CN114107400B - Construction method and application of retinal vascular disease model - Google Patents
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Abstract
The invention discloses a construction method and application of a retinal vascular disease model, and relates to the technical field of retinal vascular disease research. The invention provides a method for constructing a retinal vascular disease animal model, which can make an animal show that retinal blood vessels develop slowly and the density is reduced by inhibiting the expression of Lmbr1l gene or making the Lmbr1l gene not express, can construct the retinal vascular disease animal model with a retinal angiogenesis defect phenotype, and provides a model basis for researchers in the field to deeply research the pathogenesis of the retinal vascular disease, and to screen and screen effective target treatment medicines in the early clinical stage.
Description
Technical Field
The invention relates to the technical field of research of retinal vascular diseases, in particular to a construction method of a retinal vascular disease model and application thereof.
Background
Vascular development is critical to tissue growth and homeostasis, and abnormal vascular development leads to a variety of human diseases. The mammalian retina possesses a well organized vascular system to meet the high demand for oxygen and nutrients to maintain proper function of the retina. Retinal vascular dysfunction is one of the causes of vision deterioration, so that it is of great significance to find new genes for regulating retinal vascular development of eyes.
The Norrin/β -catenin signaling pathway is associated with a variety of retinal vascular diseases, including wet age-related macular degeneration (AMD), retinopathy of prematurity (ROP), diabetic Retinopathy (DR), and corneal neovascularization. Abnormal activation and inhibition of the Norrin/β -catenin signaling pathway leads to retinal vascular dysplasia, with inherited retinal vascular diseases including Norrin disease, familial Exudative Vitreoretinopathy (FEVR). The existing research shows that the precise regulation and control of the Norrin/beta-catenin signal path are important for the development of retinal blood vessels.
At present, the diagnosis of retinal vascular diseases mainly depends on fundus angiography and genetics screening, and early screening has important significance on the treatment and prognosis of the diseases. Early stage laser treatment of the disease can control the progress of the disease; in the later stage of the disease, the retinal detachment can be performed by scleral buckle surgery and vitrectomy, but the prognosis is poor; the inhibition effect of the anti-vascular endothelial growth factor on the new blood vessels can play a certain role in treatment. With the further identification of pathogenic genes and the intensive research on pathogenic mechanisms, selective targeted therapy against pathogenic genes of retinal vascular diseases will become a new direction for the treatment of patients with certain clinical phenotypes.
In order to better screen and research retinal vascular diseases and screen effective targeted therapeutic drugs, the identification of more new pathogenic genes of retinal vascular diseases and animal models simulating the characteristics of retinal vascular diseases are needed in the field.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a construction method of a retinal vascular disease model and application thereof to solve the technical problems.
The invention is realized by the following steps:
the invention provides a construction method of a retinal vascular disease animal model, which comprises the following steps: the Lmbr1l gene of the target animal is not expressed or the expression is inhibited by the genetic engineering technology.
The inventor firstly discovers that the expression of the Lmbr1ll gene is inhibited or the Lmbr1ll gene is not expressed through a genetic engineering technology, so that the animal can show the phenotype of retinal angiogenesis defect. Including, but not limited to: slow development of superficial blood vessels, low blood vessel density, slow development of retinal blood vessels to the deep layer, further resulting in delayed degeneration of vitreous blood vessels.
Therefore, the invention can construct a retinal vascular disease animal model with a retinal angiogenesis defect phenotype, and provides a model basis for researchers in the field to deeply research the pathogenesis of the retinal vascular disease, and to screen and screen effective targeted therapeutic drugs in early clinical.
The expression of which is suppressed means that: the expression level of the Lmbr1l gene is slightly or significantly reduced compared with the normal level (before gene knockout). Including but not limited to the level of mRNA and/or protein of the Lmbr1l gene is slightly or significantly reduced compared to the normal level (prior to gene knock-out).
In a preferred embodiment of the present invention, the above construction method includes: the exon 2 to exon 15 sequences of the Lmbr1l gene of the target animal are not expressed or the expression is inhibited by the genetic engineering technology.
The full-length cDNA of the Lmbr1l gene can be obtained from databases such as NCBI. It should be noted that, on the basis that the lack of expression or the inhibition of the expression of the Lmbr1ll gene in the target animal may cause retinal angiogenesis defect, it is within the scope of the present invention for one skilled in the art to adopt any gene manipulation technique to make the Lmbr1ll gene not expressed or the expression of the Lmbr1ll gene inhibited (i.e., not functioning normally) in the target animal.
In a preferred embodiment of the present invention, the genetic engineering technique is any one technique or a combination of techniques selected from the group consisting of a gene editing technique, a gene knockout technique, and an RNA interference technique.
