KR20190102465A - Methods for Screening Therapeutic Agents for Airway Conduct MucinSecretion Inhibitor Using the Frog Embryo - Google Patents

Abstract

The present invention relates to a screening method of an airway mucin secretion inhibitor using embryos of frogs, and more specifically, to a method for screening an airway mucin secretion inhibitor in a large quantity by using embryos of Bombina orientalis. A ciliary mucosal epithelial tissue model of embryo epidermis of Bombina orientalis of the present invention has an epidermis structure comprising a goblet cell (WGA dyeing) secreting mucus and a ciliated cell (acetylated tubulin dyeing) having cilia existed and thus, provides an epidermis structure similar to a respiratory structure of humans. In addition, the ciliary mucosal epithelial tissue model of embryo epidermis of Bombina orientalis of the present invention uses a culture medium containing only water and a small amount of salt, unlike a cell line culture medium containing plasma and various nutrients, such that the model has a relatively less noise, and as mucus amounts increase, the model increases absorbance of an embryo medium concentration-dependently, thereby being useful in screening of materials for inhibiting or promoting secretion of mucus such as mucin secreted in a respiratory organ.

Description

Methods for Screening Therapeutic Agents for Airway Conduct MucinSecretion Inhibitor Using the Frog Embryo}

The present invention relates to a method for screening an airway mucin secretion inhibitor using a frog embryo, more specifically, a sugar-frog frog ( Bombina orientalis ) relates to a method for the large-scale screening of airway mucin secretion inhibitors using embryos.

The respirator is one of the organs that the human body is exposed to come into contact with the external environment. The respiratory system consists of the airway, which forms the passage of air, and the lungs, a gas exchange location. The airway consists of nasal caviyt, pharynx, larynx, trachea and bronchus, and the lungs are made up of bronchiole and alveolus. The human body inhales about 20 kl of air per day through the action of the respiratory tract. The respiratory tract functions as a basis for life support such as oxygen inhalation and carbon dioxide emissions, and at the same time, it performs biological defense against harmful malt that enters the human body through the respiratory tract.

Mucus on the surface of the tracheal cavity plays a very important role in the body's defense against atmospheric particles and chemicals through a mechanism called mucociliary clearance. Oversecretion or change in viscosity of airway mucus is not only an important problem for many airway diseases such as chronic bronchitis, asthma and cystic fibrosis, but can also be a serious problem for people living in cities where air pollution is severe due to fine dust.

On the other hand, the experimental model used to discover the substance that controls the over secretion of mucus in respiratory diseases has been mainly used cancer cell line, ALI-culture, or mouse disease model, for in vivo study of respiratory mucus secretion epithelial cells Respiratory epithelial tissue of mouse disease model had to be extracted and cultured. In addition, the conventional respiratory disease model system is good to study the mechanisms of mucus secretion abnormalities and diseases, but there was a difficulty in high-throughput screening for the search for mucus secretion control effective substances.

Therefore, the inventors of the present invention sought to develop a new animal model that can be used to find a new respiratory disease treatment for a variety of animals to improve the shortcomings of the respiratory disease in vivo model that has been used in the prior art, It was confirmed that the present invention can be usefully used for the screening of substances that suppress or promote the secretion of mucus such as mucin secreted to the respiratory tract, thereby completing the present invention.

Patent Registration No. 10-0268576 Patent Publication No. 10-2006-0056444

Accordingly, an object of the present invention is to (a) a sugar frog ( Bombina orientalis ) treating the airway mucin secretion regulator candidate material in the embryo; And (b) measuring the level of mucus secretion by the candidate substance in the sugar-free frog embryo model.

Another object of the present invention to provide an airway mucin secretion regulator comprising narasin as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention comprises the steps of (a) treating the airway mucin secretion regulator candidate material in Bombina orientalis embryo; And (b) measuring the level of mucus secretion in the sugar-free frog embryo model, wherein the candidate substance comprises airway mucin secretion regulators.

In the method for screening an airway mucin secretion regulator of the present invention, the method comprises the steps of: (c) determining that the mucus secretion level measured in step (b) is an airway mucin secretion inhibitor when compared to a sample that is not treated with a candidate substance. May further include;

In the method for screening an airway mucin secretion regulator of the present invention, the method comprises the steps of: (c) determining that the mucus secretion level measured in step (b) is an airway mucin secretion promoter when compared to a sample that is not treated with a candidate substance; May further include;

In the screening method of the airway mucin secretion regulator of the present invention, the female frog can be injected into human and male HCG (Human Chorionic Gonadotropin: human chorionic gonadotropin), and can be artificially spawned through natural mating (natural mating) .

