JP2016209303A - Formulation for reproducing hepatic tissue - Google Patents
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本発明は、肝芽細胞等の肝細胞を生体の肝臓切離面に投与（移植）して、肝組織を再生させるための製剤および生体の肝臓切離面に肝細胞を含む製剤を投与する方法に関する。 The present invention administers (transplants) hepatocytes, such as hepatoblasts, to a living liver cut surface and administers a preparation for regenerating liver tissue and a preparation containing hepatocytes on the living liver cut surface. Regarding the method.
この近年、ＥＳ細胞やｉＰＳ細胞から試験管内で肝細胞、さらに肝組織を作る研究が加速している。例えば、肝芽細胞を作成する方法としては、アクチビン存在下で胚性幹細胞を内胚葉細胞に分化後、ＢＭＰ−４およびｂＦＧＦ存在下で培養する方法（特許文献１）や血管内皮細胞、間葉系幹細胞等と内胚葉性機関に分化を方向づけられた細胞とを培養することにより肝芽細胞を作成する方法が記載されている（特許文献２）。 In recent years, research for producing hepatocytes and further liver tissue in vitro from ES cells and iPS cells has been accelerated. For example, as a method for producing hepatoblasts, embryonic stem cells are differentiated into endoderm cells in the presence of activin and then cultured in the presence of BMP-4 and bFGF (Patent Document 1), vascular endothelial cells, mesenchyme A method for producing hepatoblasts by culturing stem cells or the like and cells whose differentiation has been directed to an endoderm organ has been described (Patent Document 2).
しかしながら、肝臓移植で用いられるような肝臓組織を作成する（再生させる）ことは成功していない。また、肝細胞移植では、通常、肝細胞を門脈内に移植するが極めて少数の移植細胞しか定着しない。その上に、門脈内に移植するような肝細胞は、試験管内では細胞の成熟が極めて遅いことが知られている。 However, creation (regeneration) of liver tissue as used in liver transplantation has not been successful. Further, in liver cell transplantation, hepatocytes are usually transplanted into the portal vein, but very few transplanted cells are established. In addition, hepatocytes that are transplanted into the portal vein are known to undergo extremely slow maturation in vitro.
肝細胞移植の生着や効果を上げるために、肝芽細胞のように移植細胞の高機能化を図れば、門脈への投与は塞栓のリスクが高まり臨床では危険である。本来、肝臓は肝動脈を介した高圧系と門脈を介した低圧系で維持されており、 特に後者が肝細胞の増殖に必要である。試験管内で新に作製した肝組織を前述の２系統の血管吻合なしに移植すると、毛細血管である動脈系が流入して、移植組織が育たない等の課題があった。 In order to increase the engraftment and effect of hepatocyte transplantation, if the transplanted cells such as hepatoblasts are made to be highly functional, administration to the portal vein increases the risk of embolism and is dangerous in the clinic. Originally, the liver is maintained in a high-pressure system via the hepatic artery and a low-pressure system via the portal vein, and the latter is particularly necessary for hepatocyte proliferation. When liver tissue newly prepared in a test tube is transplanted without the above-mentioned two-system vascular anastomosis, there is a problem that the arterial system, which is a capillary, flows into the transplanted tissue and the transplanted tissue does not grow.
本発明者らは、これらの問題を克服する方法として、ラットならびにブタモデル用いて検討を続けてきた。組織移植の場合、ラットおよびマイクロミニブタ（ＭＭＰ）の成熟肝臓を切除して、組織スライサーで薄層組織とした。肝表面、肝臓内（肝表面からの埋め込み）、大網、さらに肝断端に血管吻合なしで移植し検討したが、肝断端が最も有効であった。さらにブタモデルを用いて臨床応用を前提に肝断端作製法を開発した。本法は、門脈系ならびに胆管系を過度に焼却することなく、生’状態で肝臓断面を作製し、薄層肝チップを張り付け後、生体吸収材料で包衣した。さらに試験管内で作成される肝芽組織の移植を前提に、ラットおよびマイクロミニブタ胎児肝を用いた肝芽移植を検討した。ルシフェラーゼ胎児の肝芽移植モデルでは、経時的に移植グラフトの発育程度を非侵襲的に観察したが、肝芽グラフトでは、極めて長期に維持され、発育を続けるものも見られた。 The present inventors have continued investigations using rat and pig models as methods for overcoming these problems. In the case of tissue transplantation, mature livers of rats and microminipigs (MMP) were excised and turned into thin layers with a tissue slicer. Transplantation was performed without vascular anastomosis on the liver surface, in the liver (implanted from the liver surface), omentum, and liver stump, but the liver stump was most effective. Furthermore, we developed a liver stump preparation method on the premise of clinical application using a pig model. In this method, without excessively incinerating the portal vein system and the bile duct system, a liver cross-section was prepared in a raw state, a thin-layer liver chip was attached, and then wrapped with a bioabsorbable material. In addition, hepatoblast transplantation using rat and micromini-pig fetal liver was examined on the premise of transplantation of liver bud tissue prepared in vitro. In the luciferase fetal liver bud transplantation model, the degree of graft graft development was observed non-invasively over time, but hepatic bud grafts were found to be maintained for a very long time and continue to grow.
