JP2009007321A - Treatment of refractory enteritis with mesenchymal stem cell - Google Patents
Treatment of refractory enteritis with mesenchymal stem cell Download PDFInfo
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- JP2009007321A JP2009007321A JP2007194910A JP2007194910A JP2009007321A JP 2009007321 A JP2009007321 A JP 2009007321A JP 2007194910 A JP2007194910 A JP 2007194910A JP 2007194910 A JP2007194910 A JP 2007194910A JP 2009007321 A JP2009007321 A JP 2009007321A
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Abstract
Description
本発明は、腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（Mesenchymal Stem Cell；ＭＳＣ）を経静脈的に投与することによって炎症性腸疾患を予防・治療するための、間葉系幹細胞（ＭＳＣ）を備えた炎症性腸疾患の予防・治療剤や、腸上皮幹細胞障害剤と、間葉系幹細胞（ＭＳＣ）とを備えたことを特徴とする炎症性腸疾患の予防・治療剤や、哺乳動物に対して腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を経静脈的に前記哺乳動物に投与する炎症性腸疾患の予防・治療方法や、腸上皮幹細胞障害剤及び間葉系幹細胞（ＭＳＣ）の、炎症性腸疾患の予防・治療のための薬剤の製造における使用等に関する。 The present invention relates to a mesenchymal stem cell for preventing / treating inflammatory bowel disease by intravenously administering a mesenchymal stem cell (MSC) after intestinal epithelial stem cell disorder treatment. A prophylactic / therapeutic agent for inflammatory bowel disease comprising (MSC), a prophylactic / therapeutic agent for inflammatory bowel disease comprising an intestinal epithelial stem cell disorder agent and a mesenchymal stem cell (MSC), A method for preventing / treating inflammatory bowel disease, wherein mesenchymal stem cells (MSC) are intravenously administered to the mammal after intestinal epithelial stem cell damage treatment is performed on the mammal, or an intestinal epithelial stem cell disorder agent And the use of mesenchymal stem cells (MSC) in the manufacture of a medicament for the prevention and treatment of inflammatory bowel disease.
炎症性腸疾患（ＩＢＤ）は、最も難治性な消化器疾患の１つであり、患者の生活の質を著しく低下させ、若年患者の結腸直腸癌のリスクを高めることが知られている。現在行なわれている炎症性腸疾患の治療方法として、ヒト化抗ＴＮＦα抗体（インフリキシマブ）の投与が行なわれている（例えば、特許文献１参照）が、決定的な治療は困難なままである。その他、チロシンキナーゼ阻害剤を、そのような治療を必要とするヒトに投与する段階を含む炎症性腸疾患を治療する方法（例えば、特許文献２参照）も知られている。 Inflammatory bowel disease (IBD) is one of the most refractory gastrointestinal diseases and is known to significantly reduce the quality of life of patients and increase the risk of colorectal cancer in young patients. Currently, humanized anti-TNFα antibody (infliximab) is administered as a method for treating inflammatory bowel disease (see, for example, Patent Document 1), but definitive treatment remains difficult. In addition, a method for treating inflammatory bowel disease including a step of administering a tyrosine kinase inhibitor to a human in need of such treatment (see, for example, Patent Document 2) is also known.
他方、骨髄間葉系幹細胞は、造血幹細胞（ＨＳＣ）とともに、多分化能をもつ骨髄幹細胞の１つとして知られている。最近、成人骨髄由来細胞が、胚葉の境界すら越えてさまざまな細胞型に融合又は分化転換するという驚くべき可塑性を有しているとの報告がある（例えば、非特許文献１〜３参照）。より具体的には、成人型多能性幹細胞（ＭＡＰＣ）（例えば、非特許文献１参照）又は急速に自己再生する細胞（ＲＳ−ＭＳＣ）（例えば、非特許文献４参照）などの間葉系幹細胞（ＭＳＣ）が、骨芽細胞や軟骨細胞のような中胚葉だけでなく、肝細胞のような内胚葉や、神経前駆体のような外胚葉にも分化する能力があることが報告されている（例えば、非特許文献５〜７参照）。このように、ＭＳＣは、移植医療における慢性的なドナー（臓器）不足の問題を解決する可能性や、組織工学の可能性を高めるような貴重な細胞の供給源となる可能性を秘めているため、近年非常に注目され、広範な研究がなされるようになった（例えば、非特許文献８参照）。免疫特権細胞又はユニバーサルドナーとも呼ばれる（例えば、非特許文献９参照）ＭＳＣは、細胞治療や遺伝子治療に有用な細胞の候補と考えられており、すでに、骨形成不全症やＧＶＨＤ（移植片対宿主病）に対して臨床的に使用されてきている（例えば、非特許文献１０及び１１参照）。 On the other hand, bone marrow mesenchymal stem cells are known as one of pluripotent bone marrow stem cells together with hematopoietic stem cells (HSC). Recently, it has been reported that adult bone marrow-derived cells have surprising plasticity such as fusion or transdifferentiation into various cell types even across the germ layer boundary (see, for example, Non-Patent Documents 1 to 3). More specifically, a mesenchymal system such as adult pluripotent stem cells (MAPC) (for example, see Non-Patent Document 1) or rapidly self-regenerating cells (RS-MSC) (for example, see Non-Patent Document 4) It has been reported that stem cells (MSC) have the ability to differentiate not only in mesoderm such as osteoblasts and chondrocytes, but also in endoderm such as hepatocytes and ectoderm such as neural precursors. (For example, see Non-Patent Documents 5 to 7). In this way, MSC has the potential to solve the problem of chronic donor (organ) shortage in transplantation medicine and to become a valuable cell source that enhances the possibility of tissue engineering. For this reason, in recent years, it has attracted a great deal of attention and extensive research has been conducted (for example, see Non-Patent Document 8). MSCs, also called immune privileged cells or universal donors (see, for example, Non-Patent Document 9), are considered to be candidates for cells useful for cell therapy and gene therapy, and already have osteogenesis imperfecta and GVHD (graft versus host). (See Non-Patent Documents 10 and 11).
一方、ブスルファン（ＢＵ）は、細胞内に取り込まれた後にＤＮＡをアルキル化して、ＤＮＡ複製を阻害することで抗悪性腫瘍作用や骨髄抑制作用を示すアルキル化抗悪性腫瘍薬であり、既に経口製剤等が慢性骨髄性白血病や癌の治療薬として広く臨床使用されている。特に、慢性骨髄性白血病を治療するための造血幹細胞移植の前に、骨髄内の悪性腫瘍や異常な幹細胞を徹底的に除去する目的で用いられている。 Busulfan (BU), on the other hand, is an alkylated antineoplastic agent that exhibits an antineoplastic action and bone marrow suppression action by alkylating DNA after being taken up into cells and inhibiting DNA replication, and has already been orally formulated. Are widely used clinically as therapeutic agents for chronic myelogenous leukemia and cancer. In particular, it is used for the purpose of thoroughly removing malignant tumors and abnormal stem cells in the bone marrow before hematopoietic stem cell transplantation for treating chronic myeloid leukemia.
本発明の課題は、間葉系幹細胞（ＭＳＣ）を用いた、炎症性腸疾患の優れた予防・治療方法を提供することにある。 An object of the present invention is to provide an excellent method for preventing and treating inflammatory bowel disease using mesenchymal stem cells (MSC).
本発明者らは、決定的な治療法が存在しない炎症性腸疾患について、幹細胞移植による治療法に着目し、炎症性腸疾患モデルラットにＭＳＣ（間葉系幹細胞）を移植したが、炎症性腸疾患の改善は見られなかった。そこで、本発明者らは鋭意検討を行い、骨髄内の悪性腫瘍や異常な幹細胞を除去するのに用いられているＢＵ（ブスルファン）を併用したところ、炎症性腸疾患の症状が著しく改善することを見い出し、本発明を完成するに至った。 The inventors of the present invention focused on a treatment method using stem cell transplantation for inflammatory bowel disease for which no definitive treatment method exists, and transplanted MSC (mesenchymal stem cells) to an inflammatory bowel disease model rat. There was no improvement in bowel disease. Therefore, the present inventors have conducted intensive studies and combined use of BU (busulfan), which is used to remove malignant tumors and abnormal stem cells in the bone marrow, significantly improves the symptoms of inflammatory bowel disease. As a result, the present invention has been completed.
すなわち本発明は、（１）腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を経静脈的に投与することによって炎症性腸疾患を予防・治療するための、間葉系幹細胞（ＭＳＣ）を備えたことを特徴とする炎症性腸疾患の予防・治療剤や、（２）腸上皮幹細胞障害処理が、腸上皮幹細胞障害剤の投与又は放射線の照射であることを特徴とする上記（１）に記載の炎症性腸疾患の予防・治療剤や、（３）腸上皮幹細胞障害剤と、間葉系幹細胞（ＭＳＣ）とを備えたことを特徴とする炎症性腸疾患の予防・治療剤や、（４）腸上皮幹細胞障害剤が、抗癌剤であることを特徴とする上記（２）又は（３）に記載の炎症性腸疾患の予防・治療剤や、（５）抗癌剤が、アルキル化剤であることを特徴とす上記（４）に記載の炎症性腸疾患の予防・治療剤や、（６）アルキル化剤が、ブスルファン又はシクロホスファミドであることを特徴とする上記（５）に記載の炎症性腸疾患の予防・治療剤や、（７）間葉系幹細胞（ＭＳＣ）が、骨髄間葉系幹細胞であることを特徴とする上記（１）〜（６）のいずれかに記載の炎症性腸疾患の予防・治療剤や、（８）炎症性腸疾患が、潰瘍性大腸炎又はクローン病であることを特徴とする上記（１）〜（７）のいずれかに記載の炎症性腸疾患の予防・治療剤に関する。 That is, the present invention provides (1) a mesenchymal stem cell for preventing / treating inflammatory bowel disease by intravenously administering a mesenchymal stem cell (MSC) after intestinal epithelial stem cell disorder treatment. (MSC), a prophylactic / therapeutic agent for inflammatory bowel disease, and (2) intestinal epithelial stem cell disorder treatment is administration of intestinal epithelial stem cell disorder agent or irradiation of radiation The prophylactic / therapeutic agent for inflammatory bowel disease according to (1) above, and (3) the prophylaxis of inflammatory bowel disease comprising an intestinal epithelial stem cell disorder agent and a mesenchymal stem cell (MSC). The therapeutic agent, (4) the prophylactic / therapeutic agent for inflammatory bowel disease according to (2) or (3) above, wherein (4) the intestinal epithelial stem cell disorder agent is an anticancer agent, and (5) an anticancer agent A prophylactic agent for inflammatory bowel disease according to (4) above, which is an alkylating agent A therapeutic agent, or (6) a prophylactic / therapeutic agent for inflammatory bowel disease according to (5), wherein the alkylating agent is busulfan or cyclophosphamide, and (7) a mesenchymal system The prophylactic / therapeutic agent for inflammatory bowel disease according to any one of (1) to (6) above, wherein the stem cell (MSC) is a bone marrow mesenchymal stem cell, or (8) inflammatory bowel disease Relates to the preventive / therapeutic agent for inflammatory bowel disease according to any one of (1) to (7) above, which is ulcerative colitis or Crohn's disease.
また本発明は、（９）哺乳動物に対して腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を経静脈的に前記哺乳動物に投与する炎症性腸疾患の予防・治療方法に関する。 The present invention also provides (9) a method for preventing / treating inflammatory bowel disease, wherein mesenchymal stem cells (MSC) are intravenously administered to the mammal after intestinal epithelial stem cell damage treatment has been performed on the mammal. About.
さらに本発明は、（１０）腸上皮幹細胞障害剤及び間葉系幹細胞（ＭＳＣ）の、炎症性腸疾患の予防・治療のための薬剤の製造における使用に関する。 The present invention further relates to (10) use of an intestinal epithelial stem cell disorder agent and mesenchymal stem cell (MSC) in the manufacture of a medicament for the prevention / treatment of inflammatory bowel disease.
本発明によると、炎症性腸疾患を有する個体に、腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を移植（経静脈的に投与）することにより、炎症性腸疾患の改善効果を得ることができ、炎症性腸疾患の新しい治療体系の構築が可能となる。ＭＳＣは骨髄、骨膜、脂肪組織、末梢血から分離することが可能であり、細胞移植に自家細胞を用いる場合にはドナーの確保や拒絶反応のない、より安全な治療を行うことが可能となる。 According to the present invention, after an intestinal epithelial stem cell disorder treatment is performed on an individual having inflammatory bowel disease, mesenchymal stem cells (MSC) are transplanted (administered intravenously), thereby improving inflammatory bowel disease. The effect can be obtained, and a new treatment system for inflammatory bowel disease can be constructed. MSCs can be isolated from bone marrow, periosteum, adipose tissue, and peripheral blood, and when autologous cells are used for cell transplantation, safer treatment without securing donors or rejection is possible. .
