JP2007033041A - Examination method of neoplastic lesion or preneoplastic lesion of rat liver showing negative to antibody confirmimg enzyme, placental glutathione s-transferase - Google Patents
Examination method of neoplastic lesion or preneoplastic lesion of rat liver showing negative to antibody confirmimg enzyme, placental glutathione s-transferase Download PDFInfo
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本発明は、ラットにおける胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の検定方法等に関する。 The present invention relates to a method for assaying rat liver neoplastic lesions or preneoplastic lesions that are negative for antibodies recognizing placental glutathione-S-transferase enzyme in rats.
癌が遺伝子の関わる異常を原因とする疾病であることが次第に明らかになりつつあるが、癌に対する羅漢率、死亡率は未だ高い。このような状況において、様々な化学物質の発癌性の有無を事前に確認しておくための安全性試験は極めて重要である。従来、化学物質の発癌性を検定する代表的な方法としては、種々の脊椎動物（例えばラット）を用いた安全性試験において腫瘍マ−カ−を指標として用いることにより、発癌性の有無を評価する方法が知られている（非特許文献１及び非特許文献２参照）。 Increasingly, it is becoming clear that cancer is a disease caused by genetic abnormalities, but the rahan and mortality rates for cancer are still high. In such a situation, a safety test for confirming in advance the presence or absence of carcinogenicity of various chemical substances is extremely important. Conventionally, as a representative method for examining the carcinogenicity of chemical substances, the presence or absence of carcinogenicity is evaluated by using a tumor marker as an index in a safety test using various vertebrates (eg, rats). The method of doing is known (refer nonpatent literature 1 and nonpatent literature 2).
しかしながら、実験動物としてラットを使用する場合において、例えば、化学物質の発癌標的となる割合の最も高い器官若しくは組織の一つである肝臓の場合には、最も頻繁に用いられるラット肝臓腫瘍マ−カ−の一つである胎盤型グルタチオン−Ｓ−トランスフェラ−ゼの場合には、肝細胞中のペルオキシソ−ム数を増大させるペルオキシソ−ム増生剤により誘発される腫瘍性病変又は前腫瘍性病変を検出することはできない等の欠点があり、場合によっては必ずしも十分に満足のゆくものではなかった。
そこで、化学物質の発癌性をできるだけ正確かつ簡便に評価するためのラットにおける癌検定方法の開発が切望されている。
However, when rats are used as experimental animals, for example, in the case of the liver, which is one of the organs or tissues with the highest rate of carcinogenic targets of chemical substances, the most frequently used rat liver tumor marker In the case of placental glutathione-S-transferase, which is one of the above, a neoplastic lesion or a preneoplastic lesion induced by a peroxisome proliferator that increases the number of peroxisomes in hepatocytes There are drawbacks such as inability to detect, and in some cases, it is not always satisfactory.
Therefore, development of a cancer assay method in rats for evaluating the carcinogenicity of chemical substances as accurately and simply as possible is eagerly desired.
かかる状況の下、本発明者らは鋭意検討した結果、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変組織を構成する肝細胞において、特定の遺伝子の翻訳産物量が隣接する正常組織での肝細胞における当該遺伝子の翻訳産物量に比べ増大していることを見出し、本発明に至った。
即ち、本発明は、
１．胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変（以下、本病変と記すこともある。）の検定出方法であって、
（１）検体における、78-kDa glucose-regulated proteinの遺伝子の翻訳産物量を測定する第一工程、及び
（２）第一工程で得られた前記検体における遺伝子の翻訳産物量の測定値を当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記検体における、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の有無或いはその発生程度を評価する第二工程
を有することを特徴とする方法（以下、本発明方法と記すこともある。）
２．検体が、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変組織を構成する肝細胞或いはその内容物が含まれる可能性のある生体試料であることを特徴とする前項１記載の方法；
３．検体が、光学顕微鏡用組織標本であることを特徴とする前項１又は２記載の方法；
４．遺伝子の翻訳産物量の対照値が、当該遺伝子の正常組織又は正常細胞における翻訳産物量の値であることを特徴とする前項１、２又は３記載の方法；
５．第二工程において、78-kDa glucose-regulated proteinの翻訳産物量の測定値が対照値よりも高いことを指標とし、当該指標に基づいて前記検体における、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の有無或いはその発生程度を評価する工程であることを特徴とする前項１、２、３又は４記載の方法；
６．78-kDa glucose-regulated proteinの翻訳産物量の測定及びその測定値が、当該蛋白質を発現している細胞からなる細胞巣の面積又は当該細胞巣の数の測定及びその測定値であることを特徴とする請求項１、２、３、４又は５記載の方法。
７．物質のラット肝発癌活性の検定方法であって、
（１）ラット肝細胞と被験物質とを接触させる第一工程、
（２）第一工程で前記被験物質と接触させた前記肝細胞における、78-kDa glucose-regulated proteinの遺伝子の翻訳産物量を測定する第二工程、及び
（３）第二工程で得られた遺伝子の翻訳産物量の測定値を当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記被験物質のラット肝発癌活性の有無又はその量を評価する第三工程
を有することを特徴とする方法（以下、本発明肝発癌活性検定方法と記すこともある。）
８．検体が、光学顕微鏡用組織標本であることを特徴とする前項７記載の方法；
９．遺伝子の翻訳産物量の対照値が、当該遺伝子の正常組織又は正常細胞における翻訳産物量の値であることを特徴とする前項７又は８記載の方法；
１０．第三工程において、78-kDa glucose-regulated proteinの翻訳産物量の測定値が対照値よりも高いことを指標とし、前記被験物質のラット肝発癌活性の有無又はその量を評価することを特徴とする前項７、８又は９記載の方法；
１１．78-kDa glucose-regulated proteinの翻訳産物量の測定及びその測定値が、当該蛋白質を発現している細胞からなる細胞巣の面積又は当該細胞巣の数の測定及びその測定値であることを特徴とする請求項７、８、９又は１０記載の方法。
１２．前項７乃至１１のいずれかに記載される方法により評価されたラット肝発癌活性の有無又はその量に基づきラット肝発癌活性を有する物質を選抜する工程を有することを特徴とする肝発癌物質の探索方法等を提供するものである。
Under such circumstances, the present inventors have conducted extensive studies and, as a result, constituted rat liver neoplastic lesions or preneoplastic lesion tissues that are negative for an antibody recognizing placental glutathione-S-transferase enzyme. In the hepatocytes, the inventors have found that the amount of translation product of a specific gene is increased compared to the amount of translation product of the gene in hepatocytes in the adjacent normal tissue, leading to the present invention.
