JP6841429B2 - Undifferentiated mesenchymal stem cell markers and their uses - Google Patents
Undifferentiated mesenchymal stem cell markers and their uses Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Description
本発明は間葉系幹細胞マーカーに関する。詳しくは、未分化な状態の間葉系幹細胞に特異性の高い分子（バイオマーカー）及びその用途に関する。本出願は、２０１５年８月３日に出願された日本国特許出願第２０１５−１５３７１２号に基づく優先権を主張するものであり、当該特許出願の全内容は参照により援用される。 The present invention relates to mesenchymal stem cell markers. Specifically, the present invention relates to a molecule (biomarker) having high specificity for undifferentiated mesenchymal stem cells and its use. This application claims priority based on Japanese Patent Application No. 2015-153712 filed on August 3, 2015, and the entire contents of the patent application are incorporated by reference.
間葉系幹細胞（mesenchymal stem cell又はmesenchymal stromal cell。以下、「MSC」と略称することがある）は骨髄、脂肪組織、歯髄、臍帯、子宮内膜をはじめとして多くの臓器に存在する細胞であり、培養皿上で特定の条件下で培養すると骨、軟骨及び脂肪に分化することが知られている。また、間葉系幹細胞は他の細胞群の増殖や分化を促す作用（trophic effect）及び免疫調整能を有することも知られ、現在ではGVHD（移植片対宿主病）、脊椎損傷、腎移植後の腎不全、心筋梗塞、末梢閉塞性動脈疾患等の多様な疾患の治療への応用が期待されている。 Mesenchymal stem cells (mesenchymal stem cells or mesenchymal stromal cells, hereinafter abbreviated as "MSC") are cells that are present in many organs including bone marrow, adipose tissue, dental pulp, umbilical cord, and endometrial membrane. , It is known that when cultured on a culture dish under specific conditions, it differentiates into bone, cartilage and fat. Mesenchymal stem cells are also known to have the ability to promote proliferation and differentiation of other cell groups (trophic effect) and immunomodulatory ability, and are now GVHD (graft-versus-host disease), spinal injury, and after renal transplantation. It is expected to be applied to the treatment of various diseases such as renal failure, myocardial infarction, and peripheral occlusive arterial disease.
間葉系幹細胞は骨髄では類洞及び細小動脈等の血管周囲に、その他の臓器では毛細血管周囲に存在することが知られている（非特許文献１）。骨髄では造血幹細胞維持のためのニッチ細胞として、あるいは骨再生のための骨幹細胞として機能している（非特許文献２）。骨髄以外の臓器ではペリサイト（血管周皮細胞）あるいは血管周囲の線維芽細胞として存在しており、血管の成熟化に関わるとされている（非特許文献３）。また、癌や各種線維化疾患において間質の線維化を引き起こす原因がペリサイトの増殖であるとする学説も存在する（非特許文献４）。 It is known that mesenchymal stem cells are present around blood vessels such as sinusoids and microarteries in bone marrow, and around capillaries in other organs (Non-Patent Document 1). In the bone marrow, it functions as a niche cell for maintaining hematopoietic stem cells or as a bone stem cell for bone regeneration (Non-Patent Document 2). In organs other than bone marrow, it exists as pericytes (pericytes of blood vessels) or fibroblasts around blood vessels, and is considered to be involved in the maturation of blood vessels (Non-Patent Document 3). There is also a theory that pericyte proliferation is the cause of interstitial fibrosis in cancer and various fibrotic diseases (Non-Patent Document 4).
国際的に以下の条件（１）〜（３）を満たす細胞が間葉系幹細胞と定義されている。
（１）培養皿上で接着する細胞であること
（２）CD105陽性、CD73陽性、CD90陽性、CD45陰性、CD34陰性、CD14又はCD11bが陰性、CD79a又はCD19が陰性、HLA-DR陰性であること
（３）試験管内（in vitro）において骨芽細胞、軟骨芽細胞及び脂肪細胞への分化能を有することInternationally, cells satisfying the following conditions (1) to (3) are defined as mesenchymal stem cells.
(1) Cells that adhere on a culture dish (2) CD105 positive, CD73 positive, CD90 positive, CD45 negative, CD34 negative, CD14 or CD11b negative, CD79a or CD19 negative, HLA-DR negative (3) Having the ability to differentiate into osteoblasts, chondrocytes and adipocytes in vitro.
上記（２）に列挙された分子以外にも、Stro-1、PDGF受容体、Sca-1、CD271、CD146などが間葉系幹細胞のマーカーとして報告されており、これらのマーカーの組み合わせで間葉系幹細胞を単離する方法が開発されている（例えば特許文献１を参照）。しかしながら、上記のCD105、CD73、CD90を含め、いずれのマーカーも上皮細胞や癌細胞、あるいは神経細胞などに発現しており、間葉系幹細胞に特異的ではない。 In addition to the molecules listed in (2) above, Stro-1, PDGF receptor, Sca-1, CD271, CD146, etc. have been reported as markers for mesenchymal stem cells, and a combination of these markers has been reported to be mesenchymal. Methods for isolating mesenchymal stem cells have been developed (see, eg, Patent Document 1). However, all markers including the above-mentioned CD105, CD73, and CD90 are expressed in epithelial cells, cancer cells, nerve cells, and the like, and are not specific to mesenchymal stem cells.
間葉系幹細胞のマーカーとして数多くの分子が報告されているが、いずれも間葉系幹細胞以外の細胞にも発現しており特異性は低い。そこで本発明は、未分化な状態の間葉系幹細胞に特異性が高く、高感度での間葉系幹細胞の同定や高純度な間葉系幹細胞の調製などを可能にするマーカー分子及びその用途を提供することを課題とする。 Many molecules have been reported as markers for mesenchymal stem cells, but all of them are expressed in cells other than mesenchymal stem cells and have low specificity. Therefore, the present invention is a marker molecule having high specificity for undifferentiated mesenchymal stem cells and enabling highly sensitive identification of mesenchymal stem cells and preparation of high-purity mesenchymal stem cells and their uses. The challenge is to provide.
上記課題の下で研究を進める中、本発明者らは細胞膜結合型／分泌型の分子であるロイシンリッチリピート含有免疫グロブリンスーパーファミリー（ISLR：immunoglobulin superfamily containing leucine-rich repeat）に着眼し、各種実験を行った。ISLRは1997年に眼の網膜に発現する分子として遺伝子クローニングされたが、その機能は未だ明らかになっていない(非特許文献５、６)。2009年に発表された遺伝子発現解析の論文では、マウス胎児において鰓弓、肢芽、体節、頭部や体幹の間葉系組織に発現していることが報告されている(非特許文献７)。ISLRの相同遺伝子に相当するISLR2（別名Linx）は神経系組織を中心に発現することも報告されている（非特許文献８）。また、肺癌、乳癌、膵癌などにおいてISLRの発現が正常に比して高いとする報告もある（例えば、特許文献２、非特許文献９、１０を参照）。但し、いずれの報告においても、同様の挙動を示す多数の遺伝子の中の一つとしてISLRが掲載されているにすぎず、また、ISLRの機能に関する言及はない。一方、間葉系幹細胞における遺伝子発現を網羅的に調べた研究の報告では、発現の上昇を認める遺伝子のリストにISLRが掲載されている（非特許文献１１、１２）。しかしながら、間葉系幹細胞におけるISLRの発現の意義、その機能などは不明である。
While proceeding with research under the above issues, the present inventors focused on leucine-rich repeat-containing immunoglobulin superfamily (ISLR), which is a cell membrane-bound / secretory molecule, and conducted various experiments. Was done. ISLR was gene-cloned as a molecule expressed on the retina of the eye in 1997, but its function has not yet been clarified (Non-Patent
後述の実施例に示す通り、本発明者らの検討の結果、ISLRが間葉系幹細胞に特異的なマーカー分子であることが明らかとなった。ISLRは未分化な状態の間葉系幹細胞に特異性が高く、これまでに報告された間葉系幹細胞のマーカーとは一線を画する。また、ヒトに限らずマウスにおいてもISLRが間葉系幹細胞のマーカーになることが判明し、その利用価値は高い。 As shown in the examples described later, as a result of the studies by the present inventors, it was clarified that ISLR is a marker molecule specific to mesenchymal stem cells. ISLR is highly specific for undifferentiated mesenchymal stem cells, which sets it apart from previously reported markers for mesenchymal stem cells. In addition, ISLR has been found to be a marker for mesenchymal stem cells not only in humans but also in mice, and its utility value is high.
