WO2006004216A1 - 一酸化窒素検出用センサー細胞とそれを用いた一酸化窒素の検出・定量方法 - Google Patents
一酸化窒素検出用センサー細胞とそれを用いた一酸化窒素の検出・定量方法 Download PDFInfo
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- WO2006004216A1 WO2006004216A1 PCT/JP2005/012721 JP2005012721W WO2006004216A1 WO 2006004216 A1 WO2006004216 A1 WO 2006004216A1 JP 2005012721 W JP2005012721 W JP 2005012721W WO 2006004216 A1 WO2006004216 A1 WO 2006004216A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
Definitions
- the invention of this application relates to a sensor cell for detecting nitric oxide in a cell, and a non-human animal that has such a sensor cell as a whole cell.
- the present invention relates to a method for detecting and quantifying nitric oxide using NO, a method for monitoring nitric oxide concentration, and a method for screening a substance that affects the binding of soluble guanylate cyclase and nitric oxide.
- Nitric Oxide was reported to be a vascular relaxation factor derived from the vascular endothelium in 1987, its role as a bioactive substance has been discovered one after another, and only the cardiovascular system In addition, it has been found to be an important biomolecule in the immune system and central nervous system. Regarding the disease, NO is considered to be related to arteriosclerosis, stroke, hypertension in the cardiovascular system, infectious disease in the immune system, dementia in the central nervous system, Alzheimer's disease, etc.
- NO synthesized by NO synthase using L-arginine as a substrate is known to be released from the cell and enter neighboring cells to exert its action.
- NO synthase NO synthase; N0S
- vascular endothelial cells, macrophages, neurons (postsynapse), etc. can each be a donor cell of NO, smooth muscle cells, antigens, neurons (presynapse), etc., respectively It can be an N0 acceptor cell.
- guanylyl cyclase In acceptor cells, there is soluble guanylyl cyclase (sGC), one of the target proteins of NO.
- sGC soluble guanylyl cyclase
- D When D is coordinated to heme iron of sGC, the enzymatic activity of sGC is reduced. More than 200 times, the second messenger cyclic guanosine 3 ', 5' -—phosphate (guanosine 3 ', 5' -cycl ic Monophosphate (cGMP) is produced in large quantities and induces intracellular signal transduction.
- cGMP cyclic guanosine 3 ', 5' -cycl ic Monophosphate
- Such NO is an unstable and short-lived molecule that is susceptible to oxidation by enzymes and active oxygen in vivo. For this reason, it is difficult to detect physiological concentrations of NO, and the situation is that the dynamics of NO in the living body was unknown.
- Non-patent Document 1 a fluorescein skeleton organic molecule (diaiinof luorescein; DAF) having a diamino group that chemically reacts with NO has been reported (Non-patent Document 1).
- DAF is usually non-fluorescent, but when it reacts with NO in the presence of oxygen, it becomes a fluorescent triazole and exhibits green fluorescence. Therefore, by using such organic molecules, it became possible to visualize and analyze NO generated in the cells under a fluorescence microscope.
- a rhodamine-skeleton organic molecule (DM) that exhibits red fluorescence based on the same principle has also been reported (Non-patent Document 2).
- Non-Patent Document 1 Anal. C eE 70; 2446-2453, 1998.
- Non-Patent Document 2 Anal. CheE 73; 1967-1973, 2001.
- Non-Patent Document 3 Proc. Natl. Acad. Sci. USA 77; 7380-7384, 1980
- Patent Document 1 Japanese Patent Laid-Open No. 2 0 0 2-0 1 7 3 5 9
- Patent Document 2 PCT / JP 0 1/5 6 3 1 Disclosure of the invention
- sGC soluble guanylate cyclase
- sGC cyclic guanosine 3 ′, 5′-monophosphate
- NO nitric oxide
- the invention of this application is such that sGC and a cGMP visualization probe coexist in a cell expressing sGC by introducing a polynucleotide expressing the cGMP visualization probe into the cell.
- a sensor cell and thirdly, a sGC and cGMP visualization probe coexisting in the cell by introducing a polynucleotide expressing sGC and a polynucleotide expressing a cGMP visualization probe into the cell.
