WO2021225089A1 - Method for elucidating correlation between irritant and produced substance - Google Patents

Abstract

The present invention addresses the problem of providing a method for elucidating the correlation between an irritant for an organism and a produced substance that is produced within the organism due to the irritant, and of providing a method for screening, based on the knowledge yielded by the aforementioned method, irritants, produced substances, and their combinations. The problem is solved by the following: a method characterized by elucidating the correlation between an irritant and a produced substance by bringing the irritant into contact with the cells in the sac of a bubble eye goldfish and then quantitating the produced substance produced by these cells or the gene for the produced substance; and a method for screening, using the aforesaid method, irritants, produced substances, or "irritant/produced substance combinations".

Description

How to clarify the correlation between stimulants and produced substances

The present invention is a method for clarifying a correlation between a stimulant and a production substance produced by the cell using cells in the bubble eye goldfish, and a method for clarifying the temperature dependence of the correlation. Further, it relates to a method for screening a stimulant, a producing substance, or a "combination of a stimulating substance and a producing substance" using the method.

Humans receive various stresses and stimulants from the external environment. Examples of the stimulant include various substances such as foods, food additives, medicines, pesticides, pollutants, etc., in addition to fungi, bacteria, viruses and the like. In addition, substances produced by them, impurities contained in them, and the like can also be mentioned.

If such a stimulant is administered to a test animal such as a mouse and the "substance A generated in the test animal" is identified or quantified by the administration, the stimulant and the substance A can be combined. The correlation is clarified, and based on the correlation, the stimulant substance and the substance A to be noted (in examination, etc.) are clarified. Then, if the substance A is known, a defense method against the stimulus (substance) becomes clear, and it becomes possible to discover, develop, screen, manufacture, etc. medicines, health foods, lifestyles, etc. that are useful for the defense (method). ..

However, "method of administering the stimulant to the test animal", "administration site in the test animal", "method of collecting substance A from the test animal", "collection site of substance A in the test animal", etc. With respect to the above, depending on the stimulant or substance A, there was no or few optimal methods, administration sites, and collection sites. The administration site and collection site include tissue fluid, blood, lymph, and the like.

In addition, even when examined in vitro, there were few optimal animals for obtaining the biological substances necessary for the examination and the parts of the animals (including tissue fluid, blood, lymph, etc.).

Patent Document 1 describes an "antibody production method" for producing an antibody by administering an antigen to a bubble eye goldfish.
Further, Patent Document 2 describes a "medium additive for animal cells" containing a solution in the bubble eye of a bubble eye goldfish.

However, in Patent Documents 1 and 2, the stimulant is brought into contact with the cells in the bubble eye goldfish, and is referred to as an immune-related substance, a stress suppressant, a natural immune activating substance, etc. There is no description or suggestion about identifying or quantifying.
Therefore, Patent Documents 1 and 2 do not further describe or suggest "the breeding temperature of goldfish and the culture temperature of cells in blisters" when the produced substance is produced.

Bacterial infectious diseases, on the other hand, are affected not only by the pathogenicity of bacteria, but also by host resistance consisting of innate immunity and acquired immunity.
Further, regarding fish and the like, it has been reported that environmental factors such as water quality and water temperature have a great influence on the resistance of a host (for example, non-patent documents 1 and the like).

It is effective to use animal cells and body fluids when examining "substances that affect humans" such as immune-related substances, stress suppressors, and innate immunity activators. None of them used the internal fluid of "water bubbles that were stably generated (by mating, etc.) near the eyes of fish."

In addition, while the above studies with mammals such as humans in mind are important, the development of methods for enhancing resistance to fish infectious diseases and methods for monitoring them is also extremely important for the aquaculture industry. there were.

Japanese Unexamined Patent Publication No. 2012-12015 Japanese Unexamined Patent Publication No. 2013-082664

The present invention has been made in view of the above background technology, and the subject thereof is to provide a method for clarifying the correlation between a stimulant substance for a living body and a production substance produced in the living body by the stimulating substance. There is. Another object of the present invention is to provide a method for screening stimulants, producing substances, and combinations thereof based on the findings obtained by the method.

As a result of diligent studies to solve the above problems, the present inventor has brought the cells in the bubble eye goldfish into contact (coexistence) with the stimulant introduced from the outside, whereby the stimulant and the stimulant are brought into contact with each other. It has been found that the correlation (correspondence relationship) with the cell-producing substance can be easily clarified, and thereby the screening of various producing substances corresponding to the stimulant can be easily and accurately performed.
Further, the present invention has been completed by finding that the above-mentioned correlation (correspondence relationship) can be quantitatively obtained and that the quantitative correlation has a temperature dependence.

That is, in the present invention, after the stimulant is brought into contact with the cells in the blisters of the bubble eye goldfish, the produced substance produced by the cells or the gene of the produced substance is quantified, and the stimulant and the produced substance are used. It provides a method characterized by clarifying the correlation between the two.

Further, the method of the present invention can be divided into the following in vivo mode 1 and in vitro mode 2.

Aspect 1, which is in vivo of the present invention, is a production substance produced by cells in the bubble eye of the bubble eye goldfish after the stimulant is administered to the bubble eye goldfish and the bubble eye goldfish is bred at a specific temperature. It provides the above-mentioned method for quantifying the gene of the producing substance and clarifying the correlation between the stimulating substance and the producing substance.

Further, in the in vivo aspect 1 of the present invention, the temperature dependence of "the correlation between the stimulant and the" producing substance or the gene of the producing substance "" is further determined, and the presence or absence of the temperature dependence or the presence or absence of the temperature dependence is determined. The above-mentioned method for clarifying the critical temperature at which the producing substance is produced is provided.