That is, the present invention
1. A method for assaying rat liver neoplastic lesions or preneoplastic lesions (hereinafter sometimes referred to as this lesion) that are negative for an antibody recognizing placental glutathione-S-transferase enzyme. ,
(1) the first step of measuring the translation product amount of the 78-kDa glucose-regulated protein gene in the sample, and (2) the measured value of the translation product amount of the gene in the sample obtained in the first step Rat liver neoplastic lesion or pre-tumor that is negative for the antibody recognizing placental glutathione-S-transferase enzyme in the specimen based on the difference compared with the control value of the amount of gene translation product A method comprising a second step of evaluating the presence or absence of sexual lesions or the degree of occurrence thereof (hereinafter sometimes referred to as the method of the present invention).
2. The specimen may contain hepatocytes or their contents constituting rat liver neoplastic lesions or preneoplastic lesion tissues that are negative for antibodies recognizing placental glutathione-S-transferase enzyme. The method according to item 1 above, which is a biological sample;
3. 3. The method according to item 1 or 2 above, wherein the specimen is a tissue specimen for an optical microscope;
4). 4. The method according to item 1, 2 or 3 above, wherein the reference value of the translation product amount of the gene is the value of the translation product amount of the gene in normal tissue or normal cells;
5. In the second step, the measured value of the translation product amount of 78-kDa glucose-regulated protein is used as an index, and the placental glutathione-S-transferase enzyme in the sample is determined based on the index. The method according to 1, 2, 3 or 4 above, which is a step of evaluating the presence or the occurrence of a rat liver neoplastic lesion or preneoplastic lesion that is negative for an antibody recognizing
6. Measurement of the translation product amount of 78-kDa glucose-regulated protein and the measured value thereof are the measurement of the area of the cell nest composed of cells expressing the protein or the number of the cell nest and the measured value thereof. 6. A method according to claim 1, 2, 3, 4 or 5.
7). 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) Second step of measuring the translation product amount of the 78-kDa glucose-regulated protein gene in the hepatocytes contacted with the test substance in the first step, and (3) obtained in the second step Comparing the measured value of the translation product amount of the gene with a control value of the translation product amount of the gene, and having a third step of evaluating the presence or absence of the rat liver carcinogenic activity of the test substance based on the difference Characteristic method (hereinafter sometimes referred to as the present hepatocarcinogenic activity assay method)
8). 8. The method according to item 7 above, wherein the specimen is a tissue specimen for an optical microscope;
9. 9. The method according to item 7 or 8 above, wherein the reference value of the translation product amount of the gene is the value of the translation product amount of the gene in normal tissue or normal cells;
10. In the third step, the measured value of the translation product of 78-kDa glucose-regulated protein is used as an index, and the presence or absence of the rat liver carcinogenic activity of the test substance is evaluated. The method according to item 7, 8 or 9 above;
11. Measurement of the translation product amount of 78-kDa glucose-regulated protein and the measured value thereof are the measurement of the area of the cell nest composed of cells expressing the protein or the number of the cell nest and the measured value thereof. 11. A method according to claim 7, 8, 9 or 10.
12 A search for a liver carcinogen, comprising a step of selecting a substance having rat liver carcinogenic activity based on the presence or absence of rat liver carcinogenic activity evaluated by the method according to any one of 7 to 11 above. A method is provided.
本発明によって、遺伝子の翻訳産物量の異常を指標としたラットにおける胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の検定方法等が提供可能となる。 According to the present invention, rat liver neoplastic lesions or preneoplastic lesions that are negative for an antibody recognizing placental glutathione-S-transferase enzyme in rats, using as an index an abnormality in the amount of translation product of the gene. A method or the like can be provided.
