JP2006325567A - Method for evaluating transcription factor-expressing cell and apparatus for evaluating the cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for numerically evaluating condition of transcription factor-expressing cell expressing a specific transcription factor Oct4 from a cell group immunostained by DAB (diaminobennzidine). <P>SOLUTION: The method for evaluating the condition of the transcription factor-expressing cell expressing the specific transcription factor from a cell group immunostained by DAB comprises (a) converting a cell immunostained by DAB from a cell group containing the transcription factor-expressing cell through a microscope to computer-readable image data, (b) carrying out CMYK image conversion of resultant image data by software and reducing blue color from the CMYK-converted image, (c) obtaining a ratio of K value which is a measured value of black element from whole images and further preferably (d) setting a plurality of black element measured value ranges in which the K value is previously determined, obtaining a ratio of the set black element measured value ranges respectively and evaluating concentration distribution of the transcription factor expression cell. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method and apparatus for evaluating transcription factor-expressing cells and a program for evaluating transcription factor-expressing cells.

DAB staining is the most commonly used immunostaining method for immunostaining, and transcription factor-expressing cells are stained brown.
Therefore, it is difficult to quantitatively evaluate and identify cells from DAB-stained cells (transcription factor-expressing cells).

Oct4 is a marker for identifying the most common ES cell, but when this was used as a transcription factor for DAB staining, there was no index indicating the degree of staining numerically. For example, Non-Patent Documents 1 to 3 show that cells with different levels of Oct4 expression exist in mouse early embryos (Blastcyst), and cells with different levels of Oct4 expression in ES cells isolated therefrom. Describes molecular biological analysis results showing different fate, that is, the characteristics of whether to continue to grow in an undifferentiated state while retaining potential multipotency or to differentiate into various cells.
Okamoto K, et al. , A novel octomer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60461-472, 1990. Pesce M et al. , Differential expression of the Oct-4 transcription factor drusing mouse germ cell differentiation. Mech. Dev. 71: 89-98, 1998. Niwa H, Miyazaki J-I, Smith AG. Quantitative expression of Oct-3 / 4, differences differentiation, self-defense or self-renewal of ES cells. Nature Genetics 24: 372-376,2000

  Therefore, an object of the present invention is to provide a transcription factor that expresses a specific transcription factor Oct4 from a DAB immunostained cell group by the most routine and frequently performed immunostaining method for evaluating the state of cellular tissue. The present invention is to provide a method for numerically evaluating the state of an expressed cell. However, the present invention is not limited to Oct4, but is extended to image evaluation of other immunostaining and further the abundance of a specific substance. The

The present invention for solving the above problems relates to the following matters.
(1) A method for evaluating a transcription factor-expressing cell for evaluating a state of a transcription factor-expressing cell expressing a specific transcription factor from a DAB immunostained cell group,
(A) converting DAB immunostained cells from the cell group containing the transcription factor-expressing cells into computer-readable image data through a microscope;
(B) The obtained image data is converted into CMYK images by software, and the blue color is subtracted from the CMYK converted image.
(C) A method for evaluating a transcription factor-expressing cell, wherein a ratio of a K value that is a black element measurement value is obtained from the entire image.
(2) Furthermore,
(D) A plurality of black element measurement value ranges in which the K value is predetermined are set, a ratio of each set black element measurement value range is obtained, and a concentration distribution of transcription factor-expressing cells is evaluated. The method for evaluating a transcription factor-expressing cell according to (1).
(3) Furthermore,
(E) DAB staining of the same cell group using 2 or more transcription factors, and comparing the ratio of K value or black element measurement value range of cells immunostained with each transcription factor, or both (2) The method for evaluating a transcription factor-expressing cell according to (2).
(4) The transcription factor-expressing cell according to any one of (1) to (3), wherein the cell is an ES or ES-like cell, and the transcription factor is Oct4 or P75NTR. Evaluation methods.
(5) A computer readable medium storing a program for executing the method according to any one of (1) and (4).
(6) A transcription factor-expressing cell evaluation apparatus comprising a computer system storing the computer-readable medium according to (5).

  By comprising in this way, it becomes possible to evaluate numerically the state of the transcription factor expression cell which expresses the specific transcription factor Oct4 from the DAB immunostained cell group. This evaluation method is not limited to Oct4, and it is possible to image-evaluate other immunostaining and further the abundance of a specific substance.