- the fifth aspect of the invention of this application is that, in the fifth aspect, the cGMP visualization probe is formed by linking two marker sites capable of detecting proximity to both ends of a cGMP-binding protein.
- the sensor cell in which the cGMP binding protein in the cGMP visualization probe is cGMP-dependent kinase I ⁇
- two marker sites that can detect proximity to each other in the cGMP visualization probe Provides sensor cells that are cyan fluorescent protein and yellow fluorescent protein.
- the invention of this application is, in the eighth aspect, a polynucleotide that expresses a cGMP visualization probe in a cell that expresses sGC in a transgenic non-human animal in which all cells are the sensor cells.
- a non-human animal or a progeny animal thereof obtained by individual generation of non-human animal totipotent cells
- ninth a transgenic non-human animal in which all cells are the sensor cells described above.
- Two non-human animals are introduced into a cell, and two types of polynucleotides expressing two types of hybrid proteins formed by linking cGMP visualization probes to each of the two subunits and ⁇ of sGC are introduced.
- a non-human animal or its progeny obtained by ontogenizing human totipotent cells is provided.
- the cGMP visualization probe is formed by linking two marker sites capable of detecting proximity to each end of a cGMP-binding protein. Or a progeny animal thereof, or a non-human animal or its progeny animal whose cGMP-binding protein is cGMP-dependent kinase Ia in the cGMP visualization probe, or a cGMP visualization probe.
- Two marker sites that can detect the proximity of each other in the non-human animal or its progeny are cyan fluorescent protein and yellow fluorescent protein.
- the invention of this application is, in 14th, a method for detecting and quantifying intracellular NO, characterized by measuring a signal change in any one of the sensor cells.
- Fifteenth is a method for monitoring changes in the NO concentration in cells due to stimulation, and a signal is applied before and after the stimulus is applied to any one of the sensor cells. It provides a method for monitoring intracellular NO concentration, which is characterized by measuring changes.
- the invention of this application is a method for detecting and quantifying NO released from a cell according to the sixteenth aspect of the present invention.
- Cell discharge NO detection and quantification methods characterized by measuring signal changes in sensor cells placed in close proximity, and No. 17 are methods for screening substances that affect NO binding to sGC.
- a screening method is provided, wherein a candidate substance is introduced into any one of the sensor cells, and a signal change in the presence and absence of the candidate substance is measured.
- the 18th is to detect and quantify NO in the living body
- a method for detecting and quantifying in vivo NO characterized by measuring a signal change in any one of the above non-human animals
- a method for monitoring a change in a living body comprising: applying a stimulus to any one of the non-human animals, and measuring a signal change in the non-human animal before and after the stimulus is applied.
- a method for screening a substance that affects the binding of NO to sGC wherein a candidate substance is administered to any one of the non-human animals, and in the presence of the candidate substance and
- At least sGC and a cGMP visualization probe that recognizes cGMP and emits a signal coexist in the living cell.
- NO coordinated to the heme iron of sGC the enzyme activity of sGC increased more than 200 times, and a large amount of second messenger cGMP was produced.
- This cGMP is recognized by a cGMP visualization probe that coexists in the sensor cell, and the cGMP visualization probe emits a signal. Therefore, by measuring this signal change, the presence or intrusion of N0 can be detected with high accuracy.
- the polynucleotide expressing the cGMP visualization probe is introduced into the cell expressing sGC so that the sGC and the cGMP visualization probe coexist in the cell.
- the sensor cell according to the third aspect of the invention by introducing a polynucleotide expressing a soluble guanylate cyclase and a polynucleotide expressing a cGMP visualization probe into the cell, an sGC and a cGMP visualization probe are introduced into the cell. Coexist.
- the cGMP visualization probe is formed by linking two marker sites capable of detecting proximity to each end of a cGMP-binding protein, so NO is contained in the sensor cell.
- the cGMP binding protein is cGMP-dependent protein kinase I ⁇ (hereinafter sometimes referred to as PKG Ia), and in the seventh invention,
- the two marker sites that can detect the proximity of each other in the cGMP visualization probe are cyan fluorescent protein and yellow fluorescent protein.
