WO2006019111A1

WO2006019111A1 - System for measuring amount of gamma-aminobutyric acid released and reaction accelerator substance for use therein

Info

Publication number: WO2006019111A1
Application number: PCT/JP2005/014998
Authority: WO
Inventors: Sachiko Yoshida; Masakazu Uematsu
Original assignee: National University Corporation Toyohashi University Of Technology
Priority date: 2004-08-17
Filing date: 2005-08-17
Publication date: 2006-02-23
Also published as: JPWO2006019111A1

Abstract

A system for measuring the amount of gamma-aminobutyric acid released, in which without any stimulation of tissues by by-products, the amount of release can be measured in correlation to the release composition while reducing any background noise; and a reaction accelerator substance for use therein. The system has been so constructed that any influence of by-products upon measurement results is avoided by the use of not only an enzyme capable of specific oxidation of gamma-aminobutyric acid to thereby effect reduction of nicotinamide dinucleotide phosphate but also a dicarboxylic acid as a reaction accelerator agent. Closed-system reaction chamber (20) has been devised so as to work out such an arrangement that by means of semipermeable membrane (24) provided at a surface of the reaction chamber (20), gamma-aminobutyric acid having been released in the brain directly permeate into the reaction chamber (20). Thus, it has become feasible to find the locality of cells having released a particular gamma-aminobutyric acid.

Description

Specification

Gammaaminobutyric acid release measurement system and reaction accelerator used for it

Technical field

[0001] The present invention relates to a new method and method for quantifying gamma-aminobutyric acid, which also releases nerve tissue force, and expressing the concentration distribution as a two-dimensional image. This method can measure the amount of released gammaaminobutyric acid released from the body tissue force in a short time and identifying the released cells that have released this gammaaminobutyric acid. In addition, in this method, the by-products generated during the measurement do not affect the measurement results, which is useful for determining the state of tissues in regenerated tissues and for pathological examinations.

[0002] Neural tissue transmits information by releasing a specific molecule outside the cell, and this molecule is called a nerve transmitter. Among them, gammaaminobutyric acid is released by many higher brain tissue forces and is an important index that is deeply involved in the development of dementia, Alzheimer's disease and brain functional development.

[0003] Conventionally, the measurement of the amount of gammaaminobutyric acid released has mostly been a method in which a solution around the tissue is collected and attached with a fluorescent carrier such as ortho-phthaldialdehyde and quantified by high performance liquid chromatography. there were. Furthermore, it is known that the conventional method of measuring the amount of gammaaminobutyric acid released with an enzyme may generate a by-product glutamic acid having physiological activity during the measurement. When this conventional method is used in the vicinity of a tissue, the tissue is secondarily stimulated by glutamate produced as a by-product. In other words, the measurement result itself makes the measurement result unreliable.

[0004] In addition, since the conventional method for measuring the amount of gammaaminobutyric acid released uses a tissue peripheral solution, it is not possible to know which cell type is released due to the surrounding solution being mixed during collection. That is, if surrounding solutions are mixed during collection, it is difficult to measure the exact gamma aminobutyric acid concentration at that site. Measures gamma aminobutyric acid with low concentration In order to do so, it is necessary to add an extremely sensitive and expensive fluorescent carrier, so that one measurement is very expensive.

Patent Document 1: None

Disclosure of the invention

[0005] As described above, in the conventional method of measuring the amount of released gammaaminobutyric acid one-dimensionally with an enzyme, a by-product having physiological activity, such as glutamic acid, is generated during the measurement. If this measurement method is used in the vicinity of the tissue, the tissue is secondarily stimulated by such by-products generated by the reaction at the time of measurement, and accurate measurement cannot be performed. Therefore, it is necessary to improve the reaction materials used for measurement and to construct a reaction system that does not stimulate tissues.

[0006] Furthermore, if the concentration distribution of gammaaminobutyric acid can be expressed two-dimensionally, it is possible to identify which part of the cell has released gammaaminobutyric acid. In order to measure the amount of gamma aminobutyric acid released in two dimensions, reduce the noise of the knock ground, set the conditions for measuring the amount of gamma amino butyric acid released and its release tissue, and specify the measurement site. It is necessary to consider an appropriate system in which the reaction chamber is configured so that it can be determined.

