JP2015019637A - Method for analyzing cells for survivin, and method for analyzing cancer utilizing it, and method for screening drug efficacy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing cells for the intracellular dynamics of survivin protein, and survivin promoter activity.SOLUTION: A method for analyzing cells comprises (1) preparing a first vector comprising a survivin promoter and a gene encoding photoprotein, and a second vector that is an expression vector comprising a promoter, a survivin gene, and a gene encoding fluorescence protein; (2) introducing the first vector and the second vector into a cell; (3) exposing the cell of the (2) to a drug; (4) observing the bright field, measuring the fluorescence intensity, and measuring the luminescence intensity, respectively at at least two points in time within duration over before and after the cell is exposed to the drug; and (5) obtaining information about the cell from the result of the (4).

Description

  The present invention relates to a method for analyzing cells for expression of survivin protein.

  Survivin protein is a protein having a function of suppressing apoptosis and regulating cell division. Survivin protein is hardly observed in normal tissues, but is known to be highly expressed in fetal tissues and many cancers. It has also been proposed to evaluate the metastasis rate and prognosis of cancer by specifying the localized site of survivin.

  Under such circumstances, survivin is attracting attention as a target for cancer treatment, and the creation of survivin expression inhibitors has been started.

Chiou, S .; K. Jones, M .; K. and Tarnawaski, A. et al. S (2003). Med. Sci. Monitor. 9,125-129 Li F.R. , Ambrosini G. , Chu EY, et al. , Nature 1998; 396: 580-584. Tanaka K, et al. , Clin Cancer Res 2000; 6: 127-134. Lehner R, et al. , Appl Immunohistomol Mol Morpol 2002; 10: 134-138.

  In view of the above situation, it is considered useful to detect the intracellular dynamics and promoter activity of survivin in basic cancer research and drug screening targeting survivin.

  The subject of this invention is providing the method of performing a cell analysis about the intracellular dynamics and survivin promoter activity of survivin protein. More specifically, it is to provide a method for observing the intracellular kinetics and survivin promoter activity of survivin protein simultaneously, for example, simultaneously in one tissue and / or one cell.

  In order to solve the above problems, the present invention provides a cell analysis method. The cell analysis method includes (1) a first vector including a survivin promoter and a gene encoding a photoprotein, and an expression vector including a promoter, a survivin gene, and a gene encoding a fluorescent protein. (2) introducing the first vector and the second vector into a cell, (3) exposing the cell of (2) to a drug, (4) ) The bright field observation, the fluorescence intensity measurement, and the luminescence intensity measurement are performed at at least two time points within the period before and after the cell is exposed to the drug, respectively, and the results of (5) and (4) Obtaining information on the cell from

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for performing cell analysis on intracellular kinetics and survivin promoter activity of survivin protein. Thereby, the intracellular kinetics of survivin protein and the survivin promoter activity can be simultaneously observed, for example, in one tissue and / or one cell.

FIG. 3 shows a vector according to Embodiment 1. FIG. 4 is a schematic diagram illustrating a configuration of an observation system according to a second embodiment. FIG. 4 is a block diagram showing an observation system according to Embodiment 2. 9 is a flowchart showing a processing procedure according to the second embodiment. FIG. 6 is a schematic diagram illustrating a configuration of a microscope apparatus according to a third embodiment. 10 is a flowchart showing a processing procedure according to the third embodiment. FIG. 6 is a schematic diagram illustrating a configuration of a microscope apparatus according to a fourth embodiment. The graph which shows the excitation spectrum (Ex) and fluorescence spectrum (Em) of D-luciferin. The figure which shows the phase-difference observation image of the HeLa cell before the chemical | medical agent stimulation in Example 1. FIG. The figure which shows the fluorescence observation image of the HeLa cell before chemical | medical agent stimulation in Example 1. FIG. The figure which shows the light emission observation image of the HeLa cell before chemical | medical agent stimulation in Example 1. FIG. FIG. 4 is a schematic diagram illustrating timings of image capturing in time-lapse observation of Example 1. The figure which shows the phase-difference observation image of the HeLa cell at the time of 30-minute progress after the chemical | medical agent stimulation in Example 1. FIG. The figure which shows the fluorescence observation image of the HeLa cell 30 minutes after the chemical | medical agent stimulation in Example 1. The figure which shows the light emission observation image of the HeLa cell at the time of 30-minute progress after the chemical | medical agent stimulation in Example 1. FIG. The graph which shows the time change of the emitted light intensity in the designated ROI shown by FIG. 10 and FIG.

  Hereinafter, Embodiment 1 of the present invention will be described in detail. The first embodiment relates to a method for obtaining information on the expression level and distribution of survivin genes in cells, the degree of survivin promoter activity, and morphology.

  The expression of the survivin gene in the subject is high when the subject is in a particular condition, such as cancer. In addition, the distribution of survivin protein varies depending on the state of the tissue or cell in which it exists.

  Therefore, it is possible to determine what state a specific cell or tissue is in, or whether it is in a specific cell, from information on the degree of survivin gene expression and the distribution of survivin protein. One embodiment is a cell analysis method for survivin that utilizes this principle.

  For example, when a survivin gene is highly expressed in a specific sample, for example, a tissue or a cell, it can be determined that the sample is a cancerous sample, that is, a cancer tissue or a cancer cell. is there. A further embodiment is a cancer analysis method that utilizes this principle.

  Alternatively, whether or not the test substance has a specific action, for example, an anticancer action, using as an index how the survivin gene expression and survivin protein distribution change before and after the cancer cell is exposed to the test substance. It is possible to evaluate. Another embodiment is a drug efficacy screening method that utilizes this principle.

  By using vectors, it is possible to obtain information on survivin gene expression and survivin protein distribution.

The cell analysis method may comprise the following steps;
(1) A first vector comprising a survivin promoter and a gene encoding a photoprotein operably linked downstream thereof, a promoter, a survivin gene operably linked downstream thereof, and a downstream thereof Providing a second vector that is an expression vector comprising a gene encoding a functionally linked fluorescent protein;
(2) introducing the first vector and the second vector into the collected and / or cultured cells;
(3) exposing the cell of (2) to a drug;
(4) Observing the bright field, measuring the fluorescence intensity, and measuring the emission intensity at at least two time points within the period before and after the cell is exposed to the drug, and (5) (4 ) To obtain information on the cells from the results.

  As the vector used in the embodiment, a first vector and a second vector are used. The first vector and the second vector will be described with reference to FIGS. 1 (1) and (2).

  The first vector 1 shown in FIG. 1 (1) is a vector for measuring the degree of survivin gene promoter activity. The first vector 1 includes a survivin promoter 2 and a first reporter gene 3 operably linked downstream thereof. Here, “operably linked” means that the first reporter gene 3 is bound downstream of the survivin promoter 2 so that the reporter gene 3 is normally expressed under the control of the survivin promoter 2. It means being.

The survivin promoter 2 may be any survivin promoter known per se, and preferably a survivin promoter of a type selected depending on the sample used. For example, when analyzing human cells and tissues, it is preferable to use a human survivin promoter. The sequence of the human survivin promoter is shown in Table 1 as SEQ ID NO: 1.

  The human survivin promoter represented by SEQ ID NO: 1 used as the survivin promoter may have one to several bases deleted, substituted and / or added as long as the function as the survivin promoter is maintained. . Similarly to any other known survivin promoter, one to several bases may be deleted, substituted and / or added as long as any known promoter sequence maintains its own survivin promoter activity. It may be.

  The first reporter gene 3 may be a gene encoding any known photoprotein. Examples of the first reporter gene 3 include luciferases (for example, firefly luciferase gene, Renilla luciferase gene, aequorin gene, oberin gene, click beetle luciferase gene, hyodosievir luciferase gene) and fluorescent protein genes (for example, BFP gene, CFP gene) GFP gene, YFP gene, RFP gene), etc., preferably luciferase.

  The first vector may further comprise additional genes such as the neo gene, β-gal gene, cat gene, and hyg gene. Furthermore, a transcription termination gene such as poly A may be included downstream of the first reporter gene 3. A commercially available vector containing a gene encoding a photoprotein may be used as the first vector. For example, a survivin promoter may be incorporated upstream of the gene encoding the photoprotein of such a commercially available vector. For such integration, for example, genetic engineering techniques such as restriction enzyme digestion and ligase ligation may be used. Alternatively, the first vector may be produced from a polynucleotide comprising a sequence including the survivin promoter 2 and the first reporter gene 3 operably linked downstream thereof by genetic engineering techniques. The first vector may be produced using a technique known per se.