本発明の炎症性腸疾患の予防・治療剤としては、腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を経静脈的に投与することによって、炎症性腸疾患を予防・治療するための、間葉系幹細胞（ＭＳＣ）を備えたことを特徴とする炎症性腸疾患の予防・治療剤や、腸上皮幹細胞障害剤と、間葉系幹細胞（ＭＳＣ）とを備えたことを特徴とする炎症性腸疾患の予防・治療剤であれば特に制限されず、また、本発明の炎症性腸疾患の予防・治療方法としては、哺乳動物に対して腸上皮幹細胞障害処理を行った後に、間葉系幹細胞（ＭＳＣ）を経静脈的に前記哺乳動物に投与する方法であれば特に制限されず、そしてまた、本発明の使用（方法）としては、炎症性腸疾患の予防・治療のための薬剤の製造における、腸上皮幹細胞障害剤及び間葉系幹細胞（ＭＳＣ）の使用であれば特に制限されず、炎症性腸疾患として具体的には、潰瘍性大腸炎及びクローン病、放射線腸炎、ケモセラピーによる腸管障害等を例示することができ、特に難治性の炎症性腸疾患として、潰瘍性大腸炎及びクローン病を例示することができる。 The prophylactic / therapeutic agent for inflammatory bowel disease according to the present invention is to prevent / treat inflammatory bowel disease by intravenously administering mesenchymal stem cells (MSC) after intestinal epithelial stem cell disorder treatment. A prophylactic / therapeutic agent for inflammatory bowel disease characterized by comprising mesenchymal stem cells (MSC), an intestinal epithelial stem cell disorder agent, and mesenchymal stem cells (MSC) The agent is not particularly limited as long as it is a prophylactic / therapeutic agent for inflammatory bowel disease, and as a method for preventing / treating inflammatory bowel disease of the present invention, intestinal epithelial stem cell disorder treatment was performed on mammals. There is no particular limitation as long as it is a method for intravenously administering mesenchymal stem cells (MSC) to the mammal later, and the use (method) of the present invention includes prevention and treatment of inflammatory bowel disease. Intestinal epithelial stem cell disorder and It is not particularly limited as long as it is a mesenchymal stem cell (MSC), and specific examples of inflammatory bowel disease include ulcerative colitis and Crohn's disease, radiation enteritis, chemotherapy-induced intestinal disorders, and the like. Particularly refractory inflammatory bowel diseases include ulcerative colitis and Crohn's disease.
本発明の炎症性腸疾患の予防・治療剤によると、腸上皮幹細胞障害剤をヒト、マウス、ラット、イヌ、ウシ、サル等の哺乳動物（特にヒト）に投与した後に、哺乳動物、好ましくは、腸上皮幹細胞障害剤を投与した哺乳動物と同種の哺乳動物（特にヒト）由来の間葉系幹細胞を、腸上皮幹細胞障害剤を投与した前述の哺乳動物（特にヒト）に経静脈的に投与することにより、炎症性腸疾患を予防又は治療することができる。本発明の炎症性腸疾患の予防・治療剤の作用機作の詳細は明らかではないが、対象となる哺乳動物に腸上皮幹細胞障害処理を行った後に、哺乳動物、好ましくは、腸上皮幹細胞障害剤を投与した哺乳動物と同種の哺乳動物由来の間葉系幹細胞を経静脈的に投与することにより、間葉系幹細胞が腸管上皮に移行して腸上皮幹細胞に分化し、その結果、炎症性腸疾患の予防・治療効果が得られると考えられる。 According to the prophylactic / therapeutic agent for inflammatory bowel disease of the present invention, after the intestinal epithelial stem cell damaging agent is administered to mammals (particularly humans) such as humans, mice, rats, dogs, cows, monkeys, mammals, preferably Mesenchymal stem cells derived from mammals (especially humans) of the same type as mammals administered with intestinal epithelial stem cell disorder agent are intravenously administered to the aforementioned mammals (especially human) administered with intestinal epithelial stem cell disorder agent By doing so, inflammatory bowel disease can be prevented or treated. Although the details of the mechanism of action of the preventive / therapeutic agent for inflammatory bowel disease of the present invention are not clear, after the subject mammal is treated with intestinal epithelial stem cell disorder, the mammal, preferably intestinal epithelial stem cell disorder Mesenchymal stem cells derived from mammals of the same species as the mammal to which the agent is administered are intravenously administered, so that the mesenchymal stem cells migrate to the intestinal epithelium and differentiate into intestinal epithelial stem cells. It is thought that preventive and therapeutic effects for intestinal diseases can be obtained.
上記腸上皮幹細胞障害処理としては、腸上皮幹細胞の分化及び／又は増殖を抑制する効果を有する処理である限り特に制限されず、腸上皮幹細胞障害剤の投与や放射線の照射、腸上皮幹細胞のホメオスタシスにかかわるシグナル（ＷＮＴシグナルやＮｏｔｃｈシグナル）の阻害剤の投与、アンチセンスやｓｉＲＮＡなどの分子標的治療薬の投与を好ましく例示することができ、中でも、腸上皮幹細胞障害剤の投与や放射線の照射を特に好ましく例示することができる。 The intestinal epithelial stem cell disorder treatment is not particularly limited as long as it is a treatment having an effect of suppressing differentiation and / or proliferation of intestinal epithelial stem cells. Administration of intestinal epithelial stem cell disorder agents, irradiation of radiation, intestinal epithelial stem cell homeostasis Administration of an inhibitor of a signal related to (WNT signal or Notch signal), administration of a molecular target therapeutic agent such as antisense or siRNA can be preferably exemplified, and in particular, administration of an intestinal epithelial stem cell disorder agent or irradiation of radiation Particularly preferable examples can be given.
上記腸上皮幹細胞障害剤としては、腸上皮幹細胞の分化や増殖を抑制する効果を有する限り特に制限されないが、ＤＮＡをアルキル化してＤＮＡ複製を阻害するアルキル化剤や、代謝を阻害して細胞の増殖や分裂を阻害する代謝拮抗剤や、抗腫瘍性抗生物質や、微小管阻害剤や、白金製剤や、トポイソメラーゼ阻害剤等の抗癌剤であることが好ましく、アルキル化剤であることがさらに好ましい。 The intestinal epithelial stem cell damaging agent is not particularly limited as long as it has an effect of suppressing the differentiation and proliferation of intestinal epithelial stem cells, but an alkylating agent that alkylates DNA to inhibit DNA replication or a cell that inhibits metabolism and inhibits cell replication. It is preferably an anticancer agent such as an antimetabolite that inhibits proliferation or division, an antitumor antibiotic, a microtubule inhibitor, a platinum preparation, a topoisomerase inhibitor, and more preferably an alkylating agent.
上記アルキル化剤としては、ブスルファン、シクロホスファミド、ＢＣＮＵ（N,N'-bis(2-chloroethyl)-N-nitrosourea）、ナイトラミン、イホスファミド、メルファラン、チオテパ、カルボコン、塩酸ニムスチン等を好ましく例示することができ、ブスルファンをより好ましく例示することができる。また、上記代謝拮抗剤としては、５−ＦＵ、メトトレキセートを好ましく例示することができ、上記抗腫瘍性抗生物質としては、ダウノルビシン、アクチノマイシンＤ、マイトマイシンＣ、ブレオマイシンを好ましく例示することができ、上記微小管阻害剤としてはビンクリスチン、パクリタキセル、ドセタキセルを好ましく例示することができ、上記トポイソメラーゼ阻害剤としてはイリノテカン、エトポシドを好ましく例示することができる。 As the alkylating agent, busulfan, cyclophosphamide, BCNU (N, N'-bis (2-chloroethyl) -N-nitrosourea), nitramine, ifosfamide, melphalan, thiotepa, carbocon, nimustine hydrochloride, etc. are preferable. It can be exemplified, and busulfan can be exemplified more preferably. Examples of the antimetabolite include 5-FU and methotrexate. Preferred examples of the antitumor antibiotic include daunorubicin, actinomycin D, mitomycin C, and bleomycin. Preferred examples of the microtubule inhibitor include vincristine, paclitaxel, and docetaxel, and preferred examples of the topoisomerase inhibitor include irinotecan and etoposide.
上記腸上皮幹細胞障害剤の投与方法としては特に制限されないが、経口投与、腹腔内投与、静脈内投与等を好ましく例示することができる。腸上皮幹細胞障害剤の投与量としては、炎症性腸疾患の種類、その症状の度合い、腸上皮幹細胞障害剤の種類等によって左右されるため、一概にいうことはできないが、本発明における炎症性腸疾患の予防・改善効果が得られるような範囲で投与量を適宜調整することができる。なお、腸上皮幹細胞障害剤の投与は、１〜複数回に分けて行ってもよい。 The administration method of the intestinal epithelial stem cell damaging agent is not particularly limited, and oral administration, intraperitoneal administration, intravenous administration and the like can be preferably exemplified. The dose of the intestinal epithelial stem cell disorder agent depends on the type of inflammatory bowel disease, the degree of its symptoms, the type of intestinal epithelial stem cell disorder agent, etc., and cannot be generally stated. The dose can be appropriately adjusted within a range where the effect of preventing or improving bowel disease can be obtained. In addition, administration of the intestinal epithelial stem cell disorder agent may be performed in 1 to multiple times.
上記間葉系幹細胞（ＭＳＣ）としては、未分化の間葉系細胞である限り特に制限はされず、哺乳動物の骨髄、骨膜、脂肪組織、末梢血等から常法に従い採取した後、未分化の間葉系幹細胞をプラスチック付着性の有無等により選択することができる。即ち、骨髄等に含まれる細胞の中で付着性を有するものを選択することにより間葉系幹細胞を得ることができる。ここで、間葉系幹細胞としては、本発明のより優れた効果を得る観点から、骨髄間葉系幹細胞（骨髄間質細胞）を用いることが好ましい。また、間葉系幹細胞としては、移植対象であるレシピエントと同種の哺乳動物由来の間葉系幹細胞を用いることが好ましく、レシピエント以外の同種の哺乳動物由来の間葉系幹細胞や、レシピエント自身の間葉系幹細胞（自家細胞）を用いることができるが、レシピエント自身の間葉系幹細胞を用いることがより好ましい。 The mesenchymal stem cells (MSC) are not particularly limited as long as they are undifferentiated mesenchymal cells, and are collected from mammalian bone marrow, periosteum, adipose tissue, peripheral blood and the like according to a conventional method, and then undifferentiated. Mesenchymal stem cells can be selected based on the presence or absence of plastic adhesion. That is, mesenchymal stem cells can be obtained by selecting adherent cells among cells contained in bone marrow or the like. Here, as a mesenchymal stem cell, it is preferable to use a bone marrow mesenchymal stem cell (bone marrow stromal cell) from the viewpoint of obtaining the superior effect of the present invention. Further, as the mesenchymal stem cells, it is preferable to use mesenchymal stem cells derived from the same mammal as the recipient to be transplanted, and mesenchymal stem cells derived from the same mammal other than the recipient, Although own mesenchymal stem cells (autologous cells) can be used, it is more preferable to use the recipient's own mesenchymal stem cells.
間葉系幹細胞の投与方法としては、腸上皮幹細胞障害処理後に、経静脈的に投与する限り特に制限はされないが、腸上皮幹細胞障害処理後４、５日以内に投与することが好ましい。また、間葉系幹細胞の投与量としては、炎症性腸疾患の種類、症状の度合い等にもよるが、１×１０６〜１×１０８/ｋｇの細胞（１〜複数回に分けて投与してもよい）を挙げることができる。 The method for administering mesenchymal stem cells is not particularly limited as long as it is administered intravenously after intestinal epithelial stem cell injury treatment, but it is preferably administered within 4 to 5 days after intestinal epithelial stem cell injury treatment. The dose of mesenchymal stem cells is 1 × 10 6 to 1 × 10 8 / kg cells (1 to 10 divided doses) depending on the type of inflammatory bowel disease, the degree of symptoms, and the like. May be included).
炎症性腸疾患の予防・治療のための薬剤（炎症性腸疾患の予防・治療剤）における腸上皮幹細胞障害剤や間葉系幹細胞は、それぞれ薬学的に許容される通常の担体、賦形剤、希釈剤、ｐＨ緩衝剤、生理的食塩水等の水溶性溶剤、塩化ナトリウム、グリセリン、Ｄ−マンニトール等の等張化剤、ヒト血清アルブミン等の安定化剤、メチルパラベン等の保存剤、ベンジルアルコール等の局麻剤などの各種調剤用配合成分を添加して用いることができる。また、間葉系幹細胞と共に、他の炎症性腸疾患の予防・治療剤を併用することもできる。 Intestinal epithelial stem cell disorder agents and mesenchymal stem cells in drugs for the prevention / treatment of inflammatory bowel disease (prevention / treatment agents for inflammatory bowel disease) are pharmaceutically acceptable ordinary carriers and excipients, respectively. , Diluents, pH buffering agents, water soluble solvents such as physiological saline, isotonic agents such as sodium chloride, glycerin and D-mannitol, stabilizers such as human serum albumin, preservatives such as methylparaben, benzyl alcohol It is possible to add various compounding ingredients for preparation such as local narcotics. In addition, a prophylactic / therapeutic agent for other inflammatory bowel diseases can be used in combination with mesenchymal stem cells.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。
[参考例]
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
[Reference example]
［化学薬品及び動物］
デキストラン硫酸ナトリウム（ＤＳＳ、分子量４０，０００）、トリニトロベンゼンスルホン酸（ＴＮＢＳ）、及びブスルファン（１，４−ブタンジオールジメタンスルホネート、ＢＵ）はSigma社から購入した。実験的腸炎の研究には４〜５週齢の雌のルイスラット及び７〜８週齢の雌のＢＡＬＢ／ｃマウス（Charles River Laboratories社製）を使用し、インビボでの動的なＭＳＣの局在の分析には、ＳＤ−ＴＧ（ＣＡＧ−ＥＧＦＰ）ラット、強化緑色蛍光タンパク質（ｅＧＦＰ）を普遍的に発現しているいわゆる「グリーンラット」（JSLC社製）を使用した。全ての実験動物は札幌医科大学動物実験施設にて飼育され、餌及び水は常に供給され、札幌医科大学動物実験部会の定める指針に従って扱われた。本研究は、国際条約である生物多様性条約及びバイオセーフティーに関するカルタヘナ議定書に沿っている。
[Chemicals and animals]
Dextran sulfate sodium (DSS, molecular weight 40,000), trinitrobenzenesulfonic acid (TNBS), and busulfan (1,4-butanediol dimethanesulfonate, BU) were purchased from Sigma. For the study of experimental enteritis, 4 to 5 week old female Lewis rats and 7 to 8 week old female BALB / c mice (Charles River Laboratories) were used, and the dynamic MSC was observed in vivo. For the present analysis, SD-TG (CAG-EGFP) rat, so-called “green rat” (manufactured by JSLC) that universally expresses enhanced green fluorescent protein (eGFP) was used. All experimental animals were bred at the Sapporo Medical University Animal Experiment Facility, food and water were always supplied, and were handled according to the guidelines set by the Sapporo Medical University Animal Experiment Committee. This study is in line with the International Convention on Biological Diversity and the Cartagena Protocol on Biosafety.
