JP2006262863A - Method for evaluating external stimulation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for evaluating external stimulation, which is useful for screening a candidate medicine. <P>SOLUTION: The method for evaluating external stimulation, in which the action of external stimulation is evaluated by using the state of a yolk cardiovascular system formed by at the initial stage of bird growth as an index, comprises (1) a step for preparing a fertilized egg of bird, (2) a step for providing the fertilized egg with external stimulation of a test object, (3) a step for assaying the state of the yolk cardiovascular system of the fertilized egg and (4) a step for evaluating the action of the external stimulation on the yolk cardiovascular system by the assayed result. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for evaluating the action and influence of external stimuli in vitro. For example, the evaluation method of the present invention can be used to search for drug candidates or to evaluate drug toxicity and side effects.

In 2000, the guidelines for hypertension were lowered, and there are about 37 million people with hypertension in Japan. Many lifestyle-related diseases are accompanied by vascular disease, and if they worsen, they become fatal diseases. In strokes of typical fatal diseases, there is a report that the incidence decreases if pharmacotherapy is given before arteriosclerosis occurs. Several types of treatments for heart disease associated with lifestyle-related diseases have already been used, but considering the number of patients around the world, the development of effective treatments with fewer side effects is urgent.
It is said that the part that still requires a lot of labor and time in the development of new drugs is the screening and clinical stage to experimentally determine the effectiveness and harmfulness of drug candidates. Developing a screening system that can greatly reduce the cost of the screening work and examine the potential of more drug candidates in more detail will improve the competitiveness of new drug development.
An example of a drug screening method is disclosed in Patent Publication 1.

Japanese National Patent Publication No. 9-509829

In the current drug development, it is a normal procedure to conduct in vivo verification using experimental animals such as mice, rats, and rabbits after in vitro screening using cultured cells. Mammals such as mice are small and easy to handle, but large facilities are required to handle multiple samples under the same conditions. In addition, problems such as stable supply of laboratory animals and breeding costs are also significant.
On the other hand, screening systems using chicken eggs that are easily available have been proposed. That is, attempts have been reported to screen for substances that act on angiogenesis by quantifying angiogenesis using the serous vasculature. The serous vasculature develops around the embryonic development stage 18 and forms a stable vascular network centered on a blood vessel having a relatively large pore diameter. Since the blood vessels are relatively thick and stable as described above, the serous vasculature has been considered suitable for the purpose of observing the state change. However, in the serous vasculature, many blood vessels extend not only near the surface of the egg but also inside, so that it is difficult to actually observe, and it is difficult to accurately grasp the number and amount of blood vessels. In addition, since the embryo moves actively at the developmental stage where the serous vasculature is formed, it is necessary to take into account the fluctuation and movement of blood vessels accompanying the movement of the embryo. Therefore, measurement becomes difficult and the reliability of the obtained data is low.

  On the other hand, methods using the retinal vasculature of rabbits as a method for evaluating angiogenesis and methods using blood vessel formation at the level of cultured cells as an index have been proposed in the past. Alternatively, only the imaging and analysis of the transition process of the microscopic area of only the tube in which no blood cells flow is performed, and a quantitative observation method of true angiogenesis has not been established. In addition, the method using the rabbit retinal vasculature is accompanied by the problem that it is difficult to observe blood vessels.

