JP4831662B2 - Method for inducing differentiation into vascular endothelial progenitor cells using Notch ligand - Google Patents
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Description
本発明は、Notchリガンドを用いた未成熟幹細胞から血管内皮前駆細胞への分化誘導法、上記分化誘導法により作製される分化誘導された血管内皮前駆細胞、並びに上記血管内皮前駆細胞を含む血管治療のための細胞移植療法剤。 The present invention relates to a method for inducing differentiation from an immature stem cell to a vascular endothelial progenitor cell using a Notch ligand, a differentiation-induced vascular endothelial progenitor cell produced by the differentiation induction method, and a vascular treatment comprising the vascular endothelial progenitor cell. Cell transplantation therapy agent for.
血管系の形成は、発生段階でも初期段階の現象の一つであり、成体になった後でも一生を誕生、修復、死滅の繰り返しとする動的な構造を持つ。最近では幹細胞、また再生医学の著しい発展と共に、初期発生段階のみにとどまらず、成体になった後でも血管修復を行うといった、様々な組織損害に対し能動的に対処しながら血管形成に重要な役割を果たしている細胞が注目されるようになってきた。血管内皮前駆細胞(endothelial progenitor cell:EPC)は、末梢血液中の単核球成分の一部で、これは血管形成のため骨髄組織から効率的に成長、分化し、また血管形成が進行中の場所への移動、導入される成体新血管形成において重要な機能を持つ細胞であることが分かってきた（非特許文献１〜３）。現在臨床においては、重症の虚血性心疾患である冠動脈疾患や、下肢虚血性疾患に対する血管内皮前駆細胞移植治療による血管再生療法が試みられている。このことから心血管、また臓器の再生能力をEPCが高める可能性に期待がおかれている。血管内皮前駆細胞をさらに臨床応用するためには、ごく微量存在している血管内皮前駆細胞を機能的に改善することが必要である。 The formation of the vasculature is one of the phenomena in the developmental stage and the early stage, and has a dynamic structure in which life is born, repaired, and killed even after adulthood. Recently, with the remarkable development of stem cells and regenerative medicine, it plays an important role in angiogenesis while actively dealing with various tissue damages such as repairing blood vessels not only in the early development stage but also after adulthood The cells that play a role have been attracting attention. Endothelial progenitor cells (EPCs) are part of the mononuclear cell component in peripheral blood, which grows and differentiates efficiently from bone marrow tissue for angiogenesis and is undergoing angiogenesis. It has been found that the cell has an important function in migration to a place and formation of an introduced new blood vessel (Non-Patent Documents 1 to 3). At present, revascularization therapy using coronary artery progenitor cell transplantation treatment for coronary artery disease, which is a severe ischemic heart disease, and lower limb ischemic disease, has been attempted in the clinic. For this reason, there is hope for the possibility of EPC enhancing the ability of cardiovascular and organ regeneration. For further clinical application of vascular endothelial progenitor cells, it is necessary to functionally improve the vascular endothelial progenitor cells that are present in a very small amount.
一方、Notch 信号伝達系については以下の報告がある。
（１）発生段階における血管発生に対する Notch 信号伝達系の関与については、Notch1, Notch4, Jagged-1, DII-4 遺伝子変異マウスの解析がなされており、発生段階 E10において既に血管構造の異常が発見され、胎児の死亡も確認されている（非特許文献４〜７を参照）。
On the other hand, there are the following reports on the Notch signaling system.
(1) Notch1, Notch4, Jagged-1, and DII-4 gene mutant mice have been analyzed for the involvement of Notch signaling in blood vessel development at the developmental stage, and abnormal vascular structures have already been discovered at developmental stage E10. In addition, fetal death has also been confirmed (see Non-Patent Documents 4 to 7).
（２）ヒトの病気と Notch 信号伝達系の関連性に関する報告としては、Alagile syndromeと CADASIL 血管疾病が頻繁に報告されている。これらの二つの疾患はともに、染色体の突然変異の異常による遺伝性疾患で治療方法は報告されておらず、血管系の異常に起因する全身性異常を伴う病気である。Jagged-1遺伝子変異が起こると心血管異常を含むAlagile syndromeを起こし、Notch4の遺伝子変異が起こると退行性血管疾患である CADASIL疾病を起こすことが報告されている（非特許文献８及び非特許文献９を参照）。 (2) Alagile syndrome and CADASIL vascular disease are frequently reported as the reports on the relationship between human diseases and Notch signaling. Both of these two diseases are inherited diseases caused by abnormal chromosomal mutations and no treatment method has been reported, and are diseases accompanied by systemic abnormalities caused by abnormalities in the vascular system. It has been reported that Jagged-1 gene mutation causes Alagile syndrome including cardiovascular abnormality, and Notch4 gene mutation causes CADASIL disease, which is a degenerative vascular disease (Non-patent document 8 and Non-patent document). 9).
（３）細胞分化と Notch 信号伝達系との関連性に対する報告としては、溶解性Jagged-1タンパクを試験管内で細胞培養系に添加すると血管内皮細胞への形態変化が見られ血管新生現象を誘導するという報告がある。また、細胞増殖を誘導することも報告されている（非特許文献１０及び１１を参照）。 (3) As a report on the relationship between cell differentiation and Notch signaling system, adding soluble Jagged-1 protein to cell culture system in vitro induces angiogenesis by morphological changes to vascular endothelial cells. There is a report to do. It has also been reported to induce cell proliferation (see Non-Patent Documents 10 and 11).
本発明は、未成熟幹細胞の培養条件を改善することによって質的により良い血管内皮前駆細胞(EPC)を取得する方法を提供することを解決すべき課題とした。さらに本発明は、上記方法で得られた血管内皮前駆細胞(EPC)を用いた血管治療剤を提供することを解決すべき課題とした。 An object of the present invention is to provide a method for obtaining qualitatively better vascular endothelial progenitor cells (EPC) by improving the culture conditions of immature stem cells. Furthermore, another object of the present invention is to provide a vascular therapeutic agent using vascular endothelial progenitor cells (EPC) obtained by the above method.
本発明者らは上記課題を解決するために鋭意検討した結果、血管内皮前駆細胞を含む幹細胞の分化誘導系を、細胞運命決定因子であるNotch シグナルを増加させるJagged-1、 Delta-4分子を利用して樹立することに成功した。これらの結果に基づき、Notch シグナルを増加して分化誘導した細胞を利用することにより、下肢虚血性疾患に対する細胞移植治療が可能であることを見出した。本発明はこれらの知見に基づいて完成したものである。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that a system for inducing differentiation of stem cells including vascular endothelial progenitor cells has been developed with Jagged-1 and Delta-4 molecules that increase Notch signal, which is a cell fate determinant. We succeeded in using it and establishing it. Based on these results, it was found that cell transplantation therapy for lower limb ischemic disease is possible by using cells induced to differentiate by increasing Notch signal. The present invention has been completed based on these findings.