  The first vector may be a plasmid vector or a viral vector, but a plasmid vector is preferred.

  The second vector 4 shown in FIG. 1 (2) is an expression vector, which is a vector for showing the distribution of survivin protein. The second vector 4 includes a strong promoter, a survivin gene operably linked downstream thereof, and a gene encoding a second reporter protein operably linked downstream thereof. Here, “operably linked” means that the target gene is bound so that the target function can be expressed, that is, the survivin gene can be expressed, or the second reporter protein can be expressed. It means being.

  A strong promoter is a promoter that controls to force reading of a gene linked downstream thereof. A strong promoter may be any promoter known per se generally used in expression vectors. Examples of strong promoters may be the cytomegalovirus promoter, the Simian vacuating virus 40 (SV40) promoter, and the respiratory synchronous viral (RSV) promoter.

The survivin gene may be any survivin gene known per se, for example, human, chimpanzee, marmoset, xenopus, zebrafish, Drosophila, nematode and mouse survivin genes. Preferably, it may be a survivin gene of a type selected depending on the sample used. For example, when analyzing human cells and tissues, it is preferable to use a human survivin gene. The sequence of the human survivin gene is shown in Table 2 as SEQ ID NO: 2.

  The human survivin gene represented by SEQ ID NO: 2 used as the survivin gene has one to several bases deleted as long as the function as the survivin gene and the activity as the survivin protein to be expressed are maintained. It may be substituted and / or added. Similarly for any other known survivin gene, one to several bases may be used as long as the function as its own survivin gene and the activity as the expressed survivin protein are maintained for any known sequence. May be deleted, substituted and / or added.

The gene encoding the second reporter protein may be a gene encoding a fluorescent protein, and preferably emits fluorescence from a luminescent protein used as the first reporter protein and a luminescence-related substance such as a substrate of the luminescent protein. Fluorescent proteins with an excitation wavelength on the longer wavelength side are preferred over possible excitation and fluorescence spectra. Specifically, for example, when luciferase is used as the first reporter protein, luciferin is used as the substrate. Luciferin itself fluoresces by a short wavelength. Therefore, it is desirable to select the second reporter protein as a fluorescent protein having a wavelength that does not cause fluorescence from luciferin. Examples of preferred fluorescent proteins are yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP), Venus, PhiYFP, YsYello, mBana, KusbiraOrange, mOrm, Hsr, C And mStrawberry. As an example of the sequence of a gene encoding a preferred fluorescent protein, the sequence of EYFP is shown in Table 3.

  The second vector may further comprise additional genes such as the neo gene, β-gal gene, cat gene, and hyg gene. Furthermore, the second vector may contain a transcription termination gene such as poly A downstream of the second reporter gene. A commercially available vector containing a strong promoter and / or a gene encoding a fluorescent protein may be used as the second vector. For example, in such a commercially available vector, a survivin gene may be incorporated downstream of a strong promoter and upstream of a gene encoding a fluorescent protein. For such integration, for example, genetic engineering techniques such as restriction enzyme digestion and ligase ligation may be used. Alternatively, it comprises a sequence comprising a strong promoter, a survivin gene operably linked downstream thereof, and a gene encoding a second reporter protein operably linked downstream thereof by genetic engineering techniques. A second vector may be produced from the polynucleotide. The second vector may be produced using a technique known per se.

  The second vector may be a plasmid vector or a viral vector, but a plasmid vector is preferred.

  Introduction of the first vector and the second vector into the sample may be performed, for example, by the calcium phosphate method, lipofection method, DEAE dextran method, electroporation method, and microinjection method.

  The first vector and the second vector introduced into the sample function independently of each other. The first vector expresses the first reporter protein according to the activity of the survivin promoter depending on the environment in the sample. The second vector forcibly expresses the fusion protein of survivin and fluorescent protein by a strong promoter. The expressed fusion protein is distributed in the sample depending on the environment in the sample.

  A “sample” may be a cell and / or tissue. The tissue may be a tissue collected from a living body, a tissue collected from a living body and cultured, or a section cut from a tissue collected or collected from a living body and cultured. A cell may be a cell collected from a living body, a cell collected and isolated from a living body, a cultured cell, or a cell cultured after being collected from a living body. Alternatively, it may be a subcultured cell. The cell may be prepared by digesting a tissue collected from a living body with an enzyme. The sample may be an individual organism, an embryo, or an egg.

  After introducing the first vector and the second vector into the sample, the morphology of specific cells contained in the sample is observed in a bright field, and the luminescent signal based on the first vector, The fluorescence signal based on the vector is measured sequentially. At this time, the morphological observation and the measurement of luminescence and fluorescence signals are performed on specific cells contained in the sample. Such morphological observation and measurement of luminescence and fluorescence signal can be performed by specifying a common region of interest (Region of Interest, ROI), thereby allowing bright field observation, measurement of luminescence signal and fluorescence of the same cell. It is possible to use an observation apparatus or an observation system that can measure a signal. Bright field observation, emission intensity measurement, and fluorescence intensity measurement may be performed on an image captured by such an observation apparatus and observation system.

  As an example of such an observation system, a second embodiment will be described with reference to FIGS. In addition, this invention is not limited by this embodiment.

  As shown in FIG. 2, the expression level measuring apparatus 1000 includes a cell 1020, a container 1030 containing the cell 1020 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.), and a container 1030. , A light emitting image capturing unit 1060, a fluorescence image capturing unit 1080, and an information communication terminal 1100. In the expression level measuring apparatus 1000, the objective lens 1060a included in the luminescent image capturing unit 1060 and the objective lens 1080a included in the fluorescent image capturing unit 1080 are sandwiched between a cell 1020, a container 1030, and a stage 1040 as illustrated. It arrange | positions in the upper and lower opposing position. Note that the arrangement of the luminescent image capturing unit 1060 and the fluorescence image capturing unit 1080 may be interchanged.

  The cell 1020 was introduced with a luminescence-related gene that expresses a photoprotein (specifically, for example, luciferase) in combination with a survivin promoter, and a fluorescence-related gene that expresses a fluorescent protein (specifically, for example, YFP) in combination with a survivin gene. It is alive. Here, in this specification, the light emission is a concept including bioluminescence and chemiluminescence.

  Note that the cell 1020 may be a living cell into which a fusion gene obtained by fusing a luminescence-related gene and a fluorescence-related gene is introduced. Specifically, the cell 1020 may be a living one into which a vector in which a luminescence-related gene and a fluorescence-related gene are fused is introduced. In other words, a plurality of sets of luminescence-related genes or fluorescence-related genes combined with the survivin promoter or survivin gene to be analyzed may be introduced into the cell 1020. Thereby, the survivin promoter activity and survivin gene expression level introduced into the cell 1020 are measured together, and in addition to these, the cell morphology is observed in a bright field. That is, the expression level of survivin gene is measured by fluorescence, the degree of survivin promoter activity is measured by luminescence, and the morphology of cells is observed in a bright field. Specifically, the cell 1020 may be a living one into which the first vector is introduced and the second vector is introduced.

The luminescence image capturing unit 1060 is specifically an upright luminescence microscope and captures a luminescence image of the cell 1020. As shown in the figure, the luminescent image capturing unit 1060 includes an objective lens 1060a, a dichroic mirror 1060b, and a CCD camera 1060c. Specifically, the objective lens 1060a has a value of (numerical aperture / magnification) ² of 0.01 or more. The dichroic mirror 1060b is used when light emitted from the cell 1020 is separated by color and the light emission intensity is measured by color using two colors of light emission. The CCD camera 1060c takes a light emission image and a bright field image of the cell 1020 projected onto the chip surface of the CCD camera 1060c via the objective lens 1060a. Further, the CCD camera 1060c is connected to the information communication terminal 1100 so as to be communicable by wire or wirelessly. Here, when there are a plurality of cells 1020 in the imaging range, the CCD camera 1060c may capture a light-emitting image and a bright-field image of the plurality of cells 1020 included in the imaging range. Note that FIG. 2 shows an example in which two CCD cameras 1060c separately capture light emission images corresponding to two light emissions separated by the dichroic mirror 1060b. The image capturing unit 1060 may include an objective lens 1060a and one CCD camera 1060c.

