阅读说明：本技术 视网膜新生血管疾病动物模型、构建方法及其应用 (Retina neovascular disease animal model, construction method and application thereof ) 是由 庄菁 余克明 陈熹 陈水莲 于 2021-03-31 设计创作，主要内容包括：本发明属于生物医药领域,具体涉及视网膜新生血管疾病动物模型、构建方法及其应用。本发明采用源于M1型活化小胶质细胞的外泌体,将该外泌体注射于动物的眼球组织,诱导产生视网膜新生血管,从而构建视网膜新生血管疾病动物模型,本发明利用M1型活化小胶质细胞的外泌体直接促进新生血管产生,同时又促进原位静息的小胶质细胞活化及富集,促进血管信号而间接促进视网膜新生血管的产生。这种炎症-新生血管的病理模式符合大多数视网膜新生血管产生的病理过程,使得本发明构建的视网膜新生血管疾病动物模型具有良好的代表性、稳定性和病理持续性,具有极高的推广应用价值。(The invention belongs to the field of biological medicines, and particularly relates to an animal model of a retinal neovascular disease, a construction method and application thereof. The invention adopts an exosome derived from M1 type activated microglia, and the exosome is injected into eyeball tissues of animals to induce and generate retinal neovascularization, thereby constructing an animal model of the retinal neovascularization disease. The pathological mode of the inflammation-neovascularization accords with the pathological process of most retinal neovascularization, so that the retinal neovascularization disease animal model constructed by the invention has good representativeness, stability and pathological persistence and extremely high popularization and application values.)

1. A method for constructing an animal model of retinal neovascular disease is characterized in that exosome derived from M1 type activated microglia is injected into eyeball tissue of an animal to induce and generate retinal neovascular.

2. The method for constructing an animal model of retinal neovascular disease according to claim 1, wherein the exosome derived from M1 type activated microglia is injected into a vitreous cavity of an eyeball tissue of an animal to induce retinal neovascular.

3. The method for constructing an animal model of retinal neovascular disease according to claim 1 or 2, wherein the single injection amount of the exosome is: 0.5-1 ug/uL; the injection frequency is as follows: injecting for 1 time every 2-3 days, wherein the injection duration time is as follows: 5-6 weeks.

4. The method for constructing an animal model of retinal neovascular disease according to claim 1 or 2, wherein the M1-type activated microglia are induced by microglia through LPS.

5. The method for constructing the animal model of retinal neovascular disease according to claim 4, wherein the amount of LPS for inducing microglial cell generation M1-type activated microglia is 50-100 ng/mL, and the induction time is 24-48 h.

6. The method for constructing an animal model of retinal neovascular disease according to claim 1 or 2, wherein the exosomes of M1 type activated microglia are obtained by differential ultracentrifugation or extracted by an exosome extraction kit.

7. The method for constructing an animal model of retinal neovascular disease according to claim 1 or 2, wherein the animal is an adult mouse aged 6 to 8 weeks or an adult rabbit aged 8 to 12 weeks.

8. An animal model of retinal neovascular disease constructed by the method of claim 1 or 2.

9. Use of the animal model of retinal neovascular disease according to claim 8 in the study of retinal neovascular disease.

10. Use of the animal model of retinal neovascular disease according to claim 8 in the development of a medicament for preventing or treating retinal neovascular disease.

Technical Field

The invention belongs to the field of biological medicine, relates to the field of experimental animal models for drug screening, and particularly relates to an animal model for retinal neovascular diseases, a construction method and application thereof.

Background

Retinal neovascularization belongs to abnormal vascular proliferation which mainly occurs in central retinal vein occlusion, age-related macular degeneration, diabetic retinopathy and the like, and is one of the main causes of blindness in clinical cases. To date, the molecular mechanism of retinal neoangiogenesis is not completely understood, and therefore, the construction of animal models related thereto is an important basis for the study of the mechanism of the disease, the prevention of the disease, and the development of drugs.