In the second aspect of the present invention, which is in vitro, the stimulant is mixed with a medium containing cells collected from the water bubbles of a water bubble eye goldfish, the cells are cultured at a specific temperature, and then the production substance produced by the cells. Alternatively, the above-mentioned method for quantifying the gene of the producing substance and clarifying the correlation between the stimulating substance and the producing substance is provided.

Further, in the in vitro aspect 2 of the present invention, the temperature dependence of "correlation between the stimulant and the" producing substance or the gene of the producing substance "" is obtained, and the presence or absence of the temperature dependence or the presence or absence of the temperature dependence is determined. The above-mentioned method for clarifying the critical temperature at which the producing substance is produced is provided.

The present invention also provides a method for screening a stimulant substance and / or a producing substance, which is characterized by using the method of the above-mentioned aspect 1 or aspect 2.

Further, the present invention uses the method of the above aspect 1 or 2 to determine the temperature dependence of "correlation between the above stimulant and the" said production substance or the gene of the above producing substance "", and the temperature dependence. It provides a method for screening a combination, which comprises screening a "combination of a stimulant and a producing substance" having a large or small amount, or a low or high temperature limit at which the producing substance is produced.

According to the present invention, it is extremely easy to solve the above-mentioned problems and problems and to discover and examine "substances affecting humans" such as immune-related substances, stress-suppressing substances, and innate immunity-activating substances. , And can be done accurately.
That is, in other words, if the liquid in the blister of the bubble eye goldfish is used, for example, the above-mentioned substance which is preferable for humans is captured (discovered) extremely easily and accurately as a production substance produced by the cells in the blister. , (Quantitative) examination, screening, etc. can be performed.
Then, if the correspondence between the produced substance and the stimulant that produced the produced substance is known, the defense method against the stimulus (substance) becomes clear and is useful for the defense (method), that is, the produced substance. It enables the discovery, development, screening, manufacturing, etc. of medicines, health foods, lifestyles, etc. that promote production.

Specifically, the cells in the water bubbles of the water bubble eye goldfish are brought into contact with the cells in the water bubbles by administering a stimulant to the body of the fish (including injection, feeding, etc.) or by injecting the stimulant directly into the water bubbles. The production substance produced by the cell can be identified, the production amount of the production substance can be quantitatively determined, and the presence or absence of life or death, disease onset, or abnormality of the water bubble eye goldfish (individual) due to the stimulant substance can be determined. By confirming, "correlation between the stimulant and the producing substance", preferably "temperature dependence of the correlation", particularly preferably "life or death of the individual by administration of the stimulant". , Disease or abnormality ”, etc. can be examined extremely easily and accurately.

In general, it is effective to use animal cells; body fluids such as tissue fluid, blood, and lymph fluid; etc. for the above examination, but until now, "liquids such as lymph fluid" in the blisters of bubble eye goldfish or None of them used "cells".
Since the bubble eye goldfish can be stably produced as a result of repeated mating, it can be easily obtained in carrying out the method of the present invention. In addition, as a matter of course, the liquid and cells used for the study can be easily found and collected in the individual.
Furthermore, since the liquid in the blisters can be easily collected, it is easy to study in vitro.
The blisters and blisters are superior to other parts of animals and other body fluids in the above-mentioned various points.
Moreover, since it is a fish, the "moral problems related to experimental animals", which is a problem in mammals such as mice, can be reduced.

For example, it may be difficult to extract "substances (products) generated in the animal" such as immune-related substances, stress-suppressing substances, and innate immunity-activating substances from the body of the animal to which the stimulant is administered. At some point, it is extremely easy to take them out from the bubble eye goldfish because they can be taken out with a syringe or the like.

Lymph fluid is stored in the blister of the bubble eye goldfish, and if a substance produced in this lymph fluid is identified and quantified, the above-mentioned substance can be captured and quantified extremely easily and accurately.
Further, since the liquid in the blister can be continuously collected while keeping the goldfish (individual) alive, the cells in the blister can be easily collected many times and are produced in the goldfish (individual). The time change of the amount of product is also required.

In Patent Documents 1 and 2, the liquid in the blisters is used for a purpose and method different from that of the present invention. Furthermore, there have been no reports on the cells in the blisters, and there have been no reports on changes in the fluid in the blisters while keeping the individual alive.
According to the present invention, as compared with the conventional method, not only the lymph fluid and the like can be easily taken out, but also the cells in the blisters can be easily collected.
For the first time by the present invention, the cells in the bubble eye of a bubble eye goldfish can produce an immune-related substance against stimulants such as heat-killed bacteria of Pseudomonas aeruginosa, T cell mitogen PHA, and TLR3 agonist PolyI: C. Found.

Furthermore, the temperature sensitivity of the above production response was also found. That is, for the first time by the present invention, the temperature dependence of "correlation between the stimulant and the producing substance" was clarified.
Specifically, for example, it was found that the production of immune-related substances in bubble eye goldfish in bubble eyes increases as the temperature decreases (the production capacity increases at low temperatures). More specifically, there is a limit temperature (maximum temperature for suitable production) at which a production substance such as an immune-related substance is (suitably) produced, and the substance is not produced at a temperature higher than this temperature. Or it turned out to be difficult to produce.

This means that in a poikilotherm such as a fish, if the breeding temperature (water temperature, etc.) is high, the immunity of the poikilotherm decreases.
According to the present invention, by using the result of the above-mentioned "temperature dependence of the correlation between the stimulant substance and the producing substance", a new "examination of the immune-related substance" becomes possible, and a new immune-related substance related to the environmental temperature can be examined. Screening becomes possible.