本発明において検体とは、例えば、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変を構成する肝細胞或いはその内容物が含まれる可能性のある生体試料（例えば、光学顕微鏡用組織標本等）をあげることができ、具体的には、例えば、ラットから採取された肝臓組織等の組織或いはこれら組織から分離された細胞、又はその培養細胞等をあげることができる。これらの試料はそのまま検体として用いてもよく、また、かかる試料から分離、分画、固定化等の種々の操作により調製された試料を検体として用いてもよい。
本発明において利用される78-kDa glucose-regulated protein（以下、本蛋白質と記すこともある。）は、ヒートショック蛋白質７０（HSP70）ファミリーの一つに分類され、小胞体ストレスの際に細胞内に誘導されることが知られている蛋白質である。例えば、配列番号１で示されるアミノ酸配列で示されるアミノ酸配列等の公知のアミノ酸配列を有しており、当該蛋白は動植物に広く存在し、その分子量は約７８，０００で６５４アミノ酸残基からなる。
本発明において翻訳産物量が測定される78-kDa glucose-regulated proteinの遺伝子（以下、本遺伝子と記すこともある。）には、例えば、配列番号１で示されるアミノ酸配列と併記されている塩基配列等の公知の塩基配列と全く同一の塩基配列を有する遺伝子のほか、前記の遺伝子の塩基配列に、生物の種差（具体的には、ラットの系統差）、個体差若しくは器官、組織間の差異等により天然に生じる変異による塩基の欠失、置換若しくは付加が生じた塩基配列を有する遺伝子も含まれる。本遺伝子の翻訳産物量には、これらの遺伝子の翻訳産物量も含まれる。
In the present invention, the specimen refers to, for example, a hepatocyte constituting rat liver neoplastic lesion or preneoplastic lesion showing negative for an antibody recognizing placental glutathione-S-transferase enzyme, or a content thereof. Biological samples that may be included (for example, tissue specimens for optical microscopes) can be mentioned. Specifically, for example, tissues such as liver tissues collected from rats, or cells separated from these tissues, Or the cultured cell etc. can be mention | raise | lifted. These samples may be used as specimens as they are, or samples prepared by various operations such as separation, fractionation, and immobilization from such specimens may be used as specimens.
The 78-kDa glucose-regulated protein (hereinafter sometimes referred to as the present protein) used in the present invention is classified into one of the heat shock protein 70 (HSP70) family and is intracellular during ER stress. It is a protein known to be induced by. For example, it has a known amino acid sequence such as the amino acid sequence represented by SEQ ID NO: 1, and the protein is widely present in animals and plants, and its molecular weight is about 78,000 and consists of 654 amino acid residues. .
In the present invention, the 78-kDa glucose-regulated protein gene whose amount of translation product is measured (hereinafter sometimes referred to as the present gene) includes, for example, a base described together with the amino acid sequence represented by SEQ ID NO: 1. In addition to genes having the same nucleotide sequence as a known nucleotide sequence, such as sequences, the nucleotide sequence of the above gene includes species differences (specifically, rat strain differences), individual differences or differences between organs and tissues. A gene having a base sequence in which deletion, substitution, or addition of a base due to a naturally occurring mutation due to a difference or the like is also included. The amount of translation products of this gene includes the amount of translation products of these genes.
本遺伝子の翻訳産物量の測定は、例えば、単位量の検体当たりの本遺伝子の翻訳産物量を測定する方法等により行うことができる。
本遺伝子の翻訳産物量を測定するには、例えば、本蛋白質を発現している細胞からなる細胞巣の面積又は当該細胞巣の数を測定する。当該測定としては、例えば、免疫組織化学的検査法等を挙げることができる。
また、例えば、本遺伝子の塩基配列にコ−ドされるアミノ酸配列を有する蛋白質の量を測定する。特定の蛋白質の量の測定は、具体的には、例えば、当該蛋白質に対する特異抗体を用いた免疫学的測定法（例えば、ELISA、ウェスタンブロット、RIA、免疫組織化学的検査法等）、二次元電気泳動法、高速液体クロマトグラフィ−等により実施することができる。本遺伝子の塩基配列にコ−ドされるアミノ酸配列を有する蛋白質に対する特異抗体は、常法に準じて、本遺伝子の塩基配列にコ−ドされるアミノ酸配列を有する蛋白質を免疫抗原として調製することができる。
The amount of translation product of this gene can be measured, for example, by a method of measuring the amount of translation product of this gene per unit amount of specimen.
In order to measure the amount of translation product of this gene, for example, the area of the cell nest composed of cells expressing this protein or the number of the cell nest is measured. Examples of the measurement include an immunohistochemical examination method and the like.
In addition, for example, the amount of protein having an amino acid sequence encoded by the base sequence of this gene is measured. Specifically, the amount of a specific protein can be measured, for example, by an immunological assay using a specific antibody against the protein (for example, ELISA, Western blot, RIA, immunohistochemical test, etc.), two-dimensional It can be carried out by electrophoresis, high performance liquid chromatography or the like. A specific antibody against a protein having an amino acid sequence encoded by the base sequence of this gene should be prepared using a protein having an amino acid sequence encoded by the base sequence of this gene as an immunizing antigen according to a conventional method. Can do.