Under the above background, the present inventors have intensively studied with the aim of constructing a new evaluation system that can be used for searching and evaluating drug candidates. As a result, using the yolk vasculature formed early in the development of avian eggs, it is easy to quantitatively observe angiogenesis, and the evaluation system (measurement system) has many advantages shown below. The knowledge that it can build was obtained.
(1) The yolk vasculature is highly sensitive and has excellent responsiveness when given external stimuli. In addition, in the yolk vasculature, angiogenesis, blood vessel elongation, and blood vessel hypertrophy are prosperous. These features enable rapid evaluation. In addition, measurement and evaluation with a very small amount of sample is possible. This means, for example, that the evaluation system is particularly effective when evaluating the action of substances that are difficult to prepare in large quantities.
(2) The yolk vasculature is a vasculature that is first formed at the developmental stage and is formed near the surface of the egg, so that observation and measurement are easy. The simple structure is also advantageous for observation and measurement of the yolk vasculature.
(3) Bird eggs have small individual differences compared to individual animals, so the data obtained is highly reliable and reproducible.
(4) The same individual (egg) can be observed and measured over time.
(5) The formation time of the yolk vasculature is that the embryo is almost stationary. Therefore, there is almost no shaking or movement of blood vessels accompanying the movement of the embryo, and blood vessel observation and measurement can be performed easily and with high accuracy. In addition, the movement of the embryo is active at the time of formation of the urinary serosa vasculature, and the shaking of the blood vessel is intense.
(6) For example, if chicken eggs are used, stable supply is relatively easy, which is advantageous in terms of breeding costs.
(7) In the egg at the time of yolk blood vessel formation, the pulsation of the heart can also be confirmed with the naked eye. It is also possible to observe blood cells flowing through capillaries.
(8) Because of the many advantages described above, the evaluation system using the avian egg yolk vasculature is easy to automate the measurement and can be said to be the optimal system for realizing a high-speed screening system.

The present invention provides a method for evaluating the action of an external stimulus, which includes the following steps based on the above results and knowledge.
(1) a step of preparing a fertilized egg of a bird,
(2) applying an external stimulus of a test subject to the fertilized egg,
(3) measuring the state of the yolk cardiovascular system of the fertilized egg,
(4) A step of evaluating the effect of the external stimulus on the yolk cardiovascular system from the measurement result.

In one aspect of the present invention, step (2) is performed when the fertilized egg is in the embryonic development stage 15-22. More preferably, step (2) is performed when the fertilized egg is in the embryonic development stage 15-18. Even more preferably, step (2) is performed when the fertilized egg is in the embryonic development stage 17-18. This is because if the timing of external stimulation is too early, the embryo is damaged and affects the formation of the yolk vasculature, making it difficult to evaluate the state of the yolk vasculature as an index.
On the other hand, it is preferable to perform step (3) before the serous vasculature becomes dominant over the yolk vasculature. This is because when the serous vasculature becomes dominant, it becomes difficult to observe the state of the yolk blood vessel or the state of the embryo. For example, step (3) is preferably performed when the fertilized egg is in the embryonic development stage 17-22.

  In one embodiment of the present invention, the state of the yolk cardiovascular system is measured over time in step (3). As a result, it is possible to evaluate changes over time in the action and influence of external stimuli on the yolk cardiovascular system.

The external stimulus to be tested is, for example, one or more selected from the group consisting of test substance addition, light irradiation, electromagnetic wave irradiation, radiation irradiation, sound wave irradiation, electrical stimulation, vibration addition, and pressure addition Is an external stimulus.
On the other hand, the state of the yolk heart vasculature is, for example, the area of the blood vessel, the number of blood vessels, the width or diameter of the blood vessel, the state of branching of the blood vessel, the blood vessel elongation rate, the blood volume, the blood flow rate, the blood cell migration rate, One or more items selected from the group consisting of heart rate. In a preferred embodiment of the present invention, the measurement item is one or more selected from the group consisting of the area of the blood vessel, the number of blood vessels, the width or diameter of the blood vessel, the state of branching of the blood vessel, and the blood vessel elongation rate.
A chicken or quail is preferably selected as the type of bird. This is because these fertilized eggs are relatively easy to obtain.

(the term)
In this specification, “yolk cardiovascular system” is used as a term that comprehensively expresses yolk blood vessels and the heart at the time of yolk blood vessel formation. Therefore, in this specification, the “state of the yolk cardiovascular system” includes the state of the yolk blood vessel and the state of the heart at the time of yolk blood vessel formation (for example, the heart rate).

The present invention relates to a method for evaluating the action of an external stimulus. Based on the evaluation result according to the present invention, it is possible to screen an external stimulus effective for a cell or a living body. The present invention can also be used to evaluate the degree of adverse effects (for example, toxicity and side effects) that an external stimulus has on cells.
As used herein, `` external stimulation '' refers to (1) addition of test substances such as drug candidates, (2) irradiation with light, electromagnetic waves, radiation, sound waves, (3) electrical stimulation, (4) vibration, and (5) Such as the addition of pressure.