ところで、間葉系幹細胞は腫瘍又は癌の発生部位や転移部位に集積する特性を有するとの学説がある。しかしながら、集積することの意義、集積した細胞の役割（特に癌の進展への関与）については諸説あり、その解明が切望されている。本発明者らは、間葉系幹細胞を特異的に検出するISLRというツールを得たことにより、間葉系幹細胞の集積を視覚的に捉え、また間葉系幹細胞の存在量を検出することができるようになった。この成果を基に更に研究を進めた結果、ISLRの発現が癌の進展及び癌患者の予後に関連することが見出された。また、心筋梗塞のモデル動物を用いた実験によって心筋梗塞の梗塞部位に間葉系幹細胞が集積する現象が観察されるとともに、ISLRノックアウトマウスでは心筋梗塞後の予後が有意に悪いことが示され、心筋梗塞等の線維化疾患の進展や予後の評価ないし推定にもISLRが有用であることが判明した。
以下の発明は、主として以上の成果に基づく。
［１］ロイシンリッチリピート含有免疫グロブリンスーパーファミリー（ISLR）からなる、未分化間葉系幹細胞マーカー。
［２］ISLRが、配列番号１〜３のいずれかのアミノ酸配列を含む、［１］に記載の未分化間葉系幹細胞マーカー。
［３］間葉系幹細胞を含む細胞集団から、ISLRを発現する細胞を選別し、回収するステップを含む、未分化間葉系幹細胞を調製する方法。
［４］間葉系幹細胞を含む細胞集団が、骨髄、歯髄、脂肪組織、子宮内膜、臍帯、骨格筋又は末梢血に由来する、［３］に記載の方法。
［５］ISLRの発現状態を指標として検出することを特徴とする、未分化間葉系幹細胞を検出する方法。
［６］被検間葉系幹細胞におけるISLRの発現状態を調べるステップを含む、間葉系幹細胞の未分化性を評価する方法。
［７］間葉系幹細胞がヒト細胞である、［３］〜［６］のいずれか一項に記載の方法。
［８］抗ISLR抗体を含む、未分化間葉系幹細胞検出用試薬。
［９］［８］に記載の試薬を含む、未分化間葉系幹細胞検出用キット。
［１０］ISLRの発現状態を指標として検出することを特徴とする、癌又は線維化疾患の罹患部位に集積する未分化間葉系幹細胞を検出する方法。
［１１］癌が膵癌、大腸癌、又は乳癌であり、線維化疾患が心筋梗塞である、［１０］に記載の方法。
［１２］ISLRからなる、癌又は線維化疾患の予後推定用バイオマーカー。
［１３］癌が膵癌、大腸癌、又は乳癌であり、線維化疾患が心筋梗塞である、［１２］に記載の予後推定用バイオマーカー。
［１４］ISLRの発現量を指標とした、癌又は線維化疾患患者の予後推定法。
［１５］癌が膵癌、大腸癌、又は乳癌であり、線維化疾患が心筋梗塞である、［１４］に記載の予後推定法。
［１６］抗ISLR抗体を含む、癌又は線維化疾患患者の予後推定用試薬。
［１７］癌が膵癌、大腸癌、又は乳癌であり、線維化疾患が心筋梗塞である、［１６］に記載の予後推定用試薬。
［１８］［１６］又は［１７］に記載の試薬を含む、癌又は線維化疾患患者の予後推定用キット。By the way, there is a theory that mesenchymal stem cells have the property of accumulating at the site of tumor or cancer development or metastasis. However, there are various theories about the significance of accumulation and the role of accumulated cells (particularly their involvement in the development of cancer), and their elucidation is eagerly desired. By obtaining a tool called ISLR that specifically detects mesenchymal stem cells, the present inventors can visually capture the accumulation of mesenchymal stem cells and detect the abundance of mesenchymal stem cells. I can now do it. As a result of further research based on this result, it was found that the expression of ISLR is associated with the progression of cancer and the prognosis of cancer patients. In addition, experiments using a model animal for myocardial infarction observed the accumulation of mesenchymal stem cells at the infarcted site of myocardial infarction, and showed that the prognosis after myocardial infarction was significantly worse in ISLR knockout mice. It was found that ISLR is also useful for evaluating or estimating the progression and prognosis of fibrotic diseases such as myocardial infarction.
The following inventions are mainly based on the above results.
[1] An undifferentiated mesenchymal stem cell marker consisting of a leucine-rich repeat-containing immunoglobulin superfamily (ISLR).
[2] The undifferentiated mesenchymal stem cell marker according to [1], wherein ISLR comprises any of the amino acid sequences of SEQ ID NOs: 1 to 3.
[3] A method for preparing undifferentiated mesenchymal stem cells, which comprises a step of selecting and recovering cells expressing ISLR from a cell population containing mesenchymal stem cells.
[4] The method according to [3], wherein the cell population containing mesenchymal stem cells is derived from bone marrow, dental pulp, adipose tissue, endometrium, umbilical cord, skeletal muscle or peripheral blood.
[5] A method for detecting undifferentiated mesenchymal stem cells, which comprises detecting the expression state of ISLR as an index.
[6] A method for evaluating the undifferentiated state of mesenchymal stem cells, which comprises a step of examining the expression state of ISLR in the test mesenchymal stem cells.
[7] The method according to any one of [3] to [6], wherein the mesenchymal stem cells are human cells.
[8] A reagent for detecting undifferentiated mesenchymal stem cells, which comprises an anti-ISLR antibody.
[9] A kit for detecting undifferentiated mesenchymal stem cells, which comprises the reagent according to [8].
[10] A method for detecting undifferentiated mesenchymal stem cells accumulating at an affected site of cancer or fibrotic disease, which comprises detecting the expression state of ISLR as an index.
[11] The method according to [10], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
[12] A biomarker for estimating the prognosis of cancer or fibrotic disease, which comprises ISLR.
[13] The biomarker for prognosis estimation according to [12], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
[14] A method for estimating the prognosis of a patient with cancer or fibrotic disease using the expression level of ISLR as an index.
[15] The prognosis estimation method according to [14], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
[16] A reagent for estimating prognosis of a patient with cancer or fibrotic disease, which comprises an anti-ISLR antibody.
[17] The reagent for estimating prognosis according to [16], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
[18] A kit for estimating the prognosis of a patient with cancer or fibrotic disease, which comprises the reagent according to [16] or [17].
１．未分化間葉系幹細胞マーカー
本発明の第１の局面は未分化間葉系幹細胞に特異的発現を認めたマーカー分子、即ち「未分化間葉系幹細胞マーカー」に関する。「未分化間葉系幹細胞マーカー」とは、未分化性を保持している間葉系幹細胞であることの指標となる分子をいう。本発明の未分化間葉系幹細胞マーカー（以下、略して「本発明のマーカー」と呼ぶこともある）を利用すれば、未分化間葉系幹細胞の検出、測定、標識化、調製（分取・濃縮）、評価等が可能になる。特に、間葉系幹細胞を含む細胞集団から未分化間葉系幹細胞を調製する上で本発明のマーカーはその利用価値が高い。「未分化間葉系幹細胞」は、骨芽細胞、軟骨芽細胞、脂肪細胞への分化能を有する。未分化間葉系幹細胞を特定の誘導条件で培養すれば、誘導条件に対応した細胞系譜に沿って分化する。1. 1. Undifferentiated Mesenchymal Stem Cell Marker The first aspect of the present invention relates to a marker molecule whose specific expression is observed in undifferentiated mesenchymal stem cell, that is, "undifferentiated mesenchymal stem cell marker". The "undifferentiated mesenchymal stem cell marker" refers to a molecule that is an index of being an undifferentiated mesenchymal stem cell. By utilizing the undifferentiated mesenchymal stem cell marker of the present invention (hereinafter, may be abbreviated as "marker of the present invention"), detection, measurement, labeling, and preparation (preparation) of undifferentiated mesenchymal stem cells・ Concentration), evaluation, etc. are possible. In particular, the marker of the present invention has high utility value in preparing undifferentiated mesenchymal stem cells from a cell population containing mesenchymal stem cells. "Undifferentiated mesenchymal stem cells" have the ability to differentiate into osteoblasts, chondroblasts, and adipocytes. When undifferentiated mesenchymal stem cells are cultured under specific induction conditions, they differentiate according to the cell lineage corresponding to the induction conditions.
本発明のマーカーはロイシンリッチリピート含有免疫グロブリンスーパーファミリー（ISLR：immunoglobulin superfamily containing leucine-rich repeat）からなる。ISLRは細胞膜結合型又は分泌型の分子であり、肺癌、乳癌、膵癌などにおいて高発現が認められている（例えば、特許文献２、非特許文献９、１０を参照）。
The marker of the present invention consists of an immunoglobulin superfamily containing leucine-rich repeat (ISLR). ISLR is a cell membrane-bound or secretory molecule and is highly expressed in lung cancer, breast cancer, pancreatic cancer and the like (see, for example,
公共のデータベースに登録されているISLRのアミノ酸配列とそれをコードする遺伝子配列を添付の配列表に示す。配列番号と配列の対応関係は以下の通りである。
配列番号１：ヒトISLRのアミノ酸配列（NCBI Reference Sequence: NP_005536.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Homo sapiens].）
配列番号２：マウスISLRのアミノ酸配列（NCBI Reference Sequence: NP_036173.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Mus musculus].）
配列番号３：ラットISLRのアミノ酸配列（NCBI Reference Sequence: NP_001119772.1 immunoglobulin superfamily containing leucine-rich repeat protein precursor [Rattus norvegicus]）
配列番号４：ヒトISLRのcDNA配列（GeneID:3671、NCBI Reference Sequence: NM_005545.3, Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR), transcript variant 1, mRNA.）
配列番号５：マウスISLRのcDNA配列（GeneID: 26968、NCBI Reference Sequence: NM_012043.4, Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr), transcript variant 1, mRNA.）
配列番号６：ラットISLRのcDNA配列（GeneID: 686539、NCBI Reference Sequence: XM_006243176.2, NM_001126300.1, Rattus norvegicus immunoglobulin superfamily containing leucine-rich repeat (Islr), mRNA）The amino acid sequence of ISLR registered in the public database and the gene sequence encoding it are shown in the attached sequence listing. The correspondence between the sequence numbers and the sequences is as follows.
SEQ ID NO: 1: Human ISLR amino acid sequence (NCBI Reference Sequence: NP_005536.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Homo sapiens].)
SEQ ID NO: 2: Mouse ISLR amino acid sequence (NCBI Reference Sequence: NP_036173.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Mus musculus].)
SEQ ID NO: 3: Rat ISLR amino acid sequence (NCBI Reference Sequence: NP_001119772.1 immunoglobulin superfamily containing leucine-rich repeat protein precursor [Rattus norvegicus])
SEQ ID NO: 4: Human ISLR cDNA sequence (GeneID: 3671, NCBI Reference Sequence: NM_005545.3, Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR),
SEQ ID NO: 5: Mouse ISLR cDNA sequence (GeneID: 26968, NCBI Reference Sequence: NM_012043.4, Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr),
SEQ ID NO: 6: cDNA sequence of rat ISLR (GeneID: 686539, NCBI Reference Sequence: XM_006243176.2, NM_001126300.1, Rattus norvegicus immunoglobulin superfamily containing leucine-rich repeat (Islr), mRNA)
ヒトISLRにはバリアント（variant 2）の配列（Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR), transcript variant 2, mRNA、アミノ酸配列：NP_958934.1、cDNA配列：NM_201526.1）が知られている。当該バリアントのアミノ酸配列は上記のアミノ酸配列（配列番号１）と同一である。
A variant (variant 2) sequence (Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR),
マウスについてもバリアント（variant 2）の配列（Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr), transcript variant 2, mRNA、アミノ酸配列：NP_001182360.1、cDNA配列：NM_001195431.1）が知られている。当該バリアントのアミノ酸配列は上記のアミノ酸配列（配列番号２）と同一である。
A variant (variant 2) sequence (Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr),
尚、ヒトへの適用の場合、ヒト生体中に存在する未分化間葉系幹細胞の指標としてではなく、ヒト生体から分離された状態の未分化間葉系幹細胞の指標として本発明のマーカーは利用される。 In the case of application to humans, the marker of the present invention is used not as an index of undifferentiated mesenchymal stem cells existing in the human body but as an index of undifferentiated mesenchymal stem cells isolated from the human body. Will be done.
