阅读说明：本技术 角膜新生血管/淋巴管生成损伤模型及其构建方法和应用 (Cornea new blood vessel/lymphatic vessel generation damage model and construction method and application thereof ) 是由 李宗源 王丽强 黄一飞 于 2021-05-31 设计创作，主要内容包括：本发明提供了一种角膜新生血管/淋巴管生成损伤模型及其构建方法和应用。其中,该模型是在角膜缝线的思路基础上,采用角膜上皮机械刮除联合生物胶黏合角膜缝线于角膜基质的方式构建得到,这样既解决了角膜缝线模型应用在小型啮齿类动物时造模难度大、效率低的问题,又避免了角膜穿孔、缝线脱落等导致造模失败的常见原因,还保留了角膜缝线新生血管生长比较规则、避免化学诱导方法中化学试剂影响、损伤方式单一可控等优点。此外,基于该模型的构建方法,能够简便、有效、可控的作为小鼠、大鼠角膜新生血管或淋巴管生成的一种新型有效模型,因而,其适用性较强。(The invention provides a cornea neovascularization/lymphatic vessel generation injury model and a construction method and application thereof. The model is constructed by adopting a mode of combining corneal epithelium mechanical scraping and biological adhesive bonding of corneal sutures on corneal stroma on the basis of the idea of corneal sutures, so that the problems of high molding difficulty and low efficiency when the corneal suture model is applied to small rodents are solved, common reasons of molding failure caused by corneal perforation, suture falling and the like are avoided, and the advantages of more regular growth of neovascular corneal sutures, avoidance of influence of chemical reagents in a chemical induction method, single and controllable damage mode and the like are retained. In addition, the model-based construction method can be used as a novel effective model for mouse and rat cornea neovascularization or lymphangioleiomy, and is simple, effective and controllable, so that the model is high in applicability.)

1. A cornea neovascularization/lymphangiogenesis injury model is characterized in that: and constructing the obtained mouse/rat cornea neovascularization/lymphatic vessel generation injury model by adopting a mode of mechanically scraping corneal epithelium and combining biological adhesive with a corneal suture to be adhered to corneal stroma.

2. The model of corneal neovascularization/lymphangiogenesis injury according to claim 1, wherein the mechanical scraping of corneal epithelium is performed by a corneal scraper with a diameter of 2-2.5 mm at the center of the cornea.

3. The model of corneal neovascularization/lymphangiogenic injury of claim 1, wherein said mechanical scraping of corneal epithelium is: the corneal epithelial layer is mechanically scraped or scraped to the superficial substrate layer.

4. The model for corneal neovascularization/lymphangiogenesis damage according to claim 1, wherein said biological adhesive bonding corneal sutures to corneal stroma is: the corneal sutures were secured to the corneal stromal surface using a biological tissue adhesive.

5. The model for corneal neovascularization/lymphangiogenic damage according to claim 4, wherein said biological tissue adhesive is cyanoacrylate or human fibrin tissue glue.

6. The model for corneal neovascularization/lymphangiogenic injury of claim 4, wherein said corneal sutures are either 10-0 or 11-0 non-absorbable sutures.

7. A method for constructing a model of corneal neovascularization/lymphangiogenesis injury according to any one of claims 1 to 6, comprising:

step 1, performing general anesthesia on a mouse/rat, and then performing local corneal infiltration anesthesia;

step 2, mydriasis is carried out on the mouse/rat after local anesthesia, and the fact that the pupils of the mouse/rat after local anesthesia are completely scattered is determined;

step 3, mechanically scraping corneal epithelium in a regular circle with the central diameter of the cornea of 2-2.5 mm;

step 4, performing suture molding on the mechanically scraped cornea by using a non-absorbable suture, and fixing the non-absorbable suture after head and tail knotting on the surface of a corneal stroma by using a biological tissue adhesive;

step 5, after the biological tissue adhesive is solidified, coating antibiotic gel in a conjunctival sac, and then suturing 2-3 needles of the mouse/rat eyelid;

step 6, smearing antibiotic ointment on the eyelid;

step 7, based on the proliferation and migration mode of corneal epithelial cells, the corneal epithelium grows out again and covers the non-absorbable suture and the corneal stroma;

step 8, growing regular corneal neovascularization/lymphatics in the area within the corneal limbus, and obtaining the corneal neovascularization/lymphatics damage model of any one of claims 1-6.

8. The method of claim 7, wherein in step 1, the anesthetic used for general anesthesia is a mixture of 3.3% tribromoethanol and t-amyl alcohol in an amount of 1 g: 1ml of mixed solution; the anesthetic used for local infiltration anesthesia is oxybuprocaine eye drops;

in the step 3, the corneal epithelium mechanical scraping is corneal epithelium layer mechanical scraping or corneal scraping to a superficial substrate layer;

in step 4, the non-absorbable suture is either a 10-0 non-absorbable suture or a 11-0 non-absorbable suture; the biological tissue adhesive is cyanoacrylate adhesive or human fibrin tissue glue;

in the step 5, the suture used for suturing the mouse/rat eyelid is a 6-0 non-absorbable suture.

