CN112980879A - Construction method and application of retinal vascular disease model - Google Patents
Construction method and application of retinal vascular disease model Download PDFInfo
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Abstract
The invention discloses a construction method and application of a retinal vascular disease model, and relates to the field of eye disease research. The expression of the Jup gene in the vascular endothelial cells of the target animal is not expressed or is inhibited through genetic engineering means, so that the target animal shows typical characteristics of retinal neovascular diseases. The target animal can be used as a retinal neovascular disease model for researching the retinal neovascular disease, can help to clarify the pathogenesis process and mechanism of the retinal neovascular disease, enrich the theoretical basis of the retinal neovascular disease, provide a new target for treating or preventing the retinal neovascular disease, and assist in the research and development of a retinal neovascular treatment means.
Description
Technical Field
The invention relates to the field of eye disease research, in particular to a method for constructing a retinal vascular disease model and application thereof.
Background
At present, there is no more effective treatment method for the clinical treatment of Retinal Neovascularization (RNV) except for laser photocoagulation and intraocular injection of anti-VEGF (vascular endothelial growth factor) drugs at the early stage of the disease. The existing research shows that the anti-VEGF treatment is not effective for all patients, and meanwhile, the anti-VEGF treatment has the defects of high price, repeated injection, increased infection risk of the patients and the like, so that the clinical application of the anti-VEGF treatment is limited. Therefore, it is a medical problem to be solved urgently that an effective animal model is constructed to search for a new effective treatment means or a new effective treatment target while the research on the pathogenesis of RNV is carried out. Currently, there is a lack of animal models available for the study of methods of RNV treatment.
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 method for constructing a retinal vascular disease model, which comprises the following steps: the Jup gene is not expressed or the expression is inhibited in the vascular endothelial cells of the target animal by the genetic engineering technology.
In a preferred embodiment of the present invention, the construction method comprises using genetic engineering techniques to make the exon 1 Jup gene not be expressed or its expression be inhibited in the vascular endothelial cells of 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 gene knockout technology is Cre-loxp gene knockout technology.
In a preferred embodiment of the present invention, the above construction method comprises constructing Jup gene conditional knockout founder animal, and obtaining Jup gene conditional knockout homozygote founder animal; then, the homozygote initial animal is mated with a transgenic animal carrying Pdgfb-Cre to obtain a transgenic animal with the Jup gene conditionally knocked out.
In a preferred embodiment of the invention, the conditional knock-out of Jup gene is achieved by inducing the expression of Cre recombinase.
In a preferred embodiment of the invention, the Cre recombinase is induced by tamoxifen.
In a preferred embodiment of the invention, the construction of the Jup gene conditional knockout founder animal comprises adding loxp at two ends of the 1 st exon of Jup gene of the target animal by using DNA homologous recombination technology and CRISPR/Cas9 technology.
In a preferred embodiment of The invention, The first-constructed animal for constructing a conditional knock-out of Jup gene is purchased from Jackson Laboratory, USA (The Jackson Laboratory), under The strain name: B6.129-Jup < tm1Kem >/J.
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 is selected from any one of a mouse, a rat, a horse, a pig, a monkey, a dog and a ape.
The retinal vascular disease animal model obtained by the construction method is applied to early molecular screening of medicines and screening of medicines for targeted therapy of familial exudative vitreoretinopathy.
The invention has the following beneficial effects:
the invention ensures that Jup gene in the vascular endothelial cells of the target animal is not expressed or the expression is inhibited by a genetic engineering means, thereby ensuring that the target animal shows the typical characteristics of retinal neovascular diseases. The target animal can be used as a retinal neovascular disease model for researching the retinal neovascular disease, can help to clarify the pathogenesis process and mechanism of the retinal neovascular disease, enrich the theoretical basis of the retinal neovascular disease, provide a new target for treating or preventing the retinal neovascular disease, and assist in the research and development of a retinal neovascular treatment means.
