JP6793451B2 - Genetic markers, methods, reporter vectors, primer sets and assay kits for assessing cell characterization of test cells, and cell characterization equipment - Google Patents

Description

Embodiments of the present invention relate to genetic markers, methods, reporter vectors, primer sets and assay kits for evaluating cell characteristics of test cells, and cell characteristic evaluation devices.

Cancer is the leading cause of death in Japan. Cancer is a group of diseases that have in common the formation of malignant tumors, and to date, more than 200 different tumors have been identified. Malignant tumors have in common uncontrolled proliferation of cells, their ability to invade neighboring tissues, and their ability to form metastases to systemic tissues.

In the treatment of cancer (malignant tumor), it is important to select an appropriate treatment method according to the nature of the tumor (characteristics of tumor cells). In order to properly select a treatment method, it is desirable to evaluate the characteristics of tumor cells, that is, benign or malignant tumors, as soon as possible. In the case of malignant tumors, it is desirable to be able to evaluate their aggression, that is, infiltration and metastasis. However, at present, it is difficult to evaluate the metastatic property from the primary tumor, that is, the tumor cells obtained from the lesion in which the tumor first developed.

Tumor diagnosis includes diagnosis using an image diagnostic device such as MRI and CT, and diagnosis performed by observing cells collected from the tumor. The characteristics of cells include morphological characteristics, that is, characteristics that can be discriminated from their appearance, such as cell size, shape, and adhesiveness. Papanicolaou stain and Giemsa stain are known as methods for observing the morphological characteristics of cells. In such a staining method, the nuclei and cytoplasms of cells are dyed separately, and the characteristics of tumor cells are evaluated from their morphological characteristics.

On the other hand, there are some cell characteristics that cannot be distinguished from the morphology. Such cellular properties are associated with, for example, changes in gene expression. In gene expression, a specific protein binds to the 5'upstream promoter sequence of a gene to activate the promoter. The activated promoter promotes gene transcription to synthesize mRNA, and the mRNA is translated to synthesize a protein. Changes in gene expression are detected by examining the promoter activity of the gene, or the amount of mRNA or protein mass.

For example, if a change in the expression of a particular gene is highly correlated with the properties of tumor cells, that gene can be used as a tumor marker. By detecting tumor markers, it becomes possible to discriminate cell characteristics related to gene expression. For example, detection of a particular tumor marker in blood, in another body fluid, or in collected tumor cells suggests the presence of tumor cells that are highly correlated with that marker.

Known tumor markers include the Ki-67 gene, which evaluates the proliferative potential of tumor cells. This tumor marker is used to evaluate the proliferation of tumor cells. Further, for evaluation of tumor cell infiltration and metastasis, Arf-6, Glut1 and the like have been reported as candidates for tumor markers. However, the evaluation accuracy is not sufficient.

In such a situation, it is desired to develop a further technique for evaluating cell characteristics in order to enable more appropriate selection of a treatment method for malignant tumors.

Japanese Patent Publication No. 2011-508609 Special Table 2012-533530 International Publication No. 2004/058990

Cancer Biology & Therapy, May 2008, Vol. 7, No. 5, p663-670 Endocrinology, February 2012, Vol. 153, No. 2, p554-563

The embodiment is intended to provide a means for assessing the cellular properties of test cells.

According to embodiments, genetic markers are provided for assessing the cellular properties of test cells. The genetic marker is a polynucleotide containing the mRNA, peptide or protein of the NAMPT gene, or the NAMPT gene promoter sequence.

The schematic diagram which shows one example of an embodiment. The schematic diagram which shows one example of an embodiment. The block diagram which shows one example of an embodiment. The block diagram which shows one example of an embodiment. The flowchart which shows one example of embodiment. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result. The schematic diagram which shows the structure of the vector which is an example of the embodiment used in an experiment. The figure which shows the experimental result. The figure which shows the experimental result. The figure which shows the experimental result.

Hereinafter, embodiments will be described with reference to the drawings.

In the embodiment, the NAMEPT gene was found to be highly expressed in a highly metastatic tumor cell line, and it was clarified that it is an index gene showing a high correlation with the metastatic property of tumor cells, and the cell characteristics of the test cells were revealed. Based on the finding that it can be used as a genetic marker for evaluating.

The NAMEPT gene is a gene encoding a nicotinamide phosphoribosyltransferase and is a rate-determining enzyme in mammalian NAD biosynthesis. The NAMEPT gene is highly expressed in highly metastatic tumor cell lines.

On the other hand, cell metastasis is one of the important indicators for evaluating the cell characteristics of test cells. Therefore, by measuring the expression level of the NAMPT gene, it is possible to evaluate the cell characteristics of the test cell, for example, the presence or absence or degree of metastatic or infiltrative, and whether the cell is normal or abnormal. Is. Then, when the test cell is abnormal or may be abnormal, it is possible to evaluate the degree of the abnormality or the degree of malignancy. Therefore. Genetic markers based on the NAMEPT gene make it possible to evaluate the cell characteristics of tumor cells more accurately than before.

Evaluation of the cell characteristics of a test cell is to evaluate the cell characteristics of the test cell, for example, the presence or absence or degree of metastatic or infiltrative, whether the cell is normal or abnormal, and the test cell. If is abnormal or likely to be abnormal, it includes evaluating the degree of abnormality or the degree of malignancy. The evaluation of such cell characteristics is, for example, when the test cell is a tumor cell, the evaluation of the cell characteristics of the tumor cell is, for example, the presence or absence of metastatic or invasiveness of the tumor cell, and the metastasis of the tumor cell. It may be to assess the degree of sex, the benign or malignant degree of the tumor cells, and the normality or abnormalities of the tumor cells.

When the term "tumor cell" is used here, it can be understood as "cancer cell" or collectively "cancer cell".

The test cell to be examined may be any cell whose cell characteristics should be evaluated.

Such a method for evaluating the cell characteristics of the test cell may be characterized in that the expression level of the NAPPT gene in the test cell is used as an index. For example, in this method, using the expression level of the NAMPT gene as an index means measuring the expression level of the NAMPT gene in the test cell, comparing the measured expression level of the test cell with the threshold value, and the like. It may include assessing the cellular properties of the test cells based on the results of the comparison and based on that determination.

This evaluation may be performed in comparison with the expression level or degree of expression of the NAMEPT gene in the reference cell. Alternatively, this evaluation can be performed in consideration of the expression level of the NAMEPT gene in the reference cell and the cell characteristics of the reference cell.

Examples of reference cells may be healthy cells or normal cells, tumor cells whose cell characteristics such as the presence or absence of metastasis and / or the degree of metastasis have been clarified. The expression level or cell characteristics of the NAMPT gene in the reference cell, such as the degree of metastasis, may be measured in advance, or may be measured and evaluated at the same time when the test using the genetic marker of the embodiment is performed. .. Further, the expression level or metastatic level of the gene as a reference obtained from the reference cell may be used as a threshold value, and such a threshold value may be a predetermined threshold value, and the cell characteristics of the test cell may be determined. It may be measured at the same time as the evaluation, or may be determined before and after the evaluation of the cell characteristics of the test cells.

The gene marker for evaluating the cell characteristics of the test cell may be any substance that can be used to measure the presence or absence of expression and / or the expression level of the NAMPT gene. For example, the genetic marker may be a mRNA, peptide or protein of the NAPPT gene, or a polynucleotide containing the NAPPT gene promoter sequence.

Examples of the NAMPT gene promoter sequence are shown in Table 1-1 and Table 1-2.

This is the sequence shown in SEQ ID NO: 1 and is an example of a human-derived NAMPT gene promoter sequence. However, the NAPPT gene promoter sequence is not limited to this sequence, and may contain one to several mutations such as substitutions, deletions, and additions as long as it exhibits NAPPT gene promoter sequence activity.

A further embodiment is a method of evaluating the cell characteristics of a test cell by utilizing such a genetic marker. In this method, for example, the cell characteristics of the test cell may be evaluated using the expression level of NAMEPT gene mRNA, the expression level of peptide or protein, or the activity of NAMEPT gene promoter (NAMPT promoter activity) as an index. According to the method of the embodiment, it is possible to evaluate the cell characteristics of the test cells earlier and more accurately. With conventional methods, it is difficult to assess the metastasis of tumor cells from the primary tumor, that is, the lesion in which the tumor first developed, but according to embodiments, for example, tumor cells from the primary tumor are also present. , It becomes possible to evaluate the cell characteristics.

A further embodiment provides a method of assessing the cellular properties of a tumor using the expression of the NAMPT gene as an index. Specifically, the expression of the NAPPT gene in the test cells collected from the tumor of the subject may be quantitatively detected, and the expression of the gene may be compared with the expression of the NAPPT gene in a control healthy cell. When the expression level of the NAMPT gene in the test cell is higher than that in the NAMPT gene in the healthy cell, it can be determined that the test cell has high metastasis or high abnormality. When the expression level of the NAMPT gene in the test cell is lower or equivalent to that of the NAMPT gene in the healthy cell, it can be determined that the test cell has low metastasis or low abnormality.

The test cell may be a group of cells or a single cell. The test cells may be collected from the tumor of the subject by a collection method known per se. In the present invention, tumor cells collected from epithelial tumors are preferred. Among epithelial tumors, tumor cells collected from breast tumors and lung tumors are more preferable as test cells. However, it is not limited to these. For example, cells contained in blood, urine, stool, saliva, tissues obtained by biopsy, oral mucosa, body cavity fluid, sputum, etc., or cultured cells may be used as test cells.

In the cell characterization of the test cells according to the embodiment, the expression level of the NAPPT gene in the test cells and the expression of the NAPPT gene in the healthy cells may be compared. The average NAMEPT gene expression level in healthy cells may be detected in advance, a threshold value in cell characteristic evaluation may be set from this value, and this threshold value may be compared with the NAMEPT expression level in test cells. Therefore, it is not always necessary to detect the expression level of the NAMPT gene in healthy cells.

Expression of the NAMPT gene may be carried out by detecting the activity of the NAMPT protein, the NAMPT mRNA, or the NAMPT gene promoter. These may be used as an index of the gene expression or the expression level of the gene.

When the NAMEPT protein is detected, it may be detected quantitatively by an enzyme-linked immunosorbent assay (EIA) or a fluorescence immunoassay (FIA). Alternatively, it may be detected using an immunohistochemical staining method (ICC), an immunohistochemical staining method (IHC), or an automatic cellular analysis separation device (FACS).