In the screening method of the airway mucin secretion regulator of the present invention, the frog frog embryo can be cultured 4 to 6 days after fertilization.

In the screening method of the airway mucin secretion regulator of the present invention, the measurement of the mucus secretion level can measure the secretion amount of mucus through the enzyme reaction of WGA-HRP (Wheat Germ Agglutinin-Horse Radish Peroxidase) that binds to the mucin polysaccharide chain. .

According to another aspect of the present invention, the present invention provides a composition for inhibiting airway mucin secretion containing narasin as an active ingredient.

Hereinafter, the screening method of the airway mucin secretion regulator of the present invention will be described in more detail step by step.

In order to improve the shortcomings of the respiratory disease in vivo model, which has been used in the past, the present inventors have made efforts to develop a new animal model that can be used to find a new respiratory disease treatment for various animals. Epidermal structure consisting of secreted mucous secretion cells (WGA staining) and ciliated cells (acetylated tubulin staining) with ciliated cells, which have an epidermal structure similar to that of the human respiratory system, containing plasma and various nutrients. Unlike cell line cultures, it uses relatively little noise due to the use of culture fluids containing only water and some salts, and mucous membranes such as mucin secreted in the respiratory tract due to concentration-dependent increase in the amount of mucus. Useful for screening of substances that inhibit or promote secretion In the confirmed and completed the present invention.

In the present invention, the term “single frog ( Bombina orientalis ”is a species distributed throughout the Korean peninsula and inhabits wetlands or valleys in forested areas, or rivers, wetlands, rice fields and fields. The eggs of sugar-free frogs have a diameter of 1.5 to 2 mm and a transparent jelly layer (colloid layer) has three layers. One female spawns several times over time, spawning 30 to 150 eggs. The ladle of the female frog is yellow, dark brown or yellowish brown, with small black spots on the trunk and tail. The underside is dark brown and translucent to show the intestines. The body is round and the caudal fin starts from the middle of the body. Adult adults are about 3.5 to 5 ㎝ in length, and their color and shape vary greatly depending on their habitat and location. Dorsal side green, cyan or brown with irregular black spots. Back side red, yellowish red with irregular black spots and speckles. The head is round and flat, with a short snout, and there are black spots and spots around the snout.

Step (a) in the screening method is a frog ( Bombina orientalis ) is a step of treating candidate airway mucin secretion candidates in embryos.

In the present invention, the term "mucin" is a mucous substance secreted from the mucosa, also called mucus, mucin or mucin, the chemical body is a kind of glycoprotein. The protective function of mucus is mainly due to the physicochemical properties of mucin, a mucin glycoprotein, which shows considerable diversity in the structure of carbohydrates composed of glycoproteins with a molecular weight of millions of Daltons. Therefore, it is known that abnormalities in the quantity and quality of airway mucins not only affect the airway physiology but also affect the body's defensive action, causing more severe pathologies. For example, excessive secretion or change in viscosity of airway mucus is not only an important problem in many airway diseases such as chronic bronchitis, asthma and cystic fibrosis, but can also be a serious problem for people living in cities with severe air pollution.

In such respiratory tract airway disease, the quality and amount of mucus or mucin play an important role in mediating respiratory physiology, and thus are considered important therapeutic targets.

As used herein, the term "respiratory animal model" refers to an animal other than a human, and which is in a state that a person skilled in the art can determine is in a pathological state of the respiratory tract.

The term "candidate substance" in the present invention is a substance capable of controlling the secretion level of airway mucin by increasing or decreasing the secretion of mucus or mucin in the respiratory tract, and includes without limitation oligonucleotides, proteins, compounds, and the like.

In addition, the method of the present invention further comprises (c) determining that the mucus secretion level measured in step (b) is an airway mucin secretion inhibitor if the candidate substance is reduced compared to the sample which has not been treated, or (c) the ( If the level of mucus secretion measured in step b) is increased compared to the sample that is not treated with a candidate material may include determining the airway mucin secretion promoter. More specifically, when the level of mucus secretion is low compared to the value of the untreated sample or the amount of mucus retention in the embryonic cell is high, it may be a step of determining an airway mucin secretion inhibitor, and the level of the level of mucus secretion compared with the value of the untreated sample If it is high or the amount of intracellular mucus retention is low, it may be a step of judging the airway mucin secretion promoter.