［統計解析］
後述の実験の全ての数値データは平均±標準偏差で表した。各実験グループにおけるサイトカインの定量的ｍＲＮＡ発現量の平均値、及びグループ間の組織学的評価は、一元配置分散分析（ＡＮＯＶＡ）とそれに続くボンフェローニの（Bonferrone’s）多重比較法によって分析された。２つのグループを比較するために、それぞれunpaired-ｔ検定及びマン−フィットニー（Mann-Whittney）Ｕ検定を用いたパラメトリック及びノンパラメトリック分析を行った。カテゴリ変数はカイ２乗検定、Pearsonの統計に基づく正確なＰ値、若しくはモンテカルロ法を用いて比較した。Ｐ＜０．０５のとき、有意差があるとした。すべての検定は両側検定を行った。
[Statistical analysis]
All numerical data in the experiments described below were expressed as mean ± standard deviation. Mean values of cytokine quantitative mRNA expression in each experimental group and histological evaluation between groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferrone's multiple comparison method. To compare the two groups, parametric and nonparametric analyzes were performed using the unpaired-t test and the Mann-Whittney U test, respectively. Categorical variables were compared using chi-square test, exact P value based on Pearson's statistics, or Monte Carlo method. When P <0.05, there was a significant difference. All tests were two-sided.
［ＭＳＣの単離及び細胞培養系］
骨髄由来のラットＭＳＣ（ｒＭＳＣ：以下、単に「ＭＳＣ」という。）は、Javazon et al.（Stem Cells 19, 219-225(2001)）に記載されているように回収及び培養した。すなわち、３〜６ヶ月齢のルイスラット（ｎ＝３）の大腿骨及び頚骨から、骨幹に２１ゲージの針を挿入し、２０％ウシ胎児血清（ＦＢＳ）を含むα-modified Eagle’s medium（αＭＥＭ）完全培地３０ｍｌでflushすることでラット骨髄細胞を回収した。細胞懸濁液を７０μｍナイロンフィルター（「BD Falcon」（登録商標）、 Becton Dickinson社製）で濾過後、７５ｃｍ２のフラスコに蒔いた。αＭＥＭ（２０％ＦＢＳ）完全培地中、３７℃、５％ＣＯ２で細胞を培養した。３日後に培地を新鮮な培地（１０％ＦＢＳ）に交換し、付着細胞を培養密度が９０％になるまで成長させて得た試料を継代０と定義した。後述のＭＳＣのフローサイトメトリー解析では、継代５の細胞を用いた。
[MSC isolation and cell culture system]
Bone marrow-derived rat MSC (rMSC: hereinafter simply referred to as “MSC”) was collected and cultured as described in Javazon et al. (Stem Cells 19, 219-225 (2001)). That is, a 21-gauge needle was inserted into the diaphysis from the femur and tibia of a 3-6 month old Lewis rat (n = 3), and α-modified Eagle's medium (αMEM containing 20% fetal bovine serum (FBS) was used. ) Rat bone marrow cells were recovered by flushing with 30 ml of complete medium. The cell suspension was filtered through a 70 μm nylon filter (“BD Falcon” (registered trademark), manufactured by Becton Dickinson) and then spread on a 75 cm 2 flask. Cells were cultured at 37 ° C., 5% CO 2 in αMEM (20% FBS) complete medium. After 3 days, the medium was replaced with fresh medium (10% FBS), and a sample obtained by growing adherent cells to a culture density of 90% was defined as passage 0. In the flow cytometric analysis of MSC described later, cells at passage 5 were used.
［ＭＳＣのフローサイトメトリー解析］
製造業者の説明書に従いフルオレセインイソチアネート（ＦＩＴＣ）又はフィコエリトリン（ＰＥ）が直接結合された１次抗体（ラット表面抗原特異的抗体又はアイソタイプコントロール抗体）で標識し、FACSCalibur（登録商標）フローサイトメーター（Becton Dickinson社製）を用いてＭＳＣのフローサイトメトリー解析を行った。前述のラット表面抗原特異的抗体には、抗ＣＤ１１ｂ（ＭＡＣ−１）抗体、抗ＣＤ３１（ＰＥＣＡＭ−１）抗体、抗ＣＤ４３（ロイコシアリン）抗体、抗ＣＤ４４（Indian blood group）抗体、抗ＣＤ４５（白血球共通抗原）抗体、及び抗ＣＤ９０（Ｔｈｙ１．１）抗体（それぞれImmunotech社製）が含まれ、また、アイソタイプコントロール抗体としては、マウス抗ＩｇＧ１−ＦＩＴＣ抗体及びマウス抗ＩｇＧ１−ＰＥ抗体（Immunotech社製）を用いた。なお、２次抗体としては、ヤギ抗免疫イムノグロブリンＧ（ＩｇＧ）−ＰＥ抗体（Immunotech社製）を使用した。
[MSC flow cytometry analysis]
FACSCalibur® flow cytometer labeled with primary antibody (rat surface antigen specific antibody or isotype control antibody) directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) according to manufacturer's instructions MSC flow cytometry analysis was performed using (Becton Dickinson). The aforementioned rat surface antigen-specific antibodies include anti-CD11b (MAC-1) antibody, anti-CD31 (PECAM-1) antibody, anti-CD43 (leucosialin) antibody, anti-CD44 (Indian blood group) antibody, and anti-CD45 (leukocyte common) Antigen) antibody and anti-CD90 (Thy1.1) antibody (each manufactured by Immunotech), and mouse isoIgG1-FITC antibody and mouse anti-IgG1-PE antibody (manufactured by Immunotech) as isotype control antibodies. Using. As a secondary antibody, goat anti-immunoglobulin G (IgG) -PE antibody (manufactured by Immunotech) was used.
前述のフローサイトメトリー解析の結果を図１に示す。図１のグラフのＹ軸は細胞数を表し、Ｘ軸は平均蛍光強度（ＭＦＩ）を表す。このグラフ中の塗りつぶされたヒストグラムはアイソタイプコントロール抗体を用いた結果を示し、白抜きのヒストグラムはラット表面抗原特異的抗体を用いた結果を示す。ＣＤ１１ｂ、ＣＤ３１、ＣＤ４３、ＣＤ４４、ＣＤ４５を含むほとんどの骨髄細胞マーカーは、ＭＳＣで完全に陰性であったが、ＣＤ９０（ｔｈｙ１．１）だけがＭＳＣ由来のほとんどの細胞で陽性であった。これらのＭＳＣ免疫表現型プロフィールは、以前にStem Cells, 19, 219-225(2001)で報告されている発現様式とよく一致している。 The results of the flow cytometry analysis described above are shown in FIG. In the graph of FIG. 1, the Y axis represents the number of cells, and the X axis represents the mean fluorescence intensity (MFI). The filled histogram in this graph shows the results using the isotype control antibody, and the open histogram shows the results using the rat surface antigen-specific antibody. Most bone marrow cell markers including CD11b, CD31, CD43, CD44, CD45 were completely negative in MSC, but only CD90 (thy1.1) was positive in most cells derived from MSC. These MSC immunophenotypic profiles are in good agreement with the expression pattern previously reported in Stem Cells, 19, 219-225 (2001).
［正形成骨髄下に誘導したＤＳＳ腸炎の誘導及びその評価］
以下のように、正形成骨髄を有するラットについてＤＳＳ腸炎の誘導を行った。
（１）正形成骨髄下でのＤＳＳ腸炎の誘導実験に用いるラットの飼育計画を図２に示す。図２からも分かるように、１０％ＤＳＳを含む飲料水を０日目〜６日目までラットに適宜与えた。これらのラットを４つのグループ（治療グループ１〜３、コントロールグループ）に分けた（グループ３以外のグループはｎ＝１０、グループ３はｎ＝１２）。治療グループのうち、グループ１には１日目、グループ２には２日目、グループ３には３日目に、ＭＳＣを２×１０４個／ｇ含む５００μｌのαＭＥＭを尾静脈に注射し、コントロールグループにはＭＳＣを注射しなかった。
上述の４つのグループのラットのそれぞれの体重の経時的変化を計測し、各ラットの実験開始日（ｄａｙ０）の体重を１００としたときの体重の割合（％）を算出した。各グループの体重の割合の平均値（％）の推移を図３に示す。図３から分かるように、各グループの体重の割合の平均値には有意差が見られなかった。
また、上述の４つのグループのラットの全生存率を、Kaplan-Meier法及びlog-rank（Mantel-Cox）検定を用いて分析した。その結果を図４に示す。この結果についても、各グループ間で有意な差は見られなかった。
以上のことから、本実験においてはＭＳＣ投与による治療効果は認められなかった。
[Induction of DSS enteritis induced under normal bone marrow and its evaluation]
Induction of DSS enteritis was performed on rats with positively formed bone marrow as follows.
(1) FIG. 2 shows the breeding plan of rats used for the induction experiment of DSS enteritis under normal bone marrow. As can be seen from FIG. 2, drinking water containing 10% DSS was appropriately given to rats from day 0 to day 6. These rats were divided into four groups (treatment groups 1-3, control group) (n = 10 for groups other than group 3 and n = 12 for group 3). Of the treatment groups, group 1 was injected on the 1st day, group 2 on the 2nd day, group 3 on the 3rd day, 500 μl αMEM containing 2 × 10 4 MSC / g was injected into the tail vein, The control group was not injected with MSC.
The change over time in the body weight of each of the four groups of rats described above was measured, and the ratio (%) of the body weight when the body weight on the experiment start date (day 0) of each rat was taken as 100 was calculated. The transition of the average value (%) of the weight ratio of each group is shown in FIG. As can be seen from FIG. 3, there was no significant difference in the average value of the proportion of body weight of each group.
In addition, the overall survival rate of the above four groups of rats was analyzed using the Kaplan-Meier method and the log-rank (Mantel-Cox) test. The result is shown in FIG. Again, no significant differences were found between the groups.
Based on the above, no therapeutic effect by MSC administration was observed in this experiment.
（２）次いで、投与したＭＳＣがどの程度の期間、生着しているかを確認するため、以下の実験を行った。
まず、本実験に用いるラットの飼育計画を図５に示す。１０匹ずつからなる２つのグループ（治療グループ、コントロールグループ）のラット（すべて雌）を用意し、図５に示されるように、治療グループについては、５％ＤＳＳを含む飲料水を３日目〜６日目まで継続的にラットに与え、コントロールグループについては、ＤＳＳを含まない飲料水を与えた。また、いずれのグループについても、０日目（図５の矢頭の日）に雄のドナーラット由来のＭＳＣを２×１０４個／ｇ含む５００μｌのαＭＥＭを尾静脈に注射することにより、ＭＳＣを移植（いわゆる異性（gender-mismatched）移植）した。両グループのラットをそれぞれ、１日目、７日目及び１０日目（図５の十字線の日）に屠殺し、肺組織及び結腸組織を採取した。これらの組織について、以下のようなｓｒｙ特異的定性ＰＣＲ分析を行った。すなわち、ｓｒｙ遺伝子はＹ染色体上にのみ存在するため、ｓｒｙ遺伝子の定性によって、組織中に雄のドナーラット由来の細胞が存在するか否かを調べることができる。
(2) Next, the following experiment was conducted to confirm how long the administered MSC was engrafted.
First, a rat breeding plan used in this experiment is shown in FIG. Rats (all females) of two groups of 10 animals (treatment group, control group) were prepared. As shown in FIG. 5, the treatment group was given drinking water containing 5% DSS on the third day. Rats were fed continuously until day 6, and the control group was given drinking water without DSS. In any group, MSC was injected by injecting 500 μl of αMEM containing 2 × 10 4 MSCs derived from male donor rats into the tail vein on day 0 (day of arrowhead in FIG. 5). Transplantation (so-called gender-mismatched transplantation). Rats from both groups were sacrificed on day 1, day 7 and day 10 (cross day in FIG. 5) and lung and colon tissues were collected. These tissues were subjected to the following sry specific qualitative PCR analysis. That is, since the sry gene exists only on the Y chromosome, it is possible to examine whether or not cells derived from male donor rats are present in the tissue, based on the qualification of the sry gene.
