JP2010210290A - Method for detecting dna-fragmented apotosis cell, cell image analyzer, program, and method for evaluating stage of apotosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately detecting a DNA-fragmented apotosis cell. <P>SOLUTION: The method for detecting the DNA-fragmented apotosis cell has the step (a) for performing nucleus dyeing using a double stranded nucleic acid dyeing agent and TUNEL dyeing with respect to one or a plurality of the cells fixed to a cell observing container, the step (b) for imaging the cells dyed in the step (a) to acquire the double stranded nucleic acid dyed image and TUNEL dyed image of each cell, the step (c) for analyzing the acquired double stranded nucleic acid dyed image to discriminate whether the nucleus of each cell in the image is fragmented, the step (d) for analyzing the acquired TUNEL dyed image to discriminate whether the DNA chain of each cell in the image is cut, and the step (e) for detecting the cell, of which the DNA chain is discriminated to be cut in the step (d), being the cell of which the nucleus is discriminated to be fragmented in the step (c), as the DNA fragmented cell. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is a method for distinguishing between apoptotic cells in which DNA strands are cleaved and cracks (nicks) are occurring but DNA is not yet fragmented, and cells in which DNA is fragmented, The present invention relates to a cell image analysis apparatus used in the method, and a method for evaluating an apoptosis stage using the method.

  Apoptosis is a programmed cell death that proceeds according to a controlled cascade and plays a very important role in the development of multicellular organisms. In addition, unlike necrosis (cell necrosis), since it does not cause inflammation in principle, it is an important mechanism for maintaining homeostasis of the cellular environment in vivo. For this reason, research has been conducted all over the world to elucidate the apoptotic cascade. In addition, there are many diseases in which apoptosis is important in treatment and the like. For example, in proliferative diseases such as cancer, drug discovery research that induces cancer cells to apoptosis is active. Conversely, in neurological diseases such as Parkinson's disease and Alzheimer's disease, it is expected to suppress the progression of the disease or improve the disease state by inhibiting apoptosis. Drug development is thriving.

  Apoptosis was originally a morphologically defined concept and a mode of cell death with significant morphological changes compared to necrosis. Cells that have undergone apoptosis contract, and after the nuclei are concentrated and fragmented, apoptotic bodies are formed in which the fragmented nuclei are enveloped in the cell membrane. This apoptotic body is processed by phagocytes. In general, cell rounding (cell volume reduction), nuclear aggregation, DNA fragmentation (chromosomal DNA is cleaved every nucleosome), and formation of apoptotic bodies (cell fragmentation) It is said to be a characteristic morphological change.

  The apoptotic signal transduction system is intricately intertwined with multiple cascades and involves many factors. For this reason, there are various methods for detecting apoptosis. In addition, the stage of apoptosis is mainly classified into an early stage, a middle stage, and a late stage based on its morphological characteristics, and development of a method for distinguishing and detecting this stage is also active. Here, since apoptosis is performed in a cell from the signal induction to the cell death, a method of directly detecting the cell is preferable for detecting apoptosis. For example, conventionally, a technique has been proposed in which intracellular organs such as nuclei are stained with a fluorescent substance, imaged with a microscope, and the captured cell image is analyzed (for example, Patent Document 1). By applying microscopic image analysis, morphological changes at each stage of apoptosis can be detected with higher sensitivity.

  Representative methods for detecting apoptosis include detecting fragmented caspase induction in the cytoplasm, detecting cell membrane permeability changes, detecting DNA strand breaks (degradation), and detecting DNA fragmentation. And a method for detecting a change in mitochondrial membrane potential. Of these, methods other than the method for detecting DNA fragmentation are carried out by using a commonly used flow cytometer or microscope observation by utilizing a fluorescent marker or the like. On the other hand, the method for detecting DNA fragmentation cannot be detected using a flow cytometer, and is currently performed by microscopic observation.

  Of the four detection methods described above, DNA fragmentation, in particular, is a completely irreversible reaction. Therefore, in many papers and the like, it is an object of evaluation of apoptosis performed in parallel with other markers. Yes. As a method for detecting DNA fragmentation, a method of separating a DNA extracted from cells by length by electrophoresis or the like and confirming a ladder-like ladder image is widely used. This is observed because chromosomal DNA is cleaved in nucleosome units (cleaved to nucleosomes and nucleosomes). Since about 180 bp wraps around one nucleosome, the smallest fragment is 180 bp, and the next is 360 bp. It is also observed that the nuclei also become fragmented with DNA fragmentation. Therefore, there is also a method for detecting DNA fragmentation by staining nuclei with a double-stranded DNA staining reagent such as an intercalator and observing a morphological change of fragmentation of the nuclei under a microscope.

  On the other hand, a phenomenon that occurs before fragmentation of double-stranded DNA is DNA strand breakage (decomposition). For this reason, when detecting apoptosis using a flow cytometer, an assay for DNA strand degradation, which is a characteristic phenomenon that frequently occurs in the early stages of apoptosis, is often performed as a substitute for DNA fragmentation. Examples of the method for detecting the degradation of the DNA strand include the TUNEL (terminal transferase nick end labeling) method. In the TUNEL method, a DNA cleavable site is labeled with biotin-dUTP, which is a modified nucleotide, using terminal transferase, which is a foreign enzyme. It can detect fragmented DNA, and is more sensitive than conventional template-dependent ISNT (In Situ Translation) method. In addition, it is a detection method that is frequently used for apoptosis analysis. .

Japanese Patent No. 3576491

  However, the TUNEL method detects not only fragmented DNA but also nicks (cracks) only in one strand of double-stranded DNA, so that along with the cells in which DNA is fragmented, Even cells that have not yet been fragmented will be detected. For this reason, when detecting by the TUNEL method using a flow cytometer, cells in the DNA strand break stage and cell information in the DNA fragmentation stage coexist, and true evaluation cannot be performed. Become.

  In addition, there is a method of performing a TUNEL method using a flow cytometer and observing DNA fragmentation with a microscope, but in this case, it is difficult and time-consuming to measure two different assays using exactly the same cells. There is a problem that it takes. Furthermore, when observing DNA fragmentation with a microscope, generally darkly-stained fragmented nuclei (DNA) are observed in relatively lightly stained nuclei. When detecting the number of cells in which DNA is fragmented and measuring the number thereof, the burden on the measurer is large, and the results often vary depending on the subjectivity of the individual (measurer).

  According to the present invention, a DNA fragment is detected by distinguishing between an apoptotic cell in a stage where DNA strand breakage has not yet been fragmented and an apoptotic cell in which DNA has been fragmented by image analysis. It is an object of the present invention to provide a method for more accurately detecting apoptotic cells at the stage where they have been converted.

That is, the present invention
(1) A method for detecting apoptotic cells in which DNA is fragmented by image analysis, and (a) a fluorescent double-stranded nucleic acid against one or a plurality of cells fixed in a cell observation container A step of performing nuclear staining using a staining agent and TUNEL staining for fluorescently labeling a DNA strand break site; (b) imaging the cells stained in the step (a) using a microscope; A step of obtaining a double-stranded nucleic acid-stained image and a TUNEL-stained image; and (c) analyzing the double-stranded nucleic acid-stained image obtained in the step (b), thereby fragmenting the cell nucleus in the image. And (d) analyzing the TUNEL-stained image acquired in the step (b) to determine whether or not the DNA strand of the cell in the image has been cleaved. And (e ) DNA that has been determined in step (c) that the nucleus has been fragmented and that has been determined in step (d) that DNA strand breakage has occurred. A method for detecting apoptotic cells in which DNA is fragmented, characterized by comprising:
(2) A cell image provided with a threshold storage unit for preliminarily storing a threshold for cell nuclei, a threshold for fragmented cell nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining in the analysis of the double-stranded nucleic acid staining image in the step (c) An image input step in which the analysis device inputs the double-stranded nucleic acid stained image, and a region in which the cell image analysis device has a luminance value equal to or higher than the threshold for cell nuclei from the input double-stranded nucleic acid stained image A cell nucleus region extraction step for extracting a region having an area equal to or greater than the cell nucleus area threshold as a cell nucleus region, and the cell image analysis device generates a fragmented cell nucleus from the cell nucleus region as a fragmented cell nucleus. A fragmented cell nucleus region extraction step for extracting as a region, and the cell image analysis apparatus, when the cell image analysis device has two or more fragmented cell nucleus regions extracted from the cell nucleus region; A fragmented cell nucleus discriminating step for discriminating that the cell nucleus containing the cell nucleus region is fragmented and the cell nucleus containing the cell nucleus region is not fragmented when the number is one or zero. The analysis of the TUNEL-stained image in the step (d) includes the image input step in which the cell image analyzer inputs the TUNEL-stained image, and the cell image analyzer inputs the two TUNEL-stained images. A TUNEL staining luminance value measuring step for measuring a luminance value of a pixel included in a region corresponding to a cell nucleus region extracted in a single-stranded nucleic acid staining image; and the cell image analyzer, wherein the statistical value is the TUNEL When the cell containing the cell nucleus region is above the threshold for staining, DNA strand breakage has occurred, and when the cell nucleus region is less than the threshold for TUNEL staining, the cell nucleus region is included Detection method for apoptotic cells, wherein (1) DNA according becomes fragmented vesicles, which comprises carrying out the DNA strand break determination step of determining that no cause DNA strand breaks, by,
(3) The method for detecting apoptotic cells fragmented with DNA according to (1) or (2), wherein the cell observation container is a slide glass or a multiwell plate,
(4) The method for detecting apoptotic cells fragmented with DNA according to any one of (1) to (3), wherein microscopic image acquisition and image analysis are performed using an imaging cytometer,

