WO2020009429A1 - Method for producing retinal degeneration animal model using vitrectomy and intraperiotoneal mnu injection and removal, and retinal degeneration animal model using same - Google Patents

Method for producing retinal degeneration animal model using vitrectomy and intraperiotoneal mnu injection and removal, and retinal degeneration animal model using same Download PDF

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WO2020009429A1
WO2020009429A1 PCT/KR2019/008054 KR2019008054W WO2020009429A1 WO 2020009429 A1 WO2020009429 A1 WO 2020009429A1 KR 2019008054 W KR2019008054 W KR 2019008054W WO 2020009429 A1 WO2020009429 A1 WO 2020009429A1
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mnu
retinal degeneration
animal model
vitrectomy
eye
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PCT/KR2019/008054
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French (fr)
Korean (ko)
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김성우
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고려대학교 산학협력단
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Priority claimed from KR1020190078584A external-priority patent/KR102228401B1/en
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Priority to CN201980057857.8A priority Critical patent/CN112654243A/en
Publication of WO2020009429A1 publication Critical patent/WO2020009429A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates

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  • the present invention provides a method for preparing a retinal degeneration animal model using a method of injecting and removing N-methyl-N-nitrosourea (MNU) into an eyeball of an animal in which vitreous tissue is removed through vitrectomy and a retina manufactured using the same. It relates to a denatured animal model.
  • MNU N-methyl-N-nitrosourea
  • Retinal degeneration is a disease in which vision is deteriorated due to degeneration of visual cells and eventually blindness, and there is no effective treatment yet.
  • Retinal degeneration in order to apply gene therapy, stem cell therapy, and artificial retina, which are developed for the treatment of diseases such as hereditary retinal degeneration and retinitis pigmentosa (RP) that damage the retina's eye cells and lead to blindness Development of experimental animals with disease is essential.
  • diseases such as hereditary retinal degeneration and retinitis pigmentosa (RP) that damage the retina's eye cells and lead to blindness
  • genetically modified animal models or models for inducing degeneration with drugs have been developed to apply various treatment methods.
  • animal models of drug-induced retinal degeneration once drug degeneration protocols are established, they are less expensive than genetically modified animal models, and shorter periods of degeneration can cause degeneration in large quantities and free the investigator from various regulations. There is an advantage.
  • MNU N-methyl-N-nitrosourea
  • MNU N-methyl-N-nitrosourea
  • MNU is a nitrogen compound that is widely used in everyday environments and is an alkyl compound that can cause retinal degeneration in various animals.
  • MNU is a chemical that causes alkylation directly without metabolic activation of the p-450 system. It is a powerful mutagen, teratogenic and carcinogen.
  • a method for producing an animal model using MNU a method of inducing retinal degeneration by systemic injection of MNU into a mouse or rat is popular.
  • MNU is specifically toxic to retinal photoreceptor cells, causing apoptosis of retinal photoreceptor cells, and the extent of damage is known to be proportional to the dose and time of MNU. Specifically, a decrease in retinal thickness was observed 24 hours after MNU injection, and in a morphological study of cell necrosis using TUNEL staining, apoptosis began to be observed 12 hours after drug injection and peaked 24 hours later. It was confirmed to accomplish.
  • the present inventors have injected a certain concentration of MNU into the eye where vitreous tissue has been removed through vitrectomy, and after removing the drug after leaving it for a certain time, degeneration of the whole eye regardless of the size of the eye
  • the discovery of the ability to produce an induced animal model led to the completion of the present invention.
  • the present invention is to solve the problems of the prior art, an object of the present invention is limited to one eye, to provide a method for producing a retinal degeneration animal model in which the entire range of retinal oocytes in the eye and the animal model using the same. .
  • MNU N-methyl-N-nitrosourea
  • step a) to d) may be performed by simultaneous surgery.
  • step d) may be performed after 2 minutes to 20 minutes from the step c).
  • step b) to step d) may be performed by a separate operation from the step a).
  • step b) to step d) may be performed at least three days after the step a).
  • step d) washing the eye, filling the empty space in the eye; It may further include.
  • MNU solution may be injected into the vitreous cavity region, in front of the eye retina.
  • the MNU solution may be a concentration of 2mg / ml to 10mg / ml.
  • the mammal may be a rabbit, dog, cat, pig or monkey.
  • the entire retina of the eye may be degenerated.
  • the method of manufacturing the retinal degeneration animal model of the present invention removes the vitreous tissue through vitrectomy prior to the injection of MNU, it may cause degeneration proportional to the dose of the drug, unlike when vitrectomy is not performed. It can be predicted.
  • the method of manufacturing a retinal degeneration animal model of the present invention can induce the same degree of retinal degeneration by using a certain concentration of MNU regardless of eye size and lens presence according to differences between species.
  • using the method of the present invention can minimize the residual MNU in the eye can prevent the appearance of systemic complications due to systemic absorption of the drug.
  • a retinal degeneration-induced animal model including all of the retinas with a uniform and whole-eye area limited to only one eye can be manufactured.
  • the manufactured animal model can be used for the development of artificial retina, stem cell treatment research, and the like. It can be used advantageously and can also provide the convenience of breeding.
  • Figure 1 shows the results of the OCT test before and 3 weeks after the vitrectomy and MNU (2mg / ml) drug injection surgery.
  • Figure 2 shows the results of ERG test before and 3 weeks after vitrectomy and MNU (2mg / ml) drug injection surgery.
  • Figure 3 shows the results of the OCT test before and after 12 weeks after surgery, vitrectomy and MNU (4mg / ml) drug injection surgery.
  • Figure 4 shows the ERG test results before and after 12 weeks after surgery, vitrectomy and MNU (4 mg / ml) drug injection surgery.
  • Figure 5 shows the results of the OCT test before and 6 weeks after surgery and vitrectomy and MNU (5mg / ml) drug injection surgery.
  • Figure 6 shows the ERG test results before and after 6 weeks after the surgery and vitrectomy and MNU (5mg / ml) drug injection surgery.
  • FIG. 8 shows ERG test results before and after 12 weeks after surgery, vitrectomy and MNU (6 mg / ml) drug injection.
  • Figure 9 shows the results of the OCT test before and 3 weeks after the vitrectomy and MNU (8mg / ml) drug injection surgery.
  • 10 is a wide-field fundus image and autofluorescence image of a rabbit eye according to the presence of vitrectomy and MNU concentration.
  • FIG. 11 is a near infrared (IR) photograph, light interference tomography (OCT) photograph and H & E tissue staining photograph 1 month after MNU injection into rabbit eye.
  • IR near infrared
  • OCT light interference tomography
  • 12 is a wide-field fundus image and autofluorescence image of a rabbit eye intraocularly injected without performing vitrectomy.
  • N-methyl-N-nitrosourea is one of the drugs currently used to make a retinal degeneration animal model and causes local degeneration of the retina, depending on how local it is or is dispersed in the retina after injection into the eye. The difference will occur. Thus, in order to cause total degeneration in the retina, the MNU injected into the eye needs to be controlled to be evenly present throughout the retina.
  • the vitreous cavity in which the vitreous is removed is replaced with air, followed by injecting a certain concentration of MNU, leaving it for a certain time, and then removing the drug to induce uniform degeneration of the entire retina. I found it possible to complete it.
  • PV Pars Plana Vitrectomy
  • a vitrectomy a surgical procedure to remove the vitreous tissue by mechanically cutting and aspirating vitreous fluid through a surgical device called a vitrectomy. Vitrectomy can be classified according to the thickness of the vitrectomy needle. Currently, there are 27, 25, 23, and 20 gauge types of vitrectomy needles. 23G vitrectomy and 25 G vitrectomy is most commonly used.
  • Vitrectomy in the animal model manufacturing method of the present invention is performed to remove the vitreous tissue to uniformly spread the drug into the eye, sclerotomy (trocar insertion), insertion of infusion cannula, equilibrium Control of perfusion pressure using a balanced salt solution, phacoemulsification, PVD (Posterior Vitreous Detachment) induction, retinal detachment treatment, and some steps may be omitted as needed.
  • MNU N-methyl-N-nitrosourea
  • the steps a) to d) may be performed by simultaneous surgery, where "simultaneous surgery" means one operation including each step.
  • step d) may be performed after 2 minutes to 20 minutes after the step c).
  • step d) when the MNU solution is removed before 2 minutes have elapsed, retinal degeneration may not occur sufficiently, and when the MNU solution is removed after 20 minutes, too severe retinal degeneration may occur. It is desirable to remove within 20 minutes.
  • the method of the present invention removes the drug after injecting the MNU solution into the eye to cause degeneration, thereby minimizing the side effects that the drug may remain in the eye and is easy to control the degree of retinal degeneration.
  • the method of injecting the MNU solution into the empty space in the eye where the vitreous is removed may cause retinal detachment and cause a wide and uniform retinal degeneration including the entire width of the retina, unlike the method of injecting MNU below the retina. have.
  • the denaturation caused by the injected MNU solution is not injection dose-based, but densities based on the concentration of the injection, the difference in the size of the eye according to the species and individual differences, or the difference in the intravitreal volume, depending on the presence or absence of intraocular lens. It is not affected. Furthermore, the method of the present invention can solve problems such as binocular blindness and drug toxicity caused by systemic injection such as intraperitoneal administration, and it is useful to induce retinal degeneration only in one eye because MNU is injected directly into the eye. Can be.
  • steps b) to d) may be performed by a separate operation from step a).
  • steps b) to d) may be performed to secure a sufficient recovery period after vitreous removal. Preferably at least 3 days after the step).