The female frog is injected with HCG (Human Chorionic Gonadotropin: human chorionic gonadotropin) to females and males, and can be reared in large quantities by artificial spawning through natural mating.

In addition, the sugar-frog embryo is preferably embryos cultured 4 to 6 days after fertilization in consideration of whether the easy observation of embryo tissue and cells and the ease of malformation.

Step (b) in the screening method is a step in which the candidate substance measures the level of mucus secretion in a sugar-free frog embryo model. Detection of mucous or mucin proteins at this stage can include Northern blot, Western blot, Immunohistochemical staining, In situ hybridization, Reverse Transcriptase polymerisation. -Polymer chain reaction (RT-PCR), real-time quantitative RT-PCR (enzyme-linked immunosorbent assay), etc., but mucus or mucin has a molecular weight of 1 million Daltons Considering the fact that the structure of carbohydrates composed of the glycoproteins possesses considerable diversity, the secretion of mucus secretion is measured by the enzyme reaction of WGA-HRP (Wheat Germ Agglutinin-Horse Radish Peroxidase) which can effectively bind mucin polysaccharide chains. It is preferable.

In one embodiment of the present invention, mucus secretion can be confirmed using an enzyme reaction of WGA-HRP (Wheat Germ Agglutinin-Horse Radish Peroxidase) to easily bind to the polysaccharide chain of the mucin sugar glycoprotein, which is the main component of the mucus. A ciliary mucosal epithelial model of the embryonic epidermis was constructed, and it was confirmed that mucus secretion was inhibited when bicuculline was treated with a drug known to reduce mucus secretion in humans.

As another aspect, the present invention provides a composition for inhibiting airway mucin secretion containing narasin as an active ingredient.

The mucin secretion and respiratory related models are as described above.

As used herein, the term "substance that modulates the secretion of airway mucin" refers to the expression of airway mucin in a manner that acts directly on a gene encoding a glycoprotein or a protein thereof, or indirectly on a ligand thereof. It may include any substance that inhibits expression in a way that reduces transcriptional levels or promotes post-translational degradation into proteins, or any substance that reduces extracellular transport of mucin.

The substance that inhibits the secretion of airway mucin can be used without limitation in the form, such as a compound, a nucleic acid, a peptide, a virus or a vector containing the nucleic acid that can target the airway mucin and inhibit its expression or activity. Examples of substances that inhibit the expression or activity of the airway mucin include oligonucleotides that inhibit the expression of mRNA of airway mucin, aptamers or compounds that inhibit the synthesis of airway mucin glycoproteins, but are not limited thereto. It doesn't work.

As used herein, the term "antibody" is a protein molecule comprising an immunoglobulin molecule that is immunologically reactive with a specific antigen, and serves as an antigen receptor that specifically recognizes and reacts when a specific antigen invades the body. Refers to protein molecules. One antibody molecule consists of two heavy chains and two light chains, each of which comprises a variable region and a fixed region. The variable region includes three complementarity determining regions (CDRs) and four framework regions (FRs), and the binding regions between the antibody and the antigen are formed by the complementarity determining regions. The binding specificity of the antibody to the antigen can be generated.

The antibody includes all forms of full length antibodies or antibody fragments, and may also include both polyclonal antibodies, monoclonal antibodies, and the like. It may also include all genetically engineered antibodies, such as chimeric antibodies or heterologous binding antibodies.

In addition, the substance that inhibits the secretion of airway mucus may be in the form of a compound, for example, a narasin compound (narasin) compound, but is not limited thereto.

The narasin compound is well known as an antimicrobial agent, and it is well known that NFκB does not act as a transcription factor by interfering with phosphorylation of IκB. Narisin is also used primarily as a preventive and therapeutic agent for poultry coccidial infections and as an ionophoric antibiotic that forms ion channels in bacterial cell membranes. In addition, Narasin has been reported to have an inhibitory effect on IκBα phosphorylation. It has been reported to induce ER stress respond, but no studies on mucin and mucus secretion have been reported.