(5) A threshold storage unit for preliminarily storing a threshold for cell nuclei, a threshold for fragmented cell nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining, and cells stained with a fluorescent double-stranded nucleic acid stain were imaged A double-stranded nucleic acid stained image, a TUNEL-stained image that is fluorescently labeled at the DNA strand break site, an image input unit that inputs a TUNEL-stained image having the same field of view as the double-stranded nucleic acid stained image, and an input double-stranded nucleic acid From a stained image, a cell nucleus region extraction unit that extracts a region having an area greater than or equal to the cell nucleus area threshold as a cell nucleus region among regions having a luminance value greater than or equal to the cell nucleus threshold, and from the cell nucleus region, the fragmented cell nucleus threshold or greater When a fragmented cell nucleus region extraction unit that extracts a region having a luminance value as a fragmented cell nucleus region and two or more fragmented cell nucleus regions extracted from the cell nucleus region A fragmented cell nucleus discriminating unit for discriminating that the cell nucleus including the cell nucleus region is not fragmented when the cell nucleus including the cell nucleus region is fragmented and is 1 or 0; The TUNEL staining luminance value measurement unit that measures the statistical value of the luminance value of the pixels included in the region corresponding to the cell nucleus region extracted in the double-stranded nucleic acid staining image of the TUNEL staining image, and the statistical value, When the TUNEL staining threshold is exceeded, the cell containing the cell nucleus region has undergone DNA strand breaks, and when the TUNEL staining threshold is less than the cell, the cell containing the cell nucleus region has undergone DNA strand breaks. A DNA strand break discriminating section for discriminating that the cell has been determined that the nucleus has been fragmented and the DNA strand break has occurred, And analysis section which analyzes as a scan cell,
A cell image analysis device comprising:
(6) The fragmented cell nucleus region extraction unit extracts a region having a luminance value equal to or higher than a fragmented cell nucleus threshold value from the cell nucleus region as a candidate region; and the luminance value of each pixel in the candidate region A region determination unit that detects a boundary line by performing a boundary line detection process based on the inclination of the region, and extracts a region surrounded by the detected boundary line as a fragmented cell nucleus region, 5) The cell image analysis device according to
(7) Cells stained with a fluorescent double-stranded nucleic acid stain for a computer having a threshold storage unit that stores in advance a threshold for cell nuclei, a threshold for fragmented cell nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining An image input step for inputting a double-stranded nucleic acid stained image obtained by imaging, and a TUNEL-stained image having the same field of view as the double-stranded nucleic acid stained image that is fluorescently labeled at the DNA strand break site. A cell nucleus region extraction step for extracting, as a cell nucleus region, a region having an area equal to or greater than the cell nucleus area threshold value from the double-stranded nucleic acid stained image, and a fragmentation from the cell nucleus region. A fragmented cell nucleus region extracting step for extracting a region having a luminance value equal to or higher than a threshold value for a cell nucleus as a fragmented cell nucleus region; and a fragmentation cell extracted from the cell nucleus region The cell nucleus containing the cell nucleus region is fragmented when the cell nucleus region is 2 or more, and the cell nucleus containing the cell nucleus region is not fragmented when the cell nucleus region is 1 or 0. A fragmented cell nucleus discriminating step for discriminating between and a TUNEL staining for measuring a statistical value of luminance values of pixels included in an area corresponding to a cell nucleus area extracted in the double-stranded nucleic acid staining image of the input TUNEL stained image When the luminance value measurement step and the statistical value are equal to or greater than the threshold for TUNEL staining, cells containing the cell nucleus region have undergone DNA strand breaks, and when the statistical value is less than the threshold for TUNEL staining, the cell nucleus region A DNA strand break discriminating step for discriminating that DNA strand breakage has not occurred in the cells containing nuclei, it was determined that the nucleus was fragmented, and it was determined that DNA strand breakage had occurred Program for executing the cells, and the analysis step of analyzing the DNA is fragmented apoptotic cells, and

(8) A method for evaluating the stage of apoptosis by image analysis, using (a′1) phosphatidylserine fluorescence staining, fluorescence staining using a probe for detecting mitochondrial membrane potential, and cell membrane permeability probe A step of fixing a cell subjected to one or more fluorescent staining selected from the group consisting of fluorescent staining to a cell observation container; and (a′2) after the step (a′1), the cell observation container A step of performing nuclear staining using a fluorescent double-stranded nucleic acid stain and TUNEL staining for fluorescently labeling a DNA strand break site on the cells in the cell, (b ′) after the step (a ′ 2) A step of acquiring a stained image of cells in the cell observation container for each type of cell staining using a microscope, and (c ′) a double-stranded nucleic acid stained image acquired in the step (b ′). By analyzing the image A step of discriminating whether or not the nucleus of the cell in the cell is fragmented, and (d ′) analyzing the TUNEL-stained image obtained in the step (b ′), thereby analyzing the DNA chain of the cell in the image A step of determining whether or not cleavage has occurred; (e ′) a cell in which the nucleus is determined to be fragmented in the step (c ′), and DNA in the step (d ′) Evaluating a cell determined to have undergone strand breakage as an intermediate or late apoptotic cell in which DNA is fragmented; and (f ′) phosphatidylserine in the step (b ′). When a fluorescent stained image is obtained, the stained image is analyzed, and a cell determined that phosphatidylserine is localized outside the cell membrane is evaluated as an apoptotic cell, and the step (b ′ ) Mitocondo When a probe fluorescent staining image for membrane potential detection is obtained, the stained image is analyzed, and a cell determined to be depolarized in the mitochondrial membrane is evaluated as an apoptotic cell, and the step (b ') When the probe membrane staining dye detection image for cell membrane permeability detection is acquired, the staining image is analyzed, and the cells determined to be stained with the cell membrane permeability probe are evaluated as apoptotic cells. g ′) Among the cells whose DNA was not judged to be fragmented in the step (e ′), the cells evaluated as apoptotic cells in the step (f ′) are early apoptotic cells. An evaluation method of the stage of apoptosis, characterized by comprising:
Is provided.

By the method for detecting apoptotic cells of the present invention, DNA strand breakage and DNA fragmentation, which are very important morphological changes in apoptosis evaluation, can be distinguished and detected. Therefore, by using the method for detecting apoptotic cells of the present invention, the stage of apoptotic cells can be more accurately evaluated.
Furthermore, by using the cell image analyzer of the present invention, an apoptotic cell at a stage in which a DNA strand has been cut but has not yet been fragmented, and an apoptotic cell at a stage where DNA has been fragmented, Can be detected separately.

It is a schematic block diagram showing the functional composition of the cell image analysis device which is one mode of the cell image analysis device. It is the schematic showing the outline of the threshold value for cell nuclei and the threshold value for fragmented cell nuclei which a threshold value memory | storage part memorize | stores. It is a figure showing the example of the image of the cell nucleus which has not raise | generated apoptosis. It is a figure showing the example of the image of the cell nucleus which raise | generated apoptosis. It is a flowchart showing the operation example of a cell image analyzer. It is a flowchart showing the operation example of the fragmented cell nucleus area | region extraction part in a fragmented cell nucleus area | region extraction process. It is a figure showing the example of the result of the area | region determination part 106a among the cell image analyzer 1a. In Example 1, it is the figure which showed the result of the image analysis. In Example 1, it is the figure which showed the analysis result for every staurosporine density | concentration.

In the present invention, “DNA is fragmented” means that chromosomal DNA is cleaved between a nucleosome and a reosome to be extracted and decomposed into two or more fragments.
Further, “a DNA strand breaks” means that at least one strand of a double-stranded DNA constituting a chromosome is cut. In other words, cells in which the DNA strand is cleaved include cells in which DNA is fragmented, but chromosomal DNA is simply cleaved and not broken down into fragments, that is, the DNA is fragmented. Not including cells.
Further, “nucleus is fragmented” means a state in which chromosomal DNA derived from one cell forms two or more aggregated regions. This aggregated area is strongly stained with a double-stranded nuclear stain. That is, when double-stranded nuclear staining is performed, if there are two or more strongly stained regions in the nucleus region, it is said that the nucleus is fragmented.

