JPWO2006019111A1 - Gamma-aminobutyric acid release amount measurement system and reaction promoting substance used therefor - Google Patents

Abstract

Provided is a gamma-aminobutyric acid release amount measurement system capable of reducing background noise and measuring the release amount and release tissue in correspondence with each other without stimulating the tissue with byproducts, and a reaction promoting substance used therefor.
By using an enzyme that specifically oxidizes gamma-aminobutyric acid to reduce nicotinamide dinucleotide phosphate and dicarboxylic acid that is a reaction promoter, it was constructed so that by-products would not affect the measurement results. In addition, the closed reaction chamber 20 is devised so that the semipermeable membrane 24 provided on the surface of the reaction chamber 20 allows the gamma-aminobutyric acid released in the brain to permeate directly into the reaction chamber 20, and the cell at any site It was made possible to know whether it was gamma aminobutyric acid released by.

Description

  The present invention relates to a new method for quantifying gamma-aminobutyric acid (γ-aminobutyric acid) released from nerve tissue and expressing its concentration distribution as a two-dimensional image. This method can measure the amount of released gamma-aminobutyric acid from the biological tissue in a short time while identifying the released cells that released the gamma-aminobutyric acid. Further, in the measurement by this method, the by-product generated at the time of measurement does not affect the measurement result, and therefore it is useful for determining the state of the tissue in the regenerated tissue, pathological examination, and the like.

  Nerve tissues release specific molecules to the outside of cells to transmit information, and these molecules are called neurotransmitters. Among them, gamma aminobutyric acid is a substance that is released from many higher brain tissues and serves as an important index deeply involved in the development of dementia, Alzheimer's disease, and functional development of the brain.

  Conventionally, most of the methods for measuring the amount of released gamma-aminobutyric acid have been to collect a solution around the tissue, add a fluorescent carrier such as orthophthaldialdehyde, and quantify it by high performance liquid chromatography. Furthermore, it is known that the conventional method of measuring the amount of released gamma aminobutyric acid with an enzyme may generate a by-product glutamic acid having a physiological activity during the measurement. When this conventional method is used in the vicinity of the tissue, the tissue is secondarily stimulated by the glutamate produced as a by-product. That is, the measurement action itself makes the measurement result unreliable.

Further, in the conventional method for measuring the amount of released gamma aminobutyric acid, since the solution around the tissue is used, it is not possible to know which cell type was released by mixing the solution around the tissue. That is, if the surrounding solution is mixed at the time of collection, it is difficult to accurately measure the gamma aminobutyric acid concentration at that site. Moreover, in order to measure low-concentration gamma aminobutyric acid, it is necessary to add an expensive fluorescent carrier with extremely high sensitivity, so that one measurement is very expensive.
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  As described above, in the conventional method of one-dimensionally measuring the amount of released gamma-aminobutyric acid with an enzyme, a by-product having a physiological activity, such as glutamic acid, is generated during the measurement. When this measuring method is used in the vicinity of the tissue, the tissue is secondarily stimulated by such a by-product generated by the reaction at the time of measurement, and an accurate measurement cannot be performed. Therefore, it is necessary to improve the reaction substance used for the measurement and construct a reaction system that does not stimulate the tissue.

  Furthermore, if the concentration distribution of gamma-aminobutyric acid can be expressed two-dimensionally, it becomes possible to specify which part of the cell released gamma-aminobutyric acid. In order to measure the release amount of gamma-aminobutyric acid two-dimensionally, the background noise is reduced, and the measurement conditions are set by specifying the conditions that enable the measurement of the release amount of gamma-aminobutyric acid and the release tissue to correspond. It is necessary to think of an appropriate system that configures the reaction chamber so that it can.

  In view of the above situation, the present invention reduces the background noise without stimulating the tissue with by-products, and a gamma-aminobutyric acid release amount measurement system capable of measuring the release amount and the release tissue in association with each other, and It is intended to provide a reaction promoting substance to be used.

The present invention, in order to achieve the above object,
[1] In a measurement system for the amount of gamma-aminobutyric acid released from living tissue, the gamma-aminobutyric acid decomposing enzyme is used to oxidize gamma-aminobutyric acid to reduce nicotinamide dinucleotide phosphate. Taking advantage of the one-to-one relationship between the reaction molar amount of butyric acid and the reduced molar amount of reduced nicotinamide dinucleotide phosphate produced, fluorescence emitted by reduced nicotinamide dinucleotide phosphate produced by this enzymatic reaction is used. Is detected to measure the amount of released gamma aminobutyric acid.

[2] In the measurement system of gamma aminobutyric acid release amount according to [1] above, dicarboxylic acid is added to the reaction field as a substance that promotes an enzymatic reaction of oxidizing gamma aminobutyric acid to reduce nicotinamide dinucleotide phosphate. It is characterized by performing.