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 the construction of Jup conditional knockout mice for vascular endothelial cells;
FIG. 2 is a schematic representation of the breeding scheme of Jup vascular endothelial cell conditional knockout mice;
FIG. 3 is a graph of Jup results of studies on the efficiency of knockdown in mouse blood vessels;
FIG. 4 is a graph showing the results of examination of retinal spreads in Jup knockout mice;
FIG. 5 is a diagram showing the results of immunohistochemical staining of an eyeball section and a vitreous vessel of a mouse with a specific Jup gene knockout for vascular endothelial cells.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
The invention provides a method for constructing a retinal vascular disease model, which comprises the following steps: the Jup gene is not expressed or the expression is inhibited in the vascular endothelial cells of the target animal by the genetic engineering technology.
The inventor firstly finds that the inhibition of the expression or the non-expression of the Jup gene can lead to the retardation of the development of superficial and deep retinal vessels, local leakage and the reduction of the vitreous vascular degeneration of target animals.
The JUP (junction plakoglobin) gene maps to chromosome 17q21, and its encoded protein plakoglobin (also known as γ -Catenin) was first identified in desmoplasma by Franke et al, and was subsequently confirmed to be present in desmosomes (desmosomes) and cell adhesion junctions (adherens junctions) by a series of experiments. Studies have shown that JUP interacts not only with desmoglein, desmosomal cadherins and desmosollins in desmoplakin, but also with adhesion connexins beta-catenin, alpha-catenin and cadherins, which act as the sole component connecting desmosomes and adherens junctions, coordinating desmosomal and cell adhesion junction interactions in cells, maintaining normal growth and proliferation of cells.
In a preferred embodiment of the present invention, the construction method comprises using genetic engineering techniques to make the exon 1 Jup gene not be expressed or its expression be inhibited in the vascular endothelial cells of the target animal.
Jup gene has 14 exons. In other embodiments, other exons of the Jup gene can also be selected to be knocked out or silenced such that the Jup gene is not expressed or is inhibited in vascular endothelial cells of 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.
The gene editing technology is selected from at least one of DNA homologous recombination technology, CRISPR/Cas9 technology, ZFN technology, and TALEN technology.
In a preferred embodiment of the present invention, the gene knockout technology is Cre-loxp gene knockout technology.
In a preferred embodiment of the present invention, the above construction method comprises constructing Jup gene conditional knockout founder animal, and obtaining Jup gene conditional knockout homozygote founder animal; then, the homozygote initial animal is mated with a transgenic animal carrying Pdgfb-Cre to obtain a transgenic animal with the Jup gene conditionally knocked out.
In a preferred embodiment of the invention, the conditional knock-out of Jup gene is achieved by inducing the expression of Cre recombinase.
In a preferred embodiment of the invention, the Cre recombinase is induced by tamoxifen.
Transgenic animals of Pdgfb-Cre were donated by London University College London, UK (transgenic mouse PDGF β -Cre). The transgenic animal expresses a specially modified Cre gene expression driven by a Platelet-derived growth factor (PDGF) beta promoter specific to vascular endothelial cells. The modified Cre protein is normally positioned in cytoplasm, and can enter cell nucleus after being combined with Tamoxifen (Tamoxifen) to identify LoxP sites on genome, so that gene knockout is realized.
In other embodiments, the above construction method further comprises mating the transgenic animal with conditional knock-out Jup gene with another transgenic animal with conditional knock-out Jup gene, and screening for homozygous animals with conditional knock-out of Jup gene.
In a preferred embodiment of the invention, the construction of the Jup gene conditional knockout founder animal comprises adding loxp at two ends of the 1 st exon of Jup gene of the target animal by using DNA homologous recombination technology and CRISPR/Cas9 technology.
In a preferred embodiment of the invention, the founder animal constructed for conditional knock-out of the Jup gene is purchased from Jackson, USA.
In other embodiments, the Jup gene may be knocked out by other genetic engineering methods, and the methods are not limited to the Cre-loxp knock-out method described above. For example, the Jup gene can be knocked out by TALEN technology.
In a preferred embodiment of the present invention, the target animal is a non-human mammal. It should be noted that, for any non-human mammal, as long as it has Jup gene, the 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.
In a preferred embodiment of the present invention, the non-human mammal is selected from any one of a mouse, a rat, a horse, a pig, a monkey, a dog and a ape.