まず、採取した肺組織及び結腸組織から常法にしたがってゲノムを単離した。プライマーは以下のプライマーを用いた。
Ｙ染色体の性決定領域（sry）のゲノムＰＣＲ用フォワード：5’-catcgaagggttaaagtgcca-3’（配列番号１）
sryのゲノムＰＣＲ用リバース：5’-atagtgtgtaggttgttgtcc-3’（配列番号２）（Nucleic Acids Res, 14, 7569-7580(1986)）
本ゲノムＰＣＲは、SYBR（登録商標) Green PCR Master Mix（Applied Biosystems社製）を用いて、ABI PRISM（登録商標）7000 Sequence Detection System（Applied Biosystems社製）で、４０サイクルのゲノムＰＣＲ増幅（９４℃３０秒、６２℃３０秒、７２℃３０秒。ただし、１サイクル目に９４℃で６０秒変性させ、最後のサイクルで７２℃１０分間伸長させた。）によって行った。得られた各ＰＣＲ産物を２％アガロースゲル（Sigma社製）でそれぞれ電気泳動し、エチジウムブロマイド染色した結果を図６に示す。図６から分かるように、肺組織ではいずれのグループについてもほぼ同程度のｓｒｙ遺伝子シグナルが検出され、ｄａｙ１においては、特に強いシグナルが検出された。一方、結腸組織においては、いずれのグループについてもｄａｙ１とｄａｙ１０では、ｓｒｙ遺伝子のシグナルは同程度に弱かったが、ｄａｙ７では、ＤＳＳを投与したグループに最も強いｓｒｙ遺伝子シグナルが検出された。ＤＳＳを投与したグループにおいて、腸炎のピークであるｄａｙ１０でｓｒｙ遺伝子のシグナルが有意に低下した理由は定かではないが、この腸炎モデルラット（５％ＤＳＳ投与）においては、ＭＳＣが充分長期間といえるほどには生着を維持できず、細胞供給源又は免疫修飾物質のどちらか又は両方として適切に機能することができなかったと考えられる。
First, the genome was isolated from the collected lung tissue and colon tissue according to a conventional method. The following primers were used.
Y chromosome sex-determining region (sry) genomic PCR forward: 5'-catcgaagggttaaagtgcca-3 '(SEQ ID NO: 1)
Reverse for sry genomic PCR: 5'-atagtgtgtaggttgttgtcc-3 '(SEQ ID NO: 2) (Nucleic Acids Res, 14, 7569-7580 (1986))
This genomic PCR is performed using SYBR (registered trademark) Green PCR Master Mix (Applied Biosystems) and ABI PRISM (registered trademark) 7000 Sequence Detection System (Applied Biosystems) for 40 cycles of genomic PCR amplification (94 30 seconds at 62 ° C., 30 seconds at 62 ° C., 30 seconds at 72 ° C. However, denaturation was performed at 94 ° C. for 60 seconds in the first cycle and elongation was performed at 72 ° C. for 10 minutes in the last cycle. FIG. 6 shows the results of electrophoresis of each obtained PCR product on a 2% agarose gel (manufactured by Sigma) and staining with ethidium bromide. As can be seen from FIG. 6, almost the same level of sry gene signal was detected in the lung tissue, and particularly strong signal was detected in day1. On the other hand, in the colon tissue, the signal of the sry gene was weak in both groups of day 1 and day 10, but in day 7, the strongest sry gene signal was detected in the group administered with DSS. In the group administered DSS, the reason why the signal of the sry gene was significantly decreased at day 10 which is the peak of enteritis is not clear, but in this enteritis model rat (5% DSS administration), MSC can be said to be sufficiently long. It is believed that the engraftment could not be maintained as much and could not function properly as either a cell source or an immunomodulator or both.
［ＴＮＢＳ腸炎の誘導及びその評価］
以下のように、正形成骨髄を有するラットについてＴＮＢＳ腸炎の誘導を行った。
（１）正形成骨髄下でのＴＮＢＳ腸炎の誘導実験に用いるラットの飼育計画を図７に示す。０日目に、異性ＭＳＣ（雄のｒＭＳＣ）をラットに移植した（図７のｄａｙ０の黒矢頭）。２日目に、一般的な麻酔下（ペントバルビタールナトリウム）に、一匹当たり３０ｍｇのＴＮＢＳを含む５０％エタノール１００ｍｌを腸管内投与することで腸炎を誘導した（図７のｄａｙ２の黒矢頭）。また、対象として、異性ＭＳＣを含まない溶媒を移植した（図７のｄａｙ０の白矢頭）グループや、ＴＮＢＳを含まない５０％エタノールを腸内投与した（図７のｄａｙ２の白矢頭）グループを設けた。具体的には、２４匹のラットを以下の（１）〜（４）の４つのグループ（それぞれｎ＝６）に分けた。
（１）ＭＳＣ移植及びＴＮＢＳ投与（ＭＳＣ＋ＴＮＢＳ＋）
（２）溶媒移植及びＴＮＢＳ投与（ＭＳＣ−ＴＮＢＳ＋）
（３）ＭＳＣ移植及び５０％エタノール投与（ＭＳＣ＋ＴＮＢＳ−）
（４）溶媒移植及び５０％エタノール投与（ＭＳＣ−ＴＮＢＳ−）
上述の４つのグループのラットのそれぞれの体重の経時的変化を計測し、各ラットの実験開始日（ｄａｙ１）の体重を１００としたときの体重の割合（％）を算出した。各グループの体重の割合の平均値（％）の推移を図８に示す。図８から分かるように、ｄａｙ５においても、各グループの体重の割合の平均値には有意差が見られなかった。
[Induction of TNBS enteritis and its evaluation]
Induction of TNBS enteritis was performed on rats with positively formed bone marrow as follows.
(1) FIG. 7 shows the breeding plan of rats used for the induction experiment of TNBS enteritis under normal bone marrow. On day 0, heterosexual MSCs (male rMSCs) were transplanted into rats (black arrowhead on day 0 in FIG. 7). On the second day, enteritis was induced by intestinal administration of 100 ml of 50% ethanol containing 30 mg of TNBS per animal under general anesthesia (pentobarbital sodium) (black arrowhead of day 2 in FIG. 7). In addition, as a subject, a group transplanted with a solvent containing no isomeric MSC (day 0 in FIG. 7) and a group in which 50% ethanol not containing TNBS was administered enterally (white arrow in day 2 in FIG. 7) were provided. It was. Specifically, 24 rats were divided into the following four groups (1) to (4) (n = 6 respectively).
(1) MSC transplantation and TNBS administration (MSC + TNBS +)
(2) Solvent transplantation and TNBS administration (MSC-TNBS +)
(3) MSC transplantation and 50% ethanol administration (MSC + TNBS-)
(4) Solvent transplantation and 50% ethanol administration (MSC-TNBS-)
The change over time in the body weight of each of the four groups of rats described above was measured, and the ratio (%) of the body weight when the body weight on the experiment start date (day 1) of each rat was taken as 100 was calculated. The transition of the average value (%) of the weight ratio of each group is shown in FIG. As can be seen from FIG. 8, no significant difference was found in the average value of the weight ratio of each group even in day 5.
なお、「ＭＳＣ＋ＴＮＢＳ＋」グループのラットは、１日目、５日目及び９日目に屠殺し、肺組織及び結腸組織を採取した。採取したこれらの組織について、前述のｓｒｙ特異的定性ＰＣＲ分析を行った。得られた各ＰＣＲ産物を２％アガロースゲル（Sigma社製）でそれぞれ電気泳動し、エチジウムブロマイド染色した結果を図９に示す。図９から分かるように、肺組織のｄａｙ１ではｓｒｙ遺伝子の比較的強いシグナルが検出されたが、ｄａｙ５ではシグナルが有意に低下した。一方、結腸組織においては、ｄａｙ１ではｓｒｙ遺伝子のシグナルはほとんど見られなかったが、ｄａｙ５では比較的強いシグナルが検出され、ｄａｙ９では、シグナルの強度は有意に低下した。ＴＮＢＳを投与したグループにおいて、腸炎のピークであるｄａｙ９でｓｒｙ遺伝子のシグナルが有意に低下した理由は定かではないが、この腸炎モデルラット（ＴＮＢＳ投与）においては、ＭＳＣが充分長期間といえるほどには生着を維持できなかったために細胞供給源又は免疫修飾物質のどちらか又は両方として適切に機能することができなかったと考えられる。 The rats of the “MSC + TNBS +” group were sacrificed on the first day, the fifth day, and the ninth day, and lung tissue and colon tissue were collected. These collected tissues were subjected to the above-mentioned sry specific qualitative PCR analysis. The obtained PCR products were each electrophoresed on a 2% agarose gel (manufactured by Sigma) and stained with ethidium bromide. FIG. 9 shows the results. As can be seen from FIG. 9, a relatively strong signal of the sry gene was detected in day 1 of lung tissue, but the signal was significantly reduced in day 5. On the other hand, in the colon tissue, almost no signal of the sry gene was observed in day 1, but a relatively strong signal was detected in day 5, and the signal intensity was significantly decreased in day 9. In the group administered TNBS, the reason why the signal of the sry gene was significantly decreased at day 9 which is the peak of enteritis is not clear, but in this enteritis model rat (administration of TNBS), MSC can be said to be sufficiently long. Could not function properly as either a cell source or an immunomodulator or both because of failure to maintain engraftment.
[５％ＤＳＳ又はＢＵ２０ｍｇ／ｋｇ単回投与を受けたラットレシピエント結腸上皮組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在]
ＭＳＣの移植後３日目から５％ＤＳＳ（経口投与）を３日間投与した、移植７日目のラットレシピエント（図５）の結腸上皮組織、又はＢＵ２０ｍｇ／ｋｇ（腹腔内注射）を単回投与し７日目にＭＳＣの移植をしたラットレシピエント（図１７）の移植から３日目（ｄａｙ３）の結腸上皮組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在を調べた。具体的には、以下のような方法により局在を調べた。
０．２％グルタールアルデヒド含有ＰＢＳ及び４％パラホルムアルデヒドでラットを経心的に灌流して屠殺した。屠殺したラットの結腸を取り出し、これを灌流バッファーで４ｈｒ固定した。その後、結腸組織をパラフィンに包埋し、脱パラフィンしたパラフィン切片中でｅＧＦＰの免疫蛍光を決定するために厚さ３μｍの切片を作製した。次に、この組織切片を、２００倍希釈したヤギ抗ｅＧＦＰポリクローナル抗体（Abcam社製、英国ケンブリッジ）と共に４℃で一晩インキュベートした後、ＰＢＳで洗浄した。次いで、５００倍希釈したヤギＩｇＧ Ｈ＆Ｌに対するウサギポリクローナル抗体（ＦＩＴＣ）（Abcam社製）と共に３７℃で３０分間インキュベートした。インキュベートしたスライドをVECTASHIELD Mounting Medium及びＤＡＰＩ（Vector Laboratories社製）で対比染色した上で、蛍光顕微鏡（KEYENCE社製BZ-8000）及びZEISS Pascalレーザー走査型顕微鏡（R2100AG2、Bio-Rad社製）を用いてｅＧＦＰ発現細胞の局在を決定した。
[Localization of donor-derived eGFP-labeled MSCs in rat recipient colon epithelial tissue receiving a single dose of 5% DSS or BU 20 mg / kg]
Single dose of colon epithelial tissue of rat recipient (FIG. 5) on day 7 of transplantation, or BU 20 mg / kg (intraperitoneal injection) administered 3% from 3 days after transplantation of MSC for 3 days The localization of donor-derived eGFP-labeled MSCs in the colon epithelial tissue on the third day (day 3) from the transplantation of rat recipients (FIG. 17) who were administered and transplanted with MSCs on the seventh day was examined. Specifically, localization was examined by the following method.
Rats were transcardially perfused and sacrificed with PBS containing 0.2% glutaraldehyde and 4% paraformaldehyde. The sacrificed rat colon was removed and fixed with perfusion buffer for 4 hr. The colon tissue was then embedded in paraffin and 3 μm thick sections were made to determine eGFP immunofluorescence in deparaffinized paraffin sections. Next, this tissue section was incubated overnight at 4 ° C. with a 200-fold diluted goat anti-eGFP polyclonal antibody (Abcam, Cambridge, UK), and then washed with PBS. Subsequently, it was incubated at 37 ° C. for 30 minutes with a rabbit polyclonal antibody (FITC) (manufactured by Abcam) against goat IgG H & L diluted 500 times. The incubated slide was counterstained with VECTASHIELD Mounting Medium and DAPI (manufactured by Vector Laboratories), then using a fluorescence microscope (KEYENCE BZ-8000) and a ZEISS Pascal laser scanning microscope (R2100AG2, manufactured by Bio-Rad) The localization of eGFP expressing cells was determined.
５％ＤＳＳを経口投与されたラットにおける結腸上皮組織の切片の共焦点顕微鏡による拡大図（×１２００）を図１０のパネルａ、ｂに示し、ＢＵ２０ｍｇ／ｋｇ（腹腔内注射）を単回投与されたラットのｄａｙ７における結腸上皮組織の切片の共焦点顕微鏡による拡大図（×１２００）を図１０のパネルｃ、ｄに示す。
白矢頭はドナー由来ｅＧＦＰ標識ＭＳＣ（緑色）を表し、アスタリスクは腸上皮幹細胞ニッチと推定されている部位を示す。また、青色はＤＡＰＩによる核の対比染色を示す。ドナー由来ｅＧＦＰ標識ＭＳＣは、５％ＤＳＳ腸炎ラットにおいてはクリプトの上部上皮（図１０のパネルａ）及び被覆上皮で観察され、ＢＵ単独投与のラットにおいてはクリプト底部の幹細胞ニッチに観察された（図１０のパネルｃ及びｄ）。
このことから、ＤＳＳ処理によってレシピエントの結腸上皮で傷害を受ける細胞は、ＢＵ処理によってレシピエントの結腸上皮で傷害を受ける細胞と大きく異なっており、ＤＳＳが成熟分化した上皮細胞に損傷を与えるのに対し、ＢＵは最も未分化な腸上皮幹細胞を損傷することが示唆された。
An enlarged view (× 1200) of a section of colonic epithelial tissue in a rat orally administered with 5% DSS is shown in panels a and b of FIG. 10, and BU 20 mg / kg (intraperitoneal injection) was administered once. An enlarged view (× 1200) of a section of colonic epithelial tissue in day 7 of a given rat is shown in panels c and d of FIG.