There are three main types of retinal neovascular models: retinopathy of prematurity model, diabetic retinopathy model, retinal vein occlusion animal model.

1) Oxygen induced retinopathy model (OIR model): the model generates new-born mouse retina new vessels through a high-oxygen relatively-anoxic breeding environment, namely, a mouse born on the 7 th day and a lactating mother mouse are placed in a closed oxygen box together, the oxygen concentration is maintained at 75% +/-2%, the mouse and the lactating mother mouse are placed back to the indoor standard environment on the 12 th day and are bred under the standard environment (the oxygen concentration is 21%), the mouse retina new vessels are generated at the moment, the peak is reached after 5 days, then the new vessels begin to disappear, and the new vessels almost completely disappear after one week. The molding method is very simple in operation, high in molding success rate and good in repeatability, and is widely applied to research of retinal neovascularization in recent years. However, this model has the following drawbacks:

the OIR model induces acute retinal neovascularization in a very short period of days by short-term hyperoxia and hypoxia, and the neovascularization only resembles human retinopathy of prematurity and is inconsistent with the course of other chronic retinal neovascularization and even the molecular mechanism of the pathogenesis of the neovascularization is different.

Secondly, the model has the defects of high animal mortality, low film forming rate, instability and the like in the actual molding process and is also limited by specific equipment.

The OIR model reflects the state of the retinal neovasculature of the newborn mice more, and has poor representativeness to the retinal neovasculature of the adult mice.

The oxygen-induced retinal neovascularization model can be naturally faded away within 1-2 weeks, and the characteristic is not beneficial to partial long-time intervention research.

2) Diabetic retinopathy neovascular model: in diabetic retinopathy, retinal neovascularization is a major manifestation. At present, many animal models are used for researching diabetic retinopathy and complications, and the animal models mainly include the following 3 types: drug or diet inducible, transgenic or gene knockout, and spontaneous heritability.

Advantages and disadvantages of the evoked model: the method has the advantages of short time consumption, low cost, simple method, good repeatability and capability of batch molding in a short time, and is the most common model at present. The disadvantage is that the drug has some toxic damage to other tissues; individual differences between animals; the risk of death of animals is greater; complete retinopathy neovascularisation cannot occur.

Advantages and disadvantages of transgenic or knockout models: the advantages are that the pathological changes are caused by the transferred exogenous gene, the etiology is clear at the gene level of the disease, and the difference of the expression among model animals is small. The defects are that the model mainly aims at the pathological changes caused by certain specific gene mutation, so that the model cannot become a broad-spectrum animal model; moreover, the mold is complicated to operate, expensive, and not suitable for mass molding, thereby limiting its wide application.

The advantages and disadvantages of the spontaneous diabetes model: the model animal is not subjected to any conscious manual treatment, and most inbred line pure breed animals with spontaneous diabetes tendency are adopted and fed according to feeding conditions to form a model spontaneously, which is closest to the pathogenesis process of human diseases. The advantage of such models is that they have a homogeneous genetic background, control environmental factors, and allow genetic analysis of this multifactorial disease. The disadvantage is frequent homologus reproduction and monogenic inheritance, which makes the genetic homogeneity of diabetes development different from that of human beings. Meanwhile, the requirements on the feeding and breeding conditions are high, and the price is high.

Generally, the diabetic animal model is a systemic disease, so the induced retinal neovascularization cannot be simply regarded as simple retinal neovascularization, various mixed factors need to be considered, and the representativeness of the diabetic animal model to a specific pathological process is poor.

