JP4086204B2 - Hematopoietic stem cell growth regulator and polynucleotide encoding the same - Google Patents
Hematopoietic stem cell growth regulator and polynucleotide encoding the same Download PDFInfo
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Description
本発明は、造血微小環境(niche)を形成し造血因子・接着因子を介して造血幹細胞又は造血前駆細胞の増殖・分化を制御していると考えられている骨髄ストローマ細胞が産生する、新規な造血幹細胞増殖調節因子及びそれをコードする遺伝子(ポリヌクレオチド)等に関する。 The present invention is a novel product produced by bone marrow stromal cells that are considered to form a hematopoietic microenvironment (niche) and control the proliferation and differentiation of hematopoietic stem cells or hematopoietic progenitor cells via hematopoietic factors and adhesion factors. The present invention relates to a hematopoietic stem cell growth regulator and a gene (polynucleotide) encoding the same.
骨髄内に存在する造血幹細胞及び間葉系幹細胞は終生に渡り、それぞれ血球系細胞、骨・軟骨、脂肪細胞等に分化し、それらを供給し続けることで成体の生命を維持している。しかし近年、これら臓器特異的幹細胞がある条件下で多臓器の細胞(神経、肝臓、肺、腸管、心筋、筋肉等)にも分化し得ることが示され、その増殖機構の解明が、移植治療及び再生医療への応用という見地からも大変に重要であると考えられている。 Hematopoietic stem cells and mesenchymal stem cells present in the bone marrow are differentiated into blood cells, bone / cartilage, adipocytes, etc. throughout life, and the adult life is maintained by continuing to supply them. Recently, however, these organ-specific stem cells have been shown to be capable of differentiating into multi-organ cells (nerves, liver, lungs, intestinal tract, heart muscle, muscle, etc.) under certain conditions. It is also considered very important from the viewpoint of application to regenerative medicine.
これまで生体外にて造血幹細胞を増幅する試みが数多くなされ、一定の成果を挙げつつある。骨髄造血組織においては、骨髄ストローマ細胞が造血微小環境(niche)を形成し造血因子・接着因子を介して造血前駆細胞の増殖・分化を制御していると考えられている。ストローマ細胞の産生する造血因子として、IL−3,IL−6などのサイトカイン、SCF、Flt−3 ligandなどの受容体型チロシンキナーゼリガンド(非特許文献1)、分化抑制因子であるNotchリガンドのjagged−1(非特許文献2)が報告されている。 Many attempts have been made to amplify hematopoietic stem cells in vitro, and certain results are being achieved. In bone marrow hematopoietic tissue, it is considered that bone marrow stromal cells form a hematopoietic microenvironment (niche) and control the proliferation and differentiation of hematopoietic progenitor cells via hematopoietic factors and adhesion factors. Examples of hematopoietic factors produced by stromal cells include cytokines such as IL-3 and IL-6, receptor tyrosine kinase ligands such as SCF and Flt-3 ligand (Non-Patent Document 1), and notch ligands that are differentiation inhibitors jagged- 1 (non-patent document 2) has been reported.
しかしながら、上述の様に造血に重要な膜結合型因子・接着分子に関してはまだ多くが未同定であると考えられている。また、現時点では既知の因子の組み合わせだけで、造血幹細胞を長期に培養することは不可能といえ、一方、造血支持細胞であるストローマ細胞と共培養させることにより、それが一定期間可能となることから、ストローマ細胞には未だ同定されていない造血関連遺伝子を発現していることが期待される。
従って、本発明は、上記課題を解決する為に、ストローマ細胞の産生する造血幹細胞増殖調節因子を解析し、cDNAライブラリーを作成し、新規な造血関連遺伝子の単離を行うことによって、将来的には造血幹細胞の生体外増幅、造血器悪性腫瘍の移植治療、幹細胞による再生医学、遺伝子治療への応用を目的としたものである。 Therefore, in order to solve the above problems, the present invention analyzes the hematopoietic stem cell growth regulatory factor produced by stromal cells, creates a cDNA library, and isolates a new hematopoietic-related gene. The purpose is to apply hematopoietic stem cells in vitro, transplantation of hematopoietic malignant tumors, regenerative medicine using stem cells, and gene therapy.
本発明者は、上記の目的を達成する為に、鋭意研究の結果、造血幹細胞増殖調節活性を有する蛋白質をコードする新規な遺伝子を骨髄ストローマ細胞で見出し、本発明を完成した。 In order to achieve the above-mentioned object, the present inventor has found a novel gene encoding a protein having hematopoietic stem cell proliferation regulating activity in bone marrow stromal cells as a result of intensive studies and completed the present invention.
即ち、本発明は、以下の(a)又は(b)の蛋白質をコードする遺伝子に係る:
(a)配列番号2、4、6、8、10及び12から成る群から選択される一つの配列番号に示されるアミノ酸配列、
(b)アミノ酸配列(a)において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、造血幹細胞増殖調節活性を有する蛋白質。
That is, the present invention relates to a gene encoding the following protein (a) or (b):
(A) an amino acid sequence represented by one SEQ ID NO selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10 and 12;
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having hematopoietic stem cell growth regulatory activity.
本発明は更に、以下の(a)又は(b)のDNAを含む遺伝子に係る:
(a)配列番号1、3、5、7、9及び11から成る群から選択される一つの配列番号に示される塩基配列からなるDNA
(b)塩基配列(a)からなるDNAとストリンジェントな条件下でハイブリダイズし、且つ、造血幹細胞増殖調節活性を有する蛋白質をコードするDNA。
The present invention further relates to a gene comprising the following DNA (a) or (b):
(A) DNA comprising a base sequence represented by one SEQ ID NO selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9 and 11
(B) A DNA that hybridizes with a DNA comprising the base sequence (a) under a stringent condition and encodes a protein having a hematopoietic stem cell proliferation-regulating activity.
本明細書において、「造血幹細胞増殖調節活性」とは、例えば、造血幹細胞の増殖を増幅又は増加する能力、及び、逆に造血幹細胞の増殖を抑制する能力を含み、造血幹細胞の増殖に対して何らかの影響を与える機能を意味する。更に、「造血幹細胞」とは、狭義の造血幹細胞のみならず、例えば、後述のCobblestone area forming cell (CAFC) で検出できるような、1段階分化した骨髄球系、赤芽球系、巨核球系、リンパ球系前駆細胞を含むものである。 In the present specification, the “hematopoietic stem cell proliferation regulating activity” includes, for example, the ability to amplify or increase the proliferation of hematopoietic stem cells, and the ability to suppress the proliferation of hematopoietic stem cells. It means a function that has some influence. Furthermore, the “hematopoietic stem cell” is not limited to a hematopoietic stem cell in a narrow sense, but can be detected by, for example, a Cobblestone area forming cell (CAFC) described later, and a one-stage differentiated myeloid system, erythroblast system, megakaryocyte system Including lymphocyte progenitor cells.