[3] The system for measuring the amount of released gamma aminobutyric acid according to [2], wherein the dicarboxylic acid is any one of α-ketoglutaric acid, α-ketobutyric acid, oxaloacetic acid, or a mixture thereof.

[4] A living body having a luminescent image in which fluorescence emitted by reduced nicotinamide dinucleotide phosphate is detected by an image sensor using the measurement system for gamma aminobutyric acid release amount according to [1], [2] or [3] above. It is characterized in that the concentration distribution of gamma aminobutyric acid released from the tissue is displayed two-dimensionally.

[5] In the measurement system of the amount of released gamma aminobutyric acid according to [4], the image pickup device is a charge coupled device or a complementary metal oxide film semiconductor.

[6] A reaction promoting substance used in a measurement system for the amount of released gamma-aminobutyric acid from biological tissue, which uses gamma-aminobutyric acid degrading enzyme to oxidize gamma-aminobutyric acid to reduce nicotinamide dinucleotide phosphate In the enzymatic reaction, a dicarboxylic acid is contained as a substance that promotes the enzymatic reaction.

[7] The reaction accelerating substance according to the above [6], wherein the dicarboxylic acid is any one of α-ketoglutaric acid, α-ketobutyric acid and oxaloacetic acid, or a mixture thereof.

As described above, in the present invention, the reaction system is constituted by the substance originally existing in the living body. That is, by using an enzyme that specifically reacts with gamma aminobutyric acid released from the nerve tissue of the living body to reduce nicotinamide dinucleotide phosphate, the reduced nicotinamide dinucleotide phosphate generated at that time is emitted. The amount of fluorescence is measured to quantify the amount of released gamma aminobutyric acid. Here, since nicotinamide dinucleotide phosphate and reduced nicotinamide dinucleotide phosphate are types of vitamins existing in the living body, secondary stimulation of living tissues can be avoided.

When measuring 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 gamma aminobutyric acid is permeated through this semipermeable membrane. Excitation light is introduced by a UV light emitting element, and image pickup is performed using an image pickup element such as an artificial retina LSI.

The fluorescence of a specific wavelength emitted from the reduced nicotinamide dinucleotide phosphate is photographed by an image sensor (for example, a CCD camera) and image processing is performed to obtain a two-dimensional release amount of gamma aminobutyric acid. That is, it constitutes a two-dimensional measurement system of the amount of released gamma aminobutyric acid.

It is a block diagram of the measurement system which measures the release amount of gamma aminobutyric acid released from the nerve tissue showing the first embodiment of the present invention in vitro. It is the figure which showed the enzyme reaction used by this invention. It is a figure showing the difference in the fluorescence generation behavior when the reaction promoting substance used in the present invention is changed. It is a block diagram of the measurement system which measures the release amount of gamma aminobutyric acid released from the nerve tissue showing the second embodiment of the present invention. It is a figure which shows the mode of light emission in the Purkinje layer vicinity which shows the Example of this invention. It is a figure which shows the relationship between the gamma aminobutyric acid concentration and light intensity concerning this invention.

In a measurement system for the amount of gamma aminobutyric acid released from living tissues, the reaction of gamma aminobutyric acid is used in the enzymatic reaction that uses gamma aminobutyric acid degrading enzyme to oxidize gamma aminobutyric acid to reduce nicotinamide dinucleotide phosphate. By utilizing the one-to-one relationship between the molar amount and the generated molar amount of the reduced nicotinamide dinucleotide phosphate produced, the fluorescence emitted by the reduced nicotinamide dinucleotide phosphate produced by this enzymatic reaction is detected. The amount of gamma aminobutyric acid released is measured. Therefore, it is possible to reduce the background noise and measure the release amount and the release tissue in a corresponding manner without stimulating the tissue with a by-product.

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 release amount of gamma aminobutyric acid released from nerve tissue in vitro according to the first embodiment of the present invention, and FIG. (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 piece of nerve tissue that releases gamma-aminobutyric acid), 4 is a reaction chamber (a chamber into which the sample 3 and the enzyme solution are charged), 5 is a UV light source, 6 is a primary filter and lens, 7 is excitation UV light, 8 is a reflection mirror, 9 is a secondary filter, 10 is a CCD camera with a cooling function (that is, a light amount detector), 11 is a micropipette, and 12 is the upper surface of the chamber 2. It is an opening provided in the.

That is, the inverted culture microscope (UV light source 5 to secondary filter 9) and the CCD camera 10 with a cooling function are used to measure the amount of gamma aminobutyric acid released by the tissue. The chamber block 2 is bonded to the glass surface of the glass container 1 with, for example, putty. The chamber block 2 has a thickness of 0.5 to 2 cm, preferably 0.8 to 1.2 cm, and the reaction chamber 4 has a volume of 100 to 600 μl, preferably 200 to 400 μl.