3) Animal model of retinal vein occlusion: temporal inconsistencies and poor stability occurred in the various studies, some of which indicated that no significant retinal neovascularization could be induced even if significant venous ischemia and hemodynamic changes occurred (1.Guthoff Rainer, Meigen Thomas, Hennemann Kathrin et al, company of great importance and triammonolone for treatment of vascular disease secondary to branched vascular disease in a patient-treated analysis, [ J ]. Ophthalmologica,2010,224: 319-24.; 2.Yar Atiye Seda, Menev Seratrad, Dogan Irem et al, investigation of ocular vascular disease-treated and vascular disease in biological reaction, Fowlett-packard et al, evaluation of ocular tissue culture and experimental reaction, Fowlett-treated J.: 15. J..

Disclosure of Invention

Aiming at the problems of the existing animal model of the retinal neovascular disease, the invention aims to provide the animal model of the retinal neovascular disease with strong modeling stability, short induction period and good representativeness and the construction method and the application thereof based on the common pathological feature of the retinal neovascular disease, namely the microglial cell activation.

Based on the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a method for constructing an animal model of a retinal neovascular disease, which comprises injecting exosomes derived from M1 type activated microglia into eyeball tissues of an animal to induce and generate retinal neovessels.

The research team of the inventor finds that in various existing retinal neovascularization animal models, retinal microglia are abnormally aggregated and activated, and confirms that exosomes derived from M1 type activated microglia can directly promote the generation of neovascularization for the first time, and can indirectly promote the generation of retinal neovascularization by promoting the activation of in-situ resting microglia and enriching and promoting vascular signals, and the inflammation-neovascularization pathological mode accords with most pathological processes generated by retinal neovascularization, so that the animal model constructed by inducing the generation of the retinal neovascularization by using the exosomes derived from the M1 type activated microglia has good representativeness on the retinal pathological processes.

In addition, compared with a classical OIR model, the construction of the OIR model is limited by high oxygen box equipment, so that a plurality of research teams cannot establish the OIR model, and the application of the OIR model is limited; the invention is based on the induction of the exosome to generate the retinal neovascularization, and can separate the exosome from the M1 type activated microglia by adopting the separation mode of the conventional exosome, the acquisition mode of the exosome is simple, and the whole model construction process does not depend on specific equipment, so the invention has better popularization and application prospect.

Further, exosomes derived from M1 type activated microglia were injected into the vitreous cavity of animal eyeball tissue to induce retinal neovascularization.

The injection needle can enter the vitreous cavity only by penetrating the sclera, the pigment epithelium and the local retina at the outer side by adopting the vitreous cavity injection, and meanwhile, the vitreous cavity injection only penetrates the retina at the needle inserting part, so that the retinal detachment is avoided, the damage to retinal cells is small, the inflammatory response is light, and the vitreous cavity injection has the advantages of simple operation, small damage and wide action range.

Further, the single injection amount of exosomes was: 0.5-1 ug/uL; the injection frequency is as follows: injecting for 1 time every 2-3 days, wherein the injection duration time is as follows: 5-6 weeks.

In the invention, in about 6 weeks, the animal is induced to generate the retinal neovascular by periodically injecting exosome into animal eyeball tissues (such as a vitreous cavity), and compared with the prior retinal neovascular disease animal model, such as diabetic retinal neovascular disease animal model induced by streptozotocin, the induction period is usually more than 6 months, the method for constructing the animal model remarkably shortens the induction time of the retinal neovascular disease, namely remarkably shortens the construction period of the retinal neovascular disease animal model.

Further, M1 type activated microglia were induced by LPS from microglia.

Furthermore, the amount of LPS for inducing the microglia to generate the M1 type activated microglia is 50-100 ng/mL, and the induction time is 24-48 h.

LPS activates TLR4 receptor on microglia cell membrane, downstream activates NF-kB signal channel, and induces microglia to generate M1 type activation.

Further, exosomes of M1 type activated microglia were obtained by differential ultracentrifugation or extracted from exosome extraction kits.

The exosome for inducing the retinal neovascularization can be extracted by an exosome extraction kit or obtained by differential ultracentrifugation separation by an ultracentrifuge, and the isolated exosome can be injected into animal eyeball tissues such as vitreous bodies to construct an animal model of the retinal neovascularization diseases.