本発明は更に、これら遺伝子又はDNAにコードされる蛋白質に係る。本発明で初めて見出されたこれら6種類の蛋白質は、ストローマ細胞に由来し、後述する実施例で示されるように造血幹細胞増殖調節活性を有するものであることから、夫々、SDHF(Stromal cell Derived Hematopoietic Factor)−1、SDHF−2、SDHF−3、SDHF−4、SDHF−5及びSDHF−6と命名される。これら各蛋白質をコードする塩基配列及び対応するアミノ酸配列は、それぞれ、配列番号1及び2、配列番号3及び4、配列番号5及び6、配列番号7及び8、配列番号9及び10、並びに、配列番号11及び12で示されている。 The present invention further relates to proteins encoded by these genes or DNA. Since the first time the found these six proteins in the present invention are derived from stromal cells, and has a hematopoietic stem cell growth regulating activity, as shown in the examples below, respectively, SDHF (S tromal cell D erived H ematopoietic F actor) -1 , termed SDHF-2, SDHF-3, SDHF-4, SDHF-5 and SDHF-6. The base sequences encoding these proteins and the corresponding amino acid sequences are SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, and The numbers 11 and 12 are shown.
本発明は更に、少なくとも一種の上記のいずれかの遺伝子を含有する組換え発現ビークル、該発現ビークルによって形質転換された形質転換体、及び、該形質転換体を培養することを含む、上記のいずれかの蛋白質の製造方法に係る。 The present invention further includes a recombinant expression vehicle containing at least one of the above genes, a transformant transformed with the expression vehicle, and culturing the transformant. The present invention relates to a method for producing such a protein.
本発明は更に、少なくとも一種の上記のいずれかの遺伝子によりストローマ細胞を形質転換することから成る、該ストローマ細胞の造血幹細胞増殖活性の調節方法に係る。形質転換した該ストローマ細胞において、本発明の遺伝子が発現し、その造血幹細胞増殖調節活性によって、該ストローマ細胞の造血幹細胞増殖活性が増幅又は増加したり、抑制されたりするのである。尚、形質転換は、例えば、上記の組換え発現ビークルを使用して、当業者に公知の方法で行なうことが出来る。 The present invention further relates to a method for regulating hematopoietic stem cell proliferation activity of stromal cells, comprising transforming stromal cells with at least one of the above genes. In the transformed stromal cells, the gene of the present invention is expressed, and the hematopoietic stem cell proliferation activity of the stromal cells is amplified, increased, or suppressed by the hematopoietic stem cell proliferation regulating activity. The transformation can be performed by a method known to those skilled in the art using, for example, the above-described recombinant expression vehicle.
又、配列番号13に示されるアミノ酸配列をコードする塩基配列は既に報告されており(1999 Genomics 61 (1), pp37-43)、ISLRと呼ばれているものであるが、その機能がこれまで全くわかっておらず、今回、本発明者によって、以下の実施例に示されるように、造血幹細胞増殖調節活性を有していることが初めて実証されたものである。 The base sequence encoding the amino acid sequence shown in SEQ ID NO: 13 has already been reported (1999 Genomics 61 (1), pp37-43) and is called ISLR. At this time, the present inventors have demonstrated for the first time that the present inventor has hematopoietic stem cell proliferation-regulating activity as shown in the following Examples.
従って、本発明は、又、以下の(a)又は(b)の蛋白質をコードする遺伝子:
(a)配列番号13に示されるアミノ酸配列、
(b)アミノ酸配列(a)において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、造血幹細胞増殖調節活性を有する蛋白質、
でストローマ細胞を形質転換することから成る、該ストローマ細胞の造血幹細胞増殖活性の調節方法に係る。尚、形質転換は、例えば、上記の組換え発現ビークルを使用して、当業者に公知の方法で行なうことが出来る。
Therefore, the present invention also provides a gene encoding the following protein (a) or (b):
(A) the amino acid sequence shown in SEQ ID NO: 13,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having hematopoietic stem cell growth-regulating activity,
A method for regulating the hematopoietic stem cell proliferating activity of the stromal cell, which comprises transforming the stromal cell. The transformation can be performed by a method known to those skilled in the art using, for example, the above-described recombinant expression vehicle.
本発明は更に、上記SDHF−1ないしSDHF−6及びISLRから選択される少なくとも一種の蛋白質を有効成分として含有する造血幹細胞増殖調節用組成物に係る。 The present invention further relates to a composition for regulating hematopoietic stem cell growth containing, as an active ingredient, at least one protein selected from the above-mentioned SDHF-1 to SDHF-6 and ISLR.
本明細書中で、「造血幹細胞増殖活性」とは、造血幹細胞の増殖に対して何らかの影響を与える機能を意味し、「調節」とは、該活性を増幅又は増加すること、及び、逆に抑制すること等の何らかの変化を意味する。 In the present specification, “hematopoietic stem cell proliferation activity” means a function that has some influence on the proliferation of hematopoietic stem cells, and “modulation” means that the activity is amplified or increased, and vice versa. It means some kind of change such as suppression.
本発明は、更に、上記SDHF−1、−2、−3、−4、−5及び−6等の本発明の各蛋白質に対するポリクローナル抗体及びモノクローナル抗体等の各種抗体に係る。
尚、図23に、本発明のSDHF−1からSDHF−6の分子構造を模式的に示す。
The present invention further relates to various antibodies such as a polyclonal antibody and a monoclonal antibody against each protein of the present invention such as SDHF-1, -2, -3, -4, -5 and -6.
FIG. 23 schematically shows the molecular structure of SDHF-1 to SDHF-6 of the present invention.
本発明遺伝子又はDNAは、以下の実施例に示すように、例えば、培養中に白血病阻害因子(LIF)で刺激された、マウス・ストローマ細胞株OP9細胞のようなストローマ細胞由来のmRNAから、cDNAライブラリーを作成して、これに基づき、例えば、以下の実施例に示された方法で調製することが出来る。 As shown in the following examples, the gene or DNA of the present invention is derived from mRNA derived from stromal cells such as mouse stromal cell line OP9 cells stimulated with leukemia inhibitory factor (LIF) during culture. Based on this, a library can be prepared and prepared, for example, by the method shown in the following examples.
或いは、本明細書で開示された配列情報に基づいて、当該技術分野における周知手段を用いた化学合成等によっても調製することが可能である。当業者であれば、特定のアミノ酸配列における1若しくは数個のアミノ酸を欠失、置換若しくは付加することも、当該技術分野における周知手段を用いて容易に実施することができる。 Alternatively, it can be prepared by chemical synthesis or the like using well-known means in the technical field based on the sequence information disclosed in the present specification. A person skilled in the art can easily perform deletion, substitution or addition of one or several amino acids in a specific amino acid sequence using a well-known means in the art.
本発明において、特定の遺伝子又はDNAは、当業者に周知の緩衝液中で、適当な温度及び塩濃度等の諸条件下のストリンジェントな条件でハイブリダイズさせることができる。 In the present invention, a specific gene or DNA can be hybridized in a buffer solution well known to those skilled in the art under stringent conditions under various conditions such as an appropriate temperature and salt concentration.