According to the present invention, it is possible to suppress damage to an individual to a very small extent and easily collect cells in blisters to evaluate an immune response or the like. Bubble eye goldfish cells in bubble eye were also induced to express cytokine genes, for example, in response to in vitro immune stimulation. Moreover, the induction of the expression showed high temperature sensitivity.
Therefore, it is expected that the mechanism and control method of the immune system and its high temperature sensitivity will be clarified by using the present invention.

Further, it is extremely important for the aquaculture industry to enhance the resistance to infectious diseases of fish, but by utilizing the findings of the present invention, the productivity of aquaculture fish can be increased.

Further, according to the second aspect of the present invention, after culturing the cells collected from the bubble eye of the bubble eye goldfish in a medium, the product itself produced by the cells or the gene of the product is quantified in vitro. The correlation between the stimulant and the producing substance can be clarified.

According to the present invention, in addition to finding that the above correlation (for example, the amount of produced substance produced) has temperature dependence (high temperature sensitivity) in the in vivo aspect 1, further, in the in vitro aspect 2. In other words, it was revealed that the medium containing cells collected from the blisters of bubble eye goldfish also had the temperature dependence (high temperature sensitivity).
Also in aspect 2 (in vitro) of the present invention, by using the result of the above-mentioned "temperature dependence of the correlation between the stimulant substance and the producing substance", for example, a new "examination of immune-related substances" can be made. Furthermore, it becomes possible to screen new immune-related substances related to the environmental temperature.

It is an optical micrograph (objective 40 times, white line in the photograph is 20 μm) taken after collecting cells in the bubble eye of a bubble eye goldfish and staining with Giemsa. Blisters When viewed from above and behind the goldfish, 50 μL of saline was injected into the left blisters and 50 μL of Pseudomonas aeruginosa killed bacteria (10-fold concentrated) into the right blisters, and left indoors (22 to 24 ° C) for 20 hours after breeding. It is a photograph at the time of observation (Example 1). It is a graph which shows the time course of 4 kinds of inflammatory cytokine production response to the inoculation of the cell in the bubble eye of the bubble eye goldfish with killed Pseudomonas aeruginosa (Example 2). (A) Changes in the amount of IL1β1 and IL1β2 mRNA over time (B) Changes in the amount of mRNA in TNFα1 and TNFα2 over time It is a graph which shows the temperature dependence (high temperature sensitivity) of the production response of 4 kinds of cytokines with respect to the killed Pseudomonas aeruginosa (P) of the cell in the bubble eye of the bubble eye goldfish (Example 3). It is a graph which shows the time-dependent change of the production response (in vitro) of 4 kinds of cytokines with respect to the killed Pseudomonas aeruginosa (PAO1) of the cell in the bubble eye of the bubble eye goldfish (Example 4). It is a graph which shows the influence of the culture temperature on the production response (in vitro) of 4 kinds of cytokines with respect to the killed Pseudomonas aeruginosa (PAO1) of the cells in the bubble eye of the bubble eye goldfish (Example 5). It is a graph which shows the individual difference of "the influence of temperature on the production response (in vitro) of 4 kinds of cytokines" with respect to the killed Pseudomonas aeruginosa of the cells in the bubble eye in 3 kinds of bubble eye goldfish (Example 6). It is a graph which shows the influence of temperature on the survival time (survival rate) of a goldfish inoculated with Pseudomonas aeruginosa viable bacteria (Example 7). It is a graph which shows the cytokine production response (in vitro) by the PHA and PolyI: C stimulation of the cell in the bubble eye of a bubble eye goldfish (Example 8). It is a graph which shows the cell number dependence of the cytokine (IFNγ and IL1β1) production response (in vitro) by the PHA and PolyI: C stimulation of the cell in the bubble eye of the bubble eye goldfish (Example 9).

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific aspects, and can be arbitrarily modified within the scope of the technical idea.

In the present invention, after contacting a stimulant with cells in the blisters of a bubble eye goldfish, the produced substance produced by the cells or the gene of the produced substance is quantified, and the correlation between the stimulant and the produced substance is determined. It is a method characterized by clarifying the relationship.

<Bubble Eye Goldfish>
"Bubble eye (goldfish)" is a kind of goldfish developed in China for ornamental purposes, and is a goldfish that has a bag-shaped blisters under the eyeball. Lymph fluid and the like are stored in the blisters.
However, the "bubble eye goldfish" used in the present specification and the like is not limited to the specific variety of goldfish developed in China for ornamental use as the so-called "bubble eye", and is newly referred to as "bubble eye goldfish". Includes fish that have "blisters containing equivalent inclusions" near the eye. That is, if it is a fish, it is not limited to the varieties created for ornamental use, and is not limited to goldfish.

FIG. 1 shows a micrograph of cells in the blister of a bubble eye goldfish. Most of the cells in the bubble eye of healthy bubble eye goldfish were mononuclear cells (see Giemsa stained image in FIG. 1). Among them, there were many cells that showed adhesiveness to plastic in in vitro experiments.

<Contact>
In the present invention, it is essential to bring the stimulant into contact with the cells in the blisters of the blister eye goldfish, but the "contact" is that the stimulant is directly injected into the blisters of the blister eye goldfish in vivo. As a result of administration such as, the stimulant directly comes into contact with the cells, and the stimulant is administered to a site other than the blisters of the blister eye goldfish, for example, intraperitoneal or intrablood administration by injection; Administration into the digestive tract by feeding; administration by immersing the blister eye goldfish in water containing the stimulant (aqueous solution of the stimulant); It involves circling, reaching the cells in the blisters and contacting the cells.
In vitro, the "contact" includes bringing the stimulant into contact with the cells by blending the stimulant with a medium containing cells collected from the blisters of a bubble eye goldfish.