  • the MNU solution is injected into the vitreous cavity in front of the eye's retina, which is distinct from the subretinal injection.
  • the injected MNU solution is preferably injected at a concentration of 2 mg / ml to 10 mg / ml, and more preferably at a concentration of 4 mg / ml to 8 mg / ml.
  • Retinal degeneration is not induced when MNU is injected below 2mg / ml, and when it is injected above 10mg / ml, the toxicity of the retina is reduced due to the high toxicity. It is not preferable because it is difficult to use as (see Examples 3-1, 3-2 and FIGS. 1 to 4).
  • the method for producing an animal model of the present invention comprises the following steps d): e) washing the eye and filling an empty space in the eye; It may further include.
  • the empty space within the eyeball may be filled with an equilibrium base solution, air, a filling gas, a silicone oil, etc. according to eye conditions, but is not limited thereto.
  • the mammal used in the method for producing an animal model of the present invention may use a rabbit, a dog, a cat, a pig or a monkey, and a dog, cat, a pig or a monkey with a large eyeball is more preferable.
  • pigs are most preferred because they are similar in size to humans and are easier to perform vitrectomy than dogs and cats.
  • a retinal degeneration animal model produced by the animal model manufacturing method of the present invention.
  • the retinal degeneration animal model produced by the manufacturing method of the present invention causes degeneration uniformly over the entire retina of the eye, the researcher can easily apply the actual treatment to the animal model.
  • the retinal degeneration animal model prepared by the manufacturing method of the present invention is confirmed by the optical fundus camera, OCT, retinal potential diagram, multifocal retinal potential diagram, etc. after MNU injection to meet the study purpose. It can be used for experiments.
  • Medical piglets (Medicinetics, MICROPIG®) of 1 to 2 years of age were prepared and general anesthesia was performed. Porcine pupils were expanded with 0.5% tropicamide and 2.5% phenylephrine HCl eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.
  • MICROPIG® phenylephrine HCl eye drops
  • trocars were inserted into the pig's eye using a 23 gauge MVR knife at about 3 to 4 mm from the limbus of the cornea.
  • the trocar was injected with an equilibrium base solution to maintain the volume while maintaining the volume.
  • Vitrectomy was performed using a trocar.
  • the surgery was performed using the illumination of the surgical microscope and checking the intraocular structure using the Biom system to secure the surgical field of view.
  • MNU N-methyl-N-nitrosourea
  • BSS balanced salt solution
  • Example 3-1 (2mg / ml), Experimental Example 3-2 (4mg / ml), Experimental Example 3-3 (5mg / ml), Experimental Example 3-4 (6mg / ml) according to the concentration of the injected MNU And Experimental Example 3-5 (8 mg / ml).
  • Example 3 suggest that using the method of the present invention it is possible to induce uniform and effective retinal degeneration over the entire retina.
  • the rabbits were anesthetized with alfaxalone (5 mg / kg; Alfaxan® Vetoquinol, Westshire, UK) and xylazine (4 mg / kg; Rompun® Bayer Corp., Shawnee Mission, KA, USA). Two drugs were each injected 1 CC intramuscularly. In case of lack of anesthesia, an additional 0.5 cc was injected intramuscularly.
  • the pupil of the rabbit was expanded with 0.5% tropicamide and phenylephrine HCl 2.5% eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.
  • MNU N-methyl-N-nitrosourea
  • PBS phosphate buffered saline
  • Example 4-1 0.05mg / 0.05ml
  • Experimental Example 4-2 0.1mg / 0.05ml
  • Experimental Example 4-3 0.3mg / 0.05ml
  • Experimental 4- 4 0.5 mg / 0.05 ml
  • the group injected with MNU 0.5 mg / 0.05 ml without vitrectomy were compared to Comparative Example 4-1, and the group subjected to only vitrectomy was Comparative Example 4-2.
  • FIG. 10 One month after the MNU injection, the pattern of retinal degeneration was shown in FIG. 10 by wide-field fundus imaging and autofluorescence fundus imaging. Autofluorescence coherence tomography (OCT), near-infrared (IR), and H & E tissue staining images are shown in FIG. .
  • OCT Autofluorescence coherence tomography
  • IR near-infrared
  • H & E tissue staining images are shown in FIG. .
  • a to d are the group injected with 0.5 mg / 0.05 ml of MNU without vitrectomy (Comparative Example 4-1), where the dorsal (ear) side of the fundus is brightened white and a locally formed lesion can be observed.
  • e ⁇ h is a group performed only vitrectomy (Comparative Example 4-2), it can be confirmed that there is no change.
  • i to l are the group injected with 0.05 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-1). No significant changes were observed in both fundus and autofluorescence images.
  • m ⁇ p is a group injected with 0.1 mg / 0.05 ml MNU after vitrectomy (Experimental Example 4-2), no significant change was observed in both fundus and autofluorescence.
  • q ⁇ t is a group injected with 0.3 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-3). You can see the increase.
  • u ⁇ x is a group injected with 0.5 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-4).
  • the autosomal and autofluorescence images showed a bovine-like border with white lines at the dorsal (ear) end of the fundus. Fluorescence is present, and inside the dark focal local lesions can be observed.
  • IR near-infrared
  • OCT light interference tomography
  • a, b is a group injected with 0.5 mg / 0.05 ml of MNU without vitrectomy (Comparative Example 4-1), and the outer layer corresponding to the cell layer of the retina in the OCT image of the bright region at the top of the near-infrared (IR) image But not to the inner layer of the retina, it can be seen that the entire retinal layer is severely atrophy. The histology revealed that the cell layer was lost.
  • c and d were the only vitrectomy group (Comparative Example 4-2). IR, OCT and histological findings were all normal.
  • e and f are the group injected with 0.05 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-1). All of the IR, OCT and tissue findings were normal.
  • g and h were the group injected with 0.1 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-2), and all of the IR, OCT and tissue findings were normal.
  • i, j is a group injected with MNU 0.3mg / 0.05ml after vitrectomy (Experimental Example 4-3). It was confirmed that the outer retinal outer layer corresponding to the oocyte layer was lost. The histologic examination lost the cell layer.
  • k, l is a group injected with 0.5 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-4), and the outer layer corresponding to the retinal cell layer in the OCT image of the lower bright region connected to the optic nerve in the near infrared (IR) image. As well as the inner layer of the retina, it can be seen that the entire retinal layer was severely shrunk. Biopsy also confirmed the loss of the entire retinal layer.
  • m is a diagram of the pattern of living and dead cells according to the concentration of MNU.
  • the group injected with 0.05mg / 0.05ml and 0.1mg / 0.05ml showed a similar pattern to the group not injected with MNU, indicating that retinal degeneration was not induced, and most of the group injected with 0.5mg / 0.05ml It can be seen that the cell disappeared.
  • the group injected with 0.3mg / 0.05ml it can be seen that about 60% of the cells lost, retinal degeneration in the range of 1/3 to 2/3 was induced.
  • MNU concentrations of 0.2 mg / 0.05 ml, 0.3 mg / 0.05 ml, and 0.4 mg / 0.05 ml, respectively, was injected at about 4 mm from the limbal portion of the rabbit cornea using a 30 gauge needle without vitrectomy.
  • MNU injection retinal degeneration was confirmed and shown in FIG. 12.
  • retinal degeneration was induced at all of the concentrations of 0.2 mg / 0.05 ml, 0.3 mg / 0.05 ml, and 0.4 mg / 0.05 ml, in particular 0.3 mg / 0.05 ml when injected at a concentration of 0.2 mg / 0.05 ml.
  • the degree of retinal degeneration was more severe than that injected at 0.4mg / 0.05ml.

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Abstract

According to one embodiment of the present invention, a method for producing a retinal degeneration animal model comprises: a) removing the vitreous body from an eye of a non-human mammal by performing vitrectomy; b) replacing, with air, an empty space inside the eye from which the vitreous body has been removed; c) injecting an N-methyl-N-nitrosourea (MNU) solution into the empty space inside the eye; and d) removing the MNU solution.

Description

유리체 절제술 및 안구 내 MNU 주입과 제거를 이용한 망막 변성 동물 모델의 제조방법 및 이를 이용한 망막 변성 동물 모델Retinal Degeneration Animal Model Using Vitrectomy and Intraocular MNU Injection and Removal and Retinal Degeneration Animal Model
본 발명은 유리체 절제술을 통해 유리체 조직이 제거된 동물의 안구 내로 MNU(N-methyl-N-nitrosourea)의 주입 및 제거를 수행하는 방법을 이용한 망막 변성 동물 모델의 제조방법 및 이를 이용하여 제조된 망막 변성 동물 모델에 관한 것이다.The present invention provides a method for preparing a retinal degeneration animal model using a method of injecting and removing N-methyl-N-nitrosourea (MNU) into an eyeball of an animal in which vitreous tissue is removed through vitrectomy and a retina manufactured using the same. It relates to a denatured animal model.
망막 변성은 시세포의 변성으로 시력이 저하되어 결국 실명되는 질환으로, 아직 효과적인 치료법이 없다. 유전성 망막 변성, 망막색소변성증(retinitis pigmentosa, RP) 등 망막의 시세포가 손상되어 실명에 이르는 질환들의 치료를 위하여 개발되는 유전자 치료, 줄기세포 치료, 나아가 인공망막 등 여러 치료방법을 적용하기 위해서는 망막 변성 질환을 가진 실험동물의 개발이 필수적이다.Retinal degeneration is a disease in which vision is deteriorated due to degeneration of visual cells and eventually blindness, and there is no effective treatment yet. Retinal degeneration in order to apply gene therapy, stem cell therapy, and artificial retina, which are developed for the treatment of diseases such as hereditary retinal degeneration and retinitis pigmentosa (RP) that damage the retina's eye cells and lead to blindness Development of experimental animals with disease is essential.
