JP2007195533A - Method for evaluating cell, method for evaluating cancerization of measured component by using the same, and cancer diagnosis kit and computer-readable medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging the cancerization state of stem cells or a cell group including the stem cells as any of normal cells, pre-cancerization cells or cancerization cells, and also to provide a method for evaluating the advancing degree of the cancerization in the case of the pre-cancerization cells. <P>SOLUTION: This method for evaluating the cells is provided by applying Oct3/4 (a transcription factor correlated to a non-differentiated state) and p75NTR (a neurotrophin receptor) at the same time or separately on evaluating cells and setting the expressed state and non-expressed state of the Oct3/4 and p75NTR as evaluation criteria. When the transcription factor correlated to the non-differentiated state is not expressed and also the p75NTR is expressed, the evaluating cells are judged as the normal cells. When the transcription factor correlated to the non-differentiated state is not expressed and also the neurotrophin receptor p75NTR mRNA or protein is expressed, the obtained cells are judged in the pre-cancerization state and when the transcription factor correlated to the non-differentiated state is expressed and also the p75NTR is not expressed, the evaluating cells are judged as advanced cancerization cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell evaluation method, a canceration evaluation method for a measurement component using this method, a cancer diagnosis kit, and a computer-readable medium. More specifically, a cell evaluation method for determining whether a cell selected from the group consisting of a stem cell or a group of cells containing a stem cell is a normal cell, a precancer cell, or a cancer cell, and this method is used. The present invention relates to a method for evaluating canceration of various measurement components, a cancer diagnosis kit, and a computer-readable medium.

Recent advances in equipment such as CT and NMR have made it possible to detect minute initial cancers, and the early detection rate of cancer has improved. There is a long incubation period (usually 15 to 20 years) before cancer becomes apparent, and cells (pre-cancerous cells) with an increased risk of canceration during this period are identified, and an index of malignancy If identified, it will provide a means to prevent or prevent cancer.
As a method for detecting a cancer cell, a method for detecting a cancer-related antigen using an antibody has already been used. However, these methods have a problem that sensitivity and specificity for cancer are not necessarily high. For example, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and the like are known as cancer markers, but they are limited to only about 60% of disease sensitivity. There are problems such as non-specific positivity even in diseases. In addition, disease-related genes are detected only in a limited number of cases and have not yet reached a universal marker for detecting cancer.

Stem cells, on the other hand, are a group of cells that play a role in morphogenesis during development, homeostasis of various tissues and organs in the adult, and maintenance of germ cells. It is known to exist in the intestinal tract system and contribute to the regeneration of blood, skin and intestinal mucosa. These stem cells have the pluripotency that differentiates into different types of cells and the ability to self-renew to generate new stem cells by dividing symmetrically or asymmetrically and are usually located at specific locations in the tissue. It grows very slowly or is in a mitotic state but rapidly induces growth under specific conditions such as specific growth stimuli involved in maintaining tissue regeneration and physical scientific stimuli such as wounds and inflammation be able to.
The present inventors have proposed that such long-term surviving cells including stem cells include cells of origin of carcinogenesis, and further long-term surviving cells are low-affinity nerve growth factor receptor (hereinafter referred to as “NGFR ^p75 ”). , It is disclosed that expression of integrin β4 or bcl-2 can be identified as an index (Patent Document 1: Japanese Patent Laid-Open No. 2000-4900).

However, since cells expressing these epithelial stem cell markers are also present in normal epithelial tissues and not all of them are cancerous, the presence of these cells in the tissues causes the tissues to become cancerous. It was not possible to judge that the risk of conversion was high.
A method for identifying the risk of canceration of a certain tissue is a method for identifying the expression level of a gene related to carcinogenesis (Patent Document 2: JP 2000-510330 A), or mutation of chromosomal DNA related to carcinogenesis. A method of predicting at the individual level based on the presence or absence of the above (Patent Document 3: Japanese Patent Laid-Open No. 2002-095484) has been proposed. However, even with these methods, it was impossible to predict the time and risk of carcinogenesis.
Japanese Unexamined Patent Publication No. 2000-4900 Special Table 2000-510330 JP 2002-095484 A

The subject of the present invention is a method for determining the canceration state of an acquired stem cell or a group of cells containing a stem cell, which is a normal cell, a precancerous cell or a cancerated cell, particularly in the case of a precancerous cell. Is to provide a method for evaluating the degree of progression of canceration.
Another object of the present invention is to provide a method capable of determining the effects of foods, supplements, drugs, and these medicinal ingredients on cells, that is, whether they promote or suppress canceration. It is.
Yet another object of the present invention is to provide a cancer diagnostic kit based on a cell line that serves as a standard for determining whether a cell obtained is a cancerous state, a normal cell, a precancerous cell, or a cancerated cell. Is to provide.

The present invention that solves the above-described problems provides an evaluation of a cell that determines whether a cell selected from the group consisting of a stem cell or a group of cells containing a stem cell is a normal cell, a precancer cell, or a cancer cell. A method,
A transcription factor (preferably Oct3 or Oct4, or Oct3 / 4, particularly preferably Oct3 / 4) and a neurotrophin receptor p75NTR mRNA or protein (corresponding to the undifferentiated state and maintenance of the undifferentiated state in the cell to be determined) (Hereinafter simply referred to as p75NTR) is applied simultaneously or individually, the transcription factor and the expression and non-expression state of p75NTR are used as criteria, the transcription factor correlated with the undifferentiated state is not expressed, and p75NTR is expressed. If not, it is determined that the cell to be determined is a normal cell, a transcription factor that correlates with the undifferentiated state is expressed, and when the neurotrophin receptor p75NTR mRNA or protein is expressed, the acquired cell is It is determined that the cancer is precancerous, and a transcription factor that correlates with the undifferentiated state is expressed. And when the p75NTR is not expressed, determines cells comprise determining that a progressive cancer cell.