As used herein, the term “inhibition” means any action that inhibits or delays excessive secretion of mucus in the respiratory tract by administering a composition of the invention. As used herein, the term "treatment" refers to any action that improves the symptoms of excessive secretion of mucus in the inhaled airways by administering the composition of the present invention, or inhibits the discharge of airway mucus.

The composition for inhibiting airway mucin secretion of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier to provide food, medicine, feed additives, and drinking water additives. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be administered without stimulating the organism.

The type of the carrier usable in the present invention is not particularly limited and any carrier can be used as long as it is commonly used in the art and pharmaceutically acceptable. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more thereof. In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, and / or lubricants may be added in addition to aqueous solutions, suspensions, emulsions, and the like. It may be formulated into the same injectable formulation, pills, capsules, granules or tablets and the like.

The mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be in accordance with methods commonly used in the art. As a non-limiting example of the administration mode, the composition may be administered orally or parenterally, and may be administered by oral administration in that it is a therapeutic agent for excessive secretion of mucus in the respiratory tract.

The pharmaceutical composition may be prepared in various formulations according to the desired mode of administration. Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, and the like. Can be mentioned.

In order to formulate the composition of the present invention into a dosage form for oral administration such as tablets or capsules, lactose, Saccharose, Sorbitol, Mannitol, Starch, Amylopectin, Cellulose or Gelatin ( Binders such as Gelatin); Excipients such as dicalcium phosphate and the like; Disintegrants such as corn starch or sweet potato starch; Lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol wax, and the like. Furthermore, the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

As a method for parenteral administration of the composition of the present invention, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration may be used, and the mucus of the respiratory tract is excessively secreted. Ophthalmic administration and the like can be used in that it is a treatment for, but is not limited thereto.

Formulations for parenteral administration include, for example, injectable forms such as subcutaneous injection, intravenous injection or intramuscular injection; Suppository injection mode; Or it may be formulated for spraying, such as aerosols to enable inhalation through the respiratory tract, but is not limited thereto. To formulate such injectable formulations, the compositions of the present invention can be mixed in water with stabilizers or buffers to prepare solutions or suspensions and formulated for unit administration of ampoules or vials. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed concentrate or wet powder.

Suitable applications, sprays or dosages for the pharmaceutical compositions of the invention may include the method of formulation, mode of administration, time of administration and / or route of administration, as well as the age, weight, sex and disease symptoms of the subject to be administered. It may vary depending on factors such as degree, food ingested, rate of excretion and the like, and one of ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment.

As described above, the ciliated mucosal epithelial tissue model of the muzzle frog embryo epidermis of the present invention is epidermal with mucous secreting goblet cells (WGA staining) and ciliated cells (ciliated cells, acetylated tubulin staining). The structure is made, having an epidermal structure similar to that of a human respiratory system.

In addition, the ciliary mucosal epithelial tissue model of the muzzle frog embryo epidermis of the present invention is relatively low noise, because the culture medium containing only water and some salt, unlike the cell line culture medium containing plasma and various nutrients, the amount of mucus As the absorbance of the embryo medium increases with increasing concentration, it can be usefully used for screening of substances that inhibit or promote the secretion of mucus such as mucin secreted into the respiratory tract.

1 is a diagram showing the advantages and disadvantages between them in relation to the selection of ciliary mucosal epithelial tissue model using an open embryo epidermis collected in Korea.
Figure 2 is a photograph taken to observe whether the frog frog model is similar to the human respiratory tract.
3 is a photograph of the frog frog embryo after 4-6 days after fertilization.
Figure 4 is a photograph of the change pattern of mucous expression by treating the candidate substance in the frog frog embryo model.
5 is a diagram showing a change in mucus secretion after treatment with Bicuculline, Narasin, Noscapine, and phorbol 12,13-dibutyrate, and a photograph taken by the fluorescence staining method.
6 is a diagram showing a comparison of ciliary mucosal epithelial tissue model and respiratory mucus secretion changes of human Calu-3 cell line of the muzzle frog embryo epidermis of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Collection of Domestic Native Species Frogs and Eggs

1-1. When and where to collect

Frogs native to Korea were collected for the study of screening and screening methods for respiratory functional substances. The target species for the collection was seven species of endangered and toad family, including the endangered family and toad family, among the treeless amphibians (Korean Frog, Northern Frog, Valley Frog, True Frog, Tree Frog, Om Frog, and Lady Frog).