２．未分化間葉系幹細胞の調製
本発明の第２の局面は本発明のマーカーの用途に関し、未分化間葉系幹細胞を調製する方法（以下、「本発明の調製法」と呼ぶ）を提供する。本発明の調製法を実施する前の細胞集団（即ち、本発明の調製法に供される細胞集団）と本発明の調製法によって得られる細胞集団を比較すると、前者よりも後者の方が未分化間葉系幹細胞の含有率が高いことから、「未分化間葉系幹細胞の調製」を、「未分化間葉系幹細胞の濃縮」、或いは「未分化間葉系幹細胞の純度（比率）の向上」と言い換えることも可能である。2. 2. Preparation of Undifferentiated Mesenchymal Stem Cells The second aspect of the present invention provides a method for preparing undifferentiated mesenchymal stem cells (hereinafter referred to as "preparation method of the present invention") with respect to the use of the marker of the present invention. .. Comparing the cell population before carrying out the preparation method of the present invention (that is, the cell population used in the preparation method of the present invention) with the cell population obtained by the preparation method of the present invention, the latter is not more than the former. Since the content of differentiated mesenchymal stem cells is high, "preparation of undifferentiated mesenchymal stem cells" can be referred to as "concentration of undifferentiated mesenchymal stem cells" or "purity (ratio) of undifferentiated mesenchymal stem cells". It can be rephrased as "improvement".
未分化間葉系幹細胞はそれ自体が有用であり、例えば再生医療用の移植材料としての利用が期待される。また、それを分化誘導して得られる各種細胞（骨芽細胞、軟骨芽細胞等）も特定の組織の再建に利用され得る。更には、間葉系幹細胞の産生するケモカイン、サイトカイン、増殖因子なども、様々な疾患の治療への適用が期待される。一方、未分化間葉系幹細胞は造血幹細胞の維持に重要であることが知られており、造血幹細胞を生体外で維持する場合の支持細胞としても未分化間葉系幹細胞は利用され得る。従って、未分化間葉系幹細胞は骨髄移植の分野でも有用である。本発明の調製法によれば、特異性の高いマーカーを用いることから、このように極めて有用性の高い未分化間葉系幹細胞を高純度で調製することができる。また、効率的な調製も可能になる。 Undifferentiated mesenchymal stem cells are useful by themselves, and are expected to be used as transplant materials for regenerative medicine, for example. In addition, various cells (osteoblasts, chondroblasts, etc.) obtained by inducing differentiation thereof can also be used for reconstruction of specific tissues. Furthermore, chemokines, cytokines, growth factors, etc. produced by mesenchymal stem cells are also expected to be applied to the treatment of various diseases. On the other hand, undifferentiated mesenchymal stem cells are known to be important for the maintenance of hematopoietic stem cells, and undifferentiated mesenchymal stem cells can also be used as supporting cells when the hematopoietic stem cells are maintained in vitro. Therefore, undifferentiated mesenchymal stem cells are also useful in the field of bone marrow transplantation. According to the preparation method of the present invention, since a marker having high specificity is used, undifferentiated mesenchymal stem cells having such extremely high usefulness can be prepared with high purity. It also enables efficient preparation.
本発明の調製法は上記本発明のマーカーを利用する。具体的には、本発明の調製法では以下のステップ、即ち、「間葉系幹細胞を含む細胞集団から、ISLRを発現する細胞を選別し、回収するステップ」、を行う。以下、当該ステップの詳細を説明する。 The preparation method of the present invention utilizes the above-mentioned marker of the present invention. Specifically, in the preparation method of the present invention, the following steps, that is, "a step of selecting and recovering cells expressing ISLR from a cell population containing mesenchymal stem cells" are performed. The details of the step will be described below.
間葉系幹細胞を含む細胞集団は予め用意しておく。当該細胞集団はヒト又は非ヒト動物（例えばマウス、ラット）の骨髄、歯髄、脂肪組織、子宮内膜、臍帯、臍帯血、骨格筋、末梢血等から常法で取得することができる。通常、これらの細胞源（細胞ソース、由来）から採取した試料（例えば、骨髄液、吸引脂肪、骨格筋組織片、血液）に対して物理的処理（裁断、ピペッティング、フィルタ処理等）や酵素処理（例えば、トリプシン、ディスパーゼ、コラゲナーゼ、エラスターゼ、パパイン等を使用する）を単独又は併用して行い、細胞を分離する。好ましくは、細胞塊がない状態にしておく。末梢血などから細胞集団を用意する場合には溶血処理などによって血球成分を除去しておくことが好ましい。 A cell population containing mesenchymal stem cells is prepared in advance. The cell population can be obtained by a conventional method from bone marrow, dental pulp, adipose tissue, endometrium, umbilical cord, umbilical cord blood, skeletal muscle, peripheral blood and the like of human or non-human animals (for example, mouse, rat). Usually, samples (eg, bone marrow fluid, aspirated fat, skeletal muscle tissue fragments, blood) collected from these cell sources (cell sources, origins) are physically treated (cut, pipetting, filtering, etc.) and enzymes. Treatment (eg, trypsin, dispase, collagenase, elastase, papain, etc.) is performed alone or in combination to isolate cells. Preferably, the cell mass is left free. When a cell population is prepared from peripheral blood or the like, it is preferable to remove the blood cell component by hemolysis treatment or the like.
上記細胞源から採取した試料又はそこから分離した細胞を培養容器に播種して培養し、接着性を示した細胞を「間葉系幹細胞を含む細胞集団」として用いることにしてもよい。換言すれば、本発明の一態様では、接着性細胞を選択するという、従来の間葉系幹細胞の調製法と同様の方法で得られる細胞集団を「間葉系幹細胞を含む細胞集団」として用いる。 A sample collected from the above cell source or cells separated from the sample may be seeded in a culture vessel and cultured, and the cells exhibiting adhesiveness may be used as a "cell population containing mesenchymal stem cells". In other words, in one aspect of the present invention, a cell population obtained by a method similar to the conventional method for preparing mesenchymal stem cells, which is to select adhesive cells, is used as a "cell population containing mesenchymal stem cells". ..
本発明の調製法では、間葉系幹細胞を含む細胞集団から、ISLRを発現する細胞を選別し、回収する。即ち、ISLRの発現を指標として、目的の細胞である未分化間葉系幹細胞を得る。ISLRを指標とした選別及び回収は、例えば、抗ISLR抗体を利用したフローサイトメトリー及びセルソーティングで行うことができる。フローサイトメトリー及びセルソーティングによれば特異的且つ効率的にISLR陽性細胞を分取することが可能である。フローサイトメトリー及びセルソーティングを実施するための装置（フローサイトメーター及びセルソーター）は例えばベックマン・コールター株式会社、日本ベクトン・ディッキンソン株式会社などから販売されており、それらを利用することができる。基本的な操作法、分取条件などは装置に添付の取扱説明書に従えばよい。 In the preparation method of the present invention, cells expressing ISLR are selected and recovered from a cell population containing mesenchymal stem cells. That is, using the expression of ISLR as an index, undifferentiated mesenchymal stem cells, which are the target cells, are obtained. Selection and recovery using ISLR as an index can be performed, for example, by flow cytometry and cell sorting using an anti-ISLR antibody. According to flow cytometry and cell sorting, ISLR-positive cells can be sorted specifically and efficiently. Devices for performing flow cytometry and cell sorting (flow cytometer and cell sorter) are sold by, for example, Beckman Coulter Co., Ltd., Becton Dickinson Co., Ltd., Japan, etc., and they can be used. The basic operation method, sorting conditions, etc. may be as described in the instruction manual attached to the device.
フローサイトメトリー以外にも、抗ISLR抗体を用いたアフィニティークロマトグラフィーあるいは磁気ビーズを用いた磁気細胞分離等によって、ISLR陽性細胞を選別、回収することが可能である。 In addition to flow cytometry, ISLR-positive cells can be selected and recovered by affinity chromatography using an anti-ISLR antibody, magnetic cell separation using magnetic beads, or the like.
フローサイトメトリー、セルソーティング、或いは磁気細胞分離等に用いる抗ISLR抗体は、ISLR陽性細胞の選別及び回収に利用可能な限り、ポリクローナル抗体、オリゴクローナル抗体（数種〜数十種の抗体の混合物）、及びモノクローナル抗体のいずれでもよい。ポリクローナル抗体又はオリゴクローナル抗体としては、動物免疫して得た抗血清由来のIgG画分のほか、抗原によるアフィニティー精製抗体を使用できる。抗ISLR抗体は免疫学的手法、ファージディスプレイ法、リボソームディスプレイ法などを利用して調製することができる。免疫学的手法によるポリクローナル抗体の調製は次の手順で行うことができる。抗原（ISLR又はその一部）を調製し、これを用いてウサギ等の動物に免疫を施す。生体試料を精製することにより抗原を得ることができる。また、組換え型抗原を用いることもできる。組換え型ISLRは、例えば、ISLRをコードする遺伝子（遺伝子の一部であってもよい）を、ベクターを用いて適当な宿主に導入し、得られた組換え細胞内で発現させることにより調製することができる。 Anti-ISLR antibodies used for flow cytometry, cell sorting, magnetic cell isolation, etc. are polyclonal antibodies and oligoclonal antibodies (mixtures of several to dozens of antibodies) as long as they can be used for selection and recovery of ISLR-positive cells. , And a monoclonal antibody. As the polyclonal antibody or oligoclonal antibody, in addition to the IgG fraction derived from antiserum obtained by animal immunization, an affinity purification antibody using an antigen can be used. The anti-ISLR antibody can be prepared by utilizing an immunological method, a phage display method, a ribosome display method, or the like. Preparation of polyclonal antibody by immunological method can be carried out by the following procedure. An antigen (ISLR or a part thereof) is prepared and used to immunize an animal such as a rabbit. Antigens can be obtained by purifying biological samples. Alternatively, a recombinant antigen can be used. Recombinant ISLR is prepared, for example, by introducing a gene encoding ISLR (which may be a part of the gene) into an appropriate host using a vector and expressing it in the obtained recombinant cell. can do.