이와 같이 여러 치료방법을 적용하기 위해 유전자 변성 동물 모델이나 약물로 변성을 유발하는 모델 등이 개발되고 있다. 약물로 망막 변성을 유발한 동물 모델의 경우, 일단 약물 변성 프로토콜이 확립되면 유전자 변성 동물 모델보다 가격이 저렴하며, 변성 유발기간이 짧아 대량으로 변성을 유발할 수 있고, 연구자가 각종 규제로부터 자유로울 수 있는 장점이 있다.As such, genetically modified animal models or models for inducing degeneration with drugs have been developed to apply various treatment methods. In animal models of drug-induced retinal degeneration, once drug degeneration protocols are established, they are less expensive than genetically modified animal models, and shorter periods of degeneration can cause degeneration in large quantities and free the investigator from various regulations. There is an advantage.
현재 망막 변성 동물 모델을 만들기 위해 사용되는 약제 중 하나인 N-methyl-N-nitrosourea (MNU)는 일상 환경에 널리 퍼져있는 질소화합물로, 다양한 동물 에서 망막 변성을 유발할 수 있는 알킬 화합물이다. MNU는 p-450 system의 대사 활성화 없이 직접적으로 알킬화(alkylating)를 일으키는 화학물질로 강력한 돌연변이원이며, 기형발생원 및 발암물질이다. MNU를 이용한 동물 모델의 제조방법으로는 마우스나 래트에 MNU를 전신 주사하여 망막 변성을 유발하는 방법이 대중적이다. 한편, 망막 변성 모델에서 MNU는 망막광수용체 세포에 특이적으로 독성을 나타내어 망막광수용체 세포의 세포자멸사(apoptosis)를 유발하며, 그 손상의 정도는 MNU의 용량과 시간에 비례하는 것으로 알려져 있다. 구체적으로, MNU 주입 후 24시간이 지난 시점에서 망막두께의 감소가 관찰되고, TUNEL 염색을 이용한 세포 괴사에 대한 형태학적인 연구에서는 약물주입 후 12시간에 세포자멸사가 관찰되기 시작하여 24시간 후에 최고조를 이루는 것으로 확인되었다.N-methyl-N-nitrosourea (MNU), one of the agents currently used to make animal models of retinal degeneration, is a nitrogen compound that is widely used in everyday environments and is an alkyl compound that can cause retinal degeneration in various animals. MNU is a chemical that causes alkylation directly without metabolic activation of the p-450 system. It is a powerful mutagen, teratogenic and carcinogen. As a method for producing an animal model using MNU, a method of inducing retinal degeneration by systemic injection of MNU into a mouse or rat is popular. In the retinal degeneration model, MNU is specifically toxic to retinal photoreceptor cells, causing apoptosis of retinal photoreceptor cells, and the extent of damage is known to be proportional to the dose and time of MNU. Specifically, a decrease in retinal thickness was observed 24 hours after MNU injection, and in a morphological study of cell necrosis using TUNEL staining, apoptosis began to be observed 12 hours after drug injection and peaked 24 hours later. It was confirmed to accomplish.
한편, 종래의 MNU를 이용한 망막 변성 동물 모델의 제조는 전신 주사를 시행하여 실험체의 양안을 실명하게 만들어 장기간 실험체를 사육하여 치료효과를 보아야 하는 연구에 어려움이 있었다. 게다가, 전신 주사의 경우 약물의 독성으로 인해 실험체의 체중이 감소하고 암이 발생하며 사망에 이르는 경우까지도 발생하는 문제가 있다. 대안적으로, Rosch, S., Werner, C., Muller, F. et al. Graefes Arch Clin Exp Ophthalmol (2017) 255: 317.　 에서는 토끼의 안구에 직접 MNU를 주입하여 망막 변성을 유발하는 연구를 기술하고 있으나, MNU의 주입량을 토끼의 몸무게를 기준으로 하여 실제 임상에 적용하기 어렵고, 안구에 주사한 약물이 안구 내에서 어떻게 분포하는지 명확히 제시하지 못한 한계가 있다.On the other hand, the manufacturing of a retinal degeneration animal model using a conventional MNU has been difficult to study the long-term breeding of the test subjects to make the both eyes of the test subjects blinded by performing a systemic injection. In addition, in the case of systemic injection, the toxicity of the drug causes a problem of weight loss, cancer, and even death. Alternatively, Rosch, S., Werner, C., Muller, F. et al. Graefes Arch Clin Exp Ophthalmol (2017) 255: 317. describes a study that induces retinal degeneration by injecting MNU directly into the rabbit's eye, but it is difficult to apply the amount of MNU to clinical practice based on the weight of the rabbit. However, there are limitations that do not provide a clear picture of how the drugs injected into the eye are distributed within the eye.
이러한 배경 하에, 본 발명자는 유리체 절제술을 통해 유리체 조직이 제거된 안구 내로 특정 농도의 MNU를 주입하고 일정 시간 동안 방치한 후 약물을 제거하는 방법을 이용하면 안구의 크기에 관계없이 안구 전체의 변성이 유발된 동물 모델을 제조할 수 있다는 사실을 발견함으로써 본 발명을 완성하게 되었다.Against this background, the present inventors have injected a certain concentration of MNU into the eye where vitreous tissue has been removed through vitrectomy, and after removing the drug after leaving it for a certain time, degeneration of the whole eye regardless of the size of the eye The discovery of the ability to produce an induced animal model led to the completion of the present invention.
본 발명은 종래 기술의 문제점을 해결하기 위한 것으로, 본 발명의 목적은 한쪽 눈에 국한되며, 안구 내 전체 범위의 망막 시세포가 소실된 망막 변성 동물 모델의 제조방법 및 이를 이용한 동물 모델을 제공하는 것이다.The present invention is to solve the problems of the prior art, an object of the present invention is limited to one eye, to provide a method for producing a retinal degeneration animal model in which the entire range of retinal oocytes in the eye and the animal model using the same. .
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명의 일 실시예에 따르면, a) 인간을 제외하는 포유동물의 안구에서 유리체 절제술을 수행하여 유리체를 제거하는 단계; b) 유리체가 제거된 안구 내 빈 공간을 공기로 치환하는 단계; c) 상기 안구 내 빈 공간으로 MNU(N-methyl-N-nitrosourea) 용액을 주입하는 단계; 및 d) MNU 용액을 제거하는 단계; 를 포함하는, 망막 변성 동물 모델의 제조방법이 제공된다.According to one embodiment of the invention, a) performing a vitrectomy in the eye of a mammal excluding a human to remove the vitreous; b) replacing the empty space in the eyeball from which the vitreous is removed with air; c) injecting an N-methyl-N-nitrosourea (MNU) solution into the empty space in the eye; And d) removing the MNU solution; Provided is a method of producing a retinal degeneration animal model comprising a.
일 측에 따르면, 상기 a) 단계 내지 d) 단계는 동시 수술로 수행될 수 있다.According to one side, step a) to d) may be performed by simultaneous surgery.
일 측에 따르면, 상기 d) 단계는, 상기 c) 단계로부터 2분 내지 20분 경과 후 수행될 수 있다.According to one side, step d) may be performed after 2 minutes to 20 minutes from the step c).
일 측에 따르면, 상기 b) 단계 내지 d) 단계는, 상기 a) 단계와 별개의 수술로 수행될 수 있다. According to one side, step b) to step d) may be performed by a separate operation from the step a).
일 측에 따르면, 상기 b) 단계 내지 d) 단계는, 상기 a) 단계로부터 적어도 3일 이후에 수행될 수 있다.According to one side, step b) to step d) may be performed at least three days after the step a).
일 측에 따르면, 상기 d) 단계 이후에, e) 안구를 세척하고, 안구 내 빈 공간을 충전하는 단계; 를 더 포함할 수 있다.According to one side, after step d), e) washing the eye, filling the empty space in the eye; It may further include.
일 측에 따르면, 상기 c) 단계에서 MNU 용액은 안구의 망막 앞, 유리체강 부위로 주입될 수 있다.According to one side, in step c) MNU solution may be injected into the vitreous cavity region, in front of the eye retina.
일 측에 따르면, 상기 MNU용액은 2mg/ml 내지 10mg/ml의 농도일 수 있다.According to one side, the MNU solution may be a concentration of 2mg / ml to 10mg / ml.
일 측에 따르면, 상기 포유동물은 토끼, 개, 고양이, 돼지 또는 원숭이 일 수 있다.According to one side, the mammal may be a rabbit, dog, cat, pig or monkey.
일 측에 따르면, 안구의 망막 전체가 변성될 수 있다.According to one side, the entire retina of the eye may be degenerated.
본 발명의 망막 변성 동물 모델의 제조방법은 MNU의 주입 전 유리체 절제술을 통해 유리체 조직을 제거하므로, 유리체 절제술을 수행하지 않는 경우와 달리 약물 용량에 비례한 변성을 유발할 수 있고, 망막 변성의 정도를 예측할 수 있다.Since the method of manufacturing the retinal degeneration animal model of the present invention removes the vitreous tissue through vitrectomy prior to the injection of MNU, it may cause degeneration proportional to the dose of the drug, unlike when vitrectomy is not performed. It can be predicted.