  The fluorescence image capturing unit 1080 is specifically an inverted fluorescence microscope, and captures a fluorescence image of the cell 1020. As shown in the figure, the fluorescence image capturing unit 1080 includes an objective lens 1080a, a dichroic mirror 1080b, a xenon lamp 1080c, and a CCD camera 1080d. The CCD camera 1080d takes a fluorescent image and a bright field image of the cell 1020 projected onto the chip surface of the CCD camera 1080d via the objective lens 1080a. Further, the CCD camera 1080d is connected to the information communication terminal 1100 so as to be able to communicate with each other by wire or wirelessly. Here, when there are a plurality of cells 1020 in the imaging range, the CCD camera 1080d may capture fluorescent images and bright-field images of the plurality of cells 1020 included in the imaging range. The dichroic mirror 1080b transmits the fluorescence from the cell 102 and changes the direction of the excitation light so that the excitation light emitted from the xenon lamp 1080c is emitted to the cell 1020. The xenon lamp 1080c emits excitation light.

  Here, specifically, the luminescence image capturing unit 1060 and the fluorescence image capturing unit 1080 may be an inverted luminescence microscope and an inverted fluorescence microscope, respectively, and the stage 1040 may be rotated.

  The information communication terminal 1100 is specifically a personal computer. As shown in FIG. 3, the information communication terminal 1100 is roughly divided into a control unit 1120, a clock generation unit 1140 that measures the system time, a storage unit 1160, a communication interface unit 1180, and an input / output interface unit. The configuration includes 1200, an input unit 1220, and an output unit 1240, and these units are connected via a bus.

  The storage unit 1160 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. The storage unit 1160 stores data obtained by processing of each unit of the control unit 1120.

  The communication interface unit 1180 mediates communication between the information communication terminal 1100 and the CCD camera 1060c and the CCD camera 1080d. That is, the communication interface unit 1180 has a function of communicating data with other terminals via a wired or wireless communication line.

  The input / output interface unit 1200 is connected to the input unit 1220 and the output unit 1240. Here, in addition to a monitor (including a home TV), a speaker or a printer can be used as the output unit 1240 (hereinafter, the output unit 1240 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input unit 1220 can be a monitor that realizes a pointing device function in cooperation with the mouse.

  The control unit 1120 has an internal memory for storing control programs such as an OS (Operating System), programs that define various processing procedures, and necessary data, and executes various processes based on these programs. . The control unit 1020 is roughly divided into a fluorescence image capturing instruction unit 1120a, a luminescent image capturing instruction unit 1120b, a fluorescence image acquiring unit 1120c, a luminescent image acquiring unit 1120d, a determining unit 1120e, and a fluorescence measuring unit 1120f. , A luminescence measuring unit 1120g, a selecting unit 1120h, an expression level measuring unit 1120i, a bright field image capturing instruction unit 1120j, a bright field image acquiring unit 1120k, and a form identifying unit 1120l.

  The fluorescent image capturing instruction unit 1120a instructs the CCD camera 1080d to capture a fluorescent image via the communication interface unit 1160. The luminescent image capturing instruction unit 1120b instructs the CCD camera 1060c to capture a luminescent image via the communication interface unit 1160. The fluorescence image acquisition unit 1120c acquires the fluorescence image captured by the CCD camera 1080d via the communication interface unit 1160. The luminescent image acquisition unit 1120d acquires the luminescent image captured by the CCD camera 1060c via the communication interface unit 1160. The bright field image instruction unit 1120j instructs the CCD camera 1060c and / or the CCD camera 1080d to capture a bright field image via the communication interface unit 1160. The bright field image acquisition unit 1120k acquires a bright field image captured by the CCD camera 1060c and / or the CCD camera 1080d via the communication interface unit 1160. By reproducing simultaneously one time-lapse video or one image of one cell in the same visual field, a plurality of cells, or cells different from each other at a timing according to a preset time and interval, the time axis A moving image (or frame advance) or single image display may be displayed.

  The determination unit 1120e determines for each cell 1020 whether or not each gene has been introduced based on the fluorescence image and / or the luminescence image. The fluorescence measurement unit 1120f measures the fluorescence intensity of the fluorescence emitted from each cell 1020 based on the fluorescence image captured by the CCD camera 1080d. The luminescence measurement unit 1120g measures the luminescence intensity emitted from each cell 1020 based on the luminescence image captured by the CCD camera 1060c.

  The shape identification unit 1120h is configured to set feature points that are set in advance for each of one cell or a plurality of cells included in the bright field image obtained by the bright field image acquisition unit 1120k, that is, the contour shape of the cell and the contour of the nucleus. Is determined, and based on this shape, it is identified as having a specific feature point set in advance. Based on the identification result of the shape identification unit 1120h, the selection unit 1120h selects cells to be measured over time. Here, the determination of feature points may be performed based on the presence or absence of changes in feature points measured at a plurality of time points for one cell or a plurality of cells. That is, the identification of feature points may be performed by determining whether there is a change in the cell outline and / or the nucleus outline at least at two time points.

  The expression level measurement unit 1120j measures the expression level of the survivin gene to be analyzed based on the fluorescence intensity measured by the fluorescence measurement unit 1120f for the cell 1020 selected by the selection unit 1120i, and measures it by the luminescence measurement unit 1120g. The degree of survivin promoter activity to be analyzed is measured based on the luminescence intensity. The expression level measurement unit 1120j measures the expression level of the survivin gene to be analyzed based on the fluorescence intensity measured by the fluorescence measurement unit 1120f for the cells 1020 selected by the plurality of cells 1020 or the selection unit 1120i. Alternatively, the expression site in the cell 1020 of the gene to be analyzed may be identified based on the fluorescence image captured by the CCD camera 1080d.

  An example of processing performed by the expression level measuring apparatus 1000 in the above configuration will be described with reference to FIG. In the following description, the first vector and the second vector are introduced into a plurality of cells 1020, and the degree of survivin promoter activity at the luminescence intensity in the luminescence image targeting a specific cell 1020 among the introduced plurality of cells 1020. Is described over time, the distribution of survivin protein is measured over time with the fluorescence intensity and distribution in the fluorescence image, and an example of processing for identifying the morphology by observation in the bright field image will be described.

  First, the information communication terminal 1100 instructs the CCD camera 1080d to capture a fluorescent image through the communication interface unit 1160 by the processing of the fluorescent image capturing instruction unit 1120a, and the communication interface unit 1160 through the processing of the luminescent image capturing instruction unit 1120b. Through the communication interface unit 1160 in the process of the bright field image capturing instruction unit 1120j (step SB-1). Next, the CCD camera 1080d captures a fluorescence image of the plurality of cells 1020 existing in the imaging range (step SB-2), and transmits the fluorescence image to the information communication terminal 1100 (step SB-3). On the other hand, the CCD camera 1060c captures a light emission image of a plurality of cells 1020 existing in the imaging range (step SB-4), and transmits the light emission image to the information communication terminal 1100 (step SB-5). Further, the CCD camera 1080d or the CCD camera 1060c captures a bright field image of a plurality of cells 1020 existing in the imaging range (step SB-6), and transmits the bright field image to the information communication terminal 1100 (step SB). -7). Note that the fluorescent image capturing instruction, the luminescent image capturing instruction, and the bright field image capturing instruction may be performed at different times or time intervals. For example, imaging of luminescent images used to measure the degree of survivin promoter activity, imaging of fluorescent images used to measure the expression level of survivin gene, and imaging of bright-field images to identify cell morphology May be performed every few seconds, every few minutes, or every few hours. Further, the excitation light is applied to the cell 1020 only when a fluorescent image is captured.

  Next, the information communication terminal 1100 acquires (a) a fluorescent image through the communication interface unit 1160 through the processing of the fluorescent image acquisition unit 1120c, and (b) through the communication interface unit 1160 through the processing of the light emission image acquisition unit 1120d. (C) The bright field image is acquired via the communication interface unit 1160 by the processing of the bright field image acquisition unit 1120k, and (d) the time is acquired from the clock generation unit 1140 by the processing of the control unit 1120. (E) The acquired fluorescence image, light emission image, bright field image, and time are associated with each other and stored in a predetermined storage area of the storage unit 1160 (step SB-8).

