JP5567777B2 - Signal transduction analysis method and signal transduction analysis system - Google Patents

Description

  The present invention relates to a signal transduction analysis method and a signal transduction analysis system for analyzing intracellular signal transduction.

  Previous studies on intracellular signal transduction have shown how protein dynamics are altered by extracellular stimuli and how downstream transcription of specific genes is regulated by these stimuli. It was confirmed by a separate experimental method. In addition, when investigating gene expression suppression, experiments have been conducted to detect how the expression of other genes is affected by suppressing the expression of the target gene. For example, when analyzing intracellular signal transduction, it is investigated how the genes upstream of the signal transduction cascade affect the expression of downstream genes. Furthermore, in the analysis of the signal transduction, if a part of the function of a certain gene is suppressed using RNAi or a dominant negative mutant, the influence on the downstream gene expression can be examined.

  Non-Patent Document 1 discloses a method relating to the time-dependent measurement of the amount of ATP in mitochondria in living cells.

H. J. et al. Kennedy, A.D. E. Pauli, E .; K. Ainscow, L.A. S. Joaville, R.A. Rizzuto, G.M. A. Rutter, “Glucose generators sub-plasma membrane ATP microdomains in single islet β-cells.”, Journal of Biological Chemistry, vol. 274, pp. 13281-13291, 1999

  However, in the prior art, the change in protein dynamics and the change in gene expression were confirmed by different experimental methods, and thus there was a problem that the reproducibility of the experiment was not always good.

  The present invention has been made in view of the above problems, and can confirm protein dynamic changes and gene expression fluctuations in one continuous experimental system. As a result, the reproducibility of experiments is improved. It is an object of the present invention to provide a signal transduction analysis method and a signal transduction analysis system that can be performed.

  In order to solve the above-described problems and achieve the object, a signal transduction analysis method according to the present invention is a signal transduction analysis method for analyzing intracellular signal transduction, and includes a predetermined fluorescently labeled protein and a luminescent label. A preparation step for producing the cell containing the predetermined gene, a stimulation step for giving a predetermined stimulus from the outside of the cell to the cell produced in the preparation step, and the predetermined stimulus was given in the stimulation step An imaging step of capturing a plurality of fluorescent images and luminescent images of the cells later, and dynamics of the predetermined protein in the cells by the predetermined stimulation based on the plurality of fluorescent images captured in the imaging step Change in the expression state of the predetermined gene due to the predetermined stimulus based on the plurality of luminescent images captured in the imaging step Characterized in that it comprises a, an analysis step of analyzing.

  The signal transduction analysis method according to the present invention is the signal transduction analysis method described above, wherein in the preparation step, the specific gene in the predetermined protein is labeled with a fluorescent protein upstream of the gene. The downstream side of the gene is labeled with a luminescence-related gene.

  The signal transduction analysis method according to the present invention is characterized in that, in the signal transduction analysis method described above, the predetermined protein is protein kinase C or low molecular weight G protein Ras.

  The signal transduction analysis method according to the present invention is characterized in that, in the signal transduction analysis method described above, the predetermined protein includes a dominant negative mutant.

  The present invention also relates to a signal transduction analysis system (signal transduction analysis system, signal transduction analysis device), and the signal transduction analysis system according to the present invention is a signal transduction analysis system for analyzing intracellular signal transduction. A stage (fluorescent labeling) that includes a cell prepared so as to include a predetermined fluorescently labeled protein and a predetermined luminescently labeled gene, and that holds the object before and after applying a predetermined stimulus to the cell. A stage containing a cell prepared so as to contain a predetermined protein and a luminescent-labeled predetermined gene, held before and after applying a predetermined stimulus to the cell) or both, A video rate fluorescent image and a time-lapse luminescent image of the subject. Based on the imaging device that captures a plurality of images, and the plurality of fluorescent images captured by the imaging device, the change in dynamics of the predetermined protein in the cells due to the predetermined stimulation is analyzed, and the imaging device And an image analysis device that analyzes fluctuations in the expression state of the predetermined gene due to the predetermined stimulus based on the plurality of light emission images captured in (1).

  In the signal transmission analysis system according to the present invention, in the signal transmission analysis system described above, the imaging device acquires an image on the same optical axis of the fluorescence imaging unit and the light emitting imaging unit (the imaging device is The fluorescent imaging unit and the light emitting imaging unit are configured to acquire an image on the same optical axis).

  The signal transduction analysis system according to the present invention is the signal transduction analysis system described above, wherein the image analysis device designates a cell region or site in the captured fluorescent image and / or luminescent image. Is further provided.