即ち、本発明によれば、培養されている未成熟幹細胞にNotchリガンドを作用させることを含む、未成熟幹細胞から血管内皮前駆細胞へと分化誘導させる方法が提供される。 That is, according to the present invention, there is provided a method for inducing differentiation from an immature stem cell to a vascular endothelial progenitor cell, which comprises causing a Notch ligand to act on the cultured immature stem cell.
好ましくは、未成熟幹細胞は骨髄未分化細胞である。
好ましくは、骨髄未分化細胞はlineage negative cellである。
Preferably, the immature stem cell is a bone marrow undifferentiated cell.
Preferably, the bone marrow undifferentiated cells are lineage negative cells.
好ましくは、Notch 信号伝達系のリガンドは、Jagged-1又はDelta-4である。
好ましくは、Notchリガンドを高発現する細胞と未成熟幹細胞とを隣接した状態で共培養する。
Preferably, the ligand for the Notch signaling system is Jagged-1 or Delta-4.
Preferably, cells that highly express Notch ligand and immature stem cells are co-cultured in an adjacent state.
本発明の別の側面によれば、上記した本発明の方法により作製される分化誘導された血管内皮前駆細胞が提供される。 According to another aspect of the present invention, differentiation-induced vascular endothelial progenitor cells produced by the method of the present invention described above are provided.
本発明のさらに別の側面によれば、上記した本発明の方法により作製される分化誘導された血管内皮前駆細胞を含む、血管治療のための細胞移植療法剤が提供される。
好ましくは、本発明の細胞移植療法剤は、虚血性血管疾患の治療のために使用される。
According to still another aspect of the present invention, there is provided a cell transplantation therapeutic agent for vascular treatment, comprising differentiation-induced vascular endothelial progenitor cells produced by the above-described method of the present invention.
Preferably, the cell transplantation therapeutic agent of the present invention is used for the treatment of ischemic vascular disease.
本発明による分化誘導法を用いることにより質的に優秀な血管内皮前駆細胞を作製することが可能である。本発明の分化誘導法により作製される分化誘導された内皮前駆細胞は、糖尿病や心筋梗塞などの虚血性疾患の治療のために有用である。また、血管内皮前駆細胞は他の細胞への分化可能性が少ないため、副作用などのリスクが低いという利点がある。 By using the differentiation induction method according to the present invention, it is possible to produce qualitatively superior vascular endothelial progenitor cells. The differentiation-induced endothelial progenitor cells produced by the differentiation induction method of the present invention are useful for the treatment of ischemic diseases such as diabetes and myocardial infarction. In addition, since vascular endothelial progenitor cells are less likely to differentiate into other cells, there is an advantage that the risk of side effects and the like is low.
以下、本発明の実施の形態について詳細に説明する。
本発明による未成熟幹細胞から血管内皮前駆細胞へと分化誘導する方法は、培養されている未成熟幹細胞にNotchリガンドを作用させることを特徴とする。
Hereinafter, embodiments of the present invention will be described in detail.
The method for inducing differentiation of immature stem cells into vascular endothelial progenitor cells according to the present invention is characterized in that Notch ligand is allowed to act on cultured immature stem cells.
培養されている未成熟幹細胞としては、骨髄未分化細胞、骨髄多機能性幹細胞(Bone marrow multipotent adult progenitor cells, MAPC)、骨髄間葉系幹細胞(Mesenchymal stem cells; MSCs)、造血幹細胞(hematopoietic stem cells)、胚性幹細胞(ES細胞)、始原生殖細胞(primordial germ cells)、各組織幹細胞（心臓、肝臓、膵臓、腎臓、前立腺、乳腺、表皮、毛包、腸管上皮、骨、骨格筋、脂肪、神経、網膜、内耳）などを使用することが出来る。好ましくは、未成熟幹細胞として骨髄未分化細胞を用いることが出来る。 Cultured immature stem cells include bone marrow undifferentiated cells, bone marrow multipotent adult progenitor cells (MAPC), bone marrow mesenchymal stem cells (MSCs), hematopoietic stem cells (hematopoietic stem cells) ), Embryonic stem cells (ES cells), primordial germ cells, tissue stem cells (heart, liver, pancreas, kidney, prostate, mammary gland, epidermis, hair follicle, intestinal epithelium, bone, skeletal muscle, fat, Nerve, retina, inner ear) and the like. Preferably, bone marrow undifferentiated cells can be used as immature stem cells.
骨髄未分化細胞としては、例えば、細胞運命が決定されていないLin (-)細胞(T細胞, B細胞, Macrophage, erythrocyteなど分化した細胞を除いた未分化細胞)を用いることができる。このような未分化細胞 (lineage negative cell)（Lin (-)細胞）は、先ず骨髄細胞から骨髄を分離した後、biotin化した Cocktail抗体を反応させた後、streptoavidin beadsの反応を行った後、MidiMACS separator(Miltenyi Biotec.)を用いて分離することができる。 As bone marrow undifferentiated cells, for example, Lin (−) cells whose cell fate has not been determined (undifferentiated cells excluding differentiated cells such as T cells, B cells, Macrophage, and erythrocytes) can be used. Such undifferentiated cells (lineage negative cells) (Lin (−) cells) are first separated from bone marrow cells, reacted with biotinylated Cocktail antibody, and then reacted with streptoavidin beads. Separation can be performed using a MidiMACS separator (Miltenyi Biotec.).
本発明では、培養されている未成熟幹細胞にNotchリガンドを作用させる。未成熟幹細胞の培養は通常の動物細胞の培養法に準じて行うことができる。 In the present invention, Notch ligand is allowed to act on immature stem cells that have been cultured. Culturing of immature stem cells can be performed according to a normal method for culturing animal cells.
本明細書で言う血管内皮前駆細胞（EPC）とは、培養されている未成熟幹細胞にNotchリガンドを作用させることによって未成熟幹細胞から分化誘導された細胞のことを言う。 As used herein, vascular endothelial progenitor cells (EPC) refer to cells that have been induced to differentiate from immature stem cells by allowing Notch ligand to act on the immature stem cells that have been cultured.