このようなストリンジェントな条件下で本発明の遺伝子又はDNAとハイブリダイズし、且つ、造血幹細胞増殖調節活性を有する蛋白質をコードするDNAの例として、例えば、かかる遺伝子と相同性が70%以上、好ましくは90%以上であるようなDNAを挙げることができる。 Examples of DNA that hybridizes with the gene or DNA of the present invention under such stringent conditions and encodes a protein having hematopoietic stem cell proliferation regulatory activity include, for example, 70% or more homology with such a gene, A DNA that is preferably 90% or more can be mentioned.
本発明の遺伝子又はDNAに、遺伝子組換え操作において当業者に公知である様々な配列、例えば、プロモーター及びエンハンサーなどの調節因子、制限酵素部位、並びに選択マーカー(マーカー酵素等)遺伝子等が適宜結合されたDNA分子も本発明の範囲である。 Various sequences known to those skilled in the art in gene recombination operations, for example, regulatory factors such as promoters and enhancers, restriction enzyme sites, and selectable marker (marker enzyme etc.) genes are appropriately combined with the gene or DNA of the present invention. Modified DNA molecules are also within the scope of the present invention.
更に、本発明の遺伝子、DNA又は上記DNA分子を含有する組換えプラスミドベクター又はレトロウイルス等を使用する組換えウイルスベクター等の発現ビークル、及び該発現ビークルを含みそれによって形質転換された大腸菌等の細菌、マウスCOS−7細胞又はマウス・ストローマ細胞OP9等の真核細胞等の形質転換体も本発明に含まれる。これらのDNA分子、発現ビークル及び形質転換体は、当該技術分野における公知の手段によって、容易に調製することができる。更に、本発明の蛋白質は、当業者であれば、上記の形質転換体を当該技術分野における公知手段によって培養し、得られた培養物から精製することにより、容易に製造することができる。 Further, an expression vehicle such as a recombinant plasmid vector using a gene of the present invention, DNA or the above DNA molecule or a recombinant virus vector using a retrovirus, etc., and Escherichia coli containing the expression vehicle and transformed therewith, etc. Transformants such as bacteria, eukaryotic cells such as mouse COS-7 cells or mouse stromal cells OP9 are also included in the present invention. These DNA molecules, expression vehicles and transformants can be easily prepared by means known in the art. Furthermore, the protein of the present invention can be easily produced by those skilled in the art by culturing the above transformant by means known in the art and purifying it from the obtained culture.
上記蛋白質を有効成分として含有する、本発明の造血幹細胞増殖調節活性化組成物には、造血因子として公知のその他のサイトカイン、並びに当該技術分野で公知の適当なキャリア及び助剤等を目的に応じて適宜含有させることが出来る。蛋白質を有効成分としての本発明蛋白質及びその他の成分の含有量及び含有割合等は、使用目的などに応じて当業者が適宜選択することが出来る。 The composition for activating hematopoietic stem cell proliferation regulation of the present invention containing the above protein as an active ingredient depends on the purpose of other cytokines known as hematopoietic factors and appropriate carriers and auxiliaries known in the art. Can be contained as appropriate. The content and content ratio of the protein of the present invention containing protein as an active ingredient and other ingredients can be appropriately selected by those skilled in the art according to the purpose of use.
上記SDHF−1、−2、−3、−4、−5及び−6等の本発明の各蛋白質に対する、本発明のポリクローナル抗体及びモノクローナル抗体等の各種抗体は、当業者に公知の任意の方法で製造することができる。例えば、ポリクローナル抗体の場合には、上記各蛋白質又はその部分ペプチドを免疫源としてウサギ等の各種実験動物に接種することによって容易に製造することができる。又、モノクローナル抗体の場合には、このような免疫源を使用して同様に調製した抗体産生細胞を適当な親細胞(不死化細胞株)と当業者に周知の各種方法によって細胞融合させてハイブリドーマを作成し、この中から各蛋白質と特異的に反応するクローンを選択することによって容易に得ることができる。尚、部分ペプチドは各蛋白質の任意の部分のアミノ酸配列に相当するものであり、そのアミノ酸数に特に制限はないが、少なくとも10個以上のアミノ酸を有するものが好ましく、通常、10〜20個の範囲のものが適当である。又、各蛋白質との反応性を考慮すると、免疫源として使用する部分ペプチドは各蛋白質の細胞外領域から選択することが好ましい。 Various antibodies such as the polyclonal antibody and the monoclonal antibody of the present invention against the respective proteins of the present invention such as SDHF-1, -2, -3, -4, -5 and -6 can be prepared by any method known to those skilled in the art. Can be manufactured. For example, in the case of a polyclonal antibody, it can be easily produced by inoculating various experimental animals such as rabbits using the above-mentioned proteins or partial peptides thereof as an immunogen. In the case of a monoclonal antibody, an antibody-producing cell similarly prepared using such an immunogen is cell-fused with an appropriate parent cell (immortalized cell line) by various methods well known to those skilled in the art, and then a hybridoma. And a clone that specifically reacts with each protein is selected from these. The partial peptide corresponds to the amino acid sequence of an arbitrary part of each protein, and the number of amino acids is not particularly limited, but preferably has at least 10 amino acids, and usually 10 to 20 amino acids. A range is appropriate. In consideration of reactivity with each protein, it is preferable to select a partial peptide used as an immunogen from the extracellular region of each protein.
このような抗体は、骨髄幹細胞、間葉系幹細胞、及び神経幹細胞等の各種成体細胞、並びに骨髄造血支持細胞等の幹細胞支持細胞(ストローマ細胞)を単離又は純化する方法に使用することができる。これらの単離又は純化方法は、具体的には、当業者に公知の任意の各種手段、例えば、アフィニティカラムによる精製、及びFACSによるソーティング等により実施することができる。 Such antibodies can be used in methods for isolating or purifying various adult cells such as bone marrow stem cells, mesenchymal stem cells, and neural stem cells, and stem cell support cells (stromal cells) such as bone marrow hematopoietic support cells. . Specifically, these isolation or purification methods can be carried out by any of various means known to those skilled in the art, for example, purification by affinity column, sorting by FACS, and the like.
