CN107041947B - Protective effect and application of FSTL1 in anti-fibrosis homeostatic regulation of tissues such as liver - Google Patents

Protective effect and application of FSTL1 in anti-fibrosis homeostatic regulation of tissues such as liver Download PDF

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CN107041947B
CN107041947B CN201610081875.8A CN201610081875A CN107041947B CN 107041947 B CN107041947 B CN 107041947B CN 201610081875 A CN201610081875 A CN 201610081875A CN 107041947 B CN107041947 B CN 107041947B
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fstl1
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何玉龙
蒋海娟
张禄卿
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Abstract

The invention relates to a protective effect and application of FSTL1 in anti-fibrosis steady-state regulation of tissues such as liver and the like, in particular to application of FSTL1 protein or FSTL1 regulation compound in preparation of a medicament for treating fibrosis lesions of the tissues. The invention also relates to a conditional FSTL1 gene knocking non-human animal model closely related to the degree of the tissue fibrosis lesion, a preparation method and application thereof in screening anti-tissue fibrosis drugs. The non-human animal model of systemic or vascular endothelial cell FSTL1 gene induced knockout is obtained by combining different doses of tamoxifen and different stages of animal growth for treatment, and is used for simulating the fibrotic lesion of different tissues.

Description

Protective effect and application of FSTL1 in anti-fibrosis homeostatic regulation of tissues such as liver
Technical Field
The FSTL1 protein or the application of the adjusting compound thereof in preparing the medicine for treating anti-fibrosis homeostatic regulation and anti-right ventricular dilatation in tissues and organs such as liver, lung, kidney and the like. The invention also relates to a non-human transgenic animal model and a construction method and application thereof, in particular to a controllable FSTL1 gene conditional knockout transgenic mouse animal model induced by different doses of tamoxifen at different growth stages of animals, a construction method and application thereof in tissue fibrosis lesions.
Background
FSTL1 is a secreted glycoprotein originally cloned from the mouse osteoblast line MC3T3, which is expressed in a variety of cells other than blood cells, particularly in mesenchymal cells (Knight, C., et al, J Dent Res,2001.80(10): p.1895-902; Geng, Y., et al, Proc Natl Acad Sci U S A,2011.108(17): p.7058-63; Sylva, M., et al, PLoS One,2011.6(8): p.e22616; Tanaka, M., et al, Int Immunol,1998.10(9): p.1305-14. mu.l; Wilson, D.C., et al., Arthris Rheum,2010.62(8 p.3890-6; Chatis Y., 1084. J.2518, R.8). Although the biological function of FSTL1 is not fully understood, it appears from prior studies that its role relates to cardiovascular disease, immune regulation of the body, respiratory and skeletal muscle development (Oshima, Y., et al, Circulation,2008.117(24): p.3099-108.; Liu, S., et al, Am J Physiol Endocrinol Metab,2010.299(3): p.E351-63; Ouchi, N., et al, J Biol Chem,2008.283(47): p.32802-11.; Trojan, L., et al, Anticancer Res,2005.25(1a): p.183-91.; Sundaram, G.M., et al, Nature 2013.495(7439): p.103-6). Systemic knockdown of FSTL1 results in death of neonatal mice, and dysplasia of various tissues and organs, including the respiratory system and bones (Chaly, y., et al., Ann Rheum Dis, 2014.). However, the application of the nonspecific mouse knockout model is limited by the complex environment in vivo, so that a mouse model for specifically inducing and knocking out FSTL1 in vascular endothelial cells is established, and the mouse model is used for researching the biological effect of FSTL1 derived from the vascular endothelial cells and the application of the mouse model in researching related diseases.