本発明検出方法において、免疫組織化学的検査法を用いて本遺伝子の検体における翻訳産物量を測定する方法についてさらに説明する。
まず、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変組織を構成する肝細胞或いはその内容物が含まれる可能性のある生体試料であるラット肝臓組織を、例えば、４％パラホルムアルデヒドで４℃、一晩固定する。固定後、適当な大きさに当該組織を切り出す。切り出された組織をアルコール脱水、パラフィン包埋した後、これをミクロスライサーを用いて約４μmの厚さで薄切し、薄切された組織切片をスライドグラスに載せる。
次いで、当該組織切片からキシレンを用いてパラフィンを除去した後、必要に応じて抗原の賦活化処理を行う。賦活化処理としては、例えば、以下のように行う。0.01Mクエン酸緩衝液（pH6.0）にスライドグラスに載せた組織切片を浸漬し、これをオートクレーブで１２１℃、５分間処理する。その後、これを適当な温度まで冷却する。賦活化後、内因性ペルオキシダーゼ活性を阻止するために、例えば、組織切片を３％過酸化水素水に５分間、室温で浸漬する。次いで、例えば、PBS buffer（組成：10mM リン酸ナトリウム ［pH7.4］, 100mM NaCl）で洗浄した後、当該組織切片に、必要に応じて一次抗体反応前の前処理を行う。当該前処理としては、例えば、二次抗体作製に用いた動物と同種の動物から調製された正常血清を当該組織切片にかけ、これを完全に覆った状態で室温、３０分間反応させればよい。当該前処理後、例えば、当該組織切片をPBS bufferで５分間×３回洗浄した後、当該組織切片に抗ラット本蛋白質抗体をかけ、これを完全に覆った状態で、例えば、室温、１時間反応させる。反応後、例えば、当該組織切片をPBS bufferで５分間×３回洗浄した後、二次抗体による反応を行う。例えば、当該組織切片に一次抗体の起源と同種の動物から調製されたビオチン化されたイムノグロブリンをかけ、これを完全に覆った状態で、例えば、室温、３０分間反応させる。更に、例えば、PBS bufferで５分間×３回洗浄した後、当該組織切片に３，３’−ジアミノベンジジン四塩酸塩（ナカライ製）を用いて発色させる。その後、当該組織切片を蒸留水で洗浄し、脱水、透徹、封入等の各種処理を行う。本蛋白質の発現量の指標となる染色性が低い場合には、必要に応じて、例えば、Tyramideを用いた抗原増感法を用いることで染色性を高めることもできる。
次いで、染色された組織切片を用いて、本蛋白質を発現している細胞からなる細胞巣のの面積又は細胞巣の数を測定する。当該測定では、例えば、画像解析装置IPAP（住化テクノサービス製）を用いて染色性が確認された細胞巣の面積及び組織切片全体の面積をそれぞれ測定し、下記式により単位面積当たりの検出された細胞巣の面積を算出する。また、染色性が確認された細胞巣の数をカウントし、下記式により単位面積当たりの検出された細胞巣の数を算出する。
In the detection method of the present invention, a method for measuring the amount of translation product in the specimen of this gene using an immunohistochemical test method will be further described.
First, there is a possibility that hepatocytes constituting rat liver neoplastic lesions or preneoplastic lesion tissues showing negative for an antibody recognizing placental glutathione-S-transferase enzyme or the contents thereof are included. Rat liver tissue, which is a biological sample, is fixed overnight, for example, with 4% paraformaldehyde at 4 ° C. After fixation, the tissue is cut into an appropriate size. After the cut tissue is dehydrated with alcohol and embedded in paraffin, it is sliced to a thickness of about 4 μm using a microslicer, and the sliced tissue section is placed on a slide glass.
Subsequently, after removing paraffin from the tissue section using xylene, an antigen activation treatment is performed as necessary. For example, the activation process is performed as follows. A tissue section placed on a slide glass is immersed in 0.01 M citrate buffer (pH 6.0), and this is treated with an autoclave at 121 ° C. for 5 minutes. Thereafter, it is cooled to a suitable temperature. After activation, in order to block endogenous peroxidase activity, for example, a tissue section is immersed in 3% hydrogen peroxide for 5 minutes at room temperature. Next, for example, after washing with PBS buffer (composition: 10 mM sodium phosphate [pH 7.4], 100 mM NaCl), the tissue section is pretreated before the primary antibody reaction as necessary. As the pretreatment, for example, normal serum prepared from an animal of the same kind as the animal used for the production of the secondary antibody may be applied to the tissue section and reacted for 30 minutes at room temperature in a completely covered state. After the pretreatment, for example, the tissue section is washed with PBS buffer for 5 minutes × 3 times, and then the anti-rat protein protein antibody is applied to the tissue section and completely covered, for example, at room temperature for 1 hour. React. After the reaction, for example, the tissue section is washed with PBS buffer for 5 minutes × 3 times, and then reacted with a secondary antibody. For example, a biotinylated immunoglobulin prepared from an animal of the same species as that of the primary antibody is applied to the tissue section, and this is completely covered, for example, at room temperature for 30 minutes. Further, for example, after washing with PBS buffer for 5 minutes × 3 times, the tissue section is developed with 3,3′-diaminobenzidine tetrahydrochloride (manufactured by Nacalai). Thereafter, the tissue section is washed with distilled water and subjected to various treatments such as dehydration, penetration, and encapsulation. When the staining property serving as an index of the expression level of the present protein is low, the staining property can be enhanced by using, for example, an antigen sensitization method using Tyramide as necessary.
Next, using the stained tissue section, the area of the cell nest composed of cells expressing this protein or the number of cell nests is measured. In this measurement, for example, the area of the cell nest and the area of the entire tissue section where the staining property was confirmed were measured using an image analysis apparatus IPAP (manufactured by Sumika Techno Service), and the area per unit area was detected by the following formula. Calculate the area of the cell nest. Further, the number of cell nests confirmed to be stained is counted, and the number of cell nests detected per unit area is calculated by the following formula.