  Next, the information communication terminal 1100 determines, for each cell 1020, whether or not a vector has been introduced based on the fluorescence image and / or the luminescence image by the processing of the determination unit 1120e (step SB-9). Next, when there is at least one cell 1020 into which a vector is introduced (step SB-10: Yes), the information communication terminal 1100 emits from each cell 1020 based on the fluorescence image by the processing of the fluorescence measurement unit 1120f. The fluorescence intensity of each fluorescence obtained is measured, and the emission intensity of the emission emitted from each cell 1020 is measured based on the emission image by the process of the emission measurement unit 1120g (step SB-11).

  Next, the information communication terminal 1100 identifies the feature of the cell for each cell 1020 based on the bright-field image, and identifies the morphology of the cell for each cell 1020 by the processing of the morphology identification unit 1120l (step 1012). SB-12). In addition, the second vector containing the fluorescence-related gene is introduced into the cell 1020, and the feature point of the cell morphology is determined for each cell 1020 based on the fluorescence intensity obtained thereby, so that the cell morphology is determined for each cell 1020. May be identified. Further, the feature point of the form is determined by determining the bright field image and / or the fluorescence image with respect to the position data at a predetermined site, specifically, the bright field image and / or fluorescence image of the nucleus, cell membrane, cytoplasm, etc. And time may be associated with each other and stored in a predetermined storage area of the storage unit 1160 and compared with corresponding position data at a further time point.

  Next, the information communication terminal 1100 selects the cell 1020 to be measured from the cells 1020 whose form has been identified in Step SB-12 by the processing of the selection unit 1120h (Step SB-13). Next, the information communication terminal 1100 measures the expression level of the gene to be analyzed based on the fluorescence intensity for the cell 1020 selected in step SB-13 by the processing of the expression level measurement unit 1120j, and also displays the fluorescence image. Based on the above, the expression site in the cell 1020 of the survivin gene to be analyzed is identified (step SB-14). In addition, when using fluorescence intensity or a fluorescence image in step SB-12, in step SB-14, the expression level of the survivin gene to be analyzed is measured based on the fluorescence intensity, and the degree of survivin promoter activity based on the emission intensity May be measured.

  Then, the information communication terminal 1100 repeatedly performs the above-described processing from step SB-1 to step SB-14 by a predetermined number of times, for example, at a preset time interval in the processing of the control unit 1120, and ends the predetermined number of times. In (Step SB-15: Yes), the process ends.

  Here, only the capturing and acquisition of the luminescent image, the fluorescent image and the bright field image are repeatedly performed, and at the time of analysis, the measurement of the luminescence intensity, the measurement of the fluorescence intensity, the distribution of the fluorescence, the identification of the form, the selection of the cell 1020, You may measure the expression level and the degree of activity. In other words, only the luminescent image, fluorescent image, and bright-field image, which are the original data necessary for analysis, are acquired together, and at the time of analysis, emission intensity measurement, fluorescence intensity measurement, fluorescence distribution, and morphology identification Alternatively, cell 1020 selection, expression level measurement, and activity level measurement may be performed. Specifically, after obtaining a luminescent image, a fluorescent image, and a bright field, at the time of analysis, selection of cells 1020, measurement of expression level, measurement of distribution of expressed survivin gene, and identification of morphology may be performed. . In addition, after obtaining a luminescent image, a fluorescent image, and a bright field image, at the time of analysis, identification of the morphology and selection of the cell 1020 may be performed. Alternatively, the cells 1020 may be selected at the time of analysis after obtaining the luminescent image, fluorescent image, and bright field image.

  Moreover, after acquiring a fluorescence image, the cell 1020 to be measured may be selected, and a luminescence image may be acquired.

  As described above in detail, according to the expression level measuring apparatus 1000, the luminescence-related gene, the fluorescence-related gene, the survivin gene to be analyzed, and the living cell 1020 introduced with the survivin promoter are emitted from the cell 1020. Measuring the emission intensity of the emitted light, measuring the fluorescence intensity of the fluorescence emitted from the cell 1020, and measuring the expression level of the gene to be analyzed based on the measured emission intensity or the measured fluorescence intensity. In the identification of the morphology, the cells are used using cells in which the luminescence-related gene, the fluorescence-related gene, the survivin gene to be analyzed, and the survivin promoter are identified. By such a method, the expression level and distribution of expression of survivin gene, the degree of survivin promoter activity, and morphological information can be obtained easily and accurately for one cell. Based on a plurality of information, it becomes possible to perform cell analysis comprehensively.

  Further, according to the expression level measuring apparatus 1000, when there are a plurality of cells 1020 in the imaging range, a fluorescence image of the plurality of cells 1020 is captured, a luminescence image of the plurality of cells 1020 is captured, and the captured luminescence image is displayed. Based on the fluorescence intensity emitted from each cell 1020, the fluorescence intensity emitted from each cell 1020 is measured based on the captured fluorescence image, and the form of each cell 1020 is further determined. Measured as feature points based on bright-field images, and based on information on the measured emission intensity and / or feature points related to the measured fluorescence intensity and / or morphology, the expression level of the survivin gene to be analyzed, the activity of the survivin promoter The degree, morphological identification, or morphological change is measured for each cell 1020 and cell analysis is performed for each cell 1020. As a result, for a plurality of cells 1020, the expression level of the survivin gene to be analyzed, the degree of survivin promoter activity, and morphological identification or morphological change identification can be measured or evaluated for each cell 1020.

  Further, according to the expression level measuring apparatus 1000, in the measurement of the expression level, the expression level of the survivin gene to be analyzed is measured based on the measured fluorescence intensity for the selected cell 1020, and the captured fluorescence image is captured. Based on the above, the expression site in the cell 1020 of the survivin gene to be analyzed is identified, and the degree of the survivin promoter activity is measured based on the luminescence image. Alternatively, changes in cell morphology can be measured for each cell along the time axis. Thereby, not only can the survivin gene to be analyzed and the degree of survivin promoter activity and the state and / or change of the cell morphology be evaluated, but also the survivin gene expression site in the cell 1020 can be obtained.

  Moreover, if the expression level measuring apparatus 1000 is used, specifically, an anticancer agent and its lead compound can be evaluated. In particular, whether or how much can anticancer drugs affect the expression and / or expression site of survivin gene and the degree of survivin promoter activity and cell morphology Can be determined and / or monitored, and whether the lead compound affects the transcriptional activity of the survivin gene can be monitored simultaneously. In addition, when the expression level measuring apparatus 1000 is used, specifically, for the survivin gene, while monitoring the degree of survivinb promoter activity by the luminescence measurement and the state and / or change of the morphology of the cell 1020 by the bright field, fluorescence is monitored. By identifying the expression time and localization by detection, the usefulness of the test substance can be comprehensively evaluated.

  Hereinafter, a preferred embodiment 3 of a microscope unit and a microscope apparatus as a measuring apparatus according to the present invention will be described in detail with reference to the accompanying drawings. Note that the present invention is not limited to the embodiments. In the description of the drawings, the same parts are denoted by the same reference numerals.

  First, a microscope apparatus according to a third embodiment of the present invention will be described. FIG. 5 is a schematic diagram showing the configuration of the microscope apparatus according to the third embodiment. As shown in FIG. 5, a microscope apparatus 100a according to this embodiment includes a fluorescence microscope unit 101 that performs fluorescence observation, a bioluminescence observation unit 102a that performs bioluminescence observation, and a specimen provided with a luminescence label and a fluorescence label. A holding unit 7 as holding means for holding S, a monitor 209 for displaying a specimen image of the specimen S imaged by each microscope unit 101, 102a, and a control device PC201 for controlling the overall processing and operation of the microscope apparatus 100a And comprising. The fluorescence microscope unit 101 and the bioluminescence microscope unit 102a are disposed adjacent to each other.

  The fluorescence microscope unit 101 includes a high-magnification fluorescence imaging optical system having an objective lens 201 as a fluorescence objective lens and an imaging lens 202 as a fluorescence imaging lens, and a specimen S imaged by the fluorescence imaging optical system. An imaging device 203 serving as a fluorescence imaging unit that captures a fluorescent specimen image that is a specimen image of the specimen, an excitation light source 204 that emits excitation light that excites the specimen S, a lens 205 that collects excitation light from the excitation light source 204, and And a fluorescent cube 206 as a fluorescent unit.