<Method for detecting apoptotic cells fragmented with DNA>
In the method for detecting apoptotic cells of the present invention, DNA strands are cleaved by using nuclear staining (double-stranded nucleic acid staining) using a double-stranded nucleic acid stain and TUNEL staining by image analysis. An apoptotic cell at a stage that has not yet been fragmented (hereinafter referred to as “DNA strand break stage”) and an apoptotic cell at a stage where DNA fragmentation has been performed (hereinafter referred to as “DNA fragmentation stage”) is distinguished. It is detected separately. By TUNEL staining, it can be determined whether or not the DNA strand is cleaved. TUNEL staining positive cells (TUNEL-stained cells) are cells that are nicked only in one strand of double-stranded DNA and have not yet been fragmented (hereinafter referred to as “DNA strand-breaking cells”). )) And cells in which both strands of double-stranded DNA are cleaved and fragmented (hereinafter also referred to as “DNA fragmented cells”) are included. On the other hand, it is possible to determine whether or not nuclear fragmentation has occurred by nuclear staining using a double-stranded nucleic acid stain. However, cells in which the nucleus is fragmented (hereinafter referred to as “nuclear fragmented cells”). In some cases, DNA is also fragmented. Therefore, by combining double-stranded nucleic acid staining and TUNEL staining, among TUNEL staining positive cells, the nuclear fragmented cells are defined as DNA fragmented cells, and the cells in which no nuclear fragmentation has been observed are DNA strand cleaved cells. And can be detected separately.

  In the present invention, the presence or absence of nuclear fragmentation or the presence or absence of TUNEL staining is determined for each individual cell in a cell observation container that has been subjected to double-stranded nucleic acid staining and TUNEL staining using a microscope. At this time, by acquiring a microscope image and performing image analysis, it is simpler and quicker than the conventional method of performing visual observation under a microscope, and the operation burden at the time of discrimination can be significantly reduced.

Specifically, the method for detecting apoptotic cells of the present invention comprises the following steps (a) to (e).
(A) A step of performing nuclear staining using a fluorescent double-stranded nucleic acid stain and TUNEL staining for fluorescently labeling a DNA strand break site on one or a plurality of cells fixed in a cell observation container .
(B) A step of imaging the cells stained in the step (a) using a microscope and obtaining a double-stranded nucleic acid stained image and a TUNEL stained image of each cell.
(C) A step of determining whether or not the nucleus of the cell in the image is fragmented by analyzing the double-stranded nucleic acid stained image obtained in the step (b).
(D) A step of analyzing the TUNEL-stained image acquired in the step (b) to determine whether or not the DNA strand breakage of the cells in the image has occurred.
(E) a cell in which the nucleus has been determined to be fragmented in the step (c) and the DNA in which the DNA strand breakage has been determined in the step (d) Detecting as fragmented cells.
Hereinafter, it demonstrates for every process.

  First, as step (a), nuclear staining using a fluorescent double-stranded nucleic acid stain for one or a plurality of cells fixed in a cell observation container, and TUNEL that fluorescently labels a DNA strand break site And dyeing.

The cell observation container is not particularly limited as long as it is generally used for cell staining, but it is preferably a slide glass or a multiwell plate.
By using an imaging cytometer or the like using a slide glass or a multi-well plate, a large number of specimens can be processed quickly and easily.

  The method for fixing cells to the cell observation container is not particularly limited, and can be performed by a conventional method. For example, it may be naturally dried, and cells can be fixed to the cell observation container by presenting a cell-fixing cross-linking agent such as formaldehyde, paraformaldehyde, or methanol. After the fixing treatment, the cells are washed with PBS or the like to remove the cell fixing cross-linking agent. If cell staining is not performed immediately after the washing treatment, the cell observation container can be filled with a buffer such as PBS, and a dry prevention film can be attached and stored at 4 ° C. In addition, it is also preferable to block the cell observation container on which the cells are fixed with a skim milk-containing PBS or the like before reacting with the detection antibody.

  Double-stranded nucleic acid staining and TUNEL staining are cell stains that are widely used in the art, and can be performed by conventional methods. Moreover, both dyeing | staining may be performed simultaneously and may be performed separately.

For example, double-stranded nucleic acid staining can be performed by immersing cells that have been subjected to cell membrane permeabilization with a surfactant or the like in a double-stranded nucleic acid stain solution for a certain period of time.
The double-stranded nucleic acid stain is not particularly limited as long as it is a stain made of a fluorescent substance capable of staining double-stranded DNA, and is appropriately selected from known fluorescent nuclear stains. be able to. In the present invention, an intercalator is preferable. Known fluorescent double-stranded nucleic acid stains include, for example, DAPI (4 ′, 6-diamino-2-phenylindole), PI (propidium iodide), Hoechst, DRAQ5 (registered trademark) (manufactured by Biostatus), Sytox ( Registered trademark) (manufactured by Invitrogen), YOYO (registered trademark) (manufactured by Invitrogen), and the like.

  On the other hand, in TUNEL staining, first, cells subjected to cell membrane permeabilization are immersed in a solution containing terminal transferase and modified dUTP (dUTP modified with a modifying substance) for a certain period of time, so that 3 'Incorporate modified dUTP into -OH group. The modified dUTP is not particularly limited as long as it is incorporated into the DNA strand cleavage site by terminal transferase, and dUTP modified with a modifying substance generally used for detecting nucleotides may be used. it can. Examples of such modified dUTP include those using a ligand such as BrdUTP (brominated deoxyuridine triphosphate) or biotin-dUTP as a modifying substance. Such a ligand is not particularly limited as long as it is usually used for labeling of nucleotides and the like. (digoxin) There are sugar chains composed of 2 or more sugars, polypeptides composed of 6 or more amino acids, auxins, gibberellins, steroids, proteins, and analogs thereof.

  Thereafter, the DNA strand cleavage site can be fluorescently stained using this modifying substance as an index. For example, when BrdUTP is used as the modified dUTP, immunostaining is performed using an anti-BrdUTP antibody. The immunostaining may be a direct method of staining with an anti-BrdUTP antibody labeled with a fluorescent substance, or an indirect method of staining with a secondary antibody (antibody against an anti-BrdUTP antibody) labeled with a fluorescent substance. It may be. The fluorescent substance for labeling the antibody is not particularly limited as long as it is a fluorescent substance that is generally used for labeling biomolecules such as proteins, and may be appropriately selected from known fluorescent substances. it can. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine) and the like.

  In addition, when a modified dUTP having a ligand as a modifying substance, such as biotin-dUTP, is used, fluorescent staining is performed using a receptor labeled with a fluorescent substance that can specifically bind to the ligand. Can do. As the receptor, an antibody of the ligand, a compound usually used for detection of the ligand, and the like can be used. For example, when the ligand is biotin, it is avidin or streptavidin, and the ligand is glutathione. GST (glutathione-S-transferase), and MBP (maltose binding protein) when the ligand is amylose such as maltose.

  In the present invention, since the double-stranded nucleic acid stained image and the TUNEL-stained image are separately obtained in the step (b), the fluorescent substance used as the antibody or receptor label in TUNEL staining is double-stranded nucleic acid stained. A fluorescent material having a fluorescent property different from that of the agent is used. In the present invention, the fact that the fluorescence characteristics are different means that the wavelengths of the fluorescence emitted by the excitation light irradiation are so different that they can be distinguished and detected, such as FITC and rhodamine.

  Next, as a step (b), the stained cells are imaged using a microscope, and a double-stranded nucleic acid stained image and a TUNEL-stained image of each cell are obtained. Stained images are captured using a confocal laser microscope or the like by irradiating cells with excitation light having a wavelength suitable for the fluorescence characteristics of the fluorescent material used as a label in staining, and detecting the fluorescence emitted from the fluorescent material. Can be obtained. A double-stranded nucleic acid-stained image and a TUNEL-stained image are acquired separately for each shot (one microscope field) and accumulated.

  Next, in step (c), by analyzing the obtained double-stranded nucleic acid stained image, it is determined whether or not the cell nucleus in the image is fragmented. Fragmented nuclei have a higher density than non-fragmented nuclei and are therefore more intensely (darker) stained with double-stranded nucleic acid stains. Here, in a stained image, a region that is strongly stained has higher luminance (degree of brightness) per pixel (pixel) than a region that is weakly (thinly) stained. Therefore, by utilizing this difference in brightness, cells in the double-stranded nucleic acid stained image can be detected separately from those in which the nucleus is not fragmented. In addition, the number of fragmented nuclei per cell can be detected at the same time.

Specifically, a cell nucleus threshold and a cell nucleus area threshold for recognizing nuclei in a double-stranded nucleic acid staining image, and fragmented nuclei (hereinafter sometimes referred to as “fragmented cell nuclei”) are used. A threshold for fragmented cell nuclei for recognition is set, and cell nuclei in a region where each pixel has a luminance value (hereinafter referred to as “luminance value”) greater than or equal to the threshold for cell nuclei from a double-stranded nucleic acid stained image. A region having an area equal to or larger than the use area threshold is extracted as a cell nucleus region. In addition, the area here may mean not only the numerical value of the accurate area of the region but also the number of pixels included in the region.
Furthermore, from the extracted cell nucleus region, a region having a luminance value equal to or higher than the threshold for fragmented cell nuclei is extracted as a fragmented cell nucleus region. When there are two or more fragmented cell nucleus regions extracted from the cell nucleus region, the nucleus of the cell containing the cell nucleus region is fragmented, and one or 0 fragmented cell nucleus regions are extracted from the cell nucleus region. In the case of individual cells, it is determined that the cell nucleus including the cell nucleus region is not fragmented.