実施例
以下に実施例に即して本発明を詳述するが、これら実施例は本発明の技術的範囲を如何なる意味でも何ら限定するものではない。
EXAMPLES The present invention will be described in detail below with reference to examples, but these examples do not limit the technical scope of the present invention in any way.
cDNAライブラリーの構築とSignal Sequence Trap (SST) 法
マウス・ストローマ細胞株OP9細胞はAlpha−MEM培地に20%の胎児牛血清を添加して培養する。PLAT−E細胞(2000 森田らGene Therapy vol7 1063-6)はDMEMに10%の胎児牛血清を添加した培地にて培養した。OP9細胞よりのpolyA RNAの抽出はInvitrogen社のFAST TRACK2.0mRNA Isolation Kitを用い、その添付プロトコールに従い行った。cDNAライブラリーの材料としmouse Leukemia Inhibitory Factor (LIF) 10ng/ml37℃60分にて刺激したOP9細胞1x108個から抽出したpolyARNA5μgを用いた。cDNAライブラリーの構築はSUPERSCRIPT II (Invitrogen社)を用い、ランダムヘキサマーをプライマーとして行った。得られたcDNAライブラリーにBstXIアダプタープライマー(Invitrogen社)を付加し、レトロウィルスベクターであるpMX−SSTベクター(1999 小嶋らNat Biotech vol 17 p487)のBstXI部位に組み込みプラスミド・ライブラリーを構築した。
Construction of cDNA Library and Signal Sequence Trap (SST) Method Mouse stromal cell line OP9 cells are cultured by adding 20% fetal bovine serum to Alpha-MEM medium. PLAT-E cells (2000 Morita et al. Gene Therapy vol7 1063-6) were cultured in a medium supplemented with 10% fetal bovine serum in DMEM. PolyA RNA was extracted from OP9 cells using Invitrogen's FAST TRACK 2.0 mRNA Isolation Kit according to the attached protocol. As a material for the cDNA library, 5 μg of polyARNA extracted from 1 × 10 8 OP9 cells stimulated at 37 ° C. for 60 minutes with mouse Leukemia Inhibitory Factor (LIF) 10 ng / ml was used. The cDNA library was constructed using SUPERSCRIPT II (Invitrogen) and random hexamers as primers. A BstXI adapter primer (Invitrogen) was added to the obtained cDNA library, and an integration plasmid library was constructed at the BstXI site of the retrovirus vector pMX-SST vector (1999 Kojima et al. Nat Biotech vol 17 p487).
作製したcDNAライブラリーをPLAT−E細胞にFuGene6(Roche社)を用いて遺伝子導入して上清にレトロウィルスの形で回収した。得られたレトロウィルスcDNAライブラリーをインターロイキン3(IL-3)依存性のマウスプロB細胞であるBa/F3細胞に感染させた。pMX−SSTベクターはシグナルペプチドを欠損した恒常的活性化型c−Mpl遺伝子を有しており、ライブラリー由来の遺伝子がシグナルペプチドを有している場合にcMpl遺伝子が活性化し、Ba/F3細胞がIL−3非存在下において不死化する(1999 小嶋らNat Biotech vol 17 p487-90)。レトロウィルス・ライブラリーを感染させたBa/F3細胞からIL−3を除去し96ウェルプレートに1ウェルあたり10000個ずつ分けて培養、細胞が増殖してきたものを単離し、ゲノムDNAを抽出した。シグナルペプチドを有した遺伝子はレトロウィルスにてBa/F3細胞のゲノムDNAに挿入されているのでpMX−SSTベクターの配列をもとにして設計したプライマー(GGGGGTGGACCATCCTCTA, CGCGCAGCTGTAAACGGTAG)にてゲノムDNA PCR反応(LA-Taq, TAKARA 98℃ 20 sec 68℃ 300 sec; 35 cycles)を行い回収した。 The prepared cDNA library was introduced into PLAT-E cells using FuGene6 (Roche), and the supernatant was recovered in the form of a retrovirus. The obtained retroviral cDNA library was infected with Ba / F3 cells, which are mouse pro-B cells dependent on interleukin 3 (IL-3). The pMX-SST vector has a constitutively activated c-Mpl gene that lacks a signal peptide, and when the library-derived gene has a signal peptide, the cMpl gene is activated and Ba / F3 cells Is immortalized in the absence of IL-3 (1999 Kojima et al. Nat Biotech vol 17 p487-90). IL-3 was removed from the Ba / F3 cells infected with the retrovirus library, and cultured in a 96-well plate by dividing 10,000 cells per well, and the cells that had proliferated were isolated, and genomic DNA was extracted. Since the gene having the signal peptide is inserted into the genomic DNA of Ba / F3 cells by retrovirus, the genomic DNA PCR reaction (primary design based on the sequence of pMX-SST vector (GGGGGTGGACCATCCTCTA, CGCGCAGCTGTAAACGGTAG)) LA-Taq, TAKARA 98 ° C. 20 sec 68 ° C. 300 sec; 35 cycles).
遺伝子配列の決定
シグナルペプチドを有したクローンは上記PCR反応にてDNA断片として得られるのでこの塩基配列をABI社373A シーケンサーにて決定した。得られたPCR断片をPCR purification kit (Quiagen社)にて精製しプライマーを除去した後、ABI社のプロトコールに従いdye terminator法にて配列を決定した。得られた配列情報はNCBIのBLASTプログラム(http://www.ncbi.nlm.nih.gov/BLAST/)にて解析し、既知配列、未知配列の判定を行った。未知配列の一部は得られた配列を元に設計したプライマーを用いて3’RACE法にて決定した。3’RACE法は、上記PCRクローンの配列をもとにして設計したプライマーとオリゴdTプライマーを用いたPCR法にて未知遺伝子のpolyA領域までを同定する方法である。得られたPCR断片は上記と同様にABI社373Aシーケンサーにて塩基配列を決定した。
Determination of gene sequence Since a clone having a signal peptide was obtained as a DNA fragment by the above PCR reaction, this base sequence was determined by ABI 373A sequencer. The obtained PCR fragment was purified with a PCR purification kit (Quiagen), the primer was removed, and the sequence was determined by the dye terminator method according to the protocol of ABI. The obtained sequence information was analyzed by NCBI's BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), and known sequences and unknown sequences were determined. A part of the unknown sequence was determined by the 3′RACE method using primers designed based on the obtained sequence. The 3′RACE method is a method for identifying even the polyA region of an unknown gene by PCR using a primer designed based on the sequence of the PCR clone and an oligo dT primer. The base sequence of the obtained PCR fragment was determined by ABI 373A sequencer in the same manner as described above.
その結果、上記のSST法により既知・未知合わせて234クローンの遺伝子を単離した。その内訳は、既知遺伝子181、及び未知遺伝子42、解析できなかったもの11であった。既知遺伝子の内訳としては、増殖因子49、受容体23、接着因子71、及びその他38であった。 As a result, 234 clone genes were isolated by combining the known and unknown by the above SST method. The breakdown was the known gene 181, the unknown gene 42, and the 11 that could not be analyzed. The breakdown of known genes was growth factor 49, receptor 23, adhesion factor 71, and others 38.
未知遺伝子の中に、本発明の遺伝子である、6種類のSDHF(Stromal cell derived hematopoietic factor)遺伝子、及びISLRが含まれていた。尚、第1図〜第6図及び配列13に示されるように、6種類のSDHF及びISLR遺伝子がコードするアミノ酸配列のN末端には、シグナルペプチド(四角の枠部で示された部分)が含まれていることが判明した。以上の結果を表1及び表2に示す。 Some unknown gene is a gene of the present invention, six SDHF (S tromal cell d erived h ematopoietic f actor) gene, and ISLR were included. As shown in FIGS. 1 to 6 and Sequence 13, a signal peptide (portion indicated by a square frame) is present at the N-terminus of the amino acid sequence encoded by six types of SDHF and ISLR genes. Turned out to be included. The above results are shown in Tables 1 and 2.