Furthermore, the animal is an adult mouse of 6-8 weeks old or an adult rabbit of 8-12 weeks old, or other commonly used adult animals in animal experiments.

The invention takes the adult mouse or the rabbit as an induction object to construct the animal model of the retinal neovascular disease, and because the adult animal does not relate to a developmental factor in the induction process, the constructed animal model of the adult has better stability and representativeness and higher research value compared with the animal model of the young (such as an OIR model).

In a second aspect, the invention provides an animal model of retinal neovascular disease constructed by the above method.

The animal model of the retinal neovascular disease constructed by the method has stable model building, the success rate is over 80 percent, and the retinal neovascular disease can stably exist after continuous observation for 1-2 months, so that the method has better stability and pathological state persistence.

In a third aspect, the invention provides an application of the above animal model of retinal neovascular disease in research of retinal neovascular disease.

Aiming at the fact that the molecular mechanism of the retinal neovascular disease is not completely clear at present, the retinal neovascular disease animal model constructed by the invention has good stability and persistence of pathological states, and can provide an important basis for the research of the pathogenesis of the retinal neovascular disease.

In a fourth aspect, the invention provides an application of the above animal model of retinal neovascular disease in the development of a medicament for preventing or treating retinal neovascular disease.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention firstly induces and generates retina neovascularization by an exosome of an activated microglia to construct an animal model, the exosome directly promotes the generation of the neovascularization, simultaneously promotes the activation and enrichment of in-situ resting microglia, promotes vascular signals to indirectly promote the generation of the retina neovascularization, and the pathological mode of inflammation-neovascularization accords with most pathological processes of retina neovascularization, so that the animal model of the retina neovascularization diseases constructed by the method has good representativeness.

(2) The method for constructing the animal model of the retinal neovascular disease does not depend on specific equipment, the exosome is separated by ultracentrifugation with an exosome extraction kit or an ultra-high-speed centrifuge, and the animal model of the retinal neovascular disease can be automatically constructed in a laboratory according to the method, so that the method has good popularization and application values.

(3) The animal model takes the adult animal as a construction object, reduces the influence of development factors compared with a disease model of a young animal, has higher stability, persistence and good representativeness, and has higher research value and application value.

(4) The method for constructing the animal model of the retinal neovascular disease by using exosome induction has the induction time of 5-6 weeks, namely the animal model with good stability and representativeness can be constructed within 6 weeks, and the induction time in the model construction process is obviously shortened.

In conclusion, the invention has the advantages of short period, high success rate and good representativeness for constructing the animal model of the retinal neovascular disease, and the constructed animal model has good stability and persistence of pathological states, thereby having wide application prospects in aspects of researching pathogenesis of the retinal neovascular disease, developing drugs for preventing or treating the retinal neovascular disease and the like.

Drawings

FIG. 1 is a diagram of the identification of exosomes derived from M1 type activated BV2 cells;

FIG. 2 is a graph of the identification of retinal neovascularisation induced in vivo by exosomes derived from M1-type activated BV2 cells;

FIG. 3 is a graph showing the pathological persistence after molding.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 construction of an animal model for retinal neovascular disease

In this embodiment, a method for constructing an animal model of retinal neovascular disease is described by taking microglia BV2 as an example, which specifically includes the following steps:

1. microglial cell BV2 activation

Culturing BV2 cells in a culture solution until the cells are in a logarithmic growth phase, replacing the culture solution with a culture solution containing 100ng/mL LPS, and culturing for 24-48 h to obtain an M1 type activated BV2 cell suspension, wherein the BV2 cells are purchased from a national experimental cell resource sharing platform, and the LPS lipopolysaccharide has a sigma L4391 product number.

2. Extraction and identification of exosome

(1) Exosome extraction

Exosomes of M1 type activated BV2 cells were isolated using exosome extraction kit or exosomes were isolated from M1 type activated BV2 cell suspension by differential high-speed centrifugation as follows.

