JP2004344069A - Method for examining precancerous lesion of rat liver negative to antibody recognizing placenta-type glutathione-s-transferase enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examining cancer for maximally accurately and easily assessing the carcinogenicity of chemical substances. <P>SOLUTION: The method for examining a precancerous lesion of rat liver negative to an antibody recognizing a placenta-type glutathione-S-transferase enzyme(GSTP) comprises the 1st step(1) of assaying in a specimen the expression level of a gene having a base sequence registered in the Genbank with either one of Accession numbers given in the Accession number group 1:AA799336, and the like or one more genes selected from the ortholog thereof and the 2nd step(2) of comparing the result of the expression level obtained above with the reference expression level of the gene, and based on the difference, assessing the presence/absence of the lesion in the specimen or the degree of the onset thereof. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for assaying a rat liver precancerous lesion that is negative for an antibody that recognizes a placental glutathione-S-transferase enzyme.
[0002]
[Prior art]
Although it is becoming increasingly clear that cancer is a disease caused by abnormalities involving genes, the arrest rate and mortality rate for cancer are still high. In such a situation, a safety test for confirming in advance whether or not various chemical substances are carcinogenic is extremely important. Conventionally, as a typical method for assaying the carcinogenicity of a chemical substance, a method of evaluating the presence or absence of carcinogenicity by using a tumor marker as an index in a safety test using various vertebrates (for example, rats) is known. Are known.
[0003]
[Problems to be solved by the invention]
However, when rats are used as experimental animals, for example, in the case of placental glutathione-S-transferase, which is one of the most frequently used rat liver tumor markers, peroxisomes that increase the number of peroxisomes in hepatocytes There are drawbacks such as the inability to detect preneoplastic lesions induced by the inducer, and in some cases it has not always been fully satisfactory.
Therefore, development of a cancer assay method in rats for evaluating the carcinogenicity of a chemical substance as accurately and simply as possible has been desired.
[0004]
[Means for Solving the Problems]
Under such circumstances, the present inventors have conducted intensive studies and as a result, in hepatocytes constituting a rat liver precancerous lesion tissue that is negative for an antibody that recognizes a placental glutathione-S-transferase enzyme, The present inventors have found that the expression level is higher than the expression level of the gene in hepatocytes in the adjacent normal tissue, and have led to the present invention.
That is, the present invention relates to a method for assaying a rat liver precancerous lesion that is negative for an antibody that recognizes a placental glutathione-S-transferase enzyme (hereinafter, may be simply referred to as a rat liver precancerous lesion). hand,
(1) A first method for measuring the expression level of a gene having a nucleotide sequence registered in Genbank with one of the registration numbers shown in the following registration number group 1 or one or more genes selected from orthologs thereof in a specimen: Process, and
(2) The measured value of the expression level of the gene in the sample obtained in the first step is compared with a control value of the expression level of the gene, and the placental glutathione-S-transferase enzyme in the sample is determined based on the difference. Second step of evaluating the presence or extent of rat liver precancerous lesions that are negative for antibodies that recognize
(Hereinafter sometimes referred to as the method of the present invention).
<Registration number group 1>
AA799336, AA799481, AA799488, AA799523, AA799538, AA799539, AA799641, AA799656, AA799680, AA799711, AA799735, AA799803, AA799889, AA799891, AA799893, AA799899, AA799991, AA799995, AA800034, AA800044, AA800570, AA800593, AA800637, AA800671, AA800673, AA800768, AA800787, AA800814, AA800930, AA817854, AA817798, AA818122, AA818152, AA819500, AA819643, AA8485 5, AA852004, AA858573, AA858626, AA858640, AA859661, AA859832, AA866240, AA866302, AA874919, AA874926, AA875097, AA875471, AA891877, AA891998, AA892006, AA892012, AA892036, AA892248, AA892297, AA892394, AA892399, AA892557, AA892593, AA892616, AA892637, AA892779, AA892797, AA892799, AA892828, AA892854, AA892860, AA8929297, AA893307, AA893325, AA893338, AA89 3493, AA893664, AA893702, AA893982, AA894030, AA894193, AA894282, AA899320, AA925473, AA926149, AA926193, AA945050, AA945573, AA963449, AB000507, AF016296, AF029240, AI010371, AI010725, AI012051, AI012275, AI014135, AI069990, AI103874, AI112516, AI169372, AI169695, AI169758, AI170379, AI172293, AI176308, AI176488, AI177161, AI179445, AI180442, AI229291, A 230406, AI232321, AI235707, AI233007, AI638985, AI639102, AI6339315, AI639371, AJ001641, D13912, D14564, D14987, D14988, D14989, D38938, 28,693, 1988, M1493 M33329, M37828, M64092, M64780, M96601, S59892, U11071, U15098, U18314, U19485, U42627, U44948, U46118, U49099, U50927, U75928, U89905, X123 3410, X67859, X78855;
2. The specimen is a biological sample which may contain hepatocytes constituting rat liver precancerous lesion tissue negative for antibodies recognizing placental glutathione-S-transferase enzyme or contents thereof. The method according to the above 1;
3. 3. The method according to the above 1 or 2, wherein the measurement of the expression level of the gene is performed by measuring the amount of the transcript or the translation product of the gene;
4. The method according to the above 1, 2, or 3, wherein the control value of the gene expression level is a value of the gene expression level in a normal tissue or a normal cell;
5. In the second step, the measured value of the expression level of a gene having a nucleotide sequence registered in Genbank under any of the registration numbers shown in the registration number group 1 or its ortholog is higher than a control value as an index, and The method according to claim 1, wherein the presence or absence of a rat liver precancerous lesion that is negative for an antibody recognizing a placental glutathione-S-transferase enzyme in the sample is evaluated based on an index. , 2, 3 or 4;
6. A method for assaying rat liver carcinogenic activity of a substance, comprising:
(1) a first step of contacting rat hepatocytes with a test substance,
(2) In the hepatocytes contacted with the test substance in the first step, selected from genes having a base sequence registered in Genbank under any of the accession numbers shown in accession number group 1 below, or orthologs thereof. A second step of measuring the expression level of one or more genes to be obtained, and
(3) comparing the measured value of the expression level of the gene obtained in the second step with a control value of the expression level of the gene, and evaluating the presence or absence or the amount of the test substance in rat liver carcinogenesis based on the difference. Third step
(Hereinafter sometimes referred to as the method for assaying hepatocarcinogenesis activity of the present invention).
<Registration number group 1>
AA799336, AA799481, AA799488, AA799523, AA799538, AA799539, AA799641, AA799656, AA799680, AA799711, AA799735, AA799803, AA799889, AA799891, AA799893, AA799899, AA799991, AA799995, AA800034, AA800044, AA800570, AA800593, AA800637, AA800671, AA800673, AA800768, AA800787, AA800814, AA800930, AA817854, AA817798, AA818122, AA818152, AA819500, AA819643, AA8485 5, AA852004, AA858573, AA858626, AA858640, AA859661, AA859832, AA866240, AA866302, AA874919, AA874926, AA875097, AA875471, AA891877, AA891998, AA892006, AA892012, AA892036, AA892248, AA892297, AA892394, AA892399, AA892557, AA892593, AA892616, AA892637, AA892779, AA892797, AA892799, AA892828, AA892854, AA892860, AA8929297, AA893307, AA893325, AA893338, AA89 3493, AA893664, AA893702, AA893982, AA894030, AA894193, AA894282, AA899320, AA925473, AA926149, AA926193, AA945050, AA945573, AA963449, AB000507, AF016296, AF029240, AI010371, AI010725, AI012051, AI012275, AI014135, AI069990, AI103874, AI112516, AI169372, AI169695, AI169758, AI170379, AI172293, AI176308, AI176488, AI177161, AI179445, AI180442, AI229291, A 230406, AI232321, AI235707, AI233007, AI638985, AI639102, AI6339315, AI639371, AJ001641, D13912, D14564, D14987, D14988, D14989, D38938, 28,693, 1988, M1493 M33329, M37828, M64092, M64780, M96601, S59892, U11071, U15098, U18314, U19485, U42627, U44948, U46118, U49099, U50927, U75928, U89905, X123 3410, X67859, X78855
7. 7. The method according to the above item 6, wherein the measurement of the expression level of the gene is performed by measuring the transcript amount of the gene;
8. The method according to the above item 6 or 7, wherein the control value of the gene expression level is a value of the expression level of the gene in normal tissues or cells.
9. In the third step, the expression level of a gene having a nucleotide sequence registered in Genbank under any of the accession numbers shown in accession number group 1 or an ortholog thereof is used as an index, wherein the measured value is higher than a control value, 9. The method according to the above item 8, wherein the presence or absence of rat liver carcinogenesis activity of the test substance is evaluated.
10. Searching for a hepatocarcinogen characterized by comprising a step of selecting a substance having a rat hepatocarcinogenic activity based on the presence or absence of the rat hepatocarcinogenic activity evaluated by the method described in any one of the above items 6 to 9 Method;
And so on.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the sample is, for example, a biological sample that may contain hepatocytes constituting rat liver precancerous lesions that show a negative for an antibody recognizing placental glutathione-S-transferase enzyme or the contents thereof. Specific examples include tissues such as liver tissue collected from rats, cells isolated from these tissues, and cultured cells thereof. These samples may be used as samples as they are, or samples prepared by various operations such as separation, fractionation, and immobilization from such samples may be used as samples.