Fibrosis refers to pathological hyperplasia, sclerosis and scar repair processes of tissues and organs, and is mainly characterized by differentiation and proliferation of myofibroblasts and accumulation of extracellular matrix mainly comprising collagen. Tissue fibrosis is the result of persistent chronic inflammation due to a variety of stimuli, including persistent infection, autoimmune reactions, allergic reactions, drugs, radiation, and other physicochemical factors, as well as tissue damage, among others (Wynn, t.a., J Pathol,2008.214(2): p.199-210.). Hepatic fibrosis is common in clinic, and comprises viral hepatitis, alcoholic liver, fatty liver and the like. It has been shown that FSTL1 inhibition reduces bleomycin-induced pulmonary fibrosis (Dong, Y., et al., J Exp Med,2015.212(2): p.235-52.). In addition, in a mouse model of coronary artery ligated myocardial infarction, cardiac fibrosis near the damaged area can be reduced by providing FSTL1 to the infarcted myocardium (Wei, K., et al., Nature,2015.525(7570): p.479-85.). However, no study has shown that FSTL1 is associated with liver fibrosis. At present, in biomedical experiments, test methods for establishing a hepatic fibrosis model mainly comprise bile duct ligation, thioacetamide, carbon tetrachloride chemical induction and the like, but the animal models often cannot truly reflect the pathological process of hepatic tissue fibrosis.
On the other hand, right ventricular dilatation dysfunction is often a progressive disease caused by pulmonary hypertension, and the patient is caused by increased vascular resistance, resulting in increased right ventricular load. Vascular structure disorders caused by factors such as excessive growth of vascular wall cells and inflammatory reactions are currently considered to be one of the major causes (Schermuly, R.T., et al., Nat Rev Cardiol,2011.8(8): p.443-55.). It has been shown that over-expression of FSTL1 protects mouse hearts from ischemia-reperfusion injury (Walsh, K., Circ J,2009.73(1): p.13-8.). FSTL1 has also been reported to be involved in the development of pulmonary blood vessels (Navessa P., et al., Am J Respir Crit Care Med,2015.191: A5960). However, no studies have shown that FSTL1 is associated with aberrant 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 and the like, and a pulmonary hypertension model established by using a gene knockout technology is not widely applied.
The invention utilizes the gene knockout technology to establish a multi-tissue fibrosis model of mouse liver, lung, kidney and the like, and has important scientific significance and application value for researching the pathological mechanism of corresponding tissue fibrosis and preparing preclinical animal models for screening medicaments for treating diseases related to tissue fibrosis. Meanwhile, the mouse model established in the invention particularly uses the alpha SMA positive (marking the neovascular pericyte) blood vessel in the lung tissue as the judgment of the pathological change phenotype, and has important scientific value and application prospect for researching the pathogenesis of pulmonary hypertension and right heart dilatation and applying to research and screening of related therapeutic drugs.
Disclosure of Invention
The invention aims to provide application of FSTL1 protein or a regulatory compound thereof in preparing a medicament for promoting the protective action of tissues such as liver, lung, kidney and the like in the steady state regulation of anti-tissue fibrosis.
The invention also aims to provide the application of the FSTL1 protein or the regulatory compound thereof in preparing the medicines for promoting the right ventricular dilatation protection effect.
In some embodiments of the invention, the FSTL1 protein comprises FSTL1 protein including derivatives, analogs, or polypeptides thereof.
In embodiments of the invention, the FSTL1 modulating compound is an FSTL1 expression inducer.
In embodiments of the invention, the FSTL1 expression inducer comprises an enzyme, hormone, growth factor, cytokine or antibody that activates FSTL1 gene expression.
In some embodiments of the invention, the medicament is for a disease associated with a fibrotic condition of a tissue.
In specific embodiments of the invention, the diseases associated with tissue fibrotic disorders include: fibrosis of tissues caused by various physical, chemical, inflammatory and other immunological factors and other pathological emergencies, including fibrosis of tissues of lung, liver, kidney and heart.
In some embodiments of the invention, the medicament is for treating a disease associated with right ventricular dilation lesion.
In a specific embodiment of the present invention, the disease associated with right ventricular dilatation lesion comprises: pulmonary hypertension and other pulmonary hypertension, right ventricular or left ventricular cardiomyopathy, right ventricular ischemia or infarction, pulmonary valve or tricuspid valve lesion, and the like.
The invention also aims to provide a novel method for establishing a liver, lung and kidney tissue fibrosis model or a right ventricular dilation model, and the degree of tissue fibrosis is controlled by regulating the FSTL1 gene level, and the method can specifically regulate: inducing gene knockout at different time points, inducing the efficiency of FSTL1 gene knockout in different treatment modes and the like.
In an embodiment of the invention, the animal is a mammal.
In some embodiments of the invention, the mammal is a rodent.
In a specific embodiment of the invention, the rodent is a mouse.