The white arrowhead represents donor-derived eGFP-labeled MSC (green), and the asterisk indicates a site presumed to be an intestinal epithelial stem cell niche. The blue color indicates the nuclear counterstaining with DAPI. Donor-derived eGFP-labeled MSCs were observed in the upper epithelium of crypt (Panel a in FIG. 10) and coated epithelium in 5% DSS enteritis rats, and in the stem cell niche at the bottom of the crypt in rats treated with BU alone (FIG. 10 panels c and d).
This indicates that cells damaged in the recipient colon epithelium by DSS treatment are significantly different from those damaged in the recipient colon epithelium by BU treatment, and DSS damages mature and differentiated epithelial cells. In contrast, it was suggested that BU damages the most undifferentiated intestinal epithelial stem cells.
［急性骨髄形成不全下に誘導したＤＳＳ腸炎及びその評価］
Blood, 51, 601-610(1978)に記載の方法の変法によって、マウスにＢＵを単回投与し（２０ｍｇ／ｋｇ、腹腔内注射）、骨髄形成不全を誘導した。顕微鏡で、このマウスの骨髄組織と、正常なマウスの骨髄組織とを比較した結果を図１１に示す。図１１に示されているように、ＢＵを投与したマウスでは、組織学的に重度の骨髄形成の不全が引き起こされていることが確認された。
次に、急性骨髄形成不全下でのＤＳＳ腸炎の誘導実験に用いるマウスの飼育計画を図１２に示す。図１２からも分かるように、本実験では、前述の方法によるＢＵ投与によって骨髄形成不全を誘導したマウスにさらに１％又は４％のＤＳＳを継続的に投与したマウスや、ＢＵ投与を行わずに、１％又は４％のＤＳＳを投与したマウス（コントロール）や、ＢＵ投与は行うが、ＤＳＳ投与を行わないマウス（コントロール）を用いた。具体的には、３０匹のマウスをそれぞれ６匹からなる以下の５つのグループに分けた。
（１）ＢＵのみのグループ（ＢＵ ａｌｏｎｅ）
（２）１％ＤＳＳ腸炎及びＢＵ投与（ＢＵ＋１％ＤＳＳ）
（３）１％ＤＳＳ腸炎、ＢＵなし（１％ＤＳＳ ａｌｏｎｅ）
（４）４％ＤＳＳ腸炎及びＢＵ投与（ＢＵ＋４％ＤＳＳ）
（５）４％ＤＳＳ腸炎、ＢＵなし（４％ＤＳＳ ａｌｏｎｅ）
[DSS enteritis induced under acute myelogenesis failure and its evaluation]
By a modification of the method described in Blood, 51, 601-610 (1978), mice were administered a single dose of BU (20 mg / kg, intraperitoneal injection) to induce bone marrow dysplasia. FIG. 11 shows a result of comparison between the bone marrow tissue of this mouse and the bone marrow tissue of a normal mouse under a microscope. As shown in FIG. 11, it was confirmed that mice administered with BU caused histologically severe bone marrow failure.
Next, FIG. 12 shows the breeding plan of mice used for the induction experiment of DSS enteritis under acute myelogenesis failure. As can be seen from FIG. 12, in this experiment, mice in which bone marrow dysplasia was induced by BU administration according to the above-described method were further administered without further administration of 1% or 4% DSS, or without BU administration. Mice administered with 1% or 4% DSS (control) and mice administered with BU but not with DSS (control) were used. Specifically, 30 mice were divided into the following 5 groups each consisting of 6 mice.
(1) BU only group (BU alone)
(2) 1% DSS enteritis and BU administration (BU + 1% DSS)
(3) 1% DSS enteritis, no BU (1% DSS alone)
(4) 4% DSS enteritis and BU administration (BU + 4% DSS)
(5) 4% DSS enteritis, no BU (4% DSS alone)
上述の５つのグループのマウスのそれぞれの体重の経時的変化を計測し、各マウスの実験開始日（ｄａｙ１）の体重を１００としたときの体重の割合（％）を算出した。各グループの体重の割合の平均値（％）の推移を図１３に示す。図１３から分かるように、ＢＵ及びＤＳＳの両方を投与したグループでは、それぞれ同濃度のＤＳＳのみを投与したグループに比べて、有意な体重減少が見られた。
また、実験に用いたマウスは、９日目に屠殺解剖し、腸炎の重症度の評価に用いた。腸炎の重症度は、Gut, 50, 507-512(2002)の方法に従って、マウスの体重、結腸の長さ、組織学的スコアによって評価した。より具体的には、以下のように評価を行った。
ＴＮＦα、ＩＬ１β、ＩＦＮγ、ＩＬ１０のｍＲＮＡの発現の評価及び組織学的スコアの評価のために全結腸を取り出し、長軸方向の切片に分割した。組織を１０％中性ホルマリンで固定し、パラフィンに包埋し、薄切片を作成した。グループ分けを知らない２人の病理学者が別々に、ヘマトキシリン及びエオシン（Ｈ＆Ｅ）で染色した組織切片の組織学的スコアを評価した。この組織学的スコアを図１４に示す。図１４から分かるように、骨髄形成不全と共に誘導したグループのＤＳＳ腸炎は、それと同濃度のＤＳＳのみを投与した、骨髄形成が正常なグループ（ＢＵ不使用）の腸炎よりも重度であり、特に１％ＤＳＳ腸炎における重症度では、Ｐ＝０．０１７と顕著に異なっていた。一方、ＢＵによる骨髄形成不全と共に、１％ＤＳＳによる腸炎を誘導したグループ（ＢＵ＋１％ＤＳＳ）の重症度は、ＢＵを用いずに４％ＤＳＳで腸炎を誘導したグループ（４％ＤＳＳ ａｌｏｎｅ）のそれとほぼ同程度であった。
なお、前述の５つのグループのうち、「ＢＵ ａｌｏｎｅ」、「１％ＤＳＳ ａｌｏｎｅ」、「ＢＵ＋１％ＤＳＳ」の各グループのマウスにおける、ヘマトキシリン及びエオシン染色（Ｈ＆Ｅ染色）した結腸組織切片を顕微鏡で観察した結果を図１５に示す。「ＢＵ＋１％ＤＳＳ」のグループにおける組織では、腸炎を発症していることが明らかである。
The change over time in the weight of each of the five groups of mice described above was measured, and the ratio (%) of the weight when the weight on the experiment start date (day 1) of each mouse was defined as 100 was calculated. The transition of the average value (%) of the ratio of the weight of each group is shown in FIG. As can be seen from FIG. 13, in the group administered with both BU and DSS, significant weight loss was observed as compared to the group administered with only the same concentration of DSS.
The mice used in the experiment were sacrificed and dissected on the 9th day, and used for evaluation of the severity of enteritis. The severity of enteritis was evaluated by the weight of the mouse, the length of the colon, and the histological score according to the method of Gut, 50, 507-512 (2002). More specifically, the evaluation was performed as follows.
Whole colons were removed and divided into longitudinal sections for evaluation of TNFα, IL1β, IFNγ, IL10 mRNA expression and histological scores. The tissue was fixed with 10% neutral formalin, embedded in paraffin, and thin sections were prepared. Two pathologists who did not know the grouping separately assessed the histological scores of tissue sections stained with hematoxylin and eosin (H & E). This histological score is shown in FIG. As can be seen from FIG. 14, the DSS enteritis of the group induced with myelodysplasia was more severe than the enteritis of the group with normal bone marrow formation (BU not used) that received only the same concentration of DSS. The severity in% DSS enteritis was significantly different from P = 0.17. On the other hand, the severity of the group that induced enteritis with 1% DSS (BU + 1% DSS) along with myelogenesis due to BU was similar to that of the group that induced enteritis with 4% DSS without using BU (4% DSS alone). It was almost the same level.
Of the five groups described above, hematoxylin and eosin-stained (H & E-stained) colon tissue sections were observed with a microscope in mice of each group of “BU alone”, “1% DSS alone”, and “BU + 1% DSS”. The results are shown in FIG. It is clear that the tissues in the “BU + 1% DSS” group have developed enteritis.
なお、ＴＮＦα、ＩＬ１β、ＩＦＮγ、ＩＬ１０のｍＲＮＡの発現量の測定は以下のように行った。
TRIZOL（登録商標）（Invitrogen社製）を用いてトータルＲＮＡを抽出し、ThermoscriptRT-PCR System（Invitrogen社製）を利用して、１μｇのトータルＲＮＡ及びオリゴｄＴプライマーを用いてｃＤＮＡへの逆転写を行った。Primer Express Software（Applied Biosystems社製）を用いて、半定量的ＲＴ−ＰＣＲ用のラット特異的プライマーを作成した。配列は以下の通りである。
tnfαフォワード：5’-agaactccaggcggtgtct-3’（配列番号３）
tnfαリバース：5’-tccctcaggggtgtccttag-3’（配列番号４）
il1βフォワード：5’-cttccttgtgcaagtgtctgaagc-3’（配列番号５）
il1βリバース：5’-aagaaggtgcttgggtcctcatcc-3’（配列番号６）
ifnγフォワード：5’-agcctagaaagtctgaagaac-3’（配列番号７）
ifnγリバース：5’-accgactccttttccgcttcct-3’（配列番号８）
il10フォワード：5’-tggagtgaagaccagcaaaggc-3’（配列番号９）
il10リバース：5’-gcaacccaagtaacccttaaagtcc-3’（配列番号１０）
The expression levels of TNFα, IL1β, IFNγ, and IL10 mRNA were measured as follows.
Total RNA was extracted using TRIZOL (registered trademark) (manufactured by Invitrogen), and reverse transcription to cDNA was performed using ThermoscriptRT-PCR System (manufactured by Invitrogen) using 1 μg of total RNA and oligo dT primer. went. Rat specific primers for semi-quantitative RT-PCR were created using Primer Express Software (Applied Biosystems). The sequence is as follows.
tnfα forward: 5′-agaactccaggcggtgtct-3 ′ (SEQ ID NO: 3)
tnfα reverse: 5'-tccctcaggggtgtccttag-3 '(SEQ ID NO: 4)
il1β forward: 5′-cttccttgtgcaagtgtctgaagc-3 ′ (SEQ ID NO: 5)
il1β reverse: 5′-aagaaggtgcttgggtcctcatcc-3 ′ (SEQ ID NO: 6)
ifnγ forward: 5′-agcctagaaagtctgaagaac-3 ′ (SEQ ID NO: 7)
ifnγ reverse: 5'-accgactccttttccgcttcct-3 '(SEQ ID NO: 8)
il10 forward: 5'-tggagtgaagaccagcaaaggc-3 '(SEQ ID NO: 9)
il10 reverse: 5'-gcaacccaagtaacccttaaagtcc-3 '(SEQ ID NO: 10)
ＲＮＡの品質及び定量のための内部標準として、ｇａｐｄｈプライマーセット（TaqMan GAPDHコントロール試薬キット：Applied Biosystems社製）を用いた。このｇａｐｄｈプライマーの配列は以下の通りである。
gapdhフォワード：5’-atgggtgtgaaccacgagaaa-3’（配列番号１１）
gapdhリバース：5’-ggatacattgggggtaggaa-3’（配列番号１２）
ＲＴ−ＰＣＲは、SYBR（登録商標) Green PCR Master Mix（Applied Biosystems社製）を用いて、ABI PRISM（登録商標）7000 Sequence Detection System（Applied Biosystems社製）で、４０サイクルの２ステップＲＴ−ＰＣＲ増幅（９５℃１５秒、６０℃１分）及び４０サイクルのゲノムＰＣＲ増幅（９４℃３０秒、６２℃３０秒、７２℃３０秒。ただし、１サイクル目に９４℃で６０秒変性させ、最後のサイクルで７２℃１０分間伸長させた。）によって行った。Biochem Biophys Res Commun, 195, 769-775(1993)に記載された半定量的ＰＣＲ法に従い、飽和曲線を決定するためにサイクル数を増やしてＰＣＲを行った。同等量及び同品質のｍＲＮＡであることを推定するために、ｇａｐｄｈ遺伝子についてＰＣＲ増幅を行った。増幅直線領域の各サンプルを２％アガロースゲル（Sigma社製）で電気泳動し、エチジウムブロマイド染色したｇａｐｄｈのバンドで各サンプルを標準化後、サイトカインの発現をイメージアナライザーFluorImagerシステム（Molecular Dynamics, Inc社製）で定量した。
The gapdh primer set (TaqMan GAPDH control reagent kit: Applied Biosystems) was used as an internal standard for RNA quality and quantification. The sequence of this gapdh primer is as follows.
gapdh forward: 5'-atgggtgtgaaccacgagaaa-3 '(SEQ ID NO: 11)
gapdh reverse: 5'-ggatacattgggggtaggaa-3 '(SEQ ID NO: 12)
RT-PCR is a 2-step RT-PCR with 40 cycles using SYBR (registered trademark) Green PCR Master Mix (Applied Biosystems) and ABI PRISM (registered trademark) 7000 Sequence Detection System (Applied Biosystems). Amplification (95 ° C. for 15 seconds, 60 ° C. for 1 minute) and 40 cycles of genomic PCR amplification (94 ° C. for 30 seconds, 62 ° C. for 30 seconds, 72 ° C. for 30 seconds. For 10 minutes at 72 ° C.). In accordance with the semi-quantitative PCR method described in Biochem Biophys Res Commun, 195, 769-775 (1993), PCR was performed with an increased number of cycles to determine a saturation curve. In order to estimate that the mRNA was of the same amount and quality, PCR amplification was performed on the gapdh gene. Each sample in the linear amplification region was electrophoresed on a 2% agarose gel (Sigma), and each sample was standardized with a gapdh band stained with ethidium bromide, and then the expression of cytokine was image analyzer FluorImager system (Molecular Dynamics, Inc.) ).