すなわち、本発明は、
（１）肝細胞を含み、生体の肝臓切離面に移植して肝組織を再生するための製剤、
（２）前記生体は、ヒト肝疾患患者である、（１）の製剤、
（３）前記肝細胞は、肝組織切片に由来するか、または肝芽細胞である、（１）又（２）の製剤、
（４）前記肝芽細胞が、胎児肝芽または多能性幹細胞由来の肝芽細胞である（３）の製剤、
（５）多能性幹細胞由来の肝芽細胞が、ＥＳ細胞由来またはｉＰＳ細胞由来の肝芽細胞である（４）の製剤、
（６）前記肝臓切離面が、肝断端である（１）〜（５）のいずれかの製剤、
（７）前記ヒト肝疾患患者の疾患が、肝不全である（２）〜（６）のいずれかの製剤、
（８）前記肝不全が、先天性代謝性肝疾患、急性肝不全、肝硬変、肝がん、胆道閉鎖症、又はバッドキアリー症候群である、（７）の製剤、
（９）前記生体の肝臓切離面に移植後、生体吸収材料で包衣するための（１）〜（８）のいずれかの製剤、
（１０）生体の肝臓切離面に肝細胞を含む製剤を投与して、肝組織を再生させる方法、お及び、
（１１）前記生体がヒト肝疾患患者である、（１０）に記載の方法を提供する。
That is, the present invention
(1) A preparation containing hepatocytes and regenerating liver tissue by transplanting to a liver cut surface of a living body,
(2) The living body is a human liver disease patient, the preparation of (1),
(3) The preparation of (1) or (2), wherein the hepatocyte is derived from a liver tissue section or is a hepatoblast.
(4) The preparation according to (3), wherein the hepatoblasts are fetal hepatoblasts or hepatoblasts derived from pluripotent stem cells,
(5) The preparation of (4), wherein the pluripotent stem cell-derived hepatoblast is an ES cell-derived or iPS cell-derived hepatoblast,
(6) The preparation according to any one of (1) to (5), wherein the liver dissection surface is a liver stump,
(7) The preparation according to any one of (2) to (6), wherein the disease of the human liver disease patient is liver failure,
(8) The preparation according to (7), wherein the liver failure is innate metabolic liver disease, acute liver failure, cirrhosis, liver cancer, biliary atresia, or Bad Kiary syndrome,
(9) The preparation according to any one of (1) to (8) for wrapping with a bioabsorbable material after transplantation to the liver cut surface of the living body,
(10) A method of regenerating liver tissue by administering a preparation containing hepatocytes to a liver cut surface of a living body, and
(11) The method according to (10), wherein the living body is a human liver disease patient.
本発明の製剤により、移植肝臓組織をコラゲナーゼ等の細胞移植用に加工しなくてもよく、さらに、血管内移植と異なり塞栓等の血管合併症がないことがこれまでの肝細胞移植、肝組織移植比べ利点がある。また、肝不全等の肝疾患に関し、その病変部を切除し、そこに新たに肝臓を再生させることが治療法として有効であるため、本発明の製剤は係る疾患の治療に非常に有益である。 According to the preparation of the present invention, the transplanted liver tissue does not need to be processed for cell transplantation such as collagenase, and further, unlike the intravascular transplantation, there is no vascular complication such as embolism so far hepatocyte transplantation, liver tissue There are advantages compared to transplantation. In addition, regarding a liver disease such as liver failure, it is effective as a therapeutic method to excise the lesion and newly regenerate the liver there, so the preparation of the present invention is very useful for the treatment of the disease. .
本発明で使用される肝細胞は、肝組織切片または肝芽細胞は等の組織化された肝細胞である。肝組織切片は、生体肝臓の余剰肝臓組織を組織スライサーで薄層組織として使用することができる。肝芽細胞は、未成熟な胎児から得られるものでもよく、また、ＥＳ細胞やｉＰＳ細胞からアクチビン存在下で内胚葉細胞に分化後、ＢＭＰ−４およびｂＦＧＦ存在下で培養する方法等で得られる肝芽細胞、肝前駆細胞を含む細胞塊等や血管内皮細胞、間葉系幹細胞等と内胚葉性機関に分化を方向づけられた細胞とを培養することにより得られた肝芽細胞等が含まれる。 The hepatocytes used in the present invention are organized hepatocytes such as liver tissue sections or hepatoblasts. The liver tissue section can be used as a thin layer tissue with a tissue slicer from the excess liver tissue of the living liver. Hepatoblasts may be obtained from immature fetuses, or obtained by differentiation from ES cells or iPS cells into endoderm cells in the presence of activin and then culturing in the presence of BMP-4 and bFGF. Includes hepatoblasts, cell clusters containing hepatic progenitor cells, vascular endothelial cells, mesenchymal stem cells, etc. and hepatoblasts obtained by culturing cells that have been differentiated by endoderm .