免疫惹起作用を増強するために、キャリアタンパク質を結合させた抗原を用いてもよい。キャリアタンパク質としてはKLH（Keyhole Limpet Hemocyanin）、BSA（Bovine Serum Albumin）、OVA（Ovalbumin）などが使用される。キャリアタンパク質の結合にはカルボジイミド法、グルタルアルデヒド法、ジアゾ縮合法、MBS（マレイミドベンゾイルオキシコハク酸イミド）法などを使用できる。一方、ISLR（又はその一部）を、GST、βガラクトシダーゼ、マルトース結合タンパク、又はヒスチジン（His）タグ等との融合タンパク質として発現させた抗原を用いることもできる。このような融合タンパク質は、汎用的な方法により簡便に精製することができる。 An antigen to which a carrier protein is bound may be used to enhance the immunostimulatory effect. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. A carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimide benzoyloxysuccinimide) method, or the like can be used for binding the carrier protein. On the other hand, an antigen expressing ISLR (or a part thereof) as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general-purpose method.
必要に応じて免疫を繰り返し、十分に抗体価が上昇した時点で採血し、遠心処理などによって血清を得る。得られた抗血清をアフィニティー精製し、ポリクローナル抗体とする。 Immunization is repeated as necessary, and when the antibody titer rises sufficiently, blood is collected and serum is obtained by centrifugation or the like. The obtained antiserum is affinity-purified to obtain a polyclonal antibody.
一方、モノクローナル抗体については次の手順で調製することができる。まず、上記と同様の手順で免疫操作を実施する。必要に応じて免疫を繰り返し、十分に抗体価が上昇した時点で免疫動物から抗体産生細胞を摘出する。次に、得られた抗体産生細胞と骨髄腫細胞とを融合してハイブリドーマを得る。続いて、このハイブリドーマをモノクローナル化した後、抗原に対して高い特異性を有する抗体を産生するクローンを選択する。選択されたクローンの培養液を精製することによって目的の抗体が得られる。一方、ハイブリドーマを所望数以上に増殖させた後、これを動物（例えばマウス）の腹腔内に移植し、腹水内で増殖させて腹水を精製することにより目的の抗体を取得することもできる。上記培養液の精製又は腹水の精製には、プロテインG、プロテインA等を用いたアフィニティークロマトグラフィーが好適に用いられる。また、抗原を固相化したアフィニティークロマトグラフィーを用いることもできる。更には、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、硫安分画、及び遠心分離等の方法を用いることもできる。これらの方法は単独ないし任意に組み合わされて用いられる。 On the other hand, the monoclonal antibody can be prepared by the following procedure. First, the immune operation is performed in the same procedure as described above. Immunization is repeated as necessary, and when the antibody titer rises sufficiently, antibody-producing cells are removed from the immunized animal. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after monoclonalizing this hybridoma, a clone that produces an antibody having high specificity for the antigen is selected. The antibody of interest is obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma in a desired number or more, transplanting it into the abdominal cavity of an animal (for example, a mouse), and growing it in the ascites to purify the ascites. Affinity chromatography using protein G, protein A, or the like is preferably used for purification of the culture solution or ascites. Affinity chromatography on which an antigen is immobilized can also be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods are used alone or in any combination.
本発明の調製法は未分化間葉系幹細胞に特異性の高いマーカーを利用することから、単独でも未分化間葉系幹細胞を特異的且つ効率的に調製することを可能にする。しかしながら、未分化間葉系幹細胞の選別に利用可能な他のマーカー分子（例えば、間葉系幹細胞のマーカーとして知られているCD105、CD73、CD90、CD45、CD34、CD14、CD11b、CD79a、CD19、HLA-DR等）の併用を排除或いは制限するものではない。即ち、純度ないし均一性の更なる向上、特定の細胞集団（例えば特定のマーカー分子の発現によって特徴付けられるもの）の濃縮等の目的の下、本発明の方法を構成するステップ（ISLRを利用したステップ）に加え、他のマーカー分子による選別及び回収のステップを行うことにしてもよい。当該ステップは、ISLRを利用した選別及び回収ステップの前又は後に実施される。 Since the preparation method of the present invention utilizes a marker having high specificity for undifferentiated mesenchymal stem cells, it is possible to specifically and efficiently prepare undifferentiated mesenchymal stem cells alone. However, other marker molecules available for selection of undifferentiated mesenchymal stem cells (eg, CD105, CD73, CD90, CD45, CD34, CD14, CD11b, CD79a, CD19, known as markers for mesenchymal stem cells, It does not exclude or limit the combined use of HLA-DR, etc.). That is, the steps (using ISLR) that constitute the method of the present invention were used for the purpose of further improving purity or homogeneity, enriching a specific cell population (for example, those characterized by the expression of a specific marker molecule), and the like. In addition to step), the steps of sorting and recovery by other marker molecules may be performed. The step is performed before or after the ISLR-based sorting and recovery step.
３．未分化間葉系幹細胞の検出
本発明の第３の局面は未分化間葉系幹細胞の検出に関し、未分化間葉系幹細胞を検出する方法（以下、「本発明の検出法」と呼ぶ）、当該方法に用いられる試薬及びキットが提供される。本発明の検出法は、ISLRの発現状態を指標として未分化間葉系幹細胞を検出する点に最大の特徴を有する。本発明の検出法によれば、試料中の未分化間葉系幹細胞を特異的且つ高感度に検出できる。従って、未分化間葉系幹細胞の同定、可視化、抽出等を可能にする実験手段（リサーチツール）として本発明の検出法は有用である。3. 3. Detection of Undifferentiated Mesenchymal Stem Cells The third aspect of the present invention relates to the detection of undifferentiated mesenchymal stem cells, which is a method for detecting undifferentiated mesenchymal stem cells (hereinafter referred to as "the detection method of the present invention"). Reagents and kits used in the method are provided. The detection method of the present invention has the greatest feature in that undifferentiated mesenchymal stem cells are detected using the expression state of ISLR as an index. According to the detection method of the present invention, undifferentiated mesenchymal stem cells in a sample can be detected with specificity and high sensitivity. Therefore, the detection method of the present invention is useful as an experimental means (research tool) that enables identification, visualization, extraction, etc. of undifferentiated mesenchymal stem cells.
未分化間葉系幹細胞を含む試料に対して本発明の検出法が適用される。未分化間葉系幹細胞を含む可能性がある限り、試料は特に限定されない。例えば、生体から採取ないし分離した組織片や細胞集団、或いはそれらから抽出した細胞集団などを試料として用いることができる。 The detection method of the present invention is applied to a sample containing undifferentiated mesenchymal stem cells. The sample is not particularly limited as long as it may contain undifferentiated mesenchymal stem cells. For example, a tissue piece or cell population collected or separated from a living body, or a cell population extracted from them can be used as a sample.
本発明の検出法は典型的にはin vitroで実施されるが、非ヒト動物の試料を用いる場合や、非ヒト動物に移植したヒト試料中のISLRの発現を検出する場合には、in vivoで実施することもできる。 The detection method of the present invention is typically carried out in vitro, but when using a sample of a non-human animal or when detecting the expression of ISLR in a human sample transplanted into a non-human animal, in vivo. It can also be carried out at.
「ISLRの発現状態」を指標とした検出ではISLR mRNA又はISLRタンパク質が検出対象となる。即ち、本発明の検出法ではISLR mRNA又はISLRタンパク質の発現を検出する。本発明において「発現状態」とは発現の程度（レベル）を意味し、発現の有無と発現量を包括した用語として使用される。従って、本発明の検出法ではISLR mRNA又はISLRタンパク質の発現に関して定性的又は定量的な検出が行われることになる。 In the detection using "ISLR expression status" as an index, ISLR mRNA or ISLR protein is the detection target. That is, the detection method of the present invention detects the expression of ISLR mRNA or ISLR protein. In the present invention, the "expression state" means the degree (level) of expression, and is used as a term that includes the presence or absence of expression and the amount of expression. Therefore, the detection method of the present invention results in qualitative or quantitative detection of the expression of ISLR mRNA or ISLR protein.