Collecting of individuals was collected in the breeding season for adult and eggs. Adults and eggs were collected from Ulju-gun, Gangjin-gun, Hongcheon-gun, Chuncheon-si, Wonju-si, and Uiseong-gun from February to June, and a total of seven adult species (Korean Frog (5), Bukbangsan Frog (14), and Valley Mountain Frog (12). , Frog (4), Tree Frog (11), Om Frog (4), Sugar Frog (22)] and Five Eggs (Korean Frog (8), Northern Frog (7), Valley Frog (11), True Frog (2), Sugar Frog (1)] were collected.

 In order to effectively maintain and breed the collected frogs, three amphibian breeding facilities (18 breeding cages available for each breeding facility) were designed, manufactured, and reared.

Subsequently, adult 108 individuals of the female frog were further collected through the experimental model aptitude analysis of Example 2 below.

1-2. Breeding objects

Two types of cages were used: high cage (470 × 290 × 280mm) and low cage (460 × 300 × 170mm). Cages were drilled with drains, with no gaps, but with small caps so that only feces or other food debris could fall out. When the water rises to the height of the drain, the water is automatically drained, allowing the water to maintain a constant height during the water cycle. Inside the cage, a 20mm high and 40mm sponge was used to make the land a little higher than water. Two 40mm and one 20mm sheets were used for the high cage and one 40mm sheet for the low cage to make the land. Stones, hideouts, artificial vines and leaves were placed on top of each other so that individuals could use them as hideouts. The water and land parts were simultaneously inside the cage, allowing individuals to move and use water and land comfortably.

The collected eggs were placed in cages and allowed to naturally develop and hatch according to room temperature. Confirming that the embryos grew, dead embryos that were no longer developing decayed and removed to prevent water contamination, while developing embryos continued to grow and hatched. Incubation periods in eggs depended on temperature but took approximately 10 to 15 days.

The eggs were given tetramin baby to newborn tadpoles, and as the tadpole grew, they were fed regular tetramin. When food was left, the water was contaminated, so it was given as much as 5-10 minutes to eat. If the water became too dirty, the water was cleaned while cleaning the cage in the middle of breeding. When the tadpoles started to come out of their hind legs, they used sponges and stones to make land, so they could use the land after metamorphosis. The period from tadpole to metamorphosis took about 30-50 days.

Freshly metamorphic larvae were so small that they prevented them from escaping in the kennel. Feeding was provided two to three times a week. Freshly transformed larvae fed cricket pinheads (approximately 1-2 mm) and micro-mil worms (approximately 0.3-0.8 cm), and increased the size of the food little by little as they grew. The small crickets and wheat worms, which are fed with the subjects for continuous feeding, were kept together so that there was no shortage of food.

Sugar-frog frogs have a large number of individuals, and when the size of the individual is a little small, 8 to 9 individuals, and when the size of the large animals were separated by 5-6 individuals. In the case of other species, two to three individuals were raised depending on the species. Feeding was once or twice a week. For feeding crickets (small to medium, about 0.8 to 2.0 ㎝) and common wheat worms (about 1.2 to 2.0 ㎝), three crickets per individual and four to five wheat worms were fed. Adults kept several birds in a cage, which could lead to food competition, and they gave them three to four more feeds.

  Twice a week, a full water change was performed to clean the bowels and food waste that had sunk on the bottom of the cage. The whole cage was cleaned once a month to prevent the cage from becoming contaminated or mossy. Cricket and wheat worms, which are fed with the subjects, were kept together to ensure continued feeding.

Example 2 Selection of suitable species for the first study through scanning analysis and jelly layer removal

The suitability analysis of the experimental model related to respiratory disease was performed on domestic native species frogs collected and bred according to Example 1 above. For the information related to the Korean native frogs studied in the experiment, reference was made to the Korean amphibian and reptile ecological map (Lee et al., 2011; Lee and Park, 2016). And their ecology was observed.