9. The application of a model for generating and damaging corneal neovascularization/lymphatic vessels, which is characterized in that the model for generating and damaging corneal neovascularization/lymphatic vessels of any one of claims 1 to 6 is applied to research on the inhibition effect of a drug on corneal neovascularization or corneal lymphatic vessels; or

The model of corneal neovascularization/lymphangiogenesis damage according to any one of claims 1 to 6, for use in studying the effect of a molecule and its associated signaling pathway on corneal neovascularization or corneal lymphangiogenesis.

10. The application of a cornea angiogenesis/lymphangiogenesis damage model, which is characterized in that the cornea angiogenesis/lymphangiogenesis damage model of any claim 1-6 is applied to the research of cornea inflammatory reaction and related immune mechanisms; or

Applying the corneal neovascularization/lymphangiogenesis injury model of any one of claims 1-6 to study the interaction and interaction of corneal neovascularization and lymphangiogenesis in the generation stage and the dissipation stage; or

The model for corneal neovascularization/lymphangiogenesis damage according to any one of claims 1 to 6 is applied to study of drugs for inhibiting neovascularization or lymphangiogenesis.

Technical Field

The invention belongs to the field of organic matter synthesis, and mainly relates to a cornea neovascularization/lymphangioleiomy injury model and a construction method and application thereof.

Background

Angiogenesis is a natural physiological process, and affects the body differently depending on the site and time of occurrence.

In most tissues of the human body, it is involved in the growth of tissue cells and in the repair of damaged tissues. Under physiological conditions, the capillary network grows only around the limbus and does not extend to the cornea, which remains transparent. Under the adverse conditions of inflammation, trauma, surgery, etc., capillaries invade the cornea and produce pathological CNV (i.e., pathological corneal neovascularization). The pathological process is mainly related to cytokines acting on blood vessels: cytokines acting on blood vessels play a key regulatory role in the process of angiogenesis. Among them, cytokines include promoting factors and inhibiting factors, and the process of angiogenesis is regulated by many promoting factors and inhibiting factors. The promoting factors include Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), Platelet Activating Factor (PAF), and the like; the inhibitors include Matrix Metalloproteins (MMPs), interleukins (IL-1), and the like. Under normal physiological conditions, the inhibitory factor predominates, and the concentrations of both factors will reach equilibrium to maintain the cornea in a physiological state. However, when the concentration balance is broken, the angiogenic cytokines promote the invasion of vascular endothelial cells into the cornea to form capillaries. The research shows that the over-expression factors of the cell factors acting on blood vessels are as follows: (1) corneal edema: when the cornea is edematous, the pressure between corneal tissues is reduced, and blood vessels are easy to grow in; (2) inflammatory reaction: infiltration of inflammatory cells results in the formation of cellular debris that is visible around the nascent blood vessels and contributes to angiogenesis; (3) hypoxia: hypoxia can stimulate the large expression of angiogenesis promoting cytokines; (4) corneal nerve fiber distribution: the new research finds that the corneal sensory nerve and the neovascularization are mutually inhibited, and the corneal neovascularization and the damage of the nerve can be connected.

The cornea angiogenesis animal model (CNV animal model for short) is an animal model for induced diseases, which artificially induces the cornea angiogenesis of animals by the methods of operation, physics, chemistry, biology and the like to generate a model similar to human diseases. In the art, desirable CNV animal models need to have the following characteristics: (1) the manufacturing is simple and convenient, and the model is stable; (2) the experimental conditions and operation are simple, and the blood vessel formation is regular; (3) the difference between individuals is small; (4) other conditions are easily controlled.

At present, relevant models of corneal neovascularization and lymphatic vessels of rats and mice are mainly constructed by a physical method. The physical method induces the mouse cornea angiogenesis model, which generally comprises a cornea suture model, a thermal burn model and a chemical burn model.

However, in the prior art, the physical methods induce the mouse cornea neovascularization model, and have certain technical problems. For example, the burning force of the thermal burn model is difficult to control, and when the force is too large, corneal perforation and scar formation are easy to cause the model building failure; chemical burn models are also difficult to control, can generally cause strong immune response, have multiple interference factors, and are not beneficial to simply researching cornea neovascularization or corneal lymphatics; in the corneal suture model, whether the suture is successful or not is greatly influenced by the skill of an operator, the cornea thickness of rats and mice is small, the area is small, the difficulty of model building by using a single suture is high, the corneal perforation is easily caused, and if the suture is slightly shallow, the model fails because the suture is easily broken.

In summary, there is still a lack of reliable models for corneal lymphangiogenesis in the field, and recent international research is leading to increasing research on the interphalangeal space and immune regulation. Therefore, a reliable generation model of corneal lymphatic vessels in large and small mice is urgently needed in the technical field.

Disclosure of Invention

In order to solve the problems, the invention provides a cornea neovascularization/lymphangiogenesis injury model and a construction method and application thereof. The specific contents mainly applied to rodents such as mice and rats are as follows:

in a first aspect, the present invention provides a corneal neovascularization/lymphangiogenesis damage model, where the corneal neovascularization/lymphangiogenesis damage model is: and constructing the obtained mouse/rat cornea neovascularization/lymphatic vessel generation injury model by adopting a mode of mechanically scraping corneal epithelium and combining biological adhesive with a corneal suture to be adhered to corneal stroma.

Preferably, the corneal epithelium mechanical scraping is performed by using a corneal epithelium scraper to perform corneal epithelium mechanical scraping on the central diameter of the cornea of 2-2.5 mm.