  In one embodiment of the cell evaluation method of the present invention, a cell to be determined is a stem cell or a cell group containing a stem cell cultured under a predetermined condition, and an expression pattern of a transcription factor that correlates with an undifferentiated state before and after the culture, and Culture conditions under which cells cultured under the predetermined conditions maintain normal cells according to the expression pattern of neurotrophin receptor p75NTR mRNA or protein, culture conditions for inducing normal cells into precancerous cells, precancerous cells The culture conditions for inducing from normal to normal cells, the culture conditions for maintaining precancerous cells, and the culture conditions for inducing from precancerous cells to cancerous cells are determined. Using this one embodiment, there is provided a method for evaluating canceration of a measurement component for determining canceration suppression or promotion of canceration of a single component or a mixed component such as a medicinal component / drug, supplement, food or the like.

  The present invention further comprises a transcription factor-expressing gene product reaction material that correlates with an undifferentiated state, a p75 NTR-expressed gene product reaction material, or both reaction materials, and a transcription factor-expressing gene product reaction material that correlates with the undifferentiated state, p75 Relating to expressed gene product reaction material, or both, body fluid and cell extract, immunostaining, and a simple determination kit for determining the cancerous state or precancerous state of a cell according to the state of immunostaining in the reaction material .

  The present invention further relates to a cell determination method for determining a canceration state of a cell collected from a body fluid, which is a transcription factor expression gene correlating with an undifferentiated state, a p75 expression gene, or a body fluid and a cell extract that are samples of both. In a specimen characterized by immunostaining by reacting with a liquid, observing the state of immunostaining in the reaction region, and determining a normal state, a precancerous state, or a cancerous state based on the observation result It is related with the determination method of the cell characterized by determining that the cell contained in is either a canceration state or a precancerous state.

According to the present invention, it is possible to determine whether the acquired stem cell or the canceration state of the cell group containing the stem cell is a normal cell, a precancerous cell, or a cancerous cell, and the precancerous cell. In this case, it is possible to determine the degree of progression of canceration.
Therefore, it provides an indication of the culture environment for culturing these cells and cell lines, and suppresses the effects of foods, supplements, drugs, and these medicinal ingredients on cells, that is, whether they promote canceration It is possible to determine whether to do.
Furthermore, a cancer diagnostic kit can be provided based on this evaluation method.

Embodiments of the present invention will be described below.
In the present invention, when a transcription factor that correlates with maintenance of an undifferentiated state and an undifferentiated state and p75NTR are applied to cells simultaneously or individually, normal cells, precancerous cells, advanced cancer This is based on the finding that the expression pattern of transcription factors that correlate with the undifferentiated state and the maintenance of the undifferentiated state in the state of each of the cells differs from that of p75NTR.
That is, the present inventors have found that the state of cells can be evaluated based on the expression pattern of transcription factors and the expression pattern of p75NTR.
In general, transcription factors that correlate with undifferentiated state and maintenance of undifferentiated state are not expressed in normal cells (±: 5% or less of total cells), precancerous cells and advanced cancer cells On the other hand, p75NTR is not expressed in normal cells (±), is expressed during a precancerous state (+++ to +), and the expression intensity increases as cancer progresses. It decreases and the expression disappears for advanced cancer cells (-).

Therefore, a cell population to be determined (such as a tissue piece) does not express a transcription factor that correlates with an undifferentiated state (or below the detection limit), and does not express or detect the neurotrophin receptor protein p75NTR or mRNA. If it is below the limit, this cell population is likely to have predominantly normal cells and no cells with chronic abnormalities or characteristic abnormalities in the early stages of canceration. Very expensive.
That is, in this case, it is determined to be a normal cell under the condition that it includes a measurement problem in medical biology (always an uncertain factor).
In other words, in an environment that hardly contains medical and biological uncertainties (for example, when purified cells are used as cells to be evaluated), or a combination of other determinations and the evaluation method of the present invention. It is considered that the accuracy of the determination is increased by the determination (the same applies hereinafter).

On the other hand, when a cell population to be determined (such as a tissue piece) does not express a transcription factor that correlates with the undifferentiated state and p75NTR is expressed, it is extremely low that the obtained cells correlate with the precancerous state. Can be determined.
That is, it can be determined that a precancerous cell is included in this case under the condition that a measurement problem (uncertain factor) in medical biology is included.
In addition, when a transcription factor that correlates with the undifferentiated state is expressed and p75NTR is not expressed, the obtained cell is very likely to be an advanced cancer cell (measurement problem in medical biology (uncertainties) ), And in this case, it includes cells in a precancerous state.