[0007] In view of the above situation, the present invention reduces the noise of knock ground that does not stimulate tissue by a by-product, and reduces the amount of released gamma aminobutyric acid that can be measured in correspondence with the amount of released tissue. The purpose is to provide a measurement system and a reaction promoting substance used therefor.

In order to achieve the above object, the present invention provides

[1] In a system for measuring the amount of gammaaminobutyric acid released that also releases tissue power, an enzyme reaction that reduces gammaaminobutyric acid using gammaaminobutyric acid-degrading enzyme to reduce nicotinamide dinucleotide is used for this gammaaminobutyric acid. Using the fact that there is a one-to-one relationship between the molar amount of reaction and the molar amount of reduced nicotinamide dinucleotide phosphate produced, the fluorescence emitted by reduced nicotinamide dinucleotide phosphate produced by this enzymatic reaction is emitted. Detecting and measuring the release amount of gammaaminobutyric acid.

[2] The system for measuring the amount of gammaaminobutyric acid released according to [1] above, wherein dicarboxylic acid is used as a reaction field as a substance that promotes an enzymatic reaction that oxidizes gamma aminobutyric acid to reduce nicotinamide dinucleotide phosphate. It is characterized by being added to. [3] In the system for measuring the amount of gammaaminobutyric acid released according to the above [2], the dicarboxylic acid is a ketoglutaric acid, a ketobutyric acid, or oxalic acetic acid! It is a mixture.

[0011] [4] Light emission in which fluorescence emitted from reduced nicotinamide dinucleotide phosphate is detected by an imaging device using the gammaaminobutyric acid release amount measurement system according to [1], [2] or [3] It is characterized by two-dimensionally displaying the concentration distribution of gammaaminobutyric acid released from the body tissue force with an image.

[5] The system for measuring the amount of gammaaminobutyric acid released according to [4] above, wherein the imaging element is a charge coupled device or a complementary metal oxide semiconductor.

[6] A reaction promoting substance used in a system for measuring the amount of gammaaminobutyric acid released that also releases biological tissue force, and nicotinamide dinucleotide phosphate by acidifying gammaaminobutyric acid using gammaaminobutyric acid degrading enzyme It is characterized by containing a dicarboxylic acid as a substance that promotes this enzymatic reaction.

[7] The reaction promoting substance according to [6] above, wherein the dicarboxylic acid is any one of a ketoglutaric acid, α-ketobutyric acid, oxalic acetic acid, or a mixture thereof. To do.

[0015] Thus, in the present invention, the reaction system is constituted by substances originally present in the living body. That is, by using an enzyme that specifically reacts with gammaaminobutyric acid released by the nerve tissue in the living body to reduce nicotinamide dinucleotide phosphate, the reduced nicotinamide dinucleotide phosphate produced at that time is emitted. Measure the amount of fluorescence and quantify the amount of gammaaminobutyric acid released. Here, since nicotinamide dinucleotide phosphate and reduced nicotinamide dinucleotide phosphate are a kind of vitamins present in the living body, secondary stimulation of living tissues can be avoided.

[0016] When measurement is performed in the vicinity of a specific tissue, a closed reaction chamber is used. That is, a semipermeable membrane is placed on the surface of a closed reaction chamber, and the neurotransmitter gammaaminobutyric acid is permeated from the semipermeable membrane. Excitation light is introduced with a UV light-emitting element, and images are captured using an image sensor such as an artificial retina LSI.

[0017] Fluorescence having a specific wavelength emitted by the reduced nicotinamide dinucleotide phosphate is used as an imaging device. A two-dimensional release amount of gammaaminobutyric acid is obtained by taking an image with a CCD camera (for example, a CCD camera) and processing the image. That is, it constitutes a two-dimensional measurement system for gammaaminobutyric acid release.

Brief Description of Drawings

[0018] FIG. 1 is a configuration diagram of a measurement system for measuring the amount of gammaaminobutyric acid released from a nerve tissue according to a first embodiment of the present invention in vitro.

FIG. 2 is a diagram showing an enzyme reaction used in the present invention.

FIG. 3 is a diagram showing the difference in fluorescence generation behavior when the reaction promoting substance used in the present invention is changed.