＜単位面積当たりの検出された細胞巣の面積＞
（染色性が認められた細胞巣の面積）/（組織切片全体の面積）
<Detected cell nest area per unit area>
(Area of cell nest where staining was observed) / (area of whole tissue section)
＜単位面積当たりの検出された細胞巣の数＞
（染色性が認められた細胞巣の数）/（組織切片全体の面積）
<Number of detected cell nests per unit area>
(Number of cell nests where staining was observed) / (area of whole tissue section)
本遺伝子の翻訳産物量の測定は、上述のようにして、単位量の検体当たりの本遺伝子の翻訳産物量を測定する方法等により行うことができる。 The amount of translation product of this gene can be measured by the method of measuring the amount of translation product of this gene per unit amount of specimen as described above.
上記のようにして得られた前記検体における本遺伝子の翻訳産物量の測定値を、当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記検体における、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の有無或いはその発生程度を評価する。
当該評価において、第二工程で得られた遺伝子の翻訳産物量の測定値を当該遺伝子の翻訳産物量の対照値と比較すればよいが、この場合、例えば、上述のような免疫組織化学的検査法等によって本蛋白質を発現している細胞からなる細胞巣の面積又は当該細胞巣の数を測定する場合には、（１）前記検体において差異が認められる細胞からなる細胞巣を検出し、単位面積当たりの検出された細胞巣の面積又は検出された細胞巣の数と、（２）前記被験物質と接触させていないラット肝細胞における細胞からなる細胞巣を検出し、単位面積当たりの検出された細胞巣の面積又は検出された細胞巣の数とを比較し、その差異に基づいて前記被験物質の肝発癌活性の有無又はその量を評価してもよい。
本遺伝子の翻訳産物量の対照値としては、例えば、正常組織での細胞における当該遺伝子の翻訳産物量の値をあげることができる。ここで、正常組織とは、例えば、病理組織学的検査において異常の認められない組織を意味し、特に、翻訳産物量の測定をする方法として免疫組織化学的検査方法等を用いる場合には、翻訳産物量のシグナルが肝臓腫瘍性病変又は前腫瘍性病変を有する組織より明らかに弱い組織を示す。
かかる対照値は、正常組織での細胞における本遺伝子の翻訳産物量を、検体における当該遺伝子の翻訳産物量と併行して測定して求めてもよいし、別途測定して求めてもよい。また、複数の正常組織での細胞における本遺伝子の翻訳産物量を測定してその平均値を対照値としてもよい。
例えば、正常組織での細胞における本遺伝子の翻訳産物量の値を対照値として、検体における、本蛋白質の翻訳産物量の測定値が対照値よりも高ければ、検体中に胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変を有すると評価することができる。
The measured value of the translation product amount of the present gene in the sample obtained as described above is compared with a control value of the translation product amount of the gene, and based on the difference, placental glutathione-S- The presence or extent of rat liver neoplastic lesions or preneoplastic lesions that are negative for antibodies recognizing transferase enzymes is evaluated.
In the evaluation, the measured value of the translation product amount of the gene obtained in the second step may be compared with the control value of the translation product amount of the gene. In this case, for example, the immunohistochemical examination as described above When measuring the area of the cell nest composed of cells expressing this protein or the number of cell nests by the method or the like, (1) detecting the cell nest composed of cells in which a difference is observed in the specimen, Detected cell nest area per area or number of detected cell nests, and (2) detecting cell nests composed of cells in rat hepatocytes not in contact with the test substance, The presence or amount of hepatocarcinogenic activity of the test substance may be evaluated based on the difference between the area of the detected cell nests or the number of detected cell nests.
As a reference value of the translation product amount of this gene, for example, the value of the translation product amount 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 a histopathological examination, and particularly when an immunohistochemical examination method or the like is used as a method for measuring the amount of translation product, The translation product amount signal is clearly weaker than the tissue with liver neoplastic or preneoplastic lesions.
Such a control value may be obtained by measuring the translation product amount of the gene in cells in a normal tissue in parallel with the translation product amount of the gene in a specimen, or may be obtained by measurement separately. Alternatively, the amount of translation product of this gene in cells in a plurality of normal tissues may be measured, and the average value may be used as a control value.
For example, if the value of the translation product of the gene in cells in a normal tissue is used as a control value and the measured value of the translation product of the protein in the sample is higher than the control value, placental glutathione-S- It can be evaluated as having a rat liver neoplastic lesion or a preneoplastic lesion that is negative for an antibody recognizing a transferase enzyme.
本発明肝発癌活性検定方法においては、まず、ラット肝細胞と被験物質とを接触させる。用いられるラット肝細胞としては、ラットから採取された肝臓組織内に存在している状態にある肝細胞、或いは、当該組織から分離された細胞又はその培養細胞等があげられる。 In the method for assaying hepatocarcinogenic activity of the present invention, first, rat hepatocytes are brought into contact with a test substance. Examples of rat hepatocytes used include hepatocytes existing in liver tissues collected from rats, cells isolated from the tissues, or cultured cells thereof.