本発明の「生体」とは、生存している動物の体を指す。前記動物としては、哺乳類が好ましく、ヒトがより好ましく、ヒト肝疾患患者が最も好ましい。 The “living body” of the present invention refers to a living animal body. The animal is preferably a mammal, more preferably a human, and most preferably a human liver disease patient.
対象となる肝疾患としては、先天性代謝性肝疾患、急性肝不全、肝硬変、肝がん、胆道閉鎖症、バッドキアリー症候群等に起因する肝不全患者である。先天性代謝性肝疾患が好ましい。先天性代謝性肝疾患には、アルファ1抗トリプシン欠損症 ウィルソン病，ヘマクロマトシス、チロシン血症、家族性肝内胆汁うっ滞（FIC、胆汁酸代謝の障害）、シトリン欠損による新生児肝内胆汁うっ滞（NICCD）、高リポ蛋白血症、クリグラー-ナジャール症候群、血友病、プロテインＣ欠損症、糖原病、プロトポルフィリン症、シトルリン血症（II型）、尿素回路不全（OTCD、CPS1D、アルギノコハク酸合成酵素不全症）、クリグラー-ナジャール症候群、ガラクトース血症、シュウ酸症、有機酸血症（メチルマロン酸血症、プロピオン酸血症）が含まれる。これらの疾患に対しては、HLAの一致した同種他家の肝組織切片や、先天代謝性疾患に対応した遺伝子治療を施した自家ｉＰＳ細胞から作製した肝芽細胞を用いることが望ましい。 The target liver diseases are patients with liver failure caused by innate metabolic liver disease, acute liver failure, liver cirrhosis, liver cancer, biliary atresia, Bad Kiary syndrome, and the like. Innate metabolic liver disease is preferred. Congenital metabolic liver disease includes alpha 1 antitrypsin deficiency Wilson disease, hematochromatosis, tyrosinemia, familial intrahepatic cholestasis (FIC, impaired bile acid metabolism), newborn intrahepatic bile due to citrin deficiency Stasis (NICCD), hyperlipoproteinemia, Krigler-Najar syndrome, hemophilia, protein C deficiency, glycogenosis, protoporphyria, citrullinemia (type II), urea circuit failure (OTCD, CPS1D, Arginosuccinic acid synthase deficiency), krigler-najar syndrome, galactosemia, oxalic acid, organic acidemia (methylmalonic acidemia, propionic acidemia). For these diseases, it is desirable to use hepatic tissue cells prepared from autologous iPS cells that have been subjected to gene therapy corresponding to innate metabolic diseases, and liver tissue sections of the same type of allogeneic family that have matched HLA.
本発明の肝細胞製剤は肝臓切離面に移植され、生体吸収材料および大網等で包衣される。肝臓切離面は、肝表面、肝臓内に形成された切離面や肝断端等が挙げられる、特に肝断端が好ましい。肝断端の作成は、門脈系ならびに胆管系を過度に焼却することなく、‘生’状態で肝臓断面を作製することが望ましい。 The hepatocyte preparation of the present invention is transplanted to the liver cut surface and wrapped with a bioabsorbable material, omentum or the like. Examples of the liver cut surface include a liver surface, a cut surface formed in the liver, and a liver stump. A liver stump is particularly preferable. In producing the liver stump, it is desirable to produce the liver cross section in the “raw” state without excessively incinerating the portal vein system and the bile duct system.
生体吸収材料は、マトリゲル（登録商標）、コラーゲン、フィブリン糊、ポリグリコール酸やポリ乳酸などが単独または組み合わせて用いることができるが、これらに限定するものではない。 As the bioabsorbable material, Matrigel (registered trademark), collagen, fibrin glue, polyglycolic acid, polylactic acid and the like can be used alone or in combination, but are not limited thereto.
本発明の肝細胞製剤を肝疾患患者に移植する量は、患者の年齢、体重、性別、疾患の種類や程度により適宜選択される。 The amount of the hepatocyte preparation of the present invention to be transplanted into a liver disease patient is appropriately selected depending on the patient's age, weight, sex, and disease type and degree.