マイクロアレイ法
Signal Sequence Trap法にて得られたPCR断片は全てスライドグラス上にスポットし(以上の操作は北海道システムサイエンス社に委託した。)OP9細胞をLIF10ng/ml 37℃60分による刺激前後の条件にて調整した細胞より作製した蛍光標識cDNAプローブにて発現の変化を検出した。プローブは上記と同様に抽出したpolyA RNAよりオリゴdTプライマーにてSuperScript IIにて逆転写反応を行い、Fluorolink Cy-dUTPを取り込ませることにより標識した後Microcon30(Millipore社)にて精製し用いた。得られた結果を第7図に示した。
Microarray method
All the PCR fragments obtained by the Signal Sequence Trap method were spotted on a slide glass (the above operation was entrusted to Hokkaido System Science). OP9 cells were subjected to conditions before and after stimulation with LIF 10 ng / ml at 37 ° C. for 60 minutes. Changes in expression were detected with a fluorescently labeled cDNA probe prepared from the prepared cells. The probe was subjected to reverse transcription reaction with Superscript II using oligo dT primer extracted from polyA RNA in the same manner as described above, labeled with Fluorolink Cy-dUTP, and then purified with Microcon 30 (Millipore). The obtained results are shown in FIG.
ノーザンブロッティング
OP9細胞をLIF 10ng/ml 37℃60分にて刺激後、0分、30分、1時間、3時間、8時間、24時間後にそれぞれtotal RNAをTrizol液(Invitrogen社)を用い、そのプロトコールに従い抽出した。抽出したtotal RNAは濃度測定後10μgずつ1%アガロースゲル上に電気泳動後、Hybond N(Amersham社)上に転写し、それぞれの遺伝子のDNA断片をプローブにして標準的な方法にてノーザンブロッティングを行った。その結果、第8図に示されるように、SDHF−1、SDHF−2、及びSDHF−5遺伝子について発現の増加が観察された。一方、対照的に、SDHF−6遺伝子について発現の減少が観察された。
Northern blotting OP9 cells were stimulated with LIF 10 ng / ml at 37 ° C. for 60 minutes, and after 0 minutes, 30 minutes, 1 hour, 3 hours, 8 hours, and 24 hours, total RNA was used with Trizol solution (Invitrogen), respectively. Extraction was performed according to the protocol. The extracted total RNA was subjected to 10 μg concentration measurement, electrophoresed on a 1% agarose gel, transferred to Hybond N (Amersham), and subjected to Northern blotting by a standard method using DNA fragments of each gene as probes. went. As a result, as shown in FIG. 8, increased expression was observed for the SDHF-1, SDHF-2, and SDHF-5 genes. In contrast, a decrease in expression was observed for the SDHF-6 gene.
ウェスタン解析法
得られた遺伝子の全長配列よりアミノ酸をコードしている領域(Open Reading Frame: ORF)を決定し、そのカルボキシル末端にFLAGタグ配列(DYKDDDDK)をPCR法にて付加した。これらのcDNAを発現ベクターpUC−CAGGSに挿入して作製したプラスミドをFuGene6(ロシュ社)にてCOS−7細胞に一過性に発現した。ウェスタン解析は過去に報告した方法(1995 Ueno, JBC vol 270pp20135-42)にて行った。細胞はTriton lysis buffer (0.5% (v/v) Triton X-100, 50 mM Tris-HCl pH 7.4, 2 mM PMSF, 10 U/ml aprotinin 1mM EDTA) にて可溶化した後、50μgをSDS-polyacrylamide gel elestrophoresis (PAGE)にて展開し、Immobilon−Pメンブレインに転写後抗FLAG抗体、M2(SIGMA社)にて検出した。その結果、第9図に示されるように、本発明のいずれの遺伝子もCOS7細胞にて発現されウェスタン解析にてバンドが検出された。
Western analysis method A region encoding an amino acid (Open Reading Frame: ORF) was determined from the full-length sequence of the obtained gene, and a FLAG tag sequence (DYKDDDDK) was added to the carboxyl terminus by the PCR method. A plasmid prepared by inserting these cDNAs into the expression vector pUC-CAGGS was transiently expressed in COS-7 cells with FuGene6 (Roche). Western analysis was performed by the method reported in the past (1995 Ueno, JBC vol 270pp20135-42). Cells were solubilized with Triton lysis buffer (0.5% (v / v) Triton X-100, 50 mM Tris-HCl pH 7.4, 2 mM PMSF, 10 U / ml aprotinin 1 mM EDTA), and 50 μg of SDS-polyacrylamide. It was developed by gel elestrophoresis (PAGE), transferred to Immobilon-P membrane and then detected with anti-FLAG antibody, M2 (SIGMA). As a result, as shown in FIG. 9, any gene of the present invention was expressed in COS7 cells, and a band was detected by Western analysis.
免疫染色法
上記と同条件にてFLAGタグ配列を付加した遺伝子をCOS−7細胞にFuGene試薬を用いて一過性に発現させ(上記参照)、3.7%ホルムアルデヒドにて固定後1%NP40にてpermeabilizationを行い、抗FLAG抗体を用いて染色した(二次抗体はTexas Red標識抗マウス抗体、Jackson社)。核はDAPI(フナコシ社)染色を行った。その結果を第10図に示す。
Immunostaining method A gene with a FLAG tag sequence added under the same conditions as described above is transiently expressed in COS-7 cells using FuGene reagent (see above), fixed with 3.7% formaldehyde, and 1% NP40 And stained with anti-FLAG antibody (secondary antibody was Texas Red-labeled anti-mouse antibody, Jackson). Nuclei were stained with DAPI (Funakoshi). The result is shown in FIG.
Cobblestone Area Forming Cell Assay : CAFC(敷石状領域形成細胞アッセイ)
OP9細胞に上記にて得られた遺伝子をレトロウィルスベクター(pMX-puro) にて遺伝子導入し6wellディッシュに2.5x105ずつ蒔き、一晩培養した後、5−8週齢のC57BL/6マウス大腿骨より採取した骨髄細胞1x105まき、6日後に形成されたCAFC数を測定した。
Cobblestone Area Forming Cell Assay: CAFC
The gene obtained above was introduced into OP9 cells using a retrovirus vector (pMX-puro), seeded 2.5 × 10 5 in 6-well dishes, cultured overnight, and then 5-8 weeks old C57BL / 6 mice. Bone marrow cells collected from the femur 1 × 10 5 and the number of CAFCs formed after 6 days were measured.