The present invention relates to a method for evaluating transcription factor-expressing cells, which evaluates the state of transcription factor-expressing cells that express a specific transcription factor from a group of cells immunostained by DAB (3,3′-dianobenzidine) reaction.
In the following embodiments, an example using Oct4 as a transcription factor will be described as an example. However, the present invention is not limited to such specific embodiments, and is widely applied.

First, in specifying whether or not Oct4-expressing cells are included in a predetermined cell group, first, immunostaining is performed on cells obtained by a method known in the art, and the stained cells are computerized via a microscope. Convert to readable image data (RGB data).
The obtained image data (RGB data) is subjected to CMYK image conversion by software such as Adobe Photoshop (registered trademark). The portion stained by the DAB immunostaining method (in this embodiment, the portion immunostained with Oct4, that is, the Oct4-expressing cell) is usually represented in brown. Therefore, when the blue color is subtracted from this image, the Oct4-expressing cell in the image Will contain the black element (K) only.

  The image thus converted can be numerically evaluated by assuming that the immunostained portion is a black element (K). For example, the immunostained portions in the entire cell group can be numerically evaluated as the area ratio.

  In a preferred embodiment of the present invention, the degree of staining of the stained cells (Oct4-expressing cells) thus indicated by the K value can be indicated as a K value distribution. Although not shown, the concentration distribution of the K value (that is, the staining concentration) can be shown as a distribution curve. For example, the intensity of Oct4 expression is expressed as a predetermined section of the K value, none (n) “less than 1000”, It can be indicated by a four-level evaluation of weak (m) “1000 or more and less than 5000”, medium (m) “5000 or more and less than 9000”, and strong (s) “9000 or more”. Predetermined section of K value in this embodiment, none (n) “less than 1000”, weak (m) “1000 or more and less than 5000”, medium (m) “5000 or more and less than 9000”, strong (s) “9000 or more” "" Is determined arbitrarily and can be arbitrarily changed, and the number of sections such as three-level evaluation and five-level evaluation is also arbitrary.

  Such a configuration makes it possible to evaluate Oct4-expressing cells, which have conventionally been impossible to evaluate numerically, and in particular, it has been difficult to examine the degree of expression and its distribution.

  Further, according to another embodiment of the present invention, comparative evaluation is performed by DAB staining of the same specimen using different transcription factors. For example, various transcription factors are known as DAB transcription factors in addition to Oct4 described above as markers for ES and ES-like cells (transcription factors).

And it has also been confirmed by preliminary experiments by the present inventors that the expression of these transcription factors varies depending on the state of the cells.
For example, when the present inventors investigated skin cancer, Oct4 has no abnormality (±), initial stage (±), and terminal stage (++), and P75NTR has no abnormality (±), initial stage (+), and terminal stage (++). ) And different behavior in the early cancer stage. Esophageal cancer and cervical cancer also show similar behavior.
Therefore, it is considered possible to diagnose cancers such as skin cancer, esophageal cancer and cervical cancer by comparing the two. Furthermore, it will be possible to screen cancer stem cells. That is, cancer stem cells can be screened by immunostaining the same cell group (cell group containing cancer stem cells) using different markers (transcription factors) and evaluating by the evaluation method of the present invention. Become.

Next, the procedure will be specifically described with reference to FIG.
Open the brown image file of Oct4 protein obtained by DAB (3,3′-diaminobenzidine) reaction with image processing software such as Photoshop, and select “CMYK Color” in “Mode” from the “Image” menu. CMYK is cyan (C), magenta (M), yellow (Y). It is expressed by a black (K) color component.

Next, the eyedropper tool of the image processing software is selected to select a DAB positive brown portion (in this case, the cell nucleus showing brown staining because it is the gene transcription factor Oct4 functioning in the cell nucleus).
Next, click the drawing color palette to open the color picker window and check the K value (black% value) of CMYK. Furthermore, DAB-negative cell nuclei (blue color including nonspecific coloration of DAB present in the cytoplasm and cell membrane is dominant) Measure the K value with the color picker.
The former confirms that the K value (%) is sufficiently higher than the latter, and closes the color picker window. If the K value (%) of the former and the latter is not large at this time, it is determined that the ratio of non-specific DAB coloration is high. Therefore, in order to obtain a more reliable K value, high quality with less non-specific staining. It is preferable to obtain an immunostained specimen again.

  For example, the K value of this sample is 58-60% for the former and 22-24% for the latter. Select and click the “Black” channel from the “Window” menu to display the screen in black and white. Select all images from the "Selection" menu and copy them to the clipboard using the "Edit" to "Copy" functions.