血管内皮前駆細胞（EPC）が末梢血液中の単核球成分の一部として分離されて以来、その特質の究明が進行中である。EPCは、まず血管内皮系特性として（１）特異的表面抗原の発現（２）acetyl化LDLの導入能力（３）tube形成能力あるいは成体血管内導入、などで特定することが出来る。ヒトEPCの表面マーカーを中心とした報告を見るとCD34、CD133(AC133)、KDR (VEGFR-2、 Flk-1)、 VE-カドヘリン、 CD31(PECAM-1)が発現しており、少し遅れてFlt-1 (VEGFR-1)、Tie-1、E-セレクチンが発現していることが報告されている。マウスの場合では、血球前駆細胞とその元が似ているという視点から、Sca-1(+）細胞、c-kit(+)細胞を中心として特質の究明が進行中である。特にFlk-1 (VEGFR-2、 KDR)、 CD31 (PECAM-1)、 VE-cadherin(+)細胞はEPCの特性を現していることで重要だと考えられている。 Since vascular endothelial progenitor cells (EPCs) have been isolated as part of the mononuclear cell component in peripheral blood, investigations into their properties have been ongoing. EPC can first be identified as vascular endothelial properties by (1) expression of a specific surface antigen, (2) ability to introduce acetylated LDL, (3) ability to form a tube, or introduction into an adult blood vessel. Looking at reports centered on surface markers of human EPC, CD34, CD133 (AC133), KDR (VEGFR-2, Flk-1), VE-cadherin, CD31 (PECAM-1) are expressed, a little later It has been reported that Flt-1 (VEGFR-1), Tie-1, and E-selectin are expressed. In the case of mice, investigations of the characteristics are ongoing with a focus on Sca-1 (+) cells and c-kit (+) cells, from the viewpoint that the origin is similar to blood cell progenitor cells. In particular, Flk-1 (VEGFR-2, KDR), CD31 (PECAM-1), and VE-cadherin (+) cells are considered to be important because of their EPC characteristics.
EPCは骨髄由来の極めて少ない量の細胞で、生体内の様々な信号によって増殖、分化することが分かっている。EPCの特質究明の進行と共に、比較的初期段階で幹細胞として認められている細胞(AC133、 KSL、 SP 細胞)を中心として試験管内での増殖、あるいは分化の研究が進行中である。SDF-1 (stromal derived factor-1)は VEGFと bFGFの オートクリンシグナルによって血管内皮細胞の形成と血管形成に重要であることが報告されている。これらは虚血性疾患動物モデルと試験管内での培養実験を通して証明され、特に骨髄組織内でのEPC分化あるいは細胞移動に対する役割が報告されている（Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, et al, Circulation. 2003;107(9):1322-1328）。また、VEGFはEPCの動員、あるいは分化に極めて重要であり、出生後の新血管形成に必須な分子であることが分かっている（Isner JM,Asahara T. J Clin Invest 1999;103:1231-1236；及びIwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, et al, Circulation. 2002;105(6):732-738）。VEGF, SCF, SDF-1などのサイトカインは、信号伝達系を通じてEPCの増殖、分化、虚血組織への移動等、EPCの能動的な機能発現の為に重要だと考えられる（Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M,et al. Circ.Res 1999;83:221-228及びKalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, et al. Proc Natl Acad Sci USA 2000;97:3422-3427）。
EPC is a very small amount of cells derived from bone marrow, and is known to proliferate and differentiate by various signals in vivo. As EPC characterization progresses, in vitro proliferation or differentiation studies centered on cells (AC133, KSL, SP cells) that are recognized as stem cells at a relatively early stage are in progress. SDF-1 (stromal derived factor-1) has been reported to be important for vascular endothelial cell formation and blood vessel formation by autocrine signals of VEGF and bFGF. These have been proven through ischemic disease animal models and in vitro culture experiments, and have been reported to play a role in EPC differentiation or cell migration in bone marrow tissue (Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver). M, et al, Circulation. 2003; 107 (9): 1322-1328). In addition, VEGF is extremely important for EPC mobilization or differentiation, and is known to be an essential molecule for postnatal neovascularization (Isner JM, Asahara T. J Clin Invest 1999; 103: 1231-1236 And Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, et al, Circulation. 2002; 105 (6): 732-738). Cytokines such as VEGF, SCF, and SDF-1 are thought to be important for the active expression of EPC such as proliferation and differentiation of EPC and migration to ischemic tissues through the signal transduction system (Asahara T, Masuda). H, Takahashi T, Kalka C, Pastore C, Silver M, et al. Circ.Res 1999; 83: 221-228 and Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, et al Proc Natl Acad Sci USA 2000; 97: 3422-3427).
Notchリガンドとは、Notch受容体に結合して、Notchシグナル伝達を発生させる物質を意味する。Notchリガンドとしては、Jagged類とDelta類が知られている。例えば、DeltaがNotchに結合すると、Notchの細胞内領域が切り離され、細胞核に移行して他の遺伝子の発現を促進することにより、細胞分化を制御すると考えられている。また、DeltaはNotchに類似する構造を有し、Notchと同様に細胞膜に結合している。従って、シグナルを送るためには蛋白質同士が結合することが必要であることから、Deltaを発現する細胞とNotchを発現する細胞は隣接していることが必要である。上記の通り、Notchシグナル伝達系の特徴の一つとして、隣接する細胞の情報伝達を仲介するということが挙げられる。 A Notch ligand means a substance that binds to a Notch receptor and generates Notch signaling. As Notch ligands, Jaggeds and Deltas are known. For example, when Delta binds to Notch, it is thought that the intracellular region of Notch is cut off and transferred to the cell nucleus to promote the expression of other genes, thereby controlling cell differentiation. Delta has a structure similar to Notch and is bound to the cell membrane in the same way as Notch. Therefore, in order to send a signal, it is necessary for proteins to bind to each other. Therefore, a cell expressing Delta and a cell expressing Notch need to be adjacent to each other. As described above, one of the characteristics of the Notch signaling system is that it mediates information transmission between adjacent cells.
本発明で好ましく用いることができるNotchリガンドとしては、Jagged-1及びDelta-4を挙げることができる。 Examples of Notch ligands that can be preferably used in the present invention include Jagged-1 and Delta-4.
未成熟幹細胞にNotchリガンドを作用させる方法としては、例えば、Notchリガンドを高発現する細胞と骨髄未分化細胞とを隣接した状態で共培養することができる。具体的には、本明細書の実施例１（図１ｃも参照）に記載した方法を用いることができる。即ち、直径1μｍあるいは 0.4μmの穴が1.0 x 108/cm2の密度で存在している既存のInsert培養系(BD Falcon)を用いて、あらかじめ外側に３時間ほど2.5x105/dish のNotchリガンド遺伝子導入3T3細胞を付着させた後、裏返し、内側に骨髄由来の未分化細胞(1x106/dish)を培養する方法を用いることができる。 As a method for causing Notch ligand to act on immature stem cells, for example, cells that highly express Notch ligand and bone marrow undifferentiated cells can be co-cultured in an adjacent state. Specifically, the method described in Example 1 (see also FIG. 1c) of the present specification can be used. In other words, using an existing Insert culture system (BD Falcon) in which holes with a diameter of 1 μm or 0.4 μm are present at a density of 1.0 × 10 8 / cm 2 , 2.5 × 10 5 / dish Notch of about 3 hours in advance. A method of culturing undifferentiated cells (1 × 10 6 / dish) derived from bone marrow after turning the inside after attaching the ligand gene-introduced 3T3 cells can be used.