ISLR mRNAの発現の検出は、ISLR mRNAに特異的なプライマー又はプローブを利用した各種方法、例えばRT-PCR、定量PCR、in situハイブリダイゼーション、ノーザンブロッティング等によって実施することができる。他方、ISLRタンパク質の発現の検出には、ISLRタンパク質に対して特異的な結合性を示す物質が用いられる。当該物質として、好ましくは抗ISLR抗体を採用する。抗ISLR抗体によれば特異性の高い検出が可能になる。標識化抗体を使用すれば、標識量を指標に結合抗体量を直接検出することが可能である。従って、より簡便な検出法を構築できる。その反面、標識物質を結合させた抗ISLR抗体を用意する必要があることに加えて、検出感度が一般に低くなるという問題点がある。そこで、標識物質を結合させた二次抗体を利用する方法、二次抗体と標識物質を結合させたポリマーを利用する方法など、間接的検出方法を利用することが好ましい。ここでの二次抗体とは、抗ISLR抗体に特異的結合性を有する抗体である。例えばウサギ抗体として抗ISLR抗体を調製した場合には抗ウサギIgG抗体を使用できる。ウサギやヤギ、マウスなど様々な種の抗体に対して使用可能な標識二次抗体が市販されており（例えばフナコシ株式会社やコスモ・バイオ株式会社など）、本発明の検出法の構成に応じて適切なものを適宜選択して使用することができる。 The expression of ISLR mRNA can be detected by various methods using primers or probes specific for ISLR mRNA, for example, RT-PCR, quantitative PCR, in situ hybridization, Northern blotting and the like. On the other hand, a substance showing specific binding property to the ISLR protein is used to detect the expression of the ISLR protein. As the substance, an anti-ISLR antibody is preferably adopted. The anti-ISLR antibody enables highly specific detection. If a labeled antibody is used, it is possible to directly detect the amount of bound antibody using the labeled amount as an index. Therefore, a simpler detection method can be constructed. On the other hand, in addition to the need to prepare an anti-ISLR antibody to which a labeling substance is bound, there is a problem that the detection sensitivity is generally low. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer in which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having specific binding property to an anti-ISLR antibody. For example, when an anti-ISLR antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used. Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd. and Cosmo Bio Co., Ltd.), depending on the configuration of the detection method of the present invention. Appropriate ones can be appropriately selected and used.
上記の説明から明らかなように、抗ISLR抗体は未分化間葉系幹細胞の検出に有用である。そこで本発明は、抗ISLR抗体を含む未分化間葉系幹細胞検出用試薬も提供する。一態様では抗ISLR抗体は標識化されている。標識化に用いる標識物質としては例えば、フルオレセイン、ローダミン、テキサスレッド、オレゴングリーン等の蛍光色素、ホースラディッシュペルオキシダーゼ、マイクロペルオキシダーゼ、アルカリ性ホスファターゼ、β−Ｄ−ガラクトシダーゼ等の酵素、ルミノール、アクリジン色素等の化学又は生物発光化合物、32P、131I、125I等の放射性同位体、及びビオチンを挙げることができる。As is clear from the above description, anti-ISLR antibodies are useful for the detection of undifferentiated mesenchymal stem cells. Therefore, the present invention also provides a reagent for detecting undifferentiated mesenchymal stem cells containing an anti-ISLR antibody. In one aspect, the anti-ISLR antibody is labeled. Examples of labeling substances used for labeling include fluorescent dyes such as fluorescein, rhodamine, Texas red, and olegon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, and β-D-galactosidase, and chemistry such as luminol and acrydin dye. Alternatively, bioluminescent compounds, radioisotopes such as 32 P, 131 I, 125 I, and biotin can be mentioned.
本発明は更に、本発明の試薬を構成要素として含む、未分化間葉系幹細胞検出用キットも提供する。当該キットを用いることにより、本発明の検出法をより簡便に実施することができる。本発明の検出法を実施する際に使用するその他の試薬（緩衝液、反応用試薬、酵素、酵素の基質など）及び／又は装置ないし器具（容器、反応装置、蛍光リーダーなど）をキットに含めてもよい。尚、通常、本発明のキットには取り扱い説明書が添付される。 The present invention also provides a kit for detecting undifferentiated mesenchymal stem cells, which comprises the reagent of the present invention as a component. By using the kit, the detection method of the present invention can be carried out more easily. The kit includes other reagents (buffer solution, reaction reagent, enzyme, enzyme substrate, etc.) and / or device or instrument (container, reaction device, fluorescent reader, etc.) used in carrying out the detection method of the present invention. You may. An instruction manual is usually attached to the kit of the present invention.
４．間葉系幹細胞の未分化性評価
上記の通り、ISLRは未分化間葉系幹細胞のマーカーとして有用であり、ISLRの発現状態は未分化性の指標となる。そこで本発明の更なる局面は、ISLRの発現状態を指標として判定することを特徴とする、間葉系幹細胞の未分化性を評価する方法を提供する。本発明の評価法では、試料（被検間葉系幹細胞）についてISLRの発現状態を調べ、その結果に基づき未分化性を維持しているか否か、或いは未分化性の程度を判定する。ISLRの発現状態を調べるため、ISLR mRNA又はISLRタンパク質が検出される。本発明の評価法では、例えば、ISLR mRNA又はISLRタンパク質の発現を認める場合に未分化性を維持していると判定する。ISLR mRNA又はISLRタンパク質の発現量から未分化性のレベルを判定することにしてもよい。尚、ISLR mRNA又はISLRタンパク質の検出については上記（項目３．の欄）の説明が援用される。4. Evaluation of undifferentiated mesenchymal stem cells As described above, ISLR is useful as a marker for undifferentiated mesenchymal stem cells, and the expression state of ISLR is an index of undifferentiated state. Therefore, a further aspect of the present invention provides a method for evaluating the undifferentiated state of mesenchymal stem cells, which comprises determining the expression state of ISLR as an index. In the evaluation method of the present invention, the expression state of ISLR is examined in a sample (test mesenchymal stem cell), and based on the result, whether or not undifferentiated state is maintained or the degree of undifferentiated state is determined. ISLR mRNA or ISLR protein is detected to examine the expression status of ISLR. In the evaluation method of the present invention, for example, when the expression of ISLR mRNA or ISLR protein is observed, it is determined that the undifferentiated state is maintained. The level of undifferentiation may be determined from the expression level of ISLR mRNA or ISLR protein. Regarding the detection of ISLR mRNA or ISLR protein, the above explanation (column of item 3) is incorporated.
本発明の評価法を利用すると、未分化性を維持した間葉系幹細胞（即ち未分化間葉系幹細胞）を同定したり、選別したりすることが可能になる。即ち、間葉系幹細胞の同定、選別の手段として本発明の評価法は有用である。一方、間葉系幹細胞の未分化性は間葉系幹細胞を各種用途に適用する際の有効性、言い換えれば「品質」を表すことにもなる。従って、間葉系幹細胞の品質を評価又は担保するための手段としても本発明の評価法は有用である。 By using the evaluation method of the present invention, it becomes possible to identify and select mesenchymal stem cells (that is, undifferentiated mesenchymal stem cells) that maintain undifferentiated state. That is, the evaluation method of the present invention is useful as a means for identifying and selecting mesenchymal stem cells. On the other hand, the undifferentiated state of mesenchymal stem cells also indicates the effectiveness of applying mesenchymal stem cells to various uses, in other words, "quality". Therefore, the evaluation method of the present invention is also useful as a means for evaluating or guaranteeing the quality of mesenchymal stem cells.
５．癌又は線維化疾患の罹患部位に集積（浸潤）する間葉系幹細胞の検出と患者の予後推定（評価）
ISLRの発現が癌の進展及び癌患者の予後に関連するとの知見、及び心筋梗塞等の線維化疾患の進展や予後の評価ないし推定にもISLRが有用であるとの知見に基づき、本発明は更なる局面として、癌又は線維化疾患の罹患部位に集積する未分化間葉系幹細胞を検出する方法、並びに癌又は線維化疾患患者の予後推定法及びそれに利用される試薬・キットを提供する。5. Detection of mesenchymal stem cells accumulating (infiltrating) at the affected site of cancer or fibrotic disease and estimation of patient prognosis (evaluation)
Based on the finding that the expression of ISLR is related to the progression of cancer and the prognosis of cancer patients, and the finding that ISLR is also useful for evaluating or estimating the progression and prognosis of fibrotic diseases such as myocardial infarction, the present invention presents the present invention. As a further aspect, a method for detecting undifferentiated mesenchymal stem cells accumulating at an affected site of a cancer or fibrotic disease, a prognosis estimation method for a patient with a cancer or fibrotic disease, and a reagent / kit used therein are provided.
この局面の検出法では、試料についてISLRの発現状態を調べ、その結果に基づき罹患部位に間葉系幹細胞が集積しているか否かを判定する。検出結果は疾患の進行状態（例えば癌組織の分化度）の判定や患者の予後推定（予後推定についての詳細は後述する）に利用することができる。癌を対象にした場合、癌組織が罹患部位となる。他方、線維化疾患を対象にした場合の罹患部位は、炎症ないし線維化を生じている部位である。 In the detection method of this aspect, the expression state of ISLR is examined in the sample, and based on the result, it is determined whether or not mesenchymal stem cells are accumulated in the affected site. The detection result can be used for determining the progress state of the disease (for example, the degree of differentiation of cancer tissue) and estimating the prognosis of the patient (details of the prognosis estimation will be described later). When targeting cancer, the cancer tissue becomes the affected site. On the other hand, when targeting a fibrotic disease, the affected site is a site where inflammation or fibrosis is occurring.
典型的には、生体（患者）からバイオプシー（生検）によって或いは手術の際に分離された病理組織又は病理組織標本を試料として用いる、病理組織の抽出物や、患者由来の血清、エクソソーム等を試料として用いることにしてもよい。 Typically, a pathological tissue extract, a patient-derived serum, an exosome, etc., which uses a pathological tissue or a pathological tissue specimen isolated from a living body (patient) by biopsy (biopsy) or during surgery as a sample, are used. It may be used as a sample.
癌は特に限定されない。癌を例示すれば、膵癌、大腸癌、乳癌、肺癌、腎臓癌、前立腺癌、メラノーマである。線維化疾患も同様に限定されるものではなく、代表的なものを挙げると、心筋梗塞、肺線維症（間質性肺炎）、肝硬変、慢性腎症である。 Cancer is not particularly limited. Examples of cancers are pancreatic cancer, colon cancer, breast cancer, lung cancer, kidney cancer, prostate cancer, and melanoma. Fibrotic diseases are also not limited, and typical examples are myocardial infarction, pulmonary fibrosis (interstitial pneumonia), liver cirrhosis, and chronic nephropathy.