Expression of the NAPPT gene may detect, for example, NAPPT mRNA. In that case, NAMEPT mRNA may be detected by any method. For example, a primer set may be used to amplify a sample using a PCR amplification method, a LAMP amplification method, a SMAP amplification method, an ICAN amplification method, or the like, and the amplification product may be analyzed. In this case, preferably, a reverse transcription PCR method for directly amplifying mRNA, a reverse transcription LAMP amplification method, a reverse transcription SMAP amplification method, a reverse transcription ICAN amplification method, or the like may be used. The analysis of the amplification product may be performed by detecting the binding with the probe nucleic acid, or the base sequence of the amplification product may be directly specified. Further, the detection may be performed by determining the base sequence of the nucleic acid which is the NAMPT gene expression product of the test cell without amplification.

Examples of preferred primer sets are shown in Table 2.

The primer sets shown in Table 2 are primer sets for reverse transcription PCR amplification. SEQ ID NO: 2 and SEQ ID NO: 3 are used in combination. The primer of SEQ ID NO: 2 is a forward primer, and the primer of SEQ ID NO: 3 is a reverse primer. These primer sets are preferably used for amplifying and detecting NAPPT mRNA, but the primer sets for amplifying and detecting NAPPT mRNA are not limited thereto.

When the activity of the NAMPT gene promoter is detected, it may be detected by a method using a reporter vector incorporating the promoter of the NAMPT gene. As the reporter vector, a normal reporter vector, that is, a non-replicating (ie, non-amplifying) reporter vector in the mammalian cell may be used, or a self-amplifying reporter vector, that is, autonomously replicating (that is, not amplifying) in the mammalian cell. A reporter vector to be amplified) may be used.

The method for evaluating the cell characteristics of the test cells will be described in more detail below.

1. 1. Evaluation of cell characteristics of test cells by detection of NAMPT protein 1-1. Detection method and evaluation method of cell characteristics NAMPT protein may be detected by using an antibody specific to the NAMPT protein (that is, an anti-NAMPT antibody). Such an antibody may be newly prepared by using the full length or a part of the NAMPT protein as an antigen, or a commercially available antibody may be used.

Examples of commercially available antibodies include, but are not limited to, rabbit anti-NAMPT polyclonal antibodies (ab45890, Abcam). When a new antibody is produced, it may be produced according to a known antibody production method.

The detection of NAMPT protein using an anti-NAMPT antibody may be performed by a known protein detection method. Examples of such protein detection methods include enzyme immunization method (EIA), fluorescence immunization method (FIA), immune cell staining method (ICC), immunohistochemical staining method (IHC), and automatic cell analysis separation device (FACS). However, it is not limited to these.

In EIA and FIA, for example, a protein extracted from a test cell collected from a tumor and a protein extracted from a control healthy cell are immobilized in wells of a microtiter plate, respectively. Then, an anti-NAMPT antibody is added to specifically react with the NAMPT protein, and then the excess antibody is washed with a phosphate buffer solution or the like to remove it. Then, it is detected using a labeled antibody that recognizes the anti-NAMPT antibody.

As the labeled antibody, an enzyme-labeled antibody, an antibody labeled with a fluorescent dye, or the like may be used. When an enzyme-labeled antibody is used, the anti-NAMPT antibody is reacted with the labeled antibody, and then the excess labeled antibody is washed with a phosphate buffer solution or the like to remove it. Then, the substrate of the enzyme is added to carry out the enzymatic reaction, and then the reaction product of the enzymatic reaction may be detected. For example, when the enzyme of the labeled antibody produces a luminescent substance from the substrate, the luminescence intensity of the well may be measured with a luminometer. This makes it possible to quantitatively detect the amount of luminescent substance. Under appropriate conditions, luminescence intensity correlates with the amount of labeled antibody, the amount of labeled antibody correlates with the amount of anti-NAMPT antibody, and the amount of anti-NAMPT antibody correlates with the mass of NAMPT protein. Thereby, by quantitatively measuring the amount of the luminescent substance, it is possible to quantify the mass of NAMPT protein in the protein extracted from the cell. Similarly, when the enzyme of the labeled antibody produces a fluorescent substance from the substrate, the NAPPT protein mass can be quantified by measuring the fluorescence intensity of the well with a fluorometer. For detection by fluorescence intensity, the labeled antibody may be labeled directly with a fluorescent substance. When the enzyme of the labeled antibody produces a color-developing substance from the substrate, the NAPPT protein mass can be quantified by measuring the absorbance of the well with an absorptiometer. EIA and FIA detect the NAMPT protein mass in the test cell and the healthy cell and compare the protein mass between the two, and the expression level of the NAMPT gene in the test cell is higher than the expression level of the NAMPT gene in the healthy cell. In some cases, it may be determined that the test cells are highly abnormal, for example, the test cells are highly metastatic. However, the measurement of the NAMPT protein expression level in healthy cells does not necessarily have to be performed together with the measurement of the NAMPT gene expression level in the test cells. For example, the average NAMEPT protein mass of healthy cells may be measured in advance, and this value may be set as a threshold value in cell characterization. The cell characteristics may be evaluated by comparing this threshold value with the mass of NAMPT protein of the test cell.

For example, in ICC and IHC, tumor cells (tissues) as test cells collected from a tumor and healthy cells (tissues) as controls are immobilized with an immobilization solution, and then unreacted with a phosphate buffer solution or the like. The immobilization liquid can be removed and a blocking treatment can be performed. After adding an anti-NAMPT antibody to specifically react with the NAMPT protein, the excess antibody can be detected by washing with a phosphate buffer or the like and using a labeled antibody that recognizes the anti-NAMPT antibody. The reagents used for ICC and IHC may be appropriately selected from known reagents and used. As the labeled antibody, an enzyme-labeled antibody, an antibody labeled with a fluorescent dye, or the like can be used. In ICC and IHC, the expression of NAMPT protein can be detected by an optical microscope. For example, when the enzyme of the labeled antibody produces a luminescent substance from the substrate, the luminescence of the cell can be detected by a luminescent microscope. If the enzyme of the labeled antibody produces a fluorescent substance from the substrate, fluorescence of the cell can be detected by a fluorescence microscope. If the enzyme of the labeled antibody produces a chromogenic substance from the substrate, the chromogenicity of the cells can be detected by light microscopy. When the detection is performed by fluorescence, the labeled antibody may be directly labeled with a fluorescent substance. In ICC and IHC, the staining intensity, that is, the intensity of luminescence, fluorescence or color development may be observed or compared with a microscope as the expression level of NAMEPT protein in the test cells and healthy cells. As a result, for example, when the expression level (staining intensity) of the NAMPT gene in the test cell is higher than the expression level (staining intensity) of the NAMPT gene in the healthy cell, for example, the test cell is abnormal or abnormal. It can be determined that there is a possibility, that the abnormality is high, or that the test cells are highly metastatic.

In FACS, tumor cells as test cells collected from a tumor and healthy cells as a control are immobilized with an immobilization solution, and then the unreacted immobilization solution is removed with a phosphate buffer solution or the like to perform blocking treatment. Can be After adding an anti-NAMPT antibody to specifically react with the NAMPT protein, the excess antibody is washed with a phosphate buffer or the like to remove it, and the protein is detected using a labeled antibody that recognizes the anti-NAMPT antibody. obtain. In FACS, it is preferable to use an antibody labeled with a fluorescent substance as the labeled antibody. Cells prepared in this way can be detected by FACS. For FACS, a commercially available device such as FACSVerse sold by BD (Becton, Dickinson and Company) can be used. In FACS, the fluorescence intensity of cells is detected and compared as the expression level of NAMPT protein in test cells and healthy cells, and the expression level (fluorescence intensity) of NAMPT gene in test cells is the expression of NAMPT gene in healthy cells. When the amount (fluorescence intensity) is higher than the amount (fluorescence intensity), it can be determined, for example, that there is an abnormality or that the test cells are highly metastatic.

1-2. Cell characterization kit for test cells by NAMEPT protein detection In the present invention, as a further embodiment, a cell identification evaluation kit for test cells by NAMEPT protein detection is provided. It can be, for example, a tumor cell metastasis assessment kit by NAMEPT protein detection. The cell characterization kit contains at least one anti-NAMPT antibody and buffer. The cell characterization kit may include a container containing a known carrier and / or reporter vector carrying an anti-NAMPT antibody. Furthermore, any reagent necessary for performing a method for evaluating the cell characteristics of the test cells may be included. For example, it may further include a labeled antibody that recognizes an anti-NAMPT antibody, a substrate for detecting the labeled antibody, an excipient, a stabilizer and / or other components having a desired effect. The composition of the kit may be provided in a container.

2. 2. Evaluation of cell characteristics of test cells by detection of NAMEPT mRNA 2-1. Detection method and evaluation method of cell characteristics NAMEPT mRNA may be detected by reverse transcription PCR using a primer set specific to NAPPT mRNA. However, the method is not limited to this method, and if the mRNA can be detected, an mRNA detection method known per se may be used. Examples of such a detection method include a Northern blotting method using a probe that specifically detects NAPPT mRNA, a DNA / RNA chip method, and the like.

In the reverse transcription PCR method, for example, cDNA is synthesized by a reverse transcription reaction using a test cell collected from a tumor and RNA extracted from a control healthy cell as a template, and a primer set specific to the template is used. The PCR used can be used to amplify a part or the full length of NAMPT mRNA and detect it.

The reverse transcription PCR method refers to a method for detecting mRNA in which the above reverse transcription reaction and PCR are combined. Commercially available reagents such as enzymes, substrates and buffers used for reverse transcription reaction and / or PCR can be used. Alternatively, a kit in which the above reagents are pre-packaged may be used.

The primer set used for reverse transcription PCR may include a combination of primers having complementary base sequences to the sense strand and antisense strand that code the NAPPT protein. The primer set is preferably a combination of primers that span two or more exons so as not to amplify the genomic DNA contaminated with RNA. Examples of such primer sets can be the primer sets set forth in SEQ ID NO: 2 and SEQ ID NO: 3. However, the primer set is not limited to this, as long as it can specifically amplify a part or the full length of NAPPT mRNA. The amount of NAMEPT mRNA contained in RNA extracted from cells may be quantified by amplifying a part or the entire length of NAMEPT mRNA by reverse transcription PCR using the above primer set under appropriate reaction conditions.