FIG. 4 is a configuration diagram of a measurement system for measuring the amount of gammaaminobutyric acid released from the nerve tissue according to the second embodiment of the present invention.

FIG. 5 is a diagram showing a state of light emission in the vicinity of Purkinje layer showing an example of the present invention.

FIG. 6 is a diagram showing the relationship between the gammaaminobutyric acid concentration and the light intensity according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] In the system for measuring the amount of gammaaminobutyric acid released, the gammaaminobutyric acid release enzyme uses gammaaminobutyric acid-degrading enzyme to oxidize gammaaminobutyric acid to reduce nicotinamide dinucleotidolinic acid. Using the fact that there is a one-to-one relationship between the molar amount and the molar amount of reduced nicotinamide dinucleotide phosphate produced, the fluorescence emitted by reduced nicotinamide dinucleotide phosphate produced by this enzymatic reaction is detected. To measure the amount of gammaaminobutyric acid released. Therefore, it is possible to reduce the noise of the knock ground that does not stimulate the tissue by the by-product, and to measure the released amount and the released tissue in correspondence.

Example

Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings.

FIG. 1 is a diagram showing a measurement system for measuring the amount of gammaaminobutyric acid released from the nerve tissue according to the first embodiment of the present invention in vitro, and FIG. 1 (a) shows its overall configuration. Fig. 1 (b) is a perspective view of the reaction chamber. In FIG. 1, 1 is a glass container, 2 is a chamber block, 3 is a sample (a nerve tissue piece that releases gamma aminobutyric acid), 4 is a reaction chamber (a chamber into which sample 3 and an enzyme solution are charged) ), 5 UV light source, 6 primary filter and lens, 7 excitation UV light, 8 reflecting mirror, 9 secondary filter, 10 CCD camera with cooling function (ie light detector), 11 micropipette , 12 is an opening provided on the upper surface of the chamber 12.

That is, the gammaaminobutyric acid release amount released by the tissue is measured using an inverted culture microscope (UV light source 5 to secondary filter 9) and a CCD camera 10 with a cooling function. The chamber block 2 is adhered to the glass surface of the glass container 1 with a putty, for example. The thickness of the chamber block 2 is 0.5 to 2 cm, preferably 0.8 to 1.2 cm, and the volume of the reaction chamber 14 is 100 to 600 μ1, preferably 200 to 400 μ1.

[0024] The measurement is performed by supplying the sample 3 into the reaction chamber 4 and then reacting with the released gammaaminobutyric acid specifically to reduce nicotinamide dinucleotide phosphate, that is, a gammaaminobutyric acid degrading enzyme (for example, , Glutamic acid dehydrogenase) and nicotinamide dinucleotide phosphate solution are supplied to the reaction chamber 14 with a micropipette 11. Teflon (registered trademark) can be used as the material for the chamber block 2.

[0025] As described above, in the two-dimensional measurement of gammaaminobutyric acid released by nerve tissue force, when measuring in vitro, the present invention uses an open system chamber as shown in FIG. It is configured to measure the amount of gammaaminobutyric acid released by the nerve tissue using a (UV light source 5 to 2nd filter 9) and a CCD camera 10 with a cooling function.

Next, the enzyme reaction used in the present invention will be described with reference to FIG. The reaction used in the present invention takes place in the reaction chamber 14 as an enzymatic reaction. In the presence of gammaaminobutyric acid degrading enzyme (abbreviation: GABAase), when gammaaminobutyric acid (abbreviation: GABA) and nicotinamide dinucleoside acid (abbreviation: NADP) coexist, GABA is converted to succinic acid. NADP is reduced to reduced nicotinamide dinucleotide phosphate (abbreviation; NADPH). At this time, there is a one-to-one relationship between the reaction molar amount of GABA and the molar amount of NADPH produced. Therefore, the GABA concentration becomes clear by measuring the fluorescence intensity emitted by NADPH. Here, NADP is a type of coenzyme that supplements the function of the enzyme. Gammaaminobutyric acid also receives electrons and changes to NADPH. Therefore, gamma amino Butyric acid Other coenzymes or substrates can be used as long as they receive electrons and are reduced to emit fluorescence. Since NADPH emits fluorescence of a specific wavelength (480 nm), the fluorescence emitted by this NADPH is detected by an image sensor, for example, a CCD camera 10. As the excitation light, ultraviolet light (UV light) having a wavelength of 340 nm can be used.