또한, 본 발명의 망막 변성 동물 모델의 제조방법은 개체 간 종의 차이에 따른 안구 크기, 수정체 유무에 관계없이 일정 농도의 MNU를 이용하여 같은 정도의 망막 변성 유발이 가능하다.In addition, the method of manufacturing a retinal degeneration animal model of the present invention can induce the same degree of retinal degeneration by using a certain concentration of MNU regardless of eye size and lens presence according to differences between species.
특히, 본 발명의 제조방법을 이용하면 안구 내 잔여 MNU를 최소화함으로써 전신적인 약물의 흡수에 의해 전신적인 합병증이 나타나는 것을 방지할 수 있다. In particular, using the method of the present invention can minimize the residual MNU in the eye can prevent the appearance of systemic complications due to systemic absorption of the drug.
본 발명의 제조방법에 따르면 한쪽 눈에만 국한된 균일하고 안구 전체 넓이의 망막을 모두 포함하는 망막 변성이 유발된 동물 모델을 제조할 수 있으므로, 제조된 동물 모델은 인공 망막의 개발, 줄기세포 치료 연구 등에 유리하게 이용될 수 있으며, 사육의 편리함도 제공할 수 있다.According to the manufacturing method of the present invention, a retinal degeneration-induced animal model including all of the retinas with a uniform and whole-eye area limited to only one eye can be manufactured. Thus, the manufactured animal model can be used for the development of artificial retina, stem cell treatment research, and the like. It can be used advantageously and can also provide the convenience of breeding.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 수술 전과 유리체 절제술 및 MNU(2mg/ml) 약물 주사 수술 3주 경과 후 OCT 검사 결과이다.Figure 1 shows the results of the OCT test before and 3 weeks after the vitrectomy and MNU (2mg / ml) drug injection surgery.
도 2는 은 수술 전과 유리체 절제술 및 MNU(2mg/ml) 약물 주사 수술 3주 경과 후 ERG검사 결과이다.Figure 2 shows the results of ERG test before and 3 weeks after vitrectomy and MNU (2mg / ml) drug injection surgery.
도 3은 수술 전과 유리체 절제술 및 MNU(4mg/ml) 약물 주사 수술 3주 경과 후, 12주 후 OCT 검사 결과이다.Figure 3 shows the results of the OCT test before and after 12 weeks after surgery, vitrectomy and MNU (4mg / ml) drug injection surgery.
도 4는 수술 전과 유리체 절제술 및 MNU(4mg/ml) 약물 주사 수술 3주 경과 후, 12주 후 ERG 검사 결과이다.Figure 4 shows the ERG test results before and after 12 weeks after surgery, vitrectomy and MNU (4 mg / ml) drug injection surgery.
도 5는 수술 전과 유리체 절제술 및 MNU(5mg/ml) 약물 주사 수술 3주 경과 후, 6주 후 OCT검사 결과이다.Figure 5 shows the results of the OCT test before and 6 weeks after surgery and vitrectomy and MNU (5mg / ml) drug injection surgery.
도 6은 수술 전과 유리체 절제술 및 MNU(5mg/ml) 약물 주사 수술 3주 경과 후, 6주 후 ERG 검사 결과이다.Figure 6 shows the ERG test results before and after 6 weeks after the surgery and vitrectomy and MNU (5mg / ml) drug injection surgery.
도 7은 수술 전과 유리체 절제술 및 MNU(6mg/ml) 약물 주사 수술 2주 경과 후, 12주 후 OCT검사 결과이다.7 shows the results of the OCT test before and after 12 weeks after the surgery and vitrectomy and MNU (6 mg / ml) drug injection surgery.
도 8은 수술 전과 유리체 절제술 및 MNU(6mg/ml) 약물 주사 수술 2주 경과 후, 12주 후 ERG 검사 결과이다.FIG. 8 shows ERG test results before and after 12 weeks after surgery, vitrectomy and MNU (6 mg / ml) drug injection.
도 9는 수술 전과 유리체 절제술 및 MNU(8mg/ml) 약물 주사 수술 3주 경과 후 OCT 검사 결과이다.Figure 9 shows the results of the OCT test before and 3 weeks after the vitrectomy and MNU (8mg / ml) drug injection surgery.
도 10은 유리체 절제술의 유무 및 MNU 농도에 따른 토끼 안구의 광시야 안저영상 및 자가 형광 영상 사진이다. 10 is a wide-field fundus image and autofluorescence image of a rabbit eye according to the presence of vitrectomy and MNU concentration.
도 11은 토끼 안구 내로 MNU 주사 1달 후 근적외선(IR) 사진, 빛 간섭 단층촬영(OCT) 사진 및 H&E 조직염색사진이다.FIG. 11 is a near infrared (IR) photograph, light interference tomography (OCT) photograph and H & E tissue staining photograph 1 month after MNU injection into rabbit eye.
도 12는 유리체 절제술을 수행하지 않고 MNU를 안구 내 주사한 토끼 안구의 광시야 안저영상 및 자가 형광 영상 사진이다.12 is a wide-field fundus image and autofluorescence image of a rabbit eye intraocularly injected without performing vitrectomy.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리 범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments so that the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, and substitutes for the embodiments are included in the scope of rights.
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terminology used herein is for the purpose of description and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and duplicate description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.
MNU(N-methyl-N-nitrosourea)는 현재 망막 변성 동물 모델을 만들기 위해 사용되는 약제 중 하나로, 안구 내로 주사 후 망막에 얼마나 국소적으로 존재하는지 혹은 분산되는지 여부에 따라 망막의 국소적 변성을 유발하는 정도에 차이가 발생하게 된다. 따라서, 망막에 전체적인 변성을 유발하기 위해서는 안구 내로 주사된 MNU가 망막에 전체적으로 골고루 존재하도록 제어될 필요가 있다.N-methyl-N-nitrosourea (MNU) is one of the drugs currently used to make a retinal degeneration animal model and causes local degeneration of the retina, depending on how local it is or is dispersed in the retina after injection into the eye. The difference will occur. Thus, in order to cause total degeneration in the retina, the MNU injected into the eye needs to be controlled to be evenly present throughout the retina.
본 발명은 유리체 절제술을 수행하는 동안 유리체가 제거된 유리체강 부위를 공기로 치환한 후 특정 농도의 MNU를 주입하고 일정시간 동안 방치한 후 약물을 제거하는 방법을 통해 망막 전체에 균일한 변성을 유발할 수 있음을 발견하여 완성하게 된 것이다.In the present invention, during vitrectomy, the vitreous cavity in which the vitreous is removed is replaced with air, followed by injecting a certain concentration of MNU, leaving it for a certain time, and then removing the drug to induce uniform degeneration of the entire retina. I found it possible to complete it.
본 발명에서 용어 "유리체 절제술(Pars Plana Vitrectomy, PPV)" 은 유리체 절제침(vitrector)라 불리는 외과장치를 통해 유리체 체액을 기계적으로 절단 및 흡입함으로써 유리체 조직을 제거하는 외과 시술이다. 유리체 절제술은 유리체 절제침의 굵기(gauge)에 따라서 분류될 수 있는데, 현재 사용되고 있는 유리체 절제침의 종류는 27 게이지, 25 게이지, 23 게이지, 20 게이지가 있으며, 23 게이지 유리체 절제술(23G vitrectomy)과 25 게이지 유리체 절제술(25G vitrectomy)이 가장 많이 사용된다. The term "Pars Plana Vitrectomy (PPV)" in the present invention is a surgical procedure to remove the vitreous tissue by mechanically cutting and aspirating vitreous fluid through a surgical device called a vitrectomy. Vitrectomy can be classified according to the thickness of the vitrectomy needle. Currently, there are 27, 25, 23, and 20 gauge types of vitrectomy needles. 23G vitrectomy and 25 G vitrectomy is most commonly used.
본 발명의 동물 모델 제조방법에서 유리체 절제술은 유리체 조직을 제거하여 약물이 안구 내로 균일하게 퍼지도록 하기 위한 목적으로 수행되는 것으로, 공막 절개(trocar insertion), 주입관 삽입(insertion of infusion cannula), 평형염액(balanced salt solution)을 이용한 관류 압력의 조절, 수정체제거술, PVD(Posterior vitreous detachment, 후유리체박리) induction, 망막견인막 처치 단계를 포함할 수 있으며, 필요에 따라 일부 단계가 생략될 수 있다. Vitrectomy in the animal model manufacturing method of the present invention is performed to remove the vitreous tissue to uniformly spread the drug into the eye, sclerotomy (trocar insertion), insertion of infusion cannula, equilibrium Control of perfusion pressure using a balanced salt solution, phacoemulsification, PVD (Posterior Vitreous Detachment) induction, retinal detachment treatment, and some steps may be omitted as needed.
본 발명의 일 실시예에 따르면, a) 인간을 제외하는 포유동물의 안구에서 유리체 절제술을 수행하여 유리체를 제거하는 단계; b) 유리체가 제거된 안구 내 빈 공간을 공기로 치환하는 단계; c) 상기 안구 내 빈 공간으로 MNU(N-methyl-N-nitrosourea) 용액을 주입하는 단계; 및 d) MNU 용액을 제거하는 단계; 를 포함하는, 망막 변성 동물 모델의 제조방법이 제공된다.According to one embodiment of the invention, a) performing a vitrectomy in the eye of a mammal excluding a human to remove the vitreous; b) replacing the empty space in the eyeball from which the vitreous is removed with air; c) injecting an N-methyl-N-nitrosourea (MNU) solution into the empty space in the eye; And d) removing the MNU solution; Provided is a method of producing a retinal degeneration animal model comprising a.