For example, when the amount of NAMPT mRNA in a test cell and a healthy cell is detected by the reverse transcription PCR method and the amount of mRNA is compared between the two, and the amount of NAMPT mRNA in the test cell is higher than the expression level of NAMPT mRNA in a healthy cell. , It may be determined that the test cell is abnormal, for example, the test cell has high metastasis. At this time, the measurement of the amount of NAMPT mRNA in healthy cells does not necessarily have to be performed at the same time as the measurement of the amount of NAMPT mRNA in the test cells. The average amount of NAMEPT mRNA in healthy cells may be measured in advance, and this value may be set as a threshold value in cell characterization. Cellular characteristics can be evaluated by comparing such a threshold value with the amount of NAMPT mRNA in the test cell.

2-2. Cell characterization kit of test cells by NAMEPT mRNA detection As a further embodiment, a cell characterization kit of test cells by NAMEPT mRNA detection is provided. It can be, for example, a cell characterization kit for tumor cells by NAMEPT mRNA detection. The cell characterization kit may include the primer set and buffer solution described in 1-1 above. The cell characterization kit may include a container containing a known carrier and / or primer set carrying the primer set. Further, the cell characterization kit may include any reagent necessary for performing a method for evaluating the cell characteristics of a test cell. For example, it may contain enzymes and substrates for performing reverse transcription PCR, buffers, excipients, stabilizers and / or other components having the desired effect. The composition of the kit may be provided in a container.

3. 3. Detection of NAMPT gene promoter activity using a reporter vector 3-1. Reporter Vector An example of a reporter vector that can be used in the detection of NAMEPT gene promoter activity will be described with reference to FIG. The reporter vector shown in FIG. 1 is an example of a reporter vector that does not replicate (ie, amplify) autonomously in mammalian cells. The reporter vector 1 contains a reporter gene expression unit 2 that produces a detectable signal. The reporter gene expression unit 2 includes a NAPPT promoter sequence 4, a reporter gene 5 functionally linked downstream thereof, and a transcription termination signal sequence 6 functionally linked downstream thereof.

Here, "functionally linked" means a state in which each gene is linked in a state in which it can exert a desired function. For example, in the case of a protein-encoding sequence, it means that the amino acid sequences encoded by the respective base sequences are correctly linked. In other words, it means that the amino acid codon frame is not deviated, and a peptide having the desired activity is synthesized in the cell into which the base sequence is introduced. Also here, the term "functionally concatenated" may be used interchangeably with the term "operably concatenated".

The promoter sequence 4 of the NAPPT gene is a nucleic acid sequence from which information for controlling the transcriptional activity from the NAPPT gene to mRNA should be obtained as the promoter activity of the gene. The promoter sequence 4 of the NAMPT gene is a sequence located in the 5'upstream region of the nucleic acid sequence encoding the amino acid sequence information of the NAMPT gene, that is, the NAMPT protein, in the genome of the cell. The promoter sequence 4 of the NAPPT gene may be a nucleic acid sequence having a length sufficient to obtain information for controlling the transcriptional activity from the NAPPT gene to mRNA. For example, an example of promoter sequence 4 of the NAMPT gene is shown in SEQ ID NO: 1. However, the promoter sequence of the gene does not have to completely match the nucleotide sequence shown in SEQ ID NO: 1 if information for controlling the transcriptional activity from the NAPPT gene to mRNA can be obtained. , The sequence may have homology, and the base sequence may be a base sequence shorter than SEQ ID NO: 1, that is, only a part of the sequence shown in SEQ ID NO: 1. Further, it may be a fusion nucleic acid sequence in which a part of the nucleic acid sequence shown in SEQ ID NO: 1 and a nucleic acid sequence of another gene promoter are linked. For example, the promoter sequence of the NAMPT gene may be obtained from the human genome using a primer set containing a forward primer and a reverse primer containing SEQ ID NO: 4 and SEQ ID NO: 5, respectively, shown in Table 2.

With respect to the above description, a "sequence having homology" is a sequence that is the same as a specific sequence on the genome, or a part of a sequence that is the same as a specific sequence on the genome, for example, one or several. A sequence in which a base is substituted, deleted or added, and which has promoter activity. For example, "having homology", "homologous", and "homologous" may be sequences that are 90% or more homologous to the genomic sequence. Further, the "fusion nucleic acid sequence" refers to a sequence formed by integrating two or more non-contiguous specific sequences on different genomes or on the same genome. That is, the promoter sequence 4 of the NAPPT gene may be composed of two or more units consisting of a part of the NAMEPT gene promoter sequence and another gene promoter sequence. The promoter sequence 4 of the NAMET gene may contain any additional sequence in addition to the sequence homologous to the sequence on the genome of the test cell. When containing any additional sequence, any additional sequence called a linker sequence or the like may be included as long as the promoter sequence 4 of the NAMPT gene has promoter activity.

The reporter gene 5 may be any gene encoding a protein that produces a detectable signal. The detectable signal may be any detectable signal generated from the reporter protein itself, the reaction between the reporter protein and any substance, the binding between the reporter protein and any substance, or the like.

The reporter gene 5 encoding the reporter protein may be any reporter protein that produces a detectable signal. The reporter gene 5 may be, for example, a luciferase gene, a β-galactosidase gene, a nitrogen monoxide synthase gene, a xanthin oxidase gene, a blue fluorescent protein gene, a green fluorescent protein gene, a red fluorescent protein gene and a heavy metal binding protein gene. .. Each of these genes encodes luciferase, β-galactosidase, nitric oxide synthase, xanthine oxidase, blue fluorescent protein, green fluorescent protein, red fluorescent protein and heavy metal binding protein, and activation of the reporter vector produces these proteins. Express. A preferred example in one embodiment is a gene encoding a luminescent enzyme protein, including, for example, a luciferase gene and a β-galactosidase gene.

An example of a reporter vector expressing a heavy metal binding protein as a detectable signal is disclosed in Japanese Patent Application Laid-Open No. 2012-200245.

The signal obtained from the reporter gene may be any signal that can be detected by any device. Further, such a signal may be detected by using any device selected according to the type of the signal. Examples of the device may be a light emission measuring device, a fluorescence measuring device, an X-ray measuring device, an electron spin resonance device, a nuclear medicine diagnostic device, a magnetic resonance imaging device, and the like. Further, the result obtained by detecting the signal may indicate the presence or absence of the signal, the magnitude of the signal, or both.

Preferred examples of detectable signals are optical signals such as light emission, fluorescence and color development. As used herein, "luminescence" refers to chemiluminescence, bioluminescence or biochemical luminescence. When the signal is an optical signal, the signal may be detected by an optical detection device. For example, when the detectable signal is light emission, the signal may be detected by a light emission measuring device capable of detecting the light emission, for example, a light emission microscope.

The transcription termination signal sequence 6 may be a sequence that terminates transcription of the gene upstream thereof, and may be, for example, a poly (A) addition signal. The poly (A) addition signal may be one that functions to terminate transcription of a mammalian gene. Examples include the late poly (A) addition signal of the SV40 virus (SEQ ID NO: 6), the poly (A) addition signal of the bovine growth hormone gene (SEQ ID NO: 7), and the like. Table 3 shows an example of the transcription termination signal sequence.

The transcription termination signals shown in Table 3 may be linked upstream of the promoter sequence 4 of the NAMPT gene. By linking the transcription termination signal 6 upstream of the promoter sequence 4, expression (leakage) of the reporter gene 5 due to inappropriate transcription from any sequence of the reporter vector can be prevented. This makes it possible to reduce the background (noise) due to inappropriate transcription (expression leakage) and increase the detection sensitivity and detection accuracy of the signal generated by the expression of the reporter gene 5.

However, the poly (A) addition signal that can be used in carrying out this embodiment is not limited to this, and a modified gene sequence is used as long as the function as the poly (A) addition signal is not impaired. You may use it.

An example of a self-amplifying reporter vector will be described with reference to FIG. Two examples of self-replicating vectors are shown in FIGS. 2 (a) and 2 (b).

The self-replicating vector 21 shown in FIG. 2A contains the same configuration as the reporter vector shown in FIG. 1 except that it contains a replication initiation sequence.

The self-replication vector 21 contains a reporter gene expression unit 2 and a replication initiation unit linked downstream thereof. The example shown in FIG. 2 is an example in which the replication initiation unit includes the replication initiation sequence 3. The replication origin sequence 3 initiates self-renewal in cells into which the self-replication vector 21 has been introduced by binding of the corresponding replication initiation protein.

The self-replicating vector performs self-renewal in the nucleus of the test cell under the condition that the replication initiation protein is expressed in the test cell. The replication initiation sequence 3 may be, for example, a sequence that is activated by binding of a replication initiation protein and initiates self-renewal in a mammalian cell, and may be a replication initiation sequence known per se. The replication initiation sequence 3 may be, for example, a replication initiation sequence derived from Simian virus 40 (SEQ ID NO: 8), Epstein-Barr virus or mouse polyoma virus. Table 4 shows an example of the replication origin sequence 3.

As the self-replicating vector, for example, a plasmid vector known per se may be used. For example, an example of a plasmid vector of this self-replicating vector is disclosed in Japanese Patent Application Laid-Open No. 2013-42721.

Self-replication of the self-replication vector is initiated by the binding of the replication initiation protein to the replication initiation sequence 3. The replication origin protein may be present in the nucleus of the test cell together with the self-replication vector so that it can bind to the replication origin sequence 3. For example, the replication initiation protein may be expressed from a self-replicating vector used as a reporter vector, may be expressed from a further vector, or may be expressed by a test cell.

For example, a replication initiation protein unit may be present to supply the replication initiation protein into the nucleus of the test cell. The replication initiation protein unit may include, for example, a promoter, a replication initiation protein gene encoding a replication initiation protein functionally linked downstream thereof, and a transcription termination signal sequence functionally linked downstream thereof. Alternatively, the replication initiation protein unit may consist of these sequences.

The replication initiation protein unit may be present on the nucleic acid of the self-replicating vector 21, or may be present on a nucleic acid different from that of the self-replicating vector 21. If the replication initiation protein unit is present on a nucleic acid different from the self-replication vector, the replication initiation protein unit may be present on the chromosome of the test cell or on other nucleic acids contained in the test cell. You may.