In embodiments of the invention, the knockout inducing agent comprises tetracycline, interferon, hormone.
In some embodiments of the invention, the hormone is tamoxifen.
In some embodiments of the invention, the time point is 1-8 days of birth.
In a particular embodiment of the invention, wherein the time points are 1-4 days of birth, the dose of tamoxifen is 50-100 μ g/day.
In a specific embodiment of the invention, the time points are 1-4 days of birth and the dose of tamoxifen is 50-70 μ g/day.
In another specific embodiment of the invention, the time point is 5-8 days of birth and the dose of tamoxifen is 90-100 μ g/day.
In yet another embodiment of the invention, the time point is from 2 weeks of birth to adult life and the dose of tamoxifen is 500-.
In embodiments of the invention, the promoter comprises the receptor tyrosine kinase TEK or ubiquitin c (ubc) or vascular endothelial cell-Cadherin (VE-Cadherin).
In embodiments of the invention, the non-human transgenic animals and their progeny are used as models of tissue fibrosis in tissue organs.
In some embodiments of the invention, the tissue organ is a liver, a lung, a kidney or a heart.
It is a fourth object of the present invention to provide a method for screening a drug for treating a tissue fibrotic lesion, which comprises administering the drug to the non-human transgenic animal and its offspring as described above and monitoring the effect on pathology or behavior.
In some embodiments of the invention, the tissue organ is a liver, a lung, a kidney or a heart.
The fifth purpose of the invention is to provide a method for constructing a vascular endothelial cell FSTL1 gene knockout mouse model closely related to tissue fibrosis, wherein the method comprises the following specific preparation steps: obtaining FSTL1 conditional knockout model Fstl1 by using Cre-LoxP systemFlox/FloxA mouse; while utilizing Fstl1+/-The mice were mated with Tek-Cre mice (transgenic mice expressing Cre recombinase Tek-Cre in vascular endothelial cells) to obtain Fstl1+/-Tek-cre mice, then Fstl1 as described+/-Tek-cre mice and Fstl1Flox/FloxMice (background: 129S4x C57BL/6J, prepared by Nanjing model animals research, China) were mated to obtain Fstl1Flox/-Mouse model of FSTL1 knock-out by Tek-cre vascular endothelial cells, Fstl1-/ECKO. Endothelial cell specific knockout Fstl1 mice lead to multiple tissue fibrosis Using the vascular endothelial cell specific knockout Fstl1 mouse model, mouse liver, lung, kidney tissues were collected at different time points, and fibrosis was detected by detecting fibrosis fingersTissue fibrotic lesions are assessed.
The invention also aims to provide a method for constructing a mouse model for knocking out the FSTL1 gene closely related to tissue fibrosis and inductively induced by vascular endothelial cells, wherein the method is specifically prepared by the step of using Fstl1+/-The mice are mated with transgenic mice expressing UBC-Cre/ERT2 or VE-Cadherin-Cre/ERT2 to obtain Fstl1+/-(ii) a Cre/ERT2 mouse, followed by the Fstl1+/-(ii) a Cre/ERT2 mouse and Fstl1Flox/FloxMice are mated to obtain Fstl1 genotype respectivelyFlox/-(ii) a UBC-Cre/ERT2 or Fstl1Flox/-(ii) a VE-Cadherin-Cre/ERT2, and Fstl1Flox/+(ii) a UBC-Cre/ERT2 or Fstl1Flox/+(ii) a A mouse model of VE-Cadherin-Cre/ERT 2. And (3) establishing mouse models with different fibrosis degrees by using mouse models capable of inducing Fstl1 gene knockout and different treatment times of tamoxifen. The analysis and indices for mouse multi-tissue fibrosis were as above.
In a mouse model capable of inducing Fstl1 gene knockout, the knockout efficiency of Fstl1 gene can be induced by different doses of tamoxifen, and the correlation between the Fstl1 knockout efficiency and the mouse multi-tissue fibrosis degree is established, and the specific analysis method and indexes are the same as above.