- the non-human animal of the above-mentioned eighth invention or its progeny animal introduces a polynucleotide expressing a cGMP visualization probe into a cell expressing sGC, and ontogenizes non-human animal totipotent cells. Therefore, in such a transgenic non-human animal, all cells become sensor cells in which sGC and cGMP visualization probe coexist.
- the non-human animal of the ninth invention or its progeny animal is introduced with a polynucleotide expressing a soluble guanylate cyclase and a polynucleotide expressing a cGMP visualization probe into the cell, and the totipotent cell of the non-human animal is introduced.
- all cells become sensor cells in which sGC and cGMP visualization probes coexist.
- the non-human animal or its progeny animal comprises two types of cells in which a cGMP visualization probe is linked to each of two subunits of soluble guanylate cyclase and / or? It is obtained by introducing two types of polynucleotides expressing the hybrid protein and ontogenizing non-human animal totipotent cells. Therefore, in such a transgenic non-human animal, all cells become sensor cells in which sGC and cGMP visualization probe coexist.
- sGC and cGMP visualization probe coexist in all cells, and all the cells are the sensor cells. It can be said. Therefore, by irritating such animals or administering candidate substances, observe the changes in NO concentration in each organ or tissue in the body, the effect of NO concentration on life activity, etc. Is possible.
- the cGMP visualization probe is formed by linking two marker partial positions that can detect proximity to each other at both ends of a cGMP-binding protein.
- cGMP is generated by sGC and cGMP is generated, cGMP binds to cGMP-binding protein, and the three-dimensional structure of cGMP.-binding protein changes, so that the two marker sites linked to both ends are in close proximity. And a signal is emitted. Therefore, it is possible to accurately detect the presence or invasion of NO in a specific organ or tissue.
- the cGMP-binding protein is PKG I ⁇
- the cGMP visualization probes are close to each other.
- Two detectable marker sites are cyan fluorescent protein and yellow fluorescent protein.
- the method for monitoring intracellular NO concentration according to the above-mentioned fifteenth aspect of the invention, stimulation is applied to any of the sensor cells, and a change in signal before and after the stimulation is measured, whereby the intracellular change due to the stimulation is measured. Changes in N0 concentration can be monitored.
- the cell release NO detection / quantification method of the above-mentioned sixteenth aspect of the invention the cell whose NO release is to be detected / quantified is placed in close proximity to any one of the sensor cells, and the signal change in the sensor cell is measured. Thus, NO released from cells can be detected and quantified.
- the candidate substance is introduced into any of the above NOs, and the change in signal in the presence and absence of the candidate substance is measured, thereby affecting the binding of NO to sGC. Can be screened. Furthermore, in the method for detecting and quantifying in vivo NO of the above-described eighteenth aspect of the invention, it is possible to detect and quantify in vivo NO by measuring signal changes in any of the non-human animals or their progeny animals. It becomes.
- the monitoring method of the nineteenth aspect of the invention by applying a stimulus to any of the non-human animals and measuring a signal change in the non-human animal before and after the stimulus, the NO concentration in the living body due to the stimulus is measured. Change can be monitored.
- the candidate substance is administered to any of the non-human animals, and the change in the signal of the non-human animal in the presence and absence of the candidate substance is measured. It is possible to screen for substances that affect binding.
- FIG. 1 is a schematic diagram illustrating the configuration of a sensor cell for detecting nitric oxide according to the invention of this application.
- FIG. 2 is a schematic diagram illustrating another configuration of the sensor cell for detecting nitric oxide of the invention of this application.
- Figure 3 shows the CFP and YFP in the example of the invention of this application when the sensor cell for detecting nitric oxide was stimulated with NO donor N0C-7 (10, 50, 100 nM and l ⁇ M). It is the figure which showed the time-dependent change of the fluorescence intensity ratio (CFP / YFP).
- Figure 4 shows the relationship between the N0C-7 concentration and the fluorescence intensity ratio (CFP / YFP) change between CFP and YFP when the sensor cell for detecting nitric oxide was stimulated with N0C-7 in the example of the invention of this application.