The terms used in the present invention have the following significance.
The cells selected from the group consisting of stem cells or cell groups containing stem cells may be either human or mammalian stem cells themselves or cell groups containing these, or isolated cells, What is not refine | purified and the tissue piece containing these may be sufficient. In particular, epithelial cells are preferred. The epithelial tissue is not limited to the epithelial tissue, and any sample may be used as long as it is a sample containing epithelial cells. Specifically, for example, epithelial tissue extracted from a human individual who has not yet found cancer cells but may have carcinogenesis or a human individual who has a prognosis for tumor removal, sputum, blood, plasma, serum, Cerebrospinal fluid, pleural fluid, pancreatic fluid, urine and the like are preferably used. The epithelial tissue may be any cancerous epithelial tissue that can be biopsied. For example, lung, esophagus, stomach, milk duct, endometrium And epithelial tissues such as cervix, large intestine, colon, kidney or bladder.
The epithelial tissue is removed from the living body by a method usually used for biopsy, and a sample containing epithelial cells is obtained by a known method suitable for each. Epithelial cells are obtained from the obtained biological sample by an appropriate method and analyzed for the degree of aging described below. Specifically, for example, when the sample is an epithelial tissue, the method described in JP-A No. 2000-4900 can be used as the method for obtaining epithelial cells.

A normal cell is a cell that normally maintains cell growth control function and differentiation function and functions that constitute a normal adult in which cell death (apoptosis) is induced, and that divides and ages,
Precancerous cells are characterized as cancer cells that have acquired the ability to avoid telomere shortening in many cases that have acquired unlimited growth ability among the characteristics of cancer cells, and are characterized by the above-mentioned normal cells. It retains some or many of its properties, but to varying degrees. Although it has the ability to increase cells, it does not form carcinomas in laboratory animals such as humans and mice, or its ability is extremely low.
A cancer cell (advanced cancer) describes many cell characteristics, but most characteristically, it has lost or extremely weakened cell growth control function, and is infinitely proliferated at a high frequency (over 80%). Many have acquired abilities, and many of them also have the ability to infiltrate and metastasize. As a result, it means a cell that is positioned as a malignant neoplasm that causes human death.
The transcription pattern of the transcription factor is not particularly limited as long as the expression of the transcription pattern can be discriminated. A transcription pattern measured by a method well known in the art, for example, immunostaining or PCR can be used.

Transcription factors that are undifferentiated or correlated with an undifferentiated state are those in which cells are in a normal state, that is, expression is difficult to detect in normal cells, and in cells that are pre-cancerous and fully cancerous. It means a transcription factor whose expression is remarkably detected.
A typical transcription factor is not particularly limited as long as it exhibits such an action, but includes those expressed as Oct3, Oct4, or Oct3 / 4. All that is meant to be the same transcription factor. In the present invention, Oct3 / 4 is particularly preferably used.

  p75NTR (neurotrophin receptor p75NTR mRNA or protein) is a neurotrophin low-affinity receptor present on the membrane surface of nerve cells, but is also expressed in cells other than nerve cells. Is a patent application previously filed as a marker for pancreatic cancer (a joint application with Mr. Morimura, a marker for detection of pancreatic cancer may be acceptable). However, in this application, there is no finding that the expression of p75NTR disappears as the canceration of cells progresses, and this was first demonstrated by the present invention.

(First embodiment)
First, based on FIG. 1 to FIG. 4, a cancer development process is shown using p75NTR (P75N + in the figure) used in the present invention. FIG. 1 is a schematic diagram showing a cancer development process, FIG. 2 is a drawing showing the expression of P75NTR in a cancer tissue (exemplified by esophageal cancer), and FIG. 3 is a diagram in the process of canceration of cells. FIG. 4 is a drawing showing an expression pattern of a stem cell indicator molecule (transcription factor Oct4 mRNA), and FIG. 4 is a drawing showing p75NTR-expressing stem cells in which the number of cells is amplified by immortalized keratinocytes.

  As shown in FIG. 1, this is a schematic diagram showing that most cancer cells are generated in the process of mitotic aging of stem cells present in tissues having regenerative ability, which is disclosed in Japanese Patent Application Laid-Open No. 2000-60542 and Japanese Patent Application Laid-Open No. 2000-60542. No. 2004-267167, non-patent literature (Okumura et al., Oncogene, 2003), and the origin and path of cancer cells established by the results of the present invention. These documents are incorporated herein by reference.

  As shown in FIG. 2, the distribution of cancer cells expressing p75NTR in the grown cancer tissue is the result of immunostaining (DAB method) using a p75NTR receptor protein antibody. Cells expressing the P75NTR protein are mixed at the point of contact between the carcinoma and the normal epithelial cell region, and cancer cells expressing the p75NTR grow by interrupting the normal epithelial tissue region expressing the p75NTR. Indicates that It is clearly shown by DAB staining that p75NTR is also expressed in cancer cells in that region. It is also clearly shown that the expression of p75NTR is no longer detected in the invading cancer cells spreading downward in the drawing (this is a schematic diagram showing this on the right). P75NTR expression occurs from p75NTR-expressing epithelial stem cells and is accompanied by amplification of p75NTR-expressing cancer cells in the early stage of carcinogenesis. Means that.

  Thus, p75NTR is not an indicator that is detected in any region of tissue because it is expressed exclusively in a few stem cells present in epithelial tissue, but before it is induced by chronic inflammation or sustained stimulation. It develops early in carcinogenesis with cancerous state and unlimited growth ability (immortalization) and is easily detected, and the degree of malignancy (simply the degree of canceration) progresses with invasion and metastasis. As a result, it was found that the expression intensity of p75NTR becomes small (disappears).