After Fstl1 is knocked out by using a controllable Fstl1 knockout mouse model in anti-tissue fibrosis drug screening and by using the vascular endothelium of a mouse, fibrosis occurs in tissues 21 days after birth; meanwhile, in a mouse model capable of inducing Fstl1 gene knockout, different degrees of mouse corresponding tissue fibrosis lesion are caused by different treatment time and different treatment doses of tamoxifen. The FSTL1 protein can be used for preparing medicines for preventing or treating tissue fibrosis. The invention provides a basis for preparing a medicament for treating tissue fibrosis aiming at the effect of FSTL1 in tissue fibrosis, and is beneficial to the research of tissue fibrosis and the evaluation of the effect of an anti-hepatic fibrosis medicament.
The research on the mechanism of right heart dilated lesion is carried out by using a vascular endothelial cell specific knockout Fstl1 mouse model. Mouse model using Fstl1 vascular endothelial cell specific knockout (Fstl1)-/ECKO) At different timesAt the intermediate point, mouse heart tissue was taken to detect the extent of right heart dilated lesions. After the heart of a mouse is taken down and fixed in 4% paraformaldehyde PFA overnight, the tissues are subjected to paraffin embedding, slicing and pathological analysis, and the analysis shows that the right ventricle of the knockout mouse at 21 days after birth is severely expanded, wherein specific indexes comprise morphological analysis and H&E staining, quantitative analysis of right ventricular area, etc. The present disclosure facilitates the study of the mechanism of Fstl1 in right heart dilated lesions.
The application of the Fstl1 mouse model with endothelial cell specificity knockout in the drug screening of pulmonary hypertension and right heart dilated lesion is provided. Right ventricular dilatation occurred around 3 weeks after birth after knockout of Fstl1 by the vascular endothelium of mice. The invention aims at the role of Fstl1 in vascular remodeling, and shows that FSTL1 protein and related reagents can be used for preparing a medicine for preventing or treating cardiac dilatation lesion caused by certain vascular lesions.
The animal model (Fstl1)-/ECKO) It can also be used for screening new drugs for treating pulmonary hypertension and right heart dilatation lesion, and evaluating the drug effect of related drugs.
Advantageous effects of the invention
The beneficial effects of the invention are that a mouse model of vascular endothelial cell specificity and systemic induced knockout of FSP-1 is established for the first time, and the animal model is applied to the exploration of revealing the biological function of the protein. A mouse hepatic fibrosis model established by using controllable FSTL1 knockout efficiency can be applied to research the pathogenesis of FSTL1 in hepatic fibrosis. The FSTL1 can be applied to the development of drugs for treating liver fibrosis diseases, such as FSTL1 protein or analogues can delay or stop the degree of liver fibrosis lesion. The mouse liver fibrosis model with the FSTL1 gene knockout can be applied to the drug effect evaluation of new drugs for treating liver fibrosis.
Drawings
FIG. 1 is a graph showing the detection of the expression level of FSTL1 protein in liver tissue of a vascular endothelial specific knockout Fstl1 mouse model (postnatal day 14) using the Western Blot method.
FIG. 2 is a graph showing the analysis of Fstl1mRNA in liver tissue of a vascular endothelial-specific knockout Fstl1 mouse model (postnatal day 14) using a real-time quantitative RT-PCR method.
FIG. 3 is a graph showing the detection of the expression level of FSTL1 protein in liver tissue post-natally induced knockout of Fstl1 in mice (postnatal day 19) using the Western Blot method.
Figure 4 is a graph showing fibrotic lesions of the liver in vascular endothelial specific knockout Fstl1 mice. Graph of mouse liver collagen content (postnatal day 21) using sirius red staining.
FIG. 5 is a graph showing fibrotic lesions in the liver of vascular endothelial-specific knockout Fstl1 mice. Graph of mouse liver hydroxyproline content (postnatal day 21).
Figure 6 is a graph showing the protective effect of FSTL1 protein on vascular endothelial specific knockout FSTL1 mice in developing fibrotic lesions of the liver.
FIG. 7 is a graph showing the detection of the expression level of FSTL1 protein in lung tissue (postnatal day 21) in a vascular endothelial specific knock-out Fstl1 mouse model using the Western Blot method.
FIG. 8 is a graph showing the detection of Fstl1mRNA levels in lung tissue in a mouse model of vascular endothelial-specific knockout Fstl1 (postnatal day 14) using a real-time quantitative RT-PCR method.