When the replication initiation protein unit is contained in the self-replication vector as a reporter vector, the expression of the reporter gene expression unit 2 and the replication initiation protein unit contained therein is controlled together by one promoter sequence 4. They may be regulated by two independent promoter sequences, respectively. When their expression is regulated by two promoter sequences, for example, the replication initiation protein unit may be present on the nucleic acid of the self-replicating vector 21 independently of the reporter gene expression unit 2. In that case, the promoter that controls the expression of the replication initiation protein gene is preferably a constitutively expressed promoter.

The replication initiation protein gene may be, for example, the large T antigen gene of Simian virus 40, the EBNA-1 gene of Epstein-Barr virus, the large T antigen gene of mouse polyoma virus, or the like. Examples of preferred large T antigen genes are the nucleic acids set forth in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or one or several bases in the sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 are deleted. , It may be a nucleic acid having a DNA replication initiation function indicated by a substituted or added sequence. Here, the DNA replication initiation function means encoding a protein capable of initiating replication. Examples of the replication initiation protein gene are shown in Table 5-1 and Table 5-2, Table 6-1 and Table 6-2, Table 7-1 and Table 7-2.

The combination of the replication initiation protein gene and the replication initiation sequence may be any combination capable of initiating replication. Examples of such combinations are:
A combination in which the gene encoding the replication initiation protein is the large T antigen gene of Simian virus 40, and the replication initiation sequence is the replication initiation sequence from Simian virus 40;
A combination in which the replication initiation protein gene is the EBNA-1 gene of Epstein-Barr virus and the replication initiation sequence is the replication initiation sequence from Epstein-Barr virus;
A combination in which the replication initiation protein gene is a large T antigen gene of mouse polyoma virus and the replication initiation sequence is a replication initiation sequence from mouse polyoma virus. Combinations such as these are preferred, but not limited to.

The self-replicating vector may be a bicistronic expression vector. In the bicistronic expression vector, by using the bicistronic expression sequence, the expression of two genes contained downstream thereof, that is, the expression of the reporter gene and the expression of the replication initiation protein, are simultaneously regulated by the NAMEPT promoter sequence 4. To. That is, in such a self-replicating vector, the reporter gene expression unit 2 and the replication initiation protein unit are controlled and expressed by the same promoter. An example of such a bicistronic expression self-replicating vector will be described with reference to FIG. 2 (b). The self-replicating vector 31 contains a NAMPT promoter sequence 4, a reporter gene 5, IRES 7, a replication initiation protein gene 8, a transcription termination signal sequence 6, and a replication initiation sequence 3. The NAMEPT promoter sequence 4, the reporter gene 5, the IRES 7, the replication initiation protein gene 8, and the transcription termination signal sequence 6 are functionally linked in this order from upstream to downstream. The IRES7 is an internal ribosomal entry site and is an example of a sequence for bicistronic expression. Examples of bicistronic expression sequences are, for example, the internal ribosome entry sequence of brain myocardial virus (SEQ ID NO: 12). IRES7 is a base sequence that binds a ribosome to the inside of mRNA and initiates protein translation in mammalian cells, independent of the 5'end cap structure of mRNA. By placing IRES7 between two genes, bicistronic mRNA is synthesized. That is, by integrating IRES7 between two genes, it is possible to translate two types of proteins encoded by these two genes with one mRNA. An example of this is shown in Table 8.

Due to the presence of IRES7, the NAPPT promoter sequence 4 transcribes the sequence of the reporter gene 5, followed by the replication initiation protein gene 8. After the sequence of the replication initiation protein gene 8 is read, transcription is stopped by the presence of the transcription termination signal sequence 6. The replication initiation protein produced by being transcribed and translated from the replication initiation protein gene 8 binds to the replication initiation sequence 3, and the replication of the self-replication vector 31 is initiated.

By detecting the detectable signal generated from such a reporter vector, it is possible to evaluate the activation of the NAMEPT promoter sequence, thereby evaluating the expression level of the NAMP gene. Based on the result, it becomes possible to determine the cell characteristics of the test cells.

In the above reporter vector, promoter activity means inducing transcription of a gene operably linked downstream of the promoter sequence. When the promoter sequence 4 of the NAMPT gene has promoter activity, the promoter activity is derived from a part of the promoter sequence 4 of the NAMPT gene, even if it is derived from the entire sequence of the promoter sequence 4 of the NAMPT gene. It may be. Also, for example, the promoter sequence 4 of the NAMPT gene may contain the smallest promoter. The promoter activation of the promoter sequence 4 of the NAMPT gene is controlled by the presence or absence of binding of a transcription factor to a part of the sequence on the promoter sequence 4 of the NAMPT gene and the state of modification of the modified base.

The promoter sequence 4 of the NAMET gene contains a target nucleic acid sequence which is a sequence having the same identity as a part of the genome of the test cell. Therefore, in the case of a self-amplifying reporter vector, the self-replicating vector in the target nucleic acid sequence is characterized by the epigenic property of the state of functional group substitution in the target nucleic acid sequence contained in the test cell during self-amplification. It can be copied (ie, copied) on a base. That is, activation of the NAMPT promoter sequence may be activated or inactivated depending on the presence or absence or degree of substitution of a functional group in a modifiable base in the target nucleic acid sequence, for example, a methyl group or an acetyl group. You may.

The self-replication reporter vector 31 can perform self-renewal in the test cell under the condition that the NAPPT gene promoter 4 is activated. That is, when NAMEPT promoter 4 is activated, the reporter gene 5 and the replication initiation protein gene 8 are transcribed as bicistronic mRNA by the action of IRES7, and the reporter protein and the replication initiation protein, which are translation products of the mRNA, are produced. Will be done. The replication origin protein binds to the replication origin sequence 3 of the self-replicating reporter vector, and by binding to the protein required for DNA replication, the reporter vector self-replicates.

The self-replication reporter vector may be a self-replicating vector, for example, a plasmid vector. Examples of such a self-replicating plasmid vector are disclosed in Japanese Patent Application Laid-Open No. 2013-42721 and the like.

3-2. Detection of NAMPT Promoter Activity An example of a method for detecting the promoter activity of the NAMPR gene (that is, the NAMPT promoter activity) in a test cell or a healthy cell will be described using the reporter vector described in 3-1. Here, the detection of promoter activity may be the detection of the presence or absence of promoter activity or the quantitative measurement of promoter activation.

A reporter vector 1 or a self-amplifying reporter vector 21 or 31 having a promoter region (SEQ ID NO: 1) of the NAMET gene is prepared (a). Reporter vector 1, 21 or 31 is then introduced into live test cells and healthy cells (b). After that, the state in which the reporter gene of reporter vector 1, 21 or 31 is expressed in the cell is maintained. When the reporter vector 21 or 31 is used, the culture conditions under which the cells can divide and proliferate, that is, the reporter vector is maintained in a self-replicating state (c). In such a state, the promoter activity of the NAMPT gene in the cell genome is reflected in the activity of the NAMPT promoter (SEQ ID NO: 1) of the reporter vector (d). Next, as an index of NAMPT promoter activity, the amount of reporter protein expressed from the reporter gene is measured (e). Finally, the amount of reporter protein in the test cell and the healthy cell is compared, and when the amount of the reporter protein in the test cell is large, the test cell is a cell having an abnormality, for example, a tumor cell. Judgment (f). At this time, it is not always necessary to measure the NAMEPT promoter activity in healthy cells, that is, the introduction of the reporter vector into healthy cells and the expression level of the reporter protein. For example, the average NAMPT promoter activity of healthy cells is measured in advance, this value is set as a threshold value in cell characteristic evaluation, and the evaluation is performed by comparing this threshold value with the NAMPT promoter activity of test cells. You may.

The above-mentioned detection of NAMPT promoter activity is possible by detecting or quantifying the expression of the reporter protein. The reporter protein may be, for example, luciferase, β-galactosidase, nitric oxide synthase, xanthine oxidase, blue fluorescent protein, green fluorescent protein, red fluorescent protein and heavy metal binding protein. Appropriate detection and / or measurement methods can be selected depending on the type of reporter protein selected.

An example of detecting the reporter protein is given below. When the reporter gene is a luminescent gene, for example, a luciferase gene, the luminescent intensity of the solution can be measured using a luminometer after extracting the luciferase protein from the test cells and adding a substrate. When the reporter gene is a fluorescent protein gene, for example, the test cell is irradiated with light of a specific wavelength, and the intensity of fluorescence generated from the fluorescent protein in the test cell can be measured by a fluorescence measuring device. Alternatively, an extract containing a fluorescent protein extracted from a test cell can be irradiated with light having a specific wavelength, and the intensity of fluorescence generated from the fluorescent protein in the extract can be measured by a fluorescence measuring device.

When the reporter gene is a β-galactosidase gene, the absorbance of the solution can be measured using an absorbance measuring device after extracting the β-galactosidase protein and adding a substrate.

As the reporter gene, an active oxygen-producing enzyme gene may be used. The active oxygen-producing enzyme gene is a gene encoding an enzyme that produces active oxygen. Examples of the active oxygen-producing enzyme gene include a nitric oxide synthase gene and a xanthine oxidase gene. Nitric oxide synthase, which is a translation product of the nitric oxide synthase gene, and xanthine oxidase, which is a translation product of the xanthine oxidase gene, generate active oxygen. Active oxygen can be detected by an electron spin resonance apparatus (ESR apparatus) or the like. In the method by the ESR device, the amount of active oxygen generated may be measured directly, or it may be measured by utilizing a specific spin trapping agent. When using a spin trapping agent, first, the active oxygen-producing enzyme is trapped with the spin-trapping agent, and then the amount of active oxygen generated from the trapped active oxygen-producing enzyme is measured.