  The objective lens 201 has a large NA on the specimen side, and converts the fluorescence emitted from each point of the fluorescent label applied to the specimen S into a substantially parallel light beam. The imaging lens 202 collects the fluorescence converted into a substantially parallel light beam by the objective lens 201 and forms a fluorescent specimen image that is a specimen image of the specimen S. The fluorescence imaging optical system forms a fluorescence sample image at a high magnification of 40 times or more. The imaging device 203 includes a solid-state imaging device such as a CCD or CMOS, captures a fluorescent specimen image formed on the imaging surface of the solid-state imaging device, generates image data, and outputs the image data to the control device PC201.

  The fluorescent cube 206 selectively transmits an excitation filter 206a as an excitation light transmission filter that selectively transmits excitation light for exciting the specimen S and fluorescence emitted from the specimen S excited by the excitation light. An absorption filter 206b as a fluorescence transmission filter and a dichroic mirror 206c that reflects excitation light and transmits fluorescence are integrally provided. The excitation filter 206a is a bandpass filter that extracts excitation light in a predetermined wavelength region from light of various wavelengths emitted from the excitation light source 204, and the absorption filter 206b is a long wavepass filter having a predetermined cutoff wavelength. It is. The absorption filter 206b may be a band pass filter that extracts fluorescence in a predetermined wavelength range. The band pass filter is effective when the wavelengths of bioluminescence and fluorescence emitted from the specimen S are close.

  The excitation light source 204 is realized by a mercury lamp, a xenon lamp, a laser, or the like, and the excitation light source 4 and the lens 205 as excitation light irradiation means pass the excitation light from the excitation light source 204 through the excitation light filter 206a and the dichroic mirror 206c. The sample S is reflected by and irradiated onto the specimen S. The excitation light source 204 is turned on and off based on instructions from the control device PC201.

  The bioluminescence microscope unit 102a is connected to a low-magnification bioluminescence imaging optical system having an objective lens 211 as a bioluminescence objective lens and an imaging lens 212 as a bioluminescence imaging lens, and the bioluminescence imaging optical system. An imaging device 213 as bioluminescence imaging means for imaging a bioluminescence specimen image that is a specimen image of the specimen S to be imaged.

The objective lens 211 has a large NA on the specimen side, and converts bioluminescence emitted by self-luminescence from each point of the luminescent label given to the specimen S into a substantially parallel light beam. The imaging lens 212 collects the bioluminescence converted into a substantially parallel light beam by the objective lens 211 and forms a bioluminescence specimen image that is a specimen image of the specimen S. The bioluminescence imaging optical system forms a bioluminescence specimen image with an imaging magnification lower than the imaging magnification of the fluorescence imaging optical system. At this time, the bioluminescence imaging optical system preferably satisfies (NAo / β) ² ≧ 0.01, where NA on the specimen side is NAo and the imaging magnification is β.

  The imaging device 213 includes a solid-state imaging device such as a CCD or a CMOS, captures a bioluminescent specimen image formed on the imaging surface of the solid-state imaging device, generates image data, and outputs the image data to the control device PC201. . Note that a solid-state imaging device included in the imaging device 213 is a high-sensitivity monochrome CCD, and a cooled CCD of about 0 ° C. may be used.

  The holding unit 207 is a movable member that moves the sample S two-dimensionally together with a holding member 207a such as a slide, a slide glass, a microplate, a gel support, a fine particle carrier, and an incubator on which the sample S is directly placed. Stage 207b. The movable stage 207b is driven by the stage drive unit 208 based on an instruction from the control device PC201.

  The control device PC201 is realized by a processing device such as a computer equipped with a CPU, and electrically connects the imaging devices 203 and 213, the excitation light source 204, the stage drive unit 208, and the monitor 209, and performs operations of these components. Control. In particular, the control apparatus PC201, as an imaging switching control unit, captures a bioluminescent specimen image by the bioluminescent microscope unit 102a based on the image characteristics of the bioluminescent specimen image captured by the imaging apparatus 213, and the fluorescence microscope unit 101. The imaging switching process for switching between imaging of the fluorescent specimen image by the control is performed.

  Here, the imaging switching process controlled by the control device PC201 will be described. FIG. 6 is a flowchart illustrating a processing procedure of the imaging switching process. As shown in FIG. 6, the control device PC201 images the bioluminescence sample image of the sample S that has been moved into the field of view of the bioluminescence imaging optical system by the movable stage 207b by the imaging device 213 (step S101). Based on the imaging result, the control device PC201 determines whether or not there is a region in the bioluminescent specimen image where the image intensity as the image characteristic of the bioluminescent specimen image is larger than a preset threshold value (step). S103). When it is determined that there is no region where the image intensity is greater than the threshold value (step S103: No), the control device PC201 repeats the processing from step S101.

  On the other hand, if it is determined that there is a region where the image intensity is greater than the threshold (step S103: Yes), the control device PC201 records the bioluminescent specimen image captured in step S101 (step S105), and the movable stage 207b. The sample S is moved within the field of view of the fluorescence imaging optical system (step S107). Then, the control device PC201 captures and records the fluorescent specimen image corresponding to the region where the image intensity of the bioluminescent specimen image is larger than the threshold value (step S109), and ends the imaging switching process. When performing the follow-up of the sample S, the control device PC201 moves the sample S again into the field of view of the bioluminescence imaging optical system by the movable stage 207b after step S109, and repeats the processing from step S101. It is good to control. Further, the imaging in step S109 may be either time-lapse imaging or single image imaging. Also, different cells within the same field of view can be displayed simultaneously on a time-lapse video or one image captured at a preset timing, thereby displaying a moving image (or frame advance) or one image along the time axis. Also good.

  In steps S105 and S109, the control device PC201 stores the captured bioluminescence sample image and fluorescence sample image in a storage unit such as a RAM provided in the own device. Further, the control device PC201 may sequentially display the captured bioluminescent specimen image and fluorescent specimen image on the monitor 209 in steps S101 and S109. Further, the control device PC201 turns off the excitation light source 204 during steps S101 to S105, that is, while performing bioluminescence observation of the sample S by the bioluminescence microscope unit 102, and performs fluorescence observation of the sample S in step S109. When performing, it is good to control so that the excitation light source 204 may be turned on. Alternatively, a light shielding device such as a shutter is provided on the optical path from the excitation light source 204 to the fluorescent unit 20, and the control device PC 201 opens and closes the light shielding device as an unirradiated means instead of turning on and off the excitation light source 204. The irradiation of excitation light may be controlled accordingly.

  In step S103, the control device PC201 determines to switch to fluorescence observation based on the image intensity of a partial area in the bioluminescent specimen image. However, the entire image intensity of the bioluminescent specimen image is determined. The switching may be determined based on the above. Further, the control device PC201 may acquire these image intensities as, for example, cumulative image intensities from a predetermined time point to the present time, or may acquire them as an instantaneous image intensity at the present time. In addition, when acquiring the whole image intensity | strength of a bioluminescent sample image, it may replace with the imaging device 213, and may use highly sensitive light receiving elements, such as a photomultiplier.

  As described above, according to the microscope apparatus according to the third embodiment, the fluorescence microscope unit 101 for fluorescence observation and the bioluminescence microscope unit 102a for bioluminescence observation are provided adjacent to each other, and the fluorescence imaging optics is provided. System and the movable stage 207b for moving the specimen S within each field of view of the bioluminescence imaging optical system, it is possible to appropriately switch between the fluorescence observation and the bioluminescence observation, and the image characteristics of the bioluminescence specimen image Depending on the image intensity, it is possible to immediately switch from bioluminescence observation to fluorescence observation.

  The bioluminescence imaging optical system has been described as an infinite correction optical system that forms a sample image with the objective lens 211 and the imaging lens 212, but as a finite correction optical system that forms a sample image with only the objective lens. Also good.

  In the imaging switching process described above, the bioluminescence observation is switched to the fluorescence observation based on the image characteristics such as the image intensity of the bioluminescence specimen image. However, the excitation light intensity and the specimen irradiated to the specimen S in the fluorescence observation are changed. When the fluorescence intensity emitted from S is weak and the damage to the specimen S due to excitation light and fluorescence is small, the fluorescence observation should be switched to the bioluminescence observation based on image characteristics such as the image intensity of the fluorescence specimen image. It may be.