Fig. 9 is a graph showing that vascular endothelial-specific knockout of Fstl1 gene results in abnormal vascular remodeling in lung tissue of mutant mice. The blood vessels in lung tissues are detected by an immunofluorescence staining method (PECAM-1, green; alpha SMA, red), and the result shows that the FSTL1 knockout of vascular endothelial cells leads to the obvious increase of alpha SMA positive blood vessels (postnatal day 21).
FIG. 10 is a graph showing that vascular endothelial-specific knockout of the Fstl1 gene results in abnormal vascular remodeling in kidney tissue in mutant mice.
FIG. 11 is a graph showing morphological analysis of right heart dilated lesions of vascular endothelial-specific knockout Fstl1 mice (postnatal day 21 hearts) and showing that the heart volume of vascular endothelial cell knockout Fstl1 mice is significantly larger.
Fig. 12 is a graph showing the apparent dilation of the right ventricle in Fstl1 knockout mice, observed using H & E staining for changes in the characteristics of the postnatal 21-day heart of the mice.
Figure 13 is a graph showing that quantitative analysis showed a significant increase in the ratio of the total area of the right ventricle to the heart (postnatal day 21 heart) in mice with vascular endothelial cells knocked out of Fstl 1.
Detailed Description
The present invention will be further described below by way of specific embodiments and experimental data. Although specific terms are used below for the sake of clarity, these terms are not meant to define or limit the scope of the invention.
The term "FSTL 1 modulating compound" as used herein refers to a compound that interacts with an FSTL1 protein or gene to thereby modulate (e.g., enhance or inhibit) the activity of said FSTL1 protein. For example, compounds that modulate the expression of FSTL1 at the transcriptional or protein level.
The term "expression inducer" as used herein refers to a compound that effects conditional gene expression from a promoter, and in the present invention refers to an enzyme, hormone, growth factor, cytokine or antibody that activates FSTL1 gene expression.
The term "antibody", as used herein, refers to any immunoglobulin or intact molecule that binds a particular epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of an intact antibody, provided that such fragments or portions retain the antigen binding ability of the parent antibody. For example, in the context of the present invention, an "anti-FSTL 1 antibody" refers to monoclonal antibodies, polyclonal antibodies, single chain antibodies and immunologically active fragments or portions thereof that specifically bind to FSTL1 protein, or a functional variant or functional fragment thereof. In the present invention, terms such as "FSTL 1 antibody", "anti-FSTL 1 antibody" and "antibody against FSTL 1" are used interchangeably.
The term "polypeptide" as used herein refers to a peptide or protein comprising two or more amino acids linked to each other by peptide or amorphic peptide bonds. "Polypeptides" include short chains (often referred to as peptides, oligopeptides, and oligomers) and long chains (often referred to as proteins). A polypeptide may comprise amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include polypeptides modified by natural processes such as processing and other post-translational modifications, as well as by chemical modification techniques. Such modifications are well described in the basic literature and in more detailed monographs, as well as in a number of research papers, and are well known to those skilled in the art. It is understood that the same type of modification may be present to the same or different extent at several sites in a given polypeptide. In addition, a given polypeptide may comprise multiple types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of riboflavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation (selenoylation), sulfation, transfer-RNA mediated protein amino acid addition (e.g. arginylation) and ubiquitination. For example, in the present invention, the "FSTL 1 polypeptide" refers specifically to a polypeptide capable of specifically activating FSTL1, or a polypeptide capable of inducing FSTL1 expression.
The term "treating" as used herein refers to reversing, alleviating or inhibiting the progression of the disease to which the term applies, or one or more symptoms of the disease. As used herein, the term also includes, depending on the condition of the patient, preventing the disease, including preventing the onset of the disease or any symptoms associated therewith, and lessening the severity of the disorder or any condition thereof prior to onset.
As used herein, the term "non-human transgenic animal" is obtained by techniques known in the art, and the non-human transgenic animal of the invention can be any non-human animal that is genetically modified through the original genome and is under the control of an exogenous promoter for conditional expression of the Fstl1 mutant allele.
As used herein, the term "promoter" refers to a DNA regulatory region capable of binding RNA polymerase within a mammalian cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto, including inducible promoters, such as conditionally active promoters of cre-loxP promoters.
