JP2013094098A - Method of manufacturing parallel fibrous connective tissue - Google Patents
Method of manufacturing parallel fibrous connective tissue Download PDFInfo
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- JP2013094098A JP2013094098A JP2011239140A JP2011239140A JP2013094098A JP 2013094098 A JP2013094098 A JP 2013094098A JP 2011239140 A JP2011239140 A JP 2011239140A JP 2011239140 A JP2011239140 A JP 2011239140A JP 2013094098 A JP2013094098 A JP 2013094098A
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- mesenchymal stem
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- connective tissue
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Abstract
Description
本発明は、平行線維性結合組織の製造方法に関し、詳しくは、ＰＲＥＬＰと接触させた状態で間葉系幹細胞を培養することを含む、平行線維性結合組織の製造方法に関する。また、本発明は、平行線維性結合組織の製造に有用な、細胞、組み合わせ物、キットに関する。 The present invention relates to a method for producing parallel fibrous connective tissue, and more particularly, to a method for producing parallel fibrous connective tissue, comprising culturing mesenchymal stem cells in contact with PRELP. The present invention also relates to cells, combinations and kits useful for the production of parallel fibrous connective tissue.
靭帯損傷は、関節を支える靭帯へ、スポーツ外傷や交通事故等によって異常な力が加わり、靭帯が裂けたり、切断したりすることにより生じる損傷である。重度の靭帯損傷を治療する場合には、損傷した箇所へ靭帯を移植する必要があるが、靭帯を人工的に再生する技術は未だ開発されていないため、正常な他の靭帯の一部を摘出して移植に供する必要があり、結果として正常な靭帯をも傷めてしまうことになる。そのため、人工的にインビトロで靭帯を再生する技術の開発が望まれている。 Ligament damage is damage that occurs when an abnormal force is applied to a ligament supporting a joint due to a sports injury or a traffic accident, and the ligament is torn or cut. When treating severe ligament damage, it is necessary to transplant the ligament to the damaged part, but since a technique to artificially regenerate the ligament has not been developed yet, a part of other normal ligaments is removed. Therefore, it is necessary to use for transplantation, and as a result, a normal ligament is also damaged. Therefore, development of a technique for artificially regenerating a ligament in vitro is desired.
一方、Proline/arginine-rich end leucine-rich repeat protein（ＰＲＥＬＰ）は、結合組織細胞外マトリクスに含まれるタンパク質である。ＰＲＥＬＰは、基底膜をその下に下層の結合組織にアンカーする分子として機能することが知られている。ＰＲＥＬＰは、Ｉ型コラーゲンを基底膜に、ＩＩ型コラーゲンを軟骨へ結合させることが示されている。ＰＲＥＬＰは、基底膜ヘパラン硫酸プロテオグリカンであるパールカンへも結合することが報告されている。ＰＲＥＬＰは、Hutchinson-Gilford progeria (HGP)の病態に関与することが示唆されており、この疾患の患者では、基底膜及び軟骨におけるコラーゲンの結合が欠失していることが報告されている。 On the other hand, Proline / arginine-rich end leucine-rich repeat protein (PRELP) is a protein contained in the connective tissue extracellular matrix. PRELP is known to function as a molecule that anchors the basement membrane to underlying connective tissue. PRELP has been shown to bind type I collagen to the basement membrane and type II collagen to cartilage. PRELP has also been reported to bind to perlecan, a basement membrane heparan sulfate proteoglycan. PRELP has been suggested to be involved in the pathology of Hutchinson-Gilford progeria (HGP), and it has been reported that patients with this disease lack collagen binding in the basement membrane and cartilage.
ＰＲＥＬＰは、関節内の間葉系幹細胞において発現が亢進していることが報告されている（非特許文献１）。 PRELP has been reported to be upregulated in mesenchymal stem cells in joints (Non-patent Document 1).
本発明は、靱帯等の平行線維性結合組織をインビトロで製造する技術を提供することを目的とする。 An object of this invention is to provide the technique which manufactures parallel fibrous connective tissue, such as a ligament, in vitro.