본 발명에서, 상기 a) 단계 내지 d) 단계는 동시 수술로 수행될 수 있고, 여기서 "동시 수술"은 각 단계를 포함하는 1회의 수술을 의미한다. 본 발명의 망막 변성 동물 모델의 제조방법을 동시 수술로 진행하는 경우 상기 d) 단계는, 상기 c) 단계로부터 2분 내지 20분 경과 후 수행될 수 있다. In the present invention, the steps a) to d) may be performed by simultaneous surgery, where "simultaneous surgery" means one operation including each step. When the method of manufacturing the retinal degeneration animal model of the present invention is performed by simultaneous surgery, step d) may be performed after 2 minutes to 20 minutes after the step c).
상기 d) 단계에서, MNU 용액을 2분이 경과하기 전에 제거하는 경우 망막 변성이 충분히 발생하지 않을 수 있고, MNU 용액을 20분이 경과한 후에 제거하는 경우 너무 심한 망막 변성이 발생할 수 있으므로, 2분 내지 20분 내에 제거하는 것이 바람직하다. 이와 같이, 본 발명의 방법은 안구 내에 MNU 용액을 주입하여 변성을 유발한 후 약물을 제거하므로, 약물이 안구에 남아 발생할 수 있는 부작용을 최소화할 수 있고, 망막 변성 정도를 제어하기에도 용이하다.In step d), when the MNU solution is removed before 2 minutes have elapsed, retinal degeneration may not occur sufficiently, and when the MNU solution is removed after 20 minutes, too severe retinal degeneration may occur. It is desirable to remove within 20 minutes. As such, the method of the present invention removes the drug after injecting the MNU solution into the eye to cause degeneration, thereby minimizing the side effects that the drug may remain in the eye and is easy to control the degree of retinal degeneration.
또한, MNU 용액을 유리체가 제거된 안구 내 빈 공간으로 주입하는 방식은 망막 박리를 유발하여 망막 아래로 MNU를 주사하는 방법과는 달리 망막의 전체 넓이를 포함하는 광범위하고 균일한 망막 변성을 유발할 수 있다. In addition, the method of injecting the MNU solution into the empty space in the eye where the vitreous is removed may cause retinal detachment and cause a wide and uniform retinal degeneration including the entire width of the retina, unlike the method of injecting MNU below the retina. have.
주입된 MNU용액에 의해 유발되는 변성은 주사 용량기반의 변성이 아닌 주사액의 농도기반의 변성이기 때문에 종간 혹은 개체 차이에 따른 안구의 크기의 차이나 안구 내 수정체의 유무에 다른 유리체강 내 용적의 차이에도 영향을 받지 않는다. 더욱이, 본 발명의 방법은 복강 내 투여와 같은 전신 주사로 발생하는 양안 실명, 약물 독성 등의 문제를 해결할 수 있으며, MNU를 직접 안구에 주입하므로 한쪽 눈에만 망막 변성을 유발하는 데 유용하게 이용될 수 있다.Since the denaturation caused by the injected MNU solution is not injection dose-based, but densities based on the concentration of the injection, the difference in the size of the eye according to the species and individual differences, or the difference in the intravitreal volume, depending on the presence or absence of intraocular lens. It is not affected. Furthermore, the method of the present invention can solve problems such as binocular blindness and drug toxicity caused by systemic injection such as intraperitoneal administration, and it is useful to induce retinal degeneration only in one eye because MNU is injected directly into the eye. Can be.
한편, 상기 b) 단계 내지 d) 단계는, 상기 a) 단계와 별개의 수술로 수행될 수 있으며, 이 경우 유리체 제거 후 충분한 회복 기간을 확보하기 위해 상기 b) 단계 내지 d) 단계는, 상기 a) 단계로부터 적어도 3일 이후에 수행되는 것이 바람직하다. Meanwhile, steps b) to d) may be performed by a separate operation from step a). In this case, steps b) to d) may be performed to secure a sufficient recovery period after vitreous removal. Preferably at least 3 days after the step).
상기 c) 단계에서 MNU 용액은 안구의 망막 앞, 유리체 강 부위로 주입되는 것으로, 망막 아래 주사와는 구별된다. 주입되는 MNU용액은 2mg/ml 내지 10mg/ml의 농도로 주입되는 것이 바람직하며, 4mg/ml 내지 8mg/ml의 농도로 주입되는 것이 보다 바람직하다. MNU를 2mg/ml 미만으로 주입하는 경우 망막 변성이 충분히 유발되지 않으며, 10mg/ml를 초과하여 주입하는 경우 독성이 커서 망막의 전층이 위축되고, 시세포층이 소실되는 등의 문제로 망막 변성 동물 모델로서 사용하기 어려우므로 바람직하지 않다(실시예 3-1, 3-2 및 도 1 ~ 4 참조). In step c), the MNU solution is injected into the vitreous cavity in front of the eye's retina, which is distinct from the subretinal injection. The injected MNU solution is preferably injected at a concentration of 2 mg / ml to 10 mg / ml, and more preferably at a concentration of 4 mg / ml to 8 mg / ml. Retinal degeneration is not induced when MNU is injected below 2mg / ml, and when it is injected above 10mg / ml, the toxicity of the retina is reduced due to the high toxicity. It is not preferable because it is difficult to use as (see Examples 3-1, 3-2 and FIGS. 1 to 4).
본 발명의 동물 모델 제조방법은 상기 단계 d) 이후에, e) 안구를 세척하고, 안구내 빈 공간을 충전하는 단계; 를 더 포함할 수 있다. 여기서, 안구 내 빈 공간에는 눈의 상태에 따라 평형 염기용액, 공기, 충전가스, 실리콘 오일 등을 충전할 수 있으나, 이에 한정되는 것은 아니다.The method for producing an animal model of the present invention comprises the following steps d): e) washing the eye and filling an empty space in the eye; It may further include. Here, the empty space within the eyeball may be filled with an equilibrium base solution, air, a filling gas, a silicone oil, etc. according to eye conditions, but is not limited thereto.
본 발명의 동물 모델 제조방법에 사용되는 포유동물은 토끼, 개, 고양이, 돼지 또는 원숭이를 이용할 수 있으며, 안구가 큰 개, 고양이, 돼지 또는 원숭이가 더욱 바람직하다. 그 중에서도, 돼지는 안구의 크기가 사람에 유사하고, 개나 고양이보다 유리체 절제술을 수행하기 용이하므로 가장 바람직하다. The mammal used in the method for producing an animal model of the present invention may use a rabbit, a dog, a cat, a pig or a monkey, and a dog, cat, a pig or a monkey with a large eyeball is more preferable. Among them, pigs are most preferred because they are similar in size to humans and are easier to perform vitrectomy than dogs and cats.
본 발명의 다른 일 실시예에 따르면, 본 발명의 동물 모델 제조방법으로 제조된 망막 변성 동물 모델이 제공된다.According to another embodiment of the present invention, there is provided a retinal degeneration animal model produced by the animal model manufacturing method of the present invention.
본 발명의 제조방법에 의해 제조된 망막 변성 동물 모델은 안구의 망막 전체 범위에 균일하게 변성이 유발되므로 연구자가 동물 모델에게 실제 치료를 적용하기 용이하다. 또한, 본 발명의 제조방법에 의해 제조된 망막 변성 동물 모델은 MNU 주사 이후 나타나는 망막 변성의 패턴을 광학 안저 카메라나 OCT, 망막 전위도, 다초첨 망막 전위도 등으로 확인하여 연구 목적에 부합하는 경우 실험에 이용될 수 있다. Since the retinal degeneration animal model produced by the manufacturing method of the present invention causes degeneration uniformly over the entire retina of the eye, the researcher can easily apply the actual treatment to the animal model. In addition, the retinal degeneration animal model prepared by the manufacturing method of the present invention is confirmed by the optical fundus camera, OCT, retinal potential diagram, multifocal retinal potential diagram, etc. after MNU injection to meet the study purpose. It can be used for experiments.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하기로 한다. 하기 실시예는 본 발명을 예시하기 위한 목적으로 기술된 것으로서, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.
이하의 실시 예는 Ophthalmic and Vision Research를 위한 동물의 사용법에 대하여 규제하고 있는 ARVO 선언의 규제에 따라 실시한 것이다.The following examples were carried out in accordance with the regulations of the ARVO Declaration, which regulates the use of animals for Ophthalmic and Vision Research.
실시예 1: 유리체 절제술의 시행Example 1: Perform Vitrectomy
나이 1~2년 정도의 의료 실험용 미니돼지(메디키네틱스, MICROPIG®)를 준비하고, 전신마취를 시행했다. 돼지의 동공은 tropicamide 0.5%와 phenylephrine HCl 2.5% eye drops (Mydrin-P®)에 의해 확장되었고, proparacine HCl 0.5% (Alcaine®) 국부 안약 마취제를 처리하였다.Medical piglets (Medicinetics, MICROPIG®) of 1 to 2 years of age were prepared and general anesthesia was performed. Porcine pupils were expanded with 0.5% tropicamide and 2.5% phenylephrine HCl eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.
먼저, 돼지 안구에 23 게이지 크기의 MVR knife를 이용하여 돼지 각막 윤부로부터 약 3~4mm 지점에 세개의 트로카를 삽입한 후 트로카 한 곳으로는 평형 염기용액을 주입하여 부피를 유지하면서 나머지 두개의 트로카를 이용하여 유리체 절제술을 시행했다. 이 때, 수술현미경의 조명을 이용하고, 수술 시야의 확보를 위해 Biom system을 이용하여 안구내 구조물을 확인하면서 수술을 진행하였다.First, three trocars were inserted into the pig's eye using a 23 gauge MVR knife at about 3 to 4 mm from the limbus of the cornea. The trocar was injected with an equilibrium base solution to maintain the volume while maintaining the volume. Vitrectomy was performed using a trocar. At this time, the surgery was performed using the illumination of the surgical microscope and checking the intraocular structure using the Biom system to secure the surgical field of view.