As used herein, the term "inducible gene knockout (iKO)" or "gene knockout induction" refers to a technique of gene knockout that uses the activity of a promoter controlling Cre expression or the activity of Cre enzyme expressed to have an inducible characteristic, by giving temporal control to an inducer or by using the host cell specificity of Cre gene targeting vectors in an expression system and temporal controllability of the process of transferring the expression system into an animal, to achieve the purpose of genetic modification of a specific gene in a certain developmental stage and a certain tissue cell of a 1oxP animal. Several common types of inducibility (inducers) are as follows: tetracycline-inducible; an interferon-induced type; hormone-inducible; adenovirus-mediated type.
The present invention is further described in detail below with reference to specific examples, and various modifications thereof can be made by those skilled in the art without departing from the scope of the present invention as defined in the appended claims. The experimental procedures in the following examples are conventional unless otherwise specified.
The specific embodiment is as follows:
example 1 establishment of vascular endothelial cell-specific Fstl1 knockout mouse model
With Fstl1+/-Mice (B6 background, supplied by Nanjing animal model research institute) were mated with Tek-cre mice to obtain Fstl1+/-Tek-cre mice, which were then used with Fstl1Flox/FloxMice were mated to give Fstl1Flox/-Tek-cre, this mouse was used as a laboratory mouse (also called Fstl1 in the present invention)-/ECKO) Heterozygous knockout of the littermate gene (also known as Fstl1) was used-/WT) Or wild mouse (also known as Fstl1)WT/WT) As a controlMice.
Fresh mouse liver, lung and kidney tissues are taken, washed in PBS, frozen in liquid nitrogen and stored in a freezer at the temperature of-80 ℃ for respectively extracting RNA and preparing protein; the remaining tissues were fixed overnight in 4% PFA, embedded and sectioned for histological analysis by conventional histological methods. The knockout efficiency of Fstl1 was tested using Western Blot and real-time quantitative RT-PCR. Wherein the protein level of Fstl1 knockout mice was significantly reduced in liver tissue by Western Blot analysis for liver tissue (FIG. 1); analysis by real-time quantitative RT-PCR revealed that the residual levels of Fstl1 messenger rna (mrna) in liver tissue of Fstl1 knockout mice were approximately 34% compared to wild type mice (fig. 2).
Example 2 establishment of mouse model for systemically inducing Fstl1 Gene knockout
With Fstl1+/-The mice are mated with transgenic mice expressing UBC-CreERT2 to obtain Fstl1+/-(ii) a UBC-Cre/ERT2 mouse, and Fstl1Flox/FloxMice were mated to give Fstl1Flox/-(ii) a UBC-Cre/ERT2 (also referred to herein as Fstl1)-/iKO) Fstl1 obtained simultaneously with this mouse as a laboratory mouseFlox/+(ii) a UBC-Cre/ERT2 was used as a Control mouse (Control).
Mice can induce Fstl1 gene knockout at various stages of postnatal including neonatal mice (days 1-4 of birth, P1-P4) and various stages of growth and development including adult mice to produce animal models that can mimic clinical human hepatic fibrosis. The preparation method of the mouse Fstl1 gene induced knockout model comprises the following steps:
after birth (days 1-4 of birth, P1-P4) the mice were analyzed by sampling within 3 weeks by gastric injection of tamoxifen (60 ug per mouse). As shown in fig. 3, the Western Blot method was used to examine the knock-out efficiency of Fstl1, and the results showed that Fstl1 protein level was significantly decreased in liver. Results of a systemic induced knockout animal model (P1-4 induction) with Fstl1-/ECKOThe results were consistent.
Example 3 vascular endothelial cell-specific Fstl1 knockout mouse liver fibrosis disease model
In the process of liver fibrosis, extracellular matrix mainly containing collagen is accumulated in a large amount. Three weeks after birth, mice were fixed with liver tissue in 4% paraformaldehyde PFA for 2 hours, incubated overnight in 20% sucrose solution, and then subjected to tissue embedding and sectioning, using sirius red staining, as follows: weighing 0.5g of Sirius red dye (purchased from Sigma company), dissolving in 500mL of picric acid (purchased from Shanghai Producer, Cat. No. PB0716), and storing at room temperature for use. Knockout of Fstl1 by vascular endothelial cells resulted in a significant increase in collagen content in liver tissue as determined by sirius red staining analysis (fig. 4, red represents staining of collagen in the liver by sirius red). In addition, hydroxyproline is a main component of collagen, and the degree of tissue fibrosis can also be evaluated by measuring the content of hydroxyproline. Weighing 10mg of fresh mouse liver, homogenizing with 100uL of water, hydrolyzing with 12M HCl hydrochloride at 120 ℃ for 3 hours, finally taking 50uL of supernatant, placing in a 96-well plate, and determining the content of hydroxyproline by a colorimetric method. Analysis shows that the content of hydroxyproline in liver tissues of the Fstl1 mouse knocked out by the vascular endothelial cells is obviously increased (figure 5). The blood vessel endothelial cell Fstl1 knockout mouse shows liver fibrosis after 21 days after birth by using sirius red staining and hydroxyproline content analysis in liver tissues.