- FIG. 4 shows the relationship between the N0C-7 concentration and the fluorescence intensity ratio (CFP / YFP) change between CFP and YFP when the sensor cell for detecting nitric oxide was stimulated with N0C-7 in the example of the invention of this application.
- FIG. 5 shows an example of the invention of this application in which a nerve cell (neuron) is placed close to a sensor cell for detecting nitric oxide, and the nerve cell is glutamic acid (1, 5, 10 and) known as a neurotransmitter. It is a graph showing the change over time in the fluorescence intensity ratio (CFP / YFP) of CFP and YFP when stimulated at 100 M).
- CFP / YFP fluorescence intensity ratio
- FIG. 6 shows glutamine when a neuron (neuron) placed close to the sensor cell for detecting nitric oxide is stimulated with glutamic acid in the embodiment of the invention of this application. It is the figure which showed the relationship between the acid concentration and the fluorescence intensity ratio (CFP / YFP) change of CFP and YFP.
- Figure 7 shows the change over time in the fluorescence intensity ratio (CFP / YFP) of CFP and YFP when the hippocampal region is placed on a culture dish in which sensor cells for detecting nitric oxide are spread in the example of the invention of this application. It is a figure.
- Figure 8 shows the response of the sensor cell for detecting nitric oxide.
- A When the excitation light is squeezed and UV light is emitted in a narrow area to release NO (local uncaging).
- B The case where NO is released in a wide area without narrowing the excitation light (unifonal uncaging) is shown.
- Fig. 9 shows the change in fluorescence intensity ratio (CFP / YFP) of sensor cells for nitric oxide detection by BNN5Na stimulation as a pseudo color change, where 1 is before NO is released (before B N5Na stimulation).
- Sensor cell pseudo-color change 2 (L) is a pseudo-color change when NO is locally released, 3 is a pseudo-color change after the passage of time in 2 (L), 4 (L) is N0 in a wide area 5 shows the pseudo color change after elapse of time at 4 (L).
- Fig. 10 shows the relationship between the change in fluorescence intensity ratio (CFP / YFP) of the sensor cell for detecting nitric oxide and the passage of time (in minutes) in Region-1 and Region-3.
- Figure 11 shows the spatiotemporal analysis of NO released from vascular endothelial cells using sensor cells for nitric oxide detection.
- A is a schematic diagram illustrating an experimental system for spatiotemporal analysis.
- B- 1) is the PC image of the sensor cell on the force per glass
- B- 2 is the fluorescence image of the sensor cell on the force per glass
- C is the PC image of the vascular endothelial cell on the dish. It is.
- Figure 12 shows the change in fluorescence intensity ratio (CFP / YFP) of sensor cells for nitric oxide detection by vascular endothelial cells stimulated with bradykinin as a pseudo color change.
- CFP / YFP fluorescence intensity ratio
- Figure 13 shows the sensor cells for nitric oxide detection in region-1 and region-2. It is the figure which showed the relationship between light intensity ratio (CFP / YFP) change and time passage (unit is second).
- symbol in a figure has shown the following.
- the sensor cell for detecting nitric oxide (NO) of the invention of this application is characterized in that at least sGC and a cGMP visualization probe coexist in a living cell, as described above, and its action principle is As shown schematically in Fig. 1.
- the coordination bond of NO (2) to sGC (3) to heme iron (31) is an equilibrium reaction and is reversible, so NO (2) is sGC (3) heme iron (31
- NO (2) released in the sensor cell (1) decreases, NO (2) dissociates from heme iron (31) of sGC (3), and sGC
- the enzyme activity of (3) is lost.
- cGMP (5) is not generated in the sensor cell (1), and only degradation of cGMP (5) by phosphodiesterase (PDE) inherent in the sensor cell (1) proceeds. become. Therefore, the cGMP (5) concentration in the sensor cell (1) decreases and appears as a signal change of the cGMP visualization probe (6). Therefore, it can be said that the sensor cell (1) of the invention of this application shows an irreversible response depending on the NO (2) concentration.
- the cGMP visualization probe (6) is not particularly limited as long as it recognizes cGMP and emits a signal.