2-1. Scanning analysis

Since the size of the embryo was too large and the degree of occurrence by the pigment was not easy to see, Brightfield image analysis was used. Brightfield image analysis takes longer than scanning image analysis and can't measure the time of occurrence, but instead the exposure time and light intensity can be adjusted for clearer image analysis and higher magnification. It has the advantage of obtaining more accurate and precise pictures than analysis. Bright field image analysis was performed using PBS (Phosphate-Buffered Saline: 4.09 g NaCl ⁺ , 0.01 g KCl ⁺ , 0.72 g Na ₂ HPO ₄ ⁺ , 0.12 g KH ₂ PO ₄ ⁺ , 50 mL H ₂ ) to obtain a clean background. O) was carried out on a medium in which 2% agarose was dissolved, and image analysis of the embryos was performed in a solution containing 0.1% tween-20 in PBS.

Brightfield image analysis of domestic native frogs revealed that the stage was smaller in size than the other Korean native frogs, despite the fact that the female frog was much larger in size than the control African claw frog. This was relatively easy. On the contrary, other Korean native frogs have a high melanin pigment in embryonic epidermal cells, making it difficult to observe abnormalities (see FIG. 1).

2-2. Jelly Layer Removal

It is necessary to remove the jelly layer surrounding the eggs. It must be removed to confirm mucus secretion or cilia. Normally, mucus was removed using a solution containing cysteine, 3% cysteine solution with pH of 7.8 ~ 8.0 (3g cystein, 100ml 1/3 × Marc's modified ringer's) and pH above 9.0 Two kinds of% cystein (3g cystein, 100ml 1/9 × MMR) solution were used, and the jelly layer was removed by swirling method. This disappears and the distance between the eggs decreases, and the embryos are then washed with 1/9 × MMR or MBS solution.

As a result of removing the jelly layer, it was observed that the jelly layer was easily removed when reacted with a 3% cysteine solution having a pH of 9.0 for about 10 minutes. In addition, when the true frog tried to remove the jelly layer for about 30 minutes with a 3% cysteine solution having a pH of 9.0, it was observed that the jelly layer was removed. However, it was observed that other Korean native frogs did not sufficiently remove the jelly layer even after 30 minutes of reaction with cysteine solution (see FIG. 1).

2-3. Frog Embryo Full Image Analysis

The entire epidermal structure of frog embryos was imaged by fluorescence staining. In the present invention, to determine whether the frog has a structure similar to the human respiratory tract, the structure was analyzed through the epidermal staining of the frog.

When mucus is secreted, its major component, mucin, must be glycosylated. In order to detect the mucin protein undergoing the glycosylation process, an experiment was conducted using WGA (Wheat Germ Agglutinin, Thermo), a protein that binds to the glycosylated moiety.

Staining was performed using an acetylated tubulin (Thermo) antibody to detect murine protein, WGA and mucin, which remove secreted mucus.

As a result, the Frog embryo is composed of epidermal structures consisting of goblet cells (WGA staining) and ciliated cells (acetylated tubulin staining) with ciliated secretions, similar to human respiratory structures. It was confirmed to have (Fig. 2).

In addition, the merged image of these (tubules) and WGA fluorescence staining did not overlap each other, through which, it was confirmed that the WGA fluorescence staining was not stained on the ciliated cells.

Through the above results, it was confirmed that the Frog embryo model can be used in the mucus secretion model and the screening model of the respiratory therapeutic drug through it.

2-4. Artificial spawning

In relation to the spawning and embryonic development of the Korean native species collected above, artificial spawning was attempted to proceed with the experiment at a desired time. Artificial spawning is an in vitro fertilization method, in which only females obtain an unfertilized egg by hormonal injection, sprinkle the testes obtained after dissection of the male testis, and sprinkle it on the unfertilized egg obtained. Natural mating method of obtaining a fertilized egg after the hormone injection to all was used.

The natural mating method was to induce spawning by injecting HCG (Human Chorionic Gonadotropin) into individual individuals of female and female frogs. Injections were made just under the Dorsal sac skin and a total of three methods were used. First, HCG 500 ~ 600Unit was injected to females, and 300Unit was injected to males, and then natural inclusion was induced at 16 ° C. Second, inject female HCG 100Unit and store at 16 ℃ for 24 hours. The next day (after 24 hours) the female was injected with additional HCG 400 ~ 500 Unit, the male was injected with 300 Unit and induced natural inclusion at 16 ℃. Third, HCG hormone was injected into the female by 400Unit and 320Unit by the male, followed by spawning by maintaining natural inclusion at room temperature.