The measurement is performed by supplying the sample 3 into the reaction chamber 4 and then specifically reacting with the released gamma aminobutyric acid to reduce nicotinamide dinucleotide phosphate, that is, gamma aminobutyric acid degrading enzyme (eg, glutamic acid). A solution of dehydrogenase) and nicotinamide dinucleotide phosphate is supplied to the reaction chamber 4 with the micropipette 11. As a material for the chamber block 2, Teflon (registered trademark) can be used.

As described above, in the two-dimensional measurement of gamma aminobutyric acid released from nerve tissue, when the measurement is performed in vitro, the present invention uses an open system chamber as shown in FIG. 1 and uses an inverted culture microscope (UV light source). It is configured to measure the amount of gamma-aminobutyric acid released by the nerve tissue using the 5th-secondary filter 9) and the CCD camera 10 with a cooling function.

Next, the enzymatic reaction used in the present invention will be described based on FIG. The reaction used in the present invention is an enzymatic reaction and occurs in the reaction chamber 4. When gamma aminobutyric acid (abbreviation: GABA) and nicotinamide dinucleotide phosphate (abbreviation: NADP) coexist in the presence of gamma aminobutyric acid degrading enzyme (abbreviation: GABAase), GABA is oxidized to change to succinic acid, NADP is reduced to change 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 generated molar amount of NADPH. Therefore, the GABA concentration becomes clear by measuring the fluorescence intensity emitted by NADPH. Here, NADP is a kind of coenzyme that supplements the function of the enzyme, and receives an electron from gamma aminobutyric acid and changes into NADPH. Therefore, other coenzymes or substrates can be used as long as they receive an electron from gamma-aminobutyric acid 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, the CCD camera 10. Ultraviolet light (UV light) having a wavelength of 340 nm can be used as the excitation light.

At this time, when a reaction accelerating substance is added to gamma aminobutyric acid degrading enzyme (GABAase), the time required to detect fluorescence becomes 1/10 or less, preferably 1/100 or less. As the reaction promoting substance, a dicarboxylic acid, particularly any one of α-ketoglutaric acid, α-ketobutyric acid and oxaloacetic acid, or a mixture thereof can be used. FIG. 3 shows the difference in the fluorescence behavior when the reaction promoting substance is changed (the difference in the fluorescence intensity generated during the reaction of the gamma aminobutyric acid solution of the same concentration depending on the reaction promoting substance). In this figure, FIG. 3(a) shows that the reaction promoting substance is α-ketoglutaric acid, FIG. 3(b) shows that the reaction promoting substance is α-ketobutyric acid, and FIG. 3(c) shows that the reaction promoting substance is oxaloacetic acid. Each case is shown. In these figures, the horizontal axis represents time (seconds) and the vertical axis represents main fluorescence intensity.

Next, a second embodiment of the present invention will be described with reference to FIG. 4, which is a measurement system for measuring the amount of released gamma aminobutyric acid in a living body released from nerve tissue. 4(a) is a front view thereof, and FIG. 4(b) is a side view thereof.

When measuring the release amount of gamma aminobutyric acid in the living body, the closed reaction chamber 20 is used. The volume of the closed reaction chamber 20 is 100 to 600 μl, preferably 200 to 400 μl, so that the enzymatic reaction is carried out in a short time. A semipermeable membrane 24 that is permeable to a substance having a molecular weight of 100 or less is placed on the surface of the closed reaction chamber 20 to allow gamma aminobutyric acid, which is a neurotransmitter, to permeate.

The excitation light is introduced by the ultraviolet light emitting diode 23, and the image pickup is performed by using the image pickup device (artificial retina LSI) 26. Reference numeral 21 is an output cable of the image pickup device 26, 22 is a power cable, and 25 is a reaction chamber containing an enzyme. By using a charge-coupled device or a complementary metal oxide film semiconductor as the image pickup device, the image pickup device can be inexpensive and compact.

(1) The measurement is performed according to the following procedure.
In the closed reaction chamber 20, nicotinamide dinucleotide phosphate, a reaction accelerating substance (desirably α-ketoglutaric acid) and a buffer solution are enclosed.
(2) Gamma-aminobutyric acid is permeated through the semipermeable membrane 24 having a molecular weight of 100 or less (30 seconds).
(3) The light intensity before the reaction is measured by the image pickup device 26 (the measured value of the light intensity at this time is F0).
(4) The same enzyme as that shown in FIG. 2 is quantitatively injected into the closed reaction chamber 20 and reacted (for 10 seconds).
(5) Excitation with UV light, and measurement of the light intensity after reaction with the image sensor 26 (the measured value of the light intensity at this time is F1).
(6) Since dF/F0 (here, dF=F1-F0) is proportional to the gamma aminobutyric acid concentration, this is used to calculate the gamma aminobutyric acid release amount.

