dissected in PBS, fixed in 4% paraformaldehyde overnight at 4 °C, incubated in 10% sucrose and 30% sucrose for 24 h, and the tissues were embedded in OCT. 10 μm tissue sections were made using a cryostat. During antibody immunostaining, sections were fixed in 4% paraformaldehyde at 4°C for 10 minutes, then incubated in permeabilization solution (0.3% Triton X-100 / PBS) for 15 minutes at room temperature, blocking solution (5% FBS / Incubate in PBS), block for 1 hour a...

Embodiment 2

[0032] This example intends to explore the role of the Hedgehog signaling pathway signal transduction gene Smo in regulating the differentiation of mouse airway epithelial cells. The specific test methods are as follows:

[0033] Nkx2.1Cre; Smoflox / + mice (referred to as E18.5 control) and airway epithelium-specific knockout Smo mice (Nkx2.1Cre; Smoflox / flox, referred to as SmoCKO) were used to stain the frozen sections of mouse airway tissues. Methods as below:

[0034] Trachea and lungs of E18.5 control and SmoCKO mice were dissected in PBS, fixed in 4% paraformaldehyde overnight at 4 °C, incubated in 10% sucrose and 30% sucrose for 24 h, and the tissues were embedded in OCT. 10 μm tissue sections were made using a cryostat. During antibody immunostaining, sections were fixed in 4% paraformaldehyde at 4°C for 10 minutes, then incubated in permeabilization solution (0.3% Triton X-100 / PBS) for 15 minutes at room temperature, blocking solution (5% FBS / Incubate in PBS), block...

Embodiment 3

[0039] This example intends to explore cyclopamine as a Hedgehog signaling pathway inhibitor and its role in regulating airway epithelial cell differentiation, mucus secretion of MUC5AC and MUC5B, and to explore human SHH recombinant protein (H-SHH recombinant protein) as Hedgehog signaling pathway activation The specific test methods are as follows:

[0040] (1) Air-liquid differentiation culture of human airway epithelial cells

[0041] at 37°C with 5% CO 2 Human airway epithelial primary cells (HBE) were cultured and expanded in T75 flasks to a density of After 80%, cells were isolated by digestion with 0.05% trypsin-EDTA (Gibco) and seeded on calf skin collagen (Sigma-Aldrich) coated AEGM membrane supports (12 mm Transwell, 0.4 μm pore size, Costar). HBE cells were cultured for two days until the intercellular space was closed. The apical medium was removed, and DMEM (Sigma) medium and AEGM medium supplemented with 60 ng / ml retinoic acid (Sigma) were mixed 1:1 and added...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a Hedgehog signal channel gene and a regulator in regulating physiological functions of airway epithelial cells. The invention verifies that the Hedgehog signal channel gene Shh or Smo gene is inactivated to cause airway epithelial cell dysfunction for the first time, and further verifies that Cyclopamine or GANT 58 can be used as the Hedgehog signal channel inhibitor to inhibit the generation of epithelial ciliary cells and goblet cells and reduce mucus secretion, and also verifies that the Cyclopamine or GANT 58 can be used as the Hedgehog signal channel inhibitor to inhibit the generation of the epithelial ciliary cells and goblet cells. The H-SHH recombinant protein can be used as a Hedgehog signaling pathway activator to promote the excessive generation of airway epithelial cilia cells and goblet cells and increase mucus secretion. The invention requests to protect the application of the Hedgehog signal channel gene and the regulator in regulating the physiological function of airway epithelial cells, which is of great significance in diagnosis and treatment of lung diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the field of gene function and small molecule function identification, and particularly relates to the application of Hedgehog signaling pathway genes and regulators in regulating the physiological functions of airway epithelial cells. Background technique [0002] The airway epithelium is a major target of lung disease, providing pathways for gas exchange between the environment and the lung lobes. Abnormal airway epitheligenesis and mucus secretion are implicated in various diseases of the lung, such as asthma and chronic obstructive pulmonary disease, both of which are often accompanied by hyperdifferentiation of mucus-secreting goblet cells in the airway epithelium and hypersecretion of mucus . However, little is known about the cellular and molecular mechanisms underlying airway epithelialization and mucus secretion. [0003] In animals, Hedgehog (Hh) signaling can regulate tiss...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/58A61K31/444A61K38/19A61P11/00
CPCA61K45/00A61K31/58A61K31/444A61K38/19A61P11/00
Inventor 殷文广
Owner GUANGZHOU MEDICAL UNIV
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