The application of the retinal vascular disease animal model obtained by the construction method in the research of retinal vascular diseases aims at the diagnosis or treatment of non-diseases. Such as in retinal vascular disease flow regulation.
The retinal vascular disease animal model obtained by the construction method is applied to early molecular screening of medicines and screening of medicines for targeted therapy of familial exudative vitreoretinopathy.
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 being administered with the candidate drug has the following changes, and if any one or more of the following changes are generated, 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 proliferation of vascular endothelial cells in the animal model of retinal vascular disease is increased compared with that before the candidate drug is administered;
(3) after administration of the candidate drug, the vitreous vascular degeneration lag of the animal model of retinal vascular disease is improved compared to that before administration of the candidate drug.
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 not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
In the embodiment, a mouse is taken as a target animal, and a mouse model of the retinal neovascular disease is constructed by using a Cre-loxp conditional knockout system. The tool mice were purchased and donated by University College London, england. Jup carrying loxp sequenceloxp/loxpMice were purchased from Jackson laboratories, USA (https:// www.jax.org/strain/017575).
The construction method of the model comprises the following steps:
(1) mice carrying loxp sequence (Jup)loxp/loxp) Mating with a tool mouse carrying Pdgfb-Cre, obtaining a mouse carrying both loxp sequence and Cre, denoted Juploxp/+Pdgfb-Cre, followed by Juploxp/loxpAnd Juploxp/+-Pdgfb-Cre mating to obtain Juploxp/loxpPdgfb-Cre mice (see Panel A in FIG. 1, where Jup second exon was knocked out in mouse vascular endothelial cells using the Cre-loxp system, and FIG. 2 shows a propagation roadmap of Jup vascular endothelial cell conditional knockout mice).
Juploxp/loxpPdgfb-Cre mice require intraperitoneal injection of 50 μ g Tamoxifen (Tamoxifen) on postnatal days 1, 2, 3, 5 to induce Jup knockdown, and homozygous Jup vascular endothelial cell-specific knockdown can be expressed as JupiECKO/iECKO(see fig. 1, panel B).
(2) Tamoxifen induction method: preparing an absolute ethanol solution of tamoxifen, wherein the concentration is 10mg/mL, and storing the solution at the temperature of 4 ℃ in a dark place. Before use, a tamoxifen absolute ethanol solution was prepared: and (3) vortexing and uniformly mixing the tamoxifen corn oil solution with the corn oil volume ratio of 1:9, sucking the tamoxifen solution by using a 1mL sterile syringe, and performing intraperitoneal injection on the mice.
Example 2
This example uses Western blotting to detect the expression of JUP protein in lung tissue at 25 th day after birth of target animal.
(1) Collecting lung tissues of littermate wild type and knockout type target mice, putting the lung tissues into 1 xPBS containing protease inhibitor, and ultrasonically cracking the lung tissues on ice for 10 minutes; centrifuging at 16000g speed at 4 deg.C for 10 min, transferring the supernatant to another clean centrifuge tube, adding protein loading buffer solution, mixing, and heating at 95 deg.C for 5 min.
(2) After the samples are cooled, 20 mu l of protein samples are respectively taken, and electrophoresis is carried out for 25 minutes at constant pressure of 70v and 40 minutes at 160v by adopting 10% separation gel.
(3) The film transfer conditions are as follows: the transfer was done at constant flow 0.3A for 1 hour 30min, followed by rinsing the membrane once with deionized water and blocking with a 5% skim milk block at room temperature for 1 hour.
(4) The membrane was placed in primary antibody in 5% skim milk blocking solution and incubated overnight in shaker at 4 ℃. The next wash was performed 3 times for 5 minutes with 1 XTSST, and HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour.
(5) And (3) dropwise adding a proper amount of chemiluminescent substrate onto the membrane, and observing a protein band in a chemiluminescent detector.
Protein bands were detected by ImageJ and analyzed for statistical differences by t-test, as shown in panel a of figure 3 (JUP for experimental group, GAPDH for internal reference). As can be seen from the B-plot in FIG. 3, the expression level of JUP protein in lung was decreased by about 70%, indicating that the knockout target mouse could inhibit the expression of the target protein, i.e., the mouse model constructed in example 1 was successful.