CAFC解析の結果
ストローマ細胞と骨髄細胞を共培養した際、骨髄細胞がストローマ細胞の下部に潜り込み増殖する現象をCobblestone area forming cell (CAFC) という。CAFC形成数が多い場合、ストローマ細胞が未熟な造血支持細胞を増幅していることを意味している。ただしCAFCを形成している細胞は造血幹細胞だけでなく、1段階分化した骨髄球系、赤芽球系、巨核球系、リンパ球系前駆細胞を含んでいるため、厳密にはCAFCの増加=造血幹細胞の増幅ではない。造血支持能を有するマウス骨髄ストローマ細胞株のOP9細胞に上記遺伝子をレトロウィルスpMX−puroにて導入し、ピューロマイシン5μg/mlにて薬剤耐性となった細胞についてCAFC形成能を解析した。その結果、第11図に示されるように、ISLRが最も高値を示しSDHF−1、SDHF−2、SDHF−4、及びSDHF−5についても軽度の高値を示した。これらの遺伝子の造血前駆細胞増幅能力が示唆された。逆にSDHF−3ではCAFC形成が抑制され、造血幹細胞増殖抑制機能を有すると考えられた。
As a result of CAFC analysis, when stromal cells and bone marrow cells are co-cultured, the phenomenon in which bone marrow cells sink into the lower part of stromal cells and proliferate is called Cobblestone area forming cell (CAFC). When the number of CAFC formation is large, it means that stromal cells are amplifying immature hematopoietic support cells. However, since the cells forming CAFC include not only hematopoietic stem cells but also myeloid, erythroblastic, megakaryocyte, and lymphocyte progenitor cells differentiated in one stage, strictly speaking, an increase in CAFC = It is not an amplification of hematopoietic stem cells. The gene was introduced into OP9 cells of a mouse bone marrow stromal cell line having hematopoietic support ability with the retrovirus pMX-puro, and the CAFC-forming ability of cells that became drug resistant with puromycin 5 μg / ml was analyzed. As a result, as shown in FIG. 11, ISLR showed the highest value, and SDHF-1, SDHF-2, SDHF-4, and SDHF-5 also showed mild high values. These genes suggested the ability to amplify hematopoietic progenitor cells. On the other hand, it was considered that SDHF-3 suppressed the formation of CAFC and had a hematopoietic stem cell proliferation inhibitory function.
細胞培養上清の分泌蛋白質のメチオニン標識による検出
COS−7細胞を6cmディッシュに1x105個まき、一晩培養した。FuGene法(Roche社)を用いて遺伝子導入した。これらの遺伝子のカルボキシル末端にはFLAGタグが付加されており、前述したように、抗FLAG抗体によるウェスタン解析にて発現した蛋白質を検出することが出来る。36時間の培養後、上清を除去し、PBSにて2回洗浄後、0.5 ml DMEM(Methionine−, SIGMA社)中の0.5mCi[35S]methionine(ICN社TRAN-S)を加え、4時間培養した。培養上清から遠心にて細胞成分を除去し、Micron 10(ミリポア社)にて蛋白質を濃縮した。15%SDS−PAGEにて電気泳動後、標識された蛋白質をBAS2000(富士フィルム社)にて検出した。
Detection of secreted protein in cell culture supernatant by methionine labeling 1 × 10 5 COS-7 cells were seeded in a 6 cm dish and cultured overnight. Gene transfer was performed using the FuGene method (Roche). A FLAG tag is added to the carboxyl terminus of these genes, and as described above, proteins expressed by Western analysis using an anti-FLAG antibody can be detected. After 36 hours of culture, the supernatant was removed, washed twice with PBS, 0.5 ml DMEM (Methionine - , SIGMA Co.) 0.5 mCi in [35 S] methionine and (ICN Co. TRAN-S) In addition, the cells were cultured for 4 hours. Cell components were removed from the culture supernatant by centrifugation, and the protein was concentrated with Micron 10 (Millipore). After electrophoresis with 15% SDS-PAGE, the labeled protein was detected with BAS2000 (Fuji Film).
その結果、第12図及び第13図に示されるように、SDHF−1及びSDHF−5にて、培養上清への蛋白質分泌が検出された。これらの遺伝子は構造的に膜型蛋白質であること、分泌蛋白質の分子量がウェスタン解析で検出された成熟型蛋白質よりも約20kDa小さくなっていることから、これらの分泌蛋白質はカルボキシル末端の膜貫通領域が切断されたものと考えられる。カルボキシル末端の膜貫通領域が切断されて分泌されるタイプの増殖因子としてSCFが知られており、一方、接着蛋白質ではこうした例は認められないことから、本発明の遺伝子がコードする蛋白質は、液性因子として他の細胞に情報を伝達するタイプの蛋白質であることを強く示唆するものと考えられる。 As a result, as shown in FIGS. 12 and 13, protein secretion into the culture supernatant was detected with SDHF-1 and SDHF-5. Since these genes are structurally membrane proteins and the molecular weight of the secreted protein is about 20 kDa smaller than that of the mature protein detected by Western analysis, these secreted proteins are transmembrane regions at the carboxyl terminal. Is considered to have been cut. SCF is known as a type of growth factor that is secreted by cleavage of the transmembrane region at the carboxyl terminal. On the other hand, since such an example is not observed in the adhesion protein, the protein encoded by the gene of the present invention is It seems to strongly suggest that it is a type of protein that transmits information to other cells as a sex factor.
RT−PCR法による発現部位の検定
各SDHF遺伝子の塩基配列に基づきプライマーを設定、Multiple Tissue cDNA (MTC) Panels (クロンテック社)をtemplateとしてPCR反応(LA-Taq, TAKARA)を行った。また、骨髄内の細胞は蛍光標識したモノクローナル抗体(抗B220, 抗CD3, 抗Gr1, 抗MAC1, 抗Sca1抗体、Phamingen社)を用い、FACS Vantage(BectonDickinson社)にてソーティングしたTrizol試薬(Invitrogen社)にてRNAを抽出し、ランダムヘキサマーを用いてRT反応を行った上で、同様にPCR反応を行った。
Test of expression site by RT-PCR method Primers were set based on the base sequence of each SDHF gene, and PCR was performed using Multiple Tissue cDNA (MTC) Panels (Clontech) as a template. In addition, Trizol reagent (Invitrogen) sorted by FACS Vantage (Becton Dickinson) was used for cells in bone marrow using fluorescently labeled monoclonal antibodies (anti-B220, anti-CD3, anti-Gr1, anti-MAC1, anti-Sca1 antibody, Phamingen). ), RNA was extracted, RT reaction was performed using random hexamers, and PCR reaction was performed in the same manner.
その結果、第14図に示される様に全てのSDHF遺伝子はOP9細胞での発現が確認された。特にSDHF−4は発現が脳と骨髄に限局して発現しており、また骨髄内においてはストローマ細胞に限局して発現しているなど興味深い。 As a result, as shown in FIG. 14, the expression of all SDHF genes in OP9 cells was confirmed. In particular, SDHF-4 is expressed in a limited manner in the brain and bone marrow, and in the bone marrow, it is interesting that it is expressed exclusively in stromal cells.
Long Term Culture-Initiating Cells Assay(LTC−IC法)による幹細胞支持機能の検定
上記のCAFC法では比較的短期間の造血支持機能を反映しているにすぎないため、より長期の幹細胞支持機能評価法としてLTC−IC法を行った。上記と同様に単離した遺伝子をレトロウィルス発現ベクターpMX−puroを用いてOP9細胞に高発現させLTC−IC法を行った。
Assay of stem cell support function by Long Term Culture-Initiating Cells Assay (LTC-IC method) The above-mentioned CAFC method only reflects a relatively short-term hematopoietic support function. The LTC-IC method was performed. The gene isolated in the same manner as described above was highly expressed in OP9 cells using the retrovirus expression vector pMX-puro, and the LTC-IC method was performed.