As a result, it was confirmed that in vitro fertilization of artificial spawning failed, artificial spawning by natural mating method (about 80%). The fertilized egg was maintained at 1 / 9x MMR (0.32 g KCl, 0.65 g CaCl ₂ H ₂ O, 0.45 g MgCl ₂ H ₆ O, 24.92 g HEPES, 12.99 g NaCl, 20 L H ₂ O) and Until used, Gentamicin was used to prevent bacterial contamination. The fertilized eggs were used in the experiment after raising the embryonic stage at room temperature for about 4 to 6 days, the development stage of the sugar-frog frog used in the experiment is as shown in FIG.

Example 3 construction of mucous secretion analysis model of ciliary mucosal epithelium of frog embryo epidermis

Enzyme-Linked Lectin Assay (ELLA) technique was used to construct mucus secretion analysis model of ciliary mucosal epithelial tissues of the muzzle frog embryo epidermis.

To carry out the ELLA analysis, the fertilized embryos were washed 5 days before and after fertilization with sterile 1/9 × MMR solution so that no mucus remained on the embryonic epidermis. Five washed embryos were placed in a bottomed chamber, respectively, and incubated with a 1/9 × MMR solution. After 3 hours of incubation, a certain amount of the solution was collected and transferred to a 96-well plate, stored at 4 ° C. for at least 12 hours, and then induced to naturally adhere mucus to the plate surface. The plate was washed three times with 200 μl of PTW (100 ml PBS, 0.1 ml tween-20) solution, and then again using 200 μl of 1% BSA (1 g BSA + 100 ml PTW) to minimize errors due to nonspecific binding. Treatment was performed at room temperature for 30 minutes. Thereafter, WGA-HRP (Wheat Germ Agglutinin-Horse Radish Peroxidase) was diluted to a concentration of 1:10 ⁴ (w / w) in a 1% BSA solution and placed in 150 μl each 96 well plate, and reacted at room temperature for 1 hour 30 minutes. . After completion of the reaction, the plate was washed three times using 200 μl of PTW solution, and one capsule in 25 ml to 30 ml PCB (phosphate citrate buffer, 2.25 g sodium citrate, 3.65 g Na ₂ HPO ₄ , 500 ml H ₂ O, pH 5.0) solution. O-Phenylenediamine dihydrochloride tablet (Sigma) was added to dissolve. To this was added 30% H ₂ O ₂ solution at a concentration of 1/10 ⁴ (w / w) again, dispensed into a 96 well plate at 150 μl, and then reacted at room temperature for 10 minutes, and 50 μl sulfuric acid (36.6ml sulfuricacid). + 250 ml H ₂ O) was used to stop the reaction. Subsequently, mucosal secretion of ciliary mucosal epithelial tissue of the Frog embryonic epidermis was analyzed by measuring the absorbance (OD: optical density) at 492 nm using a microplate reader.

As a result, as shown in FIG. 4B (right), as the amount of mucus increased, it was confirmed that the absorbance of the medium increased in a concentration dependent manner. These results indicate that the culture medium for culturing sugar-free frog embryos is relatively low in noise because it contains only water and some salts, unlike the cell line culture medium containing plasma and various nutrients. The ciliary mucosal epithelial tissue model of the Frog embryonic epidermis using Enzyme-Linked Lectin Assay technique can effectively analyze the amount of secreted mucus, so screening of substances that suppress the secretion of mucus such as mucin secreted into the respiratory tract It was found to be useful.

Example 4 Screening of Mucus Secretion Regulators

Using the ciliary mucosal epithelial tissue model of the muzzle frog embryo epidermis constructed in the above embodiment, an experiment was conducted to screen for a novel substance that suppresses or enhances the secretion of mucus such as mucin secreted into the respiratory tract. To test the accuracy of the model, experiments were conducted with substances known to regulate mucus secretion in conventional respiratory mucus secreting cells. As a known mucus secretion inhibitor, bicuculline was used. The staining process was fixed at 2 hours at room temperature and 12 hours at 4 ° C using MEMFA (MOPS / EGTA / Magnesium Sulfate / FormaldehydeBuffer) solution. Thereafter, washing with TBST (Tris BufferedSaline with Tween20) solution was performed, blocking was performed at room temperature for 30 minutes using a blocking solution, and the reaction was performed by treating the blocking solution with a 1: 300 primary antibody. . After completion of the primary antibody treatment, washing was further performed with TBST solution, and the secondary antibody was reacted by treating the blocking solution at 1: 300. After completion of the reaction, washing with TBST solution was performed, followed by fluorescence photographing using a confocal microscope (Zeiss LSM 710) for analysis.

