JP6164622B1 - Evaluation method of chemotaxis behavior to odorant based on olfactory sense of nematode, and petri dish used for the evaluation method - Google Patents

Abstract

The present invention provides a method for evaluating chemotaxis behavior for odor substances based on the olfactory sense of nematodes, and a petri dish used in the evaluation method. A method for evaluating the chemotaxis behavior of a nematode with respect to the odor of a test sample, comprising: a) a petri dish in which the test sample is arranged on the bottom surface, and the bottom surface being 1 cm to 3 cm away from the test sample Providing a petri dish in which nematodes are arranged in the region or part of b), and b) placing the test sample or nematodes on the bottom of the nematodes 3 to 15 minutes after the later one is placed And a petri dish suitable for this method, comprising: observing; and c) evaluating whether the test sample has attracted or repelled from the observed nematode arrangement . [Selection figure] None

Description

  The present invention relates to a method for evaluating chemotaxis behavior with respect to an odor substance based on an olfactory sense of a nematode, and a petri dish used for the evaluation method.

  Nematodes are popular organisms that are widely bred and studied all over the world as model organisms in biological research, and are characterized by their easy breeding. Nematodes have all the nerves revealed at the cellular level, and research on behavioral analysis is actively conducted.

  For the behavioral evaluation of nematodes, a 9 cm round petri dish is used. This is because it is indispensable to maintain a long movement distance of the nematode in order to maintain the accuracy of the behavior evaluation of the nematode. The present inventors have demonstrated that cancer can be detected with high sensitivity and high accuracy by using the olfactory sense of nematodes (Non-patent Document 1 and Patent Document 1).

  As described above, conventionally, in order to evaluate the chemotaxis behavior through the olfactory sense of the nematode, the test substance and the nematode are placed on the petri dish while being separated from each other by a certain distance, and the chemotaxis behavior is nematode on the petri dish. The arrangement of was observed. The nematode's chemotaxis behavior fluctuates and does not necessarily move at the shortest distance with respect to the test substance, and may show chemotaxis while detouring. This is because, in order to improve, it was considered that a method of evaluating only nematodes that moved a certain distance over a certain distance was necessary as in general behavior evaluation. In addition, since the odor can be transmitted remotely, it is possible to test by placing the test sample and the nematode at a large distance, and the test sample and the nematode are separated from each other by a large distance. Therefore, it was believed that the scent was suitable for an evaluation system that greatly separated the distance between the test sample and the nematode. Therefore, it has been assumed that a general behavior evaluation system is used in the evaluation of the chemotaxis behavior via the sense of smell.

  By the way, in order to move a nematode to a long distance of a certain length or more, it is necessary to allow the nematode to move for a long time of a certain length or more. On the other hand, if the olfactory sensation is stimulated over a certain period of time, the olfactory sensation of the nematode causes sensitization and begins to move randomly, making it impossible to evaluate the chemotaxis behavior by the olfaction. Therefore, when evaluating the chemotaxis behavior through the olfactory sense of the nematode, a nematicide (for example, a powerful drug such as sodium azide) is used to stop the movement of the nematode exhibiting the chemotaxis behavior. (Non-patent Document 1 and Patent Document 1).

WO2015 / 88039

Hirotsu T. et al., PLOS ONE, 10 (3): e0118699, 2015

  The present invention provides a method for evaluating chemotaxis behavior with respect to odor substances based on the olfactory sense of nematodes, and a petri dish used for the evaluation method.

  In the case of chemotaxis assessment via olfactory sensation, unlike the general behavior assessment, the present inventors surprisingly have a higher accuracy than conventional methods when the test sample and the nematode are placed at a short distance. We found that sexual behavior can be evaluated. Based on this finding, the present inventors have also invented a petri dish suitable for evaluating chemotaxis behavior via the olfactory sense of nematodes. The present invention is based on these findings and inventions.

In the present invention, the following inventions are provided.
(1) A petri dish having a substantially square bottom having a longitudinal direction of 3 to 6 cm and a lateral direction of 1 to 3 cm, wherein the bottom is a first in the longitudinal direction from one end to another end. A petri dish that is divided into at least three regions of a region, a second region, and a third region, and the second region is disposed at a boundary between the first region and the third region.
(2) The petri dish according to the above (1), wherein the division is an uneven shape or printing on the upper surface of the bottom surface, or an uneven shape or printing on the lower surface.
(3) The petri dish according to (1) or (2), wherein the petri dish has a display for distinguishing the first area from the third area.
(4) A multi-plate in which a plurality of petri dishes according to any one of (1) to (3) are connected.
(5) A method for evaluating the chemotaxis behavior of a nematode with respect to the odor of a test sample,
a) providing a petri dish in which a test sample is arranged, wherein a nematode is arranged in a region or part of the bottom surface 1 cm to 3 cm away from the test sample;
b) observing the arrangement on the bottom surface of the nematode 3 to 15 minutes after the test sample and the nematode are arranged in the petri dish;
c) assessing from the observed nematode placement whether the test sample has been attracted or repelled.
(6) The method according to (5) above, wherein the test sample is a sample obtained from a subject suspected of being a cancer patient.
(7) The method according to (6) above, wherein the test sample is urine obtained from a subject suspected of being a cancer patient.
(8) When the nematode exhibits an attracting action on the test sample in c), it is determined that the test sample contains an attractant derived from cancer, or the nematode is tested The method according to (6) or (7) above, further comprising determining that the test sample does not contain an attractant derived from cancer when the sample exhibits repellent behavior.
(9) The method according to any one of (5) to (8) above, wherein no nematicide is used.
(10) In (a), the petri dish according to any one of the above (1) to (3) or any one petri dish included in the multiplate according to (4) is used. ) Any one of the methods.