Centrifuging M1 type activated BV2 cell suspension for 10min at 300 Xg, 10min at 2000 Xg, 30min at 10000 Xg and 4 ℃, filtering by a 0.22 μ M filter screen to remove residual cells, then centrifuging the suspension for 70min at 100000 Xg and 4 ℃ (BecKman Coulter, California, USA), collecting precipitate, resuspending the precipitate with PBS, and then centrifuging for 70min at 100000 Xg and 4 ℃; finally, the precipitate was resuspended in 100. mu.L of PBS to obtain an exosome suspension derived from M1 type activated microglia BV2, which was designated as M1-EXO.

The exosome isolated from this microglia cell by the above-described method was designated as M0-EXO, using the microglia cell BV2 which had not been activated with LPS as a control.

(2) Identification of exosomes

3% (v/v) glutaraldehyde was added to each of the exosome suspensions M1-EXO and M0-EXO, dropped into a carbon-coated copper grid, washed repeatedly with distilled water after 5min, stained with 4% uranyl acetate for 10min, treated with a 1% methylcellulose solution for 5min, and after the grid was dried, imaged at 80KV (JEOL-1200EX) using a TEM 1011 transmission electron microscope, and the results are shown in FIG. 1A, where M1-EXO and M0-EXO are biconcave "saucer-like" round vesicles.

The size of exosomes was analyzed using a Nanosight LM10 system (Nanosight Ltd, Navato, CA) equipped with fast video capture and particle tracking software, and the nanoparticle concentration and size distribution were calculated by measuring the rate of brownian motion, as shown in fig. 1B, with M0-EXO having a diameter of 132.2 ± 40.5nm and M1-EXO having a diameter of 136.4 ± 43.1 nm.

Further, the exosomes CD63, HSP70 and TSG101 were detected by Western blotting (Western Blot), and as a result, as shown in FIG. 1C, it was found that the exosomes CD63, HSP70 and TSG101 were expressed in M0-EXO and M1-EXO.

The above results demonstrate that both M0-EXO and M1-EXO have exosomal properties.

3. Animal model for constructing retinal neovascular disease by injecting exosome into mouse vitreous cavity

(1) Injecting an exosome M1-EXO derived from M1 activated microglia BV2 into a vitreous cavity of a mouse aged 6-8 weeks, wherein the single injection amount is 1 ug/uL; the injection frequency is 1 time per 3 days, and the injection duration is 6 weeks, so as to construct an animal model of the retinal neovascular disease. The concrete process of injecting exosome into mouse vitreous cavity is as follows:

the head of the mouse is fixed under an anatomical microscope, eyeballs are fully exposed, vascular tissues are avoided at the position 1mm behind the corneal limbus, and scleral puncture is performed by using a capsulorhexis needle. A 33G Hamilton microinjector was used to place an oblique penetration into the vitreous cavity at an angle of about 45 degrees from the puncture opening avoiding the lens. Slowly injecting the injector under the condition that the needle head does not move so as to slowly release the exosome, slowly pulling out the needle head after the injector stays for 30 seconds after the injection is finished, immediately closing the needle hole by using a sterilized cotton swab and covering a conjunctiva. Eyes were also surgically coated with a gel of hydroxymethyl cellulose to ensure wetting of the surface of the eyeball before the mice awakened. And (5) returning the mouse cage after the mouse is completely recovered. The electric blanket is used for heat preservation in the operation and the recovery process so as to ensure the smooth recovery of the experimental animal and avoid postoperative illness and death.

All animal experimental procedures strictly follow the Zhongshan university's Zhongshan ophthalmic center's regulations on animal application to ethics ', with ethics numbering SYXK (YUE) 2017-093. All animal feeding, experimental handling and sacrifice principles were carried out following the association of the society for research and ophthalmology (ARVO) with the requirements of animal experimental treatment regulations.