The gene whose expression level is measured in the present invention is a gene having a nucleotide sequence registered in Genbank under any of the accession numbers shown in accession number group 1 or an ortholog thereof (hereinafter collectively referred to as the present gene and At least one gene). The information on the nucleotide sequence registered in Genbank is given to each gene by Genbank from, for example, the WEB page (URL; http://www.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information. It can be obtained by performing a search based on the above registration number (Accession No.). By the way, all the data published by the data banks of GenBnak, DDBL and EMBL can be used by anyone without any restriction, and the data registered in INSD is permanently stored and published as scientific data. "Handling of registered data of DDBL / EMBL / GenBnak" (May 23-24, 2002) prepared by the International Advisory Committee, which is an advisory body of the International Nucleotide Sequence Databases (INSD) composed of banks. Date), and any person skilled in the art can collate, search, obtain, etc. data based on the registration number (Accession No.).
The present gene used in the present invention includes, in addition to a gene having the exact same nucleotide sequence as the above-mentioned known nucleotide sequence, a mutation that naturally occurs in the nucleotide sequence of the above-described gene due to a difference between individual differences of an organism or the like. And a gene having a base sequence in which a base is deleted, substituted or added by the above.
[0006]
The expression level of the present gene can be measured, for example, by a method of measuring the amount of a transcript of the present gene per unit amount of a sample, a method of measuring the amount of a translation product of the present gene per unit amount of a sample, or the like.
In order to measure the amount of the transcript of the present gene, for example, the amount of mRNA that is the transcript of the gene is measured. Specifically, the amount of mRNA of a specific gene is measured, for example, by a quantitative real-time polymerase chain reaction (hereinafter, referred to as quantitative RT-PCR), a Northern hybridization method [J. Sambrook, E .; F. Frisch, T .; Maniatis; Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory, 1989], DNA array method, in situ hybridization method, and the like.
In order to measure the amount of the translation product of the present gene, for example, the amount of a protein having an amino acid sequence encoded by the nucleotide sequence of the present gene is measured. Specifically, the amount of a specific protein is measured by, for example, an immunological assay using a specific antibody against the protein (eg, ELISA, Western blot, RIA, immunohistochemical test, etc.), two-dimensional electrophoresis, or the like. It can be performed by an electrophoresis method, high performance liquid chromatography, or the like. A specific antibody against the protein having the amino acid sequence encoded by the nucleotide sequence of the present gene can be prepared by using a protein having the amino acid sequence encoded by the nucleotide sequence of the gene as an immunizing antigen according to a conventional method.
[0007]
The method for measuring the amount of the transcript of the present gene will be further described.
The amount of mRNA which is a transcript of the present gene can be determined, for example, by using a probe or primer designed and prepared based on the nucleotide sequence of the present gene, using a conventional genetic engineering method such as Northern hybridization, It can be measured by using RT-PCR, DNA array method, in situ hybridization method, or the like. Specifically, for example, J.I. Sambrook, E .; F. Frisch, T .; Maniatis; Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory, 1989, and the like. At this time, a gene whose expression level in the tissue is known to be constantly constant (hereinafter referred to as a control gene), for example, a β-actin gene (Nucl. Acids. Res., Vol. 12, No. .3, p.1687, 1984) and 36B4 (Acidic Ribosomal Phosphoprotein) (Nucl. Acids. Res., Vol. 19, No. 14, p. 3998, 1991). . Then, the expression level of the present gene may be determined by calculating the amount of mRNA of the present gene per the amount of mRNA of the control gene or the index value thereof or the index value thereof.
[0008]
(1. Northern hybridization method)
First, a DNA of a gene whose mRNA amount is to be measured is prepared, and then a DNA consisting of the whole or a part thereof is labeled to prepare a probe.
The above gene can be prepared by PCR using commercially available cDNA (for example, obtained from Takara Shuzo) or cDNA prepared by the method shown below as a template. For example, first, total RNA is extracted from a tissue expressing the gene by an ordinary method such as the guanidine hydrochloride / phenol method, the SDS-phenol method, and the guanidine thiocyanate / CsCl method. For example, total RNA may be extracted using a commercially available kit such as ISOGEN (manufactured by Nippon Gene).
From the extracted total RNA, for example, mRNA is prepared as follows. First, a polyA column having oligo dT as a ligand was loaded with a loading buffer [20 mM Tris-HCl buffer (pH 7.6), 0.5 M NaCl, 1 mM EDTA, 0.1% (w / v) having a column volume of 5 times or more. ) SDS], and then apply the total RNA prepared by the method described above to a column, and wash with 10 column volumes of a loading buffer. Further, the column is washed with 5 column volumes of a washing buffer [20 mM Tris-HCl buffer (pH 7.6), 0.1 M NaCl, 1 mM EDTA, 0.1% (w / v) SDS]. Subsequently, the mRNA is obtained by eluting the mRNA with three column volumes of an elution buffer [10 mM Tris-HCl buffer (pH 7.6), 1 mM EDTA, 0.05% (w / v) SDS).
Next, the oligo dT primer is annealed to the poly A chain of the total RNA or mRNA, and a single-stranded cDNA is synthesized, for example, according to the protocol of a cDNA synthesis kit (Takara Shuzo). At this time, the RNA used as a template may be either total RNA or mRNA, but it is more preferable to use mRNA.
DNA is amplified by PCR using the single-stranded cDNA as a template and a DNA polymerase such as TaKaRa taq (Takara Shuzo). PCR conditions vary depending on the type of animal to be measured, the sequence of primers used, and the like. For example, a reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ], In the presence of 2.5 mM NTP, conditions in which the temperature is maintained at 94 ° C. for 30 seconds, then at 40 ° C. to 60 ° C. for 2 minutes, and further at 72 ° C. for 2 minutes as one cycle, and this is performed for 30 to 55 cycles. it can.
[0009]
The DNA of this gene amplified in this manner may be inserted into a vector such as pUC118 and cloned. The nucleotide sequence of the DNA can be determined by the Maxam Gilbert method (for example, described in Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci., 74, 560, 1977) or the Sanger method (for example, Sanger, F.S. & AR Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and AR Coulson., Proc. Natl. Acad. Sci., 74, 5463, 1977. Can be confirmed.
[0010]
A probe can be prepared by labeling all or a part of the DNA of the present gene thus prepared with a radioisotope, a fluorescent dye or the like as follows. For example, using the DNA prepared as described above as a template, and using an oligonucleotide having a partial sequence of the base sequence of the DNA as a primer, [α- ³² P] dCTP or [α- ³² P] dNTP containing dATP is added to the reaction solution to perform PCR. ³² A probe labeled with P is obtained. Alternatively, the DNA prepared as described above may be labeled using a commercially available labeling kit such as Random Prime Labeling Kit (Boehringer Mannheim) or MEGALABEL (Takara Shuzo).
[0011]
Next, Northern hybridization analysis is performed using the above probe. Specifically, total RNA or mRNA is prepared from a tissue or cell in which the expression level of the present gene is to be measured. 20 μg of the prepared total RNA or 2 μg of mRNA is separated on an agarose gel, washed with 10 × SSC (1.5 M NaCl, 0.35 M sodium citrate), and then subjected to a nylon membrane [for example, Hybond-N (manufactured by Amersham), etc. ]. The membrane is placed in a polyethylene bag, and the hybridization buffer [6 × SSC (0.9 M NaCl, 0.21 M sodium citrate), 5 × Denhardt's solution [0.1% (w / v) Ficoll 400, 0.1% ( w / v) polyvinylpyrrolidone, 0.1% BSA], 0.1% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, 50% formamide], and incubated at 50 ° C. for 2 hours. Discard the hybridization buffer and add a fresh 2 to 6 ml of hybridization buffer. Further, the probe obtained by the above method is added, and the mixture is incubated at 50 ° C. overnight. In addition to the above, commercially available DIG EASY Hyb (Boehringer Mannheim) can be used as the hybridization buffer. The membrane is removed, incubated in 50-100 ml of 2 × SSC, 0.1% SDS at room temperature for 15 minutes, and the same operation is repeated once, and finally, 50-100 ml of 0.1 × SSC, 0. Incubate at 68 ° C. for 30 minutes in 1% SDS. By measuring the amount of label on the membrane, the amount of mRNA that is a transcription product of the present gene can be measured.