Preferably, the mechanical scraping of the corneal epithelium is: the corneal epithelial layer is mechanically scraped or scraped to the superficial substrate layer.

Preferably, the biological glue adhering the corneal suture to the corneal stroma means: the corneal sutures were secured to the corneal stromal surface using a biological tissue adhesive.

Preferably, the biological tissue adhesive is a cyanoacrylate or human fibrin tissue glue.

Preferably, the corneal sutures are either 10-0 non-absorbable sutures or 11-0 non-absorbable sutures.

In a second aspect, the present invention provides a method for constructing a model of corneal neovascularization/lymphangiogenesis damage according to the first aspect, the method comprising:

step 1, performing general anesthesia on a mouse/rat, and then performing local corneal infiltration anesthesia;

step 2, mydriasis is carried out on the mouse/rat after local anesthesia, and the fact that the pupils of the mouse/rat after local anesthesia are completely scattered is determined;

step 3, mechanically scraping corneal epithelium in a regular circle with the central diameter of the cornea of 2-2.5 mm;

step 4, performing suture molding on the mechanically scraped cornea by using a non-absorbable suture, and fixing the non-absorbable suture after head and tail knotting on the surface of a corneal stroma by using a biological tissue adhesive;

step 5, after the biological tissue adhesive is solidified, coating antibiotic gel in a conjunctival sac, and then suturing 2-3 needles of the mouse/rat eyelid;

step 6, smearing antibiotic ointment on the eyelid;

step 7, based on the proliferation and migration mode of corneal epithelial cells, the corneal epithelium grows out again and covers the non-absorbable suture and the corneal stroma;

and 8, growing regular corneal neovascularization/lymphatic vessels in an area within the corneal limbus to obtain the corneal neovascularization/lymphatic vessel generation injury model of the first aspect.

Preferably, in step 1, the anesthetic used to perform the general anesthesia is a mixture of 3.3% tribromoethanol and t-amyl alcohol in a ratio of 1 g: 1ml of mixed solution; the anesthetic used for local infiltration anesthesia is oxybuprocaine eye drops;

in the step 3, the corneal epithelium mechanical scraping is corneal epithelium layer mechanical scraping or corneal scraping to a superficial substrate layer;

in step 4, the non-absorbable suture is either a 10-0 non-absorbable suture or a 11-0 non-absorbable suture; the biological tissue adhesive is cyanoacrylate adhesive or human fibrin tissue glue;

in the step 5, the suture used for suturing the mouse/rat eyelid is a 6-0 non-absorbable suture.

In a third aspect, the invention provides an application of a model for generating and damaging corneal neovascularization/lymphatic vessels, wherein the model for generating and damaging corneal neovascularization/lymphatic vessels in the first aspect is applied to research on the inhibitory effect of a drug on corneal neovascularization or corneal lymphatic vessels; or

The corneal neovascularization/lymphangiogenesis injury model of the first aspect is applied to research on the effect of a certain molecule and its related signal pathway on corneal neovascularization or corneal lymphangiogenesis.

In a fourth aspect, the present invention provides an application of the corneal neovascularization/lymphangiogenesis damage model, wherein the corneal neovascularization/lymphangiogenesis damage model of the first aspect is applied to research of corneal inflammatory reaction and related immune mechanisms; or

Applying the model for corneal neovascularization/lymphangiogenesis injury according to the first aspect to study the interaction and the mutual influence of corneal neovascularization and lymphangiogenesis in a generation stage and a dissipation stage; or

The corneal neovascularization/lymphangiogenesis injury model described in the first aspect is applied to study of a drug for inhibiting neovascularization or lymphangiogenesis.

The invention provides a cornea neovascularization/lymphatic vessel generation injury model and a construction method and application thereof. Wherein, the cornea new blood vessel/lymphatic vessel generation damage model is as follows: and constructing the obtained mouse/rat cornea neovascularization/lymphatic vessel generation injury model by adopting a mode of mechanically scraping corneal epithelium and combining biological adhesive with a corneal suture to be adhered to corneal stroma. The model is constructed by adopting a mode of combining mechanical scraping of corneal epithelium with biological adhesive bonding of corneal sutures on corneal stroma on the basis of the concept of corneal sutures, thus solving the problems of high molding difficulty and low efficiency when the corneal suture model is applied to small rodents, avoiding common reasons of molding failure caused by corneal perforation, suture falling and the like, and also keeping the advantages of more regular growth of neovascular corneal sutures, avoidance of influence of chemical reagents in a chemical induction method, single and controllable damage mode and the like. In addition, the model-based construction method can be used as a novel effective model for mouse and rat cornea neovascularization or lymphangioleiomy, and is simple, effective and controllable, so that the model is high in applicability.

Drawings

FIG. 1 illustrates a flow chart of a method of modeling corneal neovascularization/lymphangiogenesis damage in an embodiment of the invention;

FIG. 2 is a diagram showing the neovascularization under a slit lamp at 14d after a suture operation in a cornea neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention;

FIG. 3 shows a lymphatic immunofluorescence staining pattern at 14d after suture operation of a corneal neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention;

fig. 4 shows a diagram of a model injury process of a cornea neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The specific experimental procedures or conditions not specified in the examples can be performed according to the procedures or conditions of the conventional experimental procedures described in the prior art in this field. The reagents and other instruments used are not indicated by the manufacturer, and are all conventional reagent products or instruments that are commercially available.