When a stimulant is administered into the blisters of a bubble eye goldfish, the stimulant and the cells in the blisters come into contact with each other in the blisters, dilation and redness of blood vessels are observed on the surface of the blisters, and an inflammatory response is induced. (See Example 1 and FIG. 2)

<Stimulant>
The above-mentioned stimulant is a "substance (product) generated in the animal" such as an immune-related substance, a stress-suppressing substance, a natural immune-activating substance, etc. by contacting with cells in the bubble eye of a bubble eye goldfish. It is a substance to be produced, and refers to all substances to be evaluated.
Specifically, for example, live bacteria, killed bacteria, some fungi, fungal products, fungal derivatives such as bacteria, fungi, etc .; "substances related to (derived from) viruses or microorganisms" such as viruses themselves; general Foods such as foods and health foods; Drugs such as pharmaceuticals and pesticides; Hazardous substances such as food additives, air pollutants, marine pollutants; ; Etc. can be mentioned.

<Production substance>
In the present invention, after the stimulant is brought into contact with a cell in a blister, the produced substance produced by the cell or the gene of the produced substance is quantified.
Here, the "producing substance" is not particularly limited as long as it is a substance produced by cells in the water bubbles by contact with the stimulant, but from the viewpoint of usefulness and the like, an immune-related substance, a stress-suppressing substance, and innate immunity activation. It is preferably a "substance that affects humans" such as a substance or a pathogen-recognizing substance.
In other words, by using the method of the present invention, it is possible to capture unknown, immune-related substances, stress-suppressing substances, innate immunity-activating substances, pathogen-recognizing substances, etc., and also known as stimulants. It is possible to evaluate (find) the correlation with "immunity-related substances, stress-suppressing substances, innate immunity-activating substances, pathogen-recognizing substances, etc."
In addition, it is possible to search for unknown stimulants that produce known "immune-related substances, stress-suppressing substances, innate immunity-activating substances, pathogen-recognizing substances, etc."

Examples of the immune-related substance include immunomodulators such as cytokines and interleukins; antiviral substances such as interferon; and the like. Examples of the stress-suppressing substance include superoxide dismutase and NO synthase. Antioxidants and the like.
In addition, examples of the innate immunity activating substance include inflammatory cytokines such as TNFα1, TNFα2, IL1β1, and IL1β2; and examples of the pathogen recognizing substance include Toll-like receptor TLR.

<Correlation between qualitative and quantitative>
Here, "evaluating (obtaining) the correlation" includes qualitative evaluation and quantitative evaluation.
"Qualitative evaluation" includes evaluation of the correspondence between a specific stimulant substance and a specific producing substance.
"Quantitative evaluation" refers to the contact amount (usage amount) of a specific stimulant (dose in vivo, compounding amount in vitro) and the amount of production of a specific production substance. Correlation etc. are included.

In the evaluation of the quantitative correlation, the quantification of the produced substance may be performed directly by a known chemical method / analytical method, or may be performed by quantifying the gene corresponding to the produced substance. .. The above-mentioned "quantification of gene" is preferably "quantification of mRNA of a producing substance" extracted from the above cells, because it can be quantified even if the amount is very small at the initial stage.
More specifically, it is particularly preferable to collect cells contained in the liquid in the blisters, extract RNA, and measure the amount of mRNA by qRT-PCR.
When a gene is quantified, the substances produced such as "substances that affect humans" such as "immunity-related substances, stress suppressors, innate immunity activators, etc." are referred to as gene products of the genes. Become.

<Invention of Aspect 1 in vivo and Invention of Aspect 2 in vitro>
The present invention comprises the invention of aspect 1 which is in vivo and the invention of aspect 2 which is in vitro.
In the first aspect, the stimulant is administered to a bubble eye goldfish, the bubble eye goldfish is bred at a specific temperature, and then the produced substance produced by the cells in the bubble eye of the bubble eye goldfish or the gene of the produced substance is quantified. The above-mentioned method for clarifying the correlation between the stimulant and the producing substance, which is an in vivo method.
In the second aspect, the stimulant is blended in a medium containing cells collected from bubble eye goldfish, the cells are cultured at a specific temperature, and then the produced substance produced by the cells or the gene of the produced substance is used. It is the above-mentioned method for quantifying and clarifying the correlation between the stimulant and the producing substance, and is an in vitro method.

<< Aspect 1 (in vivo) >>
In the first aspect, as a method of administering the stimulant, the bubble eye goldfish is injected into the bubble eye, the bubble eye goldfish is injected outside the bubble eye, the bubble eye goldfish is fed, or the bubble eye is injected into an aqueous solution of the stimulant. Immersion of goldfish (that is, administration (uptake) to bubble eye goldfish by adding a stimulant to a bubble eye goldfish breeding tank) and the like can be mentioned. When the stimulant is administered outside the blister by injection, feeding, immersion, etc. outside the blister, the stimulant goes around the body of the bubble eye goldfish and comes into contact with cells in the blister.

After administration of the stimulant, the bubble eye goldfish are bred at a specific temperature. The specific temperature (breeding temperature) is important, and when the breeding temperature is high, the production amount of the produced substance may gradually decrease. In addition, if the temperature is higher than a certain limit temperature, the amount of the produced substance may decrease sharply.
Therefore, in the present invention, when clarifying the correlation between the stimulant substance and the produced substance, the specific temperature (breeding temperature such as the temperature of the aquarium) is preferably 30 ° C. or lower, preferably 28 ° C. or lower. It is more preferable to keep the temperature below 25 ° C. If the temperature is higher than the above temperature, the amount of the produced substance, which is the measurement target, may decrease too much and the measurement may not be possible.