前記課題を解決すべく本発明者らは鋭意検討した結果、ヒト脊柱靭帯骨化組織の二次元電気泳動パターンをヒト骨組織のそれと比較し、靭帯に特異的に発現するタンパク質としてＰＲＥＬＰを見出した。この遺伝子をマウス間葉系幹細胞に導入し、得られたトランスフェクタントを３Ｄゲル上で培養したところ、驚くべきことにＩ型コラーゲンを含む、靱帯組織様の平行線維性結合組織が形成されることを見出し、更に検討を進めた結果、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors compared the two-dimensional electrophoresis pattern of human vertebral ligament ossification tissue with that of human bone tissue, and found PRELP as a protein specifically expressed in the ligament. . When this gene was introduced into a mouse mesenchymal stem cell and the obtained transfectant was cultured on a 3D gel, surprisingly, a parallel fibrous connective tissue like a ligament tissue containing type I collagen was formed. As a result, the present invention was completed.
即ち、本発明は、下記の通りである。
［１］ＰＲＥＬＰと接触させた状態で間葉系幹細胞を培養すること、及び培養物から平行線維性結合組織を単離することを含む、平行線維性結合組織の製造方法。
［２］ＰＲＥＬＰと接触させた状態での間葉系幹細胞の培養が、ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞を培養することにより行われる、［１］記載の製造方法。
［３］間葉系幹細胞の培養が、３次元培養により行われる、［１］又は［２］記載の製造方法。
［４］ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞。
［５］ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞、及び３次元培養用担体を含む組み合わせ物。
［６］（１）ＰＲＥＬＰ、又はその発現ベクター；及び
（２）間葉系幹細胞
を含む組み合わせ物。
［７］更に、３次元培養用担体を含む、［６］記載の組み合わせ物。
［８］［４］記載の間葉系幹細胞、又は［５］〜［７］のいずれかに記載の組み合わせ物を含む、平行線維性結合組織製造用キット。
That is, the present invention is as follows.
[1] A method for producing parallel fibrous connective tissue, comprising culturing mesenchymal stem cells in contact with PRELP, and isolating parallel fibrous connective tissue from the culture.
[2] The production method according to [1], wherein the mesenchymal stem cells are cultured in a state of being contacted with PRELP by culturing the mesenchymal stem cells into which the PRELP expression vector has been introduced.
[3] The production method according to [1] or [2], wherein the mesenchymal stem cells are cultured by three-dimensional culture.
[4] Mesenchymal stem cells into which a PRELP expression vector has been introduced.
[5] A combination comprising a mesenchymal stem cell into which a PRELP expression vector has been introduced, and a three-dimensional culture carrier.
[6] A combination comprising (1) PRELP or an expression vector thereof; and (2) a mesenchymal stem cell.
[7] The combination according to [6], further comprising a three-dimensional culture carrier.
[8] A kit for producing a parallel fibrous connective tissue, comprising the mesenchymal stem cell according to [4] or the combination according to any one of [5] to [7].
本発明によれば、インビトロの系で靱帯様の平行線維性結合組織を製造することができる。これにより、人工靱帯の作製が可能となり、スポーツ選手などの靭帯断裂による運動障害などの治療に有用である。 According to the present invention, a ligament-like parallel fibrous connective tissue can be produced in an in vitro system. This makes it possible to produce an artificial ligament, which is useful for treating movement disorders caused by ligament rupture in athletes and the like.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
（平行線維性結合組織の製造方法）
本発明は、ＰＲＥＬＰと接触させた状態で間葉系幹細胞を培養すること、及び培養物から線維組織を単離することを含む、平行線維性結合組織の製造方法を提供する。
(Method for producing parallel fibrous connective tissue)
The present invention provides a method for producing parallel fibrous connective tissue, comprising culturing mesenchymal stem cells in contact with PRELP and isolating fibrous tissue from the culture.
Proline/arginine-rich end leucine-rich repeat protein（ＰＲＥＬＰ）は、生体内において結合組織細胞外マトリクスに含まれる公知のポリペプチドである。 Proline / arginine-rich end leucine-rich repeat protein (PRELP) is a known polypeptide contained in the connective tissue extracellular matrix in vivo.
本明細書中、ＰＲＥＬＰは通常、哺乳動物由来のものを意味する。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜、イヌ、ネコ等のペット、ヒト、サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。ＰＲＥＬＰは、好ましくは霊長類（ヒト等）又はげっ歯類（マウス等）由来のものである。 In the present specification, PRELP usually means a mammal-derived one. Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. PRELP is preferably derived from primates (such as humans) or rodents (such as mice).