In the existing physical method for inducing mouse cornea neovascularization models, which usually comprise a cornea suture model, the burning force of a heat burn model is difficult to control, and the excessive force easily causes corneal perforation, and scar formation causes molding failure; chemical burn models are also difficult to control, usually cause strong immune response, have many interference factors, and are not favorable for simply researching corneal neovascularization or corneal lymphatic vessels.

In the corneal suture model, the intrastromal suture is embedded, which can cause local edema and inflammatory cell invasion at the corneal suture part; furthermore, corneal sutures can also cause damage to the corneal epithelium and basal cells, which in turn can lead to inflammatory responses. On one hand, in the inflammatory reaction, inflammatory cells secrete cytokines for promoting the growth of blood vessels to promote angiogenesis; on the other hand, hypoxia in the corneal microenvironment resulting from corneal injury also promotes the expression of angiogenic cytokines such as VEGF, thereby promoting angiogenesis. However, the success of the corneal suture model is greatly influenced by the skill of the operator, the corneal thickness of the rat and the mouse is small, the model making by the simple suture is difficult, the corneal perforation is easy to cause, and the model fails because the suture is easy to be broken if the suture is slightly shallow.

Moreover, a reliable corneal lymphatic vessel generation model is lacked at present, and with the increasing research on lymphatic vessels and immune regulation at the frontier of recent international research, a reliable corneal lymphatic vessel generation model for large and small mice is urgently needed in the technical field.

In order to solve the above technical problems, the technical idea proposed by the inventor of the present invention mainly includes: and (3) combining the advantages of the corneal suture, and constructing a corneal neovascularization/lymphatic vessel generation injury model by adopting a mode of mechanically scraping corneal epithelium and combining biological glue with the corneal suture adhered to corneal stroma. By adopting the method to construct the model, the problem of high molding difficulty and high efficiency when the corneal suture model is applied to small rodent is solved, common molding failures such as corneal perforation, suture falling and the like are avoided, and the advantages of regular growth of neovascular corneal sutures, avoidance of influence of chemical reagents in a chemical induction method, single and controllable damage mode and the like are maintained. Based on the technical concept, the embodiment of the invention provides a cornea neovascularization/lymphatic vessel generation injury model and a construction method and application thereof. The specific implementation content is as follows:

in a first aspect, embodiments of the present invention provide a corneal neovascularization/lymphangiogenesis injury model. The cornea new blood vessel/lymphatic vessel generation damage model is as follows: and constructing the obtained mouse/rat cornea neovascularization/lymphatic vessel generation injury model by adopting a mode of mechanically scraping corneal epithelium and combining biological adhesive with a corneal suture to be adhered to corneal stroma.

Since the eyes of the rodent mouse/rat are small and difficult to operate, the existing large-eye animal model (such as a rabbit model) cannot be implemented smoothly, namely, the effectiveness of the existing large-eye animal model in the practical application process is low. The corneal neovascularization/lymphatic vessel generation injury model provided by the embodiment is a novel mouse/rat corneal neovascularization/lymphatic vessel generation injury model, and therefore, the corneal neovascularization/lymphatic vessel generation injury model provided by the embodiment can be used for improving the success rate of mouse/rat model construction.

In order to avoid damaging the limbal stem cells and to better achieve the controllability and reliability of the model, the preferred mechanical scraping range in this embodiment is 2-2.5 mm. That is, in this embodiment, the mechanical scraping of corneal epithelium is performed such that the central diameter of cornea is 2 to 2.5 mm. And wherein the mechanical scraping of the corneal epithelium is: a corneal spatula is used to mechanically scrape or scrape the corneal epithelial layer to the superficial substrate layer.

In this example, the specific reasons for mechanical scraping of corneal epithelium are as follows:

the mechanical scraping model of corneal epithelium is the closest model to the physiological repair process, and has the following characteristics: 1. the damage repairing speed is high; 2. the model is simple, reliable and easy to repeat; 3. cause a weaker inflammatory response; 4. belongs to complete physiological repair, and has no scar after repair. In this embodiment, this model is selected as the basis of the model, and its main purpose is: it is desirable to expose the corneal anterior stromal layer and firmly fix the suture knots to the stromal layer (here, it is noted that if the corneal epithelial layer cells are not tightly connected, the cells are easily detached by external force together with the knots, and thus, in this embodiment, the suture knots are firmly fixed to the tightly structured corneal stromal layer).

Based on the above reasons, the novel model (i.e. the model for generating injury of corneal neovascularization/lymphatic vessels) provided in this embodiment utilizes the simple and highly operable mechanical scraping model for corneal epithelium, does not cause other complicated immune responses such as an alkali burn model, and ensures the maximum efficacy and exerts the foreign body stimulation effect of the novel model, so that the model becomes a simple, stable, reliable and high-repetition rate model for generating injury of corneal neovascularization/lymphatic vessels in mice/rats.

In this embodiment, the biogel adheres the corneal sutures to the corneal stroma, specifically: the corneal sutures were secured to the corneal stromal surface using a biological tissue adhesive.

In this embodiment, the preferred biological tissue adhesive is cyanoacrylate or human fibrin tissue glue.