まず造血幹細胞を純化する。6週齢から8週齢のC57BL/6マウスの大腿骨より骨髄細胞を採取し、Ficoll法(比重1.100) にて単核細胞を遠心分離した。PBSにて洗浄後、ビオチン標識した1次抗体(抗Lineage抗体カクテル:抗CD3, 抗CD4, 抗CD8, 抗B220, 抗Ter119, 抗Gr1,抗 MAC1抗体、以上Phamingen社)およびストレプトアビジン標識磁気ビーズにそれぞれ4℃15分間反応させた。MACS法にてカラムを通過する分画(LIN(-)細胞)を回収し、遠心洗浄後、さらにPE標識抗Sca1抗体、FITC標識抗c−kit抗体、Per−CP−Cy5.5標識ストレプトアビジン(以上Phamingen社)にて4℃15分間反応させ、FACS Vantage(Becton Dickinson社)にてLIN(−),Sca1(+),c−kit(+)分画(以上にて得られた細胞をKSL細胞と定義する。)SDHF−1−6のカルボキシル末端にFLAGペプチドを付加したcDNAをレトロウィルスベクター(pMX-puro) にてOP9細胞に遺伝子導入して樹立した安定発現株を6穴ディッシュに2.5x105ずつ蒔き、一晩培養した後、KSL細胞を100個ずつまき、3週間培養した。その後細胞を回収し、γ線を20Gy照射したOP9細胞を1穴に3000個ずつまいておいた96穴ディッシュに、8穴ずつペアで2分の1ずつ薄めて蒔いた。5週間後、各穴から細胞を回収しそれぞれメチルセルロース培地(IMDM, メチルセルロース1%、牛胎児血清15%、牛血清アルブミン 1%、ヒトエリスロポイエチン3U/ml、ヒトIL-6 10ng/ml、マウスIL-3 10ng/ml、マウスSCF 100ng/ml牛インスリン10μg/ml)に蒔いて12日後に血球コロニー(CFU-C)の有無で判定した。幹細胞の頻度はL−Calcソフトウェア(Stemcell社)にて行った。 First, hematopoietic stem cells are purified. Bone marrow cells were collected from femurs of 6 to 8 week old C57BL / 6 mice, and mononuclear cells were centrifuged by Ficoll method (specific gravity 1.100). After washing with PBS, biotin-labeled primary antibody (anti-Lineage antibody cocktail: anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-Ter119, anti-Gr1, anti-MAC1 antibody, Phamingen) and streptavidin-labeled magnetic beads Each was reacted at 4 ° C. for 15 minutes. Fractions (LIN (−) cells) that pass through the column by the MACS method are collected, washed after centrifugation, and further PE-labeled anti-Sca1 antibody, FITC-labeled anti-c-kit antibody, and Per-CP-Cy5.5-labeled streptavidin. (Phamingen, Inc.) at 4 ° C. for 15 minutes, and FACS Vantage (Becton Dickinson) for LIN (−), Sca1 (+), c-kit (+) fractions (cells obtained above). Defined as KSL cells.) A stable expression strain established by introducing a FLAG peptide at the carboxyl terminus of SDHF-1-6 into OP9 cells using a retroviral vector (pMX-puro) in a 6-well dish After 2.5 × 10 5 cells were cultured and cultured overnight, 100 KSL cells were seeded and cultured for 3 weeks. Thereafter, the cells were collected and spread in a 96-well dish in which 3000 OP9 cells irradiated with 20 Gy of γ-rays were placed in each well, and each well was diluted 1/2 by 8 in pairs. After 5 weeks, cells were collected from each well, and methylcellulose medium (IMDM, methylcellulose 1%, fetal calf serum 15%, bovine serum albumin 1%, human erythropoietin 3U / ml, human IL-6 10ng / ml, mouse IL-3 10 ng / ml, mouse SCF 100 ng / ml bovine insulin 10 μg / ml), and 12 days later, the presence or absence of blood cell colonies (CFU-C) was determined. Stem cell frequency was determined using L-Calc software (Stemcell).
その結果、第15図に示されるようにSDHF−4遺伝子に著明な造血支持能が観察された。この遺伝子の造血支持機能が示された。 As a result, as shown in FIG. 15, a remarkable hematopoietic support ability was observed in the SDHF-4 gene. The hematopoietic support function of this gene was shown.
SDHF−4細胞外領域・リコンビナントタンパク質によるマウス造血幹細胞の増幅効果
SDHF−4の細胞膜貫通領域をPSORT IIプログラム(http://psort.ims.u-tokyo.ac.jp/form2.html)で予測し(アミノ酸524-540)、細胞外領域(アミノ酸1-523)にヒト免疫グロブリンFc領域と融合タンパク質(SDHF-4-ΔTM-Fc)を作製、発現ベクターpSSRaに組み込み、CHO−k1細胞に安定的に遺伝子導入した。無血清培地CD−CHO(Invitrogen社)にて培養することで、上清から融合リコンビナントタンパク質をProteinAカラムにて精製した(第16図)。マウス幹細胞はLTC−IC法の項に記載した様にマウス骨髄LIN(−),Sca1(+),c−kit(+)分画(KSL細胞)100個ずつ、SCF(50ng/ml),IL−3(10ng/ml),IL−6(10ng/ml)及びSDHF−4−ΔTM−Fc(100ng/ml)を添加したStem Span培地(StemCell社)にて培養した。7日後、細胞を回収し、FACS CaliburにてLIN,Sca1,c−kitの発現を解析した。
SDHF-4 extracellular region / amplification effect of mouse hematopoietic stem cells by recombinant protein SDHF-4 transmembrane region predicted by PSORT II program (http://psort.ims.u-tokyo.ac.jp/form2.html) (Amino acids 524-540), human immunoglobulin Fc region and fusion protein (SDHF-4-ΔTM-Fc) were prepared in the extracellular region (amino acids 1-523), incorporated into the expression vector pSSRa, and stable in CHO-k1 cells Gene transfer. By culturing in a serum-free medium CD-CHO (Invitrogen), the fusion recombinant protein was purified from the supernatant using a Protein A column (FIG. 16). As described in the section of the LTC-IC method, mouse bone marrow LIN (−), Sca1 (+), c-kit (+) fractions (KSL cells) 100 each, SCF (50 ng / ml), IL -3 (10 ng / ml), IL-6 (10 ng / ml), and SDHF-4-ΔTM-Fc (100 ng / ml) were added to Stem Span medium (StemCell). Seven days later, the cells were collected, and the expression of LIN, Sca1, c-kit was analyzed by FACS Calibur.