本発明肝発癌活性検定方法において、肝細胞と被験物質との接触は、例えば、ラットに被験物質を投与することにより行ってもよい。ラットは、天然の動物のほか、トランスジェニック動物、遺伝子ノックアウト動物等であってもよい。ラットへの被験物質の投与方法としては、例えば、経口（強制又は飲料水や餌に混じ）、筋肉内、静脈内、皮下、腹腔内、経気道等により行うことができる。投与量、投与回数及び投与期間は、例えば、全身状態、全身諸器官組織等に重篤な影響を及ぼさない範囲内（例えば投与量は、最大耐量）とすればよい。 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 rats. The rat may be a natural animal, a transgenic animal, a gene knockout animal, or the like. As a method for administering a test substance to rats, for example, oral (forced or mixed with drinking water or food), intramuscular, intravenous, subcutaneous, intraperitoneal, trans-airway, etc. can be used. The dose, the number of doses, and the administration period may be within a range that does not seriously affect the general state, systemic organ tissues, etc. (eg, the dose is the maximum tolerated dose).
次に、上記のようにして被験物質と接触させたラット肝細胞における、78-kDa glucose-regulated proteinの遺伝子の翻訳産物量を、上述のようにして測定する。
本遺伝子の翻訳産物量の測定は、上述のようにして、単位量の検体当たりの本遺伝子の転写物量を測定する方法、単位量の検体当たりの本遺伝子の翻訳産物量を測定する方法等により行うことができ、本遺伝子の転写物量を測定する方法を好ましくあげることができる。
Next, the amount of translation product of the 78-kDa glucose-regulated protein gene in the rat hepatocytes contacted with the test substance as described above is measured as described above.
As described above, the amount of translation product of this gene can be measured by the method of measuring the amount of transcript of this gene per unit amount of sample, the method of measuring the amount of translation product of this gene per unit amount of sample, etc. A method for measuring the amount of the transcript of this gene can be preferably mentioned.
上記のようにして被験物質と接触させた又はラット肝細胞から得られた本遺伝子の翻訳産物量の測定値を、当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記被験物質のラット肝発癌活性の有無又はその量を評価する。
本遺伝子の翻訳産物量の対照値としては、例えば、前記被験物質と接触させていないラット肝細胞における翻訳産物量の値をあげることができる。ラット肝細胞は、正常組織でのラット肝細胞における当該遺伝子の翻訳産物量の値を好ましくあげることができる。ここで正常組織とは、例えば、被験物質と接触していない肝臓組織で病理組織学的検査において異常の認められない組織を意味し、特に、翻訳産物量で測定する方法として免疫組織化学的検査方法を用いる場合には、被験物質と接触した肝臓組織であっても翻訳産物量のシグナルが、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変組織よりも明らかに弱い組織を示す。
かかる対照値は、正常組織での細胞における本遺伝子の翻訳産物量を、被験物質と接触させた肝臓組織での細胞における当該遺伝子の翻訳産物量と併行して測定して求めてもよいし、別途測定して求めてもよい。例えば、被験物質と接触させる前の肝臓組織の一部を採取して本遺伝子の翻訳産物量を測定し、得られた値を対照値とすることもできる。また、複数の正常組織での細胞における本遺伝子の翻訳産物量を測定してその平均値を対照値としてもよい。
例えば、被験物質と接触させた肝細胞における本遺伝子の翻訳産物量の測定値が、正常組織での細胞における本遺伝子の翻訳産物量の値よりも高ければ、前記被験物質との接触によるラット肝臓腫瘍性病変又は前腫瘍性病変組織を構成する肝細胞の発生を意味し、当該被験物質はラット肝発癌活性を有すると評価することができる。
The measured value of the translation product amount of this gene brought into contact with the test substance as described above or obtained from rat hepatocytes is compared with the control value of the translation product amount of the gene, and the test is performed based on the difference. The presence or amount of the substance's rat liver carcinogenic activity is evaluated.
As a reference value of the translation product amount of this gene, for example, the value of the translation product amount in rat hepatocytes not in contact with the test substance can be mentioned. Rat hepatocytes can preferably increase the amount of translation product of the gene in rat hepatocytes in normal tissues. Here, the normal tissue means, for example, a tissue that is not in contact with the test substance and has no abnormality in histopathological examination, and in particular, immunohistochemical examination as a method for measuring the amount of translation product When the method is used, rat liver tumorigenicity in which the signal of the amount of translation product is negative for the antibody recognizing the placental glutathione-S-transferase enzyme even in the liver tissue in contact with the test substance Tissues that are clearly weaker than lesions or preneoplastic lesions.
Such a control value may be obtained by measuring the translation product amount of the gene in cells in normal tissue in parallel with the translation product amount of the gene in cells in liver tissue contacted with the test substance, You may obtain | require by measuring separately. For example, a part of the liver tissue before contact with the test substance can be collected to measure the translation product amount of this gene, and the obtained value can be used as a control value. Alternatively, the amount of translation product of this gene in cells in a plurality of normal tissues may be measured, and the average value may be used as a control value.
For example, if the measured value of the translation product of this gene in hepatocytes contacted with the test substance is higher than the value of the translation product of this gene in cells in normal tissue, the rat liver due to contact with the test substance It means the generation of hepatocytes constituting a neoplastic lesion or a preneoplastic lesion tissue, and the test substance can be evaluated as having rat liver carcinogenic activity.
さらに、上記のような本発明肝発癌活性検定方法は、ラット肝発癌活性を有する物質の探索等に利用することができる。具体的には、本発明肝発癌活性検定方法により評価されたラット肝発癌活性の有無又はその量に基づきラット肝発癌活性を有する物質を選抜することによって、ラット肝発癌活性物質を探索することができる。選抜されたラット肝発癌活性物質は、例えば、肝癌モデル哺乳動物の作製等に利用することもできる。 Furthermore, the method for assaying hepatocarcinogenic activity of the present invention as described above can be used for searching for a substance having rat liver carcinogenic activity. Specifically, searching for a rat liver carcinogenic activity substance by selecting a substance having rat liver carcinogenic activity based on the presence or absence or the amount of rat liver carcinogenic activity evaluated by the method for assaying hepatocarcinogenic activity of the present invention. it can. The selected rat liver carcinogenic active substance can also be used for, for example, preparation of a liver cancer model mammal.