A heavy metal binding protein gene may be used as the reporter gene. A heavy metal binding protein gene is a gene encoding a protein that specifically binds to a heavy metal. The amount of protein bound to heavy metals may be produced by a magnetic resonance imaging device, a nuclear medicine diagnostic device, or an X-ray computed tomography device. The amount of heavy metal-binding protein generated is, for example, as follows. First, a measurable heavy metal is added to the culture medium of the test cells in advance. Next, after washing the test cells as needed, the heavy metal binding protein is extracted from the test cells. Next, the extracted heavy metal-binding protein is imaged with an image diagnostic apparatus corresponding to the heavy metal added to the culture solution, and the amount of the heavy metal-binding protein is measured. The heavy metal binding protein may be expressed inside the test cell or may be expressed on the outer surface of the test cell. An example of a heavy metal-binding protein gene may be a base sequence encoding a metal compound-binding peptide. For example, the metal compound binding protein may be a peptide, oligopeptide, polypeptide and / or protein that specifically binds to a specific metal compound. For example, the sequence encoding the peptide that binds the metal compound may utilize the base sequence of an antibody gene or a single-chain antibody gene that is known to bind to the desired metal compound, and is based on such a base sequence. You can design it. Modifications and / or modifications of such base sequences, such as substitutions, deletions and additions of one or several bases within the range that maintains binding to the metal compound, modifications and modifications depending on the subject to which they are applied. / Or may be designed by modification. The gene encoding the single chain antibody peptide can be designed from the amino acid sequence of the antibody that binds the metal compound. For example, in MRI imaging, a gadolinium compound is preferably used because of its high contrast effect. The gadolinium compound may be, for example, a metal compound consisting of gadolinium, gadolinium ion, gadolinium complex, salts thereof and derivatives thereof, derivatives containing any of these, or analogs of gadolinium compounds. It may also be a reporter gene that exhibits the ability to bind metal compounds extracellularly. Such a reporter gene may be a base sequence encoding a metal compound binding peptide presented extracellularly. Such a base sequence is, for example, transcribed and translated in a cell to produce a metal compound-binding peptide, and the produced metal compound-binding peptide moves to the cell membrane to exhibit the metal compound-binding ability extracellularly. It may be configured as follows. Examples of such reporter genes are disclosed in, for example, Japanese Patent Application Laid-Open No. 2012-200245.

For example, a method for evaluating the metastatic property of a test cell using a self-replicating reporter vector is to prepare a reporter vector, introduce the reporter vector into the test cell, and express a replication initiation protein. Under these conditions, culturing the test cells and detecting the reporter protein expressed in the test cells may be included.

3-3. Cell characterization kit for test cells using a reporter vector As a further embodiment, a cell characterization kit for test cells using a reporter vector may be provided. It may be a cell characterization kit for tumor cells. The cell characterization kit may contain at least one of the reporter vectors described in 3-1 above and a buffer solution. In addition, the cell characterization kit may further include a container containing a known carrier and / or reporter vector carrying the reporter vector. In addition, the cell characterization kit may include any of the reagents necessary to perform a method for assessing the cell properties of test cells, eg, tumor cells. Such reagents may include, for example, substrates, excipients, stabilizers and / or other components of the reporter protein produced from the reporter gene that have the desired effect. These cell characterization kits may be provided in containers.

4. Drug screening method A drug that suppresses abnormalities such as metastasis of test cells may be screened by using a method for evaluating cell characteristics of test cells by detecting NAPPT protein. Drugs that suppress tumor cell abnormalities due to the detection of NAPPT protein, such as tumor cell metastasis, may be screened. For example, if a drug-treated cell group treated with a drug to be screened and a pathological cell group not treated with a drug are prepared, the cell characteristics of each of these two cell groups are evaluated, and the obtained results are compared. Good. The pathological cell population may have anomalies that should be treated with the drug to be screened. Drug screening may be performed using whether or not such pathological cell characteristics have been treated by drug treatment as an index. In addition to the drug-treated cell group and the pathological cell group, a normal cell group may be prepared and the cell characteristics of the normal cells may be evaluated. The cellular properties of normal cells may be used as a control when screening for drugs. The cellular properties of normal cells may be determined in parallel with drug screening or may be determined in advance.

5. Cell characteristic evaluation device for test cells The cell characteristic evaluation device includes a gene transfer unit, a culture unit, an imaging unit, and a determination unit. The gene transfer unit is a unit that introduces a reporter vector containing the NAMPT gene promoter sequence into the nucleus of a test cell. The culture unit is a unit for culturing the test cells processed by the gene transfer unit, that is, the gene-introduced cells. The imaging unit captures a bright-field image of the test cells cultured in the culture unit, and further captures a luminescent image consisting of luminescence signals generated for each cell due to the expression of the reporter gene in the same field of view. It is a unit to do. The determination unit is a unit that determines the cell characteristics of the test cells for each cell based on the bright field image and the luminescent image.

In a further example, the gene transfer unit is a unit that introduces a reporter vector containing the NAMPT gene promoter sequence into the nucleus of a test cell. The culture unit is a unit for culturing the test cells processed by the gene transfer unit, that is, the gene-introduced cells. The imaging unit is a unit that captures an luminescent image of the test cells cultured in the culturing unit, which consists of luminescence signals generated for each cell due to the expression of the reporter gene. The determination unit is a unit that determines the cell characteristics of the test cells for each cell based on the luminescent image.

An example of an apparatus for determining the cell characteristics of the test cells according to the embodiment will be described with reference to FIG. FIG. 3 is a block diagram showing an outline of the device 70. The device 70 is an example of a device that detects abnormal activity of a test cell and evaluates the cell characteristics of the test cell based on the abnormal activity.

The device 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and a stage 74 continuously arranged below them. The cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 share one stage 74 and are communicated in this order by the stage 74. In other words, the cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 are arranged above the stage 74 in this order from upstream to downstream.

The cell separation unit 71 includes a separator 75 for separating cells to be tested, that is, test cells, from a sample obtained from a subject. The separator 75 separates the test cells from the receptor 76a that receives the sample obtained from the subject, the processor 76b that digests the sample received by the receptor 76a, and the processed sample processed by the processor 76b. It is provided with a separation unit 77 for the purpose. The receptor 76a, the processor 76b, and the separation unit 77 are formed by stacking three hollow tubular bodies having different inner diameters in this order from top to bottom and liquid-tightly integrating them with each other. Each boundary between the receptor 76a, the processor 76b, and the separation portion 77 is entwined with a valve so as to be openable and closable. Receptor 76a is a tapered tubular body with an open top and has a lid that can be opened and closed. At the boundary between the receptor 76a and the processor 76b, the valve closes liquid-tightly. When this valve closes, it becomes the bottom of receptor 76a. The processor 76b is a tubular body continuous with the lower end of the receptor 76a. When the valve at the boundary with the receptor 76a is closed liquidtightly, the processor 76b is covered. When the valve at the boundary with the separating portion 77 is closed, the processor 76b becomes bottomed. The separation portion 77 is a tubular body whose inner wall is liquidtightly continuous from the inner wall of the processor 76b, and a valve is provided at the lower end thereof. The valve closes liquidtightly and the separation part 77 becomes bottomed. A separating material is fixed inside the separating portion 77. When the device 70 is used, the container 78 is placed on the stage 74 below the bottom of the separation portion 77. The container 78 is a container 78 that receives a sample containing the separated test cells 79 from the separation unit 77 and stores the sample. Here, the processor 76b may be provided with a heating and / or constant temperature mechanism for performing a desired treatment such as digestion and biochemical reaction on the sample contained therein. Further, the separating material may be a member for separating and collecting a sample containing a test cell from a sample descended from the processor 76b, or a contaminant may be removed from the sample descended from the processor 76b. It may be a member to be removed. The separating material may spread along a surface intersecting the central axis of the separating portion 77 and may be fixed over the circumference of the inner wall of the separating portion 77. The separating material may be, for example, a filter, a membrane and / or a magnet.

The sample containing the test cells 79 separated by the separator 75 is housed in a container 78 placed on the stage 74. The container 78 containing the test cells is sent to the DNA introduction unit 72.

The DNA introduction unit 72 includes a control unit 80 for introducing a reporter vector into the test cells housed in the container 78, and an introduction probe 81. The introduction of the reporter vector into the test cells may be carried out by a method known per se. The control unit 80 and the introduction probe 81 may be devices that utilize any introduction mechanism for achieving the selected introduction method. For example, the DNA introduction unit 72 may be a device that performs electroporation. In that case, the control unit 80 is a DNA introducer, the introduction probe 81 is an electrode probe, and an electroporator may be composed of these. Further, the DNA introduction unit 72 may be provided with a nozzle for supplying a reagent containing a reporter vector to the container 78 (not shown). Further, the DNA introduction unit 72 may include a liquid feeding system for feeding the reagent to the nozzle and a reagent storage container for supplying the reagent to the liquid feeding system (not shown).

Further, the DNA introduction unit 72 may function as an incubator for culturing the test cells 79 in the container 78 under a predetermined culture condition for a predetermined time. The culture of the DNA introduction unit 72 may be controlled by the control unit 80.

In the DNA introduction unit 72, the container 78 containing the test cells into which the reporter vector has been introduced is then sent to the measurement unit 73.

The measurement unit 73 is installed in a dark box 82 that covers a part of the stage 74 from the upper part and the lower part, a light source 83 arranged inside the dark box 82, a window that opens on the stage surface below the light source 83, and below the window. It includes an image pickup unit 88 to which the objective lens 87 is attached, a control unit 85 that communicates with the light source 83 and the image pickup unit 88, and a display unit 84 and a storage unit 86 that communicate with the control unit 85, respectively. For example, the light source 83, the objective lens 87, and the imaging unit 88 of the measurement unit 73 may be included in a microscope equipped with a CCD camera, a CMOS camera, or the like. Further, the control unit 85, the display unit 84, and the storage unit 86 may be a computer including these.

An apparatus for evaluating the cell characteristics of the test cells according to the embodiment will be described with reference to FIG. FIG. 4 is an example of the device 70 shown in FIG. 3, which differs only in the configuration of the stage. The device 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and stages 74a, 74b, 74c arranged below each of these units, respectively. As described above, in the device 70 of FIG. 3, a stage is provided for each unit. The movement between the units, that is, between the stages may be performed manually, or each movement may be automatically performed by a robot arm or the like.

An example of evaluation of cell characteristics by the apparatus 70 shown in FIGS. 3 and 4 will be described with reference to FIG. First, the container 78 is placed on the stage 74 below the separation portion 77 of the cell separation unit 71. Next, a pretreated sample such as mince or homogenize is brought into the receptor 76a from the upper opening, if desired. Along with this, reagents necessary for processing in the processing unit 76b are added. The reagent may be added from the reagent addition port provided in the processor 76b so as to be openable and closable (S20). The lid of the receptor 76a is closed and the valve at the boundary with the processor 76b is opened. The digestion process is performed in the processor 76b according to preset conditions. The processor 76b may be provided with a temperature controller for creating an environment satisfying preset conditions (S21). Next, the valve at the boundary between the processor 76b and the separation unit 77 is released, and the sample processed in the processor 76b is sent to the inside of the separation unit 77. By passing through the separating material of the separating unit 77, impurities contained in the sample are removed (S22). The sample containing the isolated test cells is sent to the container 78 and contained. Here, a reaction stop reagent for stopping the reaction carried out in the processor 76b prior to passing through the separating material may be added to the sample. For this addition, the reaction stop reagent may be stored in the separation unit 77 in advance, and an openable / closable reagent addition port for adding such a reagent is provided on the wall surface at any position of the separator 75. It may be (S23).