[0012]
(2. Quantitative RT-PCR)
MRNA is prepared from a tissue or cell whose expression level is to be measured by the same method as described in the above (1 Northern hybridization method). A reverse transcriptase such as MMLV (Toyobo) is added to the prepared mRNA, and a reaction buffer [50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl 2 ₂ , 75 mM KCl, 10 mM DTT] in the presence of 0.5 mM dNTP and 25 μg / ml oligo dT at 42 ° C. for 15 minutes to 1 hour to prepare a corresponding cDNA. The corresponding cDNA may be prepared using a cDNA synthesis kit (Takara Shuzo). PCR is performed using the prepared cDNA as a template and DNA having a part of the nucleotide sequence of the present gene as a primer. Examples of the primer include a primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2 as a primer having a partial nucleotide sequence of the present gene having the nucleotide sequence registered in Genbank under accession number X58294. The PCR conditions include, for example, TAKARA taq (Takara Shuzo) and a reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ], 2.5 mM dNTP and [α ³² In the presence of [P] -dCTP, for example, there may be mentioned a condition in which the temperature is maintained at 94 ° C. for 30 seconds, then at 40 ° C. to 60 ° C. for 2 minutes, and further at 72 ° C. for 2 minutes as one cycle, and the cycle is performed for 30 to 55 cycles. By subjecting the amplified DNA to polyacrylamide gel electrophoresis and measuring the radioactivity of the separated DNA, the amount of mRNA of the present gene can be measured. Alternatively, for example, using TAKARA taq (Takara Shuzo), a reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ), 50 μl of a reaction solution containing 25 μl of SYBR Green PCR Reagents PCR (ABI) was prepared, and the mixture was incubated at 50 ° C. for 2 minutes, then at 95 ° C. for 10 minutes, and then at 95 ° C. The PCR is carried out under the conditions that the heat retention at 60 ° C. for 1 minute is performed as one cycle for 15 seconds and the cycle is performed for 40 cycles. The amount of mRNA of the present gene can be measured by measuring the fluorescence of the amplified DNA.
[0013]
(3. DNA array analysis)
For the measurement of the amount of the transcript of the present invention, a macroarray prepared by spotting the cDNA of the present gene on a membrane filter or the like such as a nylon membrane, a microarray prepared by spotting the cDNA of the present gene on a slide glass or the like, a slide A DNA array based on a known technique such as a probe array prepared by immobilizing an oligonucleotide having a partial sequence of the base sequence of the present gene (usually a chain length of 18 to 25 mer) on glass using a photochemical reaction is used. Can be used. These arrays can be prepared, for example, according to the method described in Genome Function Research Protocol, Experimental Medical Supplement, published by Yodosha. Genechip, which is commercially available from Affymetrix, etc., can also be used.
[0014]
Hereinafter, an example of a method for measuring the transcript amount of the present gene using a DNA array will be described. (3-1. Quantification by macro array)
MRNA is prepared from a tissue or cell whose expression level is to be measured by the same method as described in the above (1 Northern hybridization method). A reverse transcriptase such as MMLV (Toyobo) is added to the prepared mRNA, and a reaction buffer [eg, 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl 2] ₂ , 75 mM KCl, and 10 mM DTT] in 0.5 mM dNTP, [α ³² Reaction is performed at 42 ° C. for 15 minutes to 1 hour in the presence of P] -dCTP and 25 μg / ml oligo dT to prepare a labeled DNA, which is used as a probe. At this time, a cDNA synthesis kit (Takara Shuzo) or the like may be used. The macroarray prepared by spotting the cDNA of the present gene on a membrane filter was placed in a polyethylene bag, and a hybridization buffer [6 × SSC (0.9 M NaCl, 0.21 M sodium citrate), 5 × Denhardt solution [0. 1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA], 0.1% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, 50% Formamide], and incubated at 50 ° C. for 2 hours. Then, the hybridization buffer is removed, and 2 to 6 ml of a new hybridization buffer is added. Further, the above probe is added and incubated at 50 ° C. overnight. In addition to the above, commercially available DIG EASY Hyb (Boehringer Mannheim) can be used as the hybridization buffer. After taking out the macroarray, immersing it in 50 ml to 100 ml of 2 × SSC and 0.1% SDS and incubating at room temperature for about 15 minutes, the same operation is repeated once, and finally, 50 ml to 100 ml of 0.1 × SDS. Incubate at 68 ° C. for 30 minutes in SSC, 0.1% SDS. By measuring the amount of label on the macroarray, the amount of mRNA that is a transcript of the present gene, that is, the expression amount of the present gene can be measured.
[0015]
(3-2. Quantification by microarray)
MRNA is prepared from a tissue or cell whose expression level is to be measured by the same method as described in the above (1 Northern hybridization method). To the prepared mRNA, a reverse transcriptase such as MMLV (Toyobo Co., Ltd.) is added, and a reaction buffer [for example, 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl 2] ₂ , 75 mM KCl, and 10 mM DTT] in the presence of 0.5 mM dNTP, Cy3-dUTP, (or Cy5-dUTP) and 25 μg / ml oligo dT at 42 ° C. for 15 minutes to 1 hour. After adding an alkaline buffer (for example, a solution containing 1N NaOH and 20 mM EDTA), and keeping the mixture at 65 ° C. for 10 minutes, a fluorescently labeled DNA is prepared by removing free Cy3 or Cy5 using MicroconYM-30 or the like. Probe. Using the obtained probe, hybridization is performed on the microarray in the same manner as described in (3-1 Quantification by DNA macroarray). By measuring the signal amount on the array with a scanner, the amount of mRNA that is a transcript of the present gene, that is, the expression amount of the present gene can be measured.
[0016]
(3-3. Quantification by probe array)
MRNA is prepared from a tissue or cell whose expression level is to be measured by the same method as described in the above (1 Northern hybridization method). A cDNA is prepared from the prepared mRNA using, for example, a cDNA synthesis kit (GENSET). The prepared cDNA is labeled with biotin using, for example, a biotin-labeled cRNA synthesis kit (In Vitro Transcription) (Enzo) or the like, and purified using a cRNA cleanup and quantification kit (In Vitro Transcription). The resulting biotin-labeled DNA is fragmented with a fragmentation buffer (200 mM Tris acetic acid (pH 8.1), 500 mM KOAc, 150 mM MgOAc). The internal standard substance Control Oligo B2 (manufactured by Amersham), 100 × Control cRNA Cocktail, Herring sperm DNA (manufactured by Promega), Acetylated BSA (manufactured by Gibco-BRL), 2 × MES Hybridization Na ⁺ ], 40 mM EDTA, 0.02% Tween20 (Pierce), pH 6.5 to 6.7] and DEPC-treated sterilized distilled water to prepare a hybrid cocktail.
After rotating a probe array [for example, Genechip (manufactured by Affymetrix) or the like] filled with 1 × MES hybridization buffer in a hybridization oven at 45 ° C., 60 rpm for 10 minutes, the 1 × MES hybridization buffer is removed. . Thereafter, 200 μl of the above-mentioned hybridization cocktail is added to the probe array, and the mixture is rotated in a hybridization oven at 45 ° C., 60 rpm for 16 hours (hybridization). Subsequently, the hybrid cocktail was removed, and Non-Stringent Wash Buffer [6 × SSPE [diluted 20 × SSPE (manufactured by Nacalai Tesque)], 0.01% Tween 20, and 0.005% Antifoam0-30 (Sigma) were added. After that, the probe array is attached to a predetermined position of GeneChip Fluidics Station 400 (manufactured by Affymetrix), and washed according to the protocol. Next, the probe array is stained according to the staining protocol EuKGE-WS2 of MicroArray Suite (Affymetrix). By measuring the fluorescence brightness at 570 nm using an HP GeneArray Scanner (manufactured by Affymetrix), the amount of mRNA that is a transcript of the present gene, that is, the expression level of the present gene can be measured.
[0017]
(4. In situ hybridization method)
Basically, it consists of 1) tissue fixation, embedding, and preparation of a section, 2) preparation of a probe, and 3) detection by hybridization. RNA or DNA previously labeled with a radioactive or non-radioactive substance is used as a probe. Other than that, see, for example, Heiles, H .; et al. , Biotechniques, 6, 978, 1988, Genetic Engineering Handbook, Yodosha 278 1991, Cell Engineering Handbook, Yodosha, 214, 1992, Cell Engineering Handbook, Yodosha, 222, 1992, etc. It can be carried out.
When preparing an RNA probe, first, for example, a DNA of the present gene is obtained in the same manner as in the above (1 Northern hybridization method), and the DNA is used as a vector having SP6, T7, and T3 RNA polymerase promoters. (For example, Bluescript of Stratagene, pGEM of Promega, etc.), and introduced into E. coli to prepare plasmid DNA. Next, the plasmid DNA is digested with restriction enzymes so that sense (for negative control) and antisense (for hybridization) RNA can be obtained. Using these DNAs as templates, in the case of radiolabeling, α- ³⁵ In the case of a non-radioactive label such as S-UTP, digoxigenin UTP or fluorescein-modified UTP or the like is used as a substrate to label while synthesizing RNA using SP6, T7, or T3 RNA polymerase, and is alkali-hydrolyzed to a size suitable for hybridization. By cleaving, an RNA previously labeled with a radioactive or non-radioactive substance is prepared. Examples of kits based on these methods include, for example, an RNA labeling kit (Amersham) for radioactive labeling, a DIG RNA labeling kit (Boehringer Mannheim) and an RNA color kit (Amersham) for non-radioactive labeling. Is commercially available.