Furthermore, as shown in FIG. 3, p75NTR is expressed at a strong intensity in the early stage of canceration that acquires immortalization, and is established from a cancer cell that has become malignant as canceration progresses. In the strain, expression of p75NTR can no longer be detected.
Therefore, a cell determined to be in a precancerous state can grasp the progress of the canceration process based on the expression intensity of p75NTR.
Therefore, by standardizing the degree of expression, it is possible to numerically evaluate the degree of cancer progression of the acquired cells (ie, cells to be determined).
Hereinafter, an example of numerical evaluation will be described by illustrating DAB immunostaining.

  As shown in FIG. 4, flow cytometry reveals evidence that the enhancement of p75NTR expression intensity in the immortalized cell lines exemplified in the present invention, which corresponds to the early stage of canceration, is brought about by the amplification of p75NTR-expressing stem cells.

In this method, first, cells obtained by a method known in the art are immunostained, and a microscope image of the stained cells is converted into computer-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). A portion stained by DAB immunostaining (in this embodiment, a portion immunostained Oct3 / 4 or p75NTR (collectively referred to as a transcription factor in this embodiment), that is, a transcription factor-expressing cell), usually expressed in brown Therefore, when the blue color is subtracted from this image, only the transcription factor-expressing cells in the image contain the black element (K).

  The image thus converted can be numerically evaluated with the immunostained portion as a black element (K). For example, the immunostained portion 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 (transcription factor-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 also be shown as a distribution curve. For example, the intensity of transcription factor expression is determined based on a predetermined section of the K value, none (n) “less than 1000” , 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 transcription factor-expressing cells, in which it has been impossible to evaluate numerical values conventionally, and in particular, it has been difficult to examine the degree of expression and its distribution.

  More specifically, a brown image file of a transcription factor expression site obtained by a DAB (3,3′-diaminebenzidine) reaction is opened with image processing software such as Photoshop, and “CMYK” in “Mode” is selected from the “Image” menu. Select “Color”. 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, a cell nucleus showing brown staining since it is a transcription factor that functions in the cell nucleus).
Next, click the drawing color palette, open the color picker window, check the K value (black% value) of CMYK, and further, DAB negative cell nuclei (blue including non-specific color development 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 a certain sample is 58-60% for the former and 22-24% for the latter. Select and click on the “Black” channel from the “Window” menu to display the screen in black and white. Select all images from the “Select” menu and copy them to the clipboard using the “Edit” to “Copy” function.

  Select "New" from the "File" menu and set the mode to grayscale. 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 coloring of the transcription factor in different specimens (tissues, cultured 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 a cell group including the transcription factor-expressing cells, the ratio of stained cells in the whole cells, none (n), weak (w), medium (m), By printing out the ratio of each strong (s) and sticking it to a container for storing the sampled cell group, it becomes possible to use the state of the cell for subsequent research and application.
By separately evaluating Oct3 / 4 and p75NTR, which are transcription factors correlating with the undifferentiated state such as Oct3 / 4, the expression pattern of such transcription factor can be evaluated numerically or in stages.

  It should be noted that the predetermined section of K value, 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” It can be shown in a four-step evaluation 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 an example for explanation, and it can be easily understood that it is appropriately set for each cell type.

  Such evaluation of the canceration state of cells can be applied not only to diagnosis of cancer but also to evaluation of cancer stem cells that have recently attracted attention. That is, for the first time, the present invention makes it possible to subject a cell having a cancerous state identified to banking.

(Second Embodiment: Application to Culture Solution)
As described above, in the present invention, it is possible to evaluate the state of a cell based on the expression pattern of Oct3 / 4 and p75NTR, which are transcription factors. By applying this, for example, when culturing stem cells. The present invention can be applied to culture conditions.

In this embodiment, stem cells cultured under a predetermined condition or a comparative cell group containing stem cells (control cultured cell group) were cultured by changing (conditioning) parameters such as culture medium and culture conditions. It is used as a cell (sample cell) for judging a cell group.
That is, the Oct3 / 4 expression pattern and p75NTR expression pattern of the comparative cell group before and after the control culture were compared with the Oct3 / 4 expression pattern and p75NTR expression pattern after the culturing of the conditioned cultured cell group. Culture conditions for maintaining normal cells to be determined according to changes in culture conditions, culture conditions for inducing normal cells to precancerous cells, culture conditions for inducing normal cancer cells to normal cells, Provided are a method for determining culture conditions for maintaining precancerous cells and culture conditions for inducing from precancerous cells to advanced cancer cells.
In addition, the expression pattern of Oct3 / 4 and the expression pattern of p75NTR are compared with the difference in expression pattern before and after the culture, the standard expression pattern is set in advance and the ratio to the expression pattern, or for a predetermined cell line For example, the relationship between the expression pattern and the degree of progression of cancer is standardized (for example, graphed) and compared with this.

  For example, as a representative sample cell culture medium suitable for epithelial cells, Dulbecco's modified Eagle's medium (DMEM) and Ham's MCDB153 medium mixed at a volume ratio of 1: 4, hydrocortisone (0.2 · M), epithelium Growth factor (EGF) (10 ng / ml), ethanolamine (5 μM), o-phosphoethanolamine (5 μM), transferrin (10 μM), insulin (5 μg / ml), bFGF (5-10 ng / ml) bovine pituitary gland Examples include those supplemented with an extract (150 μg / ml protein amount). For example, such a medium is used as a standard medium, and the expression pattern of both transcription factors before and after culturing of a medium cultured by a standard culture method is added. Phorbo such as carcinogenic promoter, PMA (phorbol myristate acetate) It becomes possible to evaluate the promotion or suppression of canceration depending on the culture solution of sample cells by changing the culture solution by changing the addition amount of a certain component or the amount of a certain component.