上記した本発明の方法により作製される分化誘導された血管内皮前駆細胞、並びに上記血管内皮前駆細胞を含む虚血性疾患治療のための細胞移植療法剤も本発明の範囲内に含まれる。 The differentiation-induced vascular endothelial progenitor cells produced by the above-described method of the present invention and cell transplantation therapeutic agents for the treatment of ischemic diseases containing the vascular endothelial progenitor cells are also included within the scope of the present invention.
本発明の分化誘導された血管内皮前駆細胞は、Notch受容体（Notch receptor）を発現し、細胞の形態についても血管内皮前駆細胞と思われる形態を呈することが好ましい。また、本発明の分化誘導された血管内皮前駆細胞では、EPCの特異的な分化マーカーであるCD31及びFlk-1の発現が陽性であることが好ましい。 It is preferable that the differentiation-induced vascular endothelial progenitor cell of the present invention expresses a Notch receptor (Notch receptor) and exhibits a form that is considered to be a vascular endothelial progenitor cell. In addition, the differentiation-induced vascular endothelial progenitor cells of the present invention preferably have positive expression of CD31 and Flk-1, which are EPC-specific differentiation markers.
また本発明の分化誘導された血管内皮前駆細胞では、増殖能力が亢進していることが好ましく、VEGF依存的に増殖能力が亢進していることが好ましい。さらに本発明の分化誘導された血管内皮前駆細胞では、VEGF依存的に細胞移動能力が増加していることが好ましい。 In the differentiation-induced vascular endothelial progenitor cells of the present invention, the proliferation ability is preferably enhanced, and the proliferation ability is preferably enhanced in a VEGF-dependent manner. Furthermore, the differentiation-induced vascular endothelial progenitor cells of the present invention preferably have an increased cell migration ability in a VEGF-dependent manner.
次に、血管内皮前駆細胞を含む虚血性疾患治療のための細胞移植療法剤について説明する。本発明の方法により分化誘導された血管内皮前駆細胞は、好ましくは増殖能力が亢進しており、また細胞移動能力も増加しているため、質的に優れた血管内皮前駆細胞である、虚血性疾患治療のための細胞移植療法剤として使用することができる。 Next, a cell transplantation therapeutic agent for treating ischemic diseases including vascular endothelial precursor cells will be described. Vascular endothelial progenitor cells induced to differentiate by the method of the present invention preferably have an enhanced proliferation ability and an increased cell migration ability, and are therefore qualitatively superior vascular endothelial precursor cells, ischemic It can be used as a cell transplantation therapeutic agent for disease treatment.
本発明の細胞移植療法剤は、例えば公知の方法に従い、本発明の分化誘導された血管内皮前駆細胞を細胞懸濁液とすることによって調製することができる。 The cell transplantation therapeutic agent of the present invention can be prepared by, for example, using the differentiation-induced vascular endothelial progenitor cells of the present invention as a cell suspension according to a known method.
本発明の細胞移植療法剤は、例えば、虚血性疾患の治療のために安全に使用することができる。 The cell transplantation therapeutic agent of the present invention can be used safely, for example, for the treatment of ischemic diseases.
本発明の細胞移植療法剤を投与する対象は好ましくは哺乳動物（例えば、ヒト、ラット、マウス、モルモット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サルなど）であり、特に好ましくはヒトである。本発明の細胞移植療法剤の投与方法は特に限定されず、カテーテルで挿入する、直接血管に注射する、静脈に注射する、バイオプシーなどの方法により投与することができる。 The subject to which the cell transplantation therapeutic agent of the present invention is administered is preferably a mammal (eg, human, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.), particularly preferably Human. The administration method of the cell transplantation therapeutic agent of the present invention is not particularly limited, and can be administered by a method such as insertion with a catheter, direct injection into a blood vessel, injection into a vein, biopsy and the like.
本発明の細胞移植療法剤の投与量、投与回数は本発明の効果が得られる限り特に限定されない。投与量については、投与１回当たりの細胞数として、成人一人当たり一般的には、１０4〜１０8細胞程度、好ましくは、１０5〜１０7細胞程度とすることができる。投与回数は１回以上であれば、任意の回数だけ投与することができ、一般的には１〜１０回、好ましくは１〜５回程度である。 The dose and frequency of administration of the cell transplantation therapeutic agent of the present invention are not particularly limited as long as the effect of the present invention is obtained. Regarding the dose, the number of cells per administration can be generally about 10 4 to 10 8 cells, preferably about 10 5 to 10 7 cells per adult. As long as the administration frequency is 1 or more, it can be administered any number of times, generally 1 to 10 times, preferably about 1 to 5 times.
以下の実施例により本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
実施例１：
血管内皮前駆細胞の分化誘導系は、以下の方法で確立した。
Notch ligandである Jagged-1,DII-1, DII-4を大量発現している細胞を、3T3細胞株にretro-viral遺伝子導入法を用いて各遺伝子を導入して確立した（Hozumi, K. et al., J. Immunol;170;4973-4979;2003)。Notchリガンド遺伝子の導入は、 各々の3T3細胞からRNeasy Micro Kit (QIAGEN)を用いてRNAを分離精製して、特定遺伝子を増幅させる RT-PCR法によって確認した（図1a）。
Example 1:
A differentiation induction system for vascular endothelial progenitor cells was established by the following method.
Cells expressing a large amount of Jagged-1, DII-1, DII-4, which are Notch ligands, were established by introducing each gene into the 3T3 cell line using the retro-viral gene transfer method (Hozumi, K. et al., J. Immunol; 170; 4973-4979; 2003). The introduction of Notch ligand gene was confirmed by RT-PCR method in which RNA was separated and purified from each 3T3 cell using RNeasy Micro Kit (QIAGEN) and a specific gene was amplified (FIG. 1a).
また、 Notch 2を発現させた CHO細胞株を用いて、信号が入った場合に蛍光を発生するreporter遺伝子を組み込んだ実験系(Kumano K et al.,Immunity 18;699-711)を作成し、Leuciferase法により、信号伝達が可能な細胞が樹立したことを確かめた(図１ｂ) 。 In addition, using a CHO cell line expressing Notch 2, an experimental system (Kumano K et al., Immunity 18; 699-711) incorporating a reporter gene that generates fluorescence when a signal is input, It was confirmed that cells capable of signal transmission were established by the Leuciferase method (FIG. 1b).
