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The protective role and application of fstl1 in the homeostatic regulation of liver and other tissues against fibrosis

A tissue fibrosis and FSTL1 technology, applied in the fields of anti-fibrosis homeostasis regulation and anti-right ventricular dilation drugs, can solve the problems that the pulmonary hypertension model has not been widely used

Active Publication Date: 2021-05-18
何玉龙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no studies have shown that FSTL1 is associated with abnormal vascular remodeling leading to right heart dilated lesions
At present, in biomedical experiments, the experimental methods for establishing pulmonary hypertension mainly include chronic hypoxia, monocrotaline injection, shunt surgery, etc. However, the pulmonary hypertension model established by gene knockout technology has not been widely used

Method used

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  • The protective role and application of fstl1 in the homeostatic regulation of liver and other tissues against fibrosis
  • The protective role and application of fstl1 in the homeostatic regulation of liver and other tissues against fibrosis
  • The protective role and application of fstl1 in the homeostatic regulation of liver and other tissues against fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Establishment of vascular endothelial cell-specific Fstl1 knockout mouse model

[0065] with Fstl1 + / - Mice (B6 background, provided by Nanjing Animal Model Institute) were mated with Tek-cre mice to obtain Fstl1 + / - , Tek-cre mice, and then use this mouse with Fstl1 Flox / Flox Mice mated to Fstl1 Flox / - , Tek-cre, this mouse is used as experimental mouse (also known as Fstl1 in the present invention - / ECKO ), using littermate gene knockout heterozygous (also known as Fstl1 - / WT ) or wild mice (also known as Fstl1 WT / WT ) as a control mouse.

[0066] Take fresh mouse liver, lung and kidney tissues, wash them in PBS, freeze them quickly in liquid nitrogen, and store them in a -80°C freezer for RNA extraction and protein preparation; the rest of the tissues were fixed overnight in 4% PFA , Tissue embedding and sectioning were performed by conventional histological methods for histological analysis. The knockout efficiency of Fstl1 was detected by Western B...

Embodiment 2

[0067] Example 2 Establishment of a mouse model for systemic inducible Fstl1 gene knockout

[0068] with Fstl1 + / - Mice mated with transgenic mice expressing UBC-CreERT2 to obtain Fstl1 + / - ; UBC-Cre / ERT2 mice, and then use this mouse with Fstl1 Flox / Flox Mice mated to Fstl1 Flox / - ; UBC-Cre / ERT2 (also referred to as Fstl1 in the present invention - / iKO ), this genotype mouse was used as an experimental mouse, and the Fstl1 obtained at the same time Flox / + ; UBC-Cre / ERT2 as a control mouse (Control).

[0069] Mice can induce Fstl1 gene knockout at different stages after birth, including neonatal mice (birth day 1-4, P1-P4), and different stages of growth and development, including adult mice, to produce liver fibrosis that can simulate clinical human occurred in animal models. The preparation method of the mouse Fstl1 gene-induced knockout model is as follows:

[0070] After the mice were born (days 1-4, P1-P4), the mice were injected with tamoxifen (60ug per mouse) thr...

Embodiment 3

[0071] Example 3 Vascular endothelial cell-specific Fstl1 knockout mouse liver fibrotic disease model

[0072] In the process of liver fibrosis, extracellular matrix mainly composed of collagen accumulates in large quantities. Three weeks after birth, liver tissues were taken from mice and fixed in 4% paraformaldehyde PFA for 2 hours, then incubated overnight in 20% sucrose solution, and then tissue-embedded and sectioned, and stained with Sirius red. The specific method is briefly described as follows : Take Sirius red dye Sirius red (purchased from Sigma company) 0.5g, dissolve in 500mL of picric acid (purchased from Shanghai Sangong, product number PB0716), and store at room temperature until use. Using Sirius red staining analysis, it was found that knocking out Fstl1 in vascular endothelial cells led to a significant increase in collagen content in liver tissue ( Figure 4 , red represents Sirius red staining of collagen in the liver). In addition, hydroxyproline is a m...

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Abstract

The present invention relates to the protective effect and application of FSTL1 in the homeostasis regulation of tissues such as liver against fibrosis, in particular to the application of FSTL1 protein or FSTL1 regulating compound in the preparation of medicine for treating tissue fibrosis. The invention also relates to a conditional FSTL1 gene knockout non-human animal model closely related to the degree of tissue fibrosis and its preparation method, and its use in screening anti-tissue fibrosis drugs. Among them, by combining different doses of tamoxifen and different stages of animal growth, a non-human animal model of systemic or vascular endothelial cell FSTL1 gene-induced knockout is obtained to simulate different tissue fibrosis lesions.

Description

technical field [0001] Use of the FSTL1 protein or its regulating compound of the present invention in the preparation of medicines for anti-fibrosis homeostasis regulation and anti-right ventricle expansion in tissues and organs such as liver, lung, and kidney. The present invention also relates to a non-human transgenic animal model and its construction method and application, in particular to a controllable follistatin-like protein 1 (FSTL1) gene induced by different doses of tamoxifen at different growth stages of animals Conditional knockout transgenic mouse animal model, its construction method and its application in tissue fibrosis. Background technique [0002] Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein originally cloned from the mouse osteoblast cell line MC3T3, which is expressed in a variety of cells except blood cells, especially in mesenchymal lines Higher expression level in cells (Knight, C., et al., J Dent Res, 2001.80(10): p.1895-902; Gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K49/00A61P1/16A61P11/00A61P13/12A61P9/00A01K67/027A01K67/02
CPCA01K67/0276A01K2217/075A01K2217/15A01K2227/105A01K2267/03A01K2267/0375A61K49/0008C07K14/575C12N15/85C12N2800/107C12N2800/30
Inventor 何玉龙蒋海娟张禄卿
Owner 何玉龙
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