When preparing a DNA probe, for example, ³² A radioactive nucleotide labeled with P or the like or a nucleotide labeled with biotin, digoxigenin or fluorescein is nick-translated (J. Mol. Biol., 113, 237, Molecular Cloning, A Laboratory Manual 2nd ed., 10, 6-10). , 12, Cold Spring Harbor Lab.) Or the random prime method (Anal. Biochem., 132, 6, Anal. Biochem., 137, 266) to incorporate DNA previously labeled with a radioactive or non-radioactive substance. Prepare. Examples of kits based on these methods include a nick translation kit (Amersham) and a Random Prime Labeling Kit (Boehringer Mannheim) for radioactive labeling, and a DIG DNA labeling kit (Boehringer Mannheim) for non-radioactive labeling. Co., Ltd.), DNA Color Kit (Amersham) and the like are commercially available.
Specifically, a tissue or cell whose expression level of the present gene is to be measured is fixed with paraformaldehyde or the like, embedded in paraffin or the like, and then a thin section is prepared and attached to a slide glass. Alternatively, after embedding the above-described tissue or cell in an OCT compound, the tissue or cell is frozen in liquid nitrogen or isopentane cooled with liquid nitrogen, and a thin section thereof is prepared and attached to a slide glass. Thus, a slide specimen is obtained.
Next, in order to remove a substance that non-specifically reacts with the probe used in the above-mentioned tissue or cell, the slide glass section prepared as described above is treated with proteinase K and acetylated. . Next, hybridization is performed between the slide glass section and the probe prepared as described above. For example, the above probe is heated at 90 ° C. for 3 minutes, then diluted with a hybridization solution, and the solution is dropped on a slide glass section that has been pretreated, covered with a film, and placed in a moisture chamber at 45 ° C. for 16 hours. By keeping the temperature warm, a hybrid is formed. After the hybridization, the non-specifically adsorbed or unreacted probe is removed by washing or the like (when an RNA probe is used, RNase treatment is also added). The transcript amount is a transcript of the present gene, for example, by measuring the amount of labeling on a slide glass section, or by counting the area or the number of cells showing a radioisotope or fluorescent activity in a thin section. The amount of mRNA or a value corresponding thereto can be measured.
[0018]
In the detection method of the present invention, the expression level of one or more genes selected from the present genes in a specimen is measured as described above. When a plurality of genes whose expression levels are to be measured are selected, a plurality of genes having base sequences registered under accession numbers shown in accession number group 1 or orthologs thereof may be selected. An antibody recognizing a placental glutathione-S-transferase enzyme, wherein a gene having a nucleotide sequence registered under the accession number shown in accession number group 1 ′ or an ortholog thereof among accession numbers shown in accession number group 1 is A gene having a larger difference between the expression level in hepatocytes constituting a hepatic pre-cancerous lesion tissue of a rat liver and the expression level of the gene in hepatocytes in a normal tissue, which is negative for the above, can be preferably mentioned.
<Registration number group 1 '>
AA799803, AA819500, AA819643, AA852004, AA892012, AA892616, AA892854, AA8932525, AA926149, AI235707, J02852, M23566, M27156, U15098, U44618, U46118
[0019]
The expression level of the present gene is measured by a method of measuring the amount of a transcript of the present gene per unit amount of a sample, a method of measuring the amount of a translation product of the present gene per unit amount of a sample, or the like, as described above. A method for measuring the amount of the transcript or translation product of the present gene can be preferably used.
[0020]
The measured value of the expression level of the present gene in the sample obtained as described above is compared with a control value of the expression level of the gene, and the placental glutathione-S-transferase enzyme in the sample is determined based on the difference. Is evaluated for the presence or absence of rat liver precancerous lesions that are negative for antibodies that recognize
As a control value of the expression level of the present gene, for example, the value of the expression level of the gene in cells in a normal tissue can be mentioned. Here, the normal tissue means, for example, a tissue in which no abnormality is observed in histopathological examination. In particular, as a method for measuring the amount of transcript with respect to the expression level, in situ hybridization, measurement of the amount of translation product, etc. In the case where an immunohistochemical examination method or the like is used as a method for performing the method, a tissue signal whose transcript amount or translation product amount is clearly weaker than that of a preneoplastic lesion tissue is shown.
Such a control value may be determined by measuring the expression level of the present gene in cells in a normal tissue in parallel with the expression level of the gene in a sample, or may be determined separately. Alternatively, the expression level of the present gene in cells in a plurality of normal tissues may be measured, and the average value thereof may be used as a control value.
For example, using the expression level of the present gene in cells in normal tissues as a control value, a gene having the base sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or an ortholog thereof in a specimen If the measured value of the expression level is higher than the control value, it can be evaluated that the specimen has a rat liver precancerous lesion that is negative for an antibody that recognizes a placental glutathione-S-transferase enzyme.
[0021]
In the method for assaying hepatocarcinogenic activity of the present invention, first, rat hepatocytes are brought into contact with a test substance. The rat hepatocytes to be used include hepatocytes existing in liver tissues collected from rats, or cells isolated from the tissues or cultured cells thereof.
[0022]
In the method for assaying hepatocarcinogenic activity of the present invention, the contact between the hepatocytes and the test substance may be performed, for example, by administering the test substance to a rat. The rat may be a natural animal, a transgenic animal, a gene knockout animal, or the like. The method of administering the test substance to the rat can be, for example, oral (gavage or mixed with drinking water or food), intramuscular, intravenous, subcutaneous, intraperitoneal, or airway. The dosage, the number of administrations, and the administration period may be, for example, within a range that does not seriously affect the general condition, systemic organ tissues, and the like (for example, the dosage is the maximum tolerated dose).
[0023]
Next, the expression level of one or more genes selected from the present genes in the rat hepatocytes contacted with the test substance as described above is measured as described above. When a plurality of genes whose expression levels are to be measured are selected, a plurality of genes having base sequences registered under accession numbers shown in the same accession number group or orthologs thereof may be selected. The gene having the nucleotide sequence registered under the accession number shown in the accession number group 1 ′ or an ortholog thereof is expressed by the expression level of the gene in hepatocytes constituting a rat liver precancerous lesion tissue and the liver level in a normal tissue. The gene having a larger difference from the expression level of the gene in the cell can be preferably used.
The expression level of the present gene is measured by a method of measuring the amount of a transcript of the present gene per unit amount of a sample, a method of measuring the amount of a translation product of the present gene per unit amount of a sample, or the like, as described above. The method for measuring the amount of the transcript of the present gene can be preferably used.
[0024]
The measured value of the expression level of the present gene, which has been contacted with the test substance or obtained from rat hepatocytes as described above, is compared with a control value of the expression level of the gene. The presence or amount of rat liver carcinogenic activity is evaluated.
As a control value of the expression level of the present gene, for example, the value of the expression level in rat hepatocytes not contacted with the test substance can be mentioned. As for rat hepatocytes, the expression level of the gene in rat hepatocytes in normal tissues can be preferably increased. Here, a normal tissue refers to, for example, a liver tissue that has not been contacted with a test substance and has no abnormalities in histopathological examination. In particular, in situ hybridization is used as a method for measuring the expression level by the amount of transcript. When using an immunohistochemical test method as a method or a method for measuring the amount of translation product, even in the case of liver tissue contacted with a test substance, the signal of the amount of transcript or the amount of translation product is not a placental glutathione-S- The tissue is clearly weaker than the rat liver precancerous lesion tissue that is negative for the antibody recognizing the transferase enzyme.
Such a control value may be determined by measuring the expression level of the present gene in cells in normal tissues in parallel with the expression level of the gene in cells in liver tissue contacted with the test substance, or may be measured separately. You may ask for it. For example, a part of the liver tissue before contact with the test substance is collected to measure the expression level of the present gene, and the obtained value can be used as a control value. Alternatively, the expression level of the present gene in cells in a plurality of normal tissues may be measured, and the average value thereof may be used as a control value.
For example, if the measured value of the expression level of the present gene in hepatocytes contacted with the test substance is higher than the value of the expression level of the present gene in normal tissues or cells, the rat liver due to the contact with the test substance is exposed. It means the generation of hepatocytes constituting the cancerous tissue, and the test substance can be evaluated as having rat liver carcinogenic activity.
[0025]
Furthermore, the method for assaying hepatocarcinogenic activity of the present invention as described above can be used for searching for a substance having hepatocarcinogenic activity in rats. Specifically, it is possible to search for a rat hepatocarcinogen-active substance by selecting a substance having rat hepatocarcinogenic activity based on the presence or absence of the rat hepatocarcinogenic activity evaluated by the hepatocarcinogenic activity assay method of the present invention. it can. The selected rat liver carcinogenic active substance can be used, for example, for preparing a liver cancer model mammal.