  FIG. 7 is a schematic diagram illustrating a configuration of a microscope apparatus according to the fourth embodiment. As shown in FIG. 7, in addition to the microscope apparatus 100a according to the third embodiment, a microscope apparatus 600 according to the fourth embodiment includes a transmissive illumination unit 103a as an illuminating unit that performs transmitted illumination, and the transmissive illumination unit. The illumination drive part 246 which drives the shutter 243 etc. which 103a has, and control apparatus PC206 replaced with control apparatus PC201 are provided. Other configurations are the same as those of the third embodiment, and the same components are denoted by the same reference numerals.

  The transmitted illumination unit 103a condenses the white light on the sample S, a white light source 244 such as a halogen lamp that emits white light for transmitted illumination, a shutter 243 that switches between irradiation and non-irradiation of white light, and the white light source 244. And an illumination lens system 245 that is arranged on the opposite side of the specimen S from the fluorescence microscope unit 101. The illumination lens system 245 includes a collector lens 245a and a condenser lens 245b, and performs critical illumination on the specimen S. Note that the illumination lens system 245 may perform Koehler illumination on the specimen S.

  The illumination drive unit 246 drives the shutter 243 and the fluorescent illumination unit 104a based on an instruction from the control device PC206. Here, the fluorescent lighting unit 104 a includes an excitation light source 204, a lens 205, and a fluorescent cube 206 that are integrally held by a housing 226. The illumination driving unit 246 opens and closes the shutter 243 to switch between irradiation and non-irradiation of the white light on the specimen S, and inserts and removes the fluorescent cube 206 on the optical path between the objective lens 201 and the imaging lens 202. The fluorescent lighting unit 104a is moved.

  The control device PC 206 controls the operation of the illumination drive unit 246 in addition to controlling the operations of the imaging devices 203 and 213, the excitation light source 204 and the stage drive unit 8 in the same manner as the control device PC 201. When switching from fluorescence observation to observation by transmitted illumination, the control device PC206 moves the fluorescence illumination unit 104a so as to remove the fluorescence cube 206 from between the objective lens 201 and the imaging lens 202, turns off the excitation light source 204, The shutter 243 is opened to transmit illumination. When switching from observation with transmitted illumination to fluorescence observation, the control device PC206 closes the shutter 243, moves the fluorescent illumination unit 104a so that the fluorescent cube 206 is disposed between the objective lens 201 and the imaging lens 202, and The excitation light source 204 is turned on.

  In addition, when switching from bioluminescence observation to observation by transmitted illumination, the control device PC206 drives the movable stage 207b by the stage drive unit 208 in addition to the control when switching from fluorescence observation to observation by transmitted illumination. Control to move within the field of view of the fluorescence imaging optical system is performed. Note that the control device PC 206 may turn on and off the white light source 244 instead of opening and closing the shutter 243.

  Although the transmission illumination unit 103a is shown to perform illumination for bright field observation, the transmission illumination unit 103a is not limited to illumination for bright field observation, and illumination for dark field observation, differential interference observation, or phase difference observation is performed. May be. Further, these various illuminations for observation may be provided so as to be switchable. When performing illumination for differential interference observation, the transmission illumination unit 103a includes a polarizer and a polarization separation prism on the light source side of the condenser lens 245b, and polarization is applied to the pupil side of the objective lens 1 in the fluorescence imaging optical system. A synthesis prism and an analyzer may be provided. In addition, when performing illumination for phase difference observation, the transmission illumination unit 103a includes a ring slit on the light source side of the condenser lens 245b, and a phase plate is disposed at a substantially pupil position of the objective lens 201 in the fluorescence imaging optical system. The objective lens 201 may be switched to an objective lens having a phase plate. Furthermore, when performing illumination for dark field observation, the transmission illumination unit 103a is preferably provided with a ring slit or the like on the light source side of the condenser lens 245b.

  Further, although the transmission illumination unit 103a is shown to be arranged corresponding to the fluorescence imaging optical system in order to perform observation with transmission illumination at a high magnification, the bioluminescence imaging optical system is capable of observation at a low magnification. You may arrange | position corresponding to. Alternatively, it may be arranged corresponding to both of these imaging optical systems, or the arrangement may be switched as appropriate for each imaging optical system.

  By the way, in the microscope apparatus 600, the configuration of the microscope apparatus 100a is further provided with the transmission illumination unit 103a and the illumination driving unit 246. However, the present invention is not limited thereto, and for example, each configuration of the microscope apparatus includes the transmission illumination unit 103a. Further, the illumination driving unit 246 or the illumination driving unit 247 may be further provided.

  For example, the configuration of the microscope apparatus 200 may further include a transmission illumination unit 103a and an illumination driving unit 246. In this case, for example, the control device PC7 controls the imaging devices 203 and 213, the excitation light source 204, the rotation drive device 214, and the stage drive unit 218 similarly to the control device PC2, and the illumination drive unit 246 similarly to the control device PC206. To control the transmission illumination unit 103a and the fluorescence illumination unit 104a.

  That is, the control device PC209 moves the fluorescent illumination unit 105 so as to remove the mirror 234 and the fluorescent cube 206 from between the objective lens 201 and the imaging lens 202 when switching from fluorescence observation or bioluminescence observation to observation by transmitted illumination. Then, the shutter 233 is closed so that the excitation light is not irradiated, and the shutter 243 is opened to transmit illumination. When switching from observation with transmitted illumination to fluorescence observation or bioluminescence, the control device PC209 closes the shutter 243, and the fluorescent illumination so that the fluorescent cube 206 or the mirror 234 is disposed between the objective lens 201 and the imaging lens 202. The unit 105 or the mirror 234 is moved. When performing fluorescence observation, the shutter 233 is further opened to irradiate the specimen S with fluorescence.

  As described above, according to the microscope apparatus 600 according to the fourth embodiment, the transmission illumination unit corresponding to at least one of the fluorescence imaging optical system and the bioluminescence imaging optical system and performing the transmission illumination on the specimen S is provided. Since it is provided, not only fluorescence observation and bioluminescence observation, but also observation by various transmitted illuminations can be performed, and the specimen S can be observed from various angles.

  Although the above-described microscope apparatuses 100a and 600 are shown as upright microscope apparatuses, they may be inverted microscope apparatuses. Further, the above-described microscope apparatus can be suitably used for, for example, examination and treatment of various reactions (for example, drug stimulation and light irradiation).

  As described above, the embodiment has been described in detail. In the present invention, “luminescence” means that light can be generated by a chemical reaction, and is a term that particularly includes bioluminescence and chemiluminescence as suitable examples. On the other hand, fluorescence means that light can be generated by excitation light. Here, BRET (bioluminescence resonance energy transfer) excited by light energy by bioluminescence is included in light emission in the present invention because a chemical reaction with a substrate solution is a dominant factor. Light generated from a sample refers to electromagnetic radiation having a wavelength between about 400 nm and about 900 nm, which is particularly harmless to living cells. Imaging of the emitting sample requires the use of a photodetector that can detect very low levels of light (usually a single photon event) and integrate the photon emission until an image can be constructed. Examples of such sensitive photodetectors include a camera or group of cameras that can detect single photons against background noise inherent in the detection system after amplifying single photon events, such as imaging such as a CCD. A CCD camera having an element group can be exemplified. In general, in order to obtain high sensitivity, the CCD camera may be cooled with liquid nitrogen or the like. High numerical aperture (NA) In particular, when using an objective lens whose optical condition expressed by the square of numerical aperture (NA) / projection magnification (β) is 0.01 or more, the cooling temperature is minus 5 ° C. It has been confirmed by the present inventors that imaging can be carried out at -20 ° C, preferably -5 ° C to room temperature. Further, as a result of further investigation, when the square of the above optical condition (NA / β) is 0.071 or more, the cell image that can be visually recognized and analyzed within 5 minutes, and in some cases about 1 minute. I found out that I can offer you. In general, a live sample is often difficult to obtain a clear luminescent image in an imaging time exceeding 30 minutes due to a change in shape or luminescent site. Therefore, the present invention provides a method and apparatus advantageous in cooperation with fluorescence measurement, in which one luminescent image is acquired in a short time, particularly within 30 minutes, preferably from 1 minute to 10 minutes, and imaging time is fast. .