  Here, the cell nucleus threshold, the cell nucleus area threshold, and the fragmented cell nucleus threshold are appropriately set in consideration of the type of cell to be analyzed, the type of double-stranded nucleic acid stain to be used, and the like. For example, the same type of cells as the analysis target cells were previously stained with a double-stranded nucleic acid stain, and after obtaining a stained image, the luminance value and area value of the nuclear region were measured from the stained image, and obtained. It can be set based on the measured value. Similarly, the threshold for fragmented cell nuclei is the same type of cell as the analysis target cell, and after obtaining a stained image of cells that have undergone apoptosis due to treatment with an apoptosis inducer, the nucleus is fragmented from the stained image. The luminance value of the area being measured can be measured and set based on the obtained measurement value.

  In addition, the fragmented cell nucleus region may be extracted from all the extracted cell nucleus regions, but the group of cell nucleus regions to be extracted is specified by the area value of the cell nucleus region and other shape description factors. May be. In this way, debris can be excluded from the extraction target by using description factors of other shapes. Examples of the shape description factor include a circularity factor, a circumferential perimeter, a MAX ferret, and an elongation factor.

  Further, as a step (d), by analyzing the acquired TUNEL-stained image, it is determined whether or not the DNA strand of the cell in the image has been broken. TUNEL staining positive cells (cells stained by TUNEL staining) have a cleaved DNA strand, while TUNEL staining negative cells do not have a cleaved DNA strand. In the present invention, when the fluorescence intensity of TUNEL staining in the nucleus is greater than or equal to a preset threshold value, the cell containing the nucleus is assumed to be a TUNEL staining positive cell.

  Specifically, a threshold for TUNEL staining is set in advance, and for each cell nucleus region (region corresponding to each cell nucleus region extracted from the double-stranded nucleic acid staining image) in the TUNEL staining image, The statistical value of the luminance value of the pixel included in the area is measured. The statistical value of the luminance value may be a total value of the luminance values of all the pixels included in the cell nucleus region, or may be an average value per pixel. When the measured statistical value is equal to or greater than the threshold for TUNEL staining, the cell containing the cell nucleus region has undergone DNA strand breakage, and when the measured amount is less than the threshold for TUNEL staining, the cell nucleus region It is determined that the DNA chain is not broken.

  Here, the threshold for TUNEL staining can be appropriately set in consideration of the type of fluorescent material used for TUNEL staining, the type of target cell to be analyzed, and the like. For example, the statistical value of the luminance value (background) of the area where no cells exist in the TUNEL-stained image may be used as the threshold for TUNEL staining. The brightness value of an appropriate region inside and outside the cell may be measured from the stained image, and may be set based on the statistical value of the obtained measurement value.

  Similarly to the extraction of the fragmented cell nucleus region, it may be determined whether all of the cell nucleus regions extracted in step (c) are positive or negative for TUNEL staining. You may go only.

  Thereafter, in step (e), cells that have been determined that the nucleus has been fragmented in step (c) and that have been determined that DNA strand breakage has occurred in step (d) By detecting the DNA as a fragmented cell, an apoptotic cell in which the DNA is fragmented can be detected.

  Theoretically, the cells in which the nucleus is determined to be fragmented in step (c) include normal cells and cells in the M (mitosis) phase of the cell cycle. In particular, after the doubling daughter chromosomes are pulled by the spindle and each move in the polar direction, cells before cytokinesis (late M phase cells to late cells) extract two fragmented cell nucleus regions per cell. It is to be done. On the other hand, these M-phase cells are negative for TUNEL staining, and it is determined that DNA strand breakage has not occurred in step (d). That is, as in the present invention, the presence or absence of nuclear fragmentation and the presence or absence of TUNEL staining (presence or absence of DNA strand breaks) are evaluated in combination to accurately detect apoptotic cells in which DNA has been fragmented. be able to.

  In addition, for cells from which two fragmented cell nucleus regions per cell have been extracted, a double-stranded nucleic acid staining image is called for each individual cell and visually confirmed, so that the cell is a normal cell in the M phase. It is possible to more accurately determine whether the cell is an apoptotic cell. In addition, normal cells and apoptotic cells can be more accurately distinguished by using as a negative control a cell that is clearly not undergoing apoptosis (for example, a cell that has not been stimulated with an apoptosis inducer, etc.). It becomes possible.

DNA fragmentation is a morphological change observed from the mid to late stages of apoptosis. Therefore, the cells in which the DNA detected by the detection method of the present invention is fragmented can be evaluated as apoptotic cells in the middle stage or late stage.
For this reason, by using the method for detecting apoptotic cells of the invention, it is also possible to evaluate the effect on apoptosis when cells are treated with a certain test compound. Therefore, an inducer or inhibitor (inhibitor) of apoptosis, etc. Screening can be performed efficiently. It can also be applied to toxicity tests of test compounds.

In addition, the detection method of the present invention may be combined with immunostaining of biomolecules that are involved in apoptosis signals such as caspases and Bcl-2 family proteins. In addition, although immunostaining of these biomolecules may be performed separately from the TUNEL staining on the cells fixed in the cell observation container, it can also be performed simultaneously.
For example, when immunostaining is performed using a specific antibody against active caspase 3, the stained cells (positive cells) can be determined to be apoptotic cells. Thus, apoptotic cells can be detected more accurately by combining immunostaining of biomolecules related to apoptotic signals.

<Cell image analyzer>
Steps (c), (d), and (e) can be analyzed using one image analysis apparatus that can realize these steps.
FIG. 1 is a schematic block diagram showing a functional configuration of a cell image analysis apparatus 1 which is an embodiment of such a cell image analysis apparatus. The cell image analysis apparatus 1 includes an image input unit 101, a threshold storage unit 102, a cell nucleus region extraction unit 103, a fragmented cell nucleus region extraction unit 104a, a fragmented cell nucleus discrimination unit 111, a TUNEL staining luminance value measurement unit 112, and a DNA strand break discrimination. Unit 113, analysis unit 114, and analysis result output unit 107. The cell image analysis apparatus 1a may be configured using an information processing apparatus such as a personal computer or a workstation, or may be configured as a dedicated apparatus incorporated in a microscope or the like.

  The image input unit 101 inputs digital data of cell images (double-stranded nucleic acid stained image and TUNEL stained image) obtained by imaging cells stained with a fluorescent substance with a microscope to the cell image analysis apparatus 1a. The double-stranded nucleic acid stained image and the TUNEL stained image are images obtained by imaging cells in the same visual field.

  The threshold value storage unit 102 is a cell nucleus threshold value, a fragmented cell nucleus threshold value, a cell nucleus area threshold value, and a TUNEL staining used in the processing of the cell nucleus region extraction unit 103, the fragmented cell nucleus region extraction unit 104a, and the DNA strand breakage determination unit 113. The threshold value is stored in advance.

  The cell nucleus region extraction unit 103 extracts a cell nucleus region from the input double-stranded nucleic acid staining image based on the cell nucleus threshold and the cell nucleus area threshold, and acquires information representing the position and range of the cell nucleus region. In addition, the area here may mean not only the numerical value of the accurate area of the region but also the number of pixels included in the region.

  The fragmented cell nucleus region extraction unit 104a extracts the fragmented cell nucleus region from the cell nucleus region of the input double-stranded nucleic acid staining image based on the fragmented cell nucleus threshold, and displays information indicating the position and range of the fragmented cell nucleus region. get.

  Specifically, the fragmented cell nucleus region extraction unit 104a includes a candidate region extraction unit 105a and a region determination unit 106a. The candidate area extraction unit 105a extracts an area having a luminance value (brightness value) equal to or higher than the fragmented cell nucleus threshold value from the inside of the cell nucleus area as a candidate area. The region determination unit 106a detects a boundary line by performing a boundary line detection process based on the gradient of the luminance value of each pixel in the candidate region, and extracts a region surrounded by the detected boundary line as a fragmented cell nucleus region. . Note that the luminance here is a value representing light and shade in a light and shade image, for example, and is a value representing the brightness of each pixel (pixel) in the image.

  The fragmented cell nucleus discriminating unit 111 discriminates the presence or absence of nuclear fragmentation in the cell nucleus region extracted by the cell nucleus region extracting unit 103 based on the result of the fragmented cell nucleus region extracting unit 104a. Specifically, when the number of fragmented cell nucleus regions extracted from the cell nucleus region is 2 or more, the nucleus of the cell containing the cell nucleus region is fragmented, and when the number is 1 or 0, It is determined that the cell nucleus including the cell nucleus region is not fragmented.

  The TUNEL staining luminance value measurement unit 112 may be referred to as a region corresponding to the cell nucleus region extracted in the double-stranded nucleic acid staining image of the input TUNEL staining image (hereinafter simply referred to as “cell nucleus region of the TUNEL staining image”). )) To measure the statistical value of the luminance value of the pixels included. The statistical value of the luminance value may be a total value of the luminance values of all the pixels included in the cell nucleus region, or may be an average value per pixel.