The evaluation method of the present invention includes the following steps.
(1) A step of preparing fertilized eggs for birds.
(2) A step of applying an external stimulus to the test subject to the fertilized egg.
(3) A step of measuring the state of the yolk cardiovascular system of the fertilized egg.
(4) A step of evaluating the effect of the external stimulus on the yolk cardiovascular system from the measurement result.
Hereinafter, each step will be described in detail.

1. step 1)
In step (1), fertilized eggs of birds are prepared. Birds here are, for example, chickens, quails, turkeys, ducks, geese, ostriches, teabos and pigeons. Preferably, chicken or quail fertilized eggs are used. This is because it is relatively easy to obtain a fertilized egg. Most preferably, chicken fertilized eggs are used. This is because the fertilized egg supply and distribution system is the most complete. In addition, the egg is larger than the quail, so it is easy to handle and the yolk cardiovascular system is easy to observe and measure.

As will be described later, in the evaluation method of the present invention, the state of the yolk cardiovascular system is the object of observation / measurement. Therefore, it is necessary to prepare a fertilized egg in a state in which the yolk cardiovascular system can be observed and measured later. Preferably, a fertilized egg before the start of urinary serous vasculature formation is used.
In chicken eggs, egg yolk blood vessels usually begin to develop around the embryonic development stage 15 (about 2 days after fertilization) and are easily observed with the naked eye around the developmental stage 17 (about 2 to 3 days after fertilization). Form a vascular network to the extent. Then, some of the yolk blood vessels begin to disappear around the development stage 22 to 24 (about 3 to 4 days after fertilization), and the yolk blood vessels are almost completely around the development stage 30 (about 7 to 8 days after fertilization). It will disappear. On the other hand, urinary serosa blood vessels usually begin to develop around embryonic development stage 18 (about 3 days after fertilization). Therefore, in the case of using chicken eggs, the stage is usually before the embryo development stage 28, preferably before the embryo development stage 22, more preferably before the embryo development stage 17, and even more preferably before the embryo development stage 15. Prepare a fertilized egg. By using a fertilized egg in a relatively early stage of development in this way, with the addition of an external stimulus described later and the subsequent time in a timely and sufficient time margin and with little influence of serous blood vessels. Measurements can be performed.
When using eggs from birds other than chickens, fertilized eggs at the same generation stage as described above are used.
The developmental stage of the chicken embryo is in accordance with Hamburger V. and Hamilton HL (1951) J. Morphol. 88: 49-92 (“Essential Developmetal Biology, A Practical Approach, ed. CC Stein & PWH Holland), supervised by Sadao Yasugi, see Medical Science International.

  In order to promote development, fertilized eggs are tumbled. For example, the eggs are cultured in a thermostat adjusted to about 37 ° C. and a relative humidity of about 60% under the condition of turning eggs (90 °) once or twice per hour.

2. Step (2)
In step (2), an external stimulus is applied to the prepared fertilized egg. That is, in this step, the external stimulus of the test subject is applied to the fertilized egg. As described above, external stimuli in the present invention include (1) addition of test substances such as drug candidates, (2) irradiation with light, electromagnetic waves, radiation, sound waves, (3) electrical stimulation, (4) addition of vibration, and ( 5) Adding pressure.
In a preferred embodiment of the present invention, an external stimulus by adding a test substance is given to a fertilized egg. In the evaluation method of this aspect, the action of the test substance is evaluated. Such an evaluation system is effective for searching for drug candidates and evaluating the toxicity and side effects of drugs. In particular, the method of the present invention can be suitably used for evaluating the action (influence) of a substance on the vascular system. Specifically, the present invention can be used for screening for compounds that act on the vascular system (compounds that inhibit or promote angiogenesis; for example, candidates for anticancer agents).