[0026]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
[0027]
Example 1 (Measurement of expression level of this gene)
(1) Preparation of total RNA
10 ml of TRIZOL Reagent (manufactured by Gibco-BRL) was added to 1 g of wet weight of each of rat liver precancerous lesion tissues and normal tissues, which were negative for antibodies recognizing placental glutathione-S-transferase enzyme, and ice-cooled. While homogenizing with a Polytron homogenizer, and leave at room temperature for 5 minutes. Next, after centrifugation at 4 ° C. and 9,000 rpm for 10 minutes, the aqueous layer is collected in a 50 ml centrifuge tube (manufactured by IWAKI). 1/5 volume of chloroform (manufactured by Kanto Chemical Co., Ltd.) of TRIZOL Reagent is added, and the mixture is vigorously stirred up and down for 15 seconds and then left at room temperature for 5 minutes. Then, after centrifugation at 4 ° C. and 9,000 rpm for 10 minutes, the aqueous layer is collected in a new 50 ml centrifuge tube. Further, 1/2 volume of 2-propanol (manufactured by Kanto Chemical Co., Ltd.) of TRIZOL Reagent is added, mixed by inversion, and allowed to stand at room temperature for 10 minutes. After centrifugation at 9,000 rpm for 10 minutes at 4 ° C, the supernatant is removed to obtain a pellet. The obtained pellet is dissolved in 1/10 volume of DEPC-treated sterilized distilled water (manufactured by Wako Pure Chemical Industries, Ltd.) of TRIZOL Reagent. 350 μl of RLT buffer (10 μL β-mercaptoethanol / mL RLT buffer) attached to RNeasy Mini Kit (manufactured by QIAGEN) is added to 0.1 ml of the obtained RNA solution, and mixed. Further, 250 μl ethanol (manufactured by Kanto Chemical Co., Ltd.) is added and mixed. The mixture is applied to RNeasy Spin Column attached to RNeasy Mini Kit, and centrifuged at 10,000 rpm for 15 seconds at room temperature. After centrifugation, the eluate is applied again to the same RNeasy Spin Column, and centrifuged at 10,000 rpm for 15 seconds at room temperature. After centrifugation, the eluate is discarded, 500 μl of RPE buffer attached to RNeasy Mini Kit diluted 4-fold with ethanol is plied, and centrifuged at room temperature, 10,000 rpm for 15 seconds. After centrifugation, the eluate is discarded, 500 μl of RPE buffer diluted with ethanol is applied, and centrifugation is performed at room temperature at 14,000 rpm for 2 minutes. Thereafter, the column is transferred to a new Eppendorf tube, 50 μl of distilled water attached to RNeasy Mini Kit is added, and the column is left still at room temperature for 1 minute. After standing, elution is performed by centrifugation at 10,000 rpm for 1 minute at room temperature to obtain total RNA. For normal tissues, the same operation as described above is performed to prepare total RNA.
[0028]
(2) Preparation of cDNA
10 μg each of total RNA prepared from rat liver precancerous lesion tissue and total RNA prepared from normal tissue, which is negative for the antibody recognizing the placental glutathione-S-transferase enzyme in (1) above, T7- (dT) 11 μl of a mixture containing 100 pmol of 24 primers (manufactured by Amersham) is heated at 70 ° C. for 10 minutes, and then cooled on ice. After cooling, 4 μl of 5 × First Strand cDNA Buffer contained in SuperScript Choice System for cDNA Synthesis (manufactured by Gibco-BRL), 2 μl of 0.1 M DTT contained in the kit, and 10 μm of TP contained in 1 μm of TP And heat at 42 ° C. for 2 minutes. Further, 2 μl (400 U) of Super ScriptII RT included in the kit is added, heated at 42 ° C. for 1 hour, and then cooled on ice. After cooling, 91 μl of sterilized distilled water treated with DEPC, 30 μl of 5 × Second Strand Reaction Buffer included in the kit, 3 μl of 10 mM dNTP Mix, 3 μl of E. coli contained in the kit. coli DNA Ligase 1 μl (10 U), E. coli DNA Ligase E. coli DNA Polymerase I 4 μl (40 U) and E. coli DNA 1 μl (2 U) of E. coli RNase H is added, and the mixture is reacted at 16 ° C. for 2 hours. Next, 2 μl (10 U) of T4 DNA Polymerase included in the kit is added, the mixture is reacted at 16 ° C. for 5 minutes, and then 10 μl of 0.5 M EDTA is added. Next, 162 μl of a phenol / chloroform / isoamyl alcohol solution (Nippon Gene) is added and mixed. The mixed solution was transferred to Phase Lock Gel Light (manufactured by Eppendorf) which had been centrifuged at room temperature and 14,000 rpm for 30 seconds, and centrifuged at room temperature and 14,000 rpm for 2 minutes. Transfer to an Eppendorf tube. To this, 72.5 μl of a 7.5 M ammonium acetate solution and 362.5 μl of ethanol are added and mixed, and then centrifuged at 4 ° C., 14,000 rpm for 20 minutes. After centrifugation, the supernatant is discarded to obtain a DNA pellet. Thereafter, 0.5 ml of 80% ethanol is added to the DNA pellet, centrifuged at 14,000 rpm for 5 minutes at 4 ° C., and the supernatant is discarded. 0.5 ml of 80% ethanol was added to the DNA pellet again, and centrifuged at 4 ° C., 14,000 rpm for 5 minutes. The supernatant was discarded, and the pellet was dried. This was dissolved in 12 μl of DEPC-treated sterilized distilled water. By doing so, a cDNA solution is obtained. On the other hand, for total RNA prepared from normal tissues, a cDNA solution is prepared by performing the same operation as described above.
[0029]
(3) Preparation of labeled cRNA
Labeled cRNA is prepared using cDNA derived from a rat liver precancerous lesion tissue and cDNA derived from a normal tissue, each of which is negative for an antibody recognizing a placental glutathione-S-transferase enzyme. That is, 5 μl of the cDNA solution obtained in the above (2), 17 μl of sterilized distilled water treated with DEPC, 4 μl of 10 × HY Reaction Buffer contained in the BioArray High Yield RNA Transcript Labeling Kit (manufactured by ENZO), and 10 × contained in the kit 4 μl of Biotin Labeled Ribonucleotides, 4 μl of 10 × DTT included in the kit, 4 μl of 10 × RNase Inhibitor Mix included in the kit, and 2 μl of 20 × T7 RNA Polymerase included in the kit are mixed and reacted at 37 ° C. for 5 hours. After adding 60 μl of DEPC-treated sterilized distilled water to the reaction solution, the labeled cRNA is purified using the RNeasy Mini Kit described in (1) above.
[0030]
(4) Fragmentation of labeled cRNA
40 μl of a reaction solution containing 20 μg of purified labeled cRNA and 5 μl of 5 × Fragmentation Buffer (200 mM Tris-acetic acid pH 8.1 (manufactured by Sigma), 500 mM potassium acetate (manufactured by Sigma), and 150 mM magnesium acetate (manufactured by Sigma)) Is heated at 94 ° C. for 35 minutes and then cooled on ice to obtain a fragmented cRNA solution.
[0031]
(5) Hybridization between fragmented cRNA and probe array
Fragmented cRNA from a rat liver precancerous lesion tissue and a normal tissue-derived fragmented cRNA that are negative for an antibody recognizing a placental glutathione-S-transferase enzyme were respectively hybridized with a probe array as follows. Let it soy. That is, 40 μl of the fragmented cRNA solution obtained in the above (4), 4 μl of 5 nM Control Oligo B2 (manufactured by Amersham), 4 μl of 100 × Control cRNA Cocktail, 40 μg of Herring sperm DNA (manufactured by Promega), AcBetGBetAceBetRayGetBecRateBrag 200 μg, 2 × MES Hybridization Buffer (200 mM MES, 2M [Na ⁺ ), 40 mM EDTA, 0.02% Tween 20 (Pierce), pH 6.5-6.7) and 200 μl of DEPC-treated sterilized distilled water are mixed to obtain 400 μl of a hybrid cocktail. The obtained hybrid cocktail is heated at 99 ° C. for 5 minutes, and further heated at 45 ° C. for 5 minutes. After heating, the mixture is centrifuged at 14,000 rpm for 5 minutes at room temperature to obtain a hybrid cocktail supernatant.
The rat genome U34 probe array (Affymetrix) filled with 1 × MES hybridization buffer is rotated in a hybridization oven at 45 ° C., 60 rpm for 10 minutes, and then the 1 × MES hybridization buffer is removed. Thereafter, 200 μl of the hybrid cocktail supernatant is added to the probe array and rotated in a hybridization oven at 45 ° C., 60 rpm for 16 hours to obtain a hybridized probe array.
[0032]
(6) Staining of probe array
The hybrid cocktail is removed from the hybridized probe array obtained in (5) above, and contains Non-Stringent Wash Buffer [6 × SSPE (diluted 20 × SSPE (manufactured by Nacalai Tesque)) and 0.01% Tween20. ]. After mounting the probe array at a predetermined position of GeneChip Fluidics Station 400 (manufactured by Affymetrix), washing the probe array according to the protocol, and following the staining method of MicroArray Suite (Affymetrix) according to EuKGE-WS2 according to the staining method described below. Stain the array. First, a primary staining solution [containing 10 μg / ml Streptavidin Phycoerythrin (SAPE) (manufactured by Molecular Probe), 2 mg / ml Acetylated BSA, 100 mM MES, 1 M NaCl (manufactured by Ambion), and 0.05% Tween 20) was used for 45 minutes. I do. Next, a secondary staining solution [100 μg / ml Goat IgG (manufactured by Sigma), 3 μg / ml Biotinylated Anti-Streptavidinantibody (manufactured by Vector Laboratories), 2 mg / ml Acetate, 100% BSA, 100% BSAM And 0.005% Antifoam 0-30] for 10 minutes, and finally for 45 minutes in the primary staining solution.