以下、実施例を挙げてさらに詳細に本発明を説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
実施例１ （免疫組織化学的検査を用いたラット肝発癌活性検定方法）
（１）ラットへの被験物質の投与
肝中期発癌性試験モデル(Ito, N., et al. CRC Crit Rev Toxicol, 19: 385−415, 1989.)を用いた。具体的には、被験物質が変異原性物質の場合には、雄F344系ラット（投与開始時６週齢）に、Ｎ−ジエチルニトロサミン（以下、DENと記す。）200mg/kg体重を腹腔内に単回投与した群を設け、対照として被験物質の溶媒を単回投与した群をさらに設けた。被験物質が非変異原性の場合には、同じ雄F344系ラットに上記と同用量のDENを単回投与した後２週目から被験物質として、クロフィブレ−ト（3000ppm）又はＷＹ−14,643（1000ppm）を混餌投与した群をそれぞれ設け、対照として基礎飼料のみを混餌投与した群をさらに設けた。被験物質の変異原性に関わらず、試験開始から３週目に2/3部分肝切除術を全例に施し、８週目に各群動物の肝臓を摘出した。
Example 1 (rat hepatocarcinogenic activity assay method using immunohistochemical examination)
(1) Administration of test substance to rat A liver metaphase carcinogenicity test model (Ito, N., et al. CRC Crit Rev Toxicol, 19: 385-415, 1989.) was used. Specifically, when the test substance is a mutagenic substance, N-diethylnitrosamine (hereinafter referred to as DEN) 200 mg / kg body weight intraperitoneally to male F344 rats (6 weeks old at the start of administration). A group administered with a single dose was provided, and a group administered with a single solvent of the test substance as a control was further provided. If the test substance is non-mutagenic, clofibrate (3000 ppm) or WY-14,643 (1000 ppm) as the test substance from the second week after the same dose of DEN is administered to the same male F344 rat as above. ) Was mixed with each group, and as a control, a group with only basal feed was further provided as a control. Regardless of the mutagenicity of the test substance, 2/3 partial hepatectomy was performed on all patients 3 weeks after the start of the test, and the livers of each group of animals were removed on the 8th week.
（２）肝臓組織の固定、包埋及び薄切切片の作製
摘出された肝臓を、固定液（4％パラホルムアルデヒド＋0.5％グルタールアルデヒド／0.075Mリン酸緩衝液、pH7.2〜7.4）を用いて４℃、一晩浸漬固定した。固定後、適当な大きさに当該組織を切り出した。切り出された組織をアルコール脱水、パラフィン包埋した後、これをミクロスライサーを用いて２〜１０μｍの厚さで薄切し、薄切された組織切片をスライドグラスに載せることにより、光学顕微鏡用組織標本を作製した。
(2) Fixation of liver tissue, embedding and preparation of sliced slices The excised liver was treated with a fixative (4% paraformaldehyde + 0.5% glutaraldehyde / 0.075M phosphate buffer, pH 7.2 to 7.4). Was fixed by immersion at 4 ° C. overnight. After fixation, the tissue was cut into an appropriate size. After the cut tissue is dehydrated with alcohol and embedded in paraffin, the tissue is sliced to a thickness of 2 to 10 μm using a micro slicer, and the sliced tissue section is placed on a slide glass to obtain a tissue for an optical microscope. A specimen was prepared.
免疫組織化学的染色法はCSAIIキット（ダコサイトメーション製）を用いて実施した。
上記で作製された組織切片を1×Target Retrieval Solution (pH6.1)（ダコサイトメーション製）に浸漬し、１２１℃、１５分間オートクレーブした後、６０℃まで室温下冷却することにより抗原の賦活化処理を行った。その後、組織切片をCSAIIキット添付のプロトコールに従い染色した。ラット本蛋白質の特異抗体（Santacruz）としては1000倍稀釈したものを使用し、immunoglobulins/HRPとしては抗ヤギ（ダコサイトメーション製）のものを200倍希釈したものを使用した。発色反応後、当該組織切片を蒸留水で洗浄し、脱水、透徹、封入を行った。
Immunohistochemical staining was performed using a CSAII kit (manufactured by Dakocytomation).
The tissue section prepared above is immersed in 1 × Target Retrieval Solution (pH6.1) (manufactured by Dakocytomation), autoclaved at 121 ° C. for 15 minutes, and then cooled to 60 ° C. at room temperature to activate the antigen. Processed. Thereafter, the tissue sections were stained according to the protocol attached to the CSAII kit. As a specific antibody (Santacruz) of this rat protein, a 1000-fold diluted antibody was used, and as immunoglobulins / HRP, an anti-goat (manufactured by Dakocytomation) diluted 200-fold was used. After the color development reaction, the tissue section was washed with distilled water, dehydrated, transparent, and sealed.
（４）遺伝子翻訳産物の発現解析
画像解析装置IPAP（住化テクノサービス製）を用いて染色性が確認された細胞巣の面積及び組織切片全体の面積をそれぞれ測定し、単位面積当たりの検出された細胞巣の面積[＝（染色性が認められた細胞巣の面積）/（組織切片全体の面積）]を算出した。また染色性が確認された細胞巣の数をカウントし、単位面積当たりの検出された細胞巣の数[＝（染色性が認められた細胞巣の数）/（組織切片全体の面積）]を算出した。各種の検定手法を用いて統計学的解析を行い、被験物質投与群における値が基礎飼料投与群における当該値よりも有意な増加を示した場合には、被験物質はラット肝発癌性を有すると評価した。
(4) Expression analysis of gene translation product Using the image analysis device IPAP (manufactured by Sumika Techno Service), the area of the cell nest and the area of the entire tissue section confirmed to be stained are measured, and the detected per unit area is detected. The area of the cell nest [= (the area of the cell nest in which staining was observed) / (the area of the entire tissue section)] was calculated. Also, count the number of cell nests for which staining was confirmed, and the number of cell nests detected per unit area [= (number of cell nests for which staining was observed) / (area of whole tissue section)] Calculated. When statistical analysis is performed using various test methods, and 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. evaluated.
本発明によって、遺伝子の翻訳産物量の異常を指標としたラットにおける胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の検定方法等が提供可能となる。 According to the present invention, rat liver neoplastic lesions or preneoplastic lesions that are negative for an antibody recognizing placental glutathione-S-transferase enzyme in rats, using as an index an abnormality in the amount of translation product of the gene. A method or the like can be provided.
Claims (12)
（１）検体における、78-kDa glucose-regulated proteinの遺伝子の翻訳産物量を測定する第一工程、及び
（２）第一工程で得られた前記検体における遺伝子の翻訳産物量の測定値を当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記検体における、胎盤型グルタチオン−Ｓ−トランスフェラ−ゼ酵素を認識する抗体に対して陰性を示すラット肝臓腫瘍性病変又は前腫瘍性病変の有無或いはその発生程度を評価する第二工程
を有することを特徴とする方法。 A method for assaying rat liver neoplastic lesions or preneoplastic lesions that is negative for an antibody recognizing placental glutathione-S-transferase enzyme, comprising:
(1) the first step of measuring the translation product amount of the 78-kDa glucose-regulated protein gene in the sample, and (2) the measured value of the translation product amount of the gene in the sample obtained in the first step Rat liver neoplastic lesion or pre-tumor that is negative for the antibody recognizing placental glutathione-S-transferase enzyme in the specimen based on the difference compared with the control value of the amount of gene translation product A method comprising a second step of evaluating the presence or absence of a sexual lesion or the degree of occurrence thereof.
（１）ラット肝細胞と被験物質とを接触させる第一工程、
（２）第一工程で前記被験物質と接触させた前記肝細胞における、78-kDa glucose-regulated proteinの遺伝子の翻訳産物量を測定する第二工程、及び
（３）第二工程で得られた遺伝子の翻訳産物量の測定値を当該遺伝子の翻訳産物量の対照値と比較し、その差異に基づいて前記被験物質のラット肝発癌活性の有無又はその量を評価する第三工程
を有することを特徴とする方法。 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) Second step of measuring the translation product amount of the 78-kDa glucose-regulated protein gene in the hepatocytes contacted with the test substance in the first step, and (3) obtained in the second step Comparing the measured value of the translation product amount of the gene with a control value of the translation product amount of the gene, and having a third step of evaluating the presence or absence of the rat liver carcinogenic activity of the test substance based on the difference Feature method.
12. A rat liver carcinogen comprising a step of selecting a substance having rat liver carcinogenic activity based on the presence or absence of the rat liver carcinogenic activity evaluated by the method according to any one of claims 7 to 11. Search method.
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| CN115391431A (en) * | 2022-10-28 | 2022-11-25 | 山东蓝客信息科技有限公司 | Specimen information acquisition and verification system |
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| JP2004344069A (en) * | 2003-05-22 | 2004-12-09 | Sumitomo Chem Co Ltd | Method for examining precancerous lesion of rat liver negative to antibody recognizing placenta-type glutathione-s-transferase enzyme |
| WO2005045428A2 (en) * | 2003-10-27 | 2005-05-19 | University Of Southern California | Methods and compositions for modulating apoptosis |
| JP2005514015A (en) * | 2001-12-19 | 2005-05-19 | アボット・ラボラトリーズ | Use of endothelial cell receptors |
| JP2005176764A (en) * | 2003-12-22 | 2005-07-07 | Sumitomo Chemical Co Ltd | Method for assaying hepato-neoplastic lesion or pre-neoplastic lesion of rat negative to antibody recognizing placental glutathione-s-transferase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2004531713A (en) * | 2001-04-03 | 2004-10-14 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Tumor markers for renal cell carcinoma |
| JP2005514015A (en) * | 2001-12-19 | 2005-05-19 | アボット・ラボラトリーズ | Use of endothelial cell receptors |
| JP2004344069A (en) * | 2003-05-22 | 2004-12-09 | Sumitomo Chem Co Ltd | Method for examining precancerous lesion of rat liver negative to antibody recognizing placenta-type glutathione-s-transferase enzyme |
| WO2005045428A2 (en) * | 2003-10-27 | 2005-05-19 | University Of Southern California | Methods and compositions for modulating apoptosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115391431A (en) * | 2022-10-28 | 2022-11-25 | 山东蓝客信息科技有限公司 | Specimen information acquisition and verification system |
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