(2) Identification of animal model of retinal neovascular disease

To confirm whether the exosome derived from M1-type activated BV2 cell has the effect of inducing retinal neovascularization and further identify whether the constructed animal model of retinal neovascularization diseases generates retinal neovascularization, the method of injecting the exosome M1-EXO derived from M1-type activated microglia BV2 into the vitreous cavity of a mouse is referred to the step (1) above, PBS and M0-EX0 are respectively injected into the vitreous cavity of the mouse as controls, and a pseudo-surgery group is set to eliminate the operational influence, wherein the pseudo-surgery group is used for carrying out the same injection operation process but is not injected with any substance.

The four tests are PBS group, M0-EX0 group, M1-EXO group and SHAM operation group (SHAM), and the number of the experimental animals in each test is not less than 5; after the experiment was injected for 6 weeks, the mice were sacrificed and the eye tissue of the mice was examined by frozen sectioning and immunofluorescent staining as follows:

fresh eyeball tissue surface tissues such as muscle, fascia and the like are trimmed, 4 percent of freshly prepared PFA is used for fixing eyeballs, and the temperature is kept overnight at 4 ℃. The next day, after dehydration in 10% sucrose solution for 2h, dehydration was performed overnight with 30% sucrose solution. Immersing the eyeball tissue dehydrated overnight into OCT embedding medium, putting the embedding mould into a refrigerator at-80 ℃ after OCT is completely immersed into the eyeball tissue, and slicing the tissue after the tissue is completely frozen. Using a cryomicrotome, the eye tissue was cut into tissue sections of 10 μm thickness, labeled, and stored in a freezer at-80 ℃ until use.

After section elution of OCT, blocking with 5% BSA for 1 hour, the following primary antibody was used: iba-1(Abcam, ab178847), SDF-1(Abcam, ab25117), CXCR4(Abcam, ab124824), PECAM-1(Santa Cruz, sc-376764), Ki67(Cell Signaling Technology, 9129), after incubation with secondary antibodies and DAPI respectively in the absence of light, a block of anti-quenching blocking agent was used.

The analysis method of the test result comprises the following steps: the two groups are compared by adopting double-tail Student T-test detection; statistical differences between the three and more groups of results were measured using One-way ANOVA and multiple comparison Tukey's HSD. Data analysis was performed using R (version 4.0.0) and visualization was performed using GraphPad Prism 8(California) software. Results are reported as mean ± Standard Error (SEM). In all experimental data statistics, p <0.05 was considered statistically significant.

The immunofluorescence detection results are shown in FIGS. 2A and 2B, after the intravitreal injection of M1-EXO, endothelial cells which break through the inner limiting membrane and have proliferation activity appear, and the result shows that the retinal neovascularization is generated by the induction of M1-EXO. In addition, as can be seen from fig. 2C and 2D, the expression levels of the angiogenesis-related proteins SDF-1 and CXCR4 of the M1-EXO group were also significantly increased, further confirming that exosomes derived from M1-type activated microglia BV2 induces retinal neovascularization in the eyeball tissue of mice. The invention successfully constructs the animal model of the retinal neovascular disease by injecting the exosome derived from the M1 type activated microglia into the vitreous cavity of the mouse eyeball tissue.

77 mice are subjected to intravitreal injection M1-EXO according to the method to construct the retinal neovascular disease animal model, wherein the injection rate is over 80%.

TABLE 1 success rate values for the construction of animal models of retinal neovascular diseases according to the invention

And then, continuously observing the successfully constructed 67 animal models of the retinal neovascular diseases for 1-2 months, and carrying out section staining on mouse tissues, wherein the result is shown in figure 3, so that the model constructed by the method can be seen, and the retinal neovascular diseases can still stably exist in 1-2 months after the model is successfully modeled, which shows that the animal models of the retinal neovascular diseases constructed by the method have good pathological persistence and stability.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.