Two or more external stimuli may be given in combination. For example, the combined use of test substance and light irradiation, the combined use of test substance and electrical stimulation, and the like. According to this aspect, it is possible to evaluate the action on the vascular system when different types of external stimuli are combined. For example, if it is obtained that the efficacy is enhanced by the combined use of a certain drug and electrical stimulation, administration of the drug while applying electrical stimulation is effective for exerting a high therapeutic effect. Judgment can be made.
A combination of external stimuli of the same kind may be given. For example, this is the case when two or more test substances are added simultaneously. Here, “simultaneous” does not mean exact synchronicity in time, but two or more test substances may be added at intervals.

Typically, an external stimulus is applied after a portion of the eggshell has been previously removed so that the inside of the egg can be observed. The removal of part of the eggshell is performed, for example, by the following procedure.
(1) With the blunt end (temperament side) of the fertilized egg up.
(2) A hole is made in a part of the shell, and a small amount (eg, about 15 ml) of egg white is extracted using this hole. This lowers the egg liquid level.
(3) Cut the eggshell slightly above the egg liquid level and remove the blunt end (temperament side) of the shell. In addition, after removing a part of eggshell, it switches from egg-turn culture to stationary culture.

An external stimulus is applied to part or all of the egg. In the embodiment in which the test substance is added as an external stimulus, the external stimulus can be applied to a part of the egg by the following procedure.
(1) A small plate (for example, a donut-shaped small plate) partially opened is placed on the surface of an egg exposed by removing a part of the eggshell.
(2) The test substance is added to the egg surface through the opening of the platelet. Thereby, only the part corresponding to the opening of the platelet on the egg surface is directly exposed to the test substance.

  The egg may be removed from the shell and transferred to a separate container before applying an external stimulus or before measurement (step (3)). However, it is preferable to maintain the eggs in the eggshell throughout the entire operation because of complicated operations and difficulty in placing the embryo in a preferred position for measurement.

  In the evaluation method of the present invention, the effect of external stimulation is evaluated using the state of the yolk cardiovascular system as an index. Accordingly, external stimulation is applied before the yolk cardiovascular system is formed or at the stage of yolk cardiovascular system formation. Preferably, the external stimulus is applied after the stage when the embryo is morphologically completed (development stages 15 to 16 for chicken embryos). This reduces the effects of external stimuli on the embryo and makes it possible to observe the yolk blood vessels over a relatively long period of time (if the external stimulus is applied too early, the embryo is damaged and the yolk cardiovascular system is damaged. Affects the formation and makes long-term observation difficult). On the other hand, as described above, the yolk cardiovascular system is destined to disappear in a short period of time. Alternatively, it may be preferable to apply an external stimulus as early as possible in the stage of formation of the yolk vasculature. In consideration of the above circumstances, when a chicken egg is used, the timing for applying an external stimulus in the present invention is, for example, embryo development stage 15 to 28 (about 2 to 6 days after fertilization), preferably embryo development stage 15 ~ 22 (about 2 to 4 days after fertilization), more preferably embryo development stage 15 to 18 (about 2 to 3 days after fertilization), still more preferably embryo development stage 17 to 18 (about 2 after fertilization) Days to 3 days).

