Application of PAX6 gene or expression product thereof to preparation of medicines for restraining fibrosis

A technology for expressing products and gene expression, applied in the field of genes

Active Publication Date: 2020-05-29
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether PAX6 can inhibit cardiac fibrosis and become a new target for clinical

Method used

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  • Application of PAX6 gene or expression product thereof to preparation of medicines for restraining fibrosis
  • Application of PAX6 gene or expression product thereof to preparation of medicines for restraining fibrosis
  • Application of PAX6 gene or expression product thereof to preparation of medicines for restraining fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Animal pathological model experiments. A mouse cardiac fibrosis model was constructed, and cardiac tissue was collected. Real-time quantitative PCR and Western blot experiments were used to detect the expression of PAX6 mRNA and protein.

[0025]Preparation of angiotensin II-induced cardiac fibrosis model in mice: 10-week-old male C57BL / 6 mice were randomly divided into two groups, the operation group and the sham operation group, and the mice were treated with angiotensin (3 mg·kg-1· day-1) A micro-osmotic pump buried pump (Alzet MODEL 1007D, DURECT, Cupertino, CA) was used to construct the fibrosis model for 7 days. Preparation of the micro-osmotic pressure pump: 1 day before the operation, inject angiotensin II (dissolved in sterile PBS buffer) into the micro-osmotic pump with a 1mL syringe, soak the micro-osmotic pressure pump in sterile PBS buffer, and equilibrate at 37 degrees Celsius overnight. During the operation, the mice were anesthetized with 2% ...

Embodiment 2

[0032] Example 2. Using cardiac fibroblasts, the cells were treated with 1 μM angiotensin for three days, and the protein levels of PAX6, fibrosis markers fibronectin, αSMA and Col I were detected by immunofluorescence and Western blot experiments.

[0033] Isolation and culture of adult mouse cardiac fibroblasts: 8-week-old male C57 / BL6 mice were killed by neck dislocation, quickly soaked in 75% alcohol for about half a minute, and the heart was immediately removed from the chest in an ultra-clean workbench. Wash twice in PBS buffer at 4 degrees Celsius, cut off the blood vessels at the atrium and the bottom of the heart, then cut the ventricle into small pieces, and wash it once with PBS to remove residual blood. Add 0.1% type II collagenase (330U, Worthington, Columbia, NJ, USA / Sigma, St.Louis, MO, USA) prepared in PBS balanced salt solution for digestion. The entire digestion process is carried out under the condition of constant temperature stirring at 36-37 degrees Celsi...

Embodiment 3

[0039] Example 3 Knockdown of PAX6 at the level of cardiac fibroblasts, using Western blot to detect the effect of PAX6 on the protein levels of myofibroblast markers fibronectin, αSMA and Col I.

[0040] Cardiac fibroblasts transfected with small interfering RNA: The P2 generation of cardiac fibroblasts was subcultured from the P1 generation to a 6-well plate the night before the experimental transfection of small interfering RNA, and cultured overnight at 37 degrees Celsius in a 5% carbon dioxide environment to ensure Fibroblasts are morphologically spread and have not yet produced an extracellular matrix that affects transfection efficiency. On the morning of the day of transfection, warm PBS at 37 degrees Celsius to gently wash the fibroblasts in the 6-well plate, and repeat the washing three times to ensure that the culture medium is completely washed away, and then add 500 μl OPTI-MEM (Opti-MEM I Reduced Serum Medium, Opti-MEM I Reduced Serum Medium, 31985070, Life), and...

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Abstract

The invention discloses an application of a PAX6 gene or an expression product thereof to preparation of medicines for restraining fibrosis. Experiment confirms that the PAX6 can restrain a fibrosis promotion factor TGF beta and promote fibrosis restraining factors IL1R2 and CXCL10, so that the effects of protecting the heart and restraining fibrosis can be achieved, and the PAX6 is a potential bran-new important target point for treating heart fibrosis so as to prevent heart failure in the heart.

Description

technical field [0001] The invention relates to the field of genes, in particular to the application of PAX6 gene or its expression product in the preparation of medicines for inhibiting fibrosis. Background technique [0002] Cardiac fibrosis is an important pathological process common in a large number of heart diseases. The most important feature of cardiac fibrosis is the massive accumulation of extracellular matrix in the myocardium. Excessive deposition of extracellular matrix can lead to decreased cardiac compliance and diastolic dysfunction. Further development of cardiac fibrosis leads to a decrease in cardiac output and eventually heart failure. Cardiac fibroblasts are the major cell type providing structural support in myocardial tissue. Under various types of cardiac injury, cardiac fibroblasts can be activated to differentiate into myofibroblasts, and thus have special cell types that more effectively secrete extracellular matrix proteins and have both fibrob...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P9/00
CPCA61K45/00A61P9/00
Inventor 张幼怡肖晗李明喆冯晔囡
Owner PEKING UNIV THIRD HOSPITAL
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