실시예 2: MNU 약물의 준비Example 2: Preparation of MNU Drugs
분자량 103g/mol의 N-methyl-N-nitrosourea (MNU) (Oakwood Products Inc., West Columbia, SC, USA)를 105mg/ml의 농도가 되도록 DMSO 용액에 녹이고, MNU가 각각 2mg/ml, 4mg/ml, 5mg/ml, 6mg/m, 8mg/ml의 농도가 되도록 평형 염기용액 (balanced salt solution) (BSS)에 희석하여 준비한다.N-methyl-N-nitrosourea (MNU) (Oakwood Products Inc., West Columbia, SC, USA) with a molecular weight of 103 g / mol was dissolved in DMSO solution to a concentration of 105 mg / ml, with MNU of 2 mg / ml and 4 mg / ml, respectively. Prepare by diluting in balanced salt solution (BSS) to a concentration of ml, 5mg / ml, 6mg / m, 8mg / ml.
실시예 3: MNU 농도에 따른 망막 변성 패턴의 비교Example 3: Comparison of Retinal Degeneration Patterns According to MNU Concentration
상기 실시예 1의 방법으로 유리체를 제거한 후 망막 앞 유리체강 부위의 평형 염기용액을 공기로 치환하고, 상기 실시예 2에서 준비한 약물들을 공기로 치환된 부위에 주입하고 2~10분이 지난 후 약물을 제거하였다. 그 다음, 평형 염기용액을 유리체강으로 관류시켜 유리체강 내부를 세척하고 유리체강을 가득 채운다. 주입된 MNU의 농도에 따라 실험예 3-1(2mg/ml), 실험예 3-2(4mg/ml), 실험예 3-3(5mg/ml), 실험예 3-4(6mg/ml), 실험예 3-5(8mg/ml)로 하였다. After removing the vitreous by the method of Example 1, the equilibrium base solution in the vitreous cavity region in front of the retina is replaced with air, and the drugs prepared in Example 2 are injected into the air-substituted site and the drug is removed after 2 to 10 minutes. Removed. The equilibrium base solution is then perfused with vitreous steel to wash the vitreous cavity interior and fill the vitreous cavity. Experimental Example 3-1 (2mg / ml), Experimental Example 3-2 (4mg / ml), Experimental Example 3-3 (5mg / ml), Experimental Example 3-4 (6mg / ml) according to the concentration of the injected MNU And Experimental Example 3-5 (8 mg / ml).
MNU 주사 2주 이후, 망막 변성의 패턴을 빛 간섭 단층촬영(OCT), 망막 전위도(ERG), 다초점 망막 전위도(mfERG)로 측정하고, 그 결과를 도 1 내지 도 9에 나타냈다.Two weeks after the MNU scan, the pattern of retinal degeneration was measured by light interference tomography (OCT), retinal potential diagram (ERG), and multifocal retinal potential diagram (mfERG), and the results are shown in FIGS. 1 to 9.
실험예 3-1의 경우, 도 1에 나타낸 바와 같이, 수술 전에 비해 유리체 절제술 및 MNU 약물 주사 후에 망막의 시세포를 나타내는 외층이 소실된 것을 알 수 있다. 그러나 도 2의 ERG 결과에서는 MNU 주사 3주 이후에도 여전히 음순응과 명순응 후 빛자극에 대한 반응이 살아 있어 망막 변성이 완전히 이루어지지 않은 것을 알 수 있다.In Experimental Example 3-1, as shown in FIG. 1, it can be seen that the outer layer showing the retinal eye cells disappeared after vitrectomy and MNU drug injection compared to before surgery. However, the ERG result of FIG. 2 shows that the response to light stimulation is still alive after the acclimatization and bright acclimation after 3 weeks of MNU injection, and thus, retinal degeneration is not completely achieved.
실험예 3-2의 경우, 도 3에 나타낸 바와 같이, 수술 전에 비해 유리체 절제술 및 MNU 약물 주사 후에 망막의 시세포를 나타내는 외층이 소실된 것을 알 수 있다. 그러나 도 4의 ERG 결과에서는 MNU 주사 12주 이후에도 여전히 음순응과 명순응후 빛자극에 대한 반응이 살아 있어 망막 변성이 완전히 이루어지지 않은 것을 알 수 있다.In Experimental Example 3-2, as shown in FIG. 3, it can be seen that the outer layer showing the retinal eye cells disappeared after vitrectomy and MNU drug injection compared to before surgery. However, in the ERG result of FIG. 4, the response to the light stimulation after the acclimatization and the light acclimatization was still alive after 12 weeks of MNU injection, indicating that retinal degeneration was not completely achieved.
실험예 3-3의 경우, 도 5에 나타낸 바와 같이, 수술 전에 비해 유리체 절제술 및 MNU 약물 주사 3주 후, 6주 후에 망막의 시세포를 나타내는 외층이 소실된 것을 알 수 있다. 그리고 도 6의 ERG 결과에서는 MNU 주사 3주 이후부터 ERG 전 반응이 소실되고 6주 이후에도 소실되는 것을 확인할 수 있으며, 망막변성의 균질성을 확인하기 위하여 시행한 mfERG에서도 망막의 전기반응을 측정한 103영역 모두에서 MNU 주사 3주, 6주 후에 빛자극에 대한 반응이 소실되었다.In Experimental Example 3-3, as shown in Fig. 5, the outer layer showing the retinal eye cells disappeared after 6 weeks after vitrectomy and MNU drug injection compared to before surgery. In the ERG result of FIG. 6, the pre-ERG response was lost after 3 weeks after MNU injection and after 6 weeks, and 103 region in which MFERG was measured to confirm the homogeneity of retinal degeneration was also measured. In all, the response to light stimulation was lost 3 and 6 weeks after MNU injection.
실험예 3-4의 경우, 도 7에 나타낸 바와 같이, 수술 전에 비해 유리체 절제술 및 MNU 약물 주사 2주 후, 12주 후에 망막의 시세포를 나타내는 망막 외층뿐만 아니라 망막 내층도 심하게 소실되고 얇아진 것을 알 수 있다. 그리고 도 8의 ERG 결과에서는 주사 2주 이후부터 ERG 전 반응이 소실되고, 12주 이후에도 소실되어 있는 것을 확인할 수 있었다. 망막 변성의 균질성을 확인하기 위하여 시행한 mfERG에서도 망막의 전기반응을 측정한 103 영역 모두에서 주사 2주 이후, 12주 이후에 빛자극에 대한 반응이 소실된 것으로 확인되었으며, 망막의 전기자극반응은 모두 소실되었으나 OCT 상에서 손상이 되어서는 안되는 망막 내층까지도 손상되어 MNU 농도가 적합 농도를 벗어났다.In Experimental Example 3-4, as shown in FIG. 7, two weeks after the vitrectomy and MNU drug injection, and 12 weeks after the vitrectomy and MNU drug injection, the inner layer of the retina as well as the inner layer of the retina were severely lost and thinned. have. In the ERG result of FIG. 8, it was confirmed that the reaction before ERG was lost from 2 weeks after the injection, and also after 12 weeks. In the mfERG, which was performed to confirm the homogeneity of retinal degeneration, the response to light stimulation was lost after 2 weeks and 12 weeks after injection in all 103 regions where the retinal electrical response was measured. All were lost, but even the retinal lining, which should not be damaged on the OCT, was damaged and the MNU concentration was outside of the appropriate concentration.
실험예 3-5의 경우, 도 9에 나타낸 바와 같이, 수술 전에 비해 유리체 절제술 및 MNU 약물 주사 3주 이후 망막 외층의 손상이 심하여 망막이 모두 망막 상피세포에서 분리되는 망막 박리가 발생하였다. 이는 주입한 약의 농도가 매우 높아서 망막 전층의 손상이 심함을 보여주는 소견이다.In Experimental Example 3-5, as shown in Fig. 9, the retinal outer layer was severely damaged after 3 weeks of vitrectomy and MNU drug injection compared to before surgery, and retinal detachment occurred in which all retinas were separated from retinal epithelial cells. This is because the concentration of the injected drug is very high.
결론적으로, 실시예 3의 결과는 본 발명의 방법을 이용하는 경우 망막 전체 범위에 균일하고 효과적인 망막 변성을 유발하는 것이 가능함을 시사한다.In conclusion, the results of Example 3 suggest that using the method of the present invention it is possible to induce uniform and effective retinal degeneration over the entire retina.
실시예 4: 유리체 절제술 유무에 따른 망막 변성 비교Example 4: Comparison of Retinal Degeneration with and Without Vitrectomy
실시예 4-1. 유리체 절제술의 시행Example 4-1. Conduct of Vitrectomy
2.5 내지 3.5 kg 범위의 뉴질랜드 흰토끼를 준비한다. A-scan을 이용하여 측정한 토끼의 안구 길이는 평균 15.75 mm (범위: 15.0~16.5 mm)이었다. Prepare New Zealand white rabbits ranging from 2.5 to 3.5 kg. The average ocular length of rabbits measured using A-scan was 15.75 mm (range: 15.0-16.5 mm).