On the other hand, when a recombinant adenovirus expressing both FSTL1 and EGFP was used to treat vascular endothelial-specific knockout Fstl1 mice at P1 and P8, preliminary results showed that FSTL1 treatment could reduce Fstl1 compared to control virus (AdLacZ) -treated mice-/ECKOThe number of mouse hepatic alpha SMA cells, indicates improvement in hepatic fibrotic disease (fig. 6).
Example 4 vascular endothelial cell-specific Fstl1 knockout mouse pulmonary fiber disease model
With Fstl1+/-Mice (B6 background, supplied by Nanjing animal model research institute) were mated with Tek-cre mice to obtain Fstl1+/-Tek-cre mice, which were then used with Fstl1Flox/FloxMice were mated to give Fstl1Flox/-,Tek-cre(Fstl1-/ECKO) The mouse was heterozygous for the littermate gene knockout as a laboratory mouse (Fstl1)-/WT) Or a wild mouse(Fstl1WT/WT) As a control.
Fresh mouse lungs were fixed in 4% PFA overnight, and tissues were embedded and sectioned by routine histology. The knockout efficiency of Fstl1 is detected by using a Western Blot and a real-time quantitative RT-PCR method, and the protein level of Fstl1 knockout mice is obviously reduced in the lung tissues by Western Blot analysis (figure 7); the lung tissue of Fstl1 messenger rna (mrna) levels in Fstl1 knockout mice were significantly reduced as analyzed by real-time quantitative RT-PCR (fig. 8). The blood vessels (PECAM-1, green; alpha SMA, red) in the lung tissue are detected by an immunofluorescence staining method, and the result shows that the FSTL1 knockout of the vascular endothelial cells leads to the obvious increase of the muscle formation degree of the alpha SMA positive blood vessels, namely the blood vessels, in the lung tissue (figure 9), thereby leading to the increase of the resistance of the lung blood vessels.
Example 5 vascular endothelial cell-specific Fstl1 knockout mouse model of renal fibrosis
Similar to the experimental procedure of example 4, in kidney tissue, blood vessels (PECAM-1, green; α SMA, red) in lung tissue were detected by immunofluorescence staining, as shown in FIG. 10, which indicates that knockout of Fstl1 by vascular endothelial cells results in a significant increase in α SMA-positive blood vessels (postnatal day 21), indicating that knockout of Fstl1 gene specific to vascular endothelium results in abnormal remodeling of blood vessels in kidney tissue of mutant mice.
Example 6 vascular endothelial cell-specific Fstl1 knockout mice with right heart dilated lesions
The pathological analysis on the heart is as follows: three weeks after birth, lethally anesthetized with 0.8% sodium pentobarbital (10uL/g), and dissected to find that the heart volume of mice with vascular endothelial cells knocked out of Fstl1 was significantly greater than that of the control group (FIG. 11); performing heart perfusion by using PBS (phosphate buffer solution), washing blood, fixing the blood by using 4% PFA (Perfluoro fluoro-alkoxy-vinyl) and performing paraffin embedding and slicing on heart tissues according to a conventional histological method. By H & E staining analysis, Fstl1 knockout mice were found to have significant right ventricular dilation (fig. 12). In addition, mice used for ventricular quantification were suddenly killed by injection of 10% potassium chloride via the tail vein and the heart was in the systolic phase, and then subjected to tissue fixation, paraffin embedding, and section staining (H & E) for quantification of the ratio of the total area of the right ventricle to the left and right ventricles (except for the atria) to further identify the right ventricular dilated lesions of the mutant mice (fig. 13).