  According to the present invention, a cell containing a predetermined protein labeled with a fluorescent label and a predetermined gene labeled with a luminescence is prepared, and a predetermined stimulus is applied to the prepared cell from outside the cell. A plurality of fluorescence images and luminescence images of each cell are captured, and based on the captured fluorescence images, changes in the dynamics of a predetermined protein in cells due to a predetermined stimulus are analyzed, and a plurality of captured luminescence images are captured Based on the above, the variation in the expression state of a predetermined gene due to a predetermined stimulus is analyzed. Specifically, molecular dynamics that change in a short time are determined based on the fluorescence image, and then expression changes related to the desired molecular dynamics are analyzed based on the luminescence image. As a result, in the study of intracellular signal transduction, changes in protein dynamics and gene expression fluctuations can be confirmed in one continuous experimental system, and as a result, the reproducibility of experiments can be improved. Play.

  According to the present invention, for a specific gene in a predetermined protein, the upstream side of the gene is labeled with a fluorescent protein, and the downstream side of the gene is labeled with a luminescence-related gene. As a result, changes in the dynamics of a specific gene in response to a stimulus are observed in fluorescence, and changes in gene expression on the downstream side linked to the changes in the dynamics are observed in luminescence. It becomes possible to confirm simultaneously or continuously.

  According to the invention, the predetermined protein is protein kinase C or low molecular weight G protein Ras. Thereby, observation of the dynamic change of protein kinase C or low molecular weight G protein Ras and continuous observation from upstream to downstream of signal transduction in cells in response to stimulation can be performed in the same experimental system. There is an effect.

  According to the present invention, the predetermined protein includes a dominant negative mutant. Here, according to the prior art, the results of experiments on how specific genes downstream of signal transduction are transcriptionally regulated are obtained not as an analysis at the single cell level, but as the sum of gene expression levels in cell groups. It was. Therefore, in the signal transduction analysis, when transient gene introduction, which is a general technique, is performed, it is considered that the cell in which gene expression is inhibited and the luciferase reporter vector are not the same. Therefore, there is a problem that a highly accurate experimental result cannot always be obtained. Thus, in the present invention, intracellular signal transduction analysis combined with gene expression suppression is performed by putting the gene sequence of a dominant negative mutant for inhibiting gene expression into a predetermined fluorescently labeled protein. As a result, it is possible to confirm the suppression of the expression of the upstream gene in the intracellular signal transduction pathway and the expression fluctuation of the downstream gene in one continuous experimental system, and as a result, conduct an experiment that can obtain a highly accurate result. There is an effect that can be done. Specifically, in the study of intracellular signal transduction, after confirming that gene expression was suppressed by fluorescence detection, a quantitative reporter assay was performed with luminescence, so that when stimulation from outside the cell was received Experiment results on both inhibition of gene expression and effects on downstream gene expression can be seen as being in the same cell and due to one continuous experimental system, resulting in high accuracy results There is an effect that an experiment can be performed.

  Hereinafter, embodiments of a signal transmission analysis method and an analysis system according to the present invention will be described in detail based on the drawings corresponding to a microscope-based system. The present invention is not limited to the embodiments, and various modifications can be made based on the gist of the invention.

  First, the configuration of a fluorescence / luminescence observation system 100 that is an analysis system for executing a signal transmission analysis method (specifically, an imaging step and an analysis step) according to the present invention will be described with reference to FIGS. 1 and 2. . In addition, the details of the following configuration are better understood by referring to International Publication WO 2006/106882 (a predetermined site luminescence measurement method, a predetermined site luminescence measurement device, an expression level measurement method, and a measurement device) by the present applicant. Will be done. Although this international patent discloses a method for analyzing two types of medical information in the same cell using both fluorescent images and luminescent images, each information is not related to a series of related stimulus responses. It does not suggest the present invention, but is merely cited as a reference for operating methods and the like. FIG. 1 is a diagram illustrating an example of the overall configuration of the fluorescence / luminescence observation system 100.