  The DNA strand break discriminating unit 113 discriminates the presence or absence of DNA strand breaks in the cell nucleus region extracted by the cell nucleus region extracting unit 103 based on the result of the TUNEL staining luminance value measuring unit 112 and the threshold for TUNEL staining. Specifically, when the statistical value obtained by the TUNEL staining luminance value measuring unit 112 is equal to or greater than the TUNEL staining threshold, the cell including the cell nucleus region has undergone DNA strand breakage, and the TUNEL staining If it is less than the threshold value for use, it is determined that the cell containing the cell nucleus region has not undergone DNA strand breakage.

  Based on the results of the fragmented cell nucleus discriminating unit 111 and the DNA strand break discriminating unit 113, the analyzing unit 114 determines whether the cell containing the cell nucleus region extracted by the cell nucleus region extracting unit 103 is an apoptotic cell in which DNA is fragmented. Analyze whether or not. Specifically, the fragmented cell nucleus discriminating unit 111 determines that the nucleus has been fragmented, and the DNA strand breakage discriminating unit 113 determines that the DNA strand break has occurred. It is analyzed that the cells are apoptotic cells.

The analysis result output unit 107 generates and outputs an analysis result based on information representing the positions and ranges of the cell nucleus region and the fragmented cell nucleus region. The analysis result output unit 107 may output the analysis result by causing the image output apparatus to display it as a graphic such as a character or a graph, or may output the analysis result by printing it on a printer. The analysis results output by the analysis result output unit 107 are, for example, the following items.
-Number of cell nucleus regions-Number of fragmented cell nucleus regions-Number of cell nucleus regions from which fragmented cell nucleus regions were not extracted from inside-Number of cell nucleus regions from which fragmented cell nucleus regions were extracted from inside-Total area of cell nucleus regions・ Total area of fragmented cell nucleus area ・ Ratio of total area of cell nucleus area and total area of fragmented cell nucleus area ・ Average brightness value of cell nucleus area and fragmented cell nucleus area in double-stranded nucleic acid stained image Ratio of average brightness value ・ Average brightness value of cell nucleus area in TUNEL-stained image ・ Total brightness value of cell nucleus area in TUNEL-stained image ・ Presence / absence of fragmentation of nucleus in each nucleus area ・ Each cell nucleus area The presence or absence of DNA strand breaks, the number of cell nucleus regions determined to be broken, the number of cell nucleus regions determined not to be broken, the nucleus being fragmented, and The number / nucleus of cell nucleus regions determined to be NA chain cleaved are fragmented, and the number of cell nucleus regions determined to be DNA chain not cleaved are not fragmented, And the number of cell nucleus regions determined to have a DNA strand cleaved, the number of cell nucleus regions that have been determined that the nucleus is not fragmented and the DNA strand is not cleaved

  FIG. 2 is a schematic diagram illustrating the outline of the threshold for cell nuclei and the threshold for fragmented cell nuclei stored in the threshold storage unit 102. The graph of FIG. 2 represents a change in luminance value in the x-axis direction when the y-axis value is fixed to a predetermined value in a double-stranded nucleic acid stained image obtained in advance by experiments. In FIG. 2, the value of the x coordinate that becomes the boundary between the areas A1 to A3 is a value set by a designer or an experimenter. The region A1 is a region that does not correspond to either the cell nucleus region or the fragmented cell nucleus region. In the double-stranded nucleic acid stained image, since the cell nucleus is stained using a fluorescent substance that selectively stains the cell nucleus, the area where the cell nucleus and the fragmented cell nucleus do not exist, such as A1, is represented by a pixel having a low luminance value. Occupied. The region A2 is a cell nucleus region in which no fragmented cell nucleus exists. The region A3 is a region where fragmented cell nuclei are present. In a double-stranded nucleic acid-stained image, since the luminance value increases as the density of cell nuclei increases, the region where fragmented cell nuclei exist has a higher luminance value than the cell nucleus region where no fragmented cell nuclei exist.

  Based on such experimental results, the values for the cell nucleus threshold and the fragmented cell nucleus threshold are set. That is, the luminance value at the pixel that becomes the boundary between the region A1 and the region A2 is set as the threshold value for the cell nucleus, and the luminance value at the pixel that becomes the boundary between the region A2 and the region A3 is set as the threshold value for the fragmented cell nucleus. At this time, the fragmented cell nucleus threshold value is set to a value larger than the cell nucleus threshold value. Actually, the two values of the threshold for the cell nucleus and the threshold for the fragmented cell nucleus are determined according to the experimental environment by performing a plurality of experiments in advance and taking statistics of the luminance values of the cell nucleus region and the region where the fragmented cell nucleus exists. Determined optimally.

FIG. 3A is a diagram illustrating an example of an image of a cell nucleus in which apoptosis has not occurred. FIG. 3B is a diagram illustrating an example of a cell nucleus region extracted by the cell nucleus region extraction unit 103.
In FIG. 3B, a region surrounded by a broken line represented by a symbol X represents a cell nucleus region extracted by the cell nucleus region extraction unit 103. In the drawings of the present application including FIG. 3, the lower the luminance value is, the more white the color is, and the higher the luminance value is, the black, the color is for convenience of drawing.

  FIG. 4A is a diagram illustrating an example of an image of a cell nucleus in which apoptosis has occurred. As shown in FIG. 4A, in the image of the cell nucleus in which apoptosis occurs, a plurality of fragmented cell nuclei exist inside the cell nucleus. FIG. 4B is a diagram illustrating an example of a cell nucleus region extracted by the cell nucleus region extraction unit 103 and a fragmented cell nucleus region extracted by the fragmented cell nucleus region extraction unit 104a. In FIG. 4B, a region surrounded by a broken line represented by a symbol X represents a cell nucleus region extracted by the cell nucleus region extraction unit 103. In FIG. 4B, a region surrounded by a solid line represented by a symbol Y represents a fragmented cell nucleus region extracted by the fragmented cell nucleus region extraction unit 104a.

  FIG. 5 is a flowchart showing an operation example of the cell image analysis apparatus 1a. Hereinafter, an operation example of the cell image analysis apparatus 1a will be described with reference to FIG. First, the image input unit 101 inputs digital data of a cell image (double-stranded nucleic acid stained image and TUNEL stained image) to the cell image analysis apparatus 1a (step S101). Next, the cell nucleus region extraction unit 103 reads the cell nucleus threshold stored in the threshold storage unit 102, and extracts all regions having a luminance value equal to or higher than the cell nucleus threshold from the double-stranded nucleic acid stained image (step S102). Next, the cell nucleus region extraction unit 103 reads the cell nucleus area threshold value stored in the threshold value storage unit 102, and among the regions extracted in step S102, the region whose area is equal to or greater than the cell nucleus area threshold value is defined as a cell nucleus region. All are extracted (step S103).

  Next, the fragmented cell nucleus region extraction unit 104a executes a fragmented cell nucleus region extraction process (step S104). FIG. 6 is a flowchart showing an operation example of the fragmented cell nucleus region extraction unit 104a in the fragmented cell nucleus region extraction process. First, the candidate region extraction unit 105a reads the fragmented cell nucleus threshold stored in the threshold storage unit 102, and has a luminance value equal to or greater than the fragmented cell nucleus threshold from the inside of each cell nucleus region extracted in step S103. Are extracted as candidate regions (step S201).

  Next, the region determination unit 106a extracts all boundary lines by performing boundary line extraction processing based on the gradient of the luminance value of each pixel in each candidate region (step S202). The boundary line extraction process in step S202 is realized by applying the boundary line extraction process (boundary line detection process) in the conventional image processing technique. For example, the region determination unit 106a calculates a differential value related to the luminance value in each pixel of the candidate region, and extracts a boundary line by continuously connecting pixels whose differential values are locally maximum.

  Next, the region determination unit 106a extracts each region surrounded by each extracted boundary line as a fragmented cell nucleus region (step S203), and ends the fragmented cell nucleus region extraction process.

  Returning to FIG. When the fragmented cell nucleus region extraction process is completed, the fragmented cell nucleus discriminating unit 111 determines whether the nucleus is fragmented in each cell nucleus region extracted in step S103 based on the extraction result of the fragmented cell nucleus region extracting unit 104a. It is determined whether or not (step S105).

  Next, the TUNEL staining luminance value measuring unit 112 measures the luminance values of all the pixels included in each cell nucleus region extracted in step S103 from the TUNEL staining image, and the statistics for each cell nucleus region are measured from the measured luminance values. A value is calculated (step S106).

  Next, the DNA strand break discriminating unit 113 reads the TUNEL staining threshold stored in the threshold storage unit 102, and in each cell nucleus region extracted in step S103 based on the measurement result of the TUNEL staining luminance value measuring unit 112, It is determined whether or not the DNA strand is cleaved (step S107).

  Note that steps S104 to S107 may be performed in any order as long as S104 is performed before step 105 and S105 is performed before step 107. For example, steps S104 and S105 may be performed after performing steps S106 and S107.

  Next, based on the discrimination result of the fragmented cell nucleus discriminating unit 111 and the discrimination result of the DNA strand break discriminating unit 113, the analysis unit 114 causes the cell including the cell nucleus region extracted by the cell nucleus region extracting unit 103 to fragment DNA. It is analyzed whether or not the cell is an apoptotic cell (step S108).