In a preferred embodiment of the present invention, the above-mentioned gene editing technology includes at least one of, but not limited to, zinc-finger endonuclease (ZFN) technology, transcription activator-like effector nucleases (TALENs) technology, CRISPR/Cas9, and DNA homologous recombination technology.
The gene knockout technology is selected from complete gene knockout technology or conditional gene knockout technology.
It should be noted that any technique may be used as long as it utilizes a disease model in which the Lmbr1ll gene is not expressed or its expression is suppressed to obtain retinal vascular defects, and the technique falls within the scope of the present invention.
In an alternative embodiment, the conditional gene knockout technique is selected from the group consisting of Cre-LoxP gene knockout techniques. In an alternative embodiment, the complete knockout technique described above is performed in ES cells according to the principle of positive-negative selection.
In a preferred embodiment of the present invention, the above construction method comprises the step of using a DNA homologous recombination technology and a CRISPR/Cas9 technology to prevent or inhibit the expression of the exon 2 to exon 15 sequences of the Lmbr1l gene in a target animal, thereby obtaining an Lmbr1l knockout animal. In an alternative embodiment, the knockout of the gene can be evaluated by knocking out exon 2 to exon 15 sequences to evaluate the effect of the knockout on retinal angiogenesis, thereby providing a theoretical basis for exploring the mechanism of retinal angiogenesis.
In a preferred embodiment of the present invention, the Lmbr1l knockout animal is obtained by the following method: and mixing gRNA aiming at the 2 nd exon to the 15 th exon sequences of the Lmbr1l gene with the Cas9 mRNA, injecting the mixture into a fertilized egg of a target animal, and after the mixture is matured, obtaining the Lmbr1l gene knockout animal.
In a preferred embodiment of the present invention, the target animal is a non-human mammal.
In a preferred embodiment of the present invention, the non-human mammal includes, but is not limited to, a mouse, rat, horse, pig, monkey, dog, or ape.
For any non-human mammal, as long as it has the Lmbr1l gene, a corresponding retinal vascular disease model can be constructed by the method provided by the present invention. No matter what kind of non-human mammal is selected, the model of retinal vascular disease is constructed, and the model belongs to the protection scope of the invention.
The invention also provides application of the retinal vascular disease animal model obtained by the construction method of the retinal vascular disease animal model in the research of retinal vascular diseases, and the research aims at the diagnosis or treatment of non-diseases.
Including but not limited to studying pathogenic mechanisms of retinal vascular diseases, mechanisms of retinal detachment, regulatory mechanisms of Norrin/beta-catenin signaling pathway, and the like.
The invention also provides the application of the retinal vascular disease animal model obtained by the construction method of the retinal vascular disease animal model in early molecular screening medicines or screening medicines for targeted therapy of the retinal vascular disease.
The application comprises the following steps: administering a candidate agent to the animal model of retinal vascular disease; observing whether the animal model of the retinal vascular disease after the candidate drug is administered has the following changes, and if any one or more of the following changes appears, indicating that the administered candidate drug can be used as a drug for treating the retinal vascular disease:
(1) After the candidate drug is administered, the retinal vascular developmental defect of the retinal vascular disease animal model is inhibited or alleviated compared with that before the candidate drug is administered;
(2) After the candidate drug is administered, the vitreous vascular degeneration lag of the animal model of retinal vascular disease is improved compared with that before the candidate drug is administered;
(3) After the candidate drug is applied, the Norrin/beta-catenin signal channel which is abnormally activated in the retinal vascular disease animal model is recovered.
Of course, it should be noted that the above-mentioned changes are merely exemplary, and those skilled in the art can select suitable observation indexes according to actual conditions for the animal model crop research object of retinal vascular disease constructed by the method of the present invention, observe the changes of these indexes before and after the candidate drug is administered, and make reasonable judgment according to the change conditions of the indexes to indicate whether the administered candidate drug can be used as a drug for treating retinal vascular disease.
The invention has the following beneficial effects:
the invention provides a method for constructing a retinal vascular disease animal model, which can make an animal show that retinal blood vessels develop slowly and the density is reduced by inhibiting the expression of Lmbr1l gene or making the Lmbr1l gene not express, can construct the retinal vascular disease animal model with a retinal angiogenesis defect phenotype, and provides a model basis for researchers in the field to deeply research the pathogenesis of the retinal vascular disease, and to screen and screen effective target treatment medicines in the early clinical stage.