本明細書中、ポリペプチドやポリヌクレオチド等の因子について、「生物Ｘ由来」とは、該ポリペプチド又はポリヌクレオチドが、生物Ｘにおいて天然に発現しているポリペプチド又はポリヌクレオチドと同一又は実質同一のアミノ酸配列又はポリヌクレオチド配列を含むことを意味する。「実質的に同一」とは、着目したアミノ酸配列又は核酸配列が、生物Ｘにおいて天然に発現している因子のアミノ酸配列又は核酸配列と７０％以上（好ましくは８０％以上、より好ましくは９０％以上、更に好ましくは９５％以上、最も好ましくは９９％以上）の同一性を有しており、且つ当該タンパク質の活性が維持されていることを意味する。 In the present specification, with respect to factors such as polypeptides and polynucleotides, “derived from organism X” means that the polypeptide or polynucleotide is identical or substantially identical to the polypeptide or polynucleotide naturally expressed in organism X Of the amino acid sequence or polynucleotide sequence. “Substantially the same” means that the focused amino acid sequence or nucleic acid sequence is 70% or more (preferably 80% or more, more preferably 90%) of the amino acid sequence or nucleic acid sequence of the factor naturally expressed in the organism X Or more, more preferably 95% or more, most preferably 99% or more), and the activity of the protein is maintained.
ＰＲＥＬＰのヌクレオチド配列やアミノ酸配列は公知である。ヒトＰＲＥＬＰ及びマウスＰＲＥＬＰの代表的なアミノ酸配列及びそれをコードするポリヌクレオチド配列（ｃＤＮＡ配列）は以下の通りである。尚、かっこ内は、ＮＣＢＩが提供するＧＥＮＢＡＮＫのアクセッション番号を示す。
［ヒトＰＲＥＬＰ アミノ酸配列］
配列番号２（NP_002716）：配列番号１（NM_002725 REGION: 201..1349）のポリヌクレオチドによりコード
配列番号４（NP_958505）：配列番号３（NM_201348 REGION: 191..1339）のポリヌクレオチドによりコード
配列番号６（配列番号２からシグナル配列（１−２０）を削除した（成熟型））：配列番号５（配列番号１からシグナル配列コード領域を削除）のポリヌクレオチドによりコード
配列番号８（配列番号４からシグナル配列（１−２０）を削除した（成熟型））：配列番号７（配列番号３からシグナル配列コード領域を削除）のポリヌクレオチドによりコード
［マウスＰＲＥＬＰ アミノ酸配列］
配列番号１０（NP_473418）：配列番号９（NM_054077 REGION: 164..1300）のポリヌクレオチドによりコード
配列番号１２（配列番号１０からシグナル配列（１−２１）を削除した（成熟型））：配列番号１１（配列番号９からシグナル配列コード領域を削除）のポリヌクレオチドによりコード
The nucleotide sequence and amino acid sequence of PRELP are known. Representative amino acid sequences of human PRELP and mouse PRELP and polynucleotide sequences (cDNA sequences) encoding the same are as follows. The parentheses indicate GENBANK accession numbers provided by NCBI.
[Human PRELP amino acid sequence]
SEQ ID NO: 2 (NP_002716): encoded by the polynucleotide of SEQ ID NO: 1 (NM_002725 REGION: 201..1349) SEQ ID NO: 4 (NP_958505): encoded by the polynucleotide of SEQ ID NO: 3 (NM_201348 REGION: 191..1339) 6 (signal sequence (1-20) was deleted from SEQ ID NO: 2 (mature type)): encoded by SEQ ID NO: 8 (from SEQ ID NO: 4) according to the polynucleotide of SEQ ID NO: 5 (signal sequence coding region deleted from SEQ ID NO: 1) Signal sequence (1-20) was deleted (mature type)): encoded by polynucleotide of SEQ ID NO: 7 (signal sequence coding region deleted from SEQ ID NO: 3) [mouse PRELP amino acid sequence]
SEQ ID NO: 10 (NP_473418): Coding SEQ ID NO: 12 (signal sequence (1-21) was deleted from SEQ ID NO: 10 (mature)) by the polynucleotide of SEQ ID NO: 9 (NM_054077 REGION: 164..1300): SEQ ID NO: 11 (deleting the signal sequence coding region from SEQ ID NO: 9)
本明細書中、ＰＲＥＬＰの活性とは、間葉系幹細胞へ接触させた状態で該細胞を培養することにより、平行線維性結合組織の形成を誘導する活性を意味する。 In this specification, the activity of PRELP means the activity of inducing the formation of parallel fibrous connective tissue by culturing the cells in contact with mesenchymal stem cells.