  When the analysis by the analysis unit 114 is completed, the analysis result output unit 107 generates and outputs the analysis result of the analysis unit 114 (step S109), and the process illustrated in FIG.

  FIG. 7 is a diagram illustrating an example of a result of the region determination unit 106a in the cell image analysis apparatus 1a. When a plurality of cell nuclei and fragmented cell nuclei are captured in the cell image input to the cell image analysis apparatus 1a, the cell nucleus region extraction unit 103 displays all the captured cell nucleus regions (represented by the symbol X in FIG. 7). 3), the fragmented cell nucleus region extraction unit 104a extracts all of the imaged fragmented cell nucleus regions (regions within eight solid lines represented by symbol Y in FIG. 7). To extract.

  In the cell image analysis apparatus 1a configured as described above, by using the threshold for cell nuclei and the threshold for fragmented cell nuclei having a value larger than the threshold for cell nuclei, that is, by using two thresholds related to the luminance value, It becomes possible to extract the cell nucleus region and the fragmented cell nucleus region, respectively.

  Further, as shown in FIG. 2, if a region having a luminance value equal to or higher than the threshold for fragmented cell nuclei is extracted as a fragmented cell nucleus region, a plurality of fragmented cell nucleus regions are mistakenly regarded as one fragmented cell nucleus region. May be detected. In response to such a problem, the fragmented cell nucleus region extraction unit 104a performs the boundary line extraction process based on the gradient of the luminance value of each pixel in addition to the process using the fragment cell nucleus threshold. Since the region surrounded by the extracted boundary line is extracted as the fragmented cell nucleus region, the fragmented cell nucleus region can be extracted more accurately.

<Modification>
The fragmented cell nucleus region extraction unit 104a may extract all candidate regions extracted in step S201 as fragmented cell nucleus regions without performing steps S202 and S203. In other words, the fragmented cell nucleus region extraction unit 104a may be configured to extract, as a fragmented cell nucleus region, a region having a luminance value equal to or higher than the fragmented cell nucleus threshold.

  You may make it implement | achieve the function of the cell image analyzer 1a which is the one aspect | mode of the cell image analyzer of this invention mentioned above with a computer. In that case, a program for realizing each function may be recorded on a computer-readable recording medium, and the program recorded on the recording medium may be read into a computer system and executed. The “computer system” here includes an OS and hardware such as peripheral devices. The “computer-readable recording medium” refers to a storage device such as a flexible medium, a magneto-optical disk, a portable medium such as a ROM and a CD-ROM, and a hard disk incorporated in a computer system. Furthermore, the “computer-readable recording medium” dynamically holds a program for a short time like a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line. In this case, a volatile memory inside a computer system serving as a server or a client in that case may be included and a program that holds a program for a certain period of time. The program may be a program for realizing a part of the functions described above, and may be a program capable of realizing the functions described above in combination with a program already recorded in a computer system.

  The method for detecting apoptotic cells of the present invention includes such a cell image analysis apparatus, a microscope unit and an imaging unit for acquiring a stained image of the cells, and an image recording unit for accumulating and recording the captured images. It is preferable to use an imaging cytometer. In the present invention, the imaging cytometer means an image analysis apparatus capable of automatically acquiring a stained image and analyzing the obtained stained image. For example, a cell population on a glass slide is scanned with a laser focused spot, the fluorescence emitted by individual cells of this cell population is detected, and the scanned image data is image-processed. Examples thereof include a laser scanning cytometer that extracts and measures data (for example, see JP-A-3-255365), a cytometer obtained by appropriately improving the cytometer, and the like.

  In particular, it is preferable to use an imaging cytometer having means for calling up image data of a microscope image from analysis data obtained based on the acquired microscope image. Since the image data can be quickly confirmed with respect to each analysis data, for example, data such as cell nucleus region, fragmented cell nucleus region, brightness value statistics of TUNEL-stained image, a more reliable diagnosis result is obtained. Because it can. Moreover, it is preferable to have a means for displaying the analysis result by a histogram or dot plot. In the present invention, dot plot display is more desirable. In the dot plot display, each cell is displayed as a single dot. Therefore, there is an advantage that the individual cell information can be confirmed instantaneously together with the image by displaying the analysis result in the dot plot display.

<Evaluation method of apoptosis stage>
By combining the detection method of the present invention with a method for detecting other apoptotic cells, the stage of apoptosis can be evaluated for each of a plurality of cells contained in one measurement sample. In the present invention, a method for specifically detecting cells at a specific stage of apoptosis is preferably combined, and among them, a method for detecting early apoptotic cells is preferably combined.

  For example, changes in the localization of phosphatidylserine in the cell membrane, depolarization of the mitochondrial membrane, and improved permeability of the cell membrane are changes observed from the early stage of apoptosis. By combining, early apoptotic cells and intermediate or late apoptotic cells can be accurately evaluated.

  Phosphatidylserine is usually localized in the inner membrane of the cell membrane, but has been found to migrate to the outer membrane when apoptosis occurs. Changes in localization of phosphatidylserine can be detected by performing cell staining (phosphatidylserine staining) using a compound that specifically binds to phosphatidylserine or a compound labeled with a fluorescent substance. Can do. Examples of the compound that specifically binds to phosphatidylserine include annexin V and the like.

  Depolarization of the mitochondrial membrane is, for example, by fluorescent cell staining using a fluorescent mitochondrial membrane potential detection probe whose localization and fluorescence characteristics change depending on whether or not the mitochondrial membrane is depolarized, Can be detected. As such a probe for detecting mitochondrial membrane potential, any known probe may be used, and examples thereof include CMXRos, JC-1, and DiOC6.

  In apoptotic cells, the permeability of the cell membrane is improved. Therefore, cells that have not undergone apoptosis cannot permeate the cell membrane, but cell staining is performed using a fluorescent substance (probe for detecting cell membrane permeability) that is sized to penetrate the cell membrane of cells that have undergone apoptosis. Can be detected. As such a cell membrane permeability detection probe, any known probe may be used, and examples thereof include PI.

  Of the phosphatidylserine fluorescent staining, the fluorescent staining using the probe for detecting mitochondrial membrane potential, and the fluorescent staining using the cell membrane permeable probe, only one may be combined with the detection method of the present invention, or a plurality may be combined. You may combine all. As the number of staining methods to be combined increases, a more reliable evaluation can be obtained.

  Phosphatidylserine fluorescence staining, fluorescence staining using a probe for detecting mitochondrial membrane potential, and fluorescence staining using a cell membrane permeability probe are all live cells (cells that have not undergone fixation treatment or cell membrane permeabilization treatment). ) Should be used for staining. For this reason, the cells to be detected are first stained and then fixed to the cell observation container. Next, the fixed cells are subjected to nuclear staining using a fluorescent double-stranded nucleic acid stain and TUNEL staining for fluorescently labeling the DNA strand break site. Thereafter, a stained image is acquired for each type of staining.

When a phosphatidylserine stained image is acquired, the phosphatidylserine stained image is analyzed. By image analysis, cells that were identified as having phosphatidylserine localized outside the cell membrane were evaluated as apoptotic cells, and phosphatidylserine was identified as being localized inside the cell membrane. The evaluated cells are evaluated as non-apoptotic cells (non-apoptotic cells).
In addition, when a probe-stained image for detecting mitochondrial membrane potential is obtained, the probe-stained image for detecting mitochondrial membrane potential is analyzed. As a result of this image analysis, cells determined to be depolarized in the mitochondrial membrane are evaluated as apoptotic cells, and cells determined to be depolarized are evaluated as non-apoptotic cells.
When a cell membrane permeability detection probe stained image is acquired, the cell membrane permeability probe stained image is analyzed. As a result of this image analysis, cells determined to be stained with the cell membrane permeable probe are evaluated as apoptotic cells, and cells determined to be unstained are evaluated as non-apoptotic cells.

  It is a cell that has been evaluated as an apoptotic cell by analysis of at least one of these three types of stained images, and that DNA has been fragmented from the analysis by staining with a double-stranded nucleic acid stain and TUNEL staining. Cells that were not determined (DNA was determined not to be fragmented) can be evaluated as early apoptotic cells.

In addition, immunostaining of biomolecules activated at a specific stage of apoptosis may be combined. In addition, although immunostaining of these biomolecules may be performed separately from the TUNEL staining on the cells fixed in the cell observation container, it can also be performed simultaneously.
For example, active caspase 3 is detected from a relatively early stage of apoptosis. Therefore, when immunostaining of active caspase 3 is combined, it is determined that it is an active caspase 3 positive cell (a cell in which active caspase 3 is detected by immunostaining) and is not DNA fragmented. The treated cells can be evaluated as early apoptotic cells.