<<< Temperature dependence in vivo >>
In the first aspect of the present invention, the temperature dependence of "the correlation between the stimulant and the" producing substance or the gene of the producing substance "" is further determined, and the presence or absence of the temperature dependence or the production of the producing substance is performed. It is also the above-mentioned method for clarifying the limit temperature to be obtained. Here, the "marginal temperature" is a temperature at which the production amount of the produced substance extremely decreases at a temperature higher than that.
Specifically, in aspect 1 of the present invention, the temperature dependence of the amount of the produced substance produced by administering the stimulant by changing the breeding temperature of the bubble eye goldfish is further determined, and the temperature dependence is obtained. The above method for clarifying the presence or absence or magnitude of sex (high temperature sensitivity), or the (substantial) maximum temperature at which the producing substance is preferably produced.

Since fish (such as bubble eye goldfish) are poikilotherms, it is considered that the breeding temperature is equal to the temperature of the liquid in the blister and the cells in the blister.
In the present invention, it has been clarified for the first time that when the produced substance is an immune-related substance such as a cytokine, the immune function in a poikilotherm decreases when the breeding temperature is high (see in vivo examples).
Further, in the present invention, it was confirmed in vitro that the amount of cytokine (producing substance) produced against Pseudomonas aeruginosa (stimulating substance) decreases as the culture temperature (cell temperature) increases (in vitro). (See Examples).

When determining the temperature dependence, the range of the breeding temperature is naturally not limited to the above 30 ° C. or lower, and the measurement temperature range is preferably 0 ° C. or higher and 45 ° C. or lower, more preferably 5 ° C. or higher and 40 ° C. or lower. It is more preferably 10 ° C. or higher and 37 ° C. or lower, and particularly preferably 15 ° C. or higher and 34 ° C. or lower. The above-mentioned lower limit temperatures can be exchanged with each other, and the above-mentioned upper limit temperatures can be exchanged with each other.

<<< Individual changes >>
Further, in aspect 1 of the present invention, in addition to identifying and quantifying the produced substance produced in the liquid in the blister after administration of the stimulant, it is also possible to obtain a change in an individual called a bubble eye goldfish. Preferred (included). That is, it is preferable that the first aspect of the present invention is the above-mentioned method for confirming whether or not the bubble eye goldfish is alive or dead, or has a disease or abnormality.
The individual used at this time is not necessarily limited to the bubble eye goldfish in which the substances produced in the bubble eye are actually examined, and is not limited to the bubble eye goldfish. That is, a fish in which blisters have not been developed may be used to separately confirm the life or death, disease onset, abnormality, etc. of the individual.
In addition, a plurality of individuals may be used to determine the survival rate, the disease incidence rate, and the like.

By observing the life or death of an individual, or the presence or absence of illness or abnormality, the effect / effect of "stimulant and / or the product produced (in the blisters) by administration of the stimulant" on the individual It becomes clear. It is possible to know the correlation between the stimulant and / or the producing substance and the change of the individual.

Although not particularly limited, the onset of the above-mentioned individual includes mouth ulcer, tail rot, fin rot, ulcer disease, pine scab, exophthalmos, etc., and the abnormalities of the above-mentioned individual include loss of appetite and parallel ataxia. And so on.

<< Aspect 2 (in vitro) >>
In aspect 2 of the present invention, the stimulant is blended in a medium containing cells collected from water bubbles of a water bubble eye goldfish, the cells are cultured at a specific temperature, and then the production substance produced by the cells or the production substance is produced. The method is characterized in that the gene of the above is quantified and the correlation between the stimulant and the producing substance is clarified.

The medium is not particularly limited, and any medium can be used as long as it can be cultured, but RPMI1640 medium, Leibovitz's L-15 medium and the like are particularly preferable. It is also preferable to add an antibiotic, carp serum, fetal bovine serum, blister eye goldfish blister solution (autologous blister solution) or the like to the medium.
The specific temperature (culture temperature) is not particularly limited as long as it can be cultured, but preferably, the temperature (range) described as the breeding temperature in the place of the above aspect 1 (in vivo) can be mentioned.
The culture time is not particularly limited, but is preferably 1 hour or more and 48 hours or less, more preferably 1.5 hours or more and 24 hours or less, and particularly preferably 2 hours or more and 12 hours or less.

The method for preparing the medium including the above "blending" is not particularly limited, and a known method can be used. For example, the order of addition of the stimulant and the cells is not particularly limited, and either of them may come first.

As the method for quantifying the "cell-produced production substance or the gene for the production substance" in the second aspect (in vitro), the same method as the method described in the above-mentioned aspect 1 (in vivo) can be used.

<<< Temperature dependence in vitro >>
In the first aspect of the present invention, the temperature dependence of "the correlation between the stimulant and the" producing substance or the gene of the producing substance "" is further determined, and the presence or absence of the temperature dependence or the production of the producing substance is performed. It is also the above-mentioned method for clarifying the limit temperature to be obtained. Examples of the "correlation" include a qualitative combination of a stimulant and a produced substance, a relationship between a certain amount of the stimulant and the amount of the produced substance produced by the stimulant, and the like.

Regarding "temperature dependence", "limit temperature", etc., if the breeding temperature in the above aspect 1 (in vivo) is read as the cell culture temperature, it is the same as the description in the above aspect 1 (in vivo). be.

Since fish (such as bubble eye goldfish) are poikilotherms, it is considered that the in vitro culture temperature of the cells taken out from the bubble eye corresponds to the actual temperature of the aquarium in vivo.
In the present invention, it was also confirmed in vitro that the amount of cytokine (producing substance) produced against killed Pseudomonas aeruginosa (stimulant) decreases with increasing culture temperature (cell temperature) (in vitro). See example).
From this, for the first time, it was clarified that the immune function in the poikilotherm decreased when the temperature was high (see Examples).