検出手段（ISLR mRNAやISLRタンパク質の検出）については上記（項目３．の欄）の説明が援用されるが、病理組織又は病理組織標本を試料とした場合に適用可能な検出法の具体例の一つとして、In situハイブリダイゼーション法を挙げることができる。 Regarding the detection means (detection of ISLR mRNA and ISLR protein), the above explanation (column 3) is used, but a specific example of a detection method applicable when a pathological tissue or a pathological tissue specimen is used as a sample. One example is the in situ hybridization method.
本発明は上記検出法の応用として、癌又は線維化疾患患者の予後推定法も提供する。本発明の予後推定法ではISLRをバイオマーカーとして用いる。即ち、本発明ではISLRを癌又は線維化疾患予後推定用マーカーとして患者の予後を推定する。本発明の予後推定法によれば、癌患者又は線維化疾患患者の予後推定に有益な情報が得られる。当該情報は例えば治療方針の決定（効果的な治療法の選択など）に利用される。判定結果を利用することによって治療成績の向上、予後改善、患者の生活の質（ＱＯＬ）の向上などがもたらされる。 The present invention also provides a prognosis estimation method for patients with cancer or fibrotic diseases as an application of the above detection method. ISLR is used as a biomarker in the prognosis estimation method of the present invention. That is, in the present invention, the prognosis of a patient is estimated using ISLR as a marker for estimating the prognosis of cancer or fibrotic disease. According to the prognosis estimation method of the present invention, useful information can be obtained for prognosis estimation of a cancer patient or a fibrotic disease patient. The information is used, for example, to determine a treatment policy (such as selecting an effective treatment method). By using the judgment result, the treatment result is improved, the prognosis is improved, and the quality of life (QOL) of the patient is improved.
本明細書において「予後推定用バイオマーカー」とは、患者の予後推定の指標となる生体分子のことをいう。本発明の予後推定法では、患者由来の試料中のISLR発現量を検出するステップ（検出ステップ）及び、検出結果に基づき予後を推定するステップ（予後推定ステップ）を実施する。検出ステップは上記検出法に準ずる。予後推定ステップでは、検出されたISLR（即ちISLR発現量）を指標として予後を推定する。基本的には、ISLR発現量が多いと予後が良好であるとの判断基準が採用される。以下、ISLR発現量に基づく評価の具体例を示す。まず、ISLR発現量と予後とが関連付けられた複数の評価区分を予め設定しておく。そして、検出ステップで得られたISLR発現量に基づき、該当する評価区分を決定する。評価区分の設定に関する具体例として、ISLRの発現の有無に注目した例（例１）と、ISLRの発現量の程度に注目した例（例２）、発現量の変化に注目した例（例３）を以下に示す。例３の場合には、通常、少なくとも２回（異なる時点）の検出を行うことになる。尚、区分名：当該区分に関連付けられるISLR発現量：当該区分に関連付けられる評価結果の順で記載する。
＜例１＞
区分１：ISLR陽性である：予後が良い
区分２：ISLR陰性である：予後が悪い
＜例２＞
区分１：ISLRの発現を認めず：予後が悪い
区分２：ISLRの弱い発現を認める：予後が比較的悪い
区分３：ISLRの中程度の発現を認める：予後が比較的良い
区分４：ISLRの強い発現を認める：予後が良い
＜例３＞
区分１：ISLR発現量の増大を認める：予後が良い
区分２：ISLR発現量の減少を認める：予後が悪いAs used herein, the term "biomarker for prognosis estimation" refers to a biomolecule that serves as an index for prognosis estimation of a patient. In the prognosis estimation method of the present invention, a step of detecting the ISLR expression level in a sample derived from a patient (detection step) and a step of estimating the prognosis based on the detection result (prognosis estimation step) are carried out. The detection step conforms to the above detection method. In the prognosis estimation step, the prognosis is estimated using the detected ISLR (that is, the ISLR expression level) as an index. Basically, the criteria for determining that the prognosis is good when the ISLR expression level is high are adopted. The following is a specific example of evaluation based on the ISLR expression level. First, a plurality of evaluation categories in which the ISLR expression level and the prognosis are associated are set in advance. Then, the corresponding evaluation category is determined based on the ISLR expression level obtained in the detection step. Specific examples of setting the evaluation category include an example focusing on the presence or absence of ISLR expression (Example 1), an example focusing on the degree of ISLR expression level (Example 2), and an example focusing on changes in the expression level (Example 3). ) Is shown below. In the case of Example 3, the detection is usually performed at least twice (at different time points). The category name: ISLR expression level associated with the category: the evaluation result associated with the category is described in this order.
<Example 1>
Category 1: ISLR positive: good prognosis Category 2: ISLR negative: poor prognosis <Example 2>
Category 1: No expression of ISLR: Poor prognosis Category 2: Weak expression of ISLR: Relatively poor prognosis Category 3: Moderate expression of ISLR: Relatively good prognosis Category 4: ISLR Strong expression: good prognosis <Example 3>
Category 1: Increase in ISLR expression level: Good prognosis Category 2: Decrease in ISLR expression level: Poor prognosis
評価区分の数、及び各評価区分に関連付けられるISLR発現量及び評価結果はいずれも上記の例に何らとらわれることなく、予備実験等を通して任意に設定することができる。尚、本発明における判定・評価は、医師や検査技師など専門知識を有する者の判断によらずとも自動的／機械的に行うことができる。 The number of evaluation categories, the ISLR expression level associated with each evaluation category, and the evaluation results can be arbitrarily set through preliminary experiments or the like without being bound by the above example. The determination / evaluation in the present invention can be performed automatically / mechanically without the judgment of a person having specialized knowledge such as a doctor or a laboratory technician.
本発明は更に、予後推定用試薬及び予後推定用キットも提供する。予後推定用試薬はISLRの検出を可能にするものであり、具体例の一つは抗ISLR抗体である。抗ISLR抗体については上記（項目３．の欄）の説明が援用される。予後推定用キットは予後推定用を必須の構成要素とするものであり、上記の「未分化間葉系幹細胞の検出」に使用されるキットに準ずる。 The present invention also provides a reagent for prognosis estimation and a kit for prognosis estimation. Prognosis estimation reagents enable the detection of ISLR, and one specific example is an anti-ISLR antibody. For anti-ISLR antibody, the above explanation (column 3) is incorporated. The prognosis estimation kit has an essential component for prognosis estimation, and is similar to the kit used for the above-mentioned "detection of undifferentiated mesenchymal stem cells".
１．ヒト骨髄由来間葉系幹細胞及び皮膚線維芽細胞におけるISLRの発現
（１）方法と結果（図１）
ヒト骨髄由来間葉系幹細胞（以下ヒト骨髄MSC）及び皮膚線維芽細胞（いずれもロンザジャパン株式会社より入手）から細胞抽出液を調製し、当該研究室によって開発された抗ISLR抗体を用いたウェスタンブロット法によってISLRの発現を調べた。抗ISLR抗体は以下の方法で調製した。まず、以下の抗原用ペプチド１〜３を合成した。
抗原用ペプチド１：ヒトISLRの229-251番目アミノ酸（CSAPSVQLSYQPSQDGAELRPGF：配列番号７）
抗原用ペプチド２：ヒトISLRの344-368番目アミノ酸（LATPGEGGEDTLGRRFHGKAVEGKG：配列番号８
抗原用ペプチド３：マウスISLRの341-359番目アミノ酸（NVALATPGEGGEDAVGHKF：配列番号９）
次に、抗原用ペプチドをキャリアタンパク質KLHと結合後、ウサギ（抗原用ペプチド１、３）又はモルモット（抗原用ペプチド２）に免疫した。免疫後に血清を回収し、抗原用ペプチドを共有結合させたアフィニティーカラムを用いて特異的抗体を精製した。このようにして３種類の抗ISLR抗体を得た。説明の便宜上、抗原用ペプチド１の免疫により得た抗体を抗ISLR抗体１、抗原用ペプチド２の免疫により得た抗体を抗ISLR抗体２、抗原用ペプチド３の免疫により得た抗体を抗ISLR抗体３とそれぞれ呼ぶ。1. 1. Expression of ISLR in human bone marrow-derived mesenchymal stem cells and cutaneous fibroblasts (1) Methods and results (Fig. 1)
Western blotting using anti-ISLR antibody developed by the laboratory prepared cell extracts from human bone marrow-derived mesenchymal stem cells (hereinafter referred to as human bone marrow MSC) and skin fibroblasts (both obtained from Lonza Japan Co., Ltd.) The expression of ISLR was examined by blotting. The anti-ISLR antibody was prepared by the following method. First, the following
Peptide for antigen 1: Amino acid 229-251 of human ISLR (CSAPSVQLSYQPSQDGAELRPGF: SEQ ID NO: 7)
Peptide for antigen 2: Amino acid 344-368 of human ISLR (LATPGEGGEDTLGRRFHGKAVEGKG: SEQ ID NO: 8)
Peptide for antigen 3: Amino acid 341-359 of mouse ISLR (NVALATPGEGGEDAVGHKF: SEQ ID NO: 9)
Next, after binding the antigen peptide to the carrier protein KLH, the rabbit (
抗ISLR抗体２を用いたウェスタンブロットの結果、約50kDaのサイズに確認されるバンドを認め、ヒト骨髄MSC及び線維芽細胞におけるISLRの発現が証明された。ヒト骨髄MSC及び線維芽細胞の培養上清（メディウム）中にもISLRが検出され、ISLRは分泌性因子であることも示された。RNA干渉法によってISLRの発現を抑制（ノックダウン）した線維芽細胞ではISLRのバンドを認めず、本実験で用いた抗体の特異性が示された。尚、PDGF受容体α及びβ−アクチンは内因性のコントロールとして用いている。
Western blotting using
（２）考察
ヒト骨髄MSC及び皮膚線維芽細胞におけるISLRの発現が確認された。各種臓器より単離される線維芽細胞はMSCと同様に骨、軟骨、脂肪への分化能を有することも報告されており、線維芽細胞と呼ばれる細胞の少なくとも一部はMSCとしての特徴を備えていることが報告されている。今回のウェスタンブロット法の結果はヒト骨髄MSCのみならず皮膚線維芽細胞に含まれるMSCにもISLRが発現している可能性を示唆するものである。(2) Discussion The expression of ISLR in human bone marrow MSCs and skin fibroblasts was confirmed. It has also been reported that fibroblasts isolated from various organs have the ability to differentiate into bone, cartilage, and fat like MSC, and at least some of the cells called fibroblasts have the characteristics of MSC. It is reported that there is. The results of this Western blotting suggest that ISLR may be expressed not only in human bone marrow MSCs but also in MSCs contained in skin fibroblasts.