  As shown in FIG. 1, a fluorescence / luminescence observation system 100 includes a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) that contains cells 102 to be analyzed, a container The stage 104 which arrange | positions 103, the light emission image imaging unit 106, the fluorescence image imaging unit 108, and the image-analysis apparatus 110 are comprised. Further, in the fluorescence / luminescence observation system 100, the objective lens 106a included in the luminescence image capturing unit 106 and the objective lens 108a included in the fluorescence image capturing unit 108 are vertically moved with the container 103 and the stage 104 interposed therebetween as shown in the figure. It is arranged on the substantially same optical axis at the facing position. The method of the present invention includes a stage for holding a target including cells prepared before and after applying a predetermined stimulus, an imaging device, and an image analysis device that performs analysis based on the captured fluorescent image and luminescent image. Executed by a system having Here, the analysis target may be a living tissue containing cells, various organs or organs containing the living tissue, or an embryo or an individual living organism containing such living tissue, organ or organ. In addition, the stage 104 for holding the analysis target can be used to move the target from both the fluorescence and emission observation fields (preferably on the optical axis) within a desired analysis period according to the type, size, number, etc. of the analysis target. You may make it provide the design (for example, the fixing tool for objects, the follow-up mechanism of a stage) so that the specific cell (one or more) to be analyzed may not remove | deviate.

  The arrangement of the light emitting image capturing unit 106 and the fluorescence image capturing unit 108 in the vertical direction may be interchanged. By disposing the light emitting image pickup unit 106 for measuring light emission weaker than fluorescence, disturbance light from above the sample due to opening and closing of the cover can be completely blocked, and the S / N ratio of the light emission image is increased. Can do. The fluorescent image capturing unit 108 that is separate from the light emitting image capturing unit 106 may be a laser scanning optical system.

  The cell 102 includes, for example, a predetermined protein fluorescently labeled with a fluorescent protein (eg, GFP, CFP, YFP, etc.) (for example, protein kinase C, low molecular weight G protein Ras, one containing a dominant negative mutant, etc.) and a luminescence-related gene. It is a living cell containing a predetermined gene (for example, gene of transcription factor NFκB1) labeled with luminescence (for example, luciferase gene). Immediately before imaging of the cell, the cell 102 is given a predetermined stimulus (for example, addition of chemical substances such as drugs or taking, application of physical energy such as electricity, light, magnetism, and ultrasound) from the outside of the cell. .

The luminescence image capturing unit 106 is specifically an upright luminescence microscope and captures a luminescence image of the cell 102. As shown in the figure, the luminescent image capturing unit 106 includes an objective lens 106a, a dichroic mirror 106b, a CCD camera 106c, and an imaging lens 106f. Specifically, the objective lens 106a preferably satisfies the value (numerical aperture / magnification) ² of 0.01 or more as an optical condition in consideration of the imaging lens to be used. The dichroic mirror 106b is used when light emitted from the cell 102 is separated by color and the amount of light emitted is measured by color using two colors of light. The CCD camera 106c takes a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the objective lens 106a, the dichroic mirror 106b, and the imaging lens 106f. The CCD camera 106c is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. The imaging lens 106f forms an image (specifically, an image including the cell 102) incident on the imaging lens 106f via the objective lens 106a and the dichroic mirror 106b. Note that FIG. 1 shows an example in which a light emission image corresponding to two light emissions separated by the dichroic mirror 106b is separately captured by the two CCD cameras 106c. The image capturing unit 106 may include an objective lens 106a, a single CCD camera 106c, and an imaging lens 106f.

The fluorescence image capturing unit 108 is specifically an inverted fluorescence microscope, and captures a fluorescence image of the cell 102. As shown in the figure, the fluorescence image capturing unit 108 includes an objective lens 108a, a dichroic mirror 108b, a light source 108c, a CCD camera 108d, an imaging lens 108e, and a shutter 108f. The objective lens 108a can be used with an arbitrary magnification depending on the object to be imaged. In this embodiment, the objective lens 108a has the same performance as the light-emitting objective lens 106a, that is, an optical system that takes into account the imaging lens to be used. As a condition, the value of (numerical aperture / magnification) ² satisfies 0.01 or more. The dichroic mirror 108b transmits the fluorescence from the cell 102 and changes the direction of the excitation light so that the excitation light emitted from the light source 108c is emitted to the cell 102. The light source 108c is for irradiating excitation light, and specifically, is a xenon lamp, a lamp such as a halogen, a laser, an LED, or the like. The CCD camera 108d takes a fluorescent image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 108d via the objective lens 108a, the dichroic mirror 108b, and the imaging lens 108e. The CCD camera 108d is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. The imaging lens 108e forms an image (specifically, an image including the cell 102) incident on the imaging lens 108e via the objective lens 108a and the dichroic mirror 108b. The shutter 108f switches the excitation light emitted from the light source 108c. In other words, the shutter 108f switches the irradiation of the excitation light to the cell 102 by transmitting or blocking the excitation light emitted from the light source 108c. Since the objective lenses 106a and 108a are arranged on the same optical axis, it is easy to align the images between the units. In this example, the optical conditions of the luminescent image capturing unit 106 and the fluorescent image capturing unit 108 (for example, the objective lenses 106a and 108a) are the same, so the arrangement of the objective lenses 106a and 108a and the stage 104 is changed. In addition, by arranging the other units so that they can be exchanged upside down or rotated, various images can be taken in the upside down direction or both up and down directions. There are also benefits. In addition, by sharing a part of the configuration (for example, objective lens, imaging lens, CCD camera, etc.) and arranging each unit of fluorescence and light emission only upward (inverted type) or downward (upright type), If the fluorescent image and the luminescent image are switched and acquired, the imaging direction can be made the same.