一方、上述の変法と同様の方法によって、ラットにＢＵを単回投与（腹腔内注射）し、骨髄形成不全を誘導した。顕微鏡で、このラットの骨髄組織と、正常なラットの骨髄組織とを比較した結果を図１６に示す。図１６に示されているように、ＢＵを投与したラットでは、組織学的に重度の骨髄形成の不全が引き起こされていることが確認された。 On the other hand, a single administration (intraperitoneal injection) of BU to rats was induced by a method similar to the above-described modified method to induce bone marrow dysplasia. FIG. 16 shows the result of comparing the bone marrow tissue of this rat with that of a normal rat using a microscope. As shown in FIG. 16, it was confirmed that the rats administered with BU caused histologically severe bone marrow formation failure.
［骨髄形成不全下に誘導したＤＳＳ腸炎に対するＭＳＣ移植による治療効果］
上述の変法と同様の方法によって、ＢＵをラットに単回投与（腹腔内注射）し、骨髄形成不全を誘導した。得られた骨髄形成不全ラットを用いて、ＭＳＣ移植による腸炎の治療効果を調べる実験を行った。この実験に用いるラットの飼育計画を図１７に示す。図１７からも分かるように、本実験では、骨髄形成不全ラットを、さらに１％ＤＳＳの投与を行うか否か及びＭＳＣ移植を行うか否かに関して以下の３つのグループに分けた。
（１）さらに１％ＤＳＳを投与し、かつ、ＭＳＣ移植を行うグループ（ｎ＝１９）（ただし、１９個体のうち、８個体はｅＧＦＰ標識ＭＳＣを移植した）（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）
（２）さらに１％ＤＳＳを投与するが、ＭＳＣ移植を行わないグループ（ｎ＝１３：内部標準）（ＢＵ＋１％ＤＳＳ）
（３）１％ＤＳＳの投与は行わず、ＭＳＣ移植も行わないグループ（ｎ＝９）（外部標準：ＢＵ ａｌｏｎｅ）
なお、本実験では、指定量のＢＵ腹腔内投与は−７日目に行い、１％ＤＳＳ投与は−２日目〜３日目までの５日間毎日行い、そしてＭＳＣの移植は０日目に行った（図１７）。また、本実験におけるＭＳＣとしては、前述の参考例で培養したＭＳＣを用いた。
[Therapeutic effect of MSC transplantation on DSS enteritis induced under myelogenesis failure]
By a method similar to the above-mentioned modified method, BU was administered once to rats (intraperitoneal injection) to induce bone marrow dysplasia. An experiment was conducted to examine the therapeutic effect of enterocolitis caused by MSC transplantation using the obtained myelopoietic rat. A rat breeding plan used in this experiment is shown in FIG. As can be seen from FIG. 17, in this experiment, the myelodysplasia rats were further divided into the following three groups regarding whether or not to administer 1% DSS and whether to perform MSC transplantation.
(1) A group in which 1% DSS is further administered and MSC transplantation is performed (n = 19) (however, 8 of 19 individuals were transplanted with eGFP-labeled MSC) (BU + 1% DSS + MSC)
(2) A group in which 1% DSS is administered but MSC transplantation is not performed (n = 13: internal standard) (BU + 1% DSS)
(3) Group in which 1% DSS is not administered and MSC transplantation is not performed (n = 9) (external standard: BU alone)
In this experiment, the specified amount of intraperitoneal injection of BU was performed on the −7th day, 1% DSS administration was performed every day for 5 days from the −2nd day to the 3rd day, and the transplantation of MSC was performed on the 0th day. Performed (FIG. 17). Further, as the MSC in this experiment, the MSC cultured in the above-mentioned reference example was used.
上述の３つのグループのラットのそれぞれの体重の経時的変化を計測し、各マウスの実験開始日（ｄａｙ−２）の体重を１００としたときの体重の割合（％）を算出した。各グループの体重の割合の平均値（％）の推移を図１８に示す。図１８から分かるように、ＭＳＣを移植したグループ（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）では、それを移植しなかったグループ（ＢＵ＋１％ＤＳＳ）に比べて、特にｄａｙ１以降の体重減少の抑制が見られた。 The change over time in the body weight of each of the three groups of rats described above was measured, and the ratio (%) of the body weight when the body weight on the experiment start day (day-2) of each mouse was taken as 100 was calculated. The transition of the average value (%) of the ratio of the weight of each group is shown in FIG. As can be seen from FIG. 18, in the group transplanted with MSC (BU + 1% DSS + MSC), the suppression of weight loss after day 1 was particularly suppressed as compared with the group not transplanted with it (BU + 1% DSS).
また、実験に用いたラットは、３日目（ｄａｙ３）に屠殺解剖し、腸炎の重症度の評価に用いた。腸炎の重症度は、Gut, 50, 507-512(2002)の方法に従って、ラットの体重、結腸の長さ、組織学的スコアによって評価した。より具体的には、以下のように評価を行った。
ＴＮＦα、ＩＬ１β、ＩＦＮγ、ＩＬ１０のｍＲＮＡの発現の評価及び組織学的スコアの評価のために全結腸を取り出し、長軸方向の切片に分割した。組織を１０％中性ホルマリンで固定し、パラフィンに包埋し、薄切片を作成した。グループ分けを知らない２人の病理学者が別々に、ヘマトキシリン及びエオシンで染色した組織切片の組織学的スコアを評価した。ＭＳＣ移植を行ったグループ（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）と、ＭＳＣ移植を行わなかったグループ（ＢＵ＋１％ＤＳＳ）の結腸の長さを図１９に示す。ＭＳＣ移植を行ったグループ（ＭＳＣ＋）の結腸の長さ（１２８．３±７．７ｍｍ）は、ＭＳＣ移植を行わなかったグループ（ＭＳＣ−）の結腸の長さ（１０９．０±１１．０ｍｍ）に対して有意に（有意差Ｐ＝０．０００１）長いことが分かった。また、前述の３グループのラットおける、ヘマトキシリン及びエオシン染色した結腸組織切片を顕微鏡で観察した結果を図２０に示す（図２０ｅ：ＢＵ ａｌｏｎｅ； 図２０ｆ：ＢＵ＋１％ＤＳＳ； 図２０ｇ：ＢＵ＋１％ＤＳＳ＋ＭＳＣ）。ＭＳＣを移植したグループ（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）における組織では、ＭＳＣを移植しなかったグループ（ＢＵ＋１％ＤＳＳ）に比べて、腸炎が抑制されていることが分かる。さらに、ＭＳＣ＋グループとＭＳＣ−グループの間の、結腸組織におけるラットＴＮＦα、ＩＬ１β、ＩＦＮγ、ＩＬ１０等のサイトカイン遺伝子の転写産物量の比較を、図２１に示す（図２１ｉ：ｔｎｆα； 図２１ｊ：ｉｌ１β； 図２１ｋ：ｉｆｎγ； 図２１ｌ：ｉｌ１０）。いずれの遺伝子の転写産物発現量についても、ＭＳＣの移植の有無による有意差は見られなかった。
また、ＭＳＣ＋（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）のグループと、ＭＳＣ−（ＢＵ＋１％ＤＳＳ）のグループの組織学的スコアを図２２に示す。図２２から分かるように、ＭＳＣ＋（ＢＵ＋１％ＤＳＳ＋ＭＳＣ）の組織学的スコア（６．０±３．２）は、ＭＳＣ−（ＢＵ＋１％ＤＳＳ）の組織学的スコア１３．２±１．６よりも有意に（有意差Ｐ＝０．００１４）低いことが示された。このことから、ＭＳＣの移植が、腸炎の改善に有効であることが示唆された。
The rats used in the experiment were sacrificed and dissected on the third day (day 3) and used to evaluate the severity of enteritis. The severity of enteritis was evaluated by rat body weight, colon length, and histological score according to the method of Gut, 50, 507-512 (2002). More specifically, the evaluation was performed as follows.
Whole colons were removed and divided into longitudinal sections for evaluation of TNFα, IL1β, IFNγ, IL10 mRNA expression and histological scores. The tissue was fixed with 10% neutral formalin, embedded in paraffin, and thin sections were prepared. Two pathologists who did not know the grouping separately evaluated the histological scores of tissue sections stained with hematoxylin and eosin. FIG. 19 shows the colon lengths of the group that received MSC transplantation (BU + 1% DSS + MSC) and the group that did not receive MSC transplantation (BU + 1% DSS). The length of the colon (128.3 ± 7.7 mm) of the group (MSC +) that received the MSC transplant was the length of the colon (109.0 ± 11.0 mm) of the group that did not receive the MSC transplant (MSC−) Was significantly longer (significant difference P = 0.0001). In addition, the results of microscopic observation of hematoxylin and eosin-stained colon tissue sections in the aforementioned three groups of rats are shown in FIG. 20 (FIG. 20e: BU alone; FIG. 20f: BU + 1% DSS; FIG. 20g: BU + 1% DSS + MSC). . It can be seen that in the group transplanted with MSC (BU + 1% DSS + MSC), enteritis was suppressed as compared with the group not transplanted with MSC (BU + 1% DSS). Furthermore, comparison of the amount of transcripts of cytokine genes such as rat TNFα, IL1β, IFNγ, IL10, etc. in colon tissue between the MSC + group and the MSC− group is shown in FIG. 21 (FIG. 21i: tnfα; FIG. 21j: il1β; FIG. 21k: ifnγ; FIG. 21l: il10). There was no significant difference in the expression level of the transcript of any gene depending on the presence or absence of MSC transplantation.
FIG. 22 shows the histological scores of the MSC + (BU + 1% DSS + MSC) group and the MSC− (BU + 1% DSS) group. As can be seen from FIG. 22, the histological score (6.0 ± 3.2) of MSC + (BU + 1% DSS + MSC) is higher than the histological score of MSC− (BU + 1% DSS) of 13.2 ± 1.6. Significantly lower (significant difference P = 0.014). This suggested that MSC transplantation is effective in improving enteritis.
［急性骨髄形成不全下に誘導したＤＳＳ腸炎のラットレシピエント結腸組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在］
前述の「ＢＵ＋１％ＤＳＳ＋ＭＳＣ」のグループのラットにおいて、ＭＳＣを移植してから３、７、及び２８日後に、０．２％グルタールアルデヒド含有ＰＢＳ及び４％パラホルムアルデヒドでこのラットを経心的に灌流して屠殺した。屠殺したラットの結腸を取り出し、これを灌流バッファーで４ｈｒ固定した。その後、結腸組織をパラフィンに包埋し、脱パラフィンしたパラフィン切片中でｅＧＦＰ及びMusashi-1（Ｍｓｉ−１）の免疫蛍光を決定するために厚さ３μｍの切片を作製した。次に、この組織切片を、２００倍希釈したヤギ抗ｅＧＦＰポリクローナル抗体（Abcam社製、英国ケンブリッジ）及び５００倍希釈したビオチン標識ラット抗マウスＭｓｉ−１モノクローナル抗体（東京都、慶応大学のOkano教授の厚意により提供：Differentiationt, 71, 28-41(2003)）と共に４℃で一晩インキュベートした後、ＰＢＳで洗浄した。
ｅＧＦＰに関しては、５００倍希釈したヤギＩｇＧ Ｈ＆Ｌに対するウサギポリクローナル抗体（ＦＩＴＣ）（Abcam社製）と共に３７℃で３０分間インキュベートし、Ｍｓｉ−１に関してはTexas Red（登録商標）が結合したImmunoPure（登録商標） Streptavidin（PIERCE社製）と共に３７℃で３０分間インキュベートした。インキュベートしたスライドをVECTASHIELD Mounting Medium及びＤＡＰＩ（Vector Laboratories社製）で対比染色した上で、蛍光顕微鏡（KEYENCE社製BZ-8000）及びZEISS Pascalレーザー走査型顕微鏡（R2100AG2、 Bio-Rad社製）を用いてｅＧＦＰ発現細胞の分布を決定した。
[Localization of donor-derived eGFP-labeled MSCs in rat recipient colon tissue with DSS enteritis induced under acute myelogenesis failure]
In the aforementioned “BU + 1% DSS + MSC” group of rats, 3, 7, and 28 days after transplanting the MSC, the rat was transcentered with PBS containing 0.2% glutaraldehyde and 4% paraformaldehyde. Perfused and sacrificed. The sacrificed rat colon was removed and fixed with perfusion buffer for 4 hr. The colon tissue was then embedded in paraffin and 3 μm thick sections were prepared to determine the immunofluorescence of eGFP and Musashi-1 (Msi-1) in deparaffinized paraffin sections. Next, this tissue section was subjected to 200-fold diluted goat anti-eGFP polyclonal antibody (Abcam, Cambridge, UK) and 500-fold diluted biotin-labeled rat anti-mouse Msi-1 monoclonal antibody (Tokyo, Prof. Okano, Keio University). Courtesy courtesy of: Differentiationt, 71, 28-41 (2003)) overnight at 4 ° C. and then washed with PBS.