<Stimulant screening method>
The present invention is also a method for screening a stimulant, which comprises using the above method.
If the presence or absence or amount of production of immune-related substances, stress-suppressing substances, innate immunity-activating substances, etc. is confirmed, measured, and monitored using the above method, the substance administered in embodiment 1 and the compound compounded in embodiment 2 However, it becomes clear whether it can be a stimulant or whether it is useful as a stimulant, and the stimulant can be screened.

<Screening method for produced substances>
The present invention is also a method for screening a product, which comprises using the above method.
When a specific stimulant is administered in aspect 1 and blended in aspect 2 using the above method, the specific stimulant can be used to screen for production substances produced in blisters, i.e., in an individual. .. That is, "substances that affect individuals" such as immune-related substances, stress-suppressing substances, and innate immunity-activating substances are clarified, and screening thereof becomes possible.

<Combination screening method>
The present invention uses the above-mentioned method to determine the temperature dependence of "the correlation between the above-mentioned stimulant and the" said-mentioned producing substance or the gene of the above-mentioned producing substance "", and the temperature dependence is large or small, or the said. It is also a combination screening method characterized by screening a "combination of a stimulant and a producing substance" having a low or high limit temperature at which the producing substance is produced.

According to the present invention, it is possible to screen a combination of a stimulant substance and a producing substance.
For example, as described above, if the "specific producing substance produced by a specific stimulating substance" is clarified (if the correspondence (combination) is clarified), the invasion of the stimulating substance is dealt with in the body. It is possible to understand what kind of substance is used to counteract, and to contribute to the development and creation of medicines, health foods, lifestyles, etc., for example.

Also, if the temperature dependence of the correlation between the stimulant and the produced substance is known, it will be useful for research on the immune system, etc., and may be used for the development of pharmaceuticals, for example. It will also contribute to the development of basic research. It also contributes to the development of the fish farming industry.

Hereinafter, the present invention will be described in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the following Examples and the like. Hereinafter, the description "%" means "mass%" when it is related to mass.

Example 1
<Innate immunity monitoring by intrablister injection of killed Pseudomonas aeruginosa>
Blisters The autoclaved killed Pseudomonas aeruginosa (10-fold concentrated) is applied to the blisters on the right side when viewed from above and behind the goldfish (bred at a water temperature of 22 to 24 ° C), and the blisters on the left side when viewed from above and behind. 50 μL of each of the saline solutions was injected, and after breeding at a water temperature of 22 to 24 ° C. for 20 hours, the appearance was observed (see FIG. 2).
As a result, vasodilation and redness were observed on the surface of the blisters on the right side when viewed from above and behind, suggesting that an inflammatory response was induced (see FIG. 2).

Example 2
<Cytokine production response of cells in blisters by intrablister injection of killed Pseudomonas aeruginosa>
After administration (injection) of Pseudomonas aeruginosa killed bacteria into the blisters, cells in the blisters of the same individual were collected over time to examine the induction of inflammatory cytokine gene expression by the killed Pseudomonas aeruginosa (Fig. 3A, FIG. FIG. 3B).
That is, one blister (1R) is inoculated with killed Pseudomonas aeruginosa (10-fold concentrated 50 μL), and the other blister (1 L) is inoculated with physiological saline (50 μL). A graph showing the vertical axis of the values obtained by collecting cells in blisters for each blister, extracting RNA, measuring the amount of 5 types of mRNA by qRT-PCR, and normalizing the amount of EF1α mRNA is shown in FIG. 3 (Fig. 3). A) Shown in (B). FIG. 3 (A) shows the time-dependent transition of the mRNA amounts of IL1β1 and IL1β2, and FIG. 3 (B) shows the time-dependent transition of the mRNA amounts of TNFα1 and TNFα2.

In the cells collected from the blisters (1R) injected with Pseudomonas aeruginosa (right side when viewed from the top of FIG. 2), the mRNA levels of IL1β1, IL1β2, TNFα1 and TNFα2 increased, and all peaked in 2 to 4 hours. Has been reached (all figures in Fig. 3).
High levels of IL1β1 and IL1β2 mRNA persisted for more than 24 hours (FIG. 3 (A)).

After 8 hours, high mRNA levels were also observed in IL1β1 in cells collected from blisters (1 L) injected with physiological saline (left side when viewed from the top of FIG. 2) (left side of FIG. 3 (A)). graph).
In both the left and right blisters, the change in IL1β1 mRNA level was slight when saline was injected.
On the other hand, for the other inflammatory cytokines IL1β2, TNFα1 and TNFα2, the mRNA levels (changes) of the cells collected from the blisters injected with physiological saline were the same regardless of the presence or absence of injection of killed Pseudomonas aeruginosa into the contralateral blisters. It was minor (the graph on the right in FIG. 3 (A) and the graph on the left and right in FIG. 3 (B)).

These results suggest that injection of killed Pseudomonas aeruginosa into blisters induces early local expression of inflammatory cytokines. That is, it was clarified at the cytokine level that inoculation of killed Pseudomonas aeruginosa into the blisters induces a strong immune / inflammatory response locally in the blisters.
It was also found that the cells in the bubble eye of the bubble eye goldfish consist of immune / inflammatory cells, and the bubble eye goldfish is useful for the study of immunity / inflammation.

Example 3
<Effect of breeding temperature on the expression of inflammatory cytokine gene in cells in blisters>
The effect of the breeding temperature of the bubble eye goldfish on the expression of the inflammatory cytokine gene in the cells in the bubble eye was investigated. That is, the temperature sensitivity of the cytokine production response of the cells in the blister was examined using the bubble eye goldfish bred at various water temperatures (see FIG. 4).