  According to the present invention, the accuracy of evaluation of chemotaxis behavior based on the olfactory sense of nematodes is improved, and the advantage that measurement time is greatly shortened and the use of deleterious substances such as sodium azide can be eliminated. There are advantages.

FIG. 1A shows a plan view of a petri dish 100 according to the first embodiment of the present invention. The two-dot chain line in the figure indicates the boundary line of the bottom segmentation pattern. FIG. 1B shows a front view of the petri dish 100 of the first embodiment of the present invention. FIG. 1C shows a plan view of a modification 100A of the first embodiment of the present invention. The two-dot chain line in the figure indicates the boundary line of the bottom segmentation pattern. FIG. 1D shows a front view of the petri dish 100 of the first embodiment of the present invention with the lid 150 covered. FIG. 1E shows a plan view of the lid 150. FIG. 1F shows a plan view of a modification 150 </ b> A of the lid 150. FIG. 1G shows a cross-sectional view taken along a straight line passing through 151 in one aspect of the modification 150A. FIG. 1H is a cross-sectional view taken along a straight line passing through 151 in one aspect of the modification 150A. FIG. 2A shows a plan view of a petri dish 200 according to the second embodiment of the present invention. The two-dot chain line in the figure indicates the boundary line of the bottom segmentation pattern. FIG. 2B shows a plan view of a modified example 200A of the second embodiment of the present invention. The two-dot chain line in the figure indicates the boundary line of the bottom segmentation pattern. FIG. 3A shows a plan view of an example 500 of the multiplate of the present invention. FIG. 3B shows a plan view of an example 500A of the multiplate of the present invention. FIG. 3C shows a front view of an example multi-plate 500 with a lid 600 of the present invention. FIG. 4 shows a photograph of a 5 cm × 2 cm square petri dish created in this example. In the picture, nematodes are shown by white dots. FIG. 5 is a diagram showing that when the petri dish of the present invention is used, the attracting action to the attracting substance (isoamyl alcohol) and the repelling action to the repellent substance (nonanone) can be measured with high sensitivity. FIG. 6 is a diagram showing that the attracting behavior of cancer patients to urine and the avoidance behavior of healthy subjects to urine can be clearly evaluated in 5 minutes and 10 minutes using the petri dish of the present invention. FIG. 7 is a diagram showing the results of chemotaxis behavior evaluation on a urine sample based on the conventional method. FIG. 8 is a diagram showing the results of chemotaxis behavior evaluation using urine (1/200 dilution) obtained from various cancer patients and healthy individuals. FIG. 9 is a diagram showing the results of evaluation of chemotaxis behavior using a 3 cm × 1 cm square petri dish.

Detailed Description of the Invention

As used herein, “nematode” means Caenorhabditis elegans . Nematodes are popular organisms that are widely bred and studied all over the world as model organisms in biological research, and are characterized by easy breeding and excellent olfaction.

  In this specification, “cancer” refers to cancers such as stomach cancer, colorectal cancer, esophageal cancer, pancreatic cancer, prostate cancer, bile duct cancer, lung cancer, blood cancer, leukemia, and lymphoma. means.

  As used herein, “subject” means a mammal, eg, a human.

  As used herein, “running behavior” means attraction or repellent behavior. Attracting behavior means behavior that shortens the physical distance from a certain substance, and repelling behavior means behavior that widens the physical distance from a certain substance. Substances that induce attraction behavior are called attractants, and substances that induce repellent behavior are called repellent substances.

C. elegans has the property of attracting attractants by olfaction and repelling repellents. The act of attracting an attracting substance is called attracting action, and the action of avoiding repellent substances is called repelling action. In addition, attracting behavior and repelling behavior are collectively referred to as chemotaxis behavior.

  ADVANTAGE OF THE INVENTION According to this invention, the petri dish suitable for evaluating the chemotaxis behavior via the olfactory sense of a nematode is provided. The petri dish and the lid can be made of a resin such as plastic, and can be made of a transparent resin. Hereinafter, the present invention will be described for each embodiment.

First Embodiment A first embodiment of the present invention will be described with reference to FIGS. 1A and 1B.

  The petri dish 100 according to the present embodiment is a petri dish having a substantially square bottom as shown in FIG. 1A. The bottom surface of the petri dish 100 has at least two separation lines 25 and 35, and divides the region of the bottom surface into at least three regions in the longitudinal direction from one end to another end. As a result, the bottom surface is divided into at least three regions of the first region 20, the second region 30, and the third region 40. In particular, the second region 30 is divided into the first region 20 and the third region. It is arranged at the boundary with the region 40.

  The petri dish 100 of the first embodiment can be a petri dish having a substantially square bottom surface, and the length of the bottom surface in the longitudinal direction is 3 cm to 6 cm and the length in the lateral direction is in the range of 1 cm to 3 cm. obtain. The length in the longitudinal direction may preferably be 4 cm to 6 cm, preferably 4.5 cm to 5.5 cm, preferably 5 cm. The length in the transverse direction is preferably 1.5 cm to 2.5 cm, preferably 2 cm. The petri dish 100 of the first embodiment has a substantially square bottom surface, the length of the bottom surface in the longitudinal direction is 4 cm to 6 cm, preferably 4.5 cm to 5.5 cm, and The length of the bottom surface in the transverse direction may be 1.5 cm to 2.5 cm. The petri dish 100 of the first embodiment may have a height of 1 cm to 3 cm.