その結果、第17図に示されるように、SCF,IL−3,IL−6を添加したStemSpan培地にて培養したLSK細胞はSDHF−4−ΔTM−Fc100ng/mlの存在下において非存在下と比較してLIN(−)細胞の割合が増加するだけでなく、LIN(−),Sca1(+),c−kit(+)分画の割合も増加することが判明した。この結果は、SDHF−4細胞外領域・リコンビナントタンパク質にマウス造血幹細胞の未分化状態を維持する作用があることを示している。 As a result, as shown in FIG. 17, LSK cells cultured in StemSpan medium supplemented with SCF, IL-3, IL-6 were not present in the presence of SDHF-4-ΔTM-Fc 100 ng / ml. In comparison, it was found that not only the proportion of LIN (−) cells increased, but also the proportions of LIN (−), Sca1 (+), and c-kit (+) fractions increased. This result shows that the SDHF-4 extracellular region / recombinant protein has an action of maintaining the undifferentiated state of mouse hematopoietic stem cells.
脂肪細胞分化の検定とオイルレッドO染色法
SDHF−6遺伝子をレトロウィルスベクター(pMX-puro)導入した細胞とベクターのみを導入した細胞をそれぞれAlpha−MEM+20%牛胎児血清の条件にて4週間以上継代せずにConfluentの状態で培養し続けた。細胞は10%ホルマリンにて固定後オイルレッドOにて染色した。
その結果、第18図に示されるように、SDHF−6をレトロウィルス発現ベクターpMX−puroを用いてOP9細胞に高発現させると約4週間の培養の後、効率に脂肪細胞に分化させる効果があることが判明した。この遺伝子が、造血幹細胞よりもむしろ間葉系の細胞に作用して脂肪細胞への分化に関与していると考えられた。
Adipocyte differentiation assay and oil red O staining method SDHF-6 gene-introduced retrovirus vector (pMX-puro) -introduced cell and vector-only cell in Alpha-MEM + 20% fetal bovine serum for 4 weeks or more The culture was continued in the confluent state without subculture. The cells were fixed with 10% formalin and then stained with oil red O.
As a result, as shown in FIG. 18, when SDHF-6 is highly expressed in OP9 cells using the retroviral expression vector pMX-puro, it has the effect of efficiently differentiating into adipocytes after about 4 weeks of culture. It turned out to be. This gene was thought to be involved in the differentiation into adipocytes by acting on mesenchymal cells rather than hematopoietic stem cells.
SDHF−4に対するポリクローナル抗体の作製及びそれによる骨髄内造血支持細胞の単離
SDHF−4のアミノ酸配列をもとに、合成ペプチド(GYMAKDKFRRMNEGQVY(第32-48番目のアミノ酸に相当する))を設計しラビットに免疫してポリクローナル抗体を作製した。
このペプチドを Epoxy-activated Sepharose 6B(ファルマシア社)に結合させ、抗原カラムを作製、これにより、得られたポリクローナル抗体をアフィニティー精製した。この抗体を用い、CHO−k1細胞にpSSRα−bsrベクターを用いてSDHF−4を発現させた細胞(SDHF4-cl12)とベクターだけ導入した細胞(mock)を比較してウェスタン解析を施行したところ、非常に特異的にSDHF−4遺伝子産物を認識できることがわかった(第19図)。また、得られた精製抗体1mgを LinKit Fluoro-Link (ISL社) によりFITC標識し、同じ細胞をFACS calibur(Becton Dickinson社)にて解析したところ、この抗体はFlowcytemetryにも使用可能であることがわかった(第20図; ○線がmock、■線がSDHF4-cl12)。この抗体を用いてC57BL/6Jマウスの骨髄細胞を染色し、FACS Vantage(Becton Dickinson社)によりSDHF4を発現している細胞をソーティングして回収したところ(第21図)、これらの細胞は培養ディッシュに付着し、形態学的にも骨髄造血支持細胞に相当する間葉系の細胞であった(第22図)。
Preparation of polyclonal antibody against SDHF-4 and isolation of intramedullary hematopoietic feeder cells Based on the amino acid sequence of SDHF-4, a synthetic peptide (GYMAKDKFRRMNEGQVY (corresponding to amino acids 32-48)) was designed. Rabbits were immunized to produce polyclonal antibodies.
This peptide was bound to Epoxy-activated Sepharose 6B (Pharmacia) to prepare an antigen column, whereby the resulting polyclonal antibody was affinity purified. When this antibody was used, Western analysis was performed by comparing a cell in which SDHF-4 was expressed using a pSSRα-bsr vector (SDHF4-cl12) in a CHO-k1 cell and a cell in which only the vector was introduced (mock). It was found that the SDHF-4 gene product can be recognized very specifically (FIG. 19). Moreover, when 1 mg of the obtained purified antibody was labeled with FITC by LinKit Fluoro-Link (ISL) and the same cells were analyzed by FACS calibur (Becton Dickinson), this antibody could be used for Flowcytemetry. Okay (Fig. 20; ○ line is mock, ■ line is SDHF4-cl12). When this antibody was used to stain bone marrow cells of C57BL / 6J mice and cells expressing SDHF4 were sorted and collected by FACS Vantage (Becton Dickinson) (FIG. 21), these cells were cultured in a dish. The cells were mesenchymal cells that morphologically corresponded to bone marrow hematopoietic support cells (FIG. 22).
本発明によって、ストローマ細胞に由来する、新規な造血関連遺伝子の単離を行うことに成功し、それらが造血幹細胞増殖調節活性を有することが実証された。これによって、造血幹細胞の生体外増幅、造血器悪性腫瘍の移植治療、幹細胞による再生医学、遺伝子治療、免疫療法、細胞移植、遺伝子治療、神経疾患(アルツハイマー病、脳梗塞、変性神経疾患等)、肝疾患(肝硬変等)、肺疾患、心筋疾患、糖尿病、骨疾患、及び慢性腎疾患等の広範囲な応用を可能としたものである。 The present invention succeeded in isolating novel hematopoietic-related genes derived from stromal cells, and demonstrated that they have hematopoietic stem cell proliferation regulatory activity. This allows in vitro amplification of hematopoietic stem cells, transplantation treatment of hematopoietic malignant tumors, regenerative medicine with stem cells, gene therapy, immunotherapy, cell transplantation, gene therapy, neurological diseases (Alzheimer's disease, cerebral infarction, degenerative neurological diseases, etc.) It enables a wide range of applications such as liver diseases (cirrhosis, etc.), lung diseases, myocardial diseases, diabetes, bone diseases, and chronic kidney diseases.
Claims (19)
(a)配列番号10に示されるアミノ酸配列。 Genes encoding the following proteins (a):
(A) The amino acid sequence shown in SEQ ID NO: 10.
(a)配列番号9に示される塩基配列からなるDNA。 A gene containing the following DNA (a):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 9.
請求項1〜2の何れかに記載の遺伝子を有効成分として含有する、ストローマ細胞の造血幹細胞増殖活性の調節用組成物。 For use in the adjustment method according to any of claims 11 to 13,
A composition for regulating hematopoietic stem cell proliferation activity of stromal cells, comprising the gene according to claim 1 as an active ingredient.
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