3. Step (3)
In step (3), the state of the yolk cardiovascular system is measured in the fertilized egg after applying the external stimulus. The measurement is performed from the start of yolk blood vessel formation to the disappearance of the yolk blood vessel. Here, the egg at the stage where the yolk blood vessel is formed is highly transparent, and it is easy to grasp the state of the embryo from the outside. In addition, the embryo at this stage is easy to observe because it is almost stationary. Therefore, by performing the measurement at the time when the yolk blood vessel is formed, it is possible to observe and evaluate not only the state of the blood vessel but also the state of the embryo (for example, the pulsation of the heart) under good conditions.
As described above, egg yolk blood vessels usually begin to develop around the embryonic development stage 15 (about 2 days after fertilization) in the chicken egg, and easily with the naked eye around the developmental stage 17 (about 2 to 3 days after fertilization). A blood vessel network is formed to the extent observed in Then, some of the yolk blood vessels begin to disappear around the development stage 22 to 24 (about 3 to 4 days after fertilization), and the yolk blood vessels are almost completely around the development stage 30 (about 7 to 8 days after fertilization). It will disappear. On the other hand, urinary serosa blood vessels usually begin to develop around the embryonic development stage 18 (about 3 days after fertilization) and become visible to the naked eye around the developmental stage 23 (about 4 days after fertilization). It becomes more dominant than yolk blood vessels (about 6 days after fertilization). When the serous blood vessel becomes dominant, it becomes difficult to observe the state of the egg yolk cardiovascular system because the serous blood vessel becomes obstructed. Therefore, measurement is preferably performed before the serous blood vessel becomes dominant.
Considering the above-mentioned formation time of yolk blood vessel and urinary serosa blood vessel, the measurement time in the present invention when using a chicken egg is, for example, 15 to 30 (about 2 to 8 days after fertilization) at the embryonic development stage. Embryo development stage 15 to 27 (about 2 to 5 days after fertilization), more preferably embryo development stage 17 to 27 (about 3 to 5 days after fertilization), still more preferably Embryo development stages 17-22 (about 3-4 days after fertilization). In the embryonic development stages 17 to 22, the yolk blood vessels are formed to such an extent that they can be observed with the naked eye, and the serous blood vessels are hardly observed, so that the state of the yolk cardiovascular system can be observed and measured easily and satisfactorily. It is.
In addition, even when using eggs of birds other than chickens, it is preferable to carry out the measurement at a generation stage equivalent to the above-described generation stage.

In the present invention, the state of the yolk cardiovascular system, that is, the state of the yolk blood vessel and / or the state of the heart at the time of yolk blood vessel formation is the measurement object. Specifically, “the yolk heart vascular system state” includes the area of blood vessels at the time of yolk blood vessel formation (for example, the total area in the measurement region), the number of blood vessels (for example, the total number in the measurement region), the width or diameter of the blood vessels ( For example, that of the target blood vessel or the average in the measurement region), the state of branching of the blood vessel (for example, that of the target blood vessel, or the total number of branches in the measurement region or the total blood vessel length), the blood vessel extension rate, the blood volume, Includes blood flow rate, blood cell migration rate, and embryo heart rate. Therefore, in the present invention, one or more selected from the above are measurement items.
Among these, the area of the blood vessel, the number of blood vessels, the width or diameter of the blood vessel, the state of branching of the blood vessel, and the rate of blood vessel elongation are easy to measure, and are particularly useful as indicators for grasping the state of angiogenesis. It is considered effective. Accordingly, in a preferred embodiment of the present invention, the number of blood vessels, the area of the blood vessels, the width or diameter of the blood vessels, the state of branching of the blood vessels, and / or the rate of blood vessel expansion are adopted as the measurement items.
In a particularly preferred embodiment of the present invention, the total area of the blood vessel is employed as one of the measurement items.

  In addition to the state of the yolk cardiovascular system, the state of the embryo may be observed and measured. For example, the size of the embryo and the state of the embryo's tissue (eye size, etc.) can be measured.

  Measurement is performed by visual observation, observation under a microscope, camera photography, video photography, and the like. Specifically, for example, as shown in an example described later, a blood vessel portion at an arbitrary place on the yolk under a constant luminous intensity with a digital camera (the coordinate position of the blood vessel system in each yolk is defined in advance for each of them) Good) is taken over time at a close-up angle of 90 ° after drug addition.

When measuring yolk blood vessels (for example, measuring the area of the blood vessels, the number of blood vessels, etc.), the measurement area is not particularly limited, and a part or all of the egg surface exposed by removing a part of the shell is used as the measurement area. . However, when an external stimulus is applied only to a partial region of the egg, it is preferable to set the region as a measurement region or include the region in the measurement region. This is because a region to which an external stimulus is applied is a region that is directly affected / affected by the external stimulus, and the effect / effect of the external stimulus can be measured with high sensitivity if the region is a measurement target.
The region to which the external stimulus is applied may be different from the measurement region. Moreover, when an external stimulus is given to the whole egg (for example, when a test substance is added to the whole exposed part of the egg), even if the whole exposed part is set as the measurement area, only a part of the exposed part is measured. It is good.