本明細書中、「間葉系幹細胞」とは、未分化の状態で増殖し、骨細胞、軟骨細胞及び脂肪細胞の全て又はいくつかへの分化が可能な幹細胞又はその前駆細胞の集団を広義に意味する。 In the present specification, the term “mesenchymal stem cell” broadly means a population of stem cells or progenitor cells that proliferate in an undifferentiated state and can differentiate into all or some of bone cells, chondrocytes and adipocytes. Means to.
本明細書中、間葉系幹細胞は、通常、哺乳動物由来のものを意味する。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜、イヌ、ネコ等のペット、ヒト、サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。間葉系幹細胞は、好ましくは霊長類（ヒト等）又はげっ歯類（マウス等）由来のものである。 In the present specification, mesenchymal stem cells usually mean those derived from mammals. Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. The mesenchymal stem cells are preferably derived from primates (human etc.) or rodents (mouse etc.).
生体においては、間葉系幹細胞は骨髄、末梢血、臍帯血、脂肪組織等の組織中に低頻度で存在する。間葉系幹細胞は、これらの組織から公知の方法で単離することが出来る。例えば、間葉系幹細胞は、パーコールグラディエント法により骨髄液から単離することができる（Hum. Cell, vol.10, p.45-50, 1997）。或いは、骨髄穿刺後の造血幹細胞等の培養、継代により間葉系幹細胞を単離することができる（Journal of Autoimmunity, 30 (2008) 163e171）。ヒト滑膜、半月板、関節内靭帯、筋肉、脂肪組織、及び骨髄からセルソーターで間葉系幹細胞を単離する方法が報告されている（Journal of Orthopaedic Research, 27:435-441, 2009）。また、市販された間葉系幹細胞を用いてもよい。 In a living body, mesenchymal stem cells are present at a low frequency in tissues such as bone marrow, peripheral blood, umbilical cord blood, and adipose tissue. Mesenchymal stem cells can be isolated from these tissues by a known method. For example, mesenchymal stem cells can be isolated from bone marrow fluid by the Percoll gradient method (Hum. Cell, vol. 10, p. 45-50, 1997). Alternatively, mesenchymal stem cells can be isolated by culture and passage of hematopoietic stem cells after bone marrow puncture (Journal of Autoimmunity, 30 (2008) 163e171). A method of isolating mesenchymal stem cells from human synovium, meniscus, intra-articular ligament, muscle, adipose tissue, and bone marrow with a cell sorter has been reported (Journal of Orthopedic Research, 27: 435-441, 2009). Commercially available mesenchymal stem cells may also be used.
本発明において用いられる間葉系幹細胞は、好ましくは単離されている。「単離」とは、天然に存在する状態とは異なる状態に人為的に置かれること、例えば、天然に存在する状態から、目的とする成分以外の成分を除去する操作が施されていることを意味する。 The mesenchymal stem cells used in the present invention are preferably isolated. “Isolated” means that the artificially placed in a state different from the naturally occurring state, for example, an operation to remove components other than the target component from the naturally occurring state. Means.
単離された間葉系幹細胞の純度（総細胞数に占める間葉系幹細胞の百分率）は、通常７０％以上、好ましくは８０％以上、より好ましくは９０％以上、最も好ましくは実質的に１００％である。 The purity of the isolated mesenchymal stem cells (percentage of mesenchymal stem cells in the total number of cells) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably substantially 100. %.