  The second region 30 may be arranged so as to separate the first region 20 and the third region 40. As shown in FIG. 1A, the second region 30 can be preferably arranged such that the first region 20 and the third region 40 face each other with the second region 30 in between.

  The second region 30 may be 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the total area of the bottom surface.

  The segmentation may be performed in any manner as long as a human or machine can distinguish each of the at least three regions. The division is not particularly limited, and can be performed, for example, by printing on the bottom surface or an uneven shape on the bottom surface. The printing on the bottom surface or the concavo-convex shape on the bottom surface may be made on the upper surface of the bottom surface or on the lower surface. For example, as shown in FIG. 1A, the division can be performed by dividing lines that are boundary lines such as the dividing line 25 and the dividing line 35. The separator line does not necessarily have to be a straight line, but can preferably be a straight line. The dividing line does not necessarily have an uneven shape.

  The first area 20 and the third area 40 may each have a display so as to distinguish which is the first area and which is the third area. The display may be a symbol such as ○ or ×, a character, or some mark. In order to distinguish the first region and the third region, the petri dish 100 may have a tag 90. The first region and the third region may have different areas, and may be distinguished from each other by using the different areas.

  The tag 90 can be used to distinguish a plurality of petri dishes 100, for example. Although not particularly limited, the tag 90 may be provided with a display such as a barcode or a two-dimensional barcode, and the display may include management information of each petri dish 100.

  As shown in FIG. 1B, the petri dish 100 has a side wall at the edge of the bottom surface and has a container shape.

Method of using petri dish of first embodiment The petri dish 100 of the first embodiment is suitable for evaluating the chemotaxis behavior through the olfactory sense of nematodes. Below, the petri dish 100 of 1st embodiment is used and the method of evaluating the chemotaxis action via the olfactory sense of a nematode is demonstrated.

The petri dish 100 of the first embodiment can add a solid medium for nematodes inside. The solid medium is, for example, an agar medium. Those skilled in the art can prepare a medium such as a medium suitable for the survival of nematodes and add it to the petri dish.
The nematode 60 can be sown in the second region 30. The nematode may be sown on the entire surface of the second region 30 or may be sown only on a part of the second region 30. A test sample is the 1st field 20 or the 3rd field 40, for example, can be arranged in the position about 1 cm-3 cm away from the field where a nematode is seeded.

  When a nematode is seeded on the petri dish 100 of the first embodiment in which the test sample is arranged, the nematode exhibits an attracting action on the test sample when the test sample is an attracting substance, and the test sample However, when the test sample is a repellent substance, the test sample exhibits repellent behavior and moves to a location away from the test sample. Therefore, by observing the place where the nematode has moved (from the observed arrangement of the nematode), it is possible to determine whether the test sample is an attractant or a repellent. The observation can be performed, for example, visually or with a microscope. The observation may be performed by imaging the arrangement information of the nematode and using the image.

Modified Example of First Embodiment A modified example 100A of the first embodiment will be described below with reference to FIG. 1C. The modification 100A of the first embodiment is shown in FIGS. 1A and 1B with respect to other components and how to use them, except that the division patterns (segmentation shapes) of the first, second, and third regions on the bottom surface are different. This is the same as the petri dish 100 of the first embodiment. Therefore, hereinafter, only the segmentation pattern will be described, and the same parts as those in the petri dish 100 of the first embodiment will be denoted by the same reference numerals and description thereof will be omitted.

  In the modification 100A of the first embodiment, the second region 30A is the same as the petri dish 100 of the first embodiment in that the second region 30A is arranged at the boundary between the first region 20A and the third region 40A. is there. However, the modification 100A of the first embodiment differs from the petri dish 100 of the first embodiment in that the first region 20A and the third region 40A are in contact with each other.

  The second region 30A may have various shapes as long as it is a closed shape. In FIG. 1C, it is divided into a substantially circular shape, but is not limited to a circular shape, and may be a substantially polygonal shape (for example, a substantially rectangular shape, a substantially hexagonal shape, or a substantially octagonal shape).

Cover 150 for petri dish 100 of the first embodiment and its modification 100A
The petri dish 100 of the first embodiment and the modification 100A thereof may include a lid 150. Hereinafter, the lid 150 will be described with reference to FIGS. 1D and 1E.

  As shown in FIGS. 1D and 1E, the lid 150 is a lid that covers the upper surface of the petri dish 100. The shape of the lid 150 is not limited as long as it is a lid that covers the upper surface of the petri dish 100. The lid 150 is preferably a lid that covers the upper surface of the petri dish 100 and that can block the air flow between the inside and the outside of the petri dish 100 from the viewpoint of improving the accuracy of behavioral evaluation based on the sense of smell.

  The tag 90 may be provided on the lid 150.

How to use the lid 150 The lid 150 can be placed on a petri dish after the test sample and nematode are placed. Thereby, it may be possible to prevent the influence of outside air and effectively confine the odor inside the petri dish.