준비한 토끼를 alfaxalone (5 mg/kg; Alfaxan®Vetoquinol, West Sussex, UK) 과 xylazine (4 mg/kg; Rompun®Bayer Corp., Shawnee Mission, KA, USA)을 이용하여 마취를 시행했다. 두 가지 약을 각각 1 CC 근육 주사하였다. 마취가 부족한 경우 0.5 cc씩 추가로 근육 주사하였다. 토끼의 동공은 tropicamide 0.5%와 phenylephrine HCl 2.5% eye drops (Mydrin-P®)에 의해 확장되었고, proparacine HCl 0.5% (Alcaine®) 국부 안약 마취제를 처리하였다.The rabbits were anesthetized with alfaxalone (5 mg / kg; Alfaxan® Vetoquinol, West Sussex, UK) and xylazine (4 mg / kg; Rompun® Bayer Corp., Shawnee Mission, KA, USA). Two drugs were each injected 1 CC intramuscularly. In case of lack of anesthesia, an additional 0.5 cc was injected intramuscularly. The pupil of the rabbit was expanded with 0.5% tropicamide and phenylephrine HCl 2.5% eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.
먼저 토끼 안구에 23게이지 크기의 유리체 절제침을 이용하여 토끼 각막 윤부에서 약 4mm 지점에 두개의 트로카를 삽입한 후 트로카 한 곳으로는 평형 염기용액을 주입하여 부피를 유지하면서 나머지 하나의 트로카를 이용하여 유리체 절제술을 시행했다. 이 때, 수술현미경의 조명을 이용하고, 수술 시야의 확보를 위해 direct concave lens를 이용하여 안구내 구조물을 확인하면서 수술을 진행하였다.First, insert two trocars about 4 mm from the limbal portion of the rabbit's cornea using a 23-gauge vitrectomy needle in the rabbit's eyeball, and inject the equilibrium base solution into one of the trocars to maintain the volume while maintaining the volume. Vitrectomy was performed. At this time, the operation was performed by using the illumination of the surgical microscope and checking the intraocular structure using a direct concave lens to secure the surgical field of view.
실시예4-2. MNU 약물의 준비Example 4-2. Preparation of MNU Drugs
분자량 103g/mol의 N-methyl-N-nitrosourea (MNU) (Oakwood Products Inc., West Columbia, SC, USA)를 105mg/ml의 농도가 되도록 DMSO 용액에 녹이고, 0.05 ml에 MNU가 각각 0.05mg, 0.1mg, 0.3mg, 0.5mg의 양이 되도록 phosphate buffered saline (PBS)에 희석하여 준비한다.N-methyl-N-nitrosourea (MNU) (Oakwood Products Inc., West Columbia, SC, USA) with a molecular weight of 103 g / mol is dissolved in DMSO solution to a concentration of 105 mg / ml, and 0.05 mg of MNU is 0.05 mg, Prepare diluted by phosphate buffered saline (PBS) to the amount of 0.1mg, 0.3mg, 0.5mg.
실시예 4-3. MNU 농도에 따른 망막 변성 패턴의 평가Example 4-3. Evaluation of Retinal Degeneration Pattern with MNU Concentration
상기 실시예 4-1에서 시행한 유리체 절제술로부터 1주일 이후, 상기 실시예 2에서 준비된 약물들을30 게이지 바늘을 이용하여 토끼 각막 윤부로부터 약 4mm 지점에 주사하였다. 주입된 MNU의 농도에 따라 실험예 4-1(0.05mg/0.05ml), 실험예 4-2(0.1mg/0.05ml), 실험예 4-3(0.3mg/0.05ml), 실험예 4-4(0.5mg/0.05ml)로 하고, 유리체 절제술 없이 MNU 0.5mg/0.05ml를 주사한 군을 비교예 4-1, 유리체 절제술만 시행한 군을 비교예 4-2로 하였다. After one week from the vitrectomy performed in Example 4-1, the drugs prepared in Example 2 were injected at about 4 mm from the rabbit corneal limbus using a 30 gauge needle. Experimental Example 4-1 (0.05mg / 0.05ml), Experimental Example 4-2 (0.1mg / 0.05ml), Experimental Example 4-3 (0.3mg / 0.05ml), Experimental 4- 4 (0.5 mg / 0.05 ml), the group injected with MNU 0.5 mg / 0.05 ml without vitrectomy were compared to Comparative Example 4-1, and the group subjected to only vitrectomy was Comparative Example 4-2.
MNU 주사 1달 이후, 망막 변성의 패턴을 광시야 안저 촬영 및 자가 형광 안저 촬영하여 도 10에 나타내고, 자가 형광 빛 간섭 단층촬영(OCT), 근적외선(IR), H&E 조직염색 사진은 도 11에 나타냈다.One month after the MNU injection, the pattern of retinal degeneration was shown in FIG. 10 by wide-field fundus imaging and autofluorescence fundus imaging. Autofluorescence coherence tomography (OCT), near-infrared (IR), and H & E tissue staining images are shown in FIG. .
도 10에서 좌측 두 열은 주사 전 사진, 우측 두 열은 주사 1달 이후 사진이다. a ~ d는 유리체 절제술 없이 MNU 0.5mg/0.05ml를 주사한 군(비교예 4-1)으로, 안저의 등 쪽(귀쪽) 하얗게 밝아져 국소적으로 형성된 병변을 관찰할 수 있다. In FIG. 10, the left two rows are photographs before the injection and the right two columns are photographs one month after the injection. a to d are the group injected with 0.5 mg / 0.05 ml of MNU without vitrectomy (Comparative Example 4-1), where the dorsal (ear) side of the fundus is brightened white and a locally formed lesion can be observed.
e ~ h는 유리체 절제술만 시행한 군(비교예 4-2)으로, 변화가 없음을 확인할 수 있다. e ~ h is a group performed only vitrectomy (Comparative Example 4-2), it can be confirmed that there is no change.
i ~ l은 유리체 절제술 후 MNU 0.05mg/0.05ml를 주사한 군(실험예 4-1)으로, 안저사진과 자가 형광 사진 모두 큰 변화가 관찰되지 않았다. i to l are the group injected with 0.05 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-1). No significant changes were observed in both fundus and autofluorescence images.
m ~ p는 유리체 절제술 후 MNU 0.1mg/0.05ml를 주사한 군(실험예 4-2)으로 안저사진과 자가 형광 사진 모두 큰 변화가 관찰되지 않았다. m ~ p is a group injected with 0.1 mg / 0.05 ml MNU after vitrectomy (Experimental Example 4-2), no significant change was observed in both fundus and autofluorescence.
q ~ t는 유리체 절제술 후 MNU 0.3mg/0.05ml를 주사한 군(실험예 4-3)으로 안저사진과 자가 형광 사진에서 visual streak에 해당하는 시력을 담당하는 부위를 중심으로 전반적으로 자가 형광이 증가된 것을 확인할 수 있다. q ~ t is a group injected with 0.3 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-3). You can see the increase.
u ~ x는 유리체 절제술 후 MNU 0.5mg/0.05ml를 주사한 군(실험예 4-4)으로 안저사진과 자가 형광 사진에서 안저의 등쪽 (귀쪽) 끝에 부메랑 형태의 경계가 줄처럼 하얗게 증가된 자가 형광을 나타내고, 내부는 어두운 지도모양인 국소적 병변을 관찰할 수 있다. u ~ x is a group injected with 0.5 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-4). The autosomal and autofluorescence images showed a bovine-like border with white lines at the dorsal (ear) end of the fundus. Fluorescence is present, and inside the dark focal local lesions can be observed.
도 11에서는 좌측부터 순서대로 근적외선(IR) 사진, 빛 간섭 단층촬영(OCT) 사진, H&E 조직염색 사진을 나타낸다. 11 shows near-infrared (IR) photographs, light interference tomography (OCT) photographs, and H & E tissue staining photographs in order from the left.
a, b는 유리체 절제술 없이 MNU 0.5mg/0.05ml를 주사한 군(비교예 4-1)으로, 근적외선 (IR) 사진에서 상부에 밝게 나타난 부위의 OCT 사진에서는 망막의 시세포층에 해당하는 외층 뿐만 아니라 망막의 내층까지, 망막 전층이 심하게 위축된 것을 확인할 수 있다. 조직 검사에서는 시세포층이 소실된 것으로 확인되었다. a, b is a group injected with 0.5 mg / 0.05 ml of MNU without vitrectomy (Comparative Example 4-1), and the outer layer corresponding to the cell layer of the retina in the OCT image of the bright region at the top of the near-infrared (IR) image But not to the inner layer of the retina, it can be seen that the entire retinal layer is severely atrophy. The histology revealed that the cell layer was lost.
c, d는 유리체 절제술만 시행한 군(비교예 4-2)으로 IR 사진, OCT 사진 및 조직 소견 모두 정상으로 나타났다. c and d were the only vitrectomy group (Comparative Example 4-2). IR, OCT and histological findings were all normal.
e, f는 유리체 절제술 후 MNU 0.05mg/0.05ml를 주사한 군(실험예 4-1)으로 IR 사진, OCT 사진 및 조직 소견 모두 정상으로 나타났다. e and f are the group injected with 0.05 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-1). All of the IR, OCT and tissue findings were normal.
g, h는 유리체 절제술 후 MNU 0.1mg/0.05ml를 주사한 군(실험예 4-2)으로 IR 사진, OCT 사진 및 조직 소견 모두 정상으로 나타났다. g and h were the group injected with 0.1 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-2), and all of the IR, OCT and tissue findings were normal.