The container 78 is sent to the DNA introduction unit 72 while still containing the sample containing the test cells 79 (S24). A reagent containing a reporter vector is added to the container 78 from the nozzle of the DNA introduction unit 72. The introduction probe 81 is brought into contact with the sample in the container 78, and the reporter vector is introduced into the test cell 79 under the control of the control unit 80 (S25). Under the control of the control unit 80, the test cells 79 are cultured under a predetermined culture condition for a predetermined time (S26).

After that, the container 78 is sent to the measuring unit 73. The measuring unit 73 measures the mass of the reporter protein in the test cells 79 housed in the container 78. All procedures in the measurement unit 73 are controlled by the control unit 85 according to a table in which a program and a plurality of information stored in advance in the storage unit 86 are collected. For example, the measurement unit 73 controls light irradiation from the light source 83, control of irradiation conditions such as irradiation time and irradiation interval, control of imaging and imaging conditions by the imaging unit 88, image processing and image processing of the captured image, and the like. Will be done. Further, the control unit 85 also determines the cell characteristics by referring to the table including the information in which the image and the cell characteristics are associated with each other according to the program stored in the storage unit 86 in advance.

After culturing the test cells in the DNA introduction unit 72, the container 78 is sent to the measurement unit 73. After that, the light from the light source 83 is irradiated into the container 78. The light that has passed through the container 78 is collected by the objective lens 87, and the imaging unit 88 arbitrarily obtains a bright field image from the light. Then, in the dark box 82, an optical signal (here, light emission as an example) from the reporter protein introduced into the test cells is imaged by the imaging unit 88 through the objective lens 87. The light irradiation of the light source 83 is stopped, the light emitted from the test cells is collected by the objective lens 87, and the imaging unit 88 obtains a light emitting image from the light (S27). Arbitrary bright-field images and light-emitting images obtained by the imaging unit 88 are image-processed by the control unit 85, arbitrarily superimposed (merged), and displayed on the display unit 84. The control unit 85 determines the cell characteristics of the test cells based on the bright-field image and the luminescent image and the table stored in the storage unit 86 at the same time as or before and after the display of the image on the display unit 84. judge. The result of this determination is shown on the display unit 84 together with the merged image (S28).

Here, an example in which light emission is generated as an optical signal from the reporter protein is shown, but even for a detectable signal in which a reporter protein different from the light emission is generated, the detection device constituting the imaging unit 88 is replaced. It is possible to measure in the same way.

Further, although not shown in the figure, the measurement unit 73 may further include a processing unit that performs computer processing, image processing, and the like, or an analysis unit that performs characteristic determination. These processing units and analysis units may perform the computer processing and image processing performed by the control unit 85 and the characteristic determination described above.

In the above embodiment, the example in which the DNA introduction unit 72 cultures the test cells is shown, but the test cells may be cultured in the measurement unit 73. In that case, the device 70 may further include a member that enables cell culture. Alternatively, the test cells may be cultured in both the DNA introduction unit 72 and the measurement unit 73.

Each unit may have its own control unit to control the movement of each unit of the device 70, and all the units included in the device 70 may be performed by one control unit, for example, the control unit 85. .. Further, the movement between the units of the container 78 mounted on the stage 74 may be performed by the container moving mechanism mounted on the stage 74. Such a mechanism may be performed, for example, by a robot arm provided in the device 70 and whose movement is controlled by a control unit provided in the device 70. Alternatively, a belt conveyor may be attached to the stage 74.

Further, the addition of the reporter vector to the container 78 may be performed at any time before the container 78 is sent to a predetermined position of the DNA introduction unit 72. In that case, the device 70 may further provide a reporter vector addition mechanism at a desired position upstream of the DNA introduction unit 72.

With such a device, the cell characteristics of the test cells are evaluated. According to this device, it is possible to evaluate the cell characteristics of the test cells with higher accuracy. In addition, since the inspection can be performed with throughput, the inspection can be easily performed, and it is possible to prevent the sample from being mistaken and contamination.

In addition, the morphological characteristics of the test cells may be obtained based on the bright-field image, and the cell characteristics of the test cells may be evaluated together with the information from the reporter vector. Morphological features include, for example, the area of the test cell, the ratio of the area of the nucleus to the area of the test cell, the ratio of the minor axis to the major axis of the test cell, the number of nuclei in the test cell, or the side of the test cell. It may be a feature of the edge portion, for example, a contrast ratio of the edge portion. These are obtained by non-invasive optical imaging methods. For example, such information may be obtained by a hyperspectral imaging method, a phase contrast microscopy method, a differential interference contrast method, or the like.

For example, when morphological observation is performed in a bright field, it is preferable to irradiate light having a wavelength in the near infrared region. For example, the light having a wavelength in the near infrared region is preferably near infrared light having a wavelength of 700 nm to 750 nm or multispectral light having a wavelength of 350 nm to 750 nm. For example, the test cell may be irradiated with light having a wavelength in the near infrared region, and the reflection or absorption of the light, for example, the reflectance or absorption rate may be used as an index to obtain morphological information of the test cell. ..

For example, when the test cells are highly metastatic, the cell morphology tends to be spindle-shaped or other cell morphology with a high aspect ratio. For example, the morphological information of such a test cell may also be acquired in the measurement unit 73. Even in that case, the measurement unit 73 may be controlled by the control unit 85 according to the program stored in the storage unit 86 in advance and the table in which the plurality of information is collected. The table contains information about the relationship between the detectable signal from the reporter vector and the cell characteristics, as well as the relationship between the morphological characteristics of the test cell and the cell characteristics, and the detectable signal and the cell characteristics. Information indicating the relationship between and morphological features may be included. Further, for example, the measurement unit 73 controls irradiation conditions such as light irradiation from the light source 83, irradiation time and irradiation interval, control of imaging and imaging conditions by the imaging unit 88, image processing and image processing of the captured image, and the like. Is controlled by. Further, in the evaluation of the cell characteristics, the control unit refers to a table containing information in which the morphological characteristics, the information from the reporter vector, and the cell characteristics are associated with each other according to the program stored in the storage unit 86 in advance. 85 should do it. By evaluating the cell characteristics of the test cells from a plurality of aspects of promoter activity and morphological characteristics in this way, it is possible to obtain more accurate results.

[Example]
As an example of a method for evaluating the cell characteristics of the test cells, the metastatic property of the tumor cell line was evaluated. That is, a tumor cell line was used as an example of the test cells. Metastatic property was observed as an example of cell characteristics.

Example 1. Metastatic evaluation of tumor cell lines by immunostaining (detection of NAMPT protein) For metastatic evaluation, as cell lines derived from human mammary tumor, highly metastatic cell lines BT-549 and MDA-MB-231, and Low metastatic cell lines MCF-7 and T47-D were used. These cell lines were seeded on 8-well chamber slides (Thermo Scientific) and cultured at 37 ° C. in a 5% CO ₂ atmosphere. Medium was removed from the 8-well chamber slides and cells were washed with phosphate buffered buffer (PBS). The cells were then immobilized by adding a 4% paraformaldehyde solution and incubating at room temperature for 20 minutes. After removing the paraformaldehyde solution and washing with PBS, 0.2% Triton X-100-containing PBS was added, and the mixture was incubated at room temperature for 10 minutes for membrane permeation treatment. After removing the PBS containing Triton X-100 and washing with PBS, Image-IT FX signal enhancer (Life Technologies) was added and incubated at room temperature for 30 minutes for blocking treatment. The Image-IT FX signal enhancer was removed and washed with PBS. Then, Can Get Signal (TOYOBO) containing a 500-fold diluted rabbit anti-NAMPT polyclonal antibody (ab45890, Abcam) was added, and the mixture was incubated overnight at 4 ° C. The antibody solution was removed, washed with PBS, 2000-fold diluted Alexa546-labeled goat anti-rabbit antibody was added, and the mixture was incubated at room temperature for 30 minutes. The antibody solution was removed, washed with PBS, DAPI solution was added, and the mixture was incubated at room temperature for 1 minute to stain cell nuclei. After washing with PBS, stained cells were encapsulated in a slide glass with Prolonged Gold Antifade Reagent (Life Technologies). The fluorescent image of the stained cells was observed with a microscope Axio Imager 2 (Carl Zeiss) and photographed. The fluorescence of Alexa 546 was observed with a filter set having an excitation wavelength of 550 nm and a fluorescence wavelength of 605 nm. The fluorescence of DAPI was observed with a filter set having an excitation wavelength of 320 nm and a fluorescence wavelength of 490 nm. Fluorescent images of stained cells are shown in FIG.

All of the mammary tumor-derived cell lines used in the test were fluorescently stained with anti-Nampt protein. Highly metastatic cell lines: BT-549 and MDA-MB-231 detected stronger staining signals than low metastatic cell lines: MCF-7 and T47-D. That is, the highly metastatic cell line highly expressed the Nampt protein. From this result, it was shown that it is possible to evaluate metastasis as an example of cell characteristics of tumor cells using the same protein mass as an index.

Example 2. Evaluation of metastatic characteristics of tumor cell lines by reverse transcription PCR (detection of NAMPT mRNA) (1) Breast tumor cell lines In experiments, MDA-MB-231 and BT, which are highly metastatic cell lines, were used as cell lines derived from human breast tumors. -549, low metastatic cell lines MCF-7 and T47-D, and benign tumor cell lines MCF-10A were used. These cell lines were seeded on a 24-well plate and cultured at 37 ° C. in a 5% CO ₂ atmosphere. Total RNA was extracted from the cell line by RNeasy Mini Kit (Qiagen), and Nampt mRNA (mRNA transcribed from the Nampt gene) was detected by RT-PCR (reverse transcription PCR).