Modification 150A of lid 150
A modification 150A of the lid 150 will be described below with reference to FIGS. 1D and 1F. As shown in FIG. 1F, the lid 150 </ b> A is the same as the lid 150 except that the lid 150 </ b> A includes a test sample arrangement site 151. Therefore, in the following, only the test sample arrangement site 151 will be described, and the same parts as those of the lid 150 are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 1F, the lid 150A includes a test sample arrangement site 151 on a surface facing the petri dish bottom surface when the lid is closed. The test sample placement site 151 is formed in a region 1 to 3 cm away from the longitudinal center of the lid 150A. In FIG. 1F, the test sample arrangement site 151 is illustrated in a circular shape, but is not limited thereto, and the test sample arrangement site 151 may have a substantially circular shape, a substantially linear shape, or a substantially square shape.

  In one embodiment, the test sample placement site 151 has a boundary 153 between the test sample placement site 151 and the other region 152. The boundary 153 can be formed by printing or an uneven shape.

  In one embodiment, the test sample placement site 151 and the other region 152 have a step at the boundary 153. In an aspect, as shown in FIG. 1G, the test sample arrangement site 151 may protrude toward the bottom of the petri dish when the lid is closed, rather than the other region 152. As shown in 1H, the test sample arrangement site 151 may be recessed with respect to the petri dish bottom surface when the lid is closed rather than the region 152. As shown in FIG. 1G, when the test sample arrangement site 151 protrudes from the other region 152 toward the petri dish bottom surface when the lid is closed, the material of the test sample arrangement site 151 is shown. May be the same as or different from the material of the region 152 and the lid 150A.

In one embodiment, in the lid 150A, the test sample placement site 151 has a hydrophilicity different from that of the other region 152 on the surface facing the petri dish bottom surface when the lid is closed.
In one embodiment, the test sample placement site 151 has a hydrophilic surface, and the other region 152 has a hydrophobic surface. Thereby, the aqueous test sample can be accurately placed on the test sample placement portion 151. In one embodiment, the test sample placement site 151 has a hydrophobic surface, and the other region 152 has a hydrophilic surface. As a result, the oily test sample can be accurately placed on the test sample placement site 151.

How to Use Lid Modification 150A The lid 150A can be placed on a petri dish after the test sample and nematode are placed. Thereby, it may be possible to prevent the influence of outside air and effectively confine the odor inside the petri dish.

  When the lid 150A is used, a test sample can be placed on the test sample placement site 151 of the lid 150A, and a nematode can be placed on the petri dish. In the evaluation of chemotaxis behavior via olfaction, the test sample and the nematode may be spaced apart from each other, and even in this case, the odor can diffuse in the air and reach the nematode . In addition, since the test sample is placed in the test sample placement site 151 of the lid 150A and the worm component reaches the nematode, it is suitable for evaluation of a part of the chemotaxis behavior through the olfaction. . In particular, when the test sample has strong chemotaxis, it is suitable for evaluating the chemotaxis behavior via the olfactory sense of the odor component contained in the test sample.

  The test sample is placed on the lid 150A by introducing the test sample from under the lid with the surface facing the petri dish bottom surface facing downward when the lid of the lid 150A is closed. it can. With respect to the arrangement of the test sample on the lid 150A, the test sample is introduced from under the lid with the surface facing the petri dish bottom surface facing upward when the lid of the lid 150A is closed, and then inverted upside down. Can be done.

Second Embodiment A petri dish 200 according to the second embodiment will be described with reference to FIG. 2A. The petri dish 200 of the second embodiment is the same as the petri dish 100 of the first embodiment shown in FIGS. 1A and 1B, except that the bottom sectioning pattern is different. Accordingly, the same parts as those in the petri dish 100 of the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 2A, in the petri dish 200 of the second embodiment, the first region 210, the second region 220, and the third region 30 have a bottom surface in the longitudinal direction from one end to another end. , And the fourth region 40 is divided into at least four regions. The difference from the petri dish 100 of the first embodiment is that the first region 10 is further divided into two parts, a first region 210 and a second region 220 in the petri dish 200 of the second embodiment. It is a point. As shown in FIG. 2A, in the petri dish 200 of the second embodiment, the first region 210 and the second region 220 are separated by a line 215.

  In the petri dish 200 of the second embodiment, the test sample can be arranged on the first region 210 and / or the line 215, for example. Since the arrangement region of the test sample is indicated by the division, the distance between the test sample and the nematode 60 (applied to part or all of the third region 30) is made constant between different tests. Easy to keep. The first region 210 and the third region 30 may have a distance of about 1 cm to 3 cm, about 1 cm to 2 cm, or about 1.5 cm to 2.5 cm, or about 1.5 cm to 2 cm, for example.

Modification 200A of the second embodiment
A modification 200A of the second embodiment will be described below with reference to FIG. 2B. The modification 200A of the second embodiment is the same as the petri dish 200 of the second embodiment shown in FIGS. 2A and 1B, except that the division pattern (segmentation shape) on the bottom surface is different. Therefore, hereinafter, only the division pattern will be described, and the same portions as those in the petri dish 200 of the second embodiment are denoted by the same reference numerals and description thereof is omitted.

  In the modification 200A of the second embodiment, the third region 30A is the same as the petri dish 100 of the first embodiment in that the third region 30A is arranged at the boundary between the second region 220 and the fourth region 40A. is there. However, the modification 200A of the second embodiment differs from the petri dish 200 of the second embodiment in that the second region 220 and the fourth region 40A are in contact with each other.

  The third region 30A may have various shapes as long as it is a closed shape. In FIG. 2B, it is divided into a substantially circular shape, but is not limited to a circular shape, and may be a substantially polygonal shape (for example, a substantially quadrangular shape, a substantially hexagonal shape, or a substantially octagonal shape).