As a result, it was confirmed that narasin and noscapine were significantly reduced in the amount of mucus present in the solution compared to the negative control among the treated candidates. In particular, noscapine was observed as much as mucous secretion with bicuculin used as a known mucus secretion inhibitor, and naracin was observed to have a superior mucus secretion inhibitory effect compared to bicuculin (see FIG. 5A).

On the other hand, phorbol 12,13-dibutyrate, known as a known mucus secretion promoter, was observed to increase the amount of mucus present in the media compared to the negative control group.

Through the above results, it was confirmed that the ciliary mucosal epithelial tissue model of the Frog embryonic epidermis showed a similar response to the drug applied to human respiratory cells (FIG. 5).

In addition, the embryos were stained and secreted mucus remaining in the cells was confirmed by confocal microscopy (Zeiss LSM 710) using a WGA antibody. As a result, it was confirmed that the mucus remaining in the epidermal cells was not secreted in the group treated with bicuculin, naracin and noscarpine compared to the negative control group, and remained in the epidermis in the phorbol 12,13-dibutyrate treated group. Small amounts of mucus were observed.

Example 5 Comparative Verification of a Frog Embryo Model and a Human Respiratory Cell Line Model

It was verified whether the substance screened by the frog frog embryo epidermal ciliary mucosal tissue model of the present invention is effective in the actual human respiratory cell line model.

The mucosa and mucin secretion inhibitory effect of Narasin, a respiratory mucus secretion inhibitor discovered in Example 4, using calu-3 cell line, a human respiratory mucus secretion test cell line, was used in the present invention. Comparative verification with organizational model.

The amount of mucin and mucus accumulated in the calu-3 cell line treated with naracin was analyzed using Muc5ac antibody and WGA antibody, respectively. Staining process was fixed in Calu-3 cell line using 4% formaldehyde (PBS, 4% formaldehyde) for 2 hours at room temperature, 12 hours at 4 ℃. Thereafter, washing with TBST (Tris BufferedSaline with Tween20) solution was performed, blocking was performed at room temperature for 30 minutes using a blocking solution, and the reaction was performed by treating the blocking solution with a 1: 300 primary antibody. . After completion of the primary antibody treatment, washing was further performed with TBST solution, and the secondary antibody was reacted by treating the blocking solution at 1: 300. After completion of the reaction, washing with TBST solution was performed, followed by fluorescence photographing using a confocal microscope (Zeiss LSM 710) for analysis.

As a result, it was confirmed that mucin and mucous proteins are accumulated in cells in both the muzzle frog embryonic epidermal cilia epithelial tissue model and the human respiratory calu-3 cell line model of the present invention (FIG. 6C). In addition, it was confirmed through fluorescence staining that the number and size of the mucin-containing vesicles remaining in the cell was confirmed to increase (Fig. 6D).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

(a) Bombina orientalis ) treating the airway mucin secretion regulator candidate material in the embryo; And
(b) measuring the mucus secretion level in the candidate frog-free frog embryo model; screening method of airway mucin secretion regulator comprising a. The method of claim 1,
The method further comprises: (c) determining that the mucus secretion level measured in step (b) is an airway mucin secretion inhibitor if the candidate substance is reduced compared to the sample that has not been treated with the airway mucin; Screening method of secretion regulators. The method of claim 1,
The method further comprises: (c) determining that the mucus secretion level measured in step (b) is an airway mucin secretion promoter if the candidate substance is increased compared to the sample which has not been treated with the candidate substance; airway mucin further comprises Screening method of secretion regulators. The method of claim 1,
The female frog is injected with HCG (Human Chorionic Gonadotropin: human chorionic gonadotropin) to females and males, and screening method of airway mucin secretion regulator characterized in that the artificial spawning through natural mating (natural mating). The method of claim 1,
The method of screening the airway mucin secretion regulator, characterized in that the frog frog embryos are cultured 4 to 6 days after fertilization. The method of claim 1,
The mucus secretion level is measured by measuring the secretion of mucus secretion through enzyme reaction of WGA-HRP (Wheat Germ Agglutinin-Horse Radish Peroxidase) that binds to the mucin polysaccharide chain. A composition for inhibiting airway mucin secretion containing narasin as an active ingredient.

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