  In addition, it is possible to evaluate the promotion or suppression of canceration by performing irradiation with light such as ultraviolet rays, radical addition, or the like using cells containing stem cells in an initial cancer state as sample cells. Similarly, it is possible to set canceration promotion conditions using a group of cells consisting of normal cells as sample cells.

For example, radicals may promote canceration depending on the radical species and the amount thereof, and may have a positive effect on the culture. It is possible to measure the optimization of.
Similarly, normal cells (maintenance of normal cells, evaluation of normal cell to canceration direction) or early cancer cells (maintenance of initial cancer state, change from initial cancer state to normal cells (apoptosis induction)), Evaluation of cell maintenance conditions and change conditions by changing (preferably continuously changing) culture parameters such as temperature conditions and culture time using evaluation of progression from initial cancer state to canceration) It becomes possible.

  In addition, when the standard culture conditions for a certain cell and the expression pattern of Oct3 / 4 and the expression pattern of p75NTR of the comparative cell group before and after the culture are sufficiently established, the established comparative cell group Using the expression pattern of Oct3 / 4 and the expression pattern of p75NTR, the expression pattern of Oct3 / 4 and the expression pattern of p75NTR after culturing the cells was conditioned by omitting the expression pattern before and after control culture and control culture. It is also possible to set the culture conditions by comparing with the established Oct3 / 4 expression pattern and p75NTR expression pattern before and after standard culture.

(Third embodiment: Evaluation of canceration promotion / suppression effect of single component / mixed component)
Culture system using the transcription factor and p75NTR correlated with the undifferentiated state described above and its culture conditions (for example, maintenance of normal cells, culture system evaluated from normal cells to canceration direction, maintenance of initial cancer state) , Change from initial cancer state to normal cells (induction of apoptosis)), culture system evaluated for progress from initial cancer state to canceration and these culture conditions) should be evaluated as standard It is also possible to add an additive and evaluate the canceration behavior of the additive.

That is, the present invention also relates to a method for evaluating canceration of a measurement component for determining suppression of canceration or promotion of canceration using the above-described cell evaluation method for determining culture conditions.
At this time, the cells to be cultured in the second embodiment are precancerous cells, and the control culture culture conditions are such that the precancerous cells maintain a precancerous state, from precancerous cells to terminal cancer. Culture conditions that induce to cells or known culture conditions that induce from pre-cancerous cells to normal cells are standard.

  Ingredients to be evaluated (single ingredients or mixtures such as medicinal ingredients, suspected carcinogenic ingredients, supplements, foods, etc.) are added to such control culture conditions and cultured. In addition, by changing the amount of these components to be evaluated, the effect of the components to be evaluated on cells (maintenance of precancerous state, It is possible to evaluate the quantitative dependence of the change from a cancerous state to normal cells and the progression from a precancerous state to advanced cancer.

  Furthermore, it is also possible to evaluate the synergistic action and antagonistic action of other components against the canceration of cells by using a medium in which a predetermined component is added to the control conditions.

  At this time, the culture conditions under which precancerous cells progress toward canceration include, for example, ultraviolet irradiation, addition of a cancer promoter, radical addition (quantitative problem), serum from cows, humans, etc. (from 1% About 20%). Examples of culture conditions that change from a precancerous state to normal cells include a known predetermined amount of anticancer agent, a predetermined amount of radical scavenger, and the like.

  Furthermore, by adding additives such as these known drugs to the culture solution, synergism / antagonism of normal cell maintenance, canceration promotion, and suppression between these additives and the sample to be measured can be measured. Expected. And it is estimated by this that the critical point (both upper limit and lower limit) of the additive amount, the canceration promoting direction, and the apoptosis induction direction can be evaluated.

  In addition to the evaluation of cell cancer behavior in a well-established culture system by the addition of a certain component as described above, evaluation of cell cancer behavior by radical species loading and light (ultraviolet rays, gamma rays, etc.) irradiation Can also be carried out according to the invention.

(Modification: Simple cancer status test kit)
The present invention has, for example, an Oct3 / 4-expressed gene product reaction material, a p75NTR-expressed gene product reaction material, or both reaction materials. For example, an anti-Oct3 / 4 antibody molecule or an anti-p75NTR antibody molecule is adsorbed on a substrate. There is also provided a simple determination kit for determining a cancerous state or a precancerous state of a cell composed of a thing. In this case, the type of base material that specifically recognizes Oct3 / 4 and p75NTR is not limited. In this simple determination kit, a sample such as a body fluid or tissue is brought into contact with a substrate capable of specifically detecting the transcription factor contained in the kit and p75NTR, and the cancer of cells in the sample is determined from the expression pattern of the transcription factor and p75NTR. The simple state is determined.

For example, it may be composed of a bottom layer, a reaction region containing Oct3 / 4 expression gene provided in the bottom layer, and a body fluid permeable surface layer provided on the reaction region, and a water-absorbing resin layer inside A sanitary napkin or starch absorbent sheet having a reaction area can be formed by mixing an Oct3 / 4 recognition substrate in the water-absorbent resin layer.
The reaction region may be composed of a fibrous material joined to the p75NTR recognition substrate.
The cancer diagnostic kit of the present invention can also have both an Oct3 / 4-expressing gene reaction region and a p75-expressing gene reaction region in the same substrate.
Moreover, the cancer diagnostic kit of the present invention may be composed of a first cancer diagnostic kit having an Oct3 / 4-expressing gene reaction region and a second diagnostic kit having a p75-expressing gene reaction region.