２．ヒト脂肪由来幹細胞におけるISLRの発現
（１）方法と結果（図２）
脂肪組織には骨髄由来間葉系幹細胞と同等の特徴と機能を有する細胞が存在していることが知られ脂肪由来幹細胞（adipose tissue-derived stem cell、以下ADSC）あるいは単にMSCと呼ばれている。ヒトADSC（ロンザジャパン株式会社）から細胞抽出液を調製し、抗ISLR抗体２を用いたウェスタンブロット法によってISLRの発現を調べた。その結果、ヒトADSCにおけるISLRの発現が確認された。尚、GAPDHは内因性のコントロールとして用いている。2. 2. Expression of ISLR in human adipose-derived stem cells (1) Methods and results (Fig. 2)
It is known that adipose tissue contains cells having the same characteristics and functions as bone marrow-derived mesenchymal stem cells, and is called adipose tissue-derived stem cell (ADSC) or simply MSC. .. Cell extracts were prepared from human ADSC (Lonza Japan Co., Ltd.), and the expression of ISLR was examined by Western blotting using
３．マウス骨髄由来間葉系幹細胞におけるISLRの発現
（１）方法と結果（図３）
マウス骨髄由来間葉系幹細胞（Cyagen社）から細胞抽出液を調製し、抗ISLR抗体３を用いたウェスタンブロット法によってISLRの発現を調べた。その結果、マウス骨髄由来間葉系幹細胞にISLRが発現していることが確認された。また、同細胞を高密度で培養した際にはISLRの発現が上昇することも確認された。尚、β−チュブリンは内因性のコントロールとして用いている。3. 3. Expression of ISLR in mouse bone marrow-derived mesenchymal stem cells (1) Methods and results (Fig. 3)
Cell extracts were prepared from mouse bone marrow-derived mesenchymal stem cells (Cyagen), and ISLR expression was examined by Western blotting using anti-ISLR antibody 3. As a result, it was confirmed that ISLR was expressed in mouse bone marrow-derived mesenchymal stem cells. It was also confirmed that the expression of ISLR increased when the cells were cultured at high density. Β-tubulin is used as an endogenous control.
４．間葉系幹細胞の分化に伴うISLR発現の減少（マウス間葉系幹細胞株C3H10T1/2を用いた検証）
（１）方法と結果（図４）
マウス間葉系幹細胞の細胞株であるC3H10T1/2を用いて、脂肪、軟骨、及び骨への分化におけるISLRの発現の変化について検証した。各分化誘導メディウム（ロンザジャパン株式会社）を添加後、0日、1日、7日、及び14日のISLR及び各種マーカー分子の発現をウェスタンブロット法（ISLRの検出には抗ISLR抗体３を使用）で検証した。脂肪、軟骨、骨分化のいずれにおいても分化誘導後1日でISLRの発現の顕著な低下が観察された。尚、FABP4は脂肪細胞のマーカー、Collagen IIaは軟骨芽細胞のマーカー、Osteopontinは骨芽細胞のマーカーとして使用している。4. Decrease in ISLR expression associated with mesenchymal stem cell differentiation (verification using mouse mesenchymal stem cell line C3H10T1 / 2)
(1) Method and result (Fig. 4)
Using the mouse mesenchymal stem cell line C3H10T1 / 2, we examined changes in ISLR expression during differentiation into fat, cartilage, and bone. Western blotting (anti-ISLR antibody 3 was used to detect ISLR) on 0, 1, 7, and 14 days after the addition of each differentiation-inducing medium (Lonza Japan Co., Ltd.). ) Was verified. A marked decrease in ISLR expression was observed 1 day after the induction of differentiation in all of fat, cartilage, and bone differentiation. FABP4 is used as a marker for adipocytes, Collagen IIa is used as a marker for chondroblasts, and Osteopontin is used as a marker for osteoblasts.
（２）考察
脂肪、軟骨、骨分化のいずれにおいても分化誘導直後にISLRの発現の顕著な低下が観察され、ISLRは未分化な状態の間葉系幹細胞のマーカー分子であることが示唆された。(2) Discussion A marked decrease in ISLR expression was observed immediately after the induction of differentiation in all of fat, cartilage, and bone differentiation, suggesting that ISLR is a marker molecule for undifferentiated mesenchymal stem cells. ..
５．間葉系幹細胞の分化に伴うISLR発現の減少（ヒト骨髄MSCを用いた検証）
（１）方法と結果（図５）
ヒト骨髄MSCを用いて、脂肪、軟骨、及び骨への分化におけるISLRの発現の変化について検証した。各分化誘導メディウム（ロンザジャパン株式会社）を添加後、0日、1日、7日、及び14日のISLRの発現をウェスタンブロット法（抗ISLR抗体２を使用）で検証した。脂肪、軟骨、骨分化のいずれにおいても分化誘導後1日目でISLRの発現の顕著な低下が観察された。5. Decrease in ISLR expression associated with mesenchymal stem cell differentiation (verification using human bone marrow MSC)
(1) Method and result (Fig. 5)
Human bone marrow MSCs were used to examine changes in ISLR expression during fat, cartilage, and bone differentiation. After the addition of each differentiation-inducing medium (Lonza Japan Co., Ltd.), the expression of ISLR on
（２）考察
図４の実験と同様、脂肪、軟骨、骨分化のいずれにおいても分化誘導直後にISLRの発現の顕著な低下が観察され、ISLRは未分化な状態のヒト骨髄MSCのマーカー分子であることが示唆された。(2) Discussion Similar to the experiment in Fig. 4, a marked decrease in ISLR expression was observed immediately after the induction of differentiation in all of fat, cartilage, and bone differentiation, and ISLR is a marker molecule for human bone marrow MSC in an undifferentiated state. It was suggested that there was.
６．間葉系幹細胞の分化に伴うISLR遺伝子メッセンジャーRNA（mRNA）の発現の減少（間葉系幹細胞株C3H10T1/2を用いた検証）
（１）方法と結果（図６）
間葉系幹細胞株C3H10T1/2を用いて、脂肪、軟骨、及び骨への分化におけるISLRのmRNAの発現量の変化について検証した。各分化誘導メディウム（ロンザジャパン株式会社）を添加後、7日後のISLRのmRNAの発現量を定量的RT-PCR法で検証した。脂肪、軟骨、骨分化のいずれにおいても分化誘導後7日でISLRのmRNAの発現量の顕著な低下が観察された。グラフの縦軸はISLRと内因性コントロールとして用いたGAPDHのmRNAの比として示している。6. Decreased expression of ISLR gene messenger RNA (mRNA) associated with mesenchymal stem cell differentiation (verification using mesenchymal stem cell line C3H10T1 / 2)
(1) Method and result (Fig. 6)
Using the mesenchymal stem cell line C3H10T1 / 2, we examined changes in the expression level of ISLR mRNA during differentiation into fat, cartilage, and bone. The expression level of
（２）考察
脂肪、軟骨、骨分化のいずれにおいても分化誘導後にISLRのmRNAの発現量の顕著な低下が観察され、ISLRは未分化な状態の間葉系幹細胞のマーカー分子であることが示唆された。(2) Discussion A marked decrease in the expression level of ISLR mRNA was observed in all of fat, cartilage, and bone differentiation after induction of differentiation, suggesting that ISLR is a marker molecule for undifferentiated mesenchymal stem cells. Was done.
７．ISLRの高発現が間葉系幹細胞の分化に及ぼす影響の検証
（１）方法と結果（図７）
間葉系幹細胞株C3H10T1/2を用いて、ISLRを外因性に強制発現させた場合に、脂肪、軟骨、及び骨への分化に与える影響について検証した。レトロウイルス発現系を用いてISLRをC3H10T1/2に強制発現させ、その後に各分化誘導メディウム（ロンザジャパン株式会社）を添加後、0及び7日のISLR、Sox9（軟骨分化のマーカー）、Osteopontin（骨分化のマーカー）及びRunx2（骨分化のマーカー）の発現をウェスタンブロット法（ISLRの検出には抗ISLR抗体３を使用）で検証した。その結果、ISLRの強制発現がSox9、Osteopointin及びRunx2の発現を抑制することが明らかとなった。7. Verification of the effect of high ISLR expression on mesenchymal stem cell differentiation (1) Methods and results (Fig. 7)
Using the mesenchymal stem cell line C3H10T1 / 2, we examined the effect of exogenous forced expression of ISLR on adipose, cartilage, and bone differentiation. ISLR is forcibly expressed in C3H10T1 / 2 using a retrovirus expression system, and then each differentiation-inducing medium (Lonza Japan Co., Ltd.) is added, and then ISLR, Sox9 (marker of cartilage differentiation), Osteopontin (marker of cartilage differentiation) on
（２）考察
軟骨・骨分化のいずれにおいてもISLRの強制発現によってSox9、Osteopontin及びRunx2の各種分化マーカーの発現の抑制が観察され、ISLRは間葉系幹細胞が未分化な状態を維持するために必要な分子である可能性が示された。(2) Discussion Suppression of the expression of various differentiation markers of Sox9, Osteopontin and Runx2 was observed by forced expression of ISLR in both cartilage and bone differentiation, and ISLR was used to maintain the undifferentiated state of mesenchymal stem cells. It has been shown that it may be a necessary molecule.