血管内皮前駆細胞の分化誘導系は、直径1μｍあるいは 0.4μmの穴が1.0 x 108/cm2の密度で存在している既存のInsert培養系(BD Falcon)を用いて、あらかじめ外側に３時間ほど2.5x105/dish のNotchリガンド遺伝子導入3T3細胞を付着させた（10%FBSを含むD-MEM培地）後、裏返し、内側に骨髄由来の未分化細胞(1x106/dish)を浅原らの方法(Asahara T et al.、Science 275;964-967;1997)で培養するシステム（EPC分化誘導系）を確立した(図１ｃ)。 The vascular endothelial progenitor cell differentiation induction system uses the existing Insert culture system (BD Falcon) in which holes with a diameter of 1 μm or 0.4 μm are present at a density of 1.0 × 10 8 / cm 2 , and the outer 3 hours in advance After attaching 2.5x10 5 / dish of Notch ligand gene-introduced 3T3 cells (D-MEM medium containing 10% FBS), turn over and place bone marrow-derived undifferentiated cells (1x10 6 / dish) inside Asahara et al. A culture system (EPC differentiation induction system) was established by the method (Asahara T et al., Science 275; 964-967; 1997) (FIG. 1c).
骨髄由来の未分化細胞をEPC分化誘導系で４日間培養した後、Notchシグナルに応答するHes-1の遺伝子が発現していることをRT-PCR法で用いて確認した(図１ｄ) 。以上のようにして、Notchリガンドを骨髄由来の未分化細胞に作用させて、血管内皮前駆細胞に分化させる系を確立した。 After culturing undifferentiated cells derived from bone marrow for 4 days in an EPC differentiation induction system, it was confirmed by RT-PCR that the gene for Hes-1 responding to Notch signal was expressed (FIG. 1d). As described above, a system was established in which Notch ligand was allowed to act on undifferentiated cells derived from bone marrow to differentiate into vascular endothelial progenitor cells.
実施例２：
実施例１に示した血管内皮前駆細胞の分化誘導系を用いて、Notchリガンドの分化誘導性について調べた。マウス骨髄組織から、まだ細胞運命が決定されていないLin (-)細胞(T細胞, B細胞, Macrophage, erythrocyteなど分化した細胞を除いた未分化細胞)を Midi MACS Separatorを用いて分離した。分離方法は、まずマウス骨髄から骨髄細胞を分離後、biotin化した Cocktail 抗体 (B220, CD3, Gr-1, Mac-1, TER-119,BD)を 4℃で20分間反応させた後、 streptoavidin beads (Miltenyi Biotec. )を同じ条件下で反応させた。これらの細胞から、Midi MACS Separatorを用いて未分化細胞 (Lin(-)細胞)を分離した。EPC分化誘導系で分離したLin (-)細胞を４日間培養した後、 RNAをRNeasy Micro Kit (QIAGEN)を用いて精製した。Notch receptorの発現程度をRT-PCR法で確認した結果、リセプター１，２，３，４の発現は、どのNotchリガンドでも変わりがなかった（図２ａ）。また、培養４日後に細胞の形態変化を顕微鏡で確認した結果、Jagged-1と DII-4で特異的に血管内皮前駆細胞と思われる形態に変化していることが観察された（図２ｂ）。
Example 2:
Using the vascular endothelial progenitor cell differentiation induction system shown in Example 1, the differentiation induction of Notch ligand was examined. Lin (−) cells (undifferentiated cells excluding differentiated cells such as T cells, B cells, Macrophage, erythrocyte) whose cell fate has not been determined were isolated from mouse bone marrow tissue using Midi MACS Separator. First, bone marrow cells were isolated from mouse bone marrow, then reacted with a biotinylated Cocktail antibody (B220, CD3, Gr-1, Mac-1, TER-119, BD) at 4 ° C for 20 minutes, then streptoavidin The beads (Miltenyi Biotec.) were reacted under the same conditions. From these cells, undifferentiated cells (Lin (−) cells) were separated using Midi MACS Separator. Lin (−) cells isolated in the EPC differentiation induction system were cultured for 4 days, and then RNA was purified using RNeasy Micro Kit (QIAGEN). As a result of confirming the expression level of Notch receptor by RT-PCR, the expression of receptors 1, 2, 3, and 4 was not changed by any Notch ligand (FIG. 2a). In addition, as a result of confirming the morphological change of the cells with a microscope after 4 days of culture, it was observed that Jagged-1 and DII-4 specifically changed to a vascular endothelial progenitor cell (Fig. 2b). .
実施例３：
Notchシグナルにより骨髄未分化細胞を刺激する血管内皮前駆細胞の分化誘導系で、未分化細胞の血管内皮前駆細胞（EPC）への分化の状況を調べた。EPC分化誘導系で4日間培養したLin(-)細胞を、EPCの特異的な分化マーカーであるCD31とFlk-1に対する抗体（BD,E-Bio）をそれぞれ４℃２０分間反応させる方法で染色した。さらにstreptoavidin-APC抗体(e-Bio)を同じ方法で反応させた後、FACS分析を行った。その結果、Jagged-1とDII-4により、CD31/Flk-1陽性細胞が対照群より約２倍以上増加していることが見出された（図３）。このことは、NotchリガンドJagged-1とDII-4により、骨髄未分化細胞が血管内皮前駆細胞に分化していることを示す。
Example 3:
We examined the state of differentiation of undifferentiated cells into vascular endothelial progenitor cells (EPCs) in a differentiation induction system of vascular endothelial progenitor cells that stimulates bone marrow undifferentiated cells by Notch signal. Lin (-) cells cultured for 4 days in EPC differentiation induction system were stained by reacting antibodies against CD31 and Flk-1 (BD, E-Bio), which are specific differentiation markers for EPC, at 4 ° C for 20 minutes, respectively. did. Furthermore, streptoavidin-APC antibody (e-Bio) was reacted in the same manner, and then FACS analysis was performed. As a result, it was found that Jagged-1 and DII-4 increased CD31 / Flk-1-positive cells more than twice as much as the control group (FIG. 3). This shows that bone marrow undifferentiated cells are differentiated into vascular endothelial progenitor cells by Notch ligands Jagged-1 and DII-4.