  Specifically, the image analysis apparatus 110 is a personal computer. As shown in FIG. 2, the image analysis apparatus 110 is roughly divided into a control unit 112, a clock generation unit 114 that measures the system time, a storage unit 116, a communication interface unit 118, and an input / output interface unit. 120, an input device 122, and an output device 124. These units are connected via a bus.

  The storage unit 116 is a storage unit. Specifically, a memory device such as a RAM or a ROM, a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like can be used. And the memory | storage part 116 memorize | stores the data etc. which were obtained by the process of each part of the control part 112. FIG. The communication interface unit 118 mediates communication between the image analysis apparatus 110 and the CCD camera 106c and the CCD camera 108d. That is, the communication interface unit 118 has a function of communicating data with other terminals via a wired or wireless communication line. The input / output interface unit 120 is connected to the input device 122 and the output device 124. Here, in addition to a monitor (including a home television), a speaker or a printer can be used as the output device 124 (hereinafter, the output device 124 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input device 122 can be a monitor that realizes a pointing device function in cooperation with the mouse. Here, the user includes one or more specific cells (or cell groups) to be analyzed within a desired analysis period based on the fluorescence image and / or the luminescence image displayed on the monitor as the output device 124. By specifying a region of interest or a site of interest in the cell via the input device 122, position information (address) of the specified region (or site) in each observation field of the specified region (or site) is stored in the storage unit 116. Remember. This stored information enables image analysis such that a specific cell (or group of cells) is collated for each of a plurality of regions (or parts) or time series. In this example, a bright-field image is also acquired and superimposed on the luminescent image, thereby enabling identification of each cell as a luminescent image. In this case, if a CCD camera or a luminescent reagent (luciferase, luciferin, or other additives) is highly sensitive, or if an image of the whole cell can be obtained simultaneously with a fluorescent image, superimposing bright-field images is not essential.

  The control unit 112 has an internal memory for storing a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data, and executes various processes based on these programs. . The control unit 112 is roughly divided into a fluorescent image capturing instruction unit 112a, a fluorescent image acquiring unit 112b, a luminescent image capturing instruction unit 112c, a luminescent image acquiring unit 112d, an image analyzing unit 112e, and an analysis result output unit. 112f.

  The fluorescence image capturing instruction unit 112a instructs the CCD camera 108d to capture a fluorescent image and a bright field image via the communication interface unit 118. The fluorescence image acquisition unit 112b acquires the fluorescence image and the bright field image captured by the CCD camera 108d via the communication interface unit 118. The luminescent image capturing instruction unit 112c instructs the CCD camera 106c to capture a luminescent image and a bright field image via the communication interface unit 118. The luminescent image acquisition unit 112d acquires the luminescent image and bright field image captured by the CCD camera 106c via the communication interface unit 118.