  In the above, the petri dish of the present invention has been described for each embodiment, but the bottom surface of these petri dishes may be further finely divided. In the same manner as described above, the classification may be performed in any manner as long as a human or a machine can distinguish each of the at least three regions.

Hereinafter, the multi-panel of the present invention will be described with reference to FIG.

  As shown in FIG. 3A, the multi-panel 500 of the present invention can be an assembly in which the petri dishes 100 of the first embodiment of the present invention are arranged vertically or horizontally. As shown in FIG. 3B, the multi-panel 500A of the present invention may be an assembly in which the petri dishes 100 of the first embodiment of the present invention are arranged vertically and horizontally. In the multi-panel 500 or 500A of the present invention, although not particularly limited, for example, the petri dish 100 of the first embodiment of the present invention is 2 to 1000, 4 to 700, 6 to 600, 8 to 500. Or 10 to 300 pieces may be arranged.

  Moreover, in the multi-panel, the petri dishes 100 of the first embodiment adjacent to each other may be directly connected or may be connected via a spacer.

  3A and 3B show an example in which the petri dish 100 of the first embodiment is used as the petri dish, the petri dish may be the petri dish 200 of the second embodiment, or a modification 100A of the first embodiment. Or it is good also as modification 200A of 2nd embodiment.

  The multi-panel 500 or 500A of the present invention may further include a lid 600. With the side walls of the lid 600 and each petri dish, it can be expected to more effectively prevent the test sample in each petri dish from leaking to the adjacent petri dish. The lid 600 may include a test sample placement site 151 as with the lid 150A. Since the usage of the lid 600 is as described in the lid 150 and the lid 150A, the description is omitted.

Evaluation method of chemotaxis behavior via olfactory sense of nematode according to the present invention Hereinafter, a method of evaluating chemotaxis behavior via olfactory sense of nematode will be described.

According to the present invention,
A method for evaluating the chemotaxis behavior of a nematode with respect to the odor of a test sample,
a) providing a petri dish in which a test sample is arranged, wherein a nematode is arranged in a region or part of the bottom surface 1 cm to 3 cm away from the test sample;
b) observing the nematode placement 3-15 minutes after sowing;
c) assessing from the observed nematode placement whether the test sample has been attracted or repelled. A method is provided.

  Hereinafter, a) to c) will be described for each process.

a) Providing a petri dish in which a test sample is arranged, wherein the nematode is arranged in a region or part of the bottom surface 1 cm to 3 cm away from the test sample

  In a), a petri dish on which the test sample is arranged is used. The petri dish may not have a lid, but preferably a petri dish having a lid can be used. The petri dish can be added with a solid medium for nematodes. As a solid medium for nematodes, a solid medium for culturing or growing nematodes can be used. As the solid medium, a known medium such as an agar medium can be used. The test sample is not particularly limited, but may preferably be disposed on the petri dish bottom surface, and particularly disposed near the periphery of the petri dish bottom surface. Alternatively, the test sample is not particularly limited, but may preferably be disposed on a surface (or a lower surface) facing the bottom surface of the petri dish on the lid of the petri dish, and particularly in the vicinity of the peripheral portion of the lower surface of the lid. By placing the test sample on the bottom of the petri dish or on the periphery of the lid (ie, near the side wall), it is easy to ensure a certain distance from the test sample when the nematode is placed in the center of the bottom of the petri dish It becomes.

  The distance from the site where the test sample is placed to the site where the nematode is seeded is about 1 cm to 3 cm, about 1 to 2.5 cm, about 1.5 to 2 cm, about 1.5 cm to 2.5 cm, Or it can be about 2 cm.

  The number of nematodes to be seeded can be, for example, about 10 to 500, for example, about 100 to 500.

  Any sample can be used as the test sample. Examples of the test sample include a sample of a subject (eg, human) suspected of having cancer. The test sample can be, for example, a cell suspected of being a cancer cell, a tissue suspected of being a cancer tissue, or an extract or lysate thereof, or suffering from cancer. A body fluid sample of a subject suspected of being, for example, blood (eg, plasma) or urine.

  The chemotaxis evaluation via the olfactory sense of the nematode can be performed, for example, as described in WO2015 / 088039.

  In the conventional method, in the petri dish provided in a), a nematicide (for example, sodium azide) is placed in the region near the test sample and in the region opposite to the test sample when viewed from the position where the nematode is placed. Although it was necessary to arrange, nematocide does not need to be used in the method of the present invention.

The petri dish provided with a) in which the test sample is arranged and the nematode is arranged in a region or a part 1 cm to 3 cm away from the test sample is a line after the test sample is arranged. It may be obtained by arranging insects or by arranging a test sample after arranging nematodes. In the case where the test sample is arranged after the nematode is arranged, the test sample may be arranged after the nematode is arranged and cultured and increased at the place to be arranged.
As time elapses after the nematode is placed, the possibility that the nematode spreads widely on the bottom surface increases. Therefore, it is desirable to start observation immediately after placing the nematode. Therefore, it is preferable to place the test sample in a predetermined position of the petri dish, and then place the nematode in the predetermined position, and immediately start counting for 3 to 15 minutes in b) described later. . When the test sample is placed after the nematode is placed, it is desirable to place the test sample within 3 minutes, more preferably within 1 minute after placing the nematode.
In the petri dish provided in a), the nematode is preferably placed in an area that is 0.5 cm or more away from any side wall of the petri dish or its lid (however, , Does not prevent it from being placed in an area within 0.5 cm from the side wall).