[0033]
(7) Probe array scanning and analysis
A probe array derived from a rat liver precancerous lesion tissue and a normal tissue-derived probe array, which is negative for an antibody recognizing a placental glutathione-S-transferase enzyme, stained in (6) above, was used as an HP GeneArray Scanner ( Affymetrix) and the signal is read by measuring the fluorescence brightness at 570 nm. The obtained results were compared and analyzed by GeneChip Microarray Suite (manufactured by Affymetrix), and genes having different expression levels between normal tissues and the above-mentioned rat liver precancerous lesion tissues were extracted. A gene having a nucleotide sequence registered in Genbank with one of the registration numbers shown in the registration number group 1 is obtained by detecting a signal detected in the above-described probe array derived from a rat liver precancerous lesion tissue from a probe derived from a normal tissue. Exceeded the signal detected in the array.
<Registration number group 1>
AA799336, AA799481, AA799488, AA799523, AA799538, AA799539, AA799641, AA799656, AA799680, AA799711, AA799735, AA799803, AA799889, AA799891, AA799893, AA799899, AA799991, AA799995, AA800034, AA800044, AA800570, AA800593, AA800637, AA800671, AA800673, AA800768, AA800787, AA800814, AA800930, AA817854, AA817798, AA818122, AA818152, AA819500, AA819643, AA8485 5, AA852004, AA858573, AA858626, AA858640, AA859661, AA859832, AA866240, AA866302, AA874919, AA874926, AA875097, AA875471, AA891877, AA891998, AA892006, AA892012, AA892036, AA892248, AA892297, AA892394, AA892399, AA892557, AA892593, AA892616, AA892637, AA892779, AA892797, AA892799, AA892828, AA892854, AA892860, AA8929297, AA893307, AA893325, AA893338, AA89 3493, AA893664, AA893702, AA893982, AA894030, AA894193, AA894282, AA899320, AA925473, AA926149, AA926193, AA945050, AA945573, AA963449, AB000507, AF016296, AF029240, AI010371, AI010725, AI012051, AI012275, AI014135, AI069990, AI103874, AI112516, AI169372, AI169695, AI169758, AI170379, AI172293, AI176308, AI176488, AI177161, AI179445, AI180442, AI229291, A 230406, AI232321, AI235707, AI233007, AI638985, AI639102, AI6339315, AI639371, AJ001641, D13912, D14564, D14987, D14988, D14989, D38938, 28,693, 1988, M1493 M33329, M37828, M64092, M64780, M96601, S59892, U11071, U15098, U18314, U19485, U42627, U44948, U46118, U49099, U50927, U75928, U89905, X123 3410, X67859, X78855
[0034]
Example 2 (Test of liver neoplastic lesion tissue (including some preneoplastic lesion tissues) using probe array)
After preparing total RNA from two or more different liver samples including histopathologically normal liver samples of untreated rats by the same operation as in Example 1 (1), Examples 1 (2) to (1) A fragmented cRNA derived from a normal liver sample of an untreated rat and a fragmented cRNA derived from a test liver sample are prepared by the same operation as in 4). Next, a fragmented cRNA derived from a normal liver sample of a non-treated rat and a fragmented cRNA derived from a test liver sample were converted into an oligonucleotide having a partial nucleotide sequence of the nucleotide sequence of the present gene by the same operation as in Example 1 (5). After hybridization to the probe array on which is fixed, staining is performed. The obtained stained probe array is subjected to an HP GeneArray Scanner (manufactured by Affymetrix), and the signal is read out by measuring the fluorescence brightness at 570 nm, and the obtained results are analyzed and analyzed by GeneChip Microarray Suite (manufactured by Affymetrix). . The expression level of a gene having a nucleotide sequence registered in Genbank under any of the accession numbers shown in accession number group 1 or an ortholog thereof is compared between a normal liver sample and a test liver sample of an untreated rat, and the difference is shown. And detecting liver cells constituting a rat liver precancerous lesion tissue that are negative for an antibody recognizing a placental glutathione-S-transferase enzyme in a test liver sample based on the above. That is, the measured value of the expression level of a gene having a nucleotide sequence registered in Genbank under any of the registration numbers shown in the registration number group 1 or the ortholog thereof in a test liver sample is the gene in an untreated rat normal liver sample. If the expression level is higher than the expression level, it is evaluated that hepatocytes constituting the rat liver precancerous lesion tissue are present in the test biopsy sample.
[0035]
Example 3 (Test for liver neoplastic lesions (including some preneoplastic lesions) using quantitative RT-PCR)
Total RNA was prepared from two or more different liver samples, including histopathologically normal liver samples from untreated rats, by the same operation as in Example 1 (1), and then as in Example 1 (2). A cDNA derived from an untreated rat normal liver sample and a cDNA derived from a test liver sample are prepared by the above procedure. Next, PCR is performed as follows using the prepared cDNA as a template, and the amplified DNA is quantified. That is, a 50 μl reaction solution containing 1 μl of the cDNA, 20 pmol of primer 1 having the nucleotide sequence of SEQ ID NO: 1, 20 pmol of primer 2 having the nucleotide sequence of SEQ ID NO: 2, and 25 μl of SYBR Green PCR Reagents PCR (ABI) Was prepared by using ABI 7700 (ABI), and then incubated at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes, and then subjected to 40 cycles of incubation at 95 ° C. for 15 seconds and then at 60 ° C. for 1 minute as one cycle. I do. From the amount of the amplified DNA, the expression level of the present gene in the test liver sample and the expression level of the present gene in the untreated rat normal liver sample are determined. The measured value of the expression level of a gene having a nucleotide sequence registered in Genbank under one of the registration numbers shown in the registration number group 1 or the ortholog thereof in a test liver sample is the expression of the gene in an untreated rat normal liver sample If the level is higher than the level, it is evaluated that the test liver sample contains hepatocytes constituting a rat liver precancerous lesion tissue that is negative for an antibody recognizing a placental glutathione-S-transferase enzyme.
[0036]
Example 4 (Method of assaying rat liver carcinogenesis activity using probe array)
(1) Administration of test substance to rats
For example, a medium-term hepatic carcinogenicity test model (Ito, N., et al. CRC Crit Rev Toxicol, 19: 385-415, 1989.) can be used.
Specifically, when the test substance is a mutagenic substance, for example, 200 mg / kg body weight of N-diethylnitrosamine (hereinafter referred to as DEN) is given to male F344 rats (6 weeks old at the start of administration). Was intraperitoneally administered once, and as a control, a group of a single administration of the test substance solvent was further provided. When the test substance is non-mutagenic, the same dose of DEN as above is administered to the same male F344 rat as a test substance from the second week after the single administration, for example, 3000 ppm of clofibrate or 1000 ppm of WY-14,643 1000 ppm. , And a group to which only basal feed was administered as a control was further provided. Irrespective of the mutagenicity of the test substance, 2/3 partial hepatectomy was performed on all cases at 3 weeks from the start of the test, and at 8 weeks, the liver of each animal group was collected.
[0037]
(2) Gene expression analysis
Total RNA was prepared from the liver tissue derived from the test substance-administered group and the liver tissue derived from only the basal feed-administered group by the same operation as in Example 1 (1), and then the total RNA was prepared in Examples 1 (2) to (4). By the same operation, fragmented cRNA derived from liver tissue of each of the test substance administration group and the basal feed administration group is prepared. Next, an oligonucleotide having a partial nucleotide sequence of the nucleotide sequence of the present gene was fixed to the fragmented cRNA derived from the liver tissue of each of the test substance administration group and the basal feed administration group by the same operation as in Example 1 (5). After hybridization to the probe array, staining is performed. The probe arrays of each of the test substance administration group and the basal feed administration group were subjected to an HP GeneArray Scanner (manufactured by Affymetrix), and the signals were read out by measuring the fluorescence luminance at 570 nm, and the obtained results were used for GeneChip Microarray Suite ( (Affymetrix). When the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or the ortholog thereof in the test substance administration group is at least twice the expression level of the basal feed administration group. If present, the test substance is evaluated as having rat liver carcinogenic activity.
[0038]
Example 5 (Method of assaying rat liver carcinogenic activity using quantitative RT-PCR)
(1) Administration of test substance to rats
Liver tissue is collected by the same operation as in Example 4 (1).