- the inventors of this application It is desirable that the reported ones (Patent Documents 1 and 2), that is, two marker sites capable of detecting proximity to each other are linked to both ends of the cGMP-binding protein.
- Patent Documents 1 and 2 that is, two marker sites capable of detecting proximity to each other are linked to both ends of the cGMP-binding protein.
- the cGMP binding protein binds to cGMP (5), and the resulting change in the configuration of the two marker sites appears as an optical change. . Therefore, by measuring this optical change, NO (2) in the sensor cell can be detected and quantified via cGMP (5).
- the cGMP binding protein is exemplified by cGMP-dependent protein kinase I (PKG la).
- Mammalian PKG ⁇ ⁇ consists of two identical monomers with four functional domains, and the dimerization domain located on the ⁇ -terminal side consists of leucine ⁇ isoleucine zipper motif.
- PKG I a shows a closed conformation that is kinase inactive and the catalytic center is occupied by an autoinhibitory domain, but when bound to cGMP (5), The autoinhibitory domain is removed from the active center and PKG la shows open conformation. Therefore, in combination with such cGMP (5) As a result, the configuration of the marker sites at both ends of PKG Ia changes, causing an optical change, and the binding to cGMP (5) is visually detected.
- the cGMP-binding protein in the cGMP visualization probe (6) is not limited to PKG I ot, and any synthetic or natural peptide chain can be used.
- the cGMP visualization probe (6) present in the sensor cell for nitric oxide detection (1) of the invention of this application is capable of detecting proximity to both ends of the cGMP-binding protein as described above.
- various chromophores are considered as the two marker sites whose proximity can be detected.
- the chromophore must produce a wavelength change with high accuracy in response to the conformational change caused by the binding of cGMP (5) and cGMP binding protein.
- various fluorescent chromophores are generally used. However, the one that responds promptly to structural changes is that the fluorescence intensity ratio changes due to the occurrence of fluorescence resonance energy transfer (FRET).
- the two marker sites include two fluorescent chromophores with different fluorescence wavelengths, specifically, the green fluorescent protein (GFP) cyan fluorescent protein (CFP), which is a GFP-shifted mutant protein of GFP, Red shift Yellow fluorescent protein (YFP), a mutant protein, can be applied.
- GFP green fluorescent protein
- CFP red shift Yellow fluorescent protein
- YFP Red shift Yellow fluorescent protein
- these two major positions include, in addition to the combination of CFP and YFP, various fluorescent proteins, split Renilla luciferase, water luciferase, j8-galactosidase, 3-lactamase, etc. Is applicable.
- sGC (3) is a soluble guanylyl cyclase, which is widely present in general.
- cGMP visualization probe ( 6) A method of coexisting sGC (3) and cGMP visualization probe (6) in the cell by introducing a polynucleotide expressing Is mentioned.
- a plasmid vector for animal cell expression is preferably used as the expression vector.
- a known method such as an electroporation method, a calcium phosphate method, a ribosome method, or a DEAE dextran method can be employed. In this way, by using a method in which an expression vector incorporating a cGMP visualization probe (6) is introduced into a cell that inherently contains sGC (3), sGC (3) and a cGMP visualization probe ( 6) can coexist.
- sGC (3) and GMP visualization probe (6) are also introduced into the cell. (6) can coexist.
- each of the two subunits ⁇ (32) and (33) of the sGC (3) has a GMP visualization probe.
- the two subunits ⁇ (32) and sGC (3) in the cell (1) / 3 (33) may be dimerized to construct sGC (3) with GP visualization probe (6) linked to ⁇ (32) and
- the GMP visualization probe (6) in the sensor cell (1) has two marker sites that can detect proximity to each end of the cGMP-binding protein.
- the marker site is a combination of CFP and YFP
- the fluorescence intensity ratio (CFP / YFP) of CFP and YFP decreases if NO (2) concentration increases due to stimulation.
- the stimuli given at this time are hormones, endocrine disruptors, etc. It may be a biochemical stimulus or a physical stimulus such as electricity, radiation, or heat.