Using the simple kit of the present invention, the canceration state (for example, uterine cancer) of cells collected from body fluid (for example, body fluid from the vagina) is determined as follows.
The body fluid is brought into contact with each reaction region of the determination kit.
The determination kit after contact can be pretreated by a conventionally known method (for example, immobilization with formaldehyde or paraformaldehyde) and detected, but this does not exclude detection methods that do not require pretreatment. For example, a detection substrate using the base sequence Oct3 / 4box to which the Oct3 / 4 protein specifically recognizes and binds is also possible. Thus, it does not limit the use of any base material that specifically recognizes Oct3 / 4 and p75NTR, but it is simply a transcription factor expression gene that correlates with an undifferentiated state, a p75 expression gene, or both. It is characterized by immunostaining by reacting with a body fluid as a specimen, observing the state of immunostaining in the reaction region, and determining a normal state, a precancerous state, or a cancerous state based on the observed result A cell determination method characterized by determining that a cell contained in a specimen is in a cancerous state or a precancerous state.

Hereinafter, the present invention will be specifically described by way of examples.
Example 1
The example of drawing which shows the expression fluctuation | variation of P75NTR in the development process of cancer.
This is a schematic diagram showing that most cancer cells occur in the process of mitotic aging of stem cells present in tissues that have regenerative ability. This is a patent document (Nipro's method for obtaining long-term viable cells), (aging stem cells) And the non-patent literature (Okumura et al., Oncogene, 2003), and the origin and path of cancer cells established by the results of the present invention.

(Example 2)
This example is an example of the drawing showing the expression pattern of stem cell indicator molecules (transcription factors) in the process of canceration of cells (FIG. 2). The distribution of cancer cells expressing p75NTR in the grown cancer tissue is the result of immunostaining (DAB method) using a p75NTR receptor protein antibody. Cells expressing the P75NTR protein are mixed at the point of contact between the carcinoma and the normal epithelial cell region, and cancer cells expressing the p75NTR grow by interrupting the normal epithelial tissue region expressing the p75NTR. Indicates that It is clearly shown by DAB staining that p75NTR is also expressed in cancer cells in that region. It is also clearly shown that the expression of p75NTR is no longer detected in the invading cancer cells spreading downward in the drawing (this is a schematic diagram showing this on the right). P75NTR expression occurs from p75NTR-expressing epithelial stem cells and is accompanied by amplification of p75NTR-expressing cancer cells in the early stage of carcinogenesis. Pre-cancer induced by chronic inflammation or persistent irritation, although not expressed in any area of the tissue because it is expressed exclusively in a few stem cells present in epithelial tissue It is more likely to be detected and easily detected in early stages of carcinogenesis with state and unrestricted growth capacity (immortalization), and the degree of malignancy with invasion and metastasis (simply canceration) According degree) is to proceed, the expression intensity of p75NTR found that small (disappears).

(Example 3)
This example is an example of the drawings showing the expression pattern of stem cell indicator molecules (transcription factors) in the process of canceration of cells by estimating the amount of mRNA by the RT-PCR method. Activation of human epithelial cells and telomerase in the process of mitosis that progressed by the introduction of human papillomavirus type 16 tumor-inducing genes (E6 and E7, respectively) from normal cells shown in Example 1 Types of cell lines led to the immortalization process accompanied by (with one type of Nsv21 introduced with SV40 having a high ability to make immortalized cells with a higher degree of canceration) and further canceration That is, total RNA is extracted from a cancer tissue-derived cell line determined to be malignant cancer by any RNA extraction method, for example, a total RNA extraction kit (Promega, Madison WI), and RNA is extracted by agarose electrophoresis. Check the quality, 1 microliter of complementary DNA (cDNA) from 2 microgram RNA using oligo (dT) as a primer Amplification was carried out at 42 ° C. for 1 hour by using SuperScript-RT (Invitrogen). The obtained cDNA was diluted to an arbitrary concentration (for example, 1:10 dilution), and cDNA was amplified by PCR using a part (for example, 1-4 microliters). For example, PCR was carried out at 94 ° C. for 5 minutes and 1 cycle, followed by 94 ° C. for 20 seconds, 50-60 ° C. for 30 seconds, and 72 ° C. for 60 seconds. The amplified cDNA was evaluated by agarose gel electrophoresis. Human p75NTR is expressed with a strong intensity in the early stage of canceration to acquire immortalization, and expression of p75NTR in cancer cell lines established separately from cancer types that have become malignant as canceration progresses is It cannot be detected.
Therefore, a cell determined to be in a precancerous state can grasp the progress of the canceration process based on the expression intensity of p75NTR.
The PCR-primers base sequence is as follows.
p75NTR-F: 5′-TgAgTgCTgCAAAgCCTgCAA-3 ′
p75NTR-R: 5′-TCTCATCCCTggTAgTAgCCgT-3 ′
HuOCT3 / 4-F: 5'-gAAgCTggAgAAggAgAAgCTg-3 '
HuOCT3 / 4-R: 5′-CCAggCCgCAgCTTACACACATgTTC-3 ′
HuIntegrin β1-F: 5′-gTgTggCCCAAgACAgTTCT-3 ′
HuIntegrin β1-R: 5′-ggTTACCCCACCCTCTgACT-3 ′