  Select “New” from the “File” menu and set the mode to gray scale (FIG. 1, FIG. 1 “This is shown in FIG. 1-1”, inserted below FIG. 1-1, for example, 1- (2) Upper left figure 40w Am-40 week amniotic base amniotic epithelial layer. Similarly lower left figure 40wPri-cultured 40 containing curtain cells). Click OK. The DAB color image is transferred to the new file by the “Paste” function from the “Edit” menu. Display “Layer” from the “Window” menu. When both the background layer and paste image are confirmed, select “Merge Image” from the pull-down menu to merge the images.

  Save is selected from the “File” menu, and the file type is saved as TIFF (extension .tif), for example. For example, when measuring the Oct4 staining value with the image analysis software ClontechImageQuant (TM), “tone reversal” is selected from “tone correction”, and the black and white image is reversed. The measured value of the black density of individual cell nuclei given by ClontechImageQuant (TM) exemplified in this analysis was recorded as an arbitrary value from about 300 to 20000. This actual measurement value (Value) is reproducible because almost constant threshold values can be obtained for each immunostained specimen by DAB color development of Oct4 in different specimens (tissues, cultured cells).

The present inventors further examined the Oct4-expressing cells expressed as Oct4-expressing cells in detail according to the strength of the black element K as described above, and found that the behavior of the Oct4-expressing cells differs depending on the K value. I found it. That is, in the present invention, the black element measurement value is none (n) “less than 1000”, weak (m) “1000 or more and less than 5000”, medium (m) “5000 or more and less than 9000”, strong (s) “ When it is shown in a four-stage evaluation of “9000 or more”, when it is none (n) or weak (w), it shows no or almost no behavior as an ES cell or ES-like cell, and conversely when it is strong (s) It was distributed in the epithelial surface layer and tended to fall into amniotic fluid, and as ES cells or ES-like cells, it was not differentiated but rather differentiated or at the terminal stage. On the other hand, in the case of middle (m), an excellent behavior of maintaining an undifferentiated state is guaranteed as an ES cell or ES-like cell that tends to be located on the epithelial basal layer side and is advantageous for long-term survival. Therefore, not only the presence of Oct4-positive cells, but also an Oct4-positive cell population with a range in the expression level of Oct4 ensures the presence of ES or ES-like cells.
It has been experimentally and analytically found that the cell group of the present invention significantly includes Oct4-expressing cells having a medium strength exhibiting excellent behavior as such ES cells or ES-like cells.

As described above, the evaluation method of the present invention can perform a series of processing on an acquired image by a computer.
As a result of the analysis as described above, for example, in the case of the cell group including the Oct4-expressing cells, the ratio of stained cells in the whole cells, none (n), weak (w), medium (m), strong By printing out the ratio of each of (s) and attaching the sampled cell group to a container for storing it, the state of the cell can be used for subsequent research and application.

  It is drawing which shows the evaluation method of this invention.

Claims (6)

A method for evaluating a transcription factor-expressing cell for evaluating a state of a transcription factor-expressing cell that expresses a specific transcription factor from a group of DAB immunostained cells,
(A) converting DAB immunostained cells from the cell group containing the transcription factor-expressing cells into computer-readable image data through a microscope;
(B) The obtained image data is converted into CMYK images by software, and the blue color is subtracted from the CMYK converted images.
(C) A method for evaluating transcription factor-expressing cells, characterized in that a ratio of a K value that is a black element measurement value is obtained from the entire image. further,
(D) A plurality of black element measurement value ranges in which the K value is predetermined are set, a ratio of each set black element measurement value range is obtained, and a concentration distribution of transcription factor-expressing cells is evaluated. The method for evaluating a transcription factor-expressing cell according to claim 1. further,
(E) DAB staining of the same cell group using 2 or more transcription factors, and comparing the ratio of K value or black element measurement value range of cells immunostained with each transcription factor, or both The method for evaluating transcription factor-expressing cells according to claim 2.   The method for evaluating a transcription factor-expressing cell according to any one of claims 1 to 3, wherein the cell is an ES or ES-like cell, and the transcription factor is Oct4 or P75NTR.   A computer readable medium storing a program for executing the method according to any one of claims 1 to 4.   An evaluation apparatus for transcription factor-expressing cells comprising a computer system in which the computer-readable medium according to claim 5 is stored.

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