- the sensor cell (1) can also be used to detect and quantify NO (2) released from the cell. This is made possible by measuring the signal change in the sensor cell (1) by placing the sensor cell (1) in close proximity to the cell (hereinafter referred to as the donor cell) for detecting and quantifying the release of nitric oxide. Become. In other words, NO (2) released from the donor cell enters the sensor cell (1), and thus appears as a signal change in the sensor cell (1).
- sensor cells (1) can be used to screen for substances that affect NO (2) binding to sGC (3).
- a candidate substance is introduced into the sensor cell (1) and signal changes in the presence and! ⁇ Of the candidate substance are measured, or a candidate substance is introduced into the donor cell, and in the presence or absence of the candidate substance.
- this candidate substance can bind NO (2) to sGC (3). It is possible to determine whether or not to inhibit the combination.
- the invention of this application further provides a transgenic non-human animal in which all cells are sensor cells as described above. That is, sGC (3) and cGMP visualization probe (6) coexist in a cell (1) by any of the methods described above, and nonhuman totipotent cells are ontogenized, so that sGC (3 ) And cGMP visualization probe (6) can be obtained.
- a transgenic non-human animal can be produced according to a known production method (for example, Non-Patent Document 3).
- transgenic non-human animals have sGC (3) and cGMP visualization probes (6) in all somatic cells, and therefore, by measuring signal changes, in vivo concentrations of NO (2) Can be measured.
- stimulating in the living body by measuring the NO concentration in cells, tissues, organs, etc. by applying stimulation such as heat, electricity, radiation, etc. to the body, administering test substances such as pharmaceuticals, etc.
- monitoring the effects of various substances and screening various substances Is also possible.
- a transgenic non-human animal is used as a disease model animal such as a genetically disrupted animal having sGC (3) and cGMP visualization probe (6) in all somatic cells. By observing these differences, it is possible to obtain basic biological knowledge about the physiological effects of NO.
- Examples of signal measurement methods in transgene non-human animals include a fluorescence microscope, a confocal laser scanning microscope, a stereoscopic fluorescence microscope, a multiphoton laser scanning microscope, and the like.
- CGY cGMP visualization probe
- PK15 cells of Example 1 expressing sGC and CGY were placed in close proximity to neurons (neurons) prepared from the hippocampus of rat fetal brain, and the neurons were stimulated with glutamic acid known as a neurotransmitter.
- N0 donor cells when vascular endothelial cells and macrophages were used as N0 donor cells and PK15 of Example 1 expressing sGC and CGY in the same manner was placed close together, it was possible to visualize the release of N0 from these donor cells.
- the brains were removed from the 21-day-old rats and sliced to a thickness of 400 ⁇ 1, and the hippocampal region important for memory / learning was excised from there.
- N0 released from the hippocampus can be detected using the PK15 cells of Example 1 expressing sGC and CGY.
- many nerve cells form a network, and it is thought that the nerve cells are spontaneously activated to some extent without adding external stimulants.
- the method of the invention of this application has not been conventionally known except for the hippocampus, brain region, and vascular tissue. It is expected to enable spatiotemporal mapping of new NO release sites.
- Nitrogen monoxide (NO) detection sensor cells (hereinafter sometimes referred to as sensor cells) were cultured to release NO locally to verify the response of individual sensor cells.
- BN5Na As a local NO release agent, caged NO ⁇ , ⁇ '-dinitrosopiieiiylenendiaiine-N, N-diacetic acid sodium salt (BNN5Na) was added to the extracellular fluid of the sensor cells to 1 M. Since BN5Na is water-soluble, it exists outside the cell, and is characterized by releasing NO by ultraviolet light excitation.
- Fig. 8 (A) when the excitation light is focused and ultraviolet light is irradiated in a narrow region to release NO (local uncaging), Fig. 8 (B) As shown in Fig. 1, we compared the case where M) was emitted over a wide area without narrowing the excitation light (uniimalmal uncaging).
- Fig. 9 is a diagram showing changes in the fluorescence intensity ratio (CFP / YFP) of sensor cells caused by BNN5Na stimulation as pseudo color changes.
- FRET occurs and the fluorescence intensity ratio (CFP / YFP) decreases, and the pseudo color shifts blue.