From the above-mentioned total RNA, cDNA was synthesized by a reverse transcription reaction using SuperScript VILO cDNA Synthesis Kit (Life Technologies). Then, Nampt mRNA was amplified by PCR using the following primer set.

Forward primer; 5'-ACCTTACAGAGATTGTAG-3'(SEQ ID NO: 2)
Reverse primer; 5'-TATGGAGAAGATCCTCGACC-3'(SEQ ID NO: 3)
After completion of PCR, the amplified product was electrophoresed on a 0.8% agarose gel and the gel was stained with ethidium bromide. The PCR amplification product in the gel was visualized and photographed under UV irradiation (Fig. 7). Here, in each of the photographs obtained by the above shooting, the background is black and the band is white. If the image is shown in the drawing as it is, the band is difficult to see. In order to make the band easier to see, the photograph obtained by the above shooting is taken into a computer, and an image processing software is used to show an image in which black and white colors are inverted so as not to impair the band intensity and relativity with other data. It was. NAMEPT mRNA was detected in all of the breast tumor-derived cell lines tested. The highly metastatic cell lines MDA-MB-231 and BT-549 have stronger amplification signals of NAMEPT mRNA than the low metastatic cell lines MCF-7 and T47-D and the benign tumor cell lines MCF-10A. was detected. That is, the highly metastatic cell line highly expressed NAMPT mRNA. From this result, it was shown that it is possible to evaluate the metastatic property of a tumor cell, which is an example of a test cell, using the mRNA amount of NAMPT as an index.

(2) Lung tumor cell line In the lung tumor cell line experiment, A-549, which is a basal epithelial gland lung cancer cell line, and LUDLU-1, which is a flat epithelial lung cancer cell line, were used as cell lines derived from lung tumor. From these cell lines, total RNA was extracted by the same method as in Example (1) above, and NAPPT mRNA was detected by RT-PCR. As the PCR primers, the primer sets shown in SEQ ID NOs: 2 and 3 were used.

After completion of PCR, the amplified product was electrophoresed on a 0.8% agarose gel and the gel was stained with ethidium bromide. The PCR amplification product in the gel was visualized and photographed under UV irradiation (Fig. 8). Here, in each of the photographs obtained by the above shooting, the background is black and the band is white. If the image is shown in the drawing as it is, the band is difficult to see. In order to make the band easier to see, the photograph obtained by the above shooting is taken into a computer, and an image processing software is used to show an image in which black and white colors are inverted so as not to impair the band intensity and relativity with other data. It was. NAMEPT mRNA was detected in both the basal epithelial lung cancer cell line A-549 and the squamous cell lung cancer cell line LUCL U-1. In addition, it was revealed that the amount of NAMEPT mRNA was small in A-549, which is a low metastatic cell line.

Example 3. Evaluation of metastasis of tumor cells by reporter gene assay (detection of NAMPT promoter activity) (1) Breast cancer cell line (a) Construction of reporter vector Promoter region of nicotinamide phosphoribosyltransferase (Nampt) gene (-2426 to +276 bp, SEQ ID NO: A reporter vector incorporating 1) was prepared. The promoter region of the Nampt gene was amplified by PCR using the human genome as a template. PCR was performed using PrimeStar GLX DNA polymerase (TaKaRa BIO). The primer sequences used for PCR are shown below.

Forward primer; 5'-GGGACGCGTGTCCATGTTGCCCAGGCTGGTCTCA-3'(SEQ ID NO: 4)
Reverse primer; 5'-CGGCTCGAGAGCTCCCCTGGCGCGCGCTGCGAGGAA-3'(SEQ ID NO: 5)
The promoter sequence obtained by PCR was incorporated into PGV-B2 (TOYO B-Net) to prepare a reporter vector: pNampt-L. PGV-B2 is a vector in which a luciferase gene (reporter gene) and a transcription termination signal sequence are provided in this order from upstream to downstream. Specifically, the CK19 promoter region, which is a promoter sequence amplified by PCR, was integrated upstream of the luciferase gene of PGV-B2 to prepare a reporter vector: pNampt-L (FIG. 9).

As a vector for correcting the introduction efficiency of the reporter vector for each cell line, pNLuc-tk was used. pNLuc-tk was prepared by incorporating the thymidine kinase (tk) gene promoter of the human herpesvirus of pRL-tk (Promega) upstream of the NanoLuc gene of pNL1.1 (Promega).

(B) Lipofection (introduction of reporter DNA into cells)
To 40 μl of Opti-MEM (Life Technologies), 0.15 μg of reporter vector: pNampt-L and 0.05 μg of vector for correcting introduction efficiency: pNLuc-tk were added. Then, 0.2 μg of enhancer reagent (Plus reagent, Life Technologies) was added, and the mixture was incubated at room temperature for 5 minutes. To this solution was added 0.7 μl of Lipofectamine LTX (Life Technologies), which was well turbid and incubated at room temperature for 25 minutes. Transfer the solution to a 48-well plate and add 2.0 ml of cell disruption from human breast tumors (MDA-MB-231, BT-549, MCF-7, T-47D and MCF-10A). And mixed gently. Then, the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(C) Reporter Gene Assay 72 hours after the lipofection of Example 3 (b) above, the culture was removed from the 48-well plate and washed with PBS. Then, 150 μl of a cell lysate (Glo-Lysis Buffer) (Promega) was added, and the mixture was allowed to stand at −80 ° C. for 30 minutes or more to freeze. At the time of use, the cell lysate was thawed at room temperature, the lysate was collected in a centrifuge tube, and the cell residue was precipitated by centrifugation. The supernatant was collected in a new sample tube and 25 μl of the supernatant was dispensed into a 96-well plate (Nunc). Add an equal volume of Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) or NanoLuc substrate solution (Nano-Glo Luciferase Assay System) (Promega) to the wells, and then add L3Mino (Promega) to the wells. Was measured. As a result, the activity of pNampt-L and the activity of the reporter DNA for correcting the introduction efficiency: pNLuc-tk were measured. The relative luminescence intensity of pNampt-L was obtained by dividing the activity value (luminescence intensity: RLU) of the reporter DNA by the activity value (luminescence intensity) of pNLuc-tk. The results are shown in FIG. The highly metastatic cell line BT-549 exhibited stronger luminescence intensity (RLU) than the low metastatic cell lines MCF-7 and T47-D, as well as the benign tumor cell line MCF-10A. That is, it was revealed that the highly metastatic cell line has high NAMPT promoter activity. This showed that it is possible to evaluate the metastatic property of a tumor cell, which is an example of a test cell, using the NAMEPT promoter activity as an index, which is an example of cell characteristics.

(2) Lung cancer cell line (a) Reporter vector The following experiment was carried out using pNampt-L described in Examples 3 (1) and (a) above.

(B) Lipofection (introduction of reporter DNA into cells)
PNampt-L was introduced into cells A-549 and LUDLU-1, which are two types of cell lines derived from human lung cancer, by the same lipofection method as in Examples 3 (1) and (b) above.

(C) Reporter Gene Assay A cell lysate was prepared in the same manner as in Examples 3 (1) and (c) above. Then, the cell residue is precipitated by centrifugation, and 25 μl of the supernatant is dispensed into a 96-well plate (Nunc) to provide an equal volume of Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) or NanoLuc substrate solution (Nano-). Glo Luciferase Assay System (Promega) was added, and the emission intensity was measured with a luminometer (Mithras LB940, Bermuda). The relative emission intensity of pNampt-L was defined as the activity value (emission intensity) of the reporter DNA divided by the activity value (emission intensity) of pNLuc-tk. The results are shown in FIG. Both the basal epithelial lung cancer cell line A-549 and the squamous cell lung cancer cell line LUCLU-1 showed luminescence. That is, NAMPT promoter activity was detected in both cell lines. In addition, it was revealed that the low metastatic cell line A-549 had low NAMPT promoter activity.

Example 4. Detection of metastatic breast cancer cell line by cell luminescence imaging (1) Reporter vector The reporter vector prepared in Example 3 (1) above: pNampt-L was used in the experiment.

(2) Lipofection (introduction of reporter DNA into cells)
To 500 μl of Opti-MEM (Life Technologies), 2.5 μg of reporter vector: pNampt-L and 2.5 μg of enhancer reagent (Plus reagent, Life Technologies) were added. This was incubated at room temperature for 5 minutes. To this solution, 6.25 μl of Lipofectamine LTX (Life Technologies) was added and the mixture was well turbid. It was then incubated at room temperature for 25 minutes. The solution was transferred to a 35 mm culture dish to obtain BT-549, a highly metastatic malignant tumor cell line derived from human mammary tumor, MCF-7, a low metastatic malignant tumor cell line, and a benign tumor cell line derived from human mammary tumor. 2.0 ml of a suspension of MCF-10A was added and mixed gently. Then, the cells were cultured in an incubator at 37 ° C. and a 5% CO ₂ atmosphere.

(3) Luminescence imaging The luminescence imaging of the reporter vector-introduced cell line was performed by a LUMINOVIEW luminescence imaging system (LV200, Olympus). Twenty-four hours after lipofection, the luminescent substrate VivoGlo luciferin (Promega) was added to the medium of BT-549, MCF-7, and MCF-10A into which pNampt-L had been introduced (final concentration 200 μM). The culture dish was set at a predetermined position on the LUMINOVIEW, and microscopic images (× 20 times) of the cells were taken in a bright field and a dark field (emission). The shooting in the dark field was performed with an exposure time (light emission detection time) of 20 minutes. The result of luminescence imaging is shown in FIG. FIG. 12 shows a superposed image of a bright-field image and a dark-field image (emission image). The superimposition of the images was performed by the image processing software METAMORPH. In the highly metastatic malignant tumor cells BT-549, strongly luminescent cells were observed (FIG. 12 (a), BT-549). Luminescent cells were observed in the low metastatic tumor cells MCF-7 (FIG. 12 (b), MCF-7), but the luminescence was weak. In addition, no luminescent cells were observed in the benign tumor cells MCF-10A (FIG. 12 (c), MCF-10A).

According to these embodiments or examples, it is possible to provide a new technique for evaluating cell characteristics.