In this embodiment, since the damage model is constructed, it is necessary to use a non-absorbable suture; furthermore, since corneal injury is performed on mice/rats, it is necessary to use thin, non-absorbable sutures. For this reason, the preferred corneal sutures of this embodiment are either 10-0 non-absorbable sutures or 11-0 non-absorbable sutures.

Here, the inventors should point out that: the model provided by the embodiment is a further improvement of the technical problems existing in the existing traditional suture model, and provides a novel construction method different from the traditional suture model construction method, so that the technical problems existing in the existing suture model are solved, and the technical problems existing in a thermal burn model and a chemical burn model are also avoided.

Moreover, the model provided by the embodiment belongs to a damage model class. The method specifically comprises the following steps: clinical diseases are simulated, and normal cornea is damaged to form certain pathophysiological changes, so that the obtained model is constructed.

In addition, the cornea neovascularization/lymphangioleiomyomatosis damage model provided by the embodiment of the invention also has the following advantages:

A. the model for generating and damaging the corneal neovascularization/lymphangioleia provided by the embodiment of the invention is constructed by adopting a mode of mechanically scraping corneal epithelium and combining biological glue with a corneal suture which is adhered to corneal stroma, so that the construction method has the advantages of simplicity, convenience, high efficiency, easiness in implementation and the like, and is suitable for various experimental animals such as rodents, mammals and the like.

B. The cornea neovascularization/lymphatic vessel generation damage model provided by the embodiment of the invention can control the degree of damage (namely, the length and the area of foreign matters growing into the cornea) by adjusting the length and the range of the non-absorbable suture (namely, the length of the suture and the area of the loop after the suture is connected end to end), so that the cornea neovascularization/lymphatic vessel generation damage model provided by the embodiment of the invention has controllability.

C. The cornea neovascularization/lymphatic vessel generation damage model provided by the embodiment of the invention ensures high success rate of the model by combining the self-growth process of mouse/rat cornea on the basis of controlling the depth and area of each damage by adjusting the length and range of the non-absorbable suture and also avoiding the difference of each mouse damage caused by manual operation, so that the cornea neovascularization/lymphatic vessel generation damage model provided by the embodiment of the invention has stability (namely effectiveness).

In summary, the corneal neovascularization/lymphatic vessel generation injury model provided by the embodiment of the invention combines the advantages of corneal sutures, adopts a mode of corneal epithelium mechanical scraping and biological adhesive bonding the corneal sutures to corneal stroma, solves the problems of great molding difficulty and low efficiency when the corneal suture model is applied to small rodent animals, avoids common molding failure reasons such as corneal perforation and suture falling, and also retains the advantages of corneal suture neovascularization growth rule, chemical reagent influence in a chemical induction method, single and controllable injury mode and the like. The model can be used as a novel effective model for generating new blood vessels or lymphatic vessels of mouse and rat corneas simply, conveniently, effectively and controllably.

In a second aspect, an embodiment of the present invention provides a method for constructing a corneal neovascularization/lymphangiogenesis injury model according to the first aspect. As shown in fig. 1, the construction method includes:

step 1(S1), general anesthesia was performed on the mouse/rat, and then local corneal infiltration anesthesia was performed.

In step 1 of this example, general anesthesia was performed using 3.3% tribromoethanol with t-amyl alcohol at a ratio of 1 g: 1ml of mixed solution; the anesthetic used for local infiltration anesthesia is oxybuprocaine eye drops.

In the implementation step, the selection and the proportion of the types of the anesthetics are based on effectiveness and safety, and the types, the proportions and the dosages which are suitable for the model to the greatest extent are used for carrying out general anesthesia. Wherein, the effectiveness means: the dosage is completely suitable for the molding duration of the model, the deep anesthesia state of the experimental animal is kept in the effective time, and the operation is convenient. The safety refers to: the dosage control is that the animals start to recover within about 10min after the molding is finished, the recovery process is fast, and the death rate caused by anesthesia is extremely low.

Step 2(S2), mydriasis is performed on the locally anesthetized mouse/rat, and it is determined that the mydriasis is completely dispersed in the locally anesthetized mouse/rat.

After the pupil is completely scattered, the following step 3 is performed.

And step 3(S3), mechanically scraping corneal epithelium by using a corneal scraper in a regular circle with the central diameter of the cornea of 2-2.5 mm.

In step 3 of this embodiment, the mechanical scraping of the corneal epithelium is a mechanical scraping of the corneal epithelium layer or scraping to a superficial substrate layer. After the mechanical scraping operation is completed, the following step 4 is performed.

And 4(S3), performing suture molding on the mechanically scraped cornea by using the non-absorbable suture, and fixing the non-absorbable suture after head and tail knotting on the surface of the corneal stroma by using a biological tissue adhesive.

In step 4 of this example, the non-absorbable suture is either a 10-0 non-absorbable suture or a 11-0 non-absorbable suture; the biological tissue adhesive is cyanoacrylate adhesive or human fibrin tissue glue.

And 5(S5), after the biological tissue adhesive is solidified, coating antibiotic gel in the conjunctival sac, and then suturing 2-3 needles of the mouse/rat eyelid.

In step 5 of this example, the suture used to suture the mouse/rat eyelids was a 6-0 non-absorbable suture.