b) Observing the arrangement of the nematode on the bottom surface of the nematode 3 to 15 minutes after the test sample and the nematode are placed on the petri dish. In b), when the nematode places the test sample on the petri dish (or The nematode exhibits chemotaxis behavior as soon as it is placed in the petri dish where the test sample is placed. Wait about 3 to 15 minutes, preferably 3 to 12 minutes, more preferably 5 to 10 minutes, for example 5 minutes, 7.5 minutes or 10 minutes, and the nematode will have enough distance to evaluate chemotaxis behavior Can only move. Therefore, about 3 to 15 minutes, preferably 3 to 12 minutes, more preferably 5 to 10 minutes, for example, 5 minutes or 7.5 minutes after the later of the test sample or nematode is arranged. Alternatively, after 10 minutes, the arrangement of the nematode on the bottom surface can be observed, whereby the chemotaxis behavior of the nematode can be evaluated. By setting this time to 3 minutes or longer, the chemotaxis behavior of the nematode is increased to such an extent that it can be sufficiently visualized, so that the evaluation accuracy of the chemotaxis behavior is improved. Moreover, when this time is 15 minutes or less, the olfactory sense of the nematode is sensitized, and the possibility that the nematode begins to move randomly on the petri dish is reduced, and the accuracy of the chemotaxis behavior is improved. When the nematode is placed on the bottom of the petri dish and the test sample is placed on the lid, “when the test sample and the nematode are placed on the petri dish” means that the lid is placed on the petri dish. Is the time.

c) Evaluating from the observed nematode arrangement whether the test sample showed an attracting action or a repellent action. c) From the observed nematode arrangement, the nematode was applied to the test sample. On the other hand, it was possible to evaluate whether or not it showed an attracting behavior or a repelling behavior but neither. In c), if the ratio of nematodes approaching the test sample is higher than the ratio of nematodes moving away from the test sample, evaluate that the nematodes have attracted the test sample. Can do. In c), if the ratio of the nematode approaching the test sample is lower than the ratio of the nematode moving away from the test sample, it is evaluated that the nematode showed repellent behavior with respect to the test sample. be able to. In c), when the ratio of nematodes approaching the test sample is equal to the ratio of nematodes moving away from the test sample, it may be evaluated that the nematode did not attract or repel behavior.

  Further, in c), for example, a chemotaxis index may be obtained by the following formula to evaluate whether the nematode has shown an attracting action or a repelling action.

Taxis index (taxis index) =
{(Number of nematodes in the area indicated by ○) − (Number of nematodes in the area indicated by ×)} / Total number of nematodes

  The chemotaxis index takes a numerical value between −1 and +1, and takes a positive value when showing an attracting action, and takes a negative value when showing an avoidance action. And it can be interpreted that the greater the absolute value of the numerical value, the stronger the chemotaxis behavior.

  c) further comprises evaluating whether the test sample is an attractant, a repellent, or neither based on the chemotaxis behavior exhibited by the nematode on the test sample. But you can.

  In WO2015 / 088039, the present inventors show that nematodes exhibit attracting behavior against body fluids (for example, urine) of cancer patients and repellent behaviors for body fluids (for example, urine) of healthy individuals. It is clear to show. This is understood to be based on the fact that cancer-specific factors are released from the cancer cells and cancer tissues of the cancer patient into the body fluid of the cancer patient, which induces the nematode attracting behavior.

  Therefore, in the present invention, the test sample can be a cell suspected of being a cancer cell, a tissue suspected of being a cancer tissue, or an extract or lysate thereof, or cancer. When using a body fluid sample from a subject suspected of suffering from (for example, a human), for example, blood (for example, plasma) or urine, the sample may be cancer based on the chemotaxis behavior of the nematode. It can be determined whether or not an attracting substance derived from is included. The result of this determination is useful as one of basic information when the doctor diagnoses cancer, and assists the doctor in diagnosing cancer.

  Therefore, in another aspect of the present invention, there is provided a method for determining whether or not a test sample contains an attractant derived from cancer, comprising the above a) to c), and A method is provided that further comprises, based on the chemotaxis behavior, determining whether the test sample contains an attractant derived from cancer.

  In another aspect of the present invention, there is provided a method for diagnosing whether or not a subject has cancer, including obtaining a test sample from the subject, and a) to c) above, and d) When the nematode exhibits an attracting action on the test sample, it is evaluated that the subject is suffering from cancer, or when the nematode shows repellent action on the test sample Provides a method comprising assessing that the subject is free of cancer.

  The method for evaluating the chemotaxis behavior of nematodes with respect to the odor of the test sample of the present invention, the method for determining whether or not the test sample contains an attractant derived from cancer, and the subject has cancer In the method of diagnosing whether or not the disease is affected, the chemotaxis behavior via the olfactory sense of the nematode is evaluated, but a nematicide (for example, sodium azide) for stopping the movement of the nematode is evaluated. There is no need to use (drug). Because powerful drugs such as sodium azide are required to be strictly managed for storage and use (there are such restrictions), the method of the present invention that does not use these has one practical restriction. This is beneficial in that it can be removed.

  The method for evaluating the chemotaxis behavior of nematodes with respect to the odor of the test sample of the present invention, the method for determining whether or not the test sample contains an attractant derived from cancer, and the subject has cancer In the method of diagnosing whether or not the patient is afflicted, the petri dish of the present invention described above can be used, and a multiplate can also be used. The method of using the petri dish or multiplate of the present invention in the method of the present invention is as described above.