  The detection method of the present invention is characterized by using both TUNEL staining and double-stranded nucleic acid staining. However, instead of TUNEL staining, staining of living cells (for example, phosphatidylserine staining or mitochondrial membrane described above) The stage of apoptotic cells in the state of living cells by combining fluorescent cell staining using a potential detection probe) and staining with a double-stranded nucleic acid stain capable of staining nucleic acid of living cells such as Hoechst Can be evaluated. Specifically, the cells are cultured in a medium to which each stain is added, a cell image is acquired in this state, and image analysis is performed on the acquired image. In this method, since a time-dependent change of one living cell can be observed, the progress of apoptosis can also be evaluated by image analysis.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
HL60 cells in which apoptosis was induced by staurosporine were stained with TUNEL, then stained with a double-stranded nucleic acid stain, and image analysis was performed to detect cells fragmented with DNA. Specifically, it was performed as follows. In addition, culture of HL60 cells is carried out so as to be 1 to 9 × 10 ⁵ cells / mL in a 35 cm ² flask using RPMI medium (GIBCO) containing 10% FBS (fetal bovine serum, GIBCO). Seeding was performed at 37 ° C. in a CO ₂ incubator.

<Staurosporin treatment>
In HL60 cells seeded at 24 well plate so as to be 0.5 × 10 ⁶ cells / well, stauros so that the final concentrations are 0, 0.1, 0.25, 0.5, and 1.0 μM, respectively. Porin (CALBIOCHEM) was added and incubated for 5 hours.

<Cell fixation>
After incubation, the cells were transferred from the well to the 15 mL tube together with the medium, and after removing the medium, a 4% paraformaldehyde (manufactured by Wako Pure Chemical Industries) solution was added and the cells were fixed by allowing to stand at room temperature for 20 minutes. . Thereafter, centrifugation was performed to remove the 4% paraformaldehyde solution, and after washing once with PBS, PBS was added to prepare a cell suspension.
This cell suspension was added to 96 well plate so that it might become 5 * 10 < ⁴ > cells / well. The plate was centrifuged at 600 rpm for 10 minutes, then the plate direction was reversed, and the solution was removed again by centrifugation at 600 rpm for 10 minutes, followed by natural drying for 30 minutes. Thereafter, 70% cold ethanol was added in an amount of 100 μL / well, and the cells were fixed at −20 ° C. overnight. Thereafter, ethanol was removed and washed with PBS.

<Immunostaining>
1% Triton X was added to each well at 100 μL / well, allowed to stand on ice for 10 minutes, and then washed once with PBS. 50 μL of Labeling Reaction Mix was added to each well and incubated at 37 ° C. for 1 hour. Next, after washing three times with Rinsing Buffer (PBS containing 0.1% Triton X and 0.5% BSA), anti-BrdU antibody (manufactured by CALBIOCHEM) was added to each well with Incubation Buffer (1% BSA and 0.1% BSA). A solution diluted 200-fold with PBS containing% Tween 20) was added by 100 μL / well and incubated at room temperature for 1 hour. Each well was washed twice with Washing Buffer (PBS containing 0.1% Tween 20), and then anti-mouse-Alexa647 antibody (Alexa Fluor 647 goat anti-mouse IgG (H + L), manufactured by Invitrogen) was used with Incubation Buffer 250. 100 μL / well of the diluted solution was added and incubated for 30 minutes at room temperature and protected from light. Further, after washing twice with Washing Buffer, a DAPI (Roche) solution diluted to 1 μg / mL with PBS was added in an amount of 100 μL / well, incubated at room temperature for 10 minutes, and then washed twice with PBS. The Labeling Reaction Mix is 10 μL × 5 TdT Buffer (Roche), 2 μL 25 mM CoCl ₂ (Roche), 0.125 μL TdT enzyme (Roche), and 1 μL 2 mM BrdUTP (SIGMA). ) To the final volume of 50 μL.

<Image analysis>
Each well after the above operation is added with 100 μL of PBS as a sample, and CELAVIEW (manufactured by Olympus) is used to acquire and analyze DAPI-stained images (double-stranded nucleic acid-stained images) and TUNEL-stained images. did. In addition, the dyeing | staining image was acquired on the conditions shown below.

Auto focus
Type: Object-based
ZDC Sequence: Don't use ZDC
Acquisition
Color channel: DAPI
Microscope
Objective: LUCPLFLAN 20x
Filter cube: UV
Z offset (μm): 0
Camera
Exposure time (ms): 25
Prequisition delay (s): 0
Illumination
Fluorescence
Exclusion filter: UV
Intensity: 33.3
Color channel: Alexa647
Microscope
Objective: LUCPLFLAN 20x
Filter cube: Cy5
Z offset (μm): −1
Camera
Exposure time (ms): 500
Prequisition delay (s): 0
Illumination
Fluorescence
Exclusion filter: RED
Intensity: 100
Well Manager
Position: 6Columns / well 6Rows / well 36Total positions
Spacing: 550 Column spacing (μm) 550 Row spacing (μm)

  Thereafter, according to the flowchart shown in FIG. 5, a cell nucleus region was extracted from the DAPI-stained image, and a fragmented cell nucleus region was extracted from the extracted cell nucleus region. Cells in which two or more fragmented cell nucleus regions were extracted from one cell nucleus region were identified as cells in which the nucleus was fragmented. Moreover, TUNEL staining positive cells were discriminated from the TUNEL stained images as cells in which DNA strand breakage occurred.

FIG. 8 shows the result of image analysis.
FIG. 8A shows the cells recognized by extracting the cell nucleus region by dot plot (scattergram) using the area value and the circularity factor as parameters. One dot represents one cell (that is, one cell nucleus region). Note that a histogram may be used instead of the dot plot. As shown in the dot plot of FIG. 8 (a), the group including the cell nucleus region (cell) to be analyzed (the region surrounded by the line) is specified, and only for those included in the group, the following Image analysis was performed.

  FIG. 8B is a graph displaying the cells recognized by extracting the cell nucleus region for each number of the extracted fragmented cell nucleus regions. The X axis indicates the number of fragmented cell nucleus regions per cell (one cell nucleus region). In the figure, a region A shows a population of cells having two or more fragmented cell nucleus regions per cell nucleus region, and a region B shows a population of cells having one or zero fragmented cell nucleus regions. ing.

  FIG. 8C is a histogram displaying the cells recognized by extracting the cell nucleus region, using the average value of the luminance values of the pixels included in the cell nucleus region in the TUNEL-stained image as a parameter. In the figure, a region C shows a population of TUNEL staining positive cells whose average luminance value is equal to or greater than a threshold for TUNEL staining, and a region D is a population of TUNEL staining negative cells whose average is less than the threshold for TUNEL staining. Is shown.

  As shown in Table 1, the cells classified into both regions A and D shown in FIGS. 8B and 8C are normal cells that have not undergone apoptosis, and are classified into both regions B and C. The cells to be treated are apoptotic cells in which DNA is fragmented. On the other hand, cells classified into both regions A and C are apoptotic cells at a stage where the DNA strand is cleaved but the DNA is not fragmented. In the table, “+” indicates that DNA strand breakage or DNA fragmentation has occurred, and “−” indicates that DNA strand breakage or DNA fragmentation has not occurred. Yes.

  The analysis results for each staurosporine concentration are shown in Table 2 and FIG. In Table 2, “number of analyzed cells” refers to cells to be analyzed (cells surrounded by a line) among the cell nucleus regions (cells) extracted from the DAPI-stained image shown in FIG. Indicates the number of In Table 2, “nuclear fragmented cells” refers to the number of cells (cells from which two or more fragmented cell nucleus regions have been extracted) contained in region B of FIG. 8 (b), and “TUNEL positive cells” Indicates the number of cells (DNA strand-cleaved cells in which the DNA strand is cleaved) contained in region C in FIG. 8 (c), and “DNA fragmented cells” refers to region B in FIG. 8 (b). The number of cells included and included in the region C in FIG.

  FIG. 9 is a graph of the numerical values in Table 2. In the figure, “DNA body” indicates the number of cells with DNA strand breaks (number of cells in region B), “TUNEL” indicates the number of TUNEL staining positive cells (number of cells in region C), and “DNA body”. and TUNEL positive ”indicate the number of DNA fragmented cells (number of cells contained in both regions B and C). As shown in FIG. 9, when the staurosporine concentration was 0.1 μM, the number of nuclear fragmented cells and the number of DNA strand break cells were almost equal, but as the staurosporine concentration increased, the nuclear fragmented cells A tendency for the number of DNA strand break cells to be larger than the number was observed. This is presumably because cells in which nicks have occurred in the DNA strand before DNA fragmentation are also counted. That is, even from this result, DNA fragmented cells cannot be accurately detected only by TUNEL staining, but DNA fragmented cells can be detected more accurately by using double-stranded nucleic acid staining together as in the present invention. Is clear.

  By using the method for detecting apoptotic cells of the present invention, it is possible to distinguish and detect DNA strand breaks and DNA fragmentation using image analysis. It can also be used in fields such as drug development.

  DESCRIPTION OF SYMBOLS 1 ... Cell image analysis apparatus, 101 ... Image input part, 102 ... Threshold storage part, 103 ... Cell nucleus area extraction part, 104a ... Fragmented cell nucleus area extraction part, 105a ... Candidate area extraction part, 106a ... Area determination part, 107 ... Analysis result output unit 108... Peak detection unit 109 109 Fragmented cell nucleus region selection unit 111. Fragmented cell nucleus discrimination unit 112 112 TUNEL staining luminance value measurement unit 113... DNA strand breakage discrimination unit 114.