  The image analysis unit 112e analyzes changes in the dynamics of a predetermined protein in the cell 102 due to a predetermined stimulus based on the plurality of fluorescent images acquired by the fluorescent image acquisition unit 112b. In other words, the image analysis unit 112e analyzes how a predetermined protein moves in the cell 102 by a predetermined stimulus based on a plurality of fluorescent images. In addition, the image analysis unit 112e analyzes the variation in the expression state of a predetermined gene due to a predetermined stimulus based on the plurality of luminescent images acquired by the luminescent image acquisition unit 112d. Specifically, the image analysis unit 112e is configured to superimpose the acquired fluorescence image, bright field image, and luminescence image and associate and analyze the fluorescence and luminescence data for each cell, and based on the plurality of luminescence images. Then, how the expression of a predetermined gene is changed by a predetermined stimulus is quantified and analyzed. The analysis result output unit 112f outputs the analysis result of the image analysis unit 112e to the output device 124. Specifically, the analysis result output unit 112f graphs and displays on the output device 124 time-series numerical data regarding the variation in the expression state of a predetermined gene obtained by the image analysis unit 112e. In addition to the numerical data graph, the output device 124 may output a plurality of fluorescent and / or luminescent images corresponding to at least a part of the time-series numerical data to the monitor in a moving image or parallel display format. . Thus, according to the present invention, it is possible to quickly determine how protein dynamics are changed and how the transcription of a specific gene downstream is regulated by the stimulation for the same cell (or group of cells). It can also be analyzed in real time, providing fast and accurate information in medical research and clinical applications. As an analysis system for executing the method of the present invention, the fluorescence imaging unit and the luminescent image unit are arranged on different optical axes with respect to the same stage, or are separately provided on different stages. (For example, a fluorescence microscope and a light emission microscope), and imaging and analysis may be performed while sequentially moving a plurality of objects to be analyzed on the same or different stages. Further, as an analysis system, if it has at least an image analysis apparatus as shown in FIG. 2, it can be applied to an imaging system (for example, an endoscope or a fiberscope) other than the above-described microscope-based system. It is. In addition, when the target is a biological sample (cell, biological tissue, organ (or organ), etc.) isolated from a living body and cultured or artificially processed, the biological activity is maintained during a desired analysis period. It is preferable that the system is combined with an appropriate culture device. However, in the case of an individual, the same analysis can be performed by intermittently imaging while appropriately supplying or feeding oxygen and nutrients instead of the culture device. Is possible.

[Observation of PKC (Protein Kinase C) θ Membrane Translocation and NF (Nuclear Factor) κB1 Promoter Activity Changes]
PKC is a serine / threonine kinase that is involved in various cellular responses such as cell proliferation, differentiation, and apoptosis. PKC is activated cooperatively by calcium ions induced by diacylglycerol (DAG) produced from the cell membrane by receptor stimulation and inositol triphosphate produced simultaneously. It is known that PKC moves from the cytoplasm to the cell membrane (translocation) upon activation and exhibits kinase activity on the cell membrane.

  However, in the study of intracellular signal transduction so far, there is no choice but to confirm the change in the kinetics of PKC by the stimulation from outside the cell and the transcriptional regulation of the specific gene downstream by the stimulation by separate experimental methods. There was a problem in terms of sex.

  Therefore, in PKC activation signal transduction, the behavior of PKCθ labeled with GFP (Green Fluorescent Protein: green fluorescent protein) after stimulation with PMA (Phorbol Myristate Acetate) was observed, and the gene expression fluctuation of the downstream transcription factor was observed by luciferase Monitored by gene. That is, Ras signal system was used as a model of intracellular signal transduction system. After stimulation with growth factors, Ras behavior was observed by GFP, and downstream gene expression fluctuations were monitored by luciferase gene.

An experimental flow (procedure) in the first embodiment will be described below.
[Procedure 1 (corresponding to the production step of the present invention)] HeLa cells were cultured overnight in a 35 mm glass bottom dish. As the medium, DMEM (with phenol red and 10% FCS) was used.
[Procedure 2 (corresponding to the production step of the present invention)] After overnight culture, pPKCq-EGFP and pGL4-NFκB1 expression vectors were transfected into HeLa cells, which were further cultured overnight. As a transfection reagent, PolyFect (manufactured by QIAGEN) was used. The medium was replaced with DMEM (1% FCS, without phenol red) containing HEPES and cultured for 6 hours.
[Procedure 3 (corresponding to the stimulation step and imaging step of the present invention)] After stimulation with PMA (final concentration 5 ng / ml) and A2318 (final concentration 100 ng / ml), fluorescence transmission image and luminescence (chemiluminescence and / or bioluminescence) ) HeLa cells were observed using a luminescence imaging system “LV200 (manufactured by Olympus Corporation)” capable of imaging and observing three types of images and transmitted bright-field images. This luminescence imaging system has a configuration capable of culturing a sample containing cells, and further has a common imaging part (objective lens, imaging lens and CCD camera), illumination for exciting fluorescence and bright field illumination. And has a configuration that can selectively acquire the above three types of images, display various types of images individually, and perform image analysis in accordance with user instructions. Yes. Therefore, the user can output the image analysis result as described above by appropriately giving an instruction through the system interface. Here, HeLa cells were photographed at a video rate for 25 minutes at 1 minute intervals to observe the kinetics of PKCθ membrane translocation (see FIG. 3). Excitation BP470-490 and spectral BP535-555 were used for the spectral filter. As fluorescence observation conditions, the objective lens is × 40, the exposure time is 200 milliseconds, the binning is 1 × 1, and the CCD camera is ImagEM (manufactured by Hamamatsu Photonics). For each cell to be observed, the entire interface of the cells, in which the luminance distribution in the cell is seen to change, is displayed among the plurality of cells displayed in the fluorescence images before and after the stimulus displayed on the monitor by the system interface. Drag specified on the screen with the input mouse.
[Procedure 4 (corresponding to the imaging step of the present invention)] For observation of luminescence, D-luciferin (manufactured by Promega: final concentration 100 mM) was added, and images were taken at 15 minute intervals for 10 hours (see FIG. 4). As light emission observation conditions, the objective lens is × 40, the exposure time is 10 minutes, the binning is 1 × 1, and the CCD camera is ImagEM (manufactured by Hamamatsu Photonics).
[Procedure 5 (corresponding to the analysis step of the present invention)] After the time-lapse observation, the observation image captured in the procedure 3 and the procedure 4 is stored and observed using the image analysis system “AQUACOSMOS (manufactured by Hamamatsu Photonics)”. The numerical data corresponding to the luminescence intensity of each site in the cell was analyzed in time series from the image, and the analyzed numerical data was graphed (see FIG. 5). The intracellular part to be observed is specified by reading out and displaying the image after storing the luminescent image captured by time lapse on the monitor and displaying the brightness corresponding to the luminescence intensity in the cell displayed in the luminescent image displayed on the monitor. A plurality of different parts (four parts in the figure) were pointed on the screen with an input mouse (parts in a block of a fixed area surrounded by a square in the figure). For the designated point, a block having a constant area is assigned to each point (1 to 4 in the figure) as shown in FIG. 3, and the intensity value of all pixels in the block (for each point (1 to 4)) ( Arbitrary units) were totaled to obtain emission intensity data at each time point.

  As a result of the above experiments, it is possible to observe the membrane transfer of PKCθ labeled with GFP and the continuous observation from the upstream to the downstream of signal transduction in response to the stimulation in the same experimental system, and the molecular dynamics It became clear that analysis of different response speeds such as gene expression fluctuations can be carried out while following every cell. In addition, when specifying by observation or input, specify the site in the cell only from the fluorescent image displayed on the monitor and start capturing the luminescent image immediately after that, confirming the upstream gene change It is also possible to observe and / or analyze changes in expression of downstream genes in real time for the obtained cells. In addition, after capturing both the fluorescent image and the luminescent image, the stored fluorescent image and luminescent image are displayed on the monitor, so that the dynamics of the upstream gene observed at different times and the expression variation of the downstream gene can be observed. Comparison observation can be performed, and it is also possible to analyze a comprehensive stimulus response correlation regarding a cell population in the observation visual field. In addition, the imaging speed of the fluorescent image and / or the luminescent image may be changed so as to be easily observed and / or analyzed by increasing or decreasing the video rate and / or time lapse time interval. In parallel with the light emission intensity, a video image based on the fluorescent image and / or a moving image based on the light emission image (or a parallel display of frame advance) may be simultaneously output to a monitor or the like.

[Inhibition of Ras molecular gene expression and observation of changes in AP1 gene promoter activity]
Ras / Raf signals are widely known as intracellular signal transduction pathways that regulate cell proliferation and differentiation in response to extracellular stimuli. Inactive Ras is rapidly diffusing on the cell membrane, but it is known that active Ras stays on the cell membrane upon activation. Furthermore, Ras activates the MAP kinase cascade, causes an increase in downstream transcription factor activity, and ultimately controls cell differentiation and proliferation.

  However, in the study of intracellular signal transduction so far, the change in Ras kinetics caused by stimulation from outside the cell and the transcriptional regulation of specific genes downstream by the stimulation can only be confirmed by separate experimental methods. There was a problem in terms of sex.

  Therefore, in Ras signaling, N17 mutant, which is a dominant negative of Ras labeled with GFP (Green Fluorescent Protein), was introduced into cells, confirmation of gene expression inhibition by GFP, and luciferase as an index The gene expression of the transcription factor downstream of the signal transduction was observed. That is, the Ras signal system was used as a model of the intracellular signal transduction system, the behavior of Ras was observed by GFP after stimulation with a growth factor, and then the fluctuation in downstream gene expression was monitored by the luciferase gene.

The flow of the experiment (procedure) in the second embodiment will be described below.
[Procedure 1 (corresponding to the production process of the present invention)] PC12 cells were cultured overnight in a 35 mm glass bottom dish. RPMI (phenol red, containing 10% FCS) was used as the medium.
[Procedure 2 (corresponding to the production process of the present invention)] After overnight culture, pPCI-EGFP-RBD (Wild type) and pGL4-AP1 expression vector or pPCI-EGFP-RBD + N17 (dominant negative type) and pGL4-AP1 expression vector Was transfected into PC12 cells, which were further cultured overnight (see FIG. 6). Lipofectamine 2000 (manufactured by Invitrogen) was used as a transfection reagent. The medium was replaced with RPMI with HEPES (1% FCS, without phenol red) and cultured for 6 hours.
[Procedure 3 (corresponding to the stimulation step and imaging step of the present invention)] After stimulation with NGF (final concentration 5 ng / ml), fluorescence transmission image, luminescence (chemiluminescence and / or bioluminescence) image, and transmission brightfield image 3 Using a luminescence imaging system “LV200 (manufactured by Olympus)” capable of various types of imaging and observation, fluorescence observation of PC12 cells was performed. Raf membrane translocation was observed by photographing PC12 cells at 1 minute intervals for 25 minutes (see FIGS. 7, 8 and 9). Excitation BP470-490 and spectral BP535-555 were used for the spectral filter. As fluorescence observation conditions, the objective lens is × 40, the exposure time is 200 milliseconds, the binning is 1 × 1, and the CCD camera is ImagEM (manufactured by Hamamatsu Photonics). About the imaged two-dimensional fluorescence image, the fluorescence amount on the AB disconnection described in the photograph of FIG. 7 was graphed (see FIG. 7). The designation for each cell to be observed is the same as that in Example 1, but in this example, a desired cross section is dragged in a linear shape in the designated cell image. For each pixel along the designated straight line, the luminance value of each pixel (the total value for the number of columns in the case of multiple columns) was used as the fluorescence amount at each position.
[Procedure 4 (corresponding to the imaging step of the present invention)] For luminescence observation, D-luciferin (manufactured by Promega: final concentration 100 μM) was added, and luminescence images were taken by time-lapse observation at 15-minute intervals for 14 hours. As light emission observation conditions, the objective lens is × 40, the exposure time is 10 minutes, the binning is 1 × 1, and the CCD camera is ImagEM (manufactured by Hamamatsu Photonics).
[Procedure 5 (corresponding to the analysis step of the present invention)] After the time-lapse observation, the observation image taken in steps 3 and 4 is stored, and an image is obtained using a commercially available image analysis system “AQUACOSMOS (manufactured by Hamamatsu Photonics)”. A plurality of cells (9 cells) are selected on the screen, the numerical data of the luminescence intensity after stimulation for each individual cell selected from the observed image is analyzed by the system, and the analyzed numerical data is time-series (See FIG. 10). The emission intensity data relating to the designation of the intracellular site to be observed and the emission amount at each point after the designation was performed in the same manner as in Example 1. For each point, a large number of points (9 in this example) were randomly specified in the cell.

  As a result of the above experiment, in PC12 cells into which pPCI-EGFP-RBD + N17 and the pGL4-AP1 expression vector were introduced, the site of high expression level was concentrated in the central part of the cell (ie, the region in the cell membrane) before stimulation. However, the highly expressed site was transferred to both ends of the cell (ie, near the cell membrane) after stimulation (see FIG. 7). From this, it can be seen that the membrane transfer of the EGFP-labeled Raf-1 molecule occurs and a signal is transmitted downstream. On the other hand, when PC12 cells introduced with pPCI-EGFP-RBD + N17 and the pGL4-AP1 expression vector were photographed at 1 minute intervals for 25 minutes, it was confirmed that Raf membrane translocation did not occur (see FIGS. 8 and 9). .

  In addition, when the expression level of the AP1 gene, which is a signal transduction downstream gene, was monitored in cells in which the introduction of dominant negative N17 was confirmed, it was confirmed that expression suppression occurred compared to the wild type without N17 introduction. (See FIG. 10). However, even when N17 is introduced, the expression level is not completely lost, and a slight increase in the expression of the AP1 gene is observed (see FIG. 10). It is also possible that signals were transmitted to AP1 from cascades other than Ras due to duplication of intracellular signal transduction pathways.