Example 1: Construction of an evaluation system for chemotaxis behavior using the sense of smell of nematodes In this example, an attempt was made to construct a new evaluation system for chemotaxis behavior of nematodes.

First, a square petri dish having a bottom surface of 5 cm × 2 cm was produced (see FIGS. 1A and B). A photograph of the petri dish actually produced is as shown in FIG. The nematode is sown in the area divided by two lines in the central part of the square petri dish, and the attracting substance or repellent substance is applied to the left end area, so that the attracting action or repelling of the nematode attractant is performed. Observed behaviors against the substance were observed. In this example, isoamyl alcohol was used as the attracting substance, and nonanone was used as the repellent substance. Specifically, isoamyl alcohol was diluted 1000 times with water and 1 μL was applied to the left end region. Nonanon was applied to the left end region with 1 μL of the stock solution. As nematodes, wild type C. elegans (10-50 animals / experiment) was used.

  The chemotaxis index described below for attracting and repelling behavior of nematodes was obtained, and the sensitivity of the constructed evaluation system was confirmed. Specifically, nematodes that have moved to the area between the left end area where the attractant or repellent substance is applied and the area where the nematode is seeded in the center in the longitudinal direction (area indicated by ○ in FIG. 4). And the difference in the number of nematode individuals that moved to the opposite area (area indicated by x in FIG. 4), and divided by the total number of nematode individuals, the chemotaxis index was obtained.

Taxis index (taxis index) =
{(Number of nematodes in the area indicated by ○) − (Number of nematodes in the area indicated by ×)} / Total number of nematodes

  The experiment was repeated 5 times, and the average value and SEM of the chemotaxis index were determined.

The result was as shown in FIG. FIG. 5 shows the chemotaxis index and standard error (SEM) measured only 10 minutes after nematode seeding. As shown in FIG. 5, the chemotaxis index exceeded 0.8 with respect to isoamyl alcohol as an attractant in only 10 minutes of measurement. This result shows that 90% or more of the nematodes were able to be measured as exhibiting attracting behavior against the attracting substance during this short period.
In addition, the chemotaxis index was less than −0.8 for nonanone, which is a repellent substance. This result shows that more than 90% of nematodes could be measured as having shown repellent behavior against repellent substances during this short period.

  In evaluation of chemotaxis behavior using olfaction, evaluation may be performed for the purpose of determining whether a test substance is an attractant, a repellent substance, or neither. Using the evaluation system of the present invention, it has been found that it is possible to determine whether a test substance is an attractant or a repellent substance with a surprising sensitivity in a short measurement time.

  Conventionally, chemotaxis behavior based on olfaction has been evaluated using a 9 cm round petri dish. In this case, the movable area of the nematode is wide, and the olfaction is sensitized during movement and runs immediately. It is known to show no sexual behavior and move around randomly. In order to prevent the nematodes that have moved once from moving more randomly, a countermeasure is taken such that a poison such as sodium azide is applied to the destination area.

  On the other hand, in the evaluation system of the present invention, it was possible to measure the chemotaxis behavior based on the olfactory sense of nematodes without using any toxic substances. In the present invention, conventionally, the use of sodium azide, which was essential for observation of chemotaxis behavior based on olfaction, was not necessary.

  Moreover, in the conventional evaluation system, in order to evaluate the chemotaxis behavior of a nematode, it was necessary to make the nematode act over a period of 30 minutes or more. On the other hand, in the evaluation system of the present invention, it is possible not only to make the nematode act for a fraction of the conventional time but also to evaluate the chemotaxis with a higher sensitivity than in the past. I was able to.

  In this example, when the test sample and the nematode are arranged closer than before, highly accurate evaluation can be performed. The reason why the evaluation of the present invention is highly accurate in the evaluation of chemotaxis is that the behavioral ability of nematodes (for example, nutritional state, environmental temperature, culture conditions, and growth stage) The influence of the influence (which is influenced by the above) was affected as noise that could not be ignored (although this influence was not known before the present invention), the evaluation method of the present invention was able to minimize this influence. It is believed that there is.

Example 2: Evaluation of chemotaxis behavior via olfactory sense of nematode using urine of cancer patient Wild type nematode shows attracting behavior in urine of cancer patient and repellent behavior in urine of healthy subject It is clear that In this example, chemotaxis behavior with respect to urine derived from cancer patients was evaluated using the new evaluation system constructed in Example 1.

  The chemotaxis behavior was evaluated in the same manner as in Example 1 except that the attractant or repellent substance was urine of a cancer patient (1/20 dilution) or urine of a healthy person (1/20 dilution). From the results of Example 1, the measurement time was 5 minutes or 10 minutes after seeding of the nematode. The result was as shown in FIG.

  As shown in FIG. 6, despite the high dilution factor, it was possible to clearly observe the attracting behavior of cancer patients to urine and the avoidance behavior of healthy subjects to urine even after 5 minutes. The result was similar even after 10 minutes. In addition, the chemotaxis index was 0.5 after 5 minutes and 0.6 after 10 minutes with respect to urine samples of cancer patients, and 75% and 80% of nematodes showed attracting behavior, respectively. It is a good numerical value that means.

  On the other hand, when an experiment similar to Example 1 of WO2015 / 088039 was performed using a 9 cm dish, chemotaxis was performed using urine diluted to 1/10 and using sodium azide for a measurement time of 30 minutes. The index remained as low as 0.3 (see FIG. 7). That is, in the 9 cm dish system, only a low value of the chemotaxis index was observed even though the urine concentration was twice as high.