i, j는 유리체 절제술 후 MNU 0.3mg/0.05ml를 주사한 군(실험예 4-3)으로 근적외선 (IR) 사진에서 시신경 아래 밝게 찍힌 부위의 OCT 사진에서 망막 내층은 비교적 잘 보존 되어있는 반면 망막의 시세포층에 해당하는 망막 외층이 소실된 것을 확인할 수 있었다. 조직 검사에서는 시세포층이 소실되었다.i, j is a group injected with MNU 0.3mg / 0.05ml after vitrectomy (Experimental Example 4-3). It was confirmed that the outer retinal outer layer corresponding to the oocyte layer was lost. The histologic examination lost the cell layer.
k, l은 유리체 절제술 후 MNU 0.5mg/0.05ml를 주사한 군(실험예 4-4)으로 근적외선 (IR) 사진에서 시신경에 연접한 아래 밝은 부위의 OCT 사진에서 망막의 시세포층에 해당하는 외층뿐만 아니라 망막의 내층까지, 망막 전층이 심하게 위축된 것을 확인할 수 있다. 조직 검사에서도 망막 전층이 소실된 것으로 확인되었다.k, l is a group injected with 0.5 mg / 0.05 ml of MNU after vitrectomy (Experimental Example 4-4), and the outer layer corresponding to the retinal cell layer in the OCT image of the lower bright region connected to the optic nerve in the near infrared (IR) image. As well as the inner layer of the retina, it can be seen that the entire retinal layer was severely shrunk. Biopsy also confirmed the loss of the entire retinal layer.
m은 MNU의 농도에 따른 살아있는 세포와 죽은 세포의 패턴을 도식화한 것이다. 0.05mg/0.05ml, 0.1mg/0.05ml를 주사한 군에서는 MNU를 주사하지 않은 군과 유사한 패턴을 나타내어 망막 변성이 유발되지 않은 것을 확인할 수 있고, 0.5mg/0.05ml를 주사한 군에서는 대부분의 시세포가 소실된 것을 확인할 수 있다. 한편, 0.3mg/0.05ml를 주사한 군에서는 약 60%의 시세포가 소실되어 1/3 내지 2/3 범위에 해당하는 망막 변성이 유발된 것을 확인할 수 있다.m is a diagram of the pattern of living and dead cells according to the concentration of MNU. The group injected with 0.05mg / 0.05ml and 0.1mg / 0.05ml showed a similar pattern to the group not injected with MNU, indicating that retinal degeneration was not induced, and most of the group injected with 0.5mg / 0.05ml It can be seen that the cell disappeared. On the other hand, in the group injected with 0.3mg / 0.05ml it can be seen that about 60% of the cells lost, retinal degeneration in the range of 1/3 to 2/3 was induced.
이상의 결과를 통해 MNU를 너무 많이 투여하는 경우(비교예 4-1, 실험예 4-4) 망막 전층이 소실되고, MNU를 너무 적게 투여하는 경우(실험예 4-1, 4-2) 망막 변성이 유발되지 않아 망막 변성 동물 모델에 적합하지 않다는 것을 알 수 있다. Based on the above results, when too much MNU is administered (Comparative Example 4-1, Experimental Example 4-4), the entire retinal layer is lost, and when too little MNU is administered (Experimental Example 4-1, 4-2). It can be seen that it is not induced and is not suitable for retinal degeneration animal models.
실시예 4-4. 유리체 절제술을 수행하지 않는 경우의 망막 변성Example 4-4. Retinal Degeneration Without Vitrectomy
세 마리의 토끼를 준비하고, 유리체 절제술 없이 0.2mg/0.05ml, 0.3mg/0.05ml, 0.4mg/0.05ml 농도의 MNU를 각각 30 게이지 바늘을 이용하여 토끼 각막 윤부로부터 약 4mm 지점에 주사하였다. MNU 주입으로부터 4주 후, 망막 변성 발생여부를 확인하여 도 12에 나타냈다.Three rabbits were prepared, and MNU at concentrations of 0.2 mg / 0.05 ml, 0.3 mg / 0.05 ml, and 0.4 mg / 0.05 ml, respectively, was injected at about 4 mm from the limbal portion of the rabbit cornea using a 30 gauge needle without vitrectomy. Four weeks after MNU injection, retinal degeneration was confirmed and shown in FIG. 12.
도 12에 나타낸 바와 같이, 0.2mg/0.05ml, 0.3mg/0.05ml, 0.4mg/0.05ml 농도 모두에서 망막 변성이 유발되었으며, 특히 0.2mg/0.05ml의 농도로 주사한 경우 0.3mg/0.05ml나 0.4mg/0.05ml 농도로 주사한 경우에 비해 망막 변성의 정도가 심한 것으로 확인되었다. As shown in FIG. 12, retinal degeneration was induced at all of the concentrations of 0.2 mg / 0.05 ml, 0.3 mg / 0.05 ml, and 0.4 mg / 0.05 ml, in particular 0.3 mg / 0.05 ml when injected at a concentration of 0.2 mg / 0.05 ml. The degree of retinal degeneration was more severe than that injected at 0.4mg / 0.05ml.
상기의 실험결과를 통해 MNU 주사 전 유리체 절제술을 수행하지 않는 경우 MNU 농도에 관계없는 망막 변성이 유발되며, 약물이 안구 내로 주사된 이후 약물의 망막에서의 거동을 예측하거나 제어할 수 없는 문제점이 발생하게 된다는 것을 알 수 있다. According to the above experimental results, non-vitrectomy prior to MNU injection causes retinal degeneration irrespective of MNU concentration, and there is a problem that the drug's retina behavior cannot be predicted or controlled after the drug is injected into the eye. You can see that
결론적으로, 상기 실시예 4-3 및 실시예 4-4에서 나타난 결과는 약물 주입 용량에 비례하여 국소적인 망막 변성이 유발되도록 하기 위해서는 MNU를 주사하기 전 유리체 절제술이 필수적임을 시사한다.In conclusion, the results shown in Examples 4-3 and 4-4 suggest that vitrectomy is necessary before MNU injection in order to cause local retinal degeneration in proportion to the dose of drug injection.
이상과 같이 실시예들이 비록 한정된 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기를 기초로 다양한 기술적 수정 및 변형을 적용할 수 있다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.Although the embodiments have been described with reference to the accompanying drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the techniques described may be performed in a different order than the described method, and / or the described components may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (10)

  1. a) 인간을 제외하는 포유동물의 안구에서 유리체 절제술을 수행하여 유리체를 제거하는 단계;a) performing vitrectomy in the eye of a mammal, excluding a human, to remove the vitreous;
    b) 유리체가 제거된 안구 내 빈 공간을 공기로 치환하는 단계; b) replacing the empty space in the eyeball from which the vitreous is removed with air;
    c) 상기 안구 내 빈 공간으로 MNU(N-methyl-N-nitrosourea) 용액을 주입하는 단계; 및c) injecting an N-methyl-N-nitrosourea (MNU) solution into the empty space in the eye; And
    d) MNU 용액을 제거하는 단계; 를 포함하는, 망막 변성 동물 모델의 제조방법.d) removing the MNU solution; Including, retinal degeneration animal model manufacturing method.
  2. 제1항에 있어서,The method of claim 1,
    상기 a) 단계 내지 d) 단계는 동시 수술로 수행되는, 망막 변성 동물 모델의 제조방법. The step a) to d) is performed by concurrent surgery, method of producing a retinal degeneration animal model.
  3. 제2항에 있어서,The method of claim 2,
    상기 d) 단계는, 상기 c) 단계로부터 2분 내지 20분 경과 후 수행되는, 망막 변성 동물 모델의 제조방법.The step d) is performed 2 to 20 minutes after the step c), retinal degeneration animal model manufacturing method.
  4. 제1항에 있어서,The method of claim 1,
    상기 b) 단계 내지 d) 단계는, 상기 a) 단계와 별개의 수술로 수행되는, 망막 변성 동물 모델의 제조방법. Wherein b) to step d), the method of manufacturing a retinal degeneration animal model is performed by a surgery separate from the step a).
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 b) 단계 내지 d) 단계는, 상기 a) 단계로부터 적어도 3일 이후에 수행되는, 망막 변성 동물 모델의 제조방법. Wherein step b) to step d) are performed at least three days after step a).
  6. 제1항에 있어서, The method of claim 1,
    상기 d) 단계 이후에, e) 안구를 세척하고, 안구 내 빈 공간을 충전하는 단계; 를 더 포함하는, 망막 변성 동물 모델의 제조방법.After step d), e) washing the eye and filling an empty space in the eye; Further comprising, retinal degeneration animal model manufacturing method.
  7. 제1항에 있어서,The method of claim 1,
    상기 c) 단계에서 MNU 용액은 안구의 망막 앞, 유리체강 부위로 주입되는, 망막 변성 동물 모델의 제조방법.In step c) MNU solution is injected into the vitreous cavity region in front of the eye, the retinal degeneration animal model manufacturing method.
  8. 제1항에 있어서, The method of claim 1,
    상기 MNU는 2mg/ml 내지 10mg/ml의 농도인, 망막 변성 동물 모델의 제조방법.The MNU is a concentration of 2mg / ml to 10mg / ml, retinal degeneration animal model manufacturing method.
  9. 제1항에 있어서,The method of claim 1,
    상기 포유동물은 토끼, 개, 고양이, 돼지 또는 원숭이인, 망막 변성 동물 모델의 제조방법.The mammal is a rabbit, dog, cat, pig or monkey, method of producing a retinal degeneration animal model.
  10. 제1항에 있어서,The method of claim 1,
    상기 동물 모델은 안구의 망막 전체 면적이 변성되는, 망막 변성 동물 모델의 제조방법.The animal model is a method of producing a retinal degeneration animal model, the total retinal area of the eye is denatured.
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