Example 3
The present embodiment utilizes the retinal crawler (holimount) technique to detect retinal vascular development.
After the mice were sacrificed, the eyeballs were taken and fixed in a 4% PFA solution at room temperature for 20 minutes (a 24-well plate may be used as a container), followed by soaking in 1 × PBS for 15 minutes. A hole was then punctured in the center of the cornea with a 30G needle, the entire cornea was trimmed away with an ophthalmic scissors, and the lens was removed. The whole sclera was then torn open with forceps leaving the inner retina and cut to clover shape, laid flat to remove more tissue, imbibed with 4% PFA and held at 4 ℃ for a further 24 h. The next day, the whole was transferred to 0.4% PFA and stored at 4 ℃.
The method of Wholemuunt dyeing: the whole was removed and washed 3 times with 1 × PBS for 5 minutes. Followed by blocking with 100. mu.L of blocking solution (5% fetal bovine serum, 0.3% Triton X-100, 0.03% sodium azide in PBS) for 1 hour at room temperature. The erythrocyte-specific antibody Ter-119 and Isonectin B4 (a marker capable of specifically recognizing a vascular structure) are prepared into a mixed solution according to an appropriate dilution ratio, added into the blocked wholemount (100 μ L of each whoulemount), and incubated at 4 ℃ overnight. The next day, the whole was removed, washed 3 times with 1 × PBS for 5 minutes, and pre-formulated fluorescent secondary antibodies (100 μ L each for different antibodies) were added and incubated at room temperature for 4 h. Finally, the cells were washed 3 times with 1 XPBS for 5 minutes, and stained with fluorescent dye.
Results referring to fig. 4, the results of staining with isonectin B4 and Ter119 in retinal slides show that after Jup gene was knocked out specifically by vascular endothelial cells, retinal blood vessels develop slowly with peripheral neovascularization, and local red blood cell leakage of blood vessels appears, which is indicated by the appearance of a large amount of Ter119 signals outside the blood vessels.
Example 4
Frozen sections of the eyeball were stained and vitreous vessel (hyaloid) was separately stained.
(1) Preparing frozen eyeball sections: after the mice were sacrificed, the eyeballs were taken and fixed in a 4% PFA solution at room temperature for 20 minutes, followed by puncturing the center of the cornea with a 30G needle and cutting the entire cornea with an ophthalmic scissors. The fixed eyeballs were rinsed once with 1 × PBS, placed in 30% 1 × PBS sucrose solution, and dehydrated at 4 ℃ for 2 hours. Then, the crystal of the eyeball was taken out, water was sucked off, and the eyeball was placed in the same direction in a frozen section embedding cassette equipped with OCT, after solidification at-80 ℃ and storage at-20 ℃. All sections were 10 μm thick.
(2) Vitreous vessel separation: mouse eyeballs were taken and fixed in a 24-well plate filled with 4% PFA at room temperature for 15 minutes, followed by puncturing the cornea and completely removing the cornea, and further fixed in 4% PFA at 4 ℃ for 2 hours. The solid gelatin is prepared into 5% PBS solution, and boiled at 50 ℃ until the gelatin is completely dissolved. The corneal-removed eyeballs were rinsed once with 1 × PBS, soaked in 24-well plates containing 5% gelatin solution, and incubated overnight at 37 ℃. The next day, the 24-well plate was taken out and put in a refrigerator at 4 ℃ until the gelatin was solidified. Subsequently, the eyeball with the solid gel wrapped therein is extracted, and the sclera is torn away by the method for preparing a holemount, leaving the inner lens and the retina portion. The lens and retina were then carefully removed on a pre-cooled slide, leaving only the hyaloid vessels. Finally, hyaloid vessels were carefully cut into clover shapes, the entire slide was placed on a 50 ℃ metal bath, and a circle was drawn around the hyaloid vessels with an immunohistochemical pen. Hyaloid vessels were rinsed with 1x PBS on a 50 ℃ metal bath to remove as much residual gelatin as possible.