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

  Gamma-aminobutyric acid 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, and it was confirmed that gamma-aminobutyric acid specific to the vicinity of the Purkinje layer was released. confirmed. The state of light emission at that time is shown in FIG. In this figure, FIG. 5(a) is a photograph before the reaction showing the shape of the part that releases gamma aminobutyric acid taken with a halogen lamp light source, and FIG. 5(b) is the light emission near the part (=gamma amino). Butyric acid has been released), and the white portion is the fluorescent image of NADPH expressed by UV light irradiation.

  FIG. 6 is a graph showing the relationship between GABA concentration and light intensity, and μM represents the micromolar amount of gamma aminobutyric acid present in 1 liter of the solution. As is clear from this figure, there is a unique relationship between the fluorescence intensity shown in FIG. 5B and the concentration of gamma aminobutyric acid. By utilizing this relationship, the amount of released gamma aminobutyric acid can be measured from the measured value of the light intensity as described above.

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

According to the present invention, it is possible to measure the amount of gamma aminobutyric acid released from the brain tissue of a patient by utilizing an enzyme reaction and an image pickup device (for example, CCD camera). In the above measurement system of gamma aminobutyric acid release amount, since nicotinamide dinucleotide phosphate added at the time of measurement and the reduced nicotinamide dinucleotide phosphate produced are originally substances (a kind of vitamin) existing in the living body, The feature is that there is no risk of secondarily stimulating or damaging biological tissues. This eliminates the adverse effect that the measurement operation itself influences the measurement, and enables treatment while accurately predicting the state of nerve activity in the brain of a patient who has suffered trauma or cerebral infarction.

Gamma-aminobutyric acid released from brain tissue is a neurotransmitter that plays a central role in modern nerve lesions such as dementia and Alzheimer's disease, and is a powerful indicator of lesion progression and brain development. With the conventional method, it was impossible to know the distribution of gamma aminobutyric acid release amount, but according to the present invention, the distribution of gamma aminobutyric acid release amount from specific cells in the tissue was two-dimensionally measured to obtain the distribution. be able to.

By measuring the amount of gamma-aminobutyric acid released from specific cells in the tissue, the amount of gamma-aminobutyric acid released from brain tissue that may have a lesion may be measured to determine whether the lesion is due to a neural circuit. can do. In addition, it becomes possible to perform a functional test of the regenerated brain tissue.

The gamma aminobutyric acid release amount measurement system of the present invention is suitable as a tool for determining whether or not a lesion has a neural circuit structure.

Claims (7)

In the measurement system of the release amount of gamma aminobutyric acid released from biological tissue,
In the enzymatic reaction of oxidizing gamma-aminobutyric acid by using gamma-aminobutyric acid degrading enzyme to reduce nicotinamide dinucleotide phosphate, the reaction molar amount of the gamma-aminobutyric acid and the generated molar amount of reduced nicotinamide dinucleotide phosphate are generated. Gamma-aminobutyric acid release amount is measured by detecting the fluorescence emitted by reduced nicotinamide dinucleotide phosphate produced by the enzymatic reaction by utilizing the one-to-one relationship between Aminobutyric acid release measurement system.   The system for measuring the amount of released gamma aminobutyric acid according to claim 1, wherein 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. Characteristic gamma-aminobutyric acid release measurement system.   The gamma aminobutyric acid release amount measuring system according to claim 2, wherein the dicarboxylic acid is any one of α-ketoglutaric acid, α-ketobutyric acid, oxaloacetic acid, or a mixture thereof. Measuring system.   Gamma-aminobutyric acid released from living tissue with a luminescence image obtained by detecting fluorescence emitted by reduced nicotinamide dinucleotide phosphate by an image sensor using the measurement system for gamma-aminobutyric acid release according to claim 1, 2 or 3. A system for measuring the amount of released gamma-aminobutyric acid, which displays the concentration distribution of the two-dimensionally.   The gamma aminobutyric acid release amount measurement system according to claim 4, wherein the image pickup device is a charge-coupled device or a complementary metal oxide film semiconductor.   It is a reaction-promoting substance used in a measurement system for the amount of gamma-aminobutyric acid released from living tissues.It is an enzymatic reaction that uses gamma-aminobutyric acid degrading enzyme to oxidize gamma-aminobutyric acid and reduce nicotinamide dinucleotide phosphate. And a reaction-promoting substance containing a dicarboxylic acid as a substance for promoting the enzymatic reaction.   The reaction promoting substance according to claim 6, wherein the dicarboxylic acid is any one of α-ketoglutaric acid, α-ketobutyric acid, oxaloacetic acid, or a mixture thereof.

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