実施例４：
実施例１で確立したEPC分化誘導系で得た血管内皮前駆細胞（EPC）の特性分析のために以下の実験を行った。実施例２に記載した分離方法によって精製した骨髄組織由来のLin (-)細胞を4日間 EPC分化誘導系で培養後、EDTA-PBS (Sigma)を用いて細胞を分離させた後、細胞の特性分析を行った。まず、FACS法を用いてEPCの分化マーカーであるCD31と Flk-1 (VEGFR2) の発現を調べ、NotchシグナルのEPC分化マーカーの発現量に対する影響を検討した。EPC分化誘導系で得た細胞1x105個を、抗CD31抗体(BD)と抗Flk-1抗体 (e-Bio)で4℃20分間染色し、FACS分析法を用いて分析した。その結果、図４a
に示すように、Jagged-1とDII-4の誘導系において分化マーカー発現パターン（赤色）が空ベクターの発現パターン（黒色）と比べて右側へシフトしていることが観察された。このことは、Jagged-1とDII-4の誘導系のほうが対照群よりもEPC分化マーカー(CD31, VEGFR2)の平均的な発現量が著しく増加していることを示している。また、RT-PCR法によりEPC分化マーカーのRNA量を測定した結果、Jagged-1とDII-4の誘導系においてCD31と Flk-1(VEGFR2)のmRNA発現が増加していることがわかった(図４ｂ)。また、VE-cadherinのmRNA量の増加も見られた（図４ｂ）。
Example 4:
The following experiment was conducted to analyze the characteristics of vascular endothelial progenitor cells (EPC) obtained by the EPC differentiation induction system established in Example 1. Bone marrow tissue-derived Lin (−) cells purified by the separation method described in Example 2 were cultured in an EPC differentiation induction system for 4 days, and then separated using EDTA-PBS (Sigma), and then cell characteristics Analysis was carried out. First, the expression of CD31 and Flk-1 (VEGFR2), which are EPC differentiation markers, was examined using the FACS method, and the effect of Notch signal on the expression level of EPC differentiation markers was examined. Five 1 × 10 5 cells obtained by the EPC differentiation induction system were stained with anti-CD31 antibody (BD) and anti-Flk-1 antibody (e-Bio) at 4 ° C. for 20 minutes, and analyzed using FACS analysis. As a result, FIG.
As shown in Fig. 5, it was observed that the differentiation marker expression pattern (red) was shifted to the right in the induction system of Jagged-1 and DII-4 compared to the expression pattern of the empty vector (black). This indicates that the average expression level of EPC differentiation markers (CD31, VEGFR2) is significantly increased in the Jagged-1 and DII-4 induction systems than in the control group. In addition, as a result of measuring the RNA level of the EPC differentiation marker by RT-PCR, it was found that the mRNA expression of CD31 and Flk-1 (VEGFR2) was increased in the Jagged-1 and DII-4 induction systems ( FIG. 4b). An increase in the amount of VE-cadherin mRNA was also observed (FIG. 4b).
実施例５：
EPC分化誘導系でのLin(-)細胞のEPCへの分化の程度を調べるために、Ac-LDL導入実験(Takahashi T et al、Nature Med.;5;434-438;1999)を行った。EPC分化誘導系で４日間共培養した骨髄組織由来のLin (-)細胞を、Fibronectinコーティングされている4 chamber(BD)で培養後、Acetyl LDL(red) (Biomedical Technologies Inc.)を導入した。この緑色の蛍光が付着した細胞群に蛍光標識Iso-lectinGS-IB4 (Sigma)を30分間反応させた後、染色された細胞群を高倍率蛍光顕微鏡を用いて観察した。そのうちから高倍率のイメージ写真５０枚を無作為に選び、染色された細胞数を数値化した結果、Jagged-1と DII-4のEPC分化誘導系において対照群およびDII-1より多くの細胞が観察され，著しい分化の亢進が認められた（図５）。このことより、NotchシグナルのうちJagged-1とDII-4は強力にEPCへの分化を誘導することができることがEPCの機能的側面からも明らかになった。
Example 5:
In order to examine the degree of differentiation of Lin (−) cells into EPC in the EPC differentiation induction system, an Ac-LDL introduction experiment (Takahashi T et al, Nature Med .; 5; 434-438; 1999) was performed. Bone marrow tissue-derived Lin (-) cells co-cultured in an EPC differentiation induction system for 4 days were cultured in 4 chambers (BD) coated with Fibronectin, and then Acetyl LDL (red) (Biomedical Technologies Inc.) was introduced. After the cells labeled with green fluorescence were reacted with fluorescently labeled Iso-lectinGS-IB4 (Sigma) for 30 minutes, the stained cells were observed using a high-magnification fluorescent microscope. Among them, 50 high-magnification image photographs were randomly selected, and the number of stained cells was quantified. As a result, more cells were found in the Jagged-1 and DII-4 EPC differentiation induction systems than in the control group and DII-1. Observed and markedly enhanced differentiation was observed (FIG. 5). From this, it became clear from the functional aspect of EPC that Jagged-1 and DII-4 of Notch signals can induce differentiation into EPC.
実施例６：
実施例１で樹立したEPC分化誘導系で得たEPCの増殖能力を調べた。骨髄組織から得たLin(-)細胞をEPC分化誘導系で４日間共培養した後細胞数を計測したが、細胞数の変化は認められなかった。しかし、これらの細胞をWST法でその増殖能力を調べた結果、Jagged-1群のみ増殖能力の亢進が認められた（図６ａ）。
Example 6:
The proliferation ability of EPC obtained by the EPC differentiation induction system established in Example 1 was examined. Lin (-) cells obtained from bone marrow tissue were co-cultured for 4 days in an EPC differentiation induction system, and the number of cells was counted, but no change in the number of cells was observed. However, as a result of examining the proliferation ability of these cells by the WST method, only the Jagged-1 group was found to have an enhanced proliferation ability (FIG. 6a).
更にEPCの成長因子であるVEGFに対する感受性を調べた。EPC分化誘導系で得た細胞を、更に１％FBSを含む培地で１２時間 starvationさせた後、異なる濃度のVEGF(0, 4, 20, 100 ng/ml,R&D)を添加してさらに２日間培養した。それらの細胞をWST-1試薬存在下で３時間培養した後、蛍光リーダーを用いて計測した。その結果、図６ｂに示すように、NotchシグナルのうちJagged-1とDII-4は、VEGFに対するEPCの感受性を亢進させたことが明らかになった。 Furthermore, the sensitivity of EPC to VEGF, a growth factor, was examined. Cells obtained from the EPC differentiation induction system were further starved in a medium containing 1% FBS for 12 hours, and then added with different concentrations of VEGF (0, 4, 20, 100 ng / ml, R & D) for another 2 days. Cultured. These cells were cultured for 3 hours in the presence of WST-1 reagent, and then counted using a fluorescence reader. As a result, as shown in FIG. 6b, it was revealed that Jagged-1 and DII-4 among Notch signals enhanced the sensitivity of EPC to VEGF.