While the present invention has been described with reference to the embodiments, it is to be understood that the present invention is not limited thereto, and those skilled in the art will appreciate that the present invention is capable of modification and variation within the spirit and scope of the present invention, and that such modification and variation are within the scope of the present invention.

Claims (9)

1. A method for preparing a vascular endothelial cell FSTL1 gene knockout mouse model closely related to tissue fibrosis and a descendant thereof is provided, wherein the method comprises the following specific preparation steps:
obtaining FSTL1 conditional knockout model Fstl1 by using Cre-LoxP systemFlox/FloxA mouse; while utilizing Fstl1+/-The mice were mated with Tek-cre mice to obtain Fstl1+/-Tek-cre mice, then Fstl1 as described+/-Tek-cre mice and Fstl1Flox/FloxMice were mated to give Fstl1Flox/-Mouse model of FSTL1 knock-out by Tek-cre vascular endothelial cells, Fstl1-/ECKO，
Wherein the tissue is selected from at least one of lung, liver, and kidney, and the mouse model is useful for mimicking at least one of a fibrotic disease of lung tissue, a fibrotic disease of liver tissue, a fibrotic disease of kidney tissue, and dilated lesion of right ventricle.
2. A method for preparing a mouse model of systemic or vascular endothelial cell induced FSTL1 gene knockout closely related to tissue fibrosis and a descendant thereof, wherein the method comprises the following specific preparation steps:
i) fstl1+/-The mice are mated with transgenic mice expressing UBC-Cre/ERT2 or VE-Cadherin-Cre/ERT2 to obtain Fstl1+/-(ii) a Cre/ERT2 mice;
ii) reusing the Fstl1+/-(ii) a Cre/ERT2 mouse and Fstl1Flox/FloxMice are mated to obtain Fstl1 genotype respectivelyFlox/-(ii) a UBC-Cre/ERT2 or Fstl1Flox/-(ii) a VE-Cadherin-Cre/ERT2, and Fstl1Flox/+(ii) a UBC-Cre/ERT2 or Fstl1Flox/+(ii) a A mouse model of VE-Cadherin-Cre/ERT2,
iii) inducing the mice 1-4 days after birth with 50-70 μ g/day tamoxifen to allow conditional knock-out of the FSTL1 allele under the control of an exogenous promoter, creating a model of severe or lighter tissue fibrotic lesions;
wherein the tissue is selected from at least one of lung, liver, and kidney, and the mouse model is useful for mimicking at least one of a fibrotic disease of lung tissue, a fibrotic disease of liver tissue, a fibrotic disease of kidney tissue, and dilated lesion of right ventricle.
3. The method of claim 1 or 2, wherein the right ventricular dilation lesion is selected from at least one of pulmonary hypertension and other pulmonary hypertension, right ventricular or left ventricular cardiomyopathy, right ventricular ischemia or infarction, pulmonary valve, and tricuspid valve lesions.
4. The method of claim 2, wherein the exogenous promoter is selected from the group consisting of receptor tyrosine kinase TEK, ubiquitin C, or vascular endothelial cell-cadherin.
5. Use of a mouse model prepared by the method of any one of claims 1 to 4 and progeny thereof in the preparation of a tissue fibrotic disease model.
6. The use of claim 5, wherein the tissue fibrotic disease is selected from at least one of a pulmonary tissue fibrotic disease, a hepatic tissue fibrotic disease, a renal tissue fibrotic disease, and right ventricular dilated lesion.
7. The use of claim 6, wherein the right ventricular dilation lesion is selected from at least one of pulmonary hypertension and other pulmonary hypertension, right ventricular or left ventricular cardiomyopathy, right ventricular ischemia or infarction, pulmonary valve and tricuspid valve lesions.
8. The use of claim 5, wherein the tissue fibrotic disease is a severe tissue fibrotic disease or a mild tissue fibrotic disease.
9. Use of a mouse and its progeny made by the method of any one of claims 1 to 4 in the preparation of a model for in vivo screening, pharmacodynamic testing, efficacy assessment, pathology or behavioral monitoring of drugs for the treatment of lung tissue fibrotic disease, liver tissue fibrotic disease, kidney tissue fibrotic disease, and right ventricular dilation lesion.
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