[0039]
(2) Gene expression analysis
Total RNA was prepared by the same operation as in Example 1 (1) from each of the liver tissue of the test substance-administered group and the liver tissue from the group administered only the basal feed, and then tested by the same operation as in Example 1 (2). CDNAs derived from liver tissues of each of the substance administration group and the basal feed administration group are prepared. Next, using the prepared cDNA as a template, PCR is performed as follows to quantify the amplified DNA. That is, a 50 μl reaction solution containing 1 μl of the cDNA, 20 pmol of a primer 1 consisting of the nucleotide sequence shown in SEQ ID NO: 1, 20 pmol of a primer 2 consisting of the nucleotide sequence shown in SEQ ID NO: 2, and 25 μl of SYBR Green PCR Reagents PCR (ABI) was used. The PCR was carried out using ABI 7700 (ABI), and after incubating at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes, the PCR was carried out under the condition that the incubation at 95 ° C. for 15 seconds and then at 60 ° C. for 1 minute was performed as one cycle and the cycle was performed for 40 cycles. Do. From the amount of the amplified DNA, the expression level of the present gene in the liver tissue of the test substance administration group and the expression level of the present gene in the liver tissue of the basal feed administration group are determined. If the expression level in the liver tissue of the group to which the test substance is administered is at least twice the expression level of the liver in the group to which the basal feed is administered, the test substance is evaluated as having rat liver carcinogenic activity.
[0040]
Example 6 (Method of assaying rat liver carcinogenic activity using in situ hybridization)
(1) Administration of test substance to rats
Liver tissue is collected by the same operation as in Example 4 (1). However, the liver is collected as it is or after perfusion with a fixative (4% paraformaldehyde + 0.5% glutaraldehyde / 0.075M phosphate buffer, pH 7.2 to 7.4).
[0041]
(2) Fixation, embedding of liver tissue and preparation of thin sections
The collected liver is fixed with a fixing solution (4% paraformaldehyde + 0.5% glutaraldehyde / 0.075M phosphate buffer, pH 7.2 to 7.4) at 4 ° C. overnight. After fixation, the liver tissue of the test substance administration group and the liver tissue from the basal feed only administration group were each dehydrated with alcohol, embedded in paraffin, and sliced to a thickness of 2 to 10 μm using a microslicer. On a slide glass.
[0042]
(3) Preparation of cDNA
From a tissue expressing a gene having a nucleotide sequence registered in Genbank with one of the registration numbers shown in the registration number group 1 (for example, cerebral tissue in X58294), the same operation as in Example 1 (1) was performed. Then, total RNA is adjusted to 1 μg / μl. Next, about 1 kbp of a target gene (for example, X58294) is selected, and primers for increasing DNA having the base sequence by PCR are synthesized. The primers were prepared by adding a T3 promoter sequence (aattaa ccc tca cta aag gga ac) to the 5 'end of the sense primer and a T7 promoter sequence (gta ata cga ctc actagg agtag at the 5' end of the antisense primer. The synthesis is performed by a general method used in genetic engineering techniques. For RT-PCR, each kit of ThermoScript RT-PCR System (Gibco) and PLATIM taq DNA Polymerase High fidelity (Gibco) is used. First, 20 pmol of the primer of Antisense, 1 μl of total RNA and H20 in the kit are mixed, kept at 65 ° C. for 5 minutes, and then cooled on ice. Next, 4 μl of 5 × DNA Synthesis Buffer, 1 μl of 0.1 M DDT, 1 μl of RNase OUT, 1 μl of sterile water, 2 μl of 10 mM dNTP Mix, and 1 μl of ThermoScript RT were added, and the mixture was kept at 65 ° C. for 1 hour and heated at 85 ° C. for 5 minutes. And inactivate ThermoScript RT. After cooling on ice, 1 μl of RNase H (Gibco) is added and reacted at 37 ° C. for 20 minutes to produce cDNA. To 5 μl of the prepared cDNA, sense primer 20 pmol, × 10 High Fidelity PCR Buffer 5 μl, 50 mM MgSO 2 μl, 10 mM dNTPmix 1 μl, PLATINUM taq DNA Polymerase After heating, 40 cycles are carried out, each cycle consisting of 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 1 minute, and finally the reaction is performed at 68 ° C. for 5 minutes. This PCR reaction solution is subjected to 1% agarose gel electrophoresis, the target PCR product is excised from the gel, mixed with an equal amount of isopropanol, applied to a column in the kit, and centrifuged for 2 minutes. After centrifugation, a new tube is connected to the column, and 50 μl of EB buffer in the kit is applied to the column and centrifuged to obtain a target DNA product.
[0043]
(4) Preparation of labeled cRNA
Using the cDNA synthesized in Example 6 (3) as a template, a cRNA probe (FITC-labeled) is synthesized using T3 polymerase or T7 polymerase. Specifically, to 0.2 to 1 μl of cDNA, 2 μl of × 10 transcription buffer (DTT +) (Takara Shuzo), 2 μl of × 10 FITC-leveling mix (Boehringer Mannheim), 1 μl of RNase inhibitor (Nippon Gene), and H2O were added. To a total volume of 18 μl. To synthesize a sense probe, 2 μl of T3 polymerase (Takara Shuzo) is added, and to synthesize an anti-sense probe, 2 μl of T7 polymerase (Takara Shuzo) is added, and the mixture is kept at 37 ° C. for 3 to 5 hours. Next, 1 μl of RNase free DNase I (Boehringer Mannheim) is added, and the mixture is further kept warm for 20 minutes. The synthesized cRNA was precipitated with ethanol, and the resulting pellet was heated at 60 ° C. to 100 μL of 40 mM NaHCO 3. ₃ , 60 mM Na ₂ CO ₃ (PH 10.2). Thereafter, the temperature is maintained at 60 ° C. for a time obtained from the following calculation formula so that fragments of about 150 b are obtained by alkali decomposition. Warming time (min) = (cDNA length (kb)-average fragment length after hydrolysis (kb)) / (0.11 x cDNA length (kb) x average fragment length after hydrolysis (kb)). After the reaction, cool on ice and add 1 μl of acetic acid. Further, 1/10 volume of 4M LiCl is added, centrifuged at 15000 rpm for 10 minutes, and the supernatant is discarded. After adding 500 μl of ethanol to wash the pellet, 20 μl of TE buffer, 2 μl of 0.1 M DTT, and 1 μl of RNase inhibitor are added and dissolved.
[0044]
(5) Hybridization
The slide specimen prepared in Example 6 (2) is deparaffinized at room temperature and rehydrated. Next, 1 × Target Retrieve Solution (DAKO) is added as a pretreatment, and the mixture is heated at 121 ° C. for 20 minutes by an autoclave. After the temperature has dropped to 60 ° C., it is taken out and left at room temperature for 20 minutes for further cooling. After cooling, at room temperature for 2 minutes x 2 in DEPC-treated sterilized water, 3 minutes in 3% hydrogen peroxide / DEPC-treated sterilized water, 2 minutes in DEPC-treated sterilized water, 90% ethanol / DEPC-treated sterilized water After immersion in 100% ethanol for 3 minutes, air-dry. The cRNA probe prepared in Example 6 (4) was subjected to hybridization buffer (composition: 40% formamide (Nippon Gene), 4 × SCC (pH 5.0) (Gibco), 1 mM EDTA, 1 × Denhardet's (Nippon gene), Dilute 250 μg / ml yeast tRNA (Gibco), 125 μg / ml salmon sperm DNA (Gibco), 10% dextran sulfate to 0.1-0.3 μg / ml / kb, drop on a section, and add EasiSeal ( The slide glass is placed in a wet box and kept at 60 ° C. for 5 hours or more, and then immersed in 5 × SCC (pH 5.0) kept at 65 ° C. Cleaning solution A (composition: 2 × S) heated to 65 ° C. Immerse in CC [pH 5.0], 1% SDS) twice for 30 minutes, and wash solution B (composition: 10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.1% Tween 20) at room temperature. After immersion in a washing solution B heated to 37 ° C. for 3 minutes and then to a solution obtained by adding 20 μg / ml RNase A (SIGMA) to a washing solution B heated to 37 ° C., a washing solution C heated to 65 ° C. (composition: 2 × SCC [pH 5.0], 50% formamide [Wako]) × 2 for 30 minutes × 3 times with TBS buffer (composition: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) at room temperature for 3 minutes After washing, the excess solution around the section is wiped off, the tissue is surrounded with PAP PEN (Daido Sangyo Co., Ltd.), and then included in the FITC-labeled probe detection kit (DAKO). An alkaline phosphatase-labeled anti-FITC antibody diluted with 1% bovine serum albumin / TBS is dropped on a section and left in a wet box at 37 ° C. for 60 minutes.After the reaction, TBST at room temperature (composition: TBS + Immerse in TBS at room temperature for 2 to 3 minutes in 5% × 3 times for 5 minutes in 0.1% Tween 20. Excess solution around the section is wiped off, and AP substrate solution (DAKO, BCIP / NBT coloring substrate solution) is added dropwise. Then, the plate is allowed to stand in a wet box at 37 ° C. for 60 minutes to 2 days, and then the color developing solution is washed out in running water, and the liquid is sealed.
[0045]
(6) Gene expression analysis
The amount of the transcript of this gene was determined by counting the area or cell number of each of the sections exhibiting each fluorescent activity in the liver slice section of the test substance-administered group and the liver slice section of the basal feed-administered group. The amount of mRNA or a value corresponding thereto can be measured. Thereafter, by performing an appropriate statistical analysis, the presence or absence of rat liver carcinogenic activity can be evaluated.