In specific implementation, after the biological tissue adhesive is solidified, antibiotic gel is coated in a conjunctival sac, and then 2-3 needles of the eyelids of the experimental animal mice are discontinuously sutured on each eye by using 6-0 nonabsorbable threads.

In the implementation step, the eyelids of the experimental animal are sutured, so that the secondary damage caused by scratching the cornea or the falling of the adhered knots can be prevented; before the eyelids are sewed, the antibiotic gel is coated to keep the antibiotic gel in the conjunctival sac to continuously play the functions of moisturizing and bacteriostasis.

In this implementation step, the antibiotic gel may be gatifloxacin gel.

Step 6(S6), applying antibiotic ointment to the eyelid.

In specific implementation, antibiotic ointment is continuously applied to the periphery of the sutured eyelid to achieve local bacteriostasis and prevent keratitis or eyelid wound infection and the like.

In this embodiment, the antibiotic ointment may be aureomycin eye ointment or tobramycin eye ointment.

Step 7(S7), based on the proliferation migration pattern of the corneal epithelium repair, the corneal epithelium grows out again and covers the non-absorbable suture.

Step 8(S8), the corneal neovascularization/lymphatic vessels break through the limbus fence structure, and regular corneal neovascularization/lymphatic vessels grow in the area within the limbus, so as to obtain the corneal neovascularization/lymphatic vessel generation injury model of the first aspect.

The construction method provided by the embodiment solves the problems of high molding difficulty and low efficiency in the existing molding technology, avoids common molding failures such as corneal perforation, suture falling and the like, and also retains the advantages of more regular growth of corneal suture neovascularization, avoidance of influence of chemical reagents in a chemical induction method, single and controllable damage mode and the like. In addition, the construction method provided by the embodiment can simply, effectively and controllably construct a novel effective model generated as mouse/rat cornea neovascularization/lymphatic vessels, and is consistent with clinical disease characteristics.

In a third aspect, the embodiments of the present invention provide an application of a model for generating damage of corneal neovascularization/lymphatic vessels, that is, the model for generating damage of corneal neovascularization/lymphatic vessels according to the first aspect is applied to research on the inhibitory effect of a drug on corneal neovascularization or corneal lymphatic vessels; or

The corneal neovascularization/lymphangiogenesis injury model of the first aspect is applied to research on the effect of a certain molecule and its related signal pathway on corneal neovascularization or corneal lymphangiogenesis.

In a fourth aspect, the embodiment of the present invention further provides an application of a corneal neovascularization/lymphangiogenesis injury model, that is, the corneal neovascularization/lymphangiogenesis injury model according to the first aspect is applied to research of corneal inflammatory response and related immune mechanisms; or

Applying the model for corneal neovascularization/lymphangiogenesis injury according to the first aspect to study the interaction and the mutual influence of corneal neovascularization and lymphangiogenesis in a generation stage and a dissipation stage; or

The corneal neovascularization/lymphangiogenesis injury model described in the first aspect is applied to study of a drug for inhibiting neovascularization or lymphangiogenesis.

In order to make the person skilled in the art better understand the present invention, the method for constructing the corneal neovascularization/lymphangiogenesis damage model provided by the present invention is described below by using a plurality of specific examples.

Example 1

The animals required: c57 adult mouse

The instrument equipment comprises: microscope, microsurgical instrument, mechanical corneal epithelium scraper, oxybuprocaine anesthetic, biological tissue adhesive, and 10-0 non-absorbable suture

The operation process comprises the following steps:

step 1(S1), C57 adult mice were subjected to general anesthesia and then to local corneal infiltration anesthesia.

Specifically, a 3.3% mixed solution of tribromoethanol and t-amyl alcohol (1 g: 1ml) was prepared, and abdominal anesthesia was performed at a rate of 0.024ml/g body weight. C57 adult mice were injected intraperitoneally using a 1ml syringe fitted with a No. 4 needle. When the syringe is held in the right hand, the tail of the C57 adult mouse is held by the little finger and the ring finger of the left hand, and the neck of the C57 adult mouse is held by the other three fingers, so that the head of the C57 adult mouse faces downwards. Therefore, organs in the abdominal cavity can naturally fall to the chest, and the large intestine, the small intestine and other organs are prevented from being damaged when the injector is punctured. The needle insertion should be gentle to prevent stabbing abdominal organs. The needle head can pass through the abdomen for a short distance subcutaneously during the abdominal injection, preferably, the needle head is inserted from one side of the abdomen, the needle head passes through the abdominal midline and then enters the abdominal cavity from the other side of the abdomen, after the medicine is injected, the needle head is slowly pulled out, and the needle head is slightly rotated to prevent leakage.

After 5min of intraperitoneal injection of the anesthetic, the general anesthesia of animals is completed, and after the anesthesia state of C57 adult mice is confirmed, the local surface infiltration anesthesia of the cornea is carried out by utilizing oxybuprocaine eye drops, wherein about 1 drop of the anesthetic is injected into each eye, and the local anesthesia lasts for about 2 min.

Step 2(S2), mydriasis is performed on the C57 adult mouse after local anesthesia, and it is determined that the pupils of the C57 adult mouse after local anesthesia are completely scattered.

In specific implementation, the compound tropicamide eye drops are used for mydriasis for 5min, and the next test is carried out after the pupils are observed to be completely dispersed under the illumination of a body type microscope.