That is, one day after inoculating the killed bacillus (P) in one blister and inoculating the physiological saline (S) in the other blister, the cells in the blister were collected to extract RNA, and RT. FIG. 4 shows a graph in which the mRNA amounts of IL1β1, IL1β2, TNFα1, TNFα2 and EF1α were measured by −qPCR, and the values when normalized to the mRNA amount of EF1α were taken on the vertical axis. In FIG. 4, "S" indicates physiological saline inoculation, and "P" indicates killed Pseudomonas aeruginosa inoculation.

At the breeding temperature of 30 ° C. to 35 ° C., the increase in mRNA level of inflammatory cytokine was lower than that at the normal breeding temperature of 24 ° C. (see FIG. 4).
These results suggest that the inflammatory cytokine production response induced by intrablister inoculation of Pseudomonas aeruginosa is sensitive to high temperatures.
(See FIG. 4).

Example 4
<Cytokine production response of cells in blisters by in vitro stimulation of killed Pseudomonas aeruginosa>
Cells collected from water bubbles were mixed-cultured with killed Pseudomonas aeruginosa in vitro, and the expression (time change) of inflammatory cytokines was examined by qRT-PCR (see FIG. 5). In FIG. 5, "PAO1" indicates the amount of cytokine mRNA produced by the cells in the blisters containing the killed Pseudomonas aeruginosa.

That is, cells collected from the blisters of bubble eye goldfish were suspended in RPMI1640 medium containing 10% BCS, 3% autoclave, and antibiotics, and placed on a 24-well plate (8 × 10 ⁴ / hole). Autoclaved treatment In the presence / absence (-) of Pseudomonas aeruginosa (PAO1), the cells were cultured at 25 ° C. for 2 to 6 hours. As used herein, the term "autologous blister fluid" refers to the blister fluid of a bubble eye goldfish from which cells in the blister have been collected.
Then, RNA was extracted, the amount of mRNA was measured by RT-qPCR, and the result of normalization with respect to the amount of EF1α mRNA is shown in FIG.

The mRNA levels of the inflammatory cytokines IL1β1, IL1β2, TNFα1 and TNFα2 increased at any time by stimulation of killed Pseudomonas aeruginosa for 2 to 6 hours (see FIG. 5).

The cells in the bubble eye of the bubble eye goldfish consisted of immune and inflammatory cells, and it was found that the cells are useful for the study of immunity and inflammation even in vitro.

Example 5
<Culture temperature dependence of cytokine gene expression in blistering cells by in vitro stimulation of killed Pseudomonas aeruginosa>
The effect of culture temperature on the expression of inflammatory cytokine genes in cells in water bubbles by in vitro stimulation of killed Pseudomonas aeruginosa (4 hours) was investigated (see FIG. 6). In FIG. 6, "PAO1" indicates the amount of Pseudomonas aeruginosa killed bacteria, and "Saline" indicates the amount of cytokine mRNA produced by the cells in the blisters containing the physiological saline solution.

Compared to 25 ° C, the increase in the mRNA level of IL1β1, TNFα1 and TNFα2 decreased at 29 ° C or higher, and at 33 ° C, the increase in the mRNA level of IL1β2 decreased in addition to these inflammatory cytokines (see FIG. 6). ..

Example 6
<Temperature dependence of cytokine expression by in vitro stimulation in multiple bubble eye goldfish>
Next, the reproducibility of Example 5 was confirmed using three bubble eye goldfish (goldfish 1, goldfish 2, goldfish 3). That is, cells in the blisters were collected from a plurality of bubble eye goldfish, and the expression levels of inflammatory cytokines at 25 ° C. and 33 ° C. were compared (see FIG. 7).

Specifically, cells collected from the blisters of three bubble eye goldfish were suspended in RPMI1640 medium containing 10% BCS, 3% autologous blisters, and antibiotics, respectively, and autoclaved with Pseudomonas aeruginosa (PAO1). ) In the presence, cultured on a 24-well plate (8 × 10 ⁴ / hole) at 25 ° C. and 33 ° C. for 4 hours.
Then, RNA was extracted, the amount of mRNA was measured by RT-qPCR, and the result of normalization with respect to the amount of EF1α mRNA is shown in FIG.

As a result, the increase in mRNA levels of IL1β1, TNFα1 and TNFα2 was statistically significantly lower at 33 ° C than 25 ° C (see FIG. 7). The increase in the mRNA level of IL1β2 also tended to decrease at 33 ° C with respect to 25 ° C (see FIG. 7).

[Summary of results of Examples 3 to 6]
The results of Examples 4, 5 and 6 described above are that the mononuclear cells in the blisters induce the expression of inflammatory cytokines in response to Pseudomonas aeruginosa killed bacteria, and the response is in Example 3 in. Similar to vivo, it was suggested to be sensitive to high temperatures.
From this, it was speculated that innate immunity, that is, resistance to bacterial infection, was reduced by breeding at high water temperature.

Example 7
<Effect of breeding temperature on resistance to Pseudomonas aeruginosa infection in goldfish>
Therefore, live Pseudomonas aeruginosa was inoculated into the abdominal cavity of goldfish, and the effect of breeding temperature on survival time was investigated. That is, 50 μL of a full-growth suspension of live Pseudomonas aeruginosa (P. aeruginosa (PAO1)) was intraperitoneally applied to a goldfish (n = 3 / group, 2 groups) weighing 4.2 g ± 0.6 g. The survival rate (number) was measured at the breeding water temperature (water tank temperature) (see FIG. 8).

As a result, at 29 ° C, the average survival time was shortened to 1/6 or less compared to the normal breeding temperature of 21 ° C (see FIG. 8). This suggests that high temperatures reduce the resistance of goldfish to bacterial infections.