[0027] At this time, if a reaction promoting substance is added to gammaaminobutyric acid degrading enzyme (GABAase), the time required to detect fluorescence is reduced to 1/10 or less, preferably 1/100 or less. As this reaction accelerator, dicarboxylic acid, particularly α-ketoglutaric acid, α-ketobutyric acid, oxaloacetic acid, or a mixture thereof can be used. Figure 3 shows the difference in fluorescence behavior when the reaction accelerator is changed (difference in fluorescence intensity generated by the reaction of a gammaaminobutyric acid solution with the same concentration). In this figure, Fig. 3 (a) shows the case where the reaction accelerator is OC ketoglutaric acid, Fig. 3 (b) shows the case where the reaction accelerator is OC ketobutyric acid, and Fig. 3 (c) shows that the reaction accelerator is Oxaguchi. The case of acetic acid is shown respectively. In these figures, the horizontal axis represents time (seconds), and the vertical axis represents the intensity of main fluorescence.

[0028] Next, a measurement system for measuring the amount of gamma aminobutyric acid released in vivo released from the nerve tissue according to the second embodiment of the present invention will be described with reference to FIG. Fig. 4 (a) is a front view and Fig. 4 (b) is a side view.

[0029] When measuring the amount of gammaaminobutyric acid released in vivo, a closed reaction chamber 20 is used. The volume of the closed reaction chamber 20 is 100 to 600 1, preferably 200 to 400 1, so that the enzyme reaction can be performed in a short time. A semi-permeable membrane 24 that is permeable to a substance with a molecular weight of 100 or less is placed on the surface of a closed reaction chamber 20 and permeated with gamma aminobutyric acid, a neurotransmitter.

Excitation light is introduced by an ultraviolet light emitting diode 23, and image capturing is performed using an image sensor (artificial retina LSI) 26. 21 is an output cable of the image sensor 26, 22 is a power cable, and 25 is a reaction chamber containing an enzyme. It should be noted that the image pickup device can be configured inexpensively and compactly by using a charge coupled device or a complementary metal oxide film semiconductor.

[0031] (1) The measurement is performed according to the following procedure.

Nicotinamide dinucleotide phosphate and reaction accelerator (20) in a closed reaction chamber Desirably encapsulate (hyketoglutarate) and buffer.

(2) Gammaaminobutyric acid permeates through a semipermeable membrane 24 that is permeable to substances with a molecular weight of 100 or less (30 seconds).

(3) Measure the light intensity before reaction with the image sensor 26 (the measured value of the light intensity at this time is F0).

(4) The same enzyme as shown in Fig. 2 is quantitatively injected into the closed reaction chamber 20 and allowed to react (10 seconds).

(5) Excitation with UV light, and measure the light intensity after reaction with the image sensor 26 (the measured value of the light intensity at this time is F1).

(6) Since dFZF0 (where dF = Fl-F0) is proportional to the gammaaminobutyric acid concentration, the amount of gammaaminobutyric acid released is calculated.

Furthermore, specific embodiments (experimental results) of the present invention will be described in detail.

[0033] Gammaaminobutyric acid, which was also released from mouse cerebellar tissue, was measured using the closed reaction chamber shown in Fig. 4 according to the second embodiment of the present invention. The release of butyric acid was confirmed. Figure 5 shows the light emission at that time. In this figure, Fig. 5 (a) is a pre-reaction photograph showing the shape of the site releasing gammaaminobutyric acid taken with a halogen lamp light source, and Fig. 5 (b) shows the luminescence in the vicinity of the site (= gamma aminobutyric acid). The white area is the fluorescence image of NADPH that was expressed by UV light irradiation.

FIG. 6 is a graph showing the relationship between GABA concentration and light intensity, and M represents the micromolar amount of gammaaminobutyric acid present in 1 liter of solution. As is clear from this figure, the fluorescence intensity and the concentration of gammaaminobutyric acid shown in FIG. 5 (b) are uniquely related. By utilizing this relationship, it is possible to measure the measured value of the light intensity gammaaminobutyric acid released as described above.