And step 3(S3), mechanically scraping corneal epithelium in a regular circle with the central diameter of the cornea of 2-2.5 mm.

In specific implementation, mechanical scraping of the corneal epithelium is performed under a stereomicroscope with a central diameter of about 2mm of the cornea. The specific mechanical scraping operation is: the corneal epithelium was removed along the scratch with a corneal epithelium knife by scratching it with a ring-shaped marker of 2mm diameter centered on the cornea, and enlarging it to a regular circle of 2.5mm diameter with a smooth edge, and the remaining epithelial fragments were gently wiped off with a water-absorbing cotton swab while scraping the epithelium from the outside to the inside.

And 4(S3), performing suture molding on the mechanically scraped cornea by using the non-absorbable suture, and fixing the non-absorbable suture after head and tail knotting on the surface of the corneal stroma by using a biological tissue adhesive.

In specific implementation, a proper amount of biological tissue adhesive (cyanoacrylate adhesive is selected as the biological tissue adhesive in this embodiment) certified by FDA is sprayed and fixed to the surface of the corneal stroma with 10-0 non-absorbable suture that is looped end to end.

And 5(S5), after the biological tissue adhesive is solidified, coating antibiotic gel in the conjunctival sac, and then suturing the C57 adult mouse eyelid 2 needle.

In specific implementation, after the biological tissue adhesive is solidified, antibiotic gel is coated in a conjunctival sac (in the implementation step, gatifloxacin gel is selected), and then a C57 adult mouse eyelid 2 needle is discontinuously sutured by 6-0 nonabsorbable thread for each eye, so that the surface of the cornea is kept moist, and meanwhile, secondary damage caused by scratching the cornea by animals or shedding of the adhered thread knots is prevented.

Step 6(S6), applying antibiotic ointment to the eyelid.

In specific implementation, in order to prevent infection after operation, antibiotic ointment (in the implementation step, aureomycin eye ointment is selected) is coated in the conjunctival sac, and antibiotic eye liquid is dripped on the surface of the eye outside the eyelid.

Step 7(S7), based on the corneal epithelial cell proliferation migration pattern, the corneal epithelium is re-grown and covered with non-absorbable sutures.

In specific implementation, the corneal epithelium grows out again in a proliferation and migration mode and covers the injury after about 24 hours, and the foreign body suture is covered on the suture fixed on the corneal stroma by the intracutaneous biological tissue adhesive.

The corneal epithelial cells are quickly restored and repaired by self renewal, and the simple mechanical scraping injury is generally completely covered within 48 hours after the injury. Can be verified by fluorescein staining and photographing under a slit lamp cobalt blue light.

Epithelial healing is only a self-healing stage and is not central to the injury model. Epithelial healing ensures that the sutures grow into the cornea and do not easily fall off. If the epithelium can not heal as soon as possible, the local inflammation of the cornea is aggravated, the knot can be used as a foreign body stimulating factor to continuously stimulate the cornea, a large amount of continuous new blood vessels and lymphatic vessels break through the corneal limbus and grow into the cornea, and the effect of the model is better.

Step 8(S8), growing regular corneal neovascularization/lymphatic vessels in the region inside the corneal limbus, so as to obtain the corneal neovascularization/lymphatic vessel generation injury model of the first aspect.

In practice, the area within the corneal limbus begins to grow regular corneal neovascularization and lymphatics within about 72 hours.

Corneal neovascularization can be directly observed by slit lamps.

Corneal neovascularization and corneal lymphatics can also be observed by immunofluorescence staining of corneal slices: normal cornea only seen at the limbus, a placket-like distribution of CD31 (corneal vascular marker) positive vascular network, and a small number of LYVE-1 (lymphatic endothelial marker) positive lymphatic vessels appeared in the limbus with an enlarged blind end, the central corneal region being devoid of any distribution of corneal blood vessels and corneal lymphatic vessels.

Fig. 2 shows a diagram of neovascularization under a slit lamp at 14d after a suture operation in a cornea neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention, in fig. 2, a front nodal image is taken under the slit lamp at 14d, a 14d neovascularization image and a fluorescein stained image under a 14d slit lamp cobalt blue lamp are sequentially taken from left to right; FIG. 3 shows immunofluorescence staining patterns of lymphatic vessels (LYVE1) at 14d after suture operation of a cornea neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention; fig. 4 shows a diagram of a model injury process of a cornea neovascularization/lymphangiogenesis injury model constructed in example 1 of the present invention. In fig. 4, from left to right, the corneal epithelium is mechanically scraped to expose the anterior stroma layer, the bioadhesive is bonded to the knotted suture, and the fluorescein staining pattern under a slit-lamp cobalt blue lamp immediately after molding is shown.

As shown in figures 2-4, after 14 days of the novel suture (i.e. the suture shown in the above steps 3-5), the corneal blood vessels and lymphatic vessels are in a vigorous growth phase, a large number of blood vessels are seen in the anterior segment of the slit lamp to grow into the cornea in a dendritic vertical limbus direction, the tail ends of the blood vessels are in a sharp burr shape, and only a few blood vessels are in sharp anastomosis. The LYVE-1 positive lymphatic vessel stretches into the center of the cornea along with the blood vessel in a flame shape, and ends with the blind end, the lumen is thicker and uneven than the blood vessel, the shape is more irregular, the lumen distortion is seen in partial area, and the dispersed LYVE-1 weak positive dendritic cells are seen in the corneal stroma.