Example 8
Using a culture system of cells in the bubble eye of a bubble eye goldfish, the cytokine production response by in vitro stimulation was investigated.
Bubbles Cells collected from the bubbles of goldfish were suspended in RPMI1640 medium supplemented with 10% BCS and antibiotics and placed in a 24-well plate in the presence of PHA (50 μg / mL) or PolyI: C (50 μg / mL). Incubate at (2x10 ⁵ / hole) for 4 hours or 24 hours, collect cells in water bubbles to extract RNA, measure the amount of each mRNA by RT-qPCR, and normalize to the amount of EF1α mRNA. The vertical axis shows the relative amount when the amount of mRNA in the control group cultured for 4 hours is 1 (see FIG. 9).

As a result of in vitro stimulation with T cell mitogen PHA or TLR3 agonist PolyI: C, the following was found (see FIG. 9).
(1) 4-hour stimulation of PHA increased the mRNA levels of IFNγ (13-fold compared to saline control), IL1β (12-fold), and TNFα2 (15-fold).
(2) The 4-hour stimulation of PolyI: C increased the mRNA levels of IL1β1 (80 times that of the saline control) and IL1β2 (30 times that of the saline control).
(3) Upon 24-hour stimulation, the mRNA levels of these cytokines were low.

The results in FIG. 9 suggest that the cells in the bubble eye goldfish produce immune and inflammatory cytokines in response to in vitro immune stimulation, and the response is rapid within a few hours. ..

Example 9
Bubble Eye Cells collected from the bubble eye goldfish were suspended in RPMI1640 medium supplemented with 10% BCS and antibiotics, and the number of cells per hole was 2 × 10 ⁵ , 6 × 10 ⁴ , 2 on a 24-well plate. The cells were sprinkled in a size of × 10 ⁴ and 6 × 10 ³ and cultured for 4 hours in the presence of PHA (50 μg / mL) or PolyI: C (50 μg / mL).
RNA was extracted and the amount of each mRNA was measured by RT-qPCR. The vertical axis of FIG. 10, normalized to mRNA levels of EFla, indicating the relative amount when the amount of mRNA of the control group of cells Number 2 × 10 ⁵ to 1 (see FIG. 10).

The cytokine expression response of in vitro stimulated cells in the bubble eye goldfish was dependent on the number of cells (see FIG. 10).
Even with 6 × 10 ³ cells per hole (24-hole plate), a PHA-stimulated IFNγ mRNA expression response and a PolyI: C-stimulated IL1β1 mRNA expression response were observed (see FIG. 10).

According to the present invention, that is, when the liquid in the bubble eye of a bubble eye goldfish is used, it is preferable for humans to produce cells in the bubble eye, for example, an immune-related substance, a stress inhibitor, an innate immunity activating substance and the like. Since the product can be easily, accurately and quantitatively grasped, the present invention is widely used for studying various stimulants, substances produced in the human body, and the like. , It is also used in the aquatic culture industry.

Claims (15)

1. After contacting the stimulant with the cells in the bubble eye goldfish, the product produced by the cell or the gene of the product is quantified to clarify the correlation between the stimulant and the product. A method characterized by doing.
2. The stimulant is administered to a bubble eye goldfish, the bubble eye goldfish is bred at a specific temperature, and then the produced substance produced by the cells in the bubble eye of the bubble eye goldfish or the gene of the produced substance is quantified, and the substance is said to be quantified. The method according to claim 1, wherein the correlation between the stimulant and the producing substance is clarified.
3. 2. The administration is injection into the bubble eye of the bubble eye goldfish, injection of the bubble eye goldfish outside the bubble eye, feeding to the bubble eye goldfish, or immersion of the bubble eye goldfish in the aqueous solution of the stimulant. The method described in.
4. The method according to claim 2, wherein the specific temperature is 30 ° C. or lower.
5. Furthermore, the temperature dependence of "the correlation between the stimulant and the" producing substance or the gene of the producing substance "" is obtained, and the presence or absence of the temperature dependence or the critical temperature at which the producing substance is produced is clarified. The method according to any one of claims 2 to 4.
6. Further, the method according to any one of claims 2 to 5, which confirms whether or not the bubble eye goldfish is alive or dead, or has a disease or abnormality.
7. The above stimulant is blended in a medium containing cells collected from bubble eye goldfish, and after culturing the cells at a specific temperature, the produced substance produced by the cells or the gene of the produced substance is quantified. The method according to claim 1, wherein the correlation between the stimulant and the producing substance is clarified.
8. Furthermore, the temperature dependence of "the correlation between the stimulant and the" producing substance or the gene of the producing substance "" is obtained, and the presence or absence of the temperature dependence or the limit temperature at which the producing substance is produced is clarified. The method according to claim 1 or 7.
9. The stimulant is a live bacterium, a dead bacterium, a part of a bacterium, a fungal product, or a fungal origin; a virus or a microorganism, or a derivative thereof; a food; a medicine; a pesticide; a food additive. The method according to any one of claims 1 to 8, which is an air pollutant; a marine pollutant; or a lectin.
10. The method according to any one of claims 1 to 9, wherein the produced substance is an immune-related substance, a stress-suppressing substance, an innate immunity-activating substance, or a pathogen-recognizing substance.
11. The method according to claim 10, wherein the immune-related substance is a cytokine.
12. The method according to any one of claims 1 to 11, wherein the "quantification of the gene of the producing substance" is the "quantification of mRNA of the producing substance" extracted from the cells.
13. A method for screening a stimulant, which comprises using the method according to any one of claims 1 to 12.
14. A method for screening a product, which comprises using the method according to any one of claims 1 to 12.
15. Using the method according to any one of claims 1 to 12, the temperature dependence of "correlation between the stimulant and the" correlation of the product or the gene of the product "" is determined, and the temperature is determined. A method for screening a combination, which comprises screening a "combination of a stimulant and a producing substance" having a large or small dependence, or a low or high temperature limit at which the producing substance is produced.

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