Note that the present invention is not limited to the above-described embodiments, and various modifications can be made based on the spirit of the present invention, and these are not excluded from the scope of the present invention.

[0036] According to the present invention, the amount of gammaaminobutyric acid released by the brain tissue force of a patient can be measured using an enzyme reaction and an imaging device (for example, a CCD camera). Above gamma amino dairy In the system for measuring acid release, nicotinamide dinucleotide phosphate added during measurement and reduced nicotinamide dinucleotide phosphate produced are substances (a kind of vitamin) that exist in the living body. It has the characteristic that there is no fear of damaging secondary damage. As a result, the adverse effect that the measurement operation itself affects the measurement is eliminated, and it becomes possible to perform treatment while accurately predicting the state of nerve activity in the brain of a patient who has developed trauma or cerebral infarction.

[0037] Gammaaminobutyric acid released by brain tissue is a neurotransmitter that plays a central role in modern neurological lesions such as dementia and Alzheimer's disease, and is a promising indicator of lesion progression and brain development. . Although it was impossible to know the distribution of gammaaminobutyric acid release by the conventional method, the present invention can measure the gammaaminobutyric acid release from specific cells in the tissue two-dimensionally to know the distribution. it can.

[0038] By measuring the amount of gammaaminobutyric acid released from specific cells in the yarn and weave, it is possible to measure the amount of gammaaminobutyric acid released by the brain tissue force with potential lesions. It can be determined whether or not. It is also possible to conduct functional tests on the regenerated brain tissue.

Industrial applicability

[0039] The measurement system for gammaaminobutyric acid release amount of the present invention is suitable as a tool for determining whether or not a neural circuit has a lesion power.

Claims

The scope of the claims

[1] Biological tissue force In a system for measuring the amount of released gammaaminobutyric acid released, in an enzymatic reaction that uses gammaaminobutyric acid degrading enzyme to reduce nicotinamide dinucleotide phosphate by acidifying gammaaminobutyric acid, the gammaaminobutyric acid is used. Using the fact that there is a one-to-one relationship between the reaction molar amount of the produced nicotinamide dinucleotide phosphate and the reduced nicotinamide dinucleotide phosphoric acid produced, the fluorescence generated by the reduced nicotinamide dinucleotide phosphate produced by the enzyme reaction A system for measuring the amount of gammaaminobutyric acid released by detecting the amount of gammaaminobutyric acid released.

[2] In the system for measuring the amount of gammaaminobutyric acid released according to claim 1, dicarboxylic acid is added to the reaction field as a substance that promotes an enzymatic reaction that oxidizes gamma aminobutyric acid to reduce nicotinamide dinucleotide phosphate. A system for measuring the amount of gammaaminobutyric acid released.

[3] The gamma amino butyric acid release amount measuring system according to claim 2, wherein the dicarboxylic acid is a ketoglutaric acid, a ketobutyric acid, oxacin acetic acid, or a mixture thereof. Quantity measuring system.

[4] Using the gamma aminobutyric acid release amount measuring system according to claim 1, 2 or 3, biological tissue force was released with a luminescence image obtained by detecting fluorescence emitted by reduced nicotinamide dinucleotide phosphate with an imaging device. A system for measuring the amount of gammaaminobutyric acid released, which displays the concentration distribution of gammaaminobutyric acid in two dimensions.

[5] The gamma aminobutyric acid release amount measurement system according to claim 4, wherein the imaging device is a charge coupled device or a complementary metal oxide film semiconductor. system.

[6] Biological tissue force A reaction accelerator used in a system for measuring the amount of released gammaaminobutyric acid released, which reduces gammaaminobutyric acid using gammaaminobutyric acid-degrading enzyme to reduce nicotinamide dinucleotide phosphate A reaction promoting substance comprising a dicarboxylic acid as a substance that promotes the enzymatic reaction in an enzymatic reaction.

[7] The reaction accelerator according to claim 6, wherein the dicarboxylic acid is a ketoglutaric acid, a ketobutyric acid, or oxalic acetic acid, or a mixture thereof.