以下、実施例をもって本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
実施例１ マイクロミニブタから肝芽の摘出
妊娠３１から３２日令のマイクロミニブタから子宮を摘出し、クリーンベンチ内で胎児（５匹）を摘出後、顕微鏡下でそれぞれの胎児臓器を分離し、胎児肝臓（肝芽）を採取した。長径６ｍｍ×短径４ｍｍ×厚み２ｍｍ程度であった。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Removal of liver buds from microminiature pigs The uterus was removed from microminiature pigs from 31 to 32 days of gestation, and fetuses (5 animals) were removed in a clean bench, and each fetal organ was separated under a microscope. Liver (liver bud) was collected. The major axis was about 6 mm, the minor axis was 4 mm, and the thickness was about 2 mm.
実施例２ レシピエントの準備
実施例１で用いたものとは異なるマイクロミニブタの左葉に０号糸でマットレス縫合3ステッチかけ、牽引による血液遮断を行い、左葉を阻血した。結紮後、左葉を切離し、断端を止血した。次に移植床となる中葉に断端面を作成した。まず、中葉の前面に電気メスにてマーキングし、カンシにて圧座し、血管系および胆管系を残し、肝臓実質を除いた。随時生理食塩水をかけながら電気メスで丁寧に切離面を作成した。実質を強く焼かないで、出血点のみ電気凝固させた。
Example 2 Preparation of Recipients Three stitches of mattress suture were applied to the left lobe of a micromini pig different from that used in Example 1 with No. 0 thread, blood was blocked by traction, and the left lobe was blocked. After ligation, the left lobe was cut off and the stump was hemostatic. Next, stumps were created in the middle lobe that would become the transplant bed. First, the front of the middle lobe was marked with an electric scalpel, pressed with a cane, leaving the vascular system and bile duct system, and excluding the liver parenchyma. A separation surface was carefully created with an electric knife while applying physiological saline as needed. Only the bleeding point was electrocoagulated without burning the substance strongly.
実施例３
前記実施例で作成したレシピエントの肝断端に肝芽及び薄層肝チップを移植し、マトリゲルを貼り付け、レシピエントの大網による包衣を行った。移植グラフトは発育を続け長期に維持された。
Example 3
Liver buds and thin-layer liver chips were transplanted to the liver stumps of the recipients prepared in the above examples, matrigel was applied, and dressing was performed with the recipient's greater omentum. The graft graft continued to grow and was maintained for a long time.
実施例４ ラットへの胚芽の移植
肝切除を加えたレシピエントラットの残肝にルシフェラーゼ遺伝子が導入した肝芽を２個移植した。一個は、肝臓の被膜下、他は断端に移植した。両肝芽とも２週前後までいったん発育した。しかし、被膜下移植のものは、しだいにそのサイズを縮小した。一方、断端へ移植された肝芽は発育を維持し、現在１００日を越えてその機能を保った。本発明の製剤を用いることにより、肝疾患を治療できることが示された。
Example 4 Embryo Transplantation into Rats Two hepatoblasts introduced with a luciferase gene were transplanted into the remaining liver of recipient rats after hepatectomy. One was transplanted under the liver capsule and the other at the stump. Both liver buds developed once until around 2 weeks. However, the subcapsular implants gradually reduced in size. On the other hand, hepatoblasts transplanted to the stump maintained their growth and maintained their function for over 100 days. It was shown that liver diseases can be treated by using the preparation of the present invention.
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| WO2020145231A1 (en) * | 2019-01-09 | 2020-07-16 | 公立大学法人横浜市立大学 | Prophylactic and/or therapeutic agent for diseases accompanied by fibrosis |
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| JP2013526297A (en) * | 2010-05-07 | 2013-06-24 | ユニバーシティー オブ ノース カロライナ アット チャペル ヒル | Method for transplanting cells from parenchyma |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2013526297A (en) * | 2010-05-07 | 2013-06-24 | ユニバーシティー オブ ノース カロライナ アット チャペル ヒル | Method for transplanting cells from parenchyma |
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| ORGAN BIOLOGY, vol. 20, no. 2, JPN6019017782, 2013, pages 205 - 210, ISSN: 0004156899 * |
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|---|---|---|---|---|
| WO2020145231A1 (en) * | 2019-01-09 | 2020-07-16 | 公立大学法人横浜市立大学 | Prophylactic and/or therapeutic agent for diseases accompanied by fibrosis |
| JPWO2020145231A1 (en) * | 2019-01-09 | 2021-11-25 | 公立大学法人横浜市立大学 | Preventive and / or therapeutic agents for diseases associated with fibrosis |
| JP7385929B2 (en) | 2019-01-09 | 2023-11-24 | 公立大学法人横浜市立大学 | Preventive and/or therapeutic agent for diseases accompanied by fibrosis |
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