For eGFP, the rabbit polyclonal antibody (FITC) against goat IgG H & L diluted 500 times (Abcam) was incubated at 37 ° C. for 30 minutes, and for Msi-1, ImmunoPure (registered trademark) to which Texas Red (registered trademark) was bound. ) Incubated with Streptavidin (PIERCE) for 30 minutes at 37 ° C. The incubated slide was counterstained with VECTASHIELD Mounting Medium and DAPI (manufactured by Vector Laboratories), then using a fluorescence microscope (KEYENCE BZ-8000) and ZEISS Pascal laser scanning microscope (R2100AG2, Bio-Rad) The distribution of eGFP expressing cells was determined.
ＭＳＣを移植してから３日目の結腸組織を染色した結果を図２３に示す。図２３中の白矢頭は、ｅＧＦＰ発現細胞（パネルａ、ｂ、ｃ、ｆ中の緑色）、Ｍｓｉ−１発現細胞（パネルｇ中の赤色）、ｅＧＦＰとＭｓｉ−１の二重染色細胞（パネルｈ中のピンク色）を表し、これらのパネルの細胞についてはＤＡＰＩで対比染色している（青色）。パネルａ及びｂは別々の切片の蛍光顕微鏡写真（×４００）であり、パネルｃ（ｅＧＦＰ）、ｄ（Ｈ＆Ｅ）、及びｅ（ＤＡＰＩ）はパネルｂと同一切片の共焦点顕微鏡による拡大図（×１２００）である。また、図２３のパネルｆ〜ｈは共焦点顕微鏡による同一切片の拡大図を表す（パネルｆ：ｅＧＦＰ；パネルｇ：Ｍｓｉ−１；パネルｈ：パネルｆとパネルｇを重ね合わせたもの）。
移植後３日目の結腸組織を調べたところ、ｅＧＦＰ発現細胞はクリプト１つにつき１つの細胞しか検出されず（図２３のパネルａやｂ等）、調べた７７１個のクリプト中、７７個がｅＧＦＰ陽性であった（９．２％）。この際、調べる対象としたクリプトは、代表切片（図２３のパネルｃ、ｄ及びｅ）のようにクリプト底部（crypt base）を適切に評価できるクリプトに限った。
図２３のパネルｅにおいて細胞位置１を２つのアスタリスク（時計回り及び反時計回りの両方向）で示しているので、ｅＧＦＰを発現しているＭＳＣ由来細胞が、推定される腸管細胞の位置（白矢頭）に対応する細胞位置４〜５に局在していることは明確である。
FIG. 23 shows the result of staining the colon tissue on the third day after the transplantation of MSC. The white arrowheads in FIG. 23 indicate eGFP-expressing cells (green in panels a, b, c, and f), Msi-1 expressing cells (red in panel g), eGFP and Msi-1 double-stained cells (panel) (pink color in h), and the cells in these panels are counterstained with DAPI (blue). Panels a and b are fluorescence micrographs (× 400) of separate sections, and panels c (eGFP), d (H & E), and e (DAPI) are enlarged views of the same section as panel b (××). 1200). Further, panels f to h in FIG. 23 show enlarged views of the same section by a confocal microscope (panel f: eGFP; panel g: Msi-1; panel h: panel f and panel g superimposed).
When the colon tissue on the third day after transplantation was examined, only one eGFP-expressing cell was detected per crypt (panels a and b, etc. in FIG. 23). Of the 771 crypts examined, 77 were It was eGFP positive (9.2%). At this time, the cryptograms to be examined were limited to those that can appropriately evaluate the crypto base (crypt base), such as representative sections (panels c, d, and e in FIG. 23).
Since the cell position 1 is indicated by two asterisks (both clockwise and counterclockwise) in the panel e of FIG. 23, the MSC-derived cells expressing eGFP are estimated to be intestinal cell positions (white arrowheads). It is clear that it is localized at the cell positions 4 to 5 corresponding to).
ＭＳＣを移植してから７日目の結腸組織を染色した結果を図２４のパネルａ、ｂに示し、２８日目の結腸組織を染色した結果を図２４のパネルｃ、ｄに示す。
図２４中の白矢頭は、ｅＧＦＰ発現細胞（パネルａ−ｄ中の緑色）を表し、幹細胞の位置をアスタリスクで示した。また、図２４中の細胞はすべてＤＡＰＩで対比染色している（青色）。
パネルａ及びｂは移植後７日目における代表切片の共焦点顕微鏡写真（×１２００）であり、パネルｃ及びｄは移植後２８日目における代表切片の共焦点顕微鏡写真（×１２００）である。移植後７日目において、調べた９０１個のクリプトのうち、１８７個がｅＧＦＰ陽性であった（２０．８％）。一方、２８日目では、８３６個のクリプトのうち、１８０個がｅＧＦＰ陽性であった（２１．５％）。この際、調べる対象としたクリプトは、代表切片（図２３のパネルｃ、ｄ及びｅ）のようにクリプト底部（crypt base）を適切に評価できるクリプトに限った。
The results of staining the colon tissue on day 7 after transplanting the MSC are shown in panels a and b of FIG. 24, and the results of staining of the colon tissue on day 28 are shown in panels c and d of FIG.
The white arrowheads in FIG. 24 represent eGFP-expressing cells (green in panels ad), and the positions of the stem cells are indicated by asterisks. Moreover, all the cells in FIG. 24 are counterstained with DAPI (blue).
Panels a and b are confocal micrographs (x1200) of representative sections on day 7 after transplantation, and panels c and d are confocal micrographs (x1200) of representative sections on day 28 after transplantation. On day 7 after transplantation, 187 out of 901 crypts examined were eGFP positive (20.8%). On the other hand, on day 28, of 836 crypts, 180 were eGFP positive (21.5%). At this time, the cryptograms to be examined were limited to those that can appropriately evaluate the crypto base (crypt base), such as representative sections (panels c, d, and e in FIG. 23).
図２３及び図２４の結果は、ｅＧＦＰ標識したドナー由来ＭＳＣが自らMusashi-1（Ｍｓｉ−１）タンパク質を発現し、クリプト底部の推定腸上皮幹細胞ニッチとされるおおよそ細胞位置１〜４の腸上皮に生着することを初めて示したものと思われる（図２３のパネルｃ〜ｈ）。これまでの様々な証拠から、Ｍｓｉ−１の発現は推定腸上皮幹細胞及び初期細胞系譜マーカーであることが示唆されている（Differentiationt, 71, 28-41(2003)）。ＭＳＣの移植後３日目では、ｅＧＦＰ発現細胞は１つのクリプトに１つの細胞として散在して検出された（図２３のパネルａ及びｂ）。しかし、移植後２８日目においては、ドナー由来ｅＧＦＰ発現細胞は幹細胞ニッチに常在する一方で、クリプト上部に向かってその数も増加した（図２４）。Unitarianの仮説（Am J Anat, 141, 537-61(1974)）は全てのクリプト構成細胞がｅＧＦＰ陽性であることを示唆しているが、全クリプトがｅＧＦＰ陽性となりプラトーに到達するまでの「クローン安定化時間」（clonal stabilizing time）は、大腸においては数ヶ月かかる（”Essentials of stem cell biology”, Gearhart, J., et al., Elsevier Academic Press, Burlington, MA, 2006）。 23 and 24 show that the eGFP-labeled donor-derived MSCs themselves express Musashi-1 (Msi-1) protein, which is the estimated intestinal epithelial stem cell niche at the bottom of the crypt. It seems that it was shown for the first time to engraft (panels c to h in FIG. 23). Various previous evidences suggest that Msi-1 expression is a putative intestinal epithelial stem cell and early cell lineage marker (Differentiationt, 71, 28-41 (2003)). On the third day after MSC transplantation, eGFP-expressing cells were detected scattered as one cell in one crypt (panels a and b in FIG. 23). However, on the 28th day after transplantation, donor-derived eGFP-expressing cells were resident in the stem cell niche, but the number thereof increased toward the upper part of the crypt (FIG. 24). Unitarian's hypothesis (Am J Anat, 141, 537-61 (1974)) suggests that all crypt-constituting cells are eGFP positive, but the “clones” until all crypts become eGFP positive and reach a plateau. The “clonal stabilizing time” takes several months in the large intestine (“Essentials of stem cell biology”, Gearhart, J., et al., Elsevier Academic Press, Burlington, MA, 2006).
図２３及び図２４の結果は、ドナーＭＳＣが、損傷を受けたレシピエント腸上皮幹細胞の「幹性」（stemness）を細胞融合又は多分化能によって直接補うことができるだけでなく、最終的に上皮細胞に分化しうることを示唆している。腸上皮幹細胞と融合するドナー細胞はＨＳＣであるという最近の報告（Proc Natl Acad Sci USA, 103, 6321-6325(2006)）があるが、本願の図２３及び図２４の結果からは、ＭＳＣが腸上皮幹細胞の融合パートナーであることが強く示唆される。
ＭＳＣが腸上皮幹細胞としてうまく生着して腸炎が改善されたにもかかわらず、調べたサイトカインの転写レベルは炎症性結腸組織において減少していなかった。このことからＭＳＣは、ホストの免疫反応を抑制するのでなく、生着し再増殖しているＭＳＣの核が再プログラミングされ、内因性腸上皮幹細胞として腸上皮の統合の維持を助けている可能性が考えられた。これは、腸の傷害回復においてＭＳＣが潜在的ではあるが中心的な役割を果たしていること、及びＭＳＣ治療が難治性腸炎に有益であることを示唆している。
The results of FIGS. 23 and 24 show that the donor MSC can not only directly compensate for the “stemness” of damaged recipient intestinal epithelial stem cells by cell fusion or pluripotency, but also ultimately epithelial It suggests that it can differentiate into cells. There is a recent report (Proc Natl Acad Sci USA, 103, 6321-6325 (2006)) that the donor cell fused with the intestinal epithelial stem cell is HSC. From the results of FIG. 23 and FIG. It is strongly suggested that it is a fusion partner of intestinal epithelial stem cells.
Despite the successful improvement of enteritis by MSC engraftment as intestinal epithelial stem cells, the transcript levels of cytokines examined were not reduced in inflammatory colon tissue. This suggests that MSC does not suppress the host's immune response, but rather reprograms the engrafted and repopulating nucleus of MSC to help maintain intestinal epithelial integration as an endogenous intestinal epithelial stem cell Was considered. This suggests that MSC plays a potential but central role in intestinal injury recovery, and that MSC treatment is beneficial for refractory enteritis.
一方、ＢＵ処理を行わずに異性ＭＳＣを移植した、１０％ＤＳＳ致死腸炎、５％ＤＳＳ腸炎、又はＴＮＢＳ腸炎ラットにおいては、治療効果はみられなかった（図２〜９）。図６及び図９に示されているように、これらの腸炎モデルにおいては、移植したＭＳＣが充分長期間といえるほどには生着を維持できなかった。これは、ＭＳＣが細胞供給源又は免疫修飾物質のどちらか又は両方として適切に機能することができなかったことを示唆している。
図２３及び図２４の結果と、図６及び図９の結果を併せ考慮すると、以下の２つの可能性が考えられた。
（１）外因性ＭＳＣは、内因的に免疫学的特権細胞ではなく、ＮＫ細胞による細胞溶解の標的となる。
（２）ＤＳＳ処理によってレシピエントの結腸上皮で傷害を受ける細胞は、ＢＵ処理によってレシピエントの結腸上皮で傷害を受ける細胞と大きく異なっている。すなわち、ＤＳＳが成熟分化した上皮細胞に損傷を与えるのに対し、ＢＵは成熟分化していない未分化な腸上皮幹細胞を損傷する。
上記の２つの可能性のうち、前者の（１）の可能性はないと考えられた。なぜなら、最近の報告（Blood, 106, 4057-4065(2005)；Blood, 108, 2114-2120(2006)；Blood, 107, 1484-1490(2006)）によれば外因性ＭＳＣの免疫原性が問題になるのは、前処置として免疫抑制をかけていない免疫学的に正常なレシピエントに同種異系間又は異種間で移植がされた場合であるが、本実験におけるＭＳＣ移植はルイスラット間で同系移植されたものであるし、さらに、Sprague-Dawley（ＳＤ）バックグラウンドの「グリーンラット」と類遺伝子ルイスラットとの間での同系移植及び同種移植の間では違いは検出されなかった（データ非公開）からである。したがって、本実験で得られた結果を全て適切に説明できるのは後者の（２）の可能性である。より詳細に述べると、以下のようなことが言える。
ＤＳＳ処理やＴＮＢＳ処理によって、上皮のバリア機能か障害されその透過性が急速に上昇し、その後上皮の剥離と免疫性炎症反応が起こる。この場合、ドナー由来ＭＳＣは、損傷を受けた成熟分化細胞のレスキュー細胞として生着し再増殖する（図１０）と考えられるが、クリプト細胞は数日のサイクルで剥離するため、さらなる治療効果を発揮できないのだろう（図２〜９）。
On the other hand, there was no therapeutic effect in 10% DSS lethal enteritis, 5% DSS enteritis or TNBS enteritis rats transplanted with heterosexual MSCs without BU treatment (FIGS. 2-9). As shown in FIGS. 6 and 9, in these enteritis models, the transplanted MSCs could not maintain engraftment for a sufficiently long time. This suggests that MSC could not function properly as either a cell source or an immunomodulator or both.