実施例７
実施例１のEPC分化誘導で得た細胞を１％FBSを含む培地で１２時間 starvationさせた後、Boydenチェンバーの下方に様々な濃度のVEGF(0, 4, 20, 100 ng/ml, R&D)を添加後、細胞移動を調べる Boyden Chamber Migration実験を行った。その結果、図7に示すように、Jagged-1と DII-4グループで対照群およびDII-1と比較して細胞移動能力がVEGF濃度依存的に増加していることが確認された。
Example 7
Cells obtained by induction of EPC differentiation in Example 1 were starvated in a medium containing 1% FBS for 12 hours, and then various concentrations of VEGF (0, 4, 20, 100 ng / ml, R & D) below the Boyden chamber. After the addition, a Boyden Chamber Migration experiment was conducted to examine cell migration. As a result, as shown in FIG. 7, it was confirmed that the cell migration ability increased in the Jagged-1 and DII-4 groups as compared to the control group and DII-1 in a VEGF concentration-dependent manner.
実施例８：
実施例１〜７までの実験結果から、Notchシグナルによって刺激された骨髄組織由来の未分化細胞（Lin(-)細胞）はEPCに分化誘導できているということを確認した。そこで、これらのEPC分化誘導系で得たEPCの血管形成能力をin vivoで調べるために、下肢虚血動物モデルを作成した(Kalka C et al,PNAS;97;3422-3427;2000, Iwaguro H et al、Circulation 105;732-738;2002)。この下肢虚血動物モデルに2.5 x 105のEPCを静脈注射し、下肢の傷害改善程度を肉眼的な観察やMoorLDI (Moor Instruments)を用いて調べた。
Example 8:
From the experimental results of Examples 1 to 7, it was confirmed that undifferentiated cells (Lin (−) cells) derived from bone marrow tissue stimulated by Notch signal could be induced to differentiate into EPC. Therefore, in order to examine the angiogenic ability of EPC obtained by these EPC differentiation induction systems in vivo, an animal model of lower limb ischemia was created (Kalka C et al, PNAS; 97; 3422-3427; 2000, Iwaguro H et al, Circulation 105; 732-738; 2002). The lower limb ischemia animal model was intravenously injected with 2.5 × 10 5 EPCs, and the degree of lower limb injury improvement was examined by macroscopic observation and MoorLDI (Moor Instruments).
肉眼的観察では、図８aに示すように、Jagged-1やDII-4グループでは、空ベクターやDII-1と比較して顕著な下肢虚血部分の再生を認めた。MoorLDI (Moor Instruments)で下肢虚血疾患の非誘導部分との比較により再生程度を数値化して比べてみた結果、Jagged-1とDII-4グループで著しく血流の改善が認められた(図８ｂ及びｃ)。更に、再生した組織切片を蛍光標識Iso-lectinGS-IB4 (Sigma)に30分間反応させて組織染色を行った。 Iso-lectin B4陽性細胞を指標として再生組織の毛細血管の密度（IB4+cell/HPF）を調べた結果、Jagged-1とDII-4のグループは対照群およびDII-1より明らかに増加していることが明らかになった(図８ｄ)。以上の結果から、Jagged-1やDII-4等のNotchリガンドによる刺激でin vitroで生成させたEPCは、下肢虚血動物モデルにおいて虚血組織の治癒、再生を促進する能力があることが示された。 In macroscopic observation, as shown in FIG. 8a, the Jagged-1 and DII-4 groups showed marked regeneration of the lower limb ischemia as compared to the empty vector and DII-1. As a result of comparing the degree of regeneration with MoorLDI (Moor Instruments) in comparison with the non-induced part of lower limb ischemia, a marked improvement in blood flow was observed in the Jagged-1 and DII-4 groups (FIG. 8b). And c). Further, the regenerated tissue section was reacted with fluorescently labeled Iso-lectinGS-IB4 (Sigma) for 30 minutes for tissue staining. As a result of examining capillary density (IB4 + cell / HPF) of the regenerated tissue using Iso-lectin B4 positive cells as an index, the Jagged-1 and DII-4 groups clearly increased from the control group and DII-1 (Fig. 8d). These results indicate that EPCs generated in vitro by stimulation with Notch ligands such as Jagged-1 and DII-4 have the ability to promote the healing and regeneration of ischemic tissues in animal models of lower limb ischemia. It was done.
実施例９
実施例８で、EPC分化誘導系で得たEPCは下肢虚血動物モデルで虚血組織の治癒、再生を促進する能力があることが認められたので、さらにそのメカニズムを明らかにするためにEPCの虚血組織内への浸透について調べた。GFPマウス(文献)の骨髄より実施例２に示した方法で未分化細胞（Lin(-)細胞）を分離し、これらの細胞を実施例１に示したEPC分化誘導系で4日間分化誘導した。このGFPマウス由来のEPC2.5x105個を下肢虚血モデルマウスに細胞移植 (下肢虚血モデル作成と同時に静脈注射)を行った。移植１週間後、
虚血部位の組織より10umの切片を作成し免疫染色を行った。組織切片を抗CD31抗体 (purified rat anti-mouse CD31, BD)と4℃で12時間反応させた後、Alexaflour 598 を結合させた抗マウスIgG抗体(Molecular probes)を4℃で1時間反応させ、蛍光顕微鏡で観察した。血管内皮細胞の特異的マーカーであるCD31の組織染色の結果から、下肢虚血モデルマウスの血管内に導入されたGFPマウス由来のEPCは対照群およびDII-1よりJagged-1, DII-4グループで顕著に増加していることが観察された(図９ａ及びｂ)。移植したEPCの、組織での分化の状況を明らかにするために、移植から４週間後、下肢虚血モデルマウスの虚血組織の切片を毛細血管や静脈の染色が可能なIso-lectin GS-IB4(Molecular probes；図９ｃ)とalpha- smooth muscle actin抗体 (Sigma；図９ｄ）を用いて染色を行った。その結果、Jagged-1やDII-4で誘導されたEPCは対象群やDII-1に比較して、毛細血管への分化が頻繁に観察され、動静脈などの血管組織への分化にも貢献していることが認められた。以上の結果から、 Notchシグナルによって分化誘導されたEPCは、虚血組織に浸透して血管内皮細胞に分化し、血管の再生に重要な役割を果たしていることが明らかになった。
Example 9
In Example 8, EPC obtained by the EPC differentiation induction system was confirmed to have the ability to promote healing and regeneration of ischemic tissue in an animal model of lower limb ischemia. To further clarify the mechanism, EPC Permeation into the ischemic tissue. Undifferentiated cells (Lin (−) cells) were isolated from the bone marrow of GFP mice (reference) by the method shown in Example 2, and these cells were induced to differentiate for 4 days using the EPC differentiation induction system shown in Example 1. . Five GFP mouse-derived EPC2.5 × 10 5 cells were transplanted into a lower limb ischemia model mouse (intravenous injection simultaneously with the creation of the lower limb ischemia model). 1 week after transplantation
A 10-um section was prepared from the tissue at the ischemic site and immunostained. After reacting tissue sections with anti-CD31 antibody (purified rat anti-mouse CD31, BD) at 4 ° C for 12 hours, anti-mouse IgG antibody (Molecular probes) bound with Alexaflour 598 was reacted at 4 ° C for 1 hour, Observed with a fluorescence microscope. From the results of tissue staining of CD31, a specific marker of vascular endothelial cells, EPCs derived from GFP mice introduced into the blood vessels of lower limb ischemia model mice were Jagged-1, DII-4 group than DII-1 It was observed that there was a marked increase in (Figs. 9a and b). In order to clarify the state of tissue differentiation of the transplanted EPC, Iso-lectin GS-, which can stain capillaries and veins, ischemic tissue sections of ischemic model mice of the lower limb ischemia 4 weeks after transplantation. Staining was performed using IB4 (Molecular probes; Fig. 9c) and alpha-smooth muscle actin antibody (Sigma; Fig. 9d). As a result, EPCs induced by Jagged-1 and DII-4 were more frequently differentiated into capillaries compared to the target group and DII-1, and contributed to differentiation into arteriovenous and other vascular tissues. It was recognized that From the above results, it was revealed that EPCs induced to differentiate by Notch signal penetrated into ischemic tissues and differentiated into vascular endothelial cells, and played an important role in the regeneration of blood vessels.