Claims (8)

A method for detecting apoptotic cells in which DNA has been fragmented by image analysis,
(A) A step of performing nuclear staining using a fluorescent double-stranded nucleic acid stain and TUNEL staining for fluorescently labeling a DNA strand break site on one or a plurality of cells fixed in a cell observation container When,
(B) capturing the cells stained in the step (a) using a microscope and obtaining a double-stranded nucleic acid stained image and a TUNEL-stained image of each cell;
(C) analyzing the double-stranded nucleic acid staining image obtained in the step (b) to determine whether or not the cell nucleus in the image is fragmented;
(D) analyzing the TUNEL-stained image obtained in the step (b) to determine whether or not the DNA strand breakage of the cells in the image has occurred;
(E) a cell in which the nucleus has been determined to be fragmented in the step (c) and the DNA in which the DNA strand breakage has been determined in the step (d) Detecting as a fragmented cell;
A method for detecting apoptotic cells in which DNA is fragmented, comprising: Analysis of the double-stranded nucleic acid staining image in the step (c),
An image input step of inputting the double-stranded nucleic acid staining image, a cell image analysis apparatus comprising a threshold storage unit for storing in advance a threshold for cell nuclei, a threshold for fragmented nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining;
Cell nucleus region extraction in which the cell image analyzer extracts, as a cell nucleus region, a region having an area equal to or greater than the cell nucleus area threshold among regions having a luminance value equal to or greater than the cell nucleus threshold from the input double-stranded nucleic acid staining image Process,
A fragmented cell nucleus region extraction step in which the cell image analyzer extracts, as the fragmented cell nucleus region, a region having a luminance value equal to or higher than a fragmented cell nucleus threshold from the cell nucleus region;
When the cell image analysis apparatus has two or more fragmented cell nucleus regions extracted from the cell nucleus region, the cell nucleus including the cell nucleus region is fragmented, and is one or zero A fragmented cell nucleus discriminating step for discriminating that the nucleus of the cell containing the cell nucleus region is not fragmented,
Done by
Analysis of the TUNEL-stained image in the step (d)
An image input step in which the cell image analyzer inputs the TUNEL-stained image;
The TUNEL staining luminance value measurement in which the cell image analyzer measures the statistical value of the luminance value of pixels included in the region corresponding to the cell nucleus region extracted from the double-stranded nucleic acid staining image of the input TUNEL staining image. Process,
When the statistical value is greater than or equal to the threshold for TUNEL staining, the cell image analyzer has a DNA strand breakage in the cell containing the nucleus region, and the cell nucleus when the statistical value is less than the threshold for TUNEL staining. A DNA strand break discriminating step for discriminating that a cell containing the region has not undergone DNA strand breaks;
The method for detecting apoptotic cells fragmented with DNA according to claim 1, wherein   3. The method for detecting apoptotic cells fragmented with DNA according to claim 1, wherein the cell observation container is a slide glass or a multiwell plate.   The method for detecting apoptotic cells in which DNA is fragmented according to any one of claims 1 to 3, wherein microscopic image acquisition and image analysis are performed using an imaging cytometer. A threshold storage unit for storing in advance a threshold for cell nuclei, a threshold for fragmented nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining;
A double-stranded nucleic acid stained image in which cells stained with a fluorescent double-stranded nucleic acid stain are imaged and a TUNEL-stained double-stranded nucleic acid stained image that fluorescently labels a DNA strand break site are in the same field of view. An image input unit for inputting a TUNEL stained image;
From the input double-stranded nucleic acid staining image, a cell nucleus region extraction unit that extracts a region having an area greater than or equal to the cell nucleus area threshold as a cell nucleus region among regions having a luminance value greater than or equal to the cell nucleus threshold; and
From the cell nucleus region, a fragmented cell nucleus region extraction unit that extracts a region having a luminance value equal to or higher than a threshold for fragmented cell nuclei as a fragmented cell nucleus region;
When the number of fragmented cell nucleus regions extracted from the cell nucleus region is 2 or more, the nucleus of the cell containing the cell nucleus region is fragmented, and when the number is 1 or 0, the cell containing the cell nucleus region A fragmented cell nucleus discriminating unit that discriminates that the nucleus of
A TUNEL staining luminance value measuring unit for measuring a statistical value of luminance values of pixels included in a region corresponding to a cell nucleus region extracted in the double-stranded nucleic acid staining image of the input TUNEL staining image;
When the statistical value is greater than or equal to the threshold for TUNEL staining, the cell containing the cell nucleus region has undergone DNA strand breakage, and when the statistical value is less than the threshold for TUNEL staining, the cell containing the cell nucleus region is DNA strand A DNA strand break discriminating part for discriminating that no cleavage occurs;
An analysis unit for analyzing a cell that has been determined that the nucleus is fragmented and that DNA strand breaks are occurring as an apoptotic cell in which DNA is fragmented;
A cell image analysis apparatus comprising: The fragmented cell nucleus region extraction unit comprises:
A candidate region extraction unit that extracts a region having a luminance value equal to or higher than a threshold for fragmented cell nuclei from the cell nucleus region as a candidate region;
A region determining unit that detects a boundary line by performing a boundary line detection process based on a gradient of a luminance value of each pixel in the candidate region, and extracts a region surrounded by the detected boundary line as a fragmented cell nucleus region; ,
The cell image analysis apparatus according to claim 5, comprising: For a computer comprising a threshold storage unit that prestores a threshold for cell nuclei, a threshold for fragmented nuclei, an area threshold for cell nuclei, and a threshold for TUNEL staining,
A double-stranded nucleic acid stained image in which cells stained with a fluorescent double-stranded nucleic acid stain are imaged and a TUNEL-stained double-stranded nucleic acid stained image that fluorescently labels a DNA strand break site are in the same field of view. An image input process for inputting a TUNEL stained image;
From the input double-stranded nucleic acid staining image, a cell nucleus region extraction step of extracting a region having an area greater than or equal to the cell nucleus area threshold as a cell nucleus region among regions having a luminance value greater than or equal to the cell nucleus threshold; and
From the cell nucleus region, a fragmented cell nucleus region extracting step of extracting a region having a luminance value equal to or higher than a threshold for fragmented cell nuclei as a fragmented cell nucleus region;
When the number of fragmented cell nucleus regions extracted from the cell nucleus region is 2 or more, the nucleus of the cell containing the cell nucleus region is fragmented, and when the number is 1 or 0, the cell containing the cell nucleus region A fragmented cell nucleus discriminating step for discriminating that the nucleus is not fragmented,
A TUNEL staining luminance value measuring step for measuring a statistical value of luminance values of pixels included in a region corresponding to a cell nucleus region extracted in the double-stranded nucleic acid staining image of the input TUNEL staining image;
When the statistical value is greater than or equal to the threshold for TUNEL staining, the cell containing the cell nucleus region has undergone DNA strand breakage, and when the statistical value is less than the threshold for TUNEL staining, the cell containing the cell nucleus region is DNA strand A DNA strand break discriminating step for discriminating that no cleavage occurs;
An analysis step of analyzing a cell determined to have a fragmented nucleus and a DNA strand break to be an apoptotic cell having a fragmented DNA;
A program for running A method for evaluating the stage of apoptosis by image analysis,
(A′1) Cells subjected to at least one fluorescent staining selected from the group consisting of phosphatidylserine fluorescent staining, fluorescent staining using a probe for detecting mitochondrial membrane potential, and fluorescent staining using a cell membrane permeable probe Fixing the cell observation container,
(A′2) After the step (a ′ 1), nuclear staining using a fluorescent double-stranded nucleic acid stain and a DNA strand break site are fluorescently labeled on the cells in the cell observation container Performing TUNEL staining;
(B ′) after the step (a ′ 2), obtaining a stained image of the cells in the cell observation container for each type of cell staining using a microscope;
(C ′) analyzing the double-stranded nucleic acid stained image obtained in the step (b ′) to determine whether or not the nucleus of the cell in the image is fragmented;
(D ′) analyzing the TUNEL-stained image obtained in the step (b ′) to determine whether or not DNA strand breakage of cells in the image has occurred;
(E ′) a cell in which the nucleus has been determined to be fragmented in the step (c ′), and a cell in which the DNA strand break has been determined in the step (d ′) Evaluating the DNA to be a fragmented metaphase or late apoptotic cell;
(F ′) When a phosphatidylserine fluorescent staining image was acquired in the step (b ′), the staining image was analyzed, and it was determined that phosphatidylserine was localized outside the cell membrane. When the cell is evaluated as an apoptotic cell and a probe fluorescent staining image for detecting mitochondrial membrane potential is obtained in the step (b ′), the stained image is analyzed and depolarization occurs in the mitochondrial membrane. When the discriminated cell is evaluated as an apoptotic cell, and a probe fluorescent staining image for cell membrane permeability detection is obtained in the step (b ′), the stained image is analyzed and stained with the cell membrane permeability probe. Evaluating a cell identified as having been apoptotic,
(G ′) Among the cells whose DNA has not been determined to be fragmented in the step (e ′), cells evaluated as apoptotic cells in the step (f ′) are treated as early apoptotic cells. A process of evaluating that there is,
A method for evaluating the stage of apoptosis characterized by comprising: