JP7038998B2 - Cellular cancer strains and animal models for developing primary liver cancer using them - Google Patents
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本発明は、免疫不全動物等の生体内での悪性度を高め、野生型動物等に生着可能である肝細胞癌株や胆管細胞癌株として樹立された細胞癌株、及びそれを用いた肝細胞癌や胆管細胞癌である原発性肝癌の発症動物モデルに関するものである。 The present invention uses a hepatocellular carcinoma strain, a cell carcinoma strain established as an intrahepatic cell carcinoma strain, and a cell carcinoma strain established as a hepatocellular carcinoma strain and a bile duct cell carcinoma strain that can be engrafted in wild-type animals and the like by increasing the malignancy in vivo such as immunodeficient animals. It relates to an animal model of the onset of primary liver cancer, which is hepatocellular carcinoma or intrahepatic cell carcinoma.
癌細胞には、癌細胞を攻撃するリンパ球の一種の細胞傷害性Ｔ細胞（キラーＴ細胞：ＣＴＬ細胞）の活性を低下させる免疫チェックポイントがある。肝臓癌のような癌疾患の免疫治療として、免疫チェックポイントを抑制する免疫標的薬が知られている。 Cancer cells have immune checkpoints that reduce the activity of cytotoxic T cells (killer T cells: CTL cells), a type of lymphocyte that attacks cancer cells. Immune target drugs that suppress immune checkpoints are known as immunotherapies for cancer diseases such as liver cancer.
癌細胞を攻撃するため活性化したキラーＴ細胞の表面にＰＤ－１分子があり、また癌細胞にはキラーＴ細胞の活性を抑制するＰＤ－Ｌ１分子があり、両者が結合するとキラーＴ細胞の活性が抑制されてしまう。そこで、抗ＰＤ－１抗体はＰＤ－１と結合することにより、また抗ＰＤ－Ｌ１抗体はＰＤ－Ｌ１と結合するにより、ＰＤ－１分子とＰＤ－Ｌ１分子との結合を阻害し、キラーＴ細胞を活性化させ、免疫細胞が正常に機能することによって癌細胞を殺傷する。抗ＰＤ－１抗体は悪性黒色腫の治療に利用されており、治療効果が実証されている。これら免疫治療は、腎細胞癌、食道癌、肺癌での臨床試験が行われている。 There is a PD-1 molecule on the surface of the killer T cell activated to attack the cancer cell, and there is a PD-L1 molecule that suppresses the activity of the killer T cell in the cancer cell. The activity is suppressed. Therefore, the anti-PD-1 antibody binds to PD-1 and the anti-PD-L1 antibody binds to PD-L1 to inhibit the binding between the PD-1 molecule and the PD-L1 molecule, and the killer T is used. It activates cells and kills cancer cells by the normal functioning of immune cells. Anti-PD-1 antibody has been used for the treatment of malignant melanoma, and its therapeutic effect has been demonstrated. These immunotherapies have been clinically tested in renal cell carcinoma, esophageal cancer, and lung cancer.
原発性肝癌（肝癌）には、肝臓の大部分を占める肝細胞の癌である肝細胞癌と、肝臓で産生された胆汁を十二指腸へ運ぶ胆管の細胞の癌である胆管細胞癌（肝内胆管癌）とがある。肝癌の多くは、慢性肝炎や肝硬変から発症したり、感染したウィルスにより発症したＢ型肝炎やＣ型肝炎で肝細胞の遺伝子に突然変異が起こって癌化したりする。肝癌は、初期では症状に乏しく、症状が現れた時点では進行した状態である臨床症例が多い。肝癌についても、近い将来、免疫チェックポイントの抑制薬を用いた免疫治療、又は腫瘍増殖を直接抑える抗癌剤や分子標的薬と免疫チェックポイントの抑制薬との併用療法が行われるようになると言われている。 Primary liver cancer (liver cancer) includes hepatocellular carcinoma, which is a cancer of the liver cells that occupy most of the liver, and intrahepatic cell carcinoma (intrahepatic bile duct), which is a cancer of the cells of the bile duct that carries the bile produced in the liver to the duodenum. Cancer). Most liver cancers develop from chronic hepatitis or cirrhosis, or hepatitis B or C caused by an infected virus causes mutations in hepatocyte genes to cause cancer. Liver cancer has few symptoms at the initial stage, and there are many clinical cases in which the symptoms are advanced when the symptoms appear. For liver cancer, it is said that in the near future, immunotherapy using immune checkpoint inhibitors or combination therapy with anticancer agents or molecular target drugs that directly suppress tumor growth and immune checkpoint inhibitors will be performed. There is.
その治療法や治療薬の開発のためには動物試験とりわけマウスでの前臨床動物試験が重要である。これらの抗癌剤や分子標的薬と免疫チェックポイントの抑制薬の候補化合物のスクリーニングや開発、前臨床動物試験での作用メカニズムの解明等で汎用されているマウスは、主にＣ５７ＢＬ／６系統とＢＡＬＢ／Ｃ系統との２系統のマウスであり、他にＣ５７Ｌ系統マウスも知られている。Ｃ５７ＢＬ／６系統マウスは、ヒトに続いて全ゲノム配列が明らかになった動物種であり、遺伝子改変マウスモデルとして汎用されているが、癌細胞を初めとする細胞株の樹立が困難な動物種である。一方、ＢＡＬＢ／Ｃ系統マウスから、殆どの癌細胞株が作出されており、例えば市販のマウス肝細胞癌株ＢＮＬはＢＡＬＢ／Ｃ系統マウス由来の細胞株である。別なマウス肝細胞癌株Ｈｅｐａ１－６はＣ５７Ｌ系統マウス由来である。本発明者が検討したところ、ＢＮＬとＨｅｐａ１－６とのいずれもＣ５７ＢＬ／６系統マウスに生着させようとしても、腫瘍形成は認められなかった。 Animal studies, especially preclinical animal studies in mice, are important for the development of therapeutic methods and drugs. C57BL / 6 strains and BALB / It is a mouse of two strains with the C strain, and a C57L strain mouse is also known. The C57BL / 6 strain mouse is an animal species whose entire genome sequence has been clarified following humans, and is widely used as a genetically modified mouse model, but it is difficult to establish cell lines such as cancer cells. Is. On the other hand, most cancer cell lines have been produced from BALB / C strain mice. For example, the commercially available mouse hepatocellular carcinoma strain BNL is a cell line derived from BALB / C strain mice. Another mouse hepatocellular carcinoma strain Hepa1-6 is derived from C57L strain mice. As a result of examination by the present inventor, no tumor formation was observed when both BNL and Hepa1-6 were attempted to engraft in C57BL / 6 strain mice.
現在使用されているマウスの担癌モデルとして、ヒトの癌細胞を免疫不全マウスに生着させるＸｅｎｏｇｒａｆｔモデルが知られている。例えば特許文献１に、（ａ）ヒト肝癌－特異抗原を発現する癌細胞株を、非ヒト正常動物に投与して癌を誘発させる段階と、（ｂ）前記癌が誘発された動物に分析対象の樹状細胞を投与する段階と、（ｃ）前記動物から癌細胞の形成または成長を測定し、樹状細胞由来の肝癌免疫治療剤の治療効能を決定する段階とを含む、ヒト肝癌動物モデルを利用した樹状細胞由来の肝癌免疫治療剤の肝癌治療効能を分析する方法が、開示されている。このＸｅｎｏｇｒａｆｔモデルでは、免疫不全マウスを用いているため免疫チェックポイントの抑制による免疫の解析ができない。免疫不全マウスには免疫学的な細胞としてナチュラルキラー細胞（ＮＫ細胞）やマクロファージが残っているが、獲得免疫に有用なＢ細胞やＴ細胞が存在しないため、腫瘍を特異的に破壊し得る細胞傷害性Ｔ細胞（ＣＴＬ細胞）が増強されたことを評価できないという問題がある。 As a cancer-bearing model of mice currently used, a Xenograft model in which human cancer cells are engrafted in immunodeficient mice is known. For example, in Patent Document 1, (a) a stage in which a cancer cell line expressing a human liver cancer-specific antigen is administered to a non-human normal animal to induce cancer, and (b) an analysis target in the animal in which the cancer is induced. A human liver cancer animal model comprising the steps of administering dendritic cells and (c) measuring the formation or growth of cancer cells from the animal to determine the therapeutic efficacy of a dendritic cell-derived hepatocancer immunotherapeutic agent. A method for analyzing the therapeutic efficacy of a dendritic cell-derived immunotherapeutic agent for liver cancer is disclosed. Since this Xenograft model uses immunodeficient mice, it is not possible to analyze immunity by suppressing immune checkpoints. Natural killer cells (NK cells) and macrophages remain as immunological cells in immunodeficient mice, but cells that can specifically destroy tumors because there are no B cells or T cells useful for acquired immunity. There is a problem that it cannot be evaluated that the cytotoxic T cells (CTL cells) are enhanced.
また、系統の異なるマウスを用いるＡｌｌｏｇｒａｆｔモデルも知られている。このモデルは、例えばＢＡＬＢ／Ｃ由来の肝癌細胞ＢＮＬを系統の異なるＣ５７ＢＬ／６マウスに生着させるというものである。このようなモデルでは系統が異なることから主要組織適合性抗原が違うので生体拒絶を惹起するという問題がある。そうすると拒絶反応で癌細胞が成長しないのか癌免疫が活性化して癌細胞が成長しないのか分からない。 Also, an Alllograft model using mice of different strains is known. In this model, for example, BALB / C-derived liver cancer cells BNL are engrafted in C57 BL / 6 mice of different strains. In such a model, since the strains are different, the major histocompatibility complex antigens are different, which causes a problem of inducing biological rejection. Then, it is unknown whether the cancer cells do not grow due to rejection or the cancer immunity is activated and the cancer cells do not grow.
別なマウスの担癌モデルとしてＳｙｎｇｅｏｎｉｃ ｇｒａｆｔモデルも知られている。このモデルは、マウスの癌細胞を同系統のマウスに生着させるというものであり、例えば、肝癌細胞としてＢＡＬＢ／Ｃ由来のＢＮＬ癌細胞をＢＡＬＢ／Ｃに生着させるというものである。同系統移植であるため拒絶反応を惹起し難い点で前二種のモデルより優れている。しかしＢＡＬＢ／Ｃは遺伝子改変マウスが殆ど使われていないため、このモデルは実用的でなく汎用性に乏しいという問題がある。 The Syngenic graft model is also known as a cancer-bearing model of another mouse. This model engrafts mouse cancer cells in mice of the same strain, for example, engrafting BALB / C-derived BNL cancer cells as liver cancer cells in BALB / C. Since it is a homologous transplant, it is superior to the previous two models in that it is less likely to cause rejection. However, since BALB / C rarely uses genetically modified mice, this model has a problem that it is not practical and has poor versatility.
今後、免疫チェックポイントのような癌生存シグナルと患者免疫シグナルとのインタラクションを作用点とする抗癌創薬が主流になりつつあることから、従来のモデルのような異系統動物種での癌細胞株の移植組み合わせ又は遺伝子非改変動物種での癌細胞株の移植では、癌発症の微小環境／メカニズム検証に課題が残る。 In the future, anticancer drug discovery that uses the interaction between the cancer survival signal and the patient's immune signal as the point of action, such as an immune checkpoint, is becoming the mainstream. Transplantation of strains or transplantation of cancer cell lines in unmodified animal species leaves a challenge in verifying the microenvironment / mechanism of cancer onset.
そのため、遺伝子導入マウスやノックアウトマウスのような遺伝子改変マウスとして汎用されており交配が容易で頑健なＣ５７ＢＬ／６マウス又は免疫不全マウスに、同系統のＣ５７ＢＬ／６マウス由来の癌細胞を、生着させて動物モデルと成すのが好ましい。 Therefore, cancer cells derived from C57 BL / 6 mice of the same strain are used in C57 BL / 6 mice or immunodeficient mice, which are widely used as genetically modified mice such as gene-introduced mice and knockout mice and are easy to mate and robust. It is preferable to engraft to form an animal model.
また、腫瘍環境の研究をする際、腫瘍血管や宿主の免疫担当分子や血管の因子や血管壁の因子等のマーカーや生理活性物質が多量に発現したとき免疫チェックポイントの効能があるか否か分子学的に研究する際に、Ｃ５７ＢＬ／６マウス由来の癌細胞株を用いて、同系統のＣ５７ＢＬ／６トランスジェニックマウス又は免疫不全マウスで行う必要がある。 In addition, when studying the tumor environment, whether or not the immunocheckpoint is effective when a large amount of markers such as tumor blood vessels, host immunodeficient molecules, blood vessel factors, and blood vessel wall factors, and physiologically active substances are expressed. Tumor cell lines derived from C57 BL / 6 mice should be used for molecular studies in C57 BL / 6 transgenic mice or immunodeficient mice of the same strain.
それにも関わらず、肝臓癌の動物モデルは極めて限られており患者に即したデータを取り得る有用な動物モデルは、殆ど無い。 Nevertheless, animal models of liver cancer are extremely limited, and there are few useful animal models that can obtain patient-specific data.
そこでＣ５７ＢＬ／６マウスのような動物に由来するもので、免疫不全動物等の生体内での悪性度を高め、野生型マウス（ＷＴマウス）等の動物に生着可能である肝細胞癌株や胆管細胞癌株として細胞癌株を樹立すること、及びそれを用いた肝細胞癌や胆管細胞癌である原発性肝癌の発症動物モデルを確立することが、求められている。 Therefore, a hepatocellular carcinoma strain derived from an animal such as C57 BL / 6 mouse, which can increase the malignancy in vivo such as an immunodeficient animal and can engraft in an animal such as a wild type mouse (WT mouse). It is required to establish a cell carcinoma strain as a bile duct cell carcinoma strain and to establish an animal model for the onset of primary hepatocellular carcinoma, which is a hepatocellular carcinoma or a bile duct cell carcinoma, using the cell carcinoma strain.
本発明は前記の課題を解決するためになされたもので、実験動物例えば免疫不全マウスの生体内での悪性度を高め、野生型マウスのような動物に生着可能な肝細胞癌や胆管細胞癌である細胞癌株、及びその樹立方法、並びにその細胞癌株を免疫不全動物や野生型動物等に生着させた肝細胞癌や胆管細胞癌である原発性肝癌の発症動物モデルを提供することを目的とする。 The present invention has been made to solve the above-mentioned problems, and it enhances the malignancy of experimental animals such as immunodeficient mice in vivo and can engraft in animals such as wild-type mice. Hepatocellular carcinoma and bile duct cells. Provided are a cell cancer strain which is a cancer, a method for establishing the cell cancer strain, and a model of an onset animal of hepatocellular carcinoma or a primary liver cancer which is an intrahepatic cell carcinoma in which the cell cancer strain is engrafted in an immunodeficient animal, a wild-type animal, or the like. The purpose is.
前記の目的を達成するためになされた細胞癌株は、Ｃ型肝炎ウィルスを遺伝子導入した動物としてＣ５７ＢＬ／６系統マウスに、動脈硬化性高コレステロール食と高中性脂肪食とを摂取させる工程、前記動物の肝臓に肝癌を発生させる工程、発生した肝癌組織から肝癌細胞を採取する工程、採取した前記肝癌細胞を培養する工程によって、肝細胞癌株及び／又は胆管細胞癌株として、樹立したものである。 The cell cancer strain made to achieve the above-mentioned object is a step of feeding a C57BL / 6 strain mouse as an animal into which hepatocyte C virus has been gene-introduced with an arteriosclerotic high cholesterol diet and a high neutral fat diet. It was established as a hepatocellular carcinoma strain and / or an intrahepatic cell carcinoma strain by a step of causing liver cancer in the liver of an animal, a step of collecting liver cancer cells from the developed liver cancer tissue, and a step of culturing the collected liver cancer cells. be.
この細胞癌株は、前記採取する工程の後、それで得られた前記肝癌細胞を動物に摂取して腫瘍生着させてから腫瘍細胞から再度、癌細胞を採取して悪性化させる工程を行ってから、それを前記培養する工程を行うことによって、樹立したものであってもよい。 This cell cancer strain is subjected to a step of ingesting the obtained liver cancer cells into an animal to cause tumor engraftment after the step of collecting the cancer cells, and then collecting the cancer cells from the tumor cells again to make them malignant. Therefore , it may be established by performing the step of culturing it.
この細胞癌株は、好ましくは、前記動脈硬化性高コレステロール食が、コレステロールを０．１～１０質量％含んでおり、前記高中性脂肪食が、動物性脂肪及び／又は植物性脂肪を含む中性脂肪を１５～８０質量％含んでいることにより、得られるものである。 In this cell cancer strain, preferably, the arteriosclerotic high-cholesterol diet contains 0.1 to 10% by mass of cholesterol, and the high-neutral fat diet contains animal fat and / or vegetable fat. It is obtained by containing 15 to 80% by mass of sex fat.
この細胞癌株は、例えば受託番号がＮＩＴＥ Ｐ－０２３５３、又は受託番号がＮＩＴＥ Ｐ－０２５５８のものである。 This cell cancer strain is, for example, one having a consignment number of NITE P-02353 or a consignment number of NITE P-02558.
前記の目的を達成するためになされたこの細胞癌株の樹立方法は、Ｃ型肝炎ウィルスを遺伝子導入した動物としてＣ５７ＢＬ／６系統マウスに、動脈硬化性高コレステロール食と高中性脂肪食とを摂取させる工程、前記動物の肝臓に肝癌を発生させる工程、発生した肝癌組織から肝癌細胞を採取する工程、採取した前記肝癌細胞を培養する工程によって、肝細胞癌株及び／又は胆管細胞癌株として、細胞癌株を樹立するというものである。 The method for establishing this cell cancer strain to achieve the above-mentioned purpose is to ingest an arteriosclerotic high cholesterol diet and a high neutral fat diet into C57BL / 6 strain mice as an animal into which hepatitis C virus has been gene-introduced. As a hepatocellular carcinoma strain and / or an intrahepatic cell carcinoma strain by a step of causing liver cancer in the liver of the animal, a step of collecting liver cancer cells from the developed liver cancer tissue, and a step of culturing the collected liver cancer cells. It is to establish a cell cancer strain.
この細胞癌株の樹立方法は、前記採取する工程の後、それで得られた前記肝癌細胞を動物に投与して腫瘍生着させてから腫瘍細胞から再度、癌細胞を採取して悪性化させる工程を行ってから、それを前記培養する工程を行うものであってもよい。 The method for establishing this cell cancer strain is a step of administering the obtained liver cancer cells to an animal after the step of collecting the cells to cause tumor engraftment, and then collecting the cancer cells again from the tumor cells to make them malignant. Then, the step of culturing it may be performed.
前記の目的を達成するためになされた肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルは、Ｃ型肝炎ウィルスを遺伝子導入した癌細胞発生用の動物としてＣ５７ＢＬ／６系統マウスに、動脈硬化性高コレステロール食と高中性脂肪食とを摂取させる工程；前記動物の肝臓に肝癌を発生させる工程；発生した肝癌組織から肝癌細胞を採取する工程；採取した前記肝癌細胞を培養する工程によって、肝細胞癌株及び／又は胆管細胞癌株として細胞癌株を樹立した後、同系統又は異系統若しくは異種で癌細胞生着用の動物の肝臓に、前記細胞癌株を生着させて、腫瘍を形成させることによって、得られたものである。 The animal model for developing primary liver cancer, which is hepatocellular carcinoma and / or bile duct cell cancer, which was made to achieve the above objectives, is a C57BL / 6 strain mouse as an animal for developing cancer cells into which hepatitis C virus has been gene-introduced. A step of ingesting an arteriosclerotic high cholesterol diet and a high neutral fat diet; a step of causing liver cancer in the liver of the animal; a step of collecting liver cancer cells from the developed liver cancer tissue; culturing the collected liver cancer cells. After establishing a cell cancer line as a hepatocellular carcinoma line and / or a bile duct cell cancer line by a step, the cell cancer line is engrafted in the liver of an animal of the same lineage, a different lineage, or a heterologous cancer cell-bearing animal. , Obtained by forming a tumor.
この原発性肝癌の発症動物モデルは、前記癌細胞発生用の動物が前記Ｃ５７ＢＬ／６系統マウスであり、前記癌細胞生着用の動物が野生型マウス、遺伝子改変マウス、及び／又は免疫不全マウスであることが好ましい。 In this primary liver cancer onset animal model, the animal for developing cancer cells is the C57BL / 6 strain mouse, and the animal wearing the cancer cells is a wild-type mouse, a genetically modified mouse, and / or an immunodeficient mouse. It is preferable to have.
この原発性肝癌の発症動物モデルは、例えば前記癌細胞発生用の動物がＣ５７ＢＬ／６系統マウスであり、前記癌細胞生着用の動物が野生型マウス、遺伝子改変マウス、及び／又は免疫不全マウスであってもよい。 In the animal model for developing primary liver cancer, for example, the animal for developing cancer cells is a C57BL / 6 strain mouse, and the animal wearing the cancer cells is a wild-type mouse, a genetically modified mouse, and / or an immunodeficient mouse. There may be.
この原発性肝癌の発症動物モデルは、例えば被スクリーニング試料を投与し、前記腫瘍の形成の促進又は抑制を指標にしてスクリーニングするためのものであり、及び／又は、前記腫瘍の形成を観察するためのものであるというものである。 This animal model of developing primary liver cancer is for, for example, administering a sample to be screened and screening using the promotion or suppression of tumor formation as an index, and / or observing the formation of the tumor. It is a thing.
本発明の肝細胞癌株及び／又は胆管細胞癌株である細胞癌株は、移植・生着によって腫瘍を形成し易く、免疫不全動物等の生体内での悪性度が高いものである。また、野生型マウス等の動物に生着可能であって、極めて肝細胞癌や胆管癌の細胞に誘導及び／又は分化し易いものである。また、この細胞癌株は、従来の非アルコール性肝炎（ＮＡＳＨ）動物モデルよりも遥かに悪性度が高く、ヒトの肝癌に近い病態を動物実験で反映できる。 The hepatocellular carcinoma strain and / or the cell carcinoma strain which is a bile duct cell carcinoma strain of the present invention is likely to form a tumor by transplantation / engraftment, and has a high degree of malignancy in vivo such as an immunodeficient animal. In addition, it can engraft in animals such as wild-type mice, and is extremely easy to induce and / or differentiate into cells of hepatocellular carcinoma and cholangiocarcinoma. In addition, this cell cancer strain is far more malignant than the conventional non-alcoholic steatohepatitis (NASH) animal model, and can reflect the pathological condition close to human liver cancer in animal experiments.
この肝細胞癌株及び／又は胆管細胞癌株である細胞癌株の樹立方法によれば、均質で均等な悪性度の細胞癌株を得ることができる。また、この細胞癌株は、継体培養が可能で、マイコプラズマ感染などを起こさずに保管でき、各種の動物実験に用いることが可能である。 According to the method for establishing a hepatocellular carcinoma strain and / or a cell carcinoma strain which is a bile duct cell carcinoma strain, a uniform and uniform grade of cell carcinoma strain can be obtained. In addition, this cell cancer strain can be subcultured, can be stored without causing mycoplasma infection, and can be used in various animal experiments.
そのため、この細胞癌株を同系統又は異系統若しくは異種で癌細胞生着用の動物、例えば免疫不全マウスや野生型マウスの肝臓に、前記細胞癌株を生着させて、肝細胞癌や胆管癌の細胞を有する悪性腫瘍を形成することができる、肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルは、腫瘍増殖を直接抑えるのに有用な抗癌剤や分子標的薬のスクリーニングや、免疫チェックポイントの抑制薬のような免疫標的薬のスクリーニング、肝癌で産生される生体マーカーや生理活性物質の探索、微小癌環境の観察や発癌メカニズムの解析に用いることができる。 Therefore, this cell cancer strain is engrafted in the liver of an animal of the same strain, a different strain, or a heterogeneous cancer cell-bearing animal, for example, an immunodeficient mouse or a wild-type mouse, and the cell cancer strain is engrafted to cause hepatocellular carcinoma or bile duct cancer. Animal models of primary liver cancer, which is hepatocellular carcinoma and / or bile duct cell carcinoma, capable of forming malignant tumors with cells of It can be used for screening immune target drugs such as inhibitors of immune checkpoints, searching for biological markers and physiologically active substances produced in liver cancer, observing the microcancer environment, and analyzing the carcinogenic mechanism.
この原発性肝癌の発症動物モデルは、短期間の腫瘍発生によりヒトの肝癌に近い病態を作製でき、免疫チェックポイントの実験モデル、癌転移のモデルとしても有用である。 This animal model of the onset of primary liver cancer can create a pathological condition similar to that of human liver cancer by developing a tumor in a short period of time, and is also useful as an experimental model of immune checkpoint and a model of cancer metastasis.
以下、本発明を実施するための形態を詳細に説明するが、本発明の範囲はこれらの形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail, but the scope of the present invention is not limited to these embodiments.
本発明の肝細胞癌株及び／又は胆管細胞癌株である細胞癌株は、in vivoでＣ型肝炎ウィルス（ＨＣＶ）を遺伝子導入した癌細胞発生用の動物でありアレルがホモ(HCV/HCV)又はヘテロ(HCV/-)のトランスジェニックマウスに、動脈硬化性高コレステロール食（Ａｔｈ）と高中性脂肪食（ＨＦ）とのコレステロール・中性脂肪過多高カロリー食を野生型マウス(オス)に対し８週齢～６８週齢まで摂取させる工程、トランスジェニックマウスの肝臓に肝癌を発生させる工程、その摂食期間経過後、例えば６～7割以上の高頻度で発症した肝癌細胞をそのトランスジェニックマウスの肝癌組織から採取し顕微鏡検査によって肝癌細胞の存在を同定してから取り出し又は組織分散粉砕装置例えばGentle MACS Dissociation Kit(Miltenyi Biotec社の商品名)を用いて細胞分離して取り出す工程、in vitroで採取した肝癌細胞をＤＭＥＭ（ダルベッコ改変イーグル培地）のような培地にて培養皿又はＤＭＥＭのような培養液中で培養し必要に応じて継体培養する工程によって、樹立したものである。 The hepatic cell cancer strain and / or the cell cancer strain which is a bile duct cell cancer strain of the present invention is an animal for developing cancer cells into which hepatitis C virus (HCV) has been gene-introduced in vivo, and the allele is homo (HCV / HCV). ) Or hetero (HCV /-) transgenic mice, and wild-type mice (male) with a cholesterol / neutral fat-rich high-calorie diet consisting of an arteriosclerotic high-cholesterol diet (Ath) and a high-neutral fat diet (HF). On the other hand, a step of ingesting from 8 weeks to 68 weeks of age, a step of causing liver cancer in the liver of transgenic mice, and after the lapse of the feeding period, for example, liver cancer cells that develop at a high frequency of 60 to 70% or more are transgenic. A step of collecting from mouse liver cancer tissue, identifying the presence of liver cancer cells by microscopic examination, and then extracting or separating and extracting the cells using a tissue dispersion crusher, for example, Gentle MACS Dissociation Kit (trade name of Miltenyi Biotec), in vitro. It was established by a step of culturing the liver cancer cells collected in 1 in a culture dish such as DMEM (Dalveco modified eagle medium) in a culture dish or a culture solution such as DMEM and subculturing as necessary.
遺伝子導入した動物は、トランスジェニックマウス、とりわけ遺伝的背景が同一の血統であり、市販されており容易く入手可能であって、交配が容易で頑健性に優れ遺伝子操作に適したＣ５７ＢＬ／６系統マウスであると、好ましい。 The transgenic animals are transgenic mice, especially C57BL / 6 strain mice that have the same genetic background, are commercially available, easily available, are easy to mate, have excellent robustness, and are suitable for genetic manipulation. Is preferable.
ＨＣＶを遺伝子導入されたＣ５７ＢＬ／６系統トランスジェニックマウスの場合、動脈硬化性高コレステロール食（Ａｔｈ）と高中性脂肪食（ＨＦ）摂取させたことによる癌細胞前駆体のアデノーマ又は完全に癌化した肝癌細胞の発生率が７割を超えた。アデノーマからの肝細胞癌株の樹立は困難であるが、それの肝臓の腫瘍となった肝細胞癌組織から、所望の肝細胞癌株が、樹立される。 In the case of HCV-transfected C57BL / 6 strain transgenic mice, adenomas of cancer cell precursors or complete canceration due to ingestion of an arteriosclerotic high-cholesterol diet (Ath) and a high-neutral fat diet (HF) The incidence of liver cancer cells exceeded 70%. Although it is difficult to establish a hepatocellular carcinoma strain from adenoma, a desired hepatocellular carcinoma strain is established from the hepatocellular carcinoma tissue that has become a tumor of the liver.
この細胞癌株の中でも特に胆管細胞癌株である細胞癌株は、肝癌細胞を動物に投与して腫瘍生着させてから腫瘍細胞から再度、癌細胞を採取して悪性化させる工程を、複数回返し行ってから、それを培養する工程を行うによって、樹立した悪性度の高いものである。 Among these cell cancer strains, the cell cancer strain, which is an intrahepatic cell cancer strain, has a plurality of steps of administering liver cancer cells to an animal to cause tumor engraftment, and then collecting cancer cells from the tumor cells again to make them malignant. It has a high degree of malignancy established by repeating the process and then culturing it.
動脈硬化性高コレステロール食としては、通常の食餌飼料にコレステロールを０．１～１０質量％、好ましくは０．５～４質量％、より好ましくは１～３質量％、例えば１．２６９質量％含有する高コレステロール食が挙げられ、市販品として、具体的にはResearch Diets, Inc.社製の商品名D06061403やD12123101-02が好ましい。コレステロール以外の成分は、通常のトランスジェニックマウス用の食餌成分であればよく、必要に応じ、タンパク質、炭水化物、ミネラル、ビタミン等を含んでいてもよい。 The arteriosclerotic high-cholesterol diet contains 0.1 to 10% by mass, preferably 0.5 to 4% by mass, more preferably 1 to 3% by mass, for example, 1.269% by mass of cholesterol in a normal diet. As a commercial product, the trade names D06061403 and D12123101-02 manufactured by Research Diets, Inc. are preferable. The components other than cholesterol may be any dietary components for ordinary transgenic mice, and may contain proteins, carbohydrates, minerals, vitamins and the like, if necessary.
なお、Research Diets, Inc.社製の商品名D06061403やD12123101-02は、ＡｔｈとＨＦとを予め含むものであるが、別々に有するものを適宜、用時調製してもよい。 The trade names D06061403 and D12123101-02 manufactured by Research Diets, Inc. include Ath and HF in advance, but those having separately may be appropriately prepared at the time of use.
高中性脂肪食としては、トリグリセリドを主成分とする中性脂肪を１５質量％以上、好ましくは２０質量％以上で８０質量％以下、好ましくは中性脂肪を３０～８０質量％、より好ましくは中性脂肪を３０～６０質量％、例えば３４質量％の高い含有率で含有する高中性脂肪食が挙げられ、市販品として具体的にはResearch Diets, Inc.社製の商品名D06061403やD12123101-02が好ましい。中性脂肪としては、食用脂肪である動物性脂肪や植物性脂肪、例えばコーン油、大豆油、なたね油、パーム油、牛脂、豚脂（ラード）が挙げられる。また高中性脂肪食中の中性脂肪以外の成分は、通常のトランスジェニックマウス用の食餌成分であればよく、必要に応じ、モノグルセリド、ジグリセリド、ろう、セラミド、複合脂質、タンパク質、炭水化物、ミネラル、ビタミン、ココアバター、カゼイン等を含んでいてもよい。 As a high triglyceride diet, triglyceride-based neutral fat is 15% by mass or more, preferably 20% by mass or more and 80% by mass or less, preferably 30 to 80% by mass of neutral fat, more preferably medium. Examples of high-neutral fat diets containing 30 to 60% by mass, for example, 34% by mass of triglycerides are commercially available products, specifically, trade names D06061403 and D12123101-02 manufactured by Research Diets, Inc. Is preferable. Examples of the neutral fat include animal fat and vegetable fat which are edible fats such as corn oil, soybean oil, rapeseed oil, palm oil, beef fat and lard. In addition, the components other than the neutral fat in the high triglyceride diet may be any dietary components for normal transgenic mice, and if necessary, monogluceride, diglyceride, wax, ceramide, complex lipid, protein, carbohydrate, mineral, etc. It may contain vitamins, cocoa butter, casein and the like.
制限酵素とベクターとを用いてＣ型肝炎ウィルス（ＨＣＶ）をトランスジェニックマウスに遺伝子導入し、交配によってアレルがホモ（ＨＣＶ／ＨＣＶ）又はヘテロ（ＨＣＶ／－）のトランスジェニックマウスを作製した。なおＨＣＶのゲノタイピングはポリメラーゼ連鎖反応（ＰＣＲ）法により確認した。 Hepatitis C virus (HCV) was gene-introduced into transgenic mice using a limiting enzyme and a vector, and mating produced transgenic mice having homo (HCV / HCV) or hetero (HCV / −) alleles. The genotyping of HCV was confirmed by the polymerase chain reaction (PCR) method.
得られた肝細胞癌株や胆管細胞癌株である細胞癌株は、例えば、その一態様として、独立行政法人製品評価技術基盤機構特許微生物寄託センター（郵便番号292-0818 千葉県木更津市かずさ鎌足２－５－８）に、平成２８年９月２１日付けで受託番号ＮＩＴＥ Ｐ－０２３５３として寄託された細胞癌株であり、別な一態様として、同特許微生物寄託センターに平成２９年１０月１３日付けで受託番号ＮＩＴＥ Ｐ－０２５５８として寄託された細胞癌株である。これらの肝細胞癌株や胆管細胞癌株は、癌細胞前駆体のアデノーマ由来ではなく、顕微鏡観察で完全に癌化した肝癌細胞由来であり、免疫不全マウスの生体内に生着させたときの線維化が強く発現され、細胞形態が完全に癌化しており、高い増殖能・スフェロイド形成能を誘発し、肝癌細胞や胆管癌細胞への分化能を有し、各種腫瘍マーカーで染色されることから悪性度が高く、免疫不全マウスや野生型マウスのような動物に生着可能な肝細胞癌株や胆管細胞癌株とする細胞癌株である。なお、ＷＴマウスへのＡｔｈとＨＦのみの投与により肝細胞癌株のような細胞癌株のライン化を試みたが、実現不可能であった。 The obtained hepatocellular carcinoma strain and cell carcinoma strain, which is an intrahepatic cell carcinoma strain, can be used, for example, as one embodiment of the product evaluation technology infrastructure organization Patent Microbial Deposit Center (postal code 292-0818 Kazusakamatari, Kisarazu City, Chiba Prefecture). It is a cell cancer strain deposited under the accession number NITE P-02353 on September 21, 2016, on foot 2-5-8). It is a cell cancer strain deposited under the accession number NITE P-02558 on March 13. These hepatocellular carcinoma strains and intrahepatic cell carcinoma strains are not derived from the adenoma of the cancer cell precursor, but are derived from hepatocellular carcinoma cells that have become completely cancerous by microscopic observation, and when engrafted in the living body of immunodeficient mice. Fibrosis is strongly expressed, the cell morphology is completely cancerous, it induces high proliferative ability and spheroid forming ability, it has the ability to differentiate into hepatocellular carcinoma cells and bile duct cancer cells, and it is stained with various tumor markers. It is a cell cancer strain that has a high degree of malignancy and can be engrafted in animals such as immunodeficient mice and wild-type mice. An attempt was made to line up a cell cancer strain such as a hepatocellular carcinoma strain by administering only Ath and HF to WT mice, but this was not feasible.
肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルは、樹立された肝細胞癌株及び／又は胆管細胞癌株である細胞癌株を、同系統又は異系統若しくは異種で癌細胞生着用の動物、好ましくはＣ５７ＢＬ／６系統マウスや免疫不全マウス（例えばＳＣＩＤマウスＮＯＤ－ＳＣＩＤ）の肝臓に、前記細胞癌株を生着させて、腫瘍を形成させることによって、得られたものである。このモデルによれば、線維化や脂肪化や発癌や免疫チェックポイントを抑制する被スクリーニング化合物（例えばある種の分岐鎖アミノ酸や非環式レチノイド）の効果のスクリーニングや、本格的な癌発症前の微小癌転移環境の観察や癌発現分子マーカーの探索や肝癌発症メカニズムの解析に用いることができる。 The animal model for the onset of primary liver cancer, which is hepatocellular carcinoma and / or intrahepatic cell carcinoma, is an established hepatocellular carcinoma strain and / or a cell cancer strain which is a bile duct cell carcinoma strain. Obtained by engrafting the cell cancer strain in the liver of an animal wearing cell cells, preferably a C57BL / 6 strain mouse or an immunodeficient mouse (for example, SCID mouse NOD-SCID) to form a tumor. Is. According to this model, screening for the effects of compounds to be screened (for example, certain branched-chain amino acids and acyclic retinoids) that suppress fibrosis, fattening, carcinogenesis, and immune checkpoints, and before the onset of full-scale cancer. It can be used for observing the metastatic environment of microcancer, searching for cancer-expressing molecular markers, and analyzing the mechanism of liver cancer onset.
以下に、肝細胞癌株及び／又は胆管細胞癌株である細胞癌株を樹立し、肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルを作製して、その有用性について実証した例を示す。 Below, a hepatocellular carcinoma strain and / or a cell cancer strain which is a bile duct cell cancer strain is established, and an animal model for developing hepatocellular carcinoma and / or a primary liver cancer which is a bile duct cell cancer is prepared, and its usefulness is described. An example that has been demonstrated is shown.
先ず、以下のようにして、肝細胞癌株である細胞癌株を樹立し、肝細胞癌である原発性肝癌の発症動物モデルを、作製した。 First, a cell cancer strain, which is a hepatocellular carcinoma strain, was established as follows, and a model of an onset animal of primary liver cancer, which is a hepatocellular carcinoma, was prepared.
（トランスジェニックマウスの作製）
通常の食餌をさせた血小板由来成長因子遺伝子導入トランスジェニックマウス（以下、ＰＤＧＦＣ Ｔｇとも称する）群と、通常の食餌をさせたＣ型肝炎ウィルス遺伝子導入トランスジェニックマウス（以下、ＨＣＶ Ｔｇとも称する）群と、動脈硬化性高コレステロール食（Ａｔｈ）と高中性脂肪食（ＨＦ）の食餌をさせたＨＣＶ遺伝子導入トランスジェニックマウス（以下、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦとも称する）群とから、肝癌細胞を成長させ、それらから肝細胞癌株（受託番号がＮＩＴＥ Ｐ－０２３５３の細胞癌株。以下、これのシングルクローンをMHCF1とも称する）を樹立した。その具体的手法を以下に示す。
(Preparation of transgenic mouse)
Hepatitis C virus gene-introduced transgenic mouse (hereinafter, also referred to as HCV Tg) group fed with a normal diet and a hepatitis C virus gene-introduced transgenic mouse group fed with a normal diet (hereinafter, also referred to as PDGFC Tg). Hepatocellular carcinoma cells were grown from a group of HCV gene-introduced transgenic mice (hereinafter, also referred to as HCV Tg / Ath + HF) fed with an arteriosclerotic high cholesterol diet (Ath) and a high neutral fat diet (HF). From them, a hepatocellular carcinoma strain (a cell carcinoma strain having an accession number of NITE P-02353; hereinafter, a single clone thereof is also referred to as MHCF1) was established. The specific method is shown below.
Ｃ５７ＢＬ／６系統マウス（チャールズリバー社製）を開腹した後、下大静脈からリン酸緩衝食塩水（ＰＢＳ）を還流させることで脱血を行った。肝臓にできた腫瘍をGentle macs Tumor Dissociation Kit(Miltenyi Biotec社の商品名)により分離する。ＤＭＥＭで２回洗浄を行った後、塩化アンモニウムによる溶血を行った。直径１０ｃｍのｄｉｓｈに細胞を全量まき、経過観察した。約１ヶ月後にdish上での増殖能と安定性を獲得し、培養できるようになった。(i) Okada H, Honda M, Campbell JS, Sakai Y, Yamashita T, Takebuchi Y, Hada K, Shirasaki T, Takabatake R, Nakamura M, Sunagozaka H, Tanaka T, Fausto N, Kaneko S.Acyclic retinoid targets platelet-derived growth factor signaling in the prevention of hepatic fibrosis and hepatocellular carcinoma development. Cancer Res. 2012 Sep 1;72(17):4459-71. doi: 10.1158/0008-5472.CAN-12-0028. Epub 2012 May 31. (ii)Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, Okuda M, Gosert R, Xiao SY, Weinman SA, Lemon SM. : Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology. 2002 Feb;122(2):352-65.に準拠して遺伝子導入を行うことにより、Ｃ５７ＢＬ／６系統マウスから、(i)ＰＤＧＦＣ導入トランスジェニックマウスと、(ii)ＨＣＶ導入トランスジェニックマウスを作製した。その各細胞において、ＰＤＧＦＣやＨＣＶの遺伝子が存在するかをＰＣＲにより確認した時のＰＤＧＦＣ及びＨＣＶ ｃｏｒｅ（Ｎ）の各フォワードプライマー(For primer)とリバースプライマー(Rev primer)とを、図１及び配列表の配列番号１～４に示す。 After opening the abdomen of C57BL / 6 strain mice (manufactured by Charles River), blood was removed by refluxing phosphate buffered saline (PBS) from the inferior vena cava. Tumors in the liver are isolated with the Gentle macs Tumor Dissociation Kit (trade name of Miltenyi Biotec). After washing twice with DMEM, hemolysis with ammonium chloride was performed. All the cells were sprinkled on a dish having a diameter of 10 cm, and the cells were followed up. After about 1 month, the growth ability and stability on the dish were acquired, and it became possible to culture. (i) Okada H, Honda M, Campbell JS, Sakai Y, Yamashita T, Takebuchi Y, Hada K, Shirasaki T, Takabatake R, Nakamura M, Sunagozaka H, Tanaka T, Fausto N, Kaneko S.Acyclic retinoid targets platelet- derived growth factor signaling in the prevention of hepatic fibrosis and hepatocellular carcinoma development. Cancer Res. 2012 Sep 1; 72 (17): 4459-71. doi: 10.1158 / 0008-5472.CAN-12-0028. Epub 2012 May 31. (ii) Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, Okuda M, Gosert R, Xiao SY, Weinman SA, Lemon SM.: Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins. By performing gene transfer in accordance with of hepatitis C virus. Gastroenterology. 2002 Feb; 122 (2): 352-65., From C57BL / 6 strain mice, (i) PDGFC-introduced transgenic mice, and (ii). HCV-introduced transgenic mice were produced. In each of the cells, each forward primer (For primer) and reverse primer (Rev primer) of PDGFC and HCV core (N) when it was confirmed by PCR whether the gene of PDGFC or HCV was present was shown in FIG. 1 and arranged. It is shown in the sequence numbers 1 to 4 of the column table.
（肝細胞癌の組織の作製）
ＰＤＧＦＣ導入トランスジェニックマウス及びＨＣＶ導入トランスジェニックマウスに、製品名ＣＲＦ－１（日本クレア社製）を通常の食餌として、一日１２５ｇ／ｋｇずつ食べさせながら６８週間飼育し、それぞれＰＤＧＦＣ Ｔｇ群と、ＨＣＶ Ｔｇ群とした。一方、通常の食餌にコレステロールを１．２６９質量％含有させた高コレステロール食と中性脂肪を３４質量％含有させた高中性脂肪食の混合食餌を、別なＨＣＶ導入トランスジェニックマウスに一日１２５ｇ／ｋｇずつ食べさせながら６８週間飼育し、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群とした。飼育期間経過後、各群のマウスの肝臓を摘出したところ、ＰＤＧＦＣ Ｔｇ群から５例中３例の肝臓に腫瘍が生じ、ＨＣＶ Ｔｇ群から７例中３例の肝臓に腫瘍が生じ、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群から１１例中３例の肝臓に腫瘍が生じていた。その結果を、図２に示す。同図中、矢印で肝臓の腫瘍組織を指している。各群の肝臓の腫瘍が癌細胞であるか否かは病理組織の顕微鏡検査で調べた。
(Preparation of hepatocellular carcinoma tissue)
PDGFC-introduced transgenic mice and HCV-introduced transgenic mice were bred for 68 weeks while feeding the product name CRF-1 (manufactured by Claire Japan) as a normal diet at 125 g / kg per day for 68 weeks. The HCV Tg group was used. On the other hand, a mixed diet of a high-cholesterol diet containing 1.269% by mass of cholesterol and a high-neutral fat diet containing 34% by mass of triglyceride in a normal diet was added to another HCV-introduced transgenic mouse at 125 g per day. The animals were bred for 68 weeks while being fed at a rate of / kg each, and were grouped into the HCV Tg / As + HF group. After the breeding period, when the livers of the mice in each group were removed, tumors developed in the livers of 3 out of 5 cases from the PDGFC Tg group, tumors developed in the livers of 3 out of 7 cases from the HCV Tg group, and HCV Tg. Tumors were found in the liver of 3 of 11 cases from the / Ath + HF group. The results are shown in FIG. In the figure, the arrow points to the tumor tissue of the liver. Whether or not the liver tumors in each group were cancer cells were examined by microscopic examination of histopathology.
（肝細胞癌株調製）
ＰＤＧＦＣ Ｔｇ群、ＨＣＶ Ｔｇ群、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群の各群の肝臓の腫瘍から、全肝組織をメスを用いて１ｍｍ３の角状に切り、Gentle MACS Dissociation Kit(Miltenyi Biotec社の商品名)を用いて組織を分離後、dish上でＤＭＥＭ培養液中で培養することによって、肝細胞癌株を樹立した。なお、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群から樹立した肝細胞癌株は、受託番号ＮＩＴＥ Ｐ－０２３５３の動物細胞であり、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞と称することとし、このＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローンをMHCF1と称することにする。
(Preparation of hepatocellular carcinoma strain)
From liver tumors in each group of PDGFC Tg group, HCV Tg group, and HCV Tg / Ath + HF group, cut whole liver tissue into 1 mm 3 squares using a scalpel, and Gentle MACS Dissociation Kit (trade name of Miltenyi Biotec). After separating the tissue using, a hepatocellular carcinoma strain was established by culturing in a DMEM culture medium on a dish. The hepatocellular carcinoma strain established from the HCV Tg / Ath + HF group is an animal cell with accession number NITE P-02353, and is referred to as an HCV Tg / Ath + HF cell. We will call it MHCF1.
（導入遺伝子の確認）
得られた肝細胞癌株における遺伝子導入の適否について確認した。図３は、Ｈｅｐａ１－６、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株、ＰＤＧＦＣ Ｔｇ群由来の肝細胞癌株、ＨＣＶ Ｔｇ群由来の肝細胞癌株について、それぞれの遺伝子導入ゲノムＤＮＡであるＰＤＧＦＣ及びＨＣＶ ｃｏｒｅ（Ｎ）について、細胞のｔｏｔａｌ ＤＮＡをQIAamp DNA Mini Kit (QIAGEN1社製)を用いて抽出後、GoTaq Green Master Mix(Promega社製)を用いたＰＣＲ反応によりＰＤＧＦＣ及びＨＣＶ遺伝子の特異的増幅を行った。図３は、増幅産物を電気泳動で検出した結果を示す図である。なお、プライマーは図１に記載のものである。図３から明らかな通り、Ｈｅｐａ１－６の肝細胞癌株はこれら遺伝子を有しておらず、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ由来の肝細胞癌株及びＨＣＶ Ｔｇ由来の肝細胞癌株はＨＣＶが導入されており、ＰＤＧＦＣ Ｔｇ由来の肝細胞癌株にはＰＤＧＦＣが導入されていることが、示された。従って、ＨＣＶ Ｔｇ群由来及びＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株は、ＨＣＶが少なくともアレルがホモ(HCV/HCV)又はヘテロ(HCV/-)として導入されていることが示された。
(Confirmation of transgene)
We confirmed the suitability of gene transfer in the obtained hepatocellular carcinoma strain. FIG. 3 shows PDGFC, which is the gene-introduced genomic DNA of Hepa1-6, hepatocellular carcinoma strain derived from HCV Tg / Ath + HF group, hepatocellular carcinoma strain derived from PDGFC Tg group, and hepatocellular carcinoma strain derived from HCV Tg group. And HCV core (N), after extracting the total DNA of cells using QIAamp DNA Mini Kit (manufactured by QIAGEN1), specificity of PDGFC and HCV genes by PCR reaction using GoTaq Green Master Mix (manufactured by Promega). Amplification was performed. FIG. 3 is a diagram showing the results of detecting the amplification product by electrophoresis. The primer is as shown in FIG. As is clear from FIG. 3, the hepatocellular carcinoma strain of Hepa1-6 does not have these genes, and the HCV Tg / Ath + HF-derived hepatocellular carcinoma strain and the HCV Tg-derived hepatocellular carcinoma strain have HCV introduced. It was shown that PDGFC was introduced into the hepatocellular carcinoma strain derived from PDGFC Tg. Therefore, it was shown that the hepatocellular carcinoma strains derived from the HCV Tg group and the HCV Tg / At + HF group were introduced with HCV at least as homo (HCV / HCV) or hetero (HCV /-).
（肝細胞癌株の細胞形態の観察）
それら樹立肝細胞癌株、及び比較のためＨｅｐａ１－６の細胞株について、顕微鏡観察によるin vitro(dish上)での２００倍率の細胞形態を図４に示す。図４から明らかな通り、樹立肝細胞癌株、Ｈｅｐａ１－６細胞株の何れも肝細胞様の形態となっていることから、肝細胞癌株であることが示された。
(Observation of cell morphology of hepatocellular carcinoma strain)
FIG. 4 shows the cell morphology of these established hepatocellular carcinoma lines and the Hepa1-6 cell line for comparison in vitro (on dish) by microscopic observation. As is clear from FIG. 4, both the established hepatocellular carcinoma strain and the Hepa1-6 cell line have a hepatocellular-like morphology, indicating that they are hepatocellular carcinoma strains.
（肝細胞癌株の増殖能の評価）
ＰＤＧＦＣ Ｔｇ群由来、ＨＣＶ Ｔｇ群由来、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来のそれら樹立肝細胞癌株、Ｈｅｐａ１－６の細胞株について、ＭＭＴ(3-(4,5-ジメチルチアゾール-2-イル)-2,5-ジフェニルテトラゾリウム ブロミド)が培養細胞によってホルマザン色素へ還元されることと利用して、９６穴プレートに１×１０４個／ｗｅｌｌで細胞を撒き、２４、４８、７２時間と経時的に増殖能をモニターし、培養細胞の増殖能の指標として生存率を比色定量するＭＴＴアッセイの結果を図５に示す。図５から明らかな通り、樹立肝細胞癌株、Ｈｅｐａ１－６細胞株の何れも時間と共に２４、４８、７２時間の経過と共に、細胞生存率が高くなっており、in vitroで細胞増殖能を有することが分かった。
(Evaluation of proliferative capacity of hepatocellular carcinoma strain)
MMT (3- (4,5-dimethylthiazol-2-yl) -2 was used for the established hepatocellular carcinoma strains derived from the PDGFC Tg group, HCV Tg group, HCV Tg / Ath + HF group, and Hepa1-6 cell lines. , 5-Diphenyltetrazolium bromide) is reduced to formazan pigment by cultured cells, and cells are sprinkled on a 96-well plate at 1 × 10 4 cells / well and proliferated over time for 24, 48, 72 hours. FIG. 5 shows the results of the MTT assay in which the ability is monitored and the viability is colorimetrically quantified as an index of the proliferative ability of cultured cells. As is clear from FIG. 5, both the established hepatocellular carcinoma line and the Hepa1-6 cell line have increased cell viability with the passage of 24, 48, and 72 hours with time, and have cell proliferation ability in vitro. It turned out.
（スフェロイド形成能の評価）
それらＰＤＧＦＣ Ｔｇ群由来、ＨＣＶ Ｔｇ群由来、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株について、in vitroにて６穴の低粘着性表面プレート（ＩＷＡＫＩ社製；商品名EZ-BindShutII マイクロプレート；EZ-BindShutは登録商標）により、All Fee Medium（ｇｉｂｃｏ社製；商品名DMEM, High Glucose (11965-092)）３ｍｌ／穴を用いて細胞５×１０３個／穴を播種し２週間培養し、スフェアと呼ばれるコロニー数を計測して、腫瘍形成能（スフェロイド形成能）を評価するSphereアッセイを行った。その結果を、図６に示す。低粘着性表面プレートでスフェアを形成できるということは、足場非依存的に増殖可能で、生体内で腫瘍を形成し得る能力を有し、in vivoでの腫瘍形成・腫瘍増殖を反映する重要な指標である。図６（ａ）から明らかな通り、ＰＤＧＦＣ Ｔｇ群由来、ＨＣＶ Ｔｇ群由来、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の何れの肝細胞癌株からも細胞塊のスフェアを形成していた。しかし、同図（ｂ）に示すように、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株は、腫瘍形成・腫瘍増殖し得るスフェロイド形成能を有していたが、ＰＤＧＦＣ Ｔｇ群、ＨＣＶ Ｔｇ群の樹立肝細胞癌株は、スフェロイド形成能を有していなかった。
(Evaluation of spheroid forming ability)
For the established hepatocellular carcinoma strains derived from the PDGFC Tg group, HCV Tg group, and HCV Tg / Ath + HF group, a 6-hole low-adhesive surface plate (manufactured by IWAKI; trade name EZ-BindShutII microplate; EZ-Bind Shut is a registered trademark. All Fee Medium (manufactured by in vitro; trade name DMEM, High Glucose (11965-092)) 3 ml / hole is used to seed 5 × 10 3 cells / hole and cultured for 2 weeks. , A Sphere assay was performed to evaluate the tumorigenicity (spheroid formation ability) by measuring the number of colonies called spheres. The results are shown in FIG. The ability to form spheres with a low-adhesive surface plate is important because it is scaffold-independent, capable of forming tumors in vivo, and reflects in vivo tumor formation and growth. It is an index. As is clear from FIG. 6A, cell mass spheres were formed from any of the hepatocellular carcinoma strains derived from the PDGFC Tg group, the HCV Tg group, and the HCV Tg / As + HF group. However, as shown in Fig. (B), the established hepatocellular carcinoma strain derived from the HCV Tg / Ath + HF group had the ability to form spheroids capable of tumor formation and tumor growth, but the PDGFC Tg group and HCV Tg group. The established hepatocellular carcinoma strain of was not capable of forming spheroids.
（ＳＣＩＤマウスへの生着性の評価）
８週齢の重度複合免疫不全マウスＮＯＤ－ＳＣＩＤマウス（NOD.CB17-Prkdcscid/J；日本チャールス・リバー社製）４匹に、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株１×１０５個の細胞をＰＢＳに懸濁し、それぞれ皮下投与し、約２ヶ月間飼育し、生着の有無を確認した。その後、肝臓を摘出したところ、全例で、肝細胞癌株に由来し腫瘍が生着していることを確認した。さらに病理組織片を取り出し、ヘマトキシリン－エオシン（ＨＥ）染色をした後、顕微鏡観察を行って癌発現性について評価した。その結果を図７に示す。同図中、ｂはａの枠部の拡大写真、ｃはｂの枠部の拡大写真である。同図から明らかな通り、生着により、ネクローシスと思われるＮＣで示される組織の間に、ＴＴで示す肝臓細胞癌組織と、ＢＤで示す胆管細胞癌組織とが、認められた。これら肝細胞癌組織と胆管細胞癌組織とは、同じ肝細胞癌株に由来していると考えられる。
(Evaluation of engraftment in SCID mice)
Established hepatocellular carcinoma strain 1 × 10 5 derived from HCV Tg / Ath + HF group in 4 8-week-old severe combined immunodeficiency mice NOD-SCID mice (NOD.CB17-Prkdc scid / J; manufactured by Charles River Japan) Individual cells were suspended in PBS, each of them was subcutaneously administered, and the cells were bred for about 2 months to confirm the presence or absence of engraftment. After that, when the liver was removed, it was confirmed that the tumor was engrafted from the hepatocellular carcinoma strain in all cases. Further, a piece of histopathological tissue was taken out, stained with hematoxylin-eosin (HE), and then observed under a microscope to evaluate the cancer expression. The results are shown in FIG. In the figure, b is an enlarged photograph of the frame portion of a, and c is an enlarged photograph of the frame portion of b. As is clear from the figure, hepatic cell carcinoma tissue indicated by TT and bile duct cell carcinoma tissue indicated by BD were observed among the tissues indicated by NC, which were considered to be necrosis, by engraftment. It is considered that these hepatocellular carcinoma tissues and bile duct cell carcinoma tissues are derived from the same hepatocellular carcinoma strain.
一方、ＰＤＧＦＣ Ｔｇ群由来、ＨＣＶ Ｔｇ群由来の樹立肝細胞癌株についても、ＮＯＤ－ＳＣＩＤマウスに皮下投与し、同様に評価したところ、全例で全く生着していなかった。 On the other hand, the established hepatocellular carcinoma strains derived from the PDGFC Tg group and the HCV Tg group were also subcutaneously administered to NOD-SCID mice and evaluated in the same manner.
（野生型マウスへの生着性の評価）
８週齢のＣ５７ＢＬ／６（日本チャールス・リバー社製）である野生型（ＷＴ）マウス１０匹にＰＤＧＦＣ Ｔｇ群由来とＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来との樹立肝細胞癌株の１×１０５個の細胞をＰＢＳに懸濁し、それぞれ脾臓投与し、６４日間飼育し、肝臓に肝細胞癌を誘発させた。その時の生存率を図８に示す。ＰＤＧＦＣ Ｔｇ群由来の樹立肝細胞癌株を投与しても全例死亡しなかったが、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株を投与すると約２ヶ月後に生存率が約１０％にまで低下した。細胞投与から６４日目の時に肝臓・脾臓を摘出したところ、ＰＤＧＦＣ Ｔｇ群由来の樹立肝細胞癌株の投与では、肝臓、脾臓ともに肉眼的所見で異常が認められず全く正常であったことから樹立肝細胞株が生着していなかった。一方、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株の投与では、細胞投与から６４日目の時に肝臓・脾臓を摘出したところ、肝臓に白い結節様の瘢痕が複数個所で観察され、加えて脾腫も観察された。解剖所見から、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株が、脾臓で増殖し一部肝臓に転移し、血栓を生じた結果、多数のマウスが死亡したのであり、脾臓投与によるダメージで死亡したのではないことが、分かった。
(Evaluation of engraftment in wild-type mice)
10 8-week-old C57BL / 6 (manufactured by Charles River Japan) wild-type (WT) mice with 1 × 10 5 established hepatocellular carcinoma strains derived from PDGFC Tg group and HCV Tg / At + HF group The cells were suspended in PBS, administered to the spleen, and bred for 64 days to induce hepatocellular carcinoma in the liver. The survival rate at that time is shown in FIG. All cases did not die even when the established hepatocellular carcinoma strain derived from the PDGFC Tg group was administered, but the survival rate reached about 10% after about 2 months when the established hepatocellular carcinoma strain derived from the HCV Tg / Ath + HF group was administered. It has declined. When the liver and spleen were removed 64 days after the cell administration, the administration of the established hepatocellular carcinoma strain derived from the PDGFC Tg group showed no abnormalities in the liver and spleen, and the liver and spleen were completely normal. The established hepatocyte line was not engrafted. On the other hand, in the administration of the established hepatocellular carcinoma strain derived from the HCV Tg / Ath + HF group, when the liver and spleen were removed 64 days after the cell administration, white nodular scars were observed in multiple places in the liver, and in addition. Splenomegaly was also observed. From the anatomical findings, the established hepatocellular carcinoma strain derived from the HCV Tg / Ath + HF group proliferated in the spleen and partially metastasized to the liver, resulting in thrombus, and as a result, many mice died. It turned out that I didn't.
ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の樹立肝細胞癌株を脾臓投与したＣ５７ＢＬ／６マウスの肝臓と脾臓との病理組織片を作製し、ＨＥ染色をした後、顕微鏡観察を行って癌発現性について評価した。その結果をそれぞれ、図９及び図１０に示す。図９中、Ａ及びＣは肝臓の異なる部位の組織片であり、Ｂ及びＤはそれらＡ・Ｃの枠部の拡大写真である。図９から明らかな通り、ＴＴで示す肝臓細胞癌組織と、管腔が明瞭なＢＤで示す胆管細胞癌組織と、ネクローシスでないかと推察されるＮで示す組織とが、認められた。図１０中、Ｅは脾臓の正常部位の組織片及びＧは脾臓の腫瘍形成部位近傍の組織片であり、Ｆ及びＨはそれらＥ・Ｇの枠部の拡大写真である。図１０から明らかな通り、Ｅ、Ｆには正常な細胞が認められ、Ｇ、Ｈの上部には白っぽく映る腫瘍組織部位が、認められた。 A piece of histopathology between the liver and spleen of C57BL / 6 mice to which the established hepatocellular carcinoma strain derived from the HCV Tg / At + HF group was spleen-administered was prepared, HE-stained, and then microscopically observed to evaluate the cancer expression. .. The results are shown in FIGS. 9 and 10, respectively. In FIG. 9, A and C are tissue pieces of different parts of the liver, and B and D are enlarged photographs of the frames of those A and C. As is clear from FIG. 9, a liver cell carcinoma tissue indicated by TT, a bile duct cell carcinoma tissue indicated by BD having a clear lumen, and a tissue indicated by N, which is presumed to be necrosis, were observed. In FIG. 10, E is a tissue piece at a normal site of the spleen and G is a tissue piece near the tumor-forming site of the spleen, and F and H are enlarged photographs of the frame portions of the E and G. As is clear from FIG. 10, normal cells were observed in E and F, and a whitish tumor tissue site was observed in the upper part of G and H.
一方、ＰＤＧＦＣ Ｔｇ群由来の樹立肝細胞癌株についても、Ｃ５７ＢＬ／６マウスに皮下投与し、同様に評価したところ、全例で全く生着していなかった。 On the other hand, the established hepatocellular carcinoma strain derived from the PDGFC Tg group was also subcutaneously administered to C57BL / 6 mice and evaluated in the same manner.
こられの結果を表１にまとめて示す。 These results are summarized in Table 1.
このことから、Ｃ型肝炎ウィルスを遺伝子導入した動物（好ましくはＣ５７ＢＬ／６マウス）に、動脈硬化性高コレステロール食と高中性脂肪食とを摂取させ肝癌細胞を発症させて肝癌細胞を採取し培養して樹立した本発明のＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株は、in vitro及びin vivoで使用でき、増殖能、転移能、肝癌細胞・胆癌管細胞への両方への分化能が高くて、極めて悪性度が高いものであることが明らかとなった。ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株は、一旦、脱分化して前駆体へ逆戻りしてから肝癌細胞・胆管癌細胞に分化している可能性もある。ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株は、単一であるが、肝癌細胞・胆管癌細胞に分化するということは、悪性度が極めて高い。 For this reason, hepatitis C virus-introduced animals (preferably C57BL / 6 mice) were fed with an arteriosclerotic high-cholesterol diet and a high-neutral fat diet to develop liver cancer cells, and liver cancer cells were collected and cultured. The hepatocellular carcinoma strain derived from the HCV Tg / At + HF group of the present invention established in the above can be used in vitro and in vivo, and has proliferative ability, metastatic ability, and ability to differentiate into both liver cancer cells and biliary carcinoma tube cells. It became clear that it was high and extremely malignant. It is possible that the hepatocellular carcinoma strain derived from the HCV Tg / At + HF group is once dedifferentiated and reverted to the precursor, and then differentiated into hepatocellular carcinoma cells and cholangiocarcinoma cells. Although the hepatocellular carcinoma strain derived from the HCV Tg / At + HF group is single, the differentiation into hepatocellular carcinoma cells and cholangiocarcinoma cells is extremely malignant.
次に、以下のようにして、胆管細胞癌株である細胞癌株を樹立し、胆管細胞癌である原発性肝癌の発症動物モデルを、作製した。 Next, a cell cancer strain, which is a bile duct cell cancer strain, was established as follows, and a model of an onset animal of primary liver cancer, which is a bile duct cell cancer, was prepared.
前記のようにして得られたＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞をＮＯＤ－ＳＣＩＤマウスに生着させてできた腫瘍を、gentle MACSTM Dissociator (Miltenyi Biotec社の商品名)を用いて分離した。分離した細胞を再度ＮＯＤ－ＳＣＩＤマウスの皮下へ移植して腫瘍生着を待ち、前記と同様にして生着した腫瘍を再び別なＮＯＤ－ＳＣＩＤマウスの皮下へ移植した。この移植工程をさらに３回繰り返し行うことで、腫瘍細胞の悪性度を上昇させることを試みた。この工程を繰り返して樹立した細胞を４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞と称し（受託番号がＮＩＴＥ Ｐ－０２５５８に対応。）、この４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローンをMHCF5と称する。また先と同様にして、MHCF5から原発性肝癌の発症動物モデルを作製した。 Tumors formed by engrafting HCV Tg / Ath + HF cells obtained as described above in NOD-SCID mice were isolated using a gentle MACS TM Dissociator (trade name of Miltenyi Biotec). The isolated cells were transplanted subcutaneously to the NOD-SCID mouse to wait for tumor engraftment, and the engrafted tumor was transplanted subcutaneously to another NOD-SCID mouse in the same manner as described above. By repeating this transplantation step three more times, an attempt was made to increase the malignancy of the tumor cells. The cells established by repeating this step are referred to as 4-HCV Tg / As + HF cells (the accession number corresponds to NITE P-02558), and the single clone established from the 4-HCV Tg / As + HF cells is referred to as MHCF5. In the same manner as before, an animal model of primary liver cancer was prepared from MHCF5.
得られた、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞群からシングルクローンを樹立した肝細胞癌株(MHCF1)と、４-ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞群からシングルクローンを樹立した胆管細胞癌株(MHCF5細胞)とを、以下のように対比した。 The obtained hepatocellular carcinoma line (MHCF1) for which a single clone was established from the HCV Tg / Ath + HF cell group and the bile duct cell cancer line (MHCF5 cell) for which a single clone was established from the 4-HCV Tg / Ath + HF cell group were obtained. The comparison was made as follows.
図１１に、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞群からシングルクローンを樹立した肝細胞癌株(MHCF1)と、４-ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞群からシングルクローンを樹立した胆管細胞癌株(MHCF5細胞)が、シャーレ上で増殖している形態の観察像の写真を示す。この写真から、シャーレ上で培養可能な、形態的特徴の異なる２種類の細胞株が樹立できたことが確認できた。 In FIG. 11, a hepatocellular carcinoma strain (MHCF1) for which a single clone was established from the HCV Tg / Ath + HF cell group and an intrahepatic cell carcinoma strain (MHCF5 cell) for which a single clone was established from the 4-HCV Tg / Ath + HF cell group are shown. The photograph of the observation image of the morphology proliferating above is shown. From this photograph, it was confirmed that two types of cell lines having different morphological characteristics could be established that could be cultured on a petri dish.
図１２に、Ｃ５７ＢＬ／６マウスの正常肝をコントロールとし、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローン(MHCF1)の細胞と４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローン(MHCF5)の細胞との遺伝子発現解析を、GeneChip Mouse Gene 2.0 ST Array (Affymetrix社の商品名)を用いて行なった。各細胞のＲＮＡの調整は、RNeasy Mini Kit (Qiagen社の商品名)を用いて行なった。同図中の(Ａ)～(Ｄ)は、夫々、肝細胞(癌)のマーカー(Hepatocyte markers)として有用なアルブミン(Alb)、αフェトプロテイン(Afp)、肝細胞核因子４α(Hnf4a)及びトランスチレチン(Ttr)の各遺伝子発現をグラフで示したものであり、同図中の(Ｅ)～(Ｈ)は、夫々、胆管細胞マーカー(Cholangiocyte markers)として有用なb4インテグリン(Itgb4)、アクアポリン１(Aqp1)、ケラチン７(Krt7)及びケラチン１９(Krt19)の各遺伝子発現をグラフで示したものである。図１２から明らかな通り、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローン(MHCF1)細胞は肝細胞癌細胞株であること、及び４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ細胞から樹立したシングルクローン(MHCF5)細胞は胆管癌細胞株であることが、夫々示唆された。 FIG. 12 shows cells of a single clone (MHCF1) established from HCV Tg / Ath + HF cells and cells of a single clone (MHCF5) established from 4-HCV Tg / Ath + HF cells using the normal liver of C57BL / 6 mice as a control. Gene expression analysis was performed using GeneChip Mouse Gene 2.0 ST Array (trade name of Affymetrix). The RNA of each cell was adjusted using the RNeasy Mini Kit (trade name of Qiagen). In the figure, (A) to (D) are albumin (Alb), α-fetoprotein (Afp), hepatocyte nuclear factor 4α (Hnf4a) and transti, which are useful as hepatocyte markers, respectively. The expression of each gene of retin (Ttr) is shown in a graph, and (E) to (H) in the figure are b4 integulin (Itgb4) and aquaporin 1 which are useful as cholangiocyte markers, respectively. The gene expression of (Aqp1), keratin 7 (Krt7) and keratin 19 (Krt19) is shown in a graph. As is clear from FIG. 12, the single clone (MHCF1) cells established from HCV Tg / Ath + HF cells are hepatocellular carcinoma cell lines, and the single clone (MHCF5) cells established from 4-HCV Tg / Ath + HF cells are bile ducts. Each was suggested to be a cancer cell line.
図１３に、原発性肝癌の発症動物モデルによる、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来でシングルクローンを樹立した肝細胞癌株（MHCF1）由来の腫瘍で、管腔構造を有しない領域（(Ａ)及びその拡大図(Ｂ)）と管腔構造を有する領域（(Ｃ)及びその拡大図(Ｄ)）、及び４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来でシングルクローンを樹立した胆管細胞癌株（MHCF5）由来の腫瘍（(Ｅ)及びその拡大図(Ｆ)）の各腫瘍組織のＨＥ染色像の写真を示す。図１３から明らかな通り、MHCF1由来腫瘍は肝細胞癌と胆管癌領域を有する混合型肝癌、MHCF5由来腫瘍は未分化な胆管癌の形態を取っており原発性肝癌の発症動物モデルとして有用であることが分かった。 FIG. 13 shows a tumor derived from a hepatocellular carcinoma strain (MHCF1) that established a single clone derived from the HCV Tg / Ath + HF group according to an animal model of primary liver cancer, and has no luminal structure ((A) and its region). Enlarged view (B)) and region with luminal structure ((C) and its enlarged view (D)), and from the cholangiocellular carcinoma strain (MHCF5) that established a single clone from the 4-HCV Tg / Ath + HF group. A photograph of an HE-stained image of each tumor tissue of a tumor ((E) and its enlarged view (F)) is shown. As is clear from FIG. 13, the MHCF1-derived tumor takes the form of a mixed liver cancer having a hepatocellular carcinoma and a cholangiocarcinoma region, and the MHCF5-derived tumor takes the form of an undifferentiated cholangiocarcinoma, which is useful as an animal model for the onset of primary liver cancer. It turned out.
各細胞癌株の細胞1×105個をPhosphate-buffered saline (PBS)100μlに懸濁し、Matrigel 基底膜マトリックス(Corning社の商品名)100μlと混ぜ、27G×13mmシリンジ(ニプロ株式会社製)を用いて、Ｃ５７ＢＬ／６系統マウスの右側腹部より皮下移植した。それぞれ５匹ずつに腫瘍を打ち込み、9日目から２４日目まで、３日毎にそれぞれの生着腫瘍を計測した。図１４に、原発性肝癌の発症動物モデルにおける、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来でシングルクローンを樹立した肝細胞癌株（MHCF1）及び４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来でシングルクローンを樹立した胆管細胞癌株（MHCF5）とのマウスへの腫瘍生着像と、腫瘍増殖曲線を図に示す。同図の写真は、各細胞癌株を生着させた２４日目のマウスの腫瘍生着像を示す外観写真であり、外観上、問題となる所見は認められなかった。同図のグラフは、各細胞癌株の移植日からに各細胞癌株に由来する腫瘍の２４日目までの経過日数(Days after tumor cell implant)と腫瘍体積(Tumor volume; ｍｍ３)との相関を示す腫瘍増殖曲線であり、何れも腫瘍が経過日数と共に、順調に増加しており、この原発性肝癌の発症動物モデルとして有用であることが、認められた。 Suspend 1 x 10 5 cells of each cell cancer line in 100 μl of Phosphate-buffered saline (PBS), mix with 100 μl of Matrigel basement membrane matrix (trade name of Corning), and use a 27 G × 13 mm syringe (manufactured by Nipro). It was subcutaneously transplanted from the right abdomen of C57BL / 6 strain mice. Tumors were placed in 5 animals each, and each engraftment tumor was measured every 3 days from the 9th day to the 24th day. FIG. 14 shows a hepatocellular carcinoma strain (MHCF1) in which a single clone was established from the HCV Tg / As + HF group and a bile duct cell carcinoma in which a single clone was established from the 4-HCV Tg / As + HF group in an animal model of primary liver cancer. The tumor engraftment image in the mouse with the strain (MHCF5) and the tumor growth curve are shown in the figure. The photograph in the figure is an external photograph showing a tumor engraftment image of a mouse on the 24th day in which each cell cancer strain was engrafted, and no problematic findings were observed in appearance. The graph in the figure shows the number of days after tumor cell implant and the tumor volume (Tumor volume; mm 3 ) from the transplantation date of each cell cancer strain to the 24th day of the tumor derived from each cell cancer strain. It is a tumor growth curve showing a correlation, and it was confirmed that the tumors are steadily increasing with the number of days elapsed, and that they are useful as an animal model for the onset of this primary liver cancer.
このように、ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の肝細胞癌株及び４－ＨＣＶ Ｔｇ／Ａｔｈ＋ＨＦ群由来の胆管細胞癌株は、in vitro、in vivoで増殖するので、それを用いた肝細胞癌発症動物モデルを作製することができる。一方、ＰＤＧＦＣ Ｔｇ群由来及びＨＣＶ Ｔｇ群由来の肝細胞癌株は樹立でき、in vitroで増殖するが、in vivoで生着も増殖もできないので、肝細胞癌発症動物モデルを作製し得ない。 As described above, the hepatocellular carcinoma strain derived from the HCV Tg / Ath + HF group and the intrahepatic cell carcinoma strain derived from the 4-HCV Tg / Ath + HF group proliferate in vitro and in vivo. Models can be created. On the other hand, hepatocellular carcinoma strains derived from the PDGFC Tg group and the HCV Tg group can be established and proliferate in vitro, but cannot engraft or proliferate in vivo, so that an animal model for developing hepatocellular carcinoma cannot be prepared.
肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルに、前記の線維化や脂肪化や発癌を抑制する被スクリーニング化合物を投与して有効性を検討し、スクリーニングを行ったり、本格的な癌発症前の微小癌転移環境の観察や癌発現分子マーカーの探索や肝癌発症メカニズムの解析を行ったり、免疫チェックポイントの抗ＰＤ－１抗体や抗ＰＤＬ－１抗体を用いて免疫機能を増強させる検討を行ったりする。それにより、肝臓癌・肝細胞癌・胆管細胞癌の治療薬や治療方法の開発に役立てることができることが分かった。 To the onset animal model of primary liver cancer, which is hepatocellular carcinoma and / or intrahepatic cell carcinoma, the above-mentioned compound to be screened that suppresses fibrosis, fattening, and carcinogenesis is administered to examine its effectiveness, and screening is performed. Observation of the microcancer metastasis environment before the onset of full-scale cancer, search for cancer-expressing molecular markers, analysis of the mechanism of liver cancer onset, and immune function using anti-PD-1 antibody and anti-PDL-1 antibody at immune checkpoints. We will consider increasing the number of patients. It was found that this can be useful for the development of therapeutic agents and methods for liver cancer, hepatocellular carcinoma, and intrahepatic cell carcinoma.
本発明の肝細胞癌株及び／又は胆管細胞癌株である細胞癌株は、樹立方法によって調製可能で、in vitro及びin vivoでの肝細胞癌・肝細胞癌・胆管細胞癌の試験に用いることができる。この細胞癌株を用いた肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデルによれば、肝臓癌・肝細胞癌・胆管細胞癌のような腫瘍の増殖を直接抑えるのに有用な抗癌剤や分子標的薬又は免疫チェックポイントの抑制薬のような免疫標的薬のスクリーニングや、肝癌で産生される生体マーカーや生理活性物質の探索、微小癌環境の観察や発癌メカニズムの解析に用いて、肝臓癌・肝細胞癌・胆管細胞癌の治療薬や治療方法の開発に有用である。 The hepatocellular carcinoma strain and / or the cell carcinoma strain which is a bile duct cell carcinoma strain of the present invention can be prepared by an establishment method and used for in vitro and in vivo hepatocellular carcinoma / hepatocellular carcinoma / bile duct cell carcinoma test. be able to. According to an animal model of hepatocellular carcinoma and / or primary liver cancer, which is a bile duct cell cancer, using this cell carcinoma strain, it is possible to directly suppress the growth of tumors such as liver cancer, hepatocellular carcinoma, and bile duct cell carcinoma. Used for screening for immunotargeting drugs such as useful anticancer agents, molecular targeting agents, or immunocheckpoint inhibitors, searching for biological markers and physiologically active substances produced in liver cancer, observing the microcancer environment, and analyzing the carcinogenic mechanism. It is useful for developing therapeutic agents and methods for liver cancer, hepatocellular carcinoma, and bile duct cell carcinoma.
Claims (10)
前記動物の肝臓に肝癌を発生させる工程、
発生した肝癌組織から肝癌細胞を採取する工程、
採取した前記肝癌細胞を培養する工程
によって、肝細胞癌株及び／又は胆管細胞癌株として、樹立したものであることを特徴とする細胞癌株。 A step of feeding a C57BL / 6 strain mouse as an animal into which hepatitis C virus has been gene-introduced with an arteriosclerotic high-cholesterol diet and a high-neutral fat diet.
The step of causing liver cancer in the liver of the animal,
The process of collecting liver cancer cells from the developed liver cancer tissue,
A cell cancer strain characterized by being established as a hepatocellular carcinoma strain and / or a bile duct cell carcinoma strain by the step of culturing the collected liver cancer cells.
前記動物の肝臓に肝癌を発生させる工程、
発生した肝癌組織から肝癌細胞を採取する工程、
採取した前記肝癌細胞を培養する工程
を有し、それによって、肝細胞癌株及び／又は胆管細胞癌株として、細胞癌株を樹立することを特徴とする細胞癌株の樹立方法。 A step of feeding a C57BL / 6 strain mouse as an animal into which hepatitis C virus has been gene-introduced with an arteriosclerotic high-cholesterol diet and a high-neutral fat diet.
The step of causing liver cancer in the liver of the animal,
The process of collecting liver cancer cells from the developed liver cancer tissue,
A method for establishing a cell cancer line, which comprises a step of culturing the collected liver cancer cells, thereby establishing a cell cancer line as a hepatocellular carcinoma line and / or a bile duct cell cancer line.
同系統又は異系統若しくは異種で癌細胞生着用の動物の肝臓に、前記細胞癌株を生着させて、腫瘍を形成させることによって、得られた、肝細胞癌及び／又は胆管細胞癌である原発性肝癌の発症動物モデル。 A step of ingesting an arteriosclerotic high cholesterol diet and a high neutral fat diet into a C57BL / 6 strain mouse as an animal for developing cancer cells into which a hepatitis C virus has been gene-introduced; a step of causing liver cancer in the liver of the animal; A step of collecting liver cancer cells from the developed liver cancer tissue; after establishing a cell cancer strain as a hepatocellular carcinoma strain and / or a bile duct cell cancer strain by the step of culturing the collected liver cancer cells.
Hepatocellular carcinoma and / or bile duct cell carcinoma obtained by engrafting the cell cancer strain in the liver of an animal of the same lineage, a different lineage, or a heterologous cancer cell-bearing animal to form a tumor. An animal model of developing primary liver cancer.
前記腫瘍の形成を観察するためのものであることを特徴とする請求項８に記載の原発性肝癌の発症動物モデル。 8. Claim 8 is for administering a sample to be screened and screening using the promotion or suppression of tumor formation as an index, and / or for observing the formation of the tumor. An animal model of the onset of primary liver cancer described in.
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