間葉系幹細胞を採取するソースとなる個体は特に制限されないが、得られる線維組織が移植医療に使用される場合には、拒絶反応が起こらないという観点から、患畜自身又はＭＨＣの型が同一もしくは実質的に同一の他個体から間葉系幹細胞を採取することが好ましい。ここでＭＨＣの型が「実質的に同一」とは、免疫抑制剤等の使用により、該間葉系幹細胞から得られた線維組織を患畜に移植した場合に移植した線維組織中に含まれる細胞が生着可能な程度にＭＨＣの型が一致していることをいう。 The individual to be used as a source for collecting mesenchymal stem cells is not particularly limited. However, when the obtained fibrous tissue is used for transplantation medical treatment, from the viewpoint that rejection does not occur, the patient itself or the type of MHC is the same or It is preferable to collect mesenchymal stem cells from substantially the same individual. Here, the type of MHC is “substantially identical” means that cells contained in the transplanted fibrous tissue when the fibrous tissue obtained from the mesenchymal stem cells is transplanted into a patient by using an immunosuppressant or the like. This means that the MHC types match to such an extent that can be engrafted.
間葉系幹細胞は、その培養に適した自体公知の培地で前培養することができる。そのような培地としては、例えば、約５〜２０％の胎仔ウシ血清を含む最小必須培地（ＭＥＭ）、ダルベッコ改変イーグル培地（ＤＭＥＭ）、Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ、ＲＰＭＩ１６４０培地、１９９培地、Ｆ１２培地等が挙げられるが、それらに限定されない。 Mesenchymal stem cells can be pre-cultured in a medium known per se suitable for the culture. Examples of such a medium include a minimum essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), Basal Medium Eagle, RPMI 1640 medium, 199 medium, and F12 medium. However, it is not limited to them.
本発明において、ＰＲＥＬＰと接触させた状態での間葉系幹細胞の培養方法は、特に限定されることはなく、線維組織の形成を誘導可能であればいかなる方法であってもよい。例えば、（Ａ）ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞を培養する方法、（Ｂ）線維組織の形成を誘導するのに十分な量のＰＲＥＬＰ（好ましくは、単離されたＰＲＥＬＰ）を含有する培地中で間葉系幹細胞を培養する方法等を採用することができる。（Ａ）においては、導入された発現ベクターから発現したＰＲＥＬＰが間葉系幹細胞の微小環境中や培地中に放出され、これが間葉系幹細胞へ接触する。好ましくは、ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞を培養する。 In the present invention, the method for culturing mesenchymal stem cells in contact with PRELP is not particularly limited, and any method may be used as long as it can induce the formation of fibrous tissue. For example, (A) a method for culturing a mesenchymal stem cell into which a PRELP expression vector has been introduced, (B) a sufficient amount of PRELP (preferably isolated PRELP) to induce the formation of fibrous tissue A method of culturing mesenchymal stem cells in a medium to be used can be employed. In (A), PRELP expressed from the introduced expression vector is released into the microenvironment of the mesenchymal stem cell or into the medium, and this contacts the mesenchymal stem cell. Preferably, mesenchymal stem cells into which a PRELP expression vector has been introduced are cultured.
ＰＲＥＬＰ発現ベクターは、プロモーター及びＰＲＥＬＰをコードするポリヌクレオチドを含み、当該プロモーターは作動可能に当該ポリヌクレオチドに連結されている。当該プロモーターは、宿主である間葉系幹細胞内で転写を開始可能なものであれば特に限定されない。 The PRELP expression vector includes a promoter and a polynucleotide encoding PRELP, and the promoter is operably linked to the polynucleotide. The promoter is not particularly limited as long as it can initiate transcription in a mesenchymal stem cell as a host.
発現ベクターとしては、特に限定されないが、例えば、レトロウイルス（レンチウイルスを含む）、アデノウイルス、アデノ随伴ウイルス、センダイウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シルビスウイルス、ラブドウイルス、パラミクソウイルス、オルソミクソウイルス等のウイルスベクター；ＹＡＣ（Ｙｅａｓｔ ａｒｔｉｆｉｃｉａｌ ｃｈｒｏｍｏｓｏｍｅ）ベクター、ＢＡＣ（Ｂａｃｔｅｒｉａｌ ａｒｔｉｆｉｃｉａｌ ｃｈｒｏｍｏｓｏｍｅ）ベクター、ＰＡＣ（Ｐ１−ｄｅｒｉｖｅｄ ａｒｔｉｆｉｃｉａｌ ｃｈｒｏｍｏｓｏｍｅ）ベクター等の人工染色体ベクター；プラスミドベクター；