  From the above, observation of Raf-N17 labeled with GFP and continuous observation from upstream to downstream of signal transduction in the cells in response to the stimulation became possible in the same experimental system. In elucidating such complex intracellular signal transduction pathways, the degree to which downstream pathway genes are affected by suppressing specific signal transduction pathways using methods such as dominant negative mutants and RNAi. It is possible to check whether it is.

  As described above, the signal transduction analysis method according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

1 is a diagram illustrating an example of an overall configuration of a fluorescence / luminescence observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of an image analysis device 110 of the fluorescence / luminescence observation system 100. FIG. It is a figure which shows the fluorescence image which caught the film | membrane transfer of PKC (theta). It is a figure which shows the light emission image which caught the expression change of NF (kappa) B1 gene. It is a figure which shows the graph showing the change of the expression level of NF (kappa) B1 gene after PMA stimulation. It is a figure which shows typically the vector introduce | transduced into a cell. It is a figure which shows the expression level and the high expression site | part in the cell before and behind stimulation. It is a figure which shows the expression level before the stimulation in the cell in which dominant negative N17 was introduce | transduced, and the state of the membrane transfer of Raf. It is a figure which shows the expression level after stimulation in the cell in which dominant negative N17 was introduce | transduced, and the state of the membrane transfer of Raf. It is a figure which shows the graph showing the time-dependent change of AP1 promoter activity in each cell after dominant negative N17 introduction | transduction.

Explanation of symbols

100 Fluorescence / luminescence observation system 103 Container (petri dish)
104 Stage 106 Luminous imaging unit
106a Objective lens (for light emission observation)
106b Dichroic mirror
106c CCD camera
106f Imaging lens 108 Fluorescence imaging unit
108a Objective lens (for fluorescence observation)
108b Dichroic mirror
108c Light source
108d CCD camera
108e Imaging lens
108f Shutter 110 Image analysis device
112 Control unit
112a Fluorescence image capturing instruction unit
112b Fluorescence image acquisition unit
112c Luminescent image capturing instruction unit
112d Luminescent image acquisition unit
112e Image analysis unit
112f Analysis result output part
114 Clock generator
116 storage unit
118 Communication interface
120 I / O interface section
122 Input device
124 Output device

Claims (7)

A signal transduction analysis method for analyzing intracellular signal transduction,
A stimulation step of providing from the extracellular to the cells fabricated to include a predetermined gene linked to a gene encoding a given protein and luminescent labels which are fluorescent labeled predetermined stimulus,
An imaging step of capturing a plurality of fluorescence images and luminescence images of the cells after the predetermined stimulus is given in the stimulation step, respectively in time series by a video rate of the fluorescence images and a time lapse of the luminescence images ;
Based on the plurality of fluorescent images captured in the imaging step, the change in the dynamics of the predetermined protein in the cells due to the predetermined stimulus is analyzed, and the plurality of luminescent images captured in the imaging step are analyzed. Based on the analysis step of analyzing the variation of the expression state of the predetermined gene due to the predetermined stimulus,
A signal transduction analysis method comprising: The cells for a particular gene in the given protein, according to claim 1 for the upstream side of the gene Rutotomoni is labeled with a fluorescent protein, wherein Rukoto labeled downstream of the gene in emission-related genes The signal transduction analysis method according to 1. The signal transduction analysis method according to claim 1 or 2, wherein the predetermined protein is protein kinase C or low molecular weight G protein Ras. The signal transduction analysis method according to claim 1, wherein the predetermined protein includes a dominant negative mutant. A signal transduction analysis system for analyzing intracellular signal transduction,
A cell prepared so as to contain a predetermined gene linked to a gene encoding a fluorescent-labeled predetermined protein and a luminescent label, and holding a subject before or after applying a predetermined stimulus to the cell Stage,
An imaging device that captures a plurality of video rate fluorescent images and time-lapse emission images of the object held on the stage in time series according to the video rate and the time-lapse ;
Based on a plurality of the fluorescence images imaged by the imaging device, a change in dynamics of the predetermined protein in the cell due to the predetermined stimulation is analyzed, and a plurality of the luminescent images captured by the imaging device Based on the image analysis device for analyzing the variation of the expression state of the predetermined gene by the predetermined stimulus,
A signal transduction analysis system comprising: The signal transmission analysis system according to claim 5, wherein the imaging device acquires an image on the same optical axis of the fluorescence imaging unit and the light emitting imaging unit. The signal transmission analysis system according to claim 5, wherein the image analysis device further includes an input device for designating a cell region or a site in the captured fluorescence image and / or luminescence image.