実施例１０
EPCの分化増殖に対するNotchリガンドの役割を明らかにするために、Notchリガンド遺伝子のconditional 遺伝子欠損マウスを作成した。conditional 遺伝子欠損マウスの作成は、Hozumiらの方法(Hozumi K. et al., Nature Immunology;5;638-644)に準じて行った。すなわち、conditional 遺伝子欠損の方法は200μg/miceのPoly I/C(Amersham Biosciences)を４回にわたってマウス腹腔内に注射後、Cre 遺伝子特異的な酵素反応によってJagged-1や DII-1遺伝子を選択的に除去した（図１０）。
Example 10
In order to elucidate the role of Notch ligand on the differentiation and proliferation of EPCs, a conditional gene-deficient mouse of Notch ligand gene was created. Conditional gene-deficient mice were prepared according to the method of Hozumi et al. (Hozumi K. et al., Nature Immunology; 5; 638-644). That is, the method of conditional gene deletion is that 200μg / mice Poly I / C (Amersham Biosciences) is injected into the abdominal cavity of mice 4 times, and then the Jagged-1 and DII-1 genes are selectively selected by Cre gene-specific enzyme reaction. (FIG. 10).
実施例１１
conditional 遺伝子欠損マウスの骨髄組織から未分化細胞をMidiMACS separatorを用いて実施例２の方法で精製後、Sca-１陽性Beads(Miltenyi Biotec. )でさらに分離精製してFACS分析をおこなった。これらの細胞をEPC特異的なマーカーであるCD31とFlk-1の抗体で4℃、20分間反応させた後、streptoavidin APC(e-Bio)でさらに20分間反応させFACS法により分析を行った。その結果、Jagged-1 遺伝子欠損マウスでは、CD31(+)Flk-1(+)細胞がDII-1欠損マウスや対照群より少ないことがわかった(図１１ａ)。
Example 11
The undifferentiated cells were purified from the bone marrow tissue of the conditional gene-deficient mouse using the MidiMACS separator by the method of Example 2, followed by further separation and purification with Sca-1 positive Beads (Miltenyi Biotec.) and FACS analysis. These cells were reacted with CD31 and Flk-1 antibodies, which are EPC-specific markers, at 4 ° C. for 20 minutes, and further reacted with streptoavidin APC (e-Bio) for 20 minutes and analyzed by FACS method. As a result, it was found that Jagged-1 gene-deficient mice had fewer CD31 (+) Flk-1 (+) cells than DII-1-deficient mice and the control group (FIG. 11a).
また上記の方法で得られたEPCについて、実施例５の方法でAcetyl-LDL導入を行い、蛍光標識Iso-lectinGS-IB4(Sigma)で染色後、蛍光顕微鏡で観察した。その結果、Jagged-1遺伝子欠損マウスではDII-1欠損マウスや対象群と比較して、EPCに分化した細胞が著しく減少していることが明らかとなった(図１１ｂ)。 Further, the EPC obtained by the above method was introduced with Acetyl-LDL by the method of Example 5, stained with a fluorescent label Iso-lectin GS-IB4 (Sigma), and then observed with a fluorescent microscope. As a result, Jagged-1 gene-deficient mice were found to have significantly reduced EPC-differentiated cells compared to DII-1-deficient mice and the subject group (FIG. 11b).
以上の結果から、EPCの分化は生体内でJagged-1依存的におきている現象の一部であることが明らかになった。 From the above results, it became clear that EPC differentiation is part of the phenomenon that occurs in a living body depending on Jagged-1.
実施例１２
実施例１０の２種類(Jagged-1, DII-1)のconditional遺伝子欠損マウスに、実施例８の方法で下肢虚血疾患モデルを作成し、虚血組織部分の修復程度を観察した。Jagged-1遺伝子欠損マウスでは血流の改善度が著しく阻害されていたが(図１２ａ及びｂ)、DII-1遺伝子欠損マウスでは対照群マウスとほとんど差が認められなかった (図１２ｃ及びｄ)。
さらに実施例８に述べた方法で、conditional遺伝子欠損マウスの骨髄組織よりEPCを分離精製後 5 x 105の細胞を静脈注射する方法によって下肢虚血性疾患モデルヌードマウスに移植した。その結果、Jagged-1遺伝子欠損マウスからのEPC移植マウスではDII-1欠損マウスや対象群と比較して血流の修復が阻害されていた(図１２ｅ)。以上の結果、Jagged-1遺伝子の欠損によりEPCはその組織修復能力も劣ることが明らかになった。
Example 12
A lower limb ischemic disease model was prepared by the method of Example 8 in the two types (Jagged-1, DII-1) conditional gene-deficient mice of Example 10, and the degree of repair of the ischemic tissue portion was observed. In the Jagged-1 gene-deficient mice, the improvement in blood flow was remarkably inhibited (FIGS. 12a and b), but in the DII-1 gene-deficient mice, there was almost no difference from the control group mice (FIGs. 12c and d). .
Furthermore, after the EPC was separated and purified from the bone marrow tissue of the conditional gene-deficient mouse by the method described in Example 8, it was transplanted to a lower limb ischemic disease model nude mouse by intravenous injection of 5 × 10 5 cells. As a result, in the EPC transplanted mice from the Jagged-1 gene-deficient mice, blood flow repair was inhibited as compared with the DII-1-deficient mice and the subject group (FIG. 12e). As a result, it became clear that EPC is inferior in tissue repair ability due to the deletion of Jagged-1 gene.
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Claims (3)
The method according to claim 1 or 2 , wherein cells that highly express Notch ligand and immature stem cells are co-cultured in an adjacent state.
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