Example 4
This example is an example of the drawing (FIG. 4) showing p75NTR-expressing stem cells amplified with immortalized keratinocytes. The cultured cells adhering to the culture dish are treated with a proteolytic enzyme such as trypsin and the like. The detached cells 2 × 105 are suspended in 50 ml of phosphate buffer (staining buffer) containing 0.02% NaN 3 (sodium azide) and 0.5% BSA. Add p75NTR antibody at a concentration of 1 microliter / ml, react the cells for 5 minutes at room temperature (about 25 ° C.), wash the cells with staining buffer, resuspend the cells in 50 microliters, and concentrate at a concentration of 1 microliter / ml. Then, the reaction was carried out with a secondary antibody bound with FITC for 5 minutes at room temperature, and the number of stem cells expressing p75NTR on the surface membrane was measured with a flow cytometer.
Flow cytometry demonstrates evidence that the enhancement of p75NTR expression intensity in the immortalized cell lines exemplified in the present invention, which corresponds to the early stage of canceration, is brought about by the amplification of p75NTR-expressing stem cells

Schematic diagram showing the cancer development process. Drawing which shows the expression change of P75NTR in the development process of cancer. Drawing which shows the expression pattern of the stem cell indicator molecule (transcription factor) in the process of canceration of a cell. The figure which shows the p75NTR expression stem cell amplified by the immortalized keratinocyte. The figure which shows the p75NTR expression stem cell which the number of cells amplified by the immortalized keratinocyte.

Claims (19)