８．各種培養細胞におけるISLRの発現
（１）方法及び結果（図８）
ヒト線維芽細胞、血管平滑筋細胞、血管内皮細胞及び各種上皮細胞（ロンザジャパン株式会社又はZenBio社より購入）の細胞抽出液を調製し、ISLR及び各種マーカー分子に対する抗体を用いてウェスタンブロット法（ISLRの検出には抗ISLR抗体２を使用）で検証した。その結果、ISLRは線維芽細胞以外の細胞には発現していないことが明らかとなった。8. Expression of ISLR in various cultured cells (1) Methods and results (Fig. 8)
Prepare cell extracts of human fibroblasts, vascular smooth muscle cells, vascular endothelial cells and various epithelial cells (purchased from Ronza Japan Co., Ltd. or ZenBio), and use antibodies against ISLR and various marker molecules by Western blotting (Western blotting).
（２）考察
ISLRは間葉系幹細胞、線維芽細胞、脂肪由来幹細胞には発現するが、それ以外の細胞では発現していないことが確認され、ISLRがMSCに特異性の高いマーカー分子であることが示された。(2) Consideration
It was confirmed that ISLR is expressed in mesenchymal stem cells, fibroblasts, and adipose-derived stem cells, but not in other cells, indicating that ISLR is a marker molecule with high MSC specificity. It was.
９．膵癌間質に浸潤する間葉系幹細胞におけるISLRの発現
（１）方法及び結果（図９）
中分化型膵癌および低分化型膵癌におけるISLRの発現をIn situハイブリダイゼーション法で調べた。矢頭で示すように、中分化型膵癌の間質に集積あるいは浸潤する間葉系幹細胞においてISLRの発現が高く、間葉系幹細胞が集積ないし浸潤していることが検出された。一方で低分化型膵癌ではISLRの発現が見られなかった。また、右図に示すように、ISLRの発現は患者の良好な予後と相関した。尚、検体採取は次のようにして行った。9. Expression of ISLR in mesenchymal stem cells infiltrating the pancreatic cancer stroma (1) Methods and results (Fig. 9)
The expression of ISLR in moderately differentiated pancreatic cancer and poorly differentiated pancreatic cancer was examined by in situ hybridization. As shown by the arrowheads, it was detected that the expression of ISLR was high in the mesenchymal stem cells that accumulated or infiltrated into the stroma of moderately differentiated pancreatic cancer, and that the mesenchymal stem cells accumulated or infiltrated. On the other hand, no expression of ISLR was observed in poorly differentiated pancreatic cancer. In addition, as shown in the figure on the right, the expression of ISLR correlated with a good prognosis of patients. The sample was collected as follows.
膵癌の患者の手術により得た病理組織標本から鏡検下で腫瘍成分が最も多い領域を選別し、ISLRの発現をIn situハイブリダイゼーション法で調べた。調べた組織の内、中倍率視野（20倍対物レンズ）で癌間質に浸潤する線維芽細胞様形態を有する細胞の20％以上にISLRが陽性のものをISLR高発現、それより少ないものを低発現としてグループ分けし、それぞれのグループについて手術後累積無増悪生存率をプロットした。In situハイブリダイゼーション法によるメッセンジャーRNAの検出であるため、細胞質の一部でもシグナルが観察される場合に陽性と判定した。 The region with the highest tumor component was selected by microscopic examination from the histopathological specimens obtained by surgery of patients with pancreatic cancer, and the expression of ISLR was examined by in situ hybridization. Among the examined tissues, those with high ISLR expression in 20% or more of the cells having fibroblast-like morphology that infiltrate the cancer stroma in the medium-magnification visual field (20x objective lens) have high ISLR expression, and those with less They were grouped as low expression and the cumulative postoperative progression-free survival was plotted for each group. Since messenger RNA was detected by the in situ hybridization method, it was judged to be positive when a signal was observed even in a part of the cytoplasm.
（２）考察
ISLRは癌組織に浸潤する間葉系幹細胞に発現しており、ISLRにより検出される間葉系幹細胞の程度は担癌患者の良好な予後を反映することが示された。(2) Consideration
ISLR is expressed in mesenchymal stem cells that infiltrate cancer tissues, and the degree of mesenchymal stem cells detected by ISLR has been shown to reflect a good prognosis for cancer-bearing patients.
１０．大腸癌間質に浸潤する間葉系幹細胞におけるISLRの発現
（１）方法及び結果（図１０）
中分化型大腸癌および低分化型大腸癌におけるISLRの発現をIn situハイブリダイゼーション法で調べた。矢頭で示すように、中分化型大腸癌の間質に集積あるいは浸潤する間葉系幹細胞においてISLRの発現が高く、間葉系幹細胞が集積ないし浸潤していることが検出された。一方で低分化型大腸癌ではISLRの発現が見られなかった。10. Expression of ISLR in mesenchymal stem cells infiltrating colorectal cancer stroma (1) Methods and results (Fig. 10)
The expression of ISLR in moderately differentiated colorectal cancer and poorly differentiated colorectal cancer was examined by in situ hybridization. As shown by the arrowheads, it was detected that the expression of ISLR was high in the mesenchymal stem cells that accumulated or infiltrated into the stroma of moderately differentiated colorectal cancer, and that the mesenchymal stem cells accumulated or infiltrated. On the other hand, no expression of ISLR was observed in poorly differentiated colorectal cancer.
（２）考察
ISLRは膵癌に加えて大腸癌組織に浸潤する間葉系幹細胞にも発現しており、ISLRが多様な癌組織に浸潤する間葉系幹細胞を検出するために有用なマーカーである可能性が示唆された。(2) Consideration
ISLR is expressed in mesenchymal stem cells that infiltrate colorectal cancer tissues in addition to pancreatic cancer, suggesting that ISLR may be a useful marker for detecting mesenchymal stem cells that infiltrate various cancer tissues. Was done.
１０．乳癌間質に浸潤する間葉系幹細胞におけるISLRの発現
（１）方法及び結果（図１１）
浸潤性乳管癌におけるISLRの発現をIn situハイブリダイゼーション法で調べた。矢頭で示すように、浸潤性乳管癌の間質に集積ないし浸潤する間葉系幹細胞においてISLRの発現が認められた。10. Expression of ISLR in mesenchymal stem cells infiltrating breast cancer stroma (1) Methods and results (Fig. 11)
The expression of ISLR in invasive ductal carcinoma in situ was examined by in situ hybridization. As indicated by the arrowheads, ISLR expression was observed in mesenchymal stem cells that accumulated or infiltrated into the stroma of invasive ductal carcinoma in situ.
（２）考察
ISLRは膵癌や大腸癌に加えて乳癌組織に浸潤する間葉系幹細胞にも発現しており、ISLRが多様な癌組織に浸潤する間葉系幹細胞を検出するために有用なマーカーである可能性が示唆された。(2) Consideration
ISLR is also expressed in mesenchymal stem cells that infiltrate breast cancer tissue in addition to pancreatic cancer and colorectal cancer, and ISLR may be a useful marker for detecting mesenchymal stem cells that infiltrate various cancer tissues. Was suggested.
１１．マウス心筋梗塞モデルの梗塞部位におけるISLRの発現
（１）方法及び結果（図１２）
マウス心筋梗塞モデルの心筋梗塞部位におけるISLRの発現をIn situハイブリダイゼーション法（RNAscope法を用いた）で調べた。8週齢雄の心筋梗塞モデルを用いた。矢頭で示すように、発症後7日目の心外膜周辺においてISLRが認めらる。即ち、心筋梗塞部位においても間葉系幹細胞が浸潤していることが検出できた。11. Expression of ISLR at the infarct site of a mouse myocardial infarction model (1) Method and results (Fig. 12)
The expression of ISLR at the site of myocardial infarction in a mouse myocardial infarction model was examined by the in situ hybridization method (using the RNAscope method). An 8-week-old male myocardial infarction model was used. As indicated by the arrowhead, ISLR is observed around the
（２）考察
ISLRは心筋梗塞部位に集積する間葉系幹細胞に発現しており、ISLRが心筋梗塞などの線維化疾患においても有用なマーカーになることが示唆された。(2) Consideration
ISLR is expressed in mesenchymal stem cells that accumulate at the site of myocardial infarction, suggesting that ISLR is also a useful marker for fibrotic diseases such as myocardial infarction.
１２．野生型マウスとISLRノックアウトマウスの心筋梗塞後の生存率の相違
（１）方法及び結果（図１３）
ISLRノックアウトマウスを作製し、心筋梗塞後の生存率を野生型マウスと比較した。ISLRノックアウトマウスは野生型マウスに比較して、心筋梗塞後の予後が有意に悪いことが明らかとなった。12. Differences in survival rate after myocardial infarction between wild-type mice and ISLR knockout mice (1) Methods and results (Fig. 13)
ISLR knockout mice were generated and the survival rate after myocardial infarction was compared with wild-type mice. ISLR knockout mice were found to have a significantly worse prognosis after myocardial infarction than wild-type mice.
（２）考察
心筋梗塞などの線維化疾患の予後にもISLRが関連することが示唆された。(2) Discussion It was suggested that ISLR is also associated with the prognosis of fibrotic diseases such as myocardial infarction.
本発明は未分化間葉系幹細胞のマーカーを提供する。本発明のマーカーは未分化間葉系幹細胞に高い特異性を示す。当該マーカーを用いた調製法によれば、特に再生医療の分野で重要且つ有用な未分化間葉系幹細胞を高純度で調製することが可能になる。また、当該マーカーは未分化間葉系幹細胞の検出や未分化性の評価等にも有用である。 The present invention provides markers for undifferentiated mesenchymal stem cells. The markers of the present invention show high specificity for undifferentiated mesenchymal stem cells. According to the preparation method using the marker, undifferentiated mesenchymal stem cells, which are important and useful especially in the field of regenerative medicine, can be prepared with high purity. The marker is also useful for detecting undifferentiated mesenchymal stem cells and evaluating undifferentiated cells.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.
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| WO2014080381A1 (en) * | 2012-11-26 | 2014-05-30 | Ecole Polytechnique Federale De Lausanne (Epfl) | Colorectal cancer classification with differential prognosis and personalized therapeutic responses |
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