  For example, as a related aspect, if you want to track the localization of the fluorescent signal and / or the intensity of the luminescent signal over time to record the effect of a treatment on the distribution and / or localization of the selected biocompatible component, By repeating photon emission measurement or imaging at selected time intervals, a series of images can be constructed. The interval may be as short as a few minutes or as long as a few days or weeks. The luminescent image or the superimposed image of fluorescent (or transmitted light) luminescence can be expressed in various forms such as screen display, printed paper, and graphic processed image.

  In another related aspect, the present invention relates to promoter induction events after detecting the presence of a promoter induction event in an animal transgenic or chimeric with a construct comprising a gene encoding a luminescent protein under the control of an inducible promoter. Includes methods of monitoring activity. For the promoter induction event, administration of a substance that directly activates the promoter, administration of a substance that stimulates production of an endogenous promoter activator (for example, stimulation of interferon production due to RNA virus infection), endogenous promoter activator For example, heat shock or stress.

  In yet another aspect, the present invention also encompasses a method for identifying therapeutic compounds that are effective in reducing the severity of infection by a pathogen. In this method, a complex of a pathogen, a fluorescent component, and a luminescent component is administered to a control animal and an experimental animal, or their cultured tissues (or cells), and the experimental animal is treated with a therapeutic compound candidate. Then, after confirming the localization of the fluorescence signal in the sample to be measured by the above method, the luminescence signal (bioluminescence or chemiluminescence) and the bright field image are continuously measured only from the sample confirmed in the station. To do. In this way, the therapeutic efficacy of the compound can be monitored.

  As yet another aspect, the present invention includes a method of continuously measuring luminescence only from a sample whose localization has been confirmed after selecting a sample localized through a medium having various opacity by a fluorescence signal. To do. In this method, it is possible to create an image by integrating the photon signal transmitted through the medium with the luminescence signal, but only a sample whose localization has been confirmed is surgically removed from the medium (eg, organ tissue), The method can be improved so that the sample taken out can be measured for luminescence in an appropriate culture environment. Limited surgery (eg, biopsy) minimizes the physical burden on the original organism (eg, mammals, especially humans) and moves only the necessary samples to a stable laboratory environment, with various possibilities It has the advantage of long-term response to certain drugs, progress management after treatment, and preventive medical testing.

  Alternatively, such an observation apparatus or observation system may use, for example, the means disclosed in JP2009-148255A and International Publication WO2006-106882, for example, a light such as LUMINOVIEW LV200 (manufactured by Olympus). An observation system capable of performing visual field, fluorescence observation and emission observation may be used, or any known observation apparatus or observation system may be used. Such an observation apparatus is preferably capable of phase difference observation.

  Observation in the bright field, measurement of the emission signal and measurement of the fluorescence signal may be performed in any order, for example, in the order of observation in the bright field, measurement of the emission signal and measurement of the fluorescence signal. Luminescence signal measurement, fluorescence signal measurement and bright field observation order, even luminescence signal measurement, bright field observation and fluorescence signal measurement order, in any other order There may be. Moreover, you may perform the same observation or measurement in multiple times continuously.

  The observation in the bright field may be, for example, an exposure of 1 msec to 1000 msec, preferably 10 msec to 200 msec. The measurement of the fluorescence signal may be, for example, an exposure of 100 msec to 10 sec, preferably 100 msec to 1 sec. The measurement of the luminescence signal may be, for example, an exposure of 1 sec to 60 min, preferably 1 sec to 10 min.

  Following bright field observation, measurement of fluorescence and luminescence signals, the sample is exposed to the drug. Observe the change in cell morphology, the degree of survivin promoter activity and the survivin protein distribution due to this drug. This observation is preferably performed over time.

  The measurement of the fluorescence signal may be performed, for example, by measuring the fluorescence intensity and / or the fluorescence amount. The measurement of the luminescence signal may be performed, for example, by measuring the luminescence intensity and / or the luminescence amount.

  You can observe changes in the sample caused by the drug by comparing the observations obtained before the exposure of the sample to the drug with the observations obtained at a specific time after exposure. It is.

  Examples of drugs for performing cell analysis are staurosporine, mitomycin, cisplatin, actinomycin D, 5-fluorouracil, paclitaxel, etoposide, imatinib and YM-155, but are not limited thereto. A person may select a drug as desired.

  Moreover, it is possible to determine the characteristic of the cell by analyzing about the cell extract | collected from the object using an already known chemical | medical agent. For example, by using a specific anticancer agent as a drug, it may be determined whether or not the anticancer agent is effective for the target cancer.

  Alternatively, for example, the metastasis rate and / or prognosis of cancer in the subject from which the sample is derived may be determined by determining whether a specific drug is effective against the cancer cells that are the sample. Cancer may be analyzed by determining such cancer metastasis rate and / or prognosis.

  A cell line known to be in a specific state may be used as a sample. For example, by performing the above cell analysis method using a cancer cell line, it is possible to screen for the anticancer activity of the test substance. In such a screening method, a test substance may be used instead of the reagent used in the aforementioned cell analysis method.

  For example, examples of cells used for screening the anticancer effect of a test substance may include HeLa cells, Ca Ski cells, TC-1 cells, and HT1080 cells, but are not limited thereto. Absent.

  According to embodiments, information regarding morphological changes, the state of survivin protein localization and the extent of survivin promoter activity can be obtained for a single cell. By performing cell analysis based on such information, it becomes possible to analyze cells comprehensively. Further, by evaluating the drug efficacy based on the information, it is possible to comprehensively screen the drug efficacy of the test substance.

[Example]
Example 1
[Detection of Survivin Promoter Activity Using Fluorescence Imaging and Luminescence Imaging of Survivin Protein Dynamics in HeLa Cells]
In Example 1, fluorescence imaging and luminescence imaging were performed using two vectors. As the first vector, a vector expressing luciferase under the control of the survivin promoter was used. As the second vector, a vector expressing a fusion protein of survivin and Enhanced yellow fluorescein protein (EYFP) was used. These vectors were transfected into one cell. Thereafter, the intracellular dynamics of survivin protein and survivin promoter activity in the same living cells were followed over a long period of time.

  The procedure of the experiment in Example 1 will be described below. The gene (SEQ ID NO: 2) encoding survivin protein with BamHI and NheI and the EYFP gene (SEQ ID NO: 3) with NheI and EcoRI were inserted into the MCS of pcDNA3.1 (+) (manufactured by Invitrogen), and survivin-EYFP A fusion protein expression vector (Suv-EYFP) was prepared.

  In Example 1, YFP was used as a tag for detecting the dynamics of survivin protein. As can be seen from FIG. 8 showing the excitation and fluorescence spectrum of D-luciferin which is the luminescent substrate of the luciferin-luciferase reaction, when a fluorescent protein having an excitation wavelength shorter than YFP excitation is used as a tag, D- Luciferin is excited, and fluorescence derived from D-luciferin may be detected as background noise. Therefore, it was considered that fluorescent proteins (YFP, RFP, etc.) having an excitation wavelength longer than D-luciferin are suitable for simultaneous imaging of fluorescence and luminescence.

  As a reporter for monitoring the activity of the survivin promoter, the survivin promoter region (SEQ ID NO: 1) was inserted into the MCS of pGL4.14 (luc2 / Hygro) (Promega) with BglII and HindIII, and the survivin promoter :: luc2 expression vector ( pSuv :: luc2) was prepared.