  Next, in the above evaluation method of this example, the nematode run was performed under the same conditions as the experiment for obtaining the results of FIG. 6 except that the urine concentration was 1/200 dilution and the measurement time was 10 minutes. Sexual behavior was observed. As urine, urine obtained from different cancer patients was used. The result was as shown in FIG.

  As shown in FIG. 8, nematodes showed attracting behavior for urine from any cancer patient, and nematodes showed repellent behavior for urine of any healthy subject. The chemotaxis index was 0.1 to 0.2 even though urine was diluted at high magnification, and the high sensitivity of this evaluation method could be demonstrated. On the other hand, the chemotaxis index was -0.15 to -0.2 for urine of healthy subjects, and the high sensitivity of this evaluation method could be re-verified.

Example 3: Evaluation of chemotaxis behavior of nematodes using 3 cm x 1 cm square petri dish In this example, the chemotaxis behavior of nematodes was evaluated using a smaller square petri dish than in Example 1.

  Except for the use of a 3 cm × 1 cm square petri dish, the chemotaxis behavior of the nematode was observed under the same conditions as in the experiment for obtaining the results of FIG. Evaluation was performed 5 minutes and 10 minutes after nematode seeding. The result was as shown in FIG.

  As shown in FIG. 9, after 5 minutes, the urine of the cancer patient showed an attracting action, and the healthy person showed a repellent action. On the other hand, after 10 minutes, the urine of the cancer patient showed an attracting action, but the urine of the healthy person showed no repellent action.

  This result shows that it is possible to evaluate the chemotaxis behavior of nematodes even in a small square petri dish, but on the other hand, it can be said that the sensitivity may be reduced. .

  The cause of the decrease in sensitivity was examined. After 10 minutes, the nematode was climbing up the side wall of the petri dish, or the nematode dived into the culture medium from the side wall, so that the countable nematode population was It became clear that the decline was a factor. That is, it was revealed that the place where the nematodes are arranged should be at least 0.5 cm away from the side wall of the petri dish.

Claims (14)

A nematode chemotaxis behavior evaluation system having a substantially square bottom having a longitudinal direction of 3 to 6 cm and a lateral direction of 1 to 3 cm and a side wall at an edge thereof. Is divided into at least three regions, ie, a first region, a second region, and a third region, toward the other end, and the second region is arranged at the boundary between the first region and the third region Nematode chemotaxis behavior evaluation system (except for those whose entire bottom surface is inclined) . Longitudinal direction 3～6Cm, a run of behavioral assessment system nematode transverse direction having sidewalls on the bottom and its edge of the substantially square shape which is 1 to 3 cm, bottom surface, a longitudinal direction by partition line It is divided into at least three regions of a first region, a second region, and a third region from one end to another end, and the second region is divided between the first region and the third region. A nematode chemotaxis behavior evaluation system that is arranged at the boundary and does not prevent the nematode from moving between regions when a solid medium is introduced at the time of nematode chemotaxis behavior evaluation . Separators is a concavo-convex shape or printed for irregularities or printing or the lower surface, with respect to the upper surface of the bottom run of behavioral assessment system nematode according to claim 2. The nematode chemotaxis behavior evaluation system according to any one of claims 1 to 3 , comprising a display for distinguishing between the first region and the third region. The chemotactic behavior evaluation system for nematodes according to any one of claims 1 to 4, wherein the first region and the third region are in contact with each other, and the second region is in a closed shape. The nematode chemotaxis behavior evaluation system according to any one of claims 1 to 5, further comprising a lid having a test sample arrangement site on a surface facing the bottom surface when the lid is closed. The nematode chemotaxis behavior evaluation system according to any one of claims 1 to 6, wherein a nematode is disposed on the second region. The chemotactic behavior evaluation system for nematodes according to any one of claims 1 to 7, wherein the test sample is arranged in the first region or the third region. A method for evaluating the chemotaxis behavior of a nematode with respect to the odor of a test sample,
a) providing a petri dish in which a test sample is arranged, wherein a nematode is arranged in a region or part of the bottom surface 1 cm to 3 cm away from the test sample;
b) observing the arrangement on the bottom surface of the nematode 3 to 15 minutes after the test sample and the nematode are arranged in the petri dish;
c) assessing from the observed nematode placement whether the test sample has been attracted or repelled. The method according to claim 9 , wherein the test sample is a sample obtained from a subject suspected of being a cancer patient. The method according to claim 9 , wherein the test sample is urine obtained from a subject suspected of being a cancer patient. When the nematodes in c) showed attractant action against the test sample, Rukoto contain attractants derived from cancer test sample is indicated, nematodes against test sample when showing the repellency action, it does not contain attractants derived from cancer test sample is indicated, the method according to claim 10 or 11. The method according to any one of claims 9 to 12 , wherein a nematicide is not used. In a), the following petri dish:
  A petri dish having a substantially square bottom having a longitudinal direction of 3 to 6 cm and a lateral direction of 1 to 3 cm, wherein the bottom is a first region in the longitudinal direction from one end to another end, A petri dish divided into at least three regions of a second region and a third region, wherein the second region is disposed at a boundary between the first region and the third region; or
  The method according to any one of claims 9 to 13, wherein any one of the petri dishes included in a multiplate in which a plurality of the petri dishes are connected is used.