The invention provides the application of the retinal vascular disease animal model obtained by the model construction method in the research of retinal vascular diseases, the early molecular screening of drugs and the screening of drugs for targeted therapy of retinal vascular diseases, and has very wide application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of construction of Lmbr1l knockout mice and a graph of sequencing results;
FIG. 2 is a mouse genotype identification map;
FIG. 3 is a graph showing the result of staining the retinal vessel development of a mouse; (in the figure: ctrl-wild type mouse, lmbr1l -/- -Lmbr1l knockout mouse)
FIG. 4 shows the result of Western blotting of activation of Norrin/beta-catenin signal pathway.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. M.j. Goal, 1984); animal Cell Culture (Animal Cell Culture) (edited by r.i. freshney, 1987); methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
In the construction method of the mouse model of retinal vascular disease provided by this embodiment, a CRISPR/Cas9 technology is used to mix grnas for sequences from exon 2 to exon 15 of the Lmbr1l gene with Cas9 mRNA, and the mixture is injected into a fertilized egg of a target animal, and after development and maturation, the Lmbr1l knockout animal is obtained.
The construction method of the retinal vascular disease mouse model comprises the following steps in sequence:
(1) Designing a pair of gRNA targets for genome shearing:
Lmbr1l-L gRNA：5’-ACCCAGCCCTGGTACTGTGCTGG-3’；
Lmbr1l-R gRNA：5’-ACAACGGACTATGACAACGAAGG-3’。
referring to FIG. 1, the second exon to the fifteenth exon in the genomic DNA were excised. The Cas9 mRNA, lmbr1L-L gRNA, and Lmbr1L-R gRNA mixture was first injected into fertilized eggs of mice (purchased from cantonese biotechnology limited), which were then sequenced after birth to perform genotyping and identification. Obtaining a mouse heterozygote with knockout of the second exon to the fifteenth exon of Lmbr1l, namely Lmbr1l +/- A mouse.
FIG. 1B shows Lmbr1l before and after knockout +/+ Mice and Lmbr1l -/- The sequencing identification result of the mouse is shown in the figure.
(2) Mixing Lmbr1l +/- Mice and Lmbr1l +/- Mating the mice to obtain homozygous mice, i.e., lmbr1l -/- A mouse.
Experimental example 1
This experimental example identified the genotype of the mouse model with retinal vascular development defects constructed in example 1 to confirm that the Lmbr1l gene was knocked out in the constructed mouse model.
The mouse tail genome DNA is extracted by an alkaline cracking method. The mouse tail genomic DNA was genotyped by PCR amplification using the following primers:
Lmbr1l-loxp-F:5’-ATTTGCTCTCCAGATGGGATTCTT-3’；
Lmbr1l-loxp-R1:5’-GAGACTTGTATCCTTTCCTTCAGC-3’；
Lmbr1l-loxp-R2:5’-GCTGAGAGTAGGGAAGTCAGAGTA-3’。
the results of the assay are shown in FIG. 2A. FIG. 2A shows that homozygote conditioned knockout mice (Lmbr 1 l) -/- ) A477 bp PCR product was amplified in wild type mice (Lmbr 1 l) +/+ ) 544bp PCR product was amplified, in heterozygote mice (Lmbr 1 l) +/- ) Two PCR products of 477bp and 544bp were amplified.
The expression level of Lmbr1l RNA and the expression level of protein are respectively detected by RT-qPCR and Western blot:
the results of the experiments are shown in FIGS. 2B-D. In FIGS. 2B-D, lmbr1l is shown -/- Neither Lmbr1L Mrna (P < 0.0001) nor Lmbr1L protein levels (P = 0.0008) were essentially detectable in mice, with differences reaching very significant levels compared to wild type.
Experimental example 2
This experimental example was performed to phenotypically identify the mouse model of retinal vascular development defect constructed in example 1.
(1) And (3) identifying retinal vascular development defects of the Lmbr1l knockout mice.
Eyes were harvested at day five postnatal (P5) from littermate wild type and example 1Lmbr1l knockout mice and retinas were dissected for retinal plating. The vessels were stained with the vascular specific antibody Isonectin-B4 (Life technologies, 15120630) and observed for horizontal growth of vessels in the superficial retina. P0-P7 days, primary blood vessels grow from the optic disc along the Nerve Fiber Layer (NFL) to form superficial blood vessels; starting on day P7, the shallow plexus of the Ganglion Cell Layer (GCL) germinated vertically, forming the deep plexus of the Outer Plexus (OPL) (day P12), followed by the formation of the intermediate plexus (INL) (day P12-15).
Growth as shown in FIGS. 3A-D, it can be seen from FIGS. 3A-D that retinal vascular dysplasia in Lmbr1l knockout mice was observed at postnatal day 5 (P5) in mice compared to wild-type mice: the superficial blood vessels have slow development and low blood vessel density. In the figure: ctrl-wild type mouse, lmbr1l -/- Lmbr1l knockout mice.
The eyes of littermate wild type and Lmbr1l knockout mice from postnatal day 9 (P9) were harvested for cryo-sectioning, blood vessels were stained with Isonectin-B4, and deep vessel development was observed. As seen in FIG. 3E, at postnatal day 9 (P9) of the mice, retinal vessels developed vertically from the optic nerve junction to OPL and IPL in the wild type mice, while retinal vessels developed to OPL in the Lmbr1l knockout mice. Compared with wild mice, the Lmbr1l gene knockout mice have slow deep development of retinal vessels.