- FIG. 8 (A) when NO was released locally, the sensor cells responded only around the center of the visual field from which NO was released, and a large change in the pseudo-force error was observed. On the other hand, the pseudo color has hardly changed in the vicinity (Fig. 9-2 (L)).
- Figure 9-1 shows the pseudo color change of a sensor cell before releasing N0 (before BN 5Na stimulation).
- Region-1 shows the change in the fluorescence intensity ratio (CFP / YFP) of the sensor cell in the region where NO is released in both the narrow region emission and the wide region emission
- region-3 Region-3) Shows the change in the fluorescence intensity ratio (CFP / YFP) of the sensor cells in the area where NO is released only in the wide area emission.
- Vascular endothelial cells, nerve cells, immune cells, and the like are known as cells that release NO.
- NO released from endodermal cells using a sensor cell for detecting NO is used. Spatio-temporal analysis was performed.
- Fig. 11 As illustrated in Fig. 1 (A), sensor cells are cultured on a cover glass, then vascular endothelial cells are cultured in a dish, and a cover glass with sensor cells attached thereon is placed. An experimental system was constructed.
- the sensor cells on the cover glass shown in Fig. 11 (B-1) and (B-2) and the endothelial cells on the dish shown in Fig. 11 (C) are observed. did. As shown in Fig. 1 1 (B) and (C), the sensor cells are spread over the entire field of view, and as shown in Fig. 1 1 (C), the endothelial cells are thinned and 0 in the center of the field of view. There is only one (the part enclosed by the solid line in the figure).
- FIG. 13 shows the change in fluorescence intensity ratio (CFP / YFP) in this experiment.
- Region 2 region-1 is a response of a sensor cell that exists directly above an endothelial cell
- region-2 region-2 is a response of a sensor cell that exists in the vicinity thereof.
- bradykinin-dependent fluorescence intensity ratio CFP / YFP
- Fig. 13 show that the sensor cell response continued immediately above the endothelial cells (region-1) even after 100 seconds after stimulation, but in the vicinity (region-2). It was confirmed that the sensor cell response was lost.
- the sensor cell for detecting NO of the invention of this application can perform spatiotemporal visual analysis of the diffusion of NO released from each cell.
- the method of the invention of this application can be applied not only to vascular endothelial cells but also to various cells such as nerve cells and immune cells.
- a sensor cell capable of easily detecting and quantifying low concentration of NO in a cell with high accuracy is provided.
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JPH10265497A (ja) * | 1997-03-27 | 1998-10-06 | Norin Suisansyo Sanshi Konchu Nogyo Gijutsu Kenkyusho | NO−r蛋白質S、該蛋白質Sを含む血管弛緩剤及び該蛋白質Sの部分配列を持つペプチド |
JP2000224986A (ja) * | 1999-02-03 | 2000-08-15 | Shingo Tsuyama | 可溶性グアニル酸シクラーゼに特異的なモノクローナル抗体 |
JP2002017359A (ja) * | 2000-07-04 | 2002-01-22 | Japan Science & Technology Corp | cGMP可視化プローブ及びそれを利用したcGMPの検出・定量方法 |
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JPH10265497A (ja) * | 1997-03-27 | 1998-10-06 | Norin Suisansyo Sanshi Konchu Nogyo Gijutsu Kenkyusho | NO−r蛋白質S、該蛋白質Sを含む血管弛緩剤及び該蛋白質Sの部分配列を持つペプチド |
JP2000224986A (ja) * | 1999-02-03 | 2000-08-15 | Shingo Tsuyama | 可溶性グアニル酸シクラーゼに特異的なモノクローナル抗体 |
JP2002017359A (ja) * | 2000-07-04 | 2002-01-22 | Japan Science & Technology Corp | cGMP可視化プローブ及びそれを利用したcGMPの検出・定量方法 |
Non-Patent Citations (1)
Title |
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OKADA DAISUKE.: "Naizaisei Phosphodiesterase Kassei o Mochiita Purkinje Saibonai Cyclic GMP Gosei no Kashika.", BIO IMAGING., vol. 6, no. 3, October 1997 (1997-10-01), pages 72 - 73, XP003005657 * |
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