  The measurement in this step may be performed at a plurality of times. For example, the measurement is continuously performed for a predetermined time from a certain time point. According to such measurement over time, the state of the yolk cardiovascular system can be monitored. Therefore, it is possible to evaluate changes over time in the action and influence of external stimulation on the yolk cardiovascular system. One feature of the evaluation method of the present invention in which the yolk cardiovascular system is a measurement target is that such time-dependent measurement can be performed using the same individual (egg). That is, according to the evaluation method of the present invention, data at a plurality of measurement points can be obtained from the same individual. Therefore, the evaluation system is free from errors caused by individual differences and a decrease in data reliability, which is a problem in an evaluation system in which different individuals are used for each measurement time point.

4). Step (4)
In step (4), the effect of external stimulation on the yolk cardiovascular system is evaluated from the measurement result of step (3). In this step, the data obtained in step (3) is subjected to analysis according to the nature of the data. For example, when image data is obtained by a digital camera or the like, image analysis is performed using appropriate software.

Usually, the effects of external stimuli are compared by comparing the data obtained from eggs with external stimuli (test group) and the data obtained from eggs with the same conditions (control group) except that no external stimuli are given. evaluate.
For example, when the blood vessel area (total blood vessel area in the measurement region) is adopted as the measurement item, when the measurement value of the test group is smaller than the measurement value of the control group (that is, a decrease in the measurement value is recognized as a result of external stimulation) ), It can be determined that an external stimulus (for example, a test substance) has an anti-angiogenic effect. On the contrary, when the measured value of the test group is larger than the measured value of the control group, it can be determined that the external stimulus has an angiogenesis promoting action.
Similarly, when the number of blood vessels is employed as the measurement item, the effect of the test substance on angiogenesis can be determined based on the increase or decrease in the number of blood vessels.
When the temporal measurement is performed in step 3, the evaluation may be performed based on the temporal change amount (speed) of the measurement result (data).

<Angiogenesis quantification using early embryonic egg yolk cardiovascular system>
Angiogenesis is associated with many diseases, and methods and drugs for regulating angiogenesis are energetically developed. Diseases involving angiogenesis include retinopathy as a complication of diabetes, inflammatory diseases (for example, rheumatoid arthritis), hyperlipidemia, tumors and the like. Angiogenesis also plays an important role in the wound healing process.
Angiogenesis consists of the development of a completely new vascular system from tuberoblasts at the developmental stage and the formation of new vascular branches based on the proliferation of endothelial cells from the vascular system already present in the tissue (narrow definition angiogenesis). Broadly divided.

An angiogenesis quantitative experiment using the early chicken embryonic yolk cardiovascular system was carried out by the following procedure. FIG. 1 illustrates the outline of the experimental procedure.
1. Egg culture (several days after incubation)
Fertilized eggs immediately after egg laying were cultured for 2.8 days under conditions of a temperature of 37.5 ° C. and a relative humidity of 60% (twice an hour at an egg turning angle of 90 °).
2. Eggshell cutting and stationary culture (after several days after incubation)
After the turnover culture, the circumference of the eggshell lateral surface on the blunt end side was cut, and an opening for observation was provided in the upper part of the eggshell. After placing a ring-shaped plastic plate (inner diameter of about 5 mm) on the egg surface, the egg was covered with a lid so that the egg surface was not dried, and cultured at a temperature of 37.5 ° C. and a relative humidity of 60%.
3. Twenty-four hours after the start of stationary culture, a drug (angiogenesis inhibitor captopril) dissolved in PBS was added to the egg surface at the central opening position (measurement region) of the plastic platelet as follows. The static culture was continued after the addition of the drug.
Experimental group 1: Add 100 μl of 5 mg / ml (in PBS) captopril (Angiotensin converting enzyme (ACE) inhibitor, Sigma-Aldrich) Experimental group 2: Add 100 μl of 0.5 mg / ml (in PBS) captopril Control group: 100 μl of PBS added No additive group: No addition Photographing The measurement area was photographed at 10-minute intervals from 0 minutes after addition to 120 minutes after addition (digital camera), and the state of the yolk cardiovascular system was observed over time.
5. Image analysis The obtained image data was analyzed by the following procedure, and the total area of yolk blood vessels present in the measurement region was calculated (FIG. 2).
(1) Grayscale conversion of image data
(2) Black spot detection
(3) Calculate black ratio from the number of black spots and the total number of black spots
(4) Convert black ratio to blood vessel area A graph of the image analysis result (blood vessel area change rate) is shown in FIG. From 60 minutes after the addition, a significant difference is observed between the drug addition group (experiment group 1, experiment group 2) and the solvent addition group or the non-addition group (risk rate 0.5%). Thus, the action of the drug could be clearly determined in a short time of only 60 minutes. In addition, if a similar experiment is performed using the urinary serosa vasculature of a chick fertilized egg, it usually takes 10 days or more to obtain a result. In addition, since blood vessels overlap in many places, the measurement data varies greatly. On the other hand, even in an evaluation system using cultured cells, it usually takes 7 days or more to obtain a result. Moreover, the measurement data greatly depends on the state of the cell, and it is difficult to obtain a reliable result.