[0046]
Example 7 (Method for assaying rat liver carcinogenic activity using immunohistochemical test)
(1) Administration of test substance to rats
Liver tissue is collected by the same operation as in Example 4 (1).
[0047]
(2) Tissue fixation, embedding, and section preparation
After the tissue is cut out to an appropriate size from the collected liver, it is embedded in OCT compound and frozen in isopentane cooled with liquid nitrogen. This is sliced to a thickness of about 5 μm in a cryostat cooled to −20 ° C., placed on a slide glass, and dried.
[0048]
(3) Immunohistochemical staining of thin sections
After fixing the thin section prepared in the above (2) with 70% ethanol for 1 minute, the thin section was washed with PBS buffer (composition: 10 mM sodium phosphate [pH 7.4], 100 mM NaCl) to remove the OCT compound. Treat with 3% hydrogen peroxide solution for 5 minutes. Wash 3 times with PBS buffer for 5 minutes and treat with normal serum for 20 minutes at room temperature. Next, the cells are treated with an antibody against a translation product of a gene having a base sequence registered in Genbank with one of the accession numbers shown in accession number group 1 appropriately diluted with PBS buffer (hereinafter referred to as a primary antibody) for 60 minutes at room temperature. . After the reaction, the plate is washed three times for 5 minutes with a PBS buffer, and treated with a VECTASTAIN / ABC reagent (Funakoshi) of the same immunized animal as the primary antibody appropriately diluted with the PBS buffer for 30 minutes at room temperature. Further, after washing with PBS buffer for 5 minutes × 3 times, the color is developed using 3,3′-diaminobenzidine tetrahydrochloride (Nacalai). After that, it is washed with distilled water, dehydrated, transparent, and sealed.
[0049]
(4) Expression analysis of gene translation products
Using an image analyzer IPAP (Sumitomo Techno Service Co., Ltd.), the area of the hepatic cell nest where staining was confirmed and the area of the whole liver thin section were measured. Calculate (area) / (total area of liver thin section). As another method, the number of hepatocyte nests confirmed to be stainable is counted, and the (number of hepatocyte nests confirmed to be stainable) / (total liver slice section area) is calculated.
Next, a statistical analysis is performed using an appropriate test method, and when the value in the test substance administration group shows a significant increase from the value in the basal feed administration group, the test substance has rat liver carcinogenicity. Then evaluate.
[0050]
【The invention's effect】
According to the present invention, it is possible to provide a method for assaying a rat liver precancerous lesion that is negative for an antibody recognizing a placental glutathione-S-transferase enzyme using an abnormality in the expression level of a gene as an index.
[0051]
[Sequence List Free Text]
SEQ ID NO: 1
Oligonucleotide primers designed for PCR
SEQ ID NO: 2
Oligonucleotide primers designed for PCR
[0052]
[Sequence list]

Claims (10)

A method for assaying a rat liver precancerous lesion that is negative for an antibody that recognizes a placental glutathione-S-transferase enzyme,
(1) A first method for measuring the expression level of a gene having a nucleotide sequence registered in Genbank with one of the registration numbers shown in the following registration number group 1 or one or more genes selected from orthologs thereof in a specimen: And (2) comparing the measured value of the expression level of the gene in the sample obtained in the first step with a control value of the expression level of the gene, and determining the placental glutathione-S in the sample based on the difference. -A method comprising a second step of evaluating the presence or absence of a rat liver precancerous lesion that is negative for an antibody recognizing a transferase enzyme.
<Registration number group 1>
AA799336, AA799481, AA799488, AA799523, AA799538, AA799539, AA799641, AA799656, AA799680, AA799711, AA799735, AA799803, AA799889, AA799891, AA799893, AA799899, AA799991, AA799995, AA800034, AA800044, AA800570, AA800593, AA800637, AA800671, AA800673, AA800768, AA800787, AA800814, AA800930, AA817854, AA817798, AA818122, AA818152, AA819500, AA819643, AA8485 5, AA852004, AA858573, AA858626, AA858640, AA859661, AA859832, AA866240, AA866302, AA874919, AA874926, AA875097, AA875471, AA891877, AA891998, AA892006, AA892012, AA892036, AA892248, AA892297, AA892394, AA892399, AA892557, AA892593, AA892616, AA892637, AA892779, AA892797, AA892799, AA892828, AA892854, AA892860, AA8929297, AA893307, AA893325, AA893338, AA89 3493, AA893664, AA893702, AA893982, AA894030, AA894193, AA894282, AA899320, AA925473, AA926149, AA926193, AA945050, AA945573, AA963449, AB000507, AF016296, AF029240, AI010371, AI010725, AI012051, AI012275, AI014135, AI069990, AI103874, AI112516, AI169372, AI169695, AI169758, AI170379, AI172293, AI176308, AI176488, AI177161, AI179445, AI180442, AI229291, A 230406, AI232321, AI235707, AI233007, AI638985, AI639102, AI6339315, AI639371, AJ001641, D13912, D14564, D14987, D14988, D14989, D38938, 28,693, 1988, M1493 M33329, M37828, M64092, M64780, M96601, S59892, U11071, U15098, U18314, U19485, U42627, U44948, U46118, U49099, U50927, U75928, U89905, X123 3410, X67859, X78855 The specimen is a biological sample which may contain hepatocytes constituting rat liver precancerous lesion tissue negative for antibodies recognizing placental glutathione-S-transferase enzyme or contents thereof. The method according to claim 1, wherein 3. The method according to claim 1, wherein the measurement of the expression level of the gene is performed by measuring the amount of a transcript or a translation product of the gene. 4. The method according to claim 1, wherein the control value of the expression level of the gene is a value of the expression level of the gene in normal tissues or cells. In the second step, the measured value of the expression level of a gene having a nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or its ortholog is used as an index, and A step of evaluating the presence or absence of rat liver precancerous lesions that are negative for an antibody recognizing a placental glutathione-S-transferase enzyme in the sample based on an index, wherein the evaluation is performed. The method according to 1, 2, 3 or 4. A method for assaying rat liver carcinogenic activity of a substance, comprising:
(1) a first step of contacting rat hepatocytes with a test substance,
(2) In the hepatocytes contacted with the test substance in the first step, selected from genes having a base sequence registered in Genbank under any of the accession numbers shown in accession number group 1 below, or orthologs thereof. And (3) comparing the measured value of the expression level of the gene obtained in the second step with a control value of the expression level of the gene, based on the difference. Evaluating the presence or absence or the amount of the test substance in rat liver carcinogenesis by a test method.
<Registration number group 1>
AA799336, AA799481, AA799488, AA799523, AA799538, AA799539, AA799641, AA799656, AA799680, AA799711, AA799735, AA799803, AA799889, AA799891, AA799893, AA799899, AA799991, AA799995, AA800034, AA800044, AA800570, AA800593, AA800637, AA800671, AA800673, AA800768, AA800787, AA800814, AA800930, AA817854, AA817798, AA818122, AA818152, AA819500, AA819643, AA8485 5, AA852004, AA858573, AA858626, AA858640, AA859661, AA859832, AA866240, AA866302, AA874919, AA874926, AA875097, AA875471, AA891877, AA891998, AA892006, AA892012, AA892036, AA892248, AA892297, AA892394, AA892399, AA892557, AA892593, AA892616, AA892637, AA892779, AA892797, AA892799, AA892828, AA892854, AA892860, AA8929297, AA893307, AA893325, AA893338, AA89 3493, AA893664, AA893702, AA893982, AA894030, AA894193, AA894282, AA899320, AA925473, AA926149, AA926193, AA945050, AA945573, AA963449, AB000507, AF016296, AF029240, AI010371, AI010725, AI012051, AI012275, AI014135, AI069990, AI103874, AI112516, AI169372, AI169695, AI169758, AI170379, AI172293, AI176308, AI176488, AI177161, AI179445, AI180442, AI229291, A 230406, AI232321, AI235707, AI233007, AI638985, AI639102, AI6339315, AI639371, AJ001641, D13912, D14564, D14987, D14988, D14989, D38938, 28,693, 1988, M1493 M33329, M37828, M64092, M64780, M96601, S59892, U11071, U15098, U18314, U19485, U42627, U44948, U46118, U49099, U50927, U75928, U89905, X123 3410, X67859, X78855 The method according to claim 6, wherein the measurement of the expression level of the gene is performed by measuring the transcript amount of the gene. 8. The method according to claim 6, wherein the control value of the expression level of the gene is a value of the expression level of the gene in normal tissues or cells. In the third step, the expression level of a gene having a base sequence registered in Genbank under any of the registration numbers shown in the registration number group 1 or an ortholog thereof is used as an index, wherein the measured value is higher than a control value, 9. The method according to claim 8, wherein the presence or absence or the amount of the test substance in rat liver carcinogenesis is evaluated. A rat hepatocarcinogen comprising a step of selecting a substance having rat hepatocarcinogenic activity based on the presence or absence of rat hepatocarcinogenic activity evaluated by the method according to any one of claims 6 to 9. Search method.