(3) Frozen sections and hyaloid staining method: circle the section/hyaloid with immunohistochemical pen, dry at 37 deg.C, put into immunohistochemical wet box, rinse 3 times with 1 × PBS, once for 5 minutes. 50 μ L of blocking solution (same as in the staining of retinal crawl plates) was added dropwise to each section and blocked at room temperature for half an hour. Primary antibody in blocking solution was then added and incubated overnight at 4 ℃. The next day the wet box was removed, the primary antibody discarded, and rinsed 3 times with 1 × PBS for 5 minutes each time. Fluorescent secondary antibody in the blocking solution was added and incubated at room temperature for 1 hour. The sections were taken out, the secondary antibody was discarded, rinsed 3 times with 1 × PBS for 5 minutes each time, 10 μ L fluorocount encapsulated tablets were dropped on each section, and the fluorescence was observed by covering with a cover glass.
The staining results of the eyeball sections are shown in a graph A in FIG. 5, and it can be seen that at the 9 th postnatal day of the mice, retinal vessels of wild-type mice begin to develop deeply, while vascular development of knockout mice is limited to the surface of vessels, and development is obviously retarded.
The vitreous vessel staining results are shown in panel B of fig. 5, and at postnatal day 9 in mice, the vitreal vessels were more abundant in knockout mice than in wild type mice, indicating a slower regression.
Panel A in FIG. 5: results of retinal sections from wild type and Jup vascular endothelial cell-specific knockout mice at day nine postnatal. Wherein, red is the blood vessel marker IsonectinB 4, and blue is the cell nucleus marker DAPI.
Panel B in fig. 5: the vitreal vascular degeneration results of wild type and Jup vascular endothelial cell-specific knockout mice at day nine postnatal.
In summary, in the embodiment of the present invention, a mouse is taken as an example, Jup gene is specifically knocked out in vascular endothelial cells of the mouse by Cre-loxP knock-out technology, so that the mouse shows phenotypes such as delayed development of superficial and deep retinal vessels, local leakage, slowed degradation of vitreous vessels, and the like. These features are typical of retinal neovascular disease. It is fully demonstrated that conditional knock-out of the Jup gene in vascular endothelial cells can cause target animals to exhibit retinal neovascular disease. The vascular endothelial cell conditional knock-out Jup gene animal can be used as a retina neovascular disease model. The disease model can be used in the fields of research on retinal neovascular diseases and the like, and provides a new model for research on the diseases, such as the pathogenesis process, mechanism and screening of related medicines.
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 method for constructing a retinal vascular disease model is characterized by comprising the following steps: the Jup gene is not expressed or the expression is inhibited in the vascular endothelial cells of the target animal by the genetic engineering technology.
2. The method of claim 1, wherein the method comprises genetically engineering the exon 1 sequence of Jup gene to be non-expressed or repressed in vascular endothelial cells of the target animal.
3. The method of claim 1 or 2, wherein 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.
4. The method of claim 3, wherein the gene knockout technique is a Cre-loxp gene knockout technique.
5. The method of claim 4, wherein the method comprises constructing Jup gene conditional knockout founder animals and obtaining Jup gene conditional knockout homozygous founder animals; then, the homozygote initial animal is mated with a transgenic animal carrying Pdgfb-Cre to obtain a transgenic animal with the Jup gene conditionally knocked out.
6. The method of claim 5, wherein the method of construction is a conditional knock-out of Jup gene by inducing Cre recombinase expression;
preferably, the expression of Cre recombinase is induced by tamoxifen.
7. The construction method of claim 5, wherein constructing Jup conditional knockout founder comprises adding loxp at two ends of the 1 st exon of Jup gene of target animal by using DNA homologous recombination technology and CRISPR/Cas9 technology.
8. The method of claim 7, wherein the target animal is a non-human mammal.
9. The method according to claim 8, wherein the non-human mammal is selected from the group consisting of a mouse, a rat, a horse, a pig, a monkey, a dog and a ape.
10. Use of the animal model of retinal vascular disease obtained by the construction method according to any one of claims 1 to 9 in early molecular screening of drugs and in screening of drugs for targeted therapy of familial exudative vitreoretinopathy.
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