According to the modeling process, the modeling process of the model provided by the embodiment is convenient, the range and the degree of modeling are controllable, and the slit lamp graph of 14d shows that the suture line is well combined with the corneal epithelium-stroma layer, the corneal epithelium is healed, and the model is successfully created.

Meanwhile, as can be seen from fig. 2-4, at 14d, a large number of new blood vessels and corneal lymphatic vessels break through the corneal limbus in a dendritic direction perpendicular to the corneal limbus and extend to the center of the cornea, grow into the cornea, have sharp burr-shaped ends, and only a few blood vessels are anastomosed with the visible tips; moreover, LYVE-1 positive lymphatic vessels flame-shaped along with blood vessels and extend into the center of the cornea, ending with the blind end, and the lumens are thicker and uneven than the blood vessels, and are more irregular in shape, and the lumens are distorted in partial areas. From this, it can be seen that the model constructed by the present embodiment is more preferable. .

Example 2

The construction method of this embodiment is similar to that of embodiment 1, and the differences are specifically as follows:

in step 1, general anesthesia of the animals is completed 3min after the intraperitoneal injection of the anesthetic.

In the step 2, the mydriasis time is 3min, and the mydriasis can be observed under a body microscope after 3min and can be completely dispersed under illumination.

In step 3, when the cornea is mechanically scraped, the cornea is scraped to the shallow substrate layer; the specific mechanical scraping operation is: placing the central impression of cornea with 2mm mark ring with alcohol assistance, injecting 20% ethanol 30ul, sucking off residual ethanol with dry blood-sucking sponge after 30s, immediately washing corneal epithelium with balancing solution or normal saline, mechanically scraping off corneal epithelium with diameter of 2.5mm with corneal epithelium knife, and exposing superficial stroma.

In step 4, the biological tissue adhesive selected in this embodiment is human fibrin tissue glue, and the non-absorbable suture selected in this embodiment is 11-0 non-absorbable suture.

In step 5, the eyelid of the adult mouse C57 was sutured with 3 needles.

In step 6, the tobramycin eye ointment is applied to the external ocular surface of the eyelid.

In step 7, the corneal epithelium will re-grow by means of proliferation and migration and cover the lesion for about 48 h.

The corneal neovascularization/lymphangiogenesis injury model obtained by the construction method provided in this embodiment is the same as the corneal neovascularization/lymphangiogenesis injury model obtained in embodiment 1.

Example 3

The construction method of this embodiment is similar to that of embodiment 1, and the differences are specifically as follows:

in the embodiment, SD adult rats are selected, and a rat cornea neovascularization/lymphangiogenesis injury model is constructed.

The rat corneal neovascularization/lymphangiogenesis injury model constructed in this example, which includes a map of neovascularization under slit lamp at 14d after the suture operation, a map of immunofluorescent staining of lymphatic vessels (LYVE1) at 14d after the suture operation, and a map during injury of the model, is the same as the one shown in example 1, and is not repeated in this example.

Example 4

The construction method of this embodiment is similar to that of embodiment 3, and the differences are specifically as follows:

in step 1, general anesthesia of the animals is completed 3min after the intraperitoneal injection of the anesthetic.

In the step 2, the mydriasis time is 3min, and the mydriasis can be observed under a body microscope after 3min and can be completely dispersed under illumination.

In step 3, when the cornea is mechanically scraped, the cornea is scraped to the shallow substrate layer; the specific mechanical scraping operation is: placing the central impression of cornea with 2mm mark ring with alcohol assistance, injecting 20% ethanol 30ul, sucking off residual ethanol with dry blood-sucking sponge after 30s, immediately washing corneal epithelium with balancing solution or normal saline, mechanically scraping off corneal epithelium with diameter of 2.5mm with corneal epithelium knife, and exposing superficial stroma.

In step 4, the biological tissue adhesive selected in this embodiment is human fibrin tissue glue, and the non-absorbable suture selected in this embodiment is 11-0 non-absorbable suture.

In step 5, the eyelid of the adult mouse C57 was sutured with 3 needles.

In step 6, the antibiotic ophthalmic gel eye ointment is applied to the external eye surface of the eyelid.

In step 7, the corneal epithelium will re-grow by means of proliferation and migration and cover the lesion for about 36 h.

It should be noted that the steps and methods in the embodiments of the present application are not limited to the corresponding embodiments, and the details of the operations and the cautions of the embodiments are all corresponding to each other. The value ranges of all the substances and the value ranges of all the parameters are only the preferable scheme of the invention, the invention does not limit the value, and all the value ranges applicable to the invention are feasible.

For simplicity of explanation, the method embodiments are described as a series of acts or combinations, but those skilled in the art will appreciate that the present invention is not limited by the order of acts, as some steps may occur in other orders or concurrently in accordance with the invention. Further, those skilled in the art will appreciate that the embodiments described in the specification are preferred embodiments and that the acts and elements referred to are not necessarily required to practice the invention.

The cornea neovascularization/lymphangiogenesis damage model provided by the invention and the construction method and application thereof are described in detail, the principle and the implementation mode of the invention are explained by applying specific examples, and the description of the examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.