The Suv-EYFP and pSuv :: luc2 vectors were introduced into HeLa cells seeded on a φ35 mm-glass bottom dish using FuGene reagent (Promega). After culturing for 24 hours, the medium was replaced with D-MEM medium containing 10% FBS, D-luciferin (manufactured by Promega) was added at a final concentration of 500 μM, and the mixture was allowed to stand for 1 hour. After the dish was set on LUMINOVIEW LV200 (Olympus), bright field images, fluorescent images, and luminescent images were taken. The HeLa cells being imaged are placed in an incubator under a 5% CO ₂ atmosphere, the magnification of the objective lens during observation is 100 times, the CCD camera is ImagEM (manufactured by Hamamatsu Photonics), Binning is 1 × 1, and the exposure time is 200 msec. (Bright field observation) 1 sec (fluorescence observation), 9 minutes (luminescence observation). Bright field images were acquired by phase contrast observation. BP490-500HQ was used as an excitation filter during fluorescence observation, and BP515-560HQ was used as a spectral filter. A spectral filter was not used during emission observation. The phase difference image, the fluorescence image, and the luminescence image before drug stimulation obtained as a result of the observation are shown in FIGS. 9, 10 and 11, respectively. In FIG. 9, it was clearly observed that the HeLa cells had an elliptical cell membrane and a nucleus contained therein before stimulation. The out-of-focus cell was shown as a circle by the light reflected on its surface. As shown in FIG. 10, in the fluorescence image before stimulation, a fluorescence signal that was uniformly spread and distributed in the cytoplasm was observed, the cell outline was clear, and the nucleus was shown in black. As shown in FIG. 11, the inside of the cell was generally shown brighter than the part other than the inside of the cell. The inside of the cell shown in FIG. 12 was shown to be uniformly and faintly bright as a whole, as compared to a portion other than a portion where the focused cell was present.

After image acquisition after drug stimulation, time-lapse imaging of bright field images, fluorescent images, and luminescent images was performed. Type lapse photography was started after addition of staurosporine (STS), an agent that induces apoptosis, at a final concentration of 1 uM. The HeLa cells after addition of 1 uM STS were allowed to stand in an incubator under a 5% CO ₂ atmosphere for observation. The magnification of the objective lens during time-lapse observation is 100 times, the CCD camera is ImagEM (manufactured by Hamamatsu Photonics), Binning is 1 × 1, exposure time is 200 msec (bright field observation), 1 sec (fluorescence observation), 9 minutes (luminescence observation), The interval time was 10 minutes and the total imaging time was 10 hours. Bright field images were acquired by phase contrast observation. BP490-500HQ was used as an excitation filter during fluorescence observation during time-lapse observation, and BP515-560HQ was used as a spectral filter. A spectral filter was not used during emission observation. FIG. 12 schematically shows the imaging timing at the time-lapse observation for such bright field imaging, fluorescence imaging, and luminescence imaging. FIG. 13, FIG. 14, and FIG. 15 show the phase difference image, fluorescence image, and luminescence image after drug stimulation obtained as a result of time lapse observation, respectively. As shown in FIG. 13, in the cells after STS stimulation, the nuclei contracted, and many protrusions that appeared to be blebs from the cell membrane were observed. As shown in FIG. 14, the outline of the cell was dim, and a fluorescent signal was observed even from the nucleus and skeleton. Moreover, as shown in FIG. 14, the brightness in the cell after stimulation became brighter than before stimulation.

  A region of interest (ROI) was designated for the obtained luminescent image (FIGS. 11 and 15). The emission intensity of the designated ROI was measured based on each emission image obtained as a result of time-lapse observation, and the change in the emission intensity over time was displayed in a graph (FIG. 16).

  As shown in FIGS. 9 and 13, rapid contraction of HeLa cells was observed after drug stimulation. As shown in FIG. 10, it is shown that survivin protein is localized in the cytoplasm in HeLa cells before drug stimulation, and hardly exists in the nucleus. However, as shown in FIG. 14, the localization of survivin protein changed after drug stimulation, and survivin protein was transferred to parts other than the cytoplasm. As shown in FIGS. 11 and 15, it was observed that the survivin promoter activity rapidly increased 30 minutes after drug stimulation. From the graph of FIG. 16, it was found that the increase in survivin promoter activity peaked at 30 minutes after drug stimulation and at 1 hour 20 minutes, and then rapidly decayed. From these results, it was suggested that the drug STS has the effect of contracting HeLa cells, delocalizing intracellular survivin protein, and temporarily increasing survivin promoter activity.

  From the above experimental results, bright field, fluorescence, and luminescence imaging using LUMINOVVIEW were performed, and morphological changes, localization of survivin protein, and survivin promoter activity in the same cell could be captured over time and quantitatively. As a result, it was shown that detailed analysis of the effect of drugs on cells is possible. Survivin is a protein having a function of suppressing apoptosis and regulating cell division, and is rarely observed in normal tissues, and has been reported to be highly expressed in fetal tissues and many human cancers. It is also known that the prognostic evaluation of cancer differs depending on whether survivin is localized in the nucleus or cytoplasm. From these reports, the expression level of survivin protein and its localization site are considered promising as cancer markers and prognostic markers for cancer. This method, which can detect survivin promoter activity and localization site at the single cell level, is considered to be particularly useful in the screening of drug efficacy for survivin and the prognostic evaluation of cancer.

Claims (17)

(1) A first vector comprising a survivin promoter and a gene encoding a photoprotein operably linked downstream thereof, a promoter, a survivin gene operably linked downstream thereof, and a downstream thereof Providing a second vector that is an expression vector comprising a gene encoding a functionally linked fluorescent protein;
(2) introducing the first vector and the second vector into the collected and / or cultured cells;
(3) exposing the cell of (2) to a reagent;
(4) performing bright field observation, fluorescence intensity measurement, and luminescence intensity measurement at each of at least one time point before and after the cell is exposed to the reagent; and (5) (4) Obtaining information on the cells from the results of
Cell analysis method for survivin including   The method according to claim 1, wherein the observation of the bright field, the measurement of the fluorescence intensity, and the measurement of the emission intensity are each performed over time.   The method according to claim 1, wherein the bright field observation, the fluorescence intensity measurement, and the emission intensity measurement are repeatedly performed in a predetermined order.   The method according to any one of claims 1 to 3, wherein the observation of the bright field, the measurement of the fluorescence intensity, and the measurement of the emission intensity are performed for a common specific region of interest.   The method according to claim 4, wherein the specific region of interest is a region including one cell.   The evaluation is to determine the morphological change of the cells from the information obtained by observation of the bright field, to identify the localization site of survivin protein from the results obtained by the measurement of the fluorescence intensity, and The method according to any one of claims 1 to 5, wherein the method is carried out based on judging the degree of survivin promoter activity from the result obtained by measuring the luminescence intensity.   The method according to claim 1, wherein the bright field observation is phase difference observation.   The method according to any one of claims 1 to 7, wherein the observation of the bright field, the measurement of the fluorescence intensity, and the measurement of the emission intensity are each performed using images formed by imaging means and formed.   The method according to any one of claims 1 to 8, wherein an anticancer agent is used as the reagent, and based on the results obtained thereby, the cancer metastasis rate in the subject from which the cells are derived and / or A method for analyzing cancer, comprising evaluating prognosis. (1) A first vector comprising a survivin promoter and a gene encoding a photoprotein operably linked downstream thereof, a promoter, a survivin gene operably linked downstream thereof, and a downstream thereof Providing a second vector that is an expression vector comprising a gene encoding a functionally linked fluorescent protein;
(2) introducing the first vector and the second vector into the collected and / or cultured cells;
(3) exposing the cell of (2) to a test substance;
(4) performing observation of bright field, measurement of fluorescence intensity, and measurement of emission intensity at each of at least one time point before and after the cell is exposed to the test substance, and (5) ( 4) evaluate the effect of the test substance from the result of
For screening the efficacy of a test substance comprising   The method according to claim 10, wherein observation of the bright field, measurement of the fluorescence intensity, and measurement of the emission intensity are each performed over time.   The method according to claim 10 or 11, wherein the bright field observation, the fluorescence intensity measurement, and the emission intensity measurement are repeatedly performed in a predetermined order.   The method according to any one of claims 10 to 12, wherein the observation of the bright field, the measurement of the fluorescence intensity, and the measurement of the emission intensity are performed for a specific region of interest in common.   The method according to claim 13, wherein the specific region of interest is a region including one cell.   The evaluation is to determine the morphological change of the cells from the information obtained by observation of the bright field, to identify the localization site of survivin protein from the results obtained by the measurement of the fluorescence intensity, and The method according to any one of claims 10 to 14, which is performed based on determining the degree of survivin promoter activity from the result obtained by the measurement of the luminescence intensity.   The method according to claim 10, wherein the bright field observation is phase difference observation.   The method according to any one of claims 10 to 16, wherein the observation of the bright field, the measurement of the fluorescence intensity, and the measurement of the emission intensity are each performed by using images formed by imaging means.

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