  As is clear from the above experimental examples, according to the evaluation method using egg yolk blood vessels, evaluation results can be obtained in a very short time. Therefore, this method is an evaluation system suitable for high throughput.

According to the present invention, it is possible to evaluate the effect of external stimulation such as addition of a drug in a short time. For example, the evaluation method of the present invention can be used for the following purposes.
(1) Search for drug candidates, especially search for drug candidates that act (suppress or promote) angiogenesis, blood vessel elongation, hypertrophy, etc.
(2) Evaluation of the effects (toxicity, side effects) of test substances (for example, drugs) on angiogenesis, blood vessel elongation, hypertrophy, etc.
(3) Search for external stimuli preferred as cell or tissue culture conditions

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

It is a figure which shows the evaluation system using a chicken egg yolk cardiovascular system. It is a figure which shows the analysis procedure (an example) of image data. It is a graph (blood vessel area change rate) of the experimental result obtained by the evaluation system using a chicken egg yolk cardiovascular system.

Claims (9)

A method for evaluating the action of an external stimulus, comprising the following steps:
(1) a step of preparing a fertilized egg of a bird,
(2) applying an external stimulus of a test subject to the fertilized egg,
(3) measuring the state of the yolk cardiovascular system of the fertilized egg,
(4) A step of evaluating the effect of the external stimulus on the yolk cardiovascular system from the measurement result.   The evaluation method according to claim 1, wherein step (2) is performed when the fertilized egg is in the embryonic development stage 15-22.   The evaluation method according to claim 1, wherein step (2) is performed when the fertilized egg is in the embryonic development stage 15-18.   The evaluation method according to any one of claims 1 to 3, wherein step (3) is performed before the serous vasculature becomes more dominant than the yolk vasculature.   The evaluation method according to any one of claims 1 to 4, wherein in step (3), the state of the yolk cardiovascular system is measured over time.   The external stimulus is one or more external stimuli selected from the group consisting of test substance addition, light irradiation, electromagnetic wave irradiation, radiation irradiation, sound wave irradiation, electrical stimulation, vibration addition, and pressure addition. The evaluation method according to any one of claims 1 to 5.   As the state of the yolk heart vasculature, the area of the blood vessel, the number of blood vessels, the width or diameter of the blood vessel, the state of the branching of the blood vessel, the blood vessel elongation rate, the blood volume, the blood flow rate, the blood cell migration rate, and the heart rate of the embryo The evaluation method according to claim 1, wherein at least one item selected from the group consisting of: is measured in step (3).   Step (3) selected from the group consisting of the area of blood vessels, the number of blood vessels, the width or diameter of blood vessels, the state of branching of blood vessels, and the rate of blood vessel expansion as the state of the yolk heart vasculature The evaluation method according to claim 1, which is measured in step 1.   The evaluation method according to claim 1, wherein the birds are chickens or quails.