Considering the results of FIGS. 23 and 24 together with the results of FIGS. 6 and 9, the following two possibilities were considered.
(1) Exogenous MSCs are not intrinsically immunologically privileged cells but are targets for cell lysis by NK cells.
(2) Cells that are damaged in the recipient colon epithelium by DSS treatment are significantly different from those that are damaged in the recipient colon epithelium by BU treatment. That is, while DSS damages mature and differentiated epithelial cells, BU damages undifferentiated intestinal epithelial stem cells that are not maturely differentiated.
Of the above two possibilities, the former (1) was not considered to be possible. According to recent reports (Blood, 106, 4057-4065 (2005); Blood, 108, 2114-2120 (2006); Blood, 107, 1484-1490 (2006)), the immunogenicity of exogenous MSCs The problem arises when allograft or xenogeneic transplantation is performed in an immunologically normal recipient without immunosuppression as a pretreatment, but MSC transplantation in this experiment is between Lewis rats. Furthermore, no difference was detected between syngeneic and allogeneic transplants between “green rats” in the Sprague-Dawley (SD) background and similar gene Lewis rats ( (Data not disclosed). Therefore, it is the possibility of the latter (2) that can properly explain all the results obtained in this experiment. In more detail, the following can be said.
DSS treatment or TNBS treatment impairs the epithelial barrier function and rapidly increases its permeability, followed by epithelial detachment and an immune inflammatory reaction. In this case, donor-derived MSCs are considered to engraft and re-grow as rescue cells of damaged mature differentiated cells (Fig. 10). However, since crypt cells detach in a cycle of several days, a further therapeutic effect is obtained. It will not be able to demonstrate (Figures 2-9).
一方、ＢＵは抗腫瘍アルキル化剤であり、ホストにおいて活発に細胞分裂していない原始幹細胞を枯渇させるという性質がある（Cancer Res, 60, 5470-5478(2000)）。つまり、ＢＵ投与は骨髄幹細胞のみならず腸上皮幹細胞を損傷させるのだろう。骨髄形成不全下に誘導したＤＳＳ腸炎において、ドナー由来ＭＳＣは、未知の優先順位選択機構により、分化成熟した上皮細胞をレスキューする代わりに、腸上皮幹細胞に取って代わるのだろう。ＢＵ単回投与によって病理学的な腸炎は惹起されないが、上皮統合性が直接損なわれることで生じるＤＳＳに対する脆弱性（図１５及び図２０のパネルｅ）、及び／又は、ＢＵは、ＭＳＣにとって毒性が強く（Immunopharmacol, 46, 103-112(2000)）、したがって骨髄形成不全下においては内因性ＭＳＣがＤＳＳ暴露によるストレスから腸をレスキューすることができないことで生じるＤＳＳに対する脆弱性、これらの脆弱性は両方とも、機能的な外因性ＭＳＣを十分に供給することで抑制される（図１６〜２２）。
結論として、ＭＳＣ由来腸上皮幹細胞は、傷害を受けた結腸の再生において骨髄細胞の中心的役割を担っているようである。レシピエントの前処置の必要性の理論的根拠を得るには、実験的腸炎におけるＭＳＣ由来腸上皮幹細胞の性質のさらなる解明が必要であり、それは「免疫学的特権細胞」とされるＭＳＣの場合でもそうである。骨髄におけるＨＳＣ及び他の前駆細胞の、腸炎に対する具体的役割の正確な詳細が明らかにされなければならないが、ＭＳＣ治療の可能性により、より効果的なＩＢＤ治療への新たな道が開かれた。
On the other hand, BU is an antitumor alkylating agent and has the property of depleting primitive stem cells that are not actively dividing in the host (Cancer Res, 60, 5470-5478 (2000)). That is, BU administration will damage not only bone marrow stem cells but also intestinal epithelial stem cells. In DSS enteritis induced under myelodysplasia, donor-derived MSCs will replace intestinal epithelial stem cells instead of rescued differentiated and mature epithelial cells by an unknown priority selection mechanism. A single dose of BU does not cause pathological enterocolitis, but is vulnerable to DSS caused by a direct loss of epithelial integrity (FIG. 15 and FIG. 20, panel e) and / or BU is toxic to MSC (Immunopharmacol, 46, 103-112 (2000)), and thus the vulnerability to DSS caused by the failure of endogenous MSC to rescue the intestine from stress caused by exposure to DSS under myelination failure, these vulnerabilities Both are suppressed by providing sufficient functional exogenous MSCs (FIGS. 16-22).
In conclusion, MSC-derived intestinal epithelial stem cells appear to play a central role for bone marrow cells in the regeneration of damaged colon. Obtaining the rationale for the need for pretreatment of recipients requires further elucidation of the nature of MSC-derived intestinal epithelial stem cells in experimental enteritis, which is the case for MSCs that are considered “immunologically privileged cells” But it is. Although the exact details of the specific role of HSCs and other progenitor cells in the bone marrow for enteritis must be clarified, the potential for MSC treatment has opened a new avenue for more effective IBD treatment .
［放射線照射を受けたラットレシピエント結腸組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在］
まず、ラットの全身にγ線を照射して、腸上皮幹細胞障害処理を行った。具体的には、６０Ｃｏ回転治療装置 ＲＣＲ−１２０−Ｃ３型（東芝メディカル株式会社製）を用いて、線量率 ０．５１Ｇｙ／ｍｉｎ、照射野 ２０×３０ｃｍにて、線源から８５ｃｍ離れたラットに８Ｇｙのγ線を照射した。その後、そのラットの尾静脈に、前述の参考例で培養したＭＳＣを２×１０４個／ｇ含む５００μｌのαＭＥＭを注射することにより、ＭＳＣを移植した。ＭＳＣ移植３日目のラットの結腸組織を染色した結果を図２５のパネルａ（ＤＡＰＩ染色）、ｂ（ｅＧＦＰ抗体染色）、ｃ（Ｍｓｉ抗体染色）、ｄ（ａ〜ｃのパネルを重ね合わせたもの）に示す。図２５の結果から分かるように、ｅＧＦＰ陽性細胞（矢頭）がクリプト底部の細胞位置１（アスタリスクの部分）に存在し、かつ、腸上皮幹細胞マーカーであるＭｓｉ発現細胞（矢頭）とｅＧＦＰ陽性細胞（矢頭）が一致していた。この結果から、８Ｇｙのγ線の全身照射により腸上皮幹細胞が傷害され、ドナーＭＳＣがそれを補うことが示された。
[Localization of donor-derived eGFP-labeled MSCs in irradiated rat recipient colon tissue]
First, the whole body of the rat was irradiated with γ-rays to treat intestinal epithelial stem cell damage. Specifically, using a 60 Co rotation therapy apparatus RCR-120-C3 (manufactured by Toshiba Medical Ltd.), dose rate 0.51Gy / min, an irradiation field 20 × 30 cm, away 85cm from the source rats Were irradiated with 8 Gy of gamma rays. Thereafter, MSCs were transplanted by injecting 500 μl of αMEM containing 2 × 10 4 MSCs cultured in the above-mentioned reference example into the tail vein of the rats. The results of staining the rat colon tissue on day 3 of MSC transplantation were overlaid on panels a (DAPI staining), b (eGFP antibody staining), c (Msi antibody staining), and d (ac panels). Stuff). As can be seen from the results of FIG. 25, eGFP positive cells (arrowheads) are present at cell position 1 (asterisk part) at the bottom of the cryptogram, and Msi-expressing cells (arrowheads) that are intestinal epithelial stem cell markers and eGFP positive cells (arrowheads) Yahead) was in agreement. From this result, it was shown that intestinal epithelial stem cells were injured by whole body irradiation with 8 Gy γ-rays, and the donor MSC compensated for it.
また、同じ手順でＭＳＣ移植し、移植から１０日目のラットの結腸組織を染色した結果を図２６に示す。図２６のパネルａ及びｂは、それぞれ別のクリプトにおけるＤＡＰＩ染色及びｅＧＦＰ抗体染色の結果を表し、図中の矢頭は、ｅＧＦＰ陽性細胞を表す。図２６の結果から分かるように、移植１０日目では、すでに１クリプトに数個のｅＧＦＰ陽性細胞が連続して認められ、ドナーＭＳＣがレシピエント腸上皮細胞へ分化する可能性が認められた。以上より、放射線はＢＵと匹敵する優れた腸上皮幹細胞障害処理であり、かつ、放射線性腸障害の治療にＭＳＣ移植が有効である可能性が示唆された。 Further, FIG. 26 shows the result of staining the rat colon tissue on the 10th day after transplantation by MSC transplantation by the same procedure. Panels a and b in FIG. 26 represent the results of DAPI staining and eGFP antibody staining in different crypts, respectively, and the arrowheads in the figure represent eGFP positive cells. As can be seen from the results of FIG. 26, several eGFP positive cells were already continuously observed in one crypt on the 10th day of transplantation, and the possibility that the donor MSC differentiated into recipient intestinal epithelial cells was confirmed. From the above, it was suggested that radiation is an excellent treatment for intestinal epithelial stem cell injury comparable to BU, and that MSC transplantation may be effective for the treatment of radiation intestinal injury.
［シクロホスファミド３００ｍｇ／ｋｇ単回投与を受けたラットレシピエント結腸組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在］
シクロホスファミド（「エンドキサン」：Sigma社製）３００ｍｇ／ｋｇを腹腔内注射によりラットに単回投与した後、そのラットの尾静脈に、前述の参考例で培養したＭＳＣを２×１０４個／ｇ含む５００μｌのαＭＥＭを注射することにより、ＭＳＣを移植した。ＭＳＣの移植から３日目の結腸上皮組織におけるドナー由来ｅＧＦＰ標識ＭＳＣの局在を調べた。その結果を図２７に示す。図２７の左パネル及び右パネルは、それぞれ別のクリプトにおけるＤＡＰＩ染色及びｅＧＦＰ抗体染色の結果を表す。図２７から分かるように、ｅＧＦＰ陽性細胞（矢頭）がクリプト底部の腸上皮幹細胞域（細胞配置１：アスタリスクの部分）（左パネル）および一過性増殖細胞域（transient amplifying cell）（右パネル）に存在していることが示された。すなわち、シクロホスファミド（ＣＹ）により腸上皮幹細胞及び一過性増殖細胞が傷害され、ドナーＭＳＣがそれを補うことが示された。以上より、ＣＹはＢＵと同様に腸上皮幹細胞障害能を示し、且つＣＹ腸障害にＭＳＣ治療が有効である可能性が示唆された。
[Localization of donor-derived eGFP-labeled MSCs in rat recipient colon tissue receiving a single dose of 300 mg / kg cyclophosphamide]
After a single dose of 300 mg / kg of cyclophosphamide (“Endoxan”: Sigma) was administered to a rat by intraperitoneal injection, 2 × 10 4 MSCs cultured in the above-mentioned reference example were placed in the tail vein of the rat. MSCs were implanted by injecting 500 μl αMEM containing / g. The localization of donor-derived eGFP-labeled MSC in the colonic epithelial tissue on day 3 after MSC transplantation was examined. The result is shown in FIG. The left panel and right panel of FIG. 27 represent the results of DAPI staining and eGFP antibody staining in different crypts, respectively. As can be seen from FIG. 27, eGFP positive cells (arrowheads) are intestinal epithelial stem cell area (cell arrangement 1: asterisk part) (left panel) and transient amplifying cell area (right panel) at the bottom of crypto. It was shown to exist. That is, it was shown that intestinal epithelial stem cells and transiently proliferating cells were damaged by cyclophosphamide (CY), and donor MSC compensated for it. From the above, it was suggested that CY showed intestinal epithelial stem cell damage ability like BU, and that MSC treatment may be effective for CY bowel damage.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019044991A1 (en) | 2017-08-30 | 2019-03-07 | 富士フイルム株式会社 | Angiogenic agent and method for producing same |
| US10980858B2 (en) | 2016-06-20 | 2021-04-20 | Fujifilm Corporation | Trophic factor releasing agent and inflammatory disease treating agent |
| WO2021075525A1 (en) | 2019-10-17 | 2021-04-22 | テルモ株式会社 | Cell culture for treating inflammatory disease |
| US11439960B2 (en) | 2017-08-30 | 2022-09-13 | Fujifilm Corporation | Cell transplant device and method of manufacturing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10980858B2 (en) | 2016-06-20 | 2021-04-20 | Fujifilm Corporation | Trophic factor releasing agent and inflammatory disease treating agent |
| WO2019044991A1 (en) | 2017-08-30 | 2019-03-07 | 富士フイルム株式会社 | Angiogenic agent and method for producing same |
| US11439960B2 (en) | 2017-08-30 | 2022-09-13 | Fujifilm Corporation | Cell transplant device and method of manufacturing the same |
| US11471564B2 (en) | 2017-08-30 | 2022-10-18 | Fujifilm Corporation | Angiogenic agent and method of manufacturing the same |
| WO2021075525A1 (en) | 2019-10-17 | 2021-04-22 | テルモ株式会社 | Cell culture for treating inflammatory disease |
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