Although some embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are also included in the scope of the invention described in the claims and the equivalent scope thereof.
The inventions described in the claims of the original application of the present application are described below.
[1]
A gene marker for evaluating the cell characteristics of a test cell, wherein the gene marker is a mRNA, peptide or protein of the NAMPT gene, or a polynucleotide containing a NAMPT gene promoter sequence.
[2]
The genetic marker according to [1], wherein the test cell is an epithelial tumor.
[3]
The genetic marker according to [1], wherein the test cell is a tumor cell derived from mammary gland tissue or lung tissue.
[4]
A method for evaluating the cell characteristics of a test cell, which comprises using the expression level of the NAMPT gene in the test cell as an index.
[5]
The method according to [4], wherein the test cell is an epithelial tumor.
[6]
The method according to [4], wherein the test cell is a tumor cell derived from mammary gland tissue or lung tissue.
[7]
Using the expression level of the NAMPT gene as an index
To measure the expression level of the NAMPT gene in the test cells,
Comparing the measured expression level of the test cells with the threshold value,
Based on the results of the comparison, the degree of expression of the NAMPT gene in the test cells is determined, and
To evaluate the cell characteristics of the test cells based on the determination.
The method according to any one of [4] to [6], which comprises.
[8]
The method according to [7], wherein the threshold value is determined based on the expression level of the NAMPT gene from healthy cells.
[9]
The expression level of the NAMPT gene from the healthy cells is
It is obtained by measuring the expression level of the NAPPT gene in the healthy cells prior to the measurement of the expression level of the test cells.
Obtained by measuring the expression level of the NAPPT gene in the healthy cell after measuring the expression level of the test cell, or
The method according to [8], which is obtained by measuring the expression level of the NAMPT gene in the healthy cell in parallel with the measurement of the expression level of the test cell.
[10]
The method according to any one of [7] to [9], wherein the expression level of the NAMPT gene is measured by measuring the NAMPT protein mass, the NAMPT mRNA level, or the NAMPT gene promoter activity level. ..
[11]
The method according to [10], wherein the measurement of the NAMPT protein mass is performed by an immunological measurement method using an anti-NAMPT antibody.
[12]
That the immunoassay is an enzyme immunoassay (EIA), a fluorescence immunotherapy (FIA), an automatic cell analysis / separation device (FACS), an immunocell staining method (ICC) or an immunohistochemical staining method (IHC). The method according to [11], which is a feature.
[13]
The method according to [10], wherein the measurement of the amount of NAMPT mRNA is performed by a reverse transcription PCR method.
[14]
In the reverse transcription PCR method, an amplification reaction is carried out using a primer set containing the first primer represented by SEQ ID NO: 2 or its complementary sequence and the second primer represented by SEQ ID NO: 3 or its complementary sequence. The method according to [13].
[15]
The method according to [10], wherein the measurement of the NAMPT gene promoter activity amount is performed using a reporter vector containing the NAMPT gene promoter sequence.
[16]
The reporter vector contains a reporter gene expression unit.
The reporter gene expression unit
(A-a) 5'upstream promoter sequence of NAPPT gene;
(Ab) A reporter gene encoding a detectable reporter protein operably linked downstream of the promoter sequence; and
(Ac) A transcription termination signal sequence functionally linked downstream of the reporter gene;
The method according to [15], which comprises.
[17]
The reporter vector further comprises a replication initiation unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit is
(B) A replication initiation sequence that initiates self-replication of the reporter vector by binding of the replication initiation protein;
The measurement of the expression level of the NAMPT gene, including
(1) To prepare the reporter vector;
(2) Introducing the reporter vector into the test cell:
(3) Culturing the test cells under the condition that the replication initiation protein is expressed:
(4) To detect the reporter protein expressed in the test cell:
The method according to [16].
[18]
A reporter vector for evaluating the cell characteristics of a test cell, wherein the reporter vector contains a reporter gene expression unit, and the reporter gene expression unit is
(A-a) 5'upstream promoter sequence of NAPPT gene;
(Ab) A reporter gene encoding a detectable reporter protein operably linked downstream of the promoter sequence; and
(Ac) A transcription termination signal sequence functionally linked downstream of the reporter gene;
A reporter vector comprising.
[19]
The reporter vector further contains a replication initiation unit and a replication initiation protein unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit is
(B) A replication initiation sequence that initiates self-replication of the reporter vector by binding of the replication initiation protein;
The replication-initiating protein unit comprises
(CA) IRES sequence operably linked downstream of the reporter gene; and
(C-b) A replication initiation protein gene encoding a replication initiation protein operably linked downstream of the IRES sequence;
Including
The reporter vector according to [18], wherein the transcription termination signal sequence (A-c) is functionally linked downstream of the replication-initiating protein gene (C-b).
[20]
The reporter vector according to [18], wherein the IRES sequence is an IRES sequence of a cerebral myocarditis virus.
[21]
The reporter vector further contains a replication initiation unit and a replication initiation protein unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit is
(B) A replication initiation sequence that initiates self-replication of the reporter vector by binding of the replication initiation protein;
The replication-initiating protein unit comprises
(Da) Constitutively expressed constitutive promoter;
(Db) The replication initiation protein gene encoding the replication initiation protein operably linked downstream of the constitutive promoter; and
(D-c) A further transcription termination signal sequence operably linked downstream of the replication initiation protein gene;
The reporter vector according to [18], which comprises.
[22]
Any one of [19] or [21], wherein the replication initiation protein gene is a large T antigen gene of Simian virus 40, and the replication initiation sequence is a replication initiation sequence of Simian virus 40. Reporter vector described in.
[23]
The reporter vector according to any one of [18] to [22], wherein the 5'upstream promoter sequence of the NAMPT gene contains the sequence represented by SEQ ID NO: 1.
[24]
The reporter vector according to any one of [18] to [23], wherein the reporter gene is a luminescent gene, a fluorescent gene, a coloring gene, a heavy metal binding gene, or a nitrogen monoxide synthesis gene.
[25]
A set of primers for evaluating the cell characteristics of a test cell, which comprises a first primer represented by SEQ ID NO: 2 or its complementary sequence and a second primer represented by SEQ ID NO: 3 or its complementary sequence. Primer set.
[26]
An assay kit for evaluating the cell characteristics of test cells.
(1) The reporter vector and buffer according to any one of [18] to [24]; or
(2) The primer set and buffer solution according to [25];
An assay kit comprising.
[27]
(1) A gene transfer section for introducing a reporter vector containing a NAMEPT gene promoter sequence into the nucleus of a test cell, and
(2) A culture unit for culturing the test cells treated by the gene transfer unit, and a culture unit.
(3) A bright-field image of the test cells cultured in the culture unit is imaged, and further, a luminescence image consisting of luminescence signals generated for each cell due to the expression of the reporter gene is imaged in the same field of view. Imaging unit and
(4) A determination unit for determining the cell characteristics of each cell of the test cell based on the bright field image and the luminescent image.
A cell characterization device comprising.

Claims (11)

A method for determining the metastatic basal epithelial gland lung cancer cell or squamous lung cancer cells the level of expression of NAMPT gene in the basal epithelial gland lung cancer cell or squamous lung cancer cells as an index,
Using the expression level of the NAMPT gene as an index,
To measure the expression level of the NAMEPT gene in the basal epithelial gland lung cancer cells or squamous cell lung cancer cells.
Comparing the measured expression levels of the basal epithelial gland lung cancer cells or squamous epithelial lung cancer cells with the threshold, and based on the result of the comparison, in the basal epithelial gland lung cancer cells or squamous lung cancer cells than the threshold . When the expression level of the NAMEPT gene is high, it is determined that the basal epithelial aden lung cancer cells or the squamous epithelial lung cancer cells are highly metastatic.
When the expression level of the NAMEPT gene in the basal epithelial gland lung cancer cell or the squamous epithelial lung cancer cell is lower than the threshold value, it is determined that the metastatic property of the basal epithelial gland lung cancer cell or the flat epithelial lung cancer cell is low. A method characterized by including. The method according to claim 1, wherein the threshold value is determined based on the expression level of the NAPPT gene from healthy cells. The expression level of the NAMPT gene from the healthy cells is
It is obtained by measuring the expression level of the NAPPT gene in the healthy cells prior to the measurement of the expression level.
Obtained by measuring the expression level of the NAPPT gene in the healthy cells after measuring the expression level of the basal epithelial gland lung cancer cell or the squamous epithelial lung cancer cell, or the expression of the basal epithelial gland lung cancer cell or the squamous epithelial lung cancer cell. The method according to claim 2, wherein the method is obtained by measuring the expression level of the NAMPT gene in the healthy cell in parallel with the measurement of the amount. The method according to any one of claims 1 to 3, wherein the expression level of the NAMPT gene is measured by measuring the NAMPT protein mass, the NAMPT mRNA level, or the NAMPT gene promoter activity level. The method according to claim 4, wherein the NAMEPT protein mass is measured by an immunological measurement method using an anti-NAPPT antibody. That the immunoassay is an enzyme immunoassay (EIA), a fluorescence immunotherapy (FIA), an automatic cell analysis / separation device (FACS), an immunocell staining method (ICC) or an immunohistochemical staining method (IHC). The method according to claim 5, which is characterized. The method according to claim 4, wherein the measurement of the amount of NAMPT mRNA is performed by a reverse transcription PCR method. In the reverse transcription PCR method, an amplification reaction is carried out using a primer set containing the first primer represented by SEQ ID NO: 2 or its complementary sequence and the second primer represented by SEQ ID NO: 3 or its complementary sequence. 7. The method according to claim 7. The method according to claim 4, wherein the NAPPT gene promoter activity amount is measured using a reporter vector containing the NAPPT gene promoter sequence. The reporter vector contains a reporter gene expression unit.
The reporter gene expression unit
(A-a) 5'upstream promoter sequence of NAPPT gene;
(A-b) A reporter gene encoding a detectable reporter protein operably linked downstream of the promoter sequence; and (A-c) a transcription termination signal sequence operably linked downstream of the reporter gene. ;
9. The method according to claim 9, wherein the method comprises. The reporter vector further comprises a replication initiation unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit is
(B) A replication initiation sequence that initiates self-replication of the reporter vector by binding of the replication initiation protein;
The measurement of the expression level of the NAMPT gene, including
(1) To prepare the reporter vector;
(2) Introducing the reporter vector into the basal epithelial gland lung cancer cells or squamous cell lung cancer cells:
(3) Culturing the basal epithelial gland lung cancer cells or squamous cell lung cancer cells under the condition that the replication initiation protein is expressed:
(4) To detect the reporter protein expressed in the basal epithelial gland lung cancer cells or squamous cell lung cancer cells:
10. The method of claim 10.

Images