The retina is a high-metabolic tissue, consumes high oxygen, and forms complex blood vessels to provide oxygen and nutrients during the development of eyes. Retinal development occurs late in development, with the oxygen and nutrient requirements of early retinal development being dependent on choroidal and vitreous vessels. The development of the vitreous vessels reaches a maximum at the third day of birth (P3) of the mouse, and as the retinal vessels mature, they begin to gradually and completely degenerate.
The eyes of littermate wild type and Lmbr1l knockout mice were harvested at postnatal day 10 (P10) and dissected for vitreal vascular staining. The degeneration of the vitreous vessels was observed using DAPI (Cell Signaling Technology, 4083S).
The experimental results are shown in fig. 3F, and it can be seen from fig. 3F that: compared with a wild mouse, the Lmbr1l gene knockout mouse has slow retinal vascular development; and results in delayed vitreous vascular degeneration.
Example 3
This experiment example was conducted to study the pathogenic mechanism of the mouse model for retinal vascular development defect constructed in example 1, and to elucidate the mechanism of retinal vascular development defect caused by deletion of Lmbr1l gene.
The experimental results are shown in fig. 4, and it can be seen from fig. 4 that: compared with wild mice, the Lmbr1l knockout mouse Norrin/beta-catenin signal channel is activated. Lmbr1l -/- The expression of mouse Norrin/beta-catenin signal channel receptor FZD4 and co-receptor LRP5 is up-regulated; meanwhile, the level of P-GSK3 beta (Ser 9) is down-regulated, so that a degradation complex is disintegrated, and beta-catenin is accumulated in cytoplasm, so that the nucleus is entered to activate the expression of downstream genes, such as CyclinD1 and Glut1 genes.
In conclusion, through the CRISPR/Cas9 technology, an Lmbr1l gene knockout mouse model is constructed for the first time, and the pathogenesis of retinal vascular development defect diseases is explored at the gene level. The model mouse shows a phenotype of retinal vascular development defect.
The invention constructs the mouse model: the Lmbr1l knockout mouse can be used as an animal model of retinal vascular diseases and used for early molecular screening, mechanism research and screening of targeted therapeutic drugs of the retinal vascular diseases.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A construction method of a retinal vascular disease animal model is characterized by comprising the following steps: making non-human mammals by genetic engineering techniquesLmbr1lThe gene is not expressed or expression is inhibited.
2. The method for constructing an animal model of retinal vascular disease according to claim 1, comprising: making non-human mammals by genetic engineering techniquesLmbr1lExon 2 to exon 15 sequences of the gene are not expressed or are inhibited.
3. The method for constructing an animal model of retinal vascular disease according to claim 1 or 2, wherein the genetic engineering technique is any one technique or a combination of techniques of a gene editing technique, a gene knockout technique, and an RNA interference technique.
4. The method for constructing an animal model of retinal vascular disease according to claim 3, wherein the gene editing technology is selected from at least one of ZFN technology, TALEN technology, and DNA homologous recombination technology;
the gene knockout technology is selected from complete gene knockout technology or conditional gene knockout technology.
5. The method for constructing an animal model of retinal vascular disease according to claim 4, wherein the conditional gene knockout technique is selected from Cre-LoxP gene knockout technique.
6. The method of claim 4The construction method of the retinal vascular disease animal model is characterized in that the construction method comprises the steps of utilizing a DNA homologous recombination technology and a CRISPR/Cas9 technology to ensure thatLmbr1lExon 2 to exon 15 sequences of the gene are not expressed or are suppressed in the non-human mammal to obtainLmbr1lA knockout animal.
7. The method for constructing an animal model of retinal vascular disease according to claim 6, wherein the retinal vascular disease is expressed byLmbr1lThe gene knockout animal is obtained by the following method: will be directed toLmbr1lMixing gRNA of exon 2 to exon 15 sequences of gene with Cas9 mRNA, injecting the mixture into fertilized eggs of non-human mammals, and obtaining the mature gRNA after development and maturationLmbr1lA knockout animal.
8. The method for constructing an animal model of retinal vascular disease according to claim 1, wherein the non-human mammal is a mouse, rat, horse, pig, monkey, dog, or ape.
9. Use of the retinal vascular disease animal model constructed by the method for constructing the retinal vascular disease animal model according to any one of claims 1 to 8 in research of retinal vascular diseases, which is aimed at diagnosis or treatment of non-diseases.
10. The use of the animal model of retinal vascular disease constructed by the method for constructing the animal model of retinal vascular disease according to any one of claims 1 to 8 in the early molecular screening of drugs or in the screening of drugs for the targeted therapy of retinal vascular disease.
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