A cell evaluation method for determining whether a cell selected from a group consisting of a stem cell or a cell group containing a stem cell is a normal cell, a precancer cell or a cancer cell,
A transcription factor exemplified by Oct3 / 4, which correlates with maintenance of an undifferentiated state and an undifferentiated state, and a neurotrophin receptor p75NTR mRNA or protein are applied to a cell to be judged simultaneously or individually, and the transcription factor, Based on the expression of the fin receptor p75NTR mRNA or protein and the non-expressed state,
When the transcription factor correlating with the undifferentiated state is not expressed (+/−) and the neurotrophin receptor protein p75NTR or mRNA is not expressed (+/−), the cell to be determined is a normal cell. Judgment,
When a transcription factor that correlates with the undifferentiated state is expressed and the neurotrophin receptor p75NTR mRNA or protein is expressed, it is determined that the obtained cell is in a precancerous state,
A cell evaluation method comprising determining that a cell to be determined is an advanced cancer cell when a transcription factor correlated with the undifferentiated state is expressed and p75NTR is not expressed.   The method for evaluating a cell according to claim 1, wherein the transcription factor correlated with the undifferentiated state is Oct3, Oct4, or Oct3 / 4.   When the cell to be determined is determined to be a precancerous cell, the degree of canceration of the cell is changed to the expression intensity of the neurotrophin receptor p75NTR mRNA or protein in which canceration progresses as the expression intensity decreases. The cell evaluation method according to claim 1 or 2, wherein the determination is made based on the determination. The expression pattern is an expression pattern by DAB staining,
(A) converting the intensity of staining of a cell expressing a transcription factor that correlates with the DAB-stained undifferentiated state into computer-readable image data by optical reading means;
(B) The obtained image data is converted into CMYK images by software, and the blue color is subtracted from the CMYK converted images.
(C) Obtain the ratio of the _Kx value, which is the black element measurement value, from the entire image,
(D) converting the DAB-stained neurotrophin receptor p75NTR mRNA or protein-expressing cell into computer-readable image data by optical reading means;
(E) CMYK image conversion is performed on the obtained image data by software, and blue color is subtracted from the CMYK converted image;
(F) _Obtain the ratio of the K _p75NTR value, which is the black element measurement value, from the entire image,
(G) The method for evaluating a cell according to any one of claims 1 to 3, wherein the canceration state of the cell determined from the obtained K _x value and the K _p75NTR value is determined. .   The method according to any one of claims 1 to 4, wherein the cell to be determined is a cell subjected to cell banking.   A cell to be determined is a stem cell or a group of cells containing a stem cell cultured under a predetermined condition, depending on an expression pattern of a transcription factor and a neurotrophin receptor p75NTR mRNA or protein that correlate with an undifferentiated state before and after the culture. Culturing conditions under which the cells cultured under the predetermined conditions maintain normal cells, culturing conditions for inducing from normal cells to precancerous cells, culturing conditions for inducing from precancerous cells to normal cells, precancerous cells The method for evaluating a cell according to any one of claims 1 to 4, wherein a culture condition for maintaining cell culture conditions and a culture condition for inducing cancer cells from precancerous cells are determined.   The cell evaluation method according to any one of claims 1 to 6, wherein the cell to be determined is a human-derived cell. A method for evaluating canceration of a measurement component for determining canceration suppression or promotion of canceration of a measurement component using the cell evaluation method according to claim 6 or 7,
The cell to be cultured is a precancer cell, and the predetermined culture condition is a condition for the precancer cell to maintain a precancerous state, a culture condition for inducing from a precancerous cell to an advanced cancer cell, or Culture using any one of the known culture conditions for inducing from precancerous cells to normal cells as a control,
Incubate under the same conditions as the control except adding a predetermined amount of measurement component to the control,
The control except that a predetermined amount of a measurement component is added to the control using the expression pattern of a transcription factor and the expression pattern of a neurotrophin receptor p75NTR mRNA or protein correlated with the differentiation state obtained when culturing as the control. Inhibition of canceration or promotion of canceration is determined by the expression pattern of transcription factors correlated with the undifferentiated state obtained by culturing under the same conditions as above and the expression pattern of neurotrophin receptor p75NTR mRNA or protein A method for evaluating canceration of a measurement component, which comprises a step. A method for evaluating canceration of a measurement component for determining canceration suppression or promotion of canceration of a measurement component using the cell evaluation method according to claim 6 or 7,
The cell to be cultured is a precancer cell, and the predetermined culture condition is a condition for the precancer cell to maintain a precancerous state, a culture condition for inducing from a precancerous cell to an advanced cancer cell, or Culture using any one of the known culture conditions for inducing from precancerous cells to normal cells as a control,
Incubate under the same conditions as the control except adding a predetermined amount of measurement component to the control,
Using the expression pattern of transcription factors correlating with the differentiation state obtained when cultured as the control and the expression pattern of the expression pattern of neurotrophin receptor p75NTR mRNA or protein as a standard, a predetermined amount of measurement component is added to the control By comparing the expression pattern of transcription factors and the expression pattern of neurotrophin receptor p75NTR mRNA or protein correlated with the undifferentiated state obtained by culturing under the same conditions as those of the control other than the above standard cancer A method for evaluating canceration of a measurement component, comprising a step of determining whether to suppress canceration or promote canceration. A method for evaluating canceration of a measurement component for determining canceration suppression or promotion of canceration of a measurement component using the cell evaluation method according to claim 6 or 7,
The cell to be cultured is a precancer cell, and the predetermined culture condition is a condition for the precancer cell to maintain a precancerous state, a culture condition for inducing from a precancerous cell to an advanced cancer cell, or Expression patterns of transcription factors correlating with the obtained differentiation state and neurotrophin receptor p75NTR mRNA when culturing using any one of the known culture conditions from precancerous cells to normal cells as a control Or using the expression pattern of the protein as a standard,
Transcription correlating with the undifferentiated state obtained by culturing under the same conditions as the control except adding a predetermined amount of measurement component to the control and culturing under the same condition as the control except adding a predetermined amount of measurement component to the control A measurement component comprising the step of determining whether the measurement component is cancerated or promoted by comparing the expression pattern of the factor and the expression pattern of the neurotrophin receptor p75NTR mRNA or protein with the standard Canceration evaluation method. A cancer cell evaluation method for determining the degree of progression of cancer cells, wherein neurotrophin receptor p75NTR mRNA or protein is applied to the cancer cells to be determined,
The method comprises the step of determining the degree of cancer progression of the cell to be determined based on the expression intensity of the neurotrophin receptor p75NTR mRNA or protein whose canceration progresses as the expression intensity decreases. To evaluate cancer cells.   Transcription factor expression gene product reaction material correlating with undifferentiated state, neurotrophin receptor p75NTR mRNA or protein-derived p75NTR expression gene product reaction material, or both reaction materials, and transcription factor expression correlating with the undifferentiated state The gene product reaction material, the p75 expression gene product reaction material, or both, body fluid and cell extract are reacted and immunostained, and the cancerous state or precancerous state of the cell is determined depending on the state of immunostaining in the reaction material. Simple judgment kit for judging.   It is composed of a bottom layer, a reaction region including a transcription factor expression gene correlated with an undifferentiated state provided in the bottom layer, and a body fluid permeable surface layer provided on the reaction region. The cancer diagnosis kit according to claim 12.   The cancer diagnostic kit is a sanitary napkin or starch absorbent sheet having a water-absorbing resin layer inside, and a reaction region is formed by mixing a transcription factor expression gene correlated with an undifferentiated state in the water-absorbing resin layer The cancer diagnosis kit according to claim 12 or 13, wherein the kit is a cancer diagnosis kit.   The cancer diagnostic kit according to claim 12 or 13, wherein the reaction region is composed of a fibrous substance conjugated with a neurotrophin receptor p75NTR mRNA or protein.   The cancer diagnostic kit has both a transcription factor-expressing gene reaction region correlating with an undifferentiated state and a neurotrophin receptor p75NTR mRNA or protein reaction region in the same substrate. The cancer diagnostic kit of Claim 10 or Claim 11.   The cancer diagnostic kit comprises a first cancer diagnostic kit having a transcription factor-expressing gene reaction region correlated with an undifferentiated state, and a second diagnostic kit having a neurotrophin receptor p75NTR mRNA or protein reaction region. The cancer diagnosis kit according to claim 10, wherein the kit is a cancer diagnosis kit. A method for determining a cell by determining a cancerous state of a cell in a living tissue from a specimen sample collected from a body fluid and a cell extract, wherein the transcription factor expression gene correlates with an undifferentiated state, a neurotrophin receptor p75NTR mRNA Alternatively, the protein, or both, and the body fluid that is the sample are reacted and immunostained,
Observe the state of immunostaining in the reaction area,
The cell contained in the sample is in a cancerous state or a precancerous state, comprising a step of determining a normal state, a precancerous state, or a cancerous state based on the observed result A method for determining a cell, characterized by being determined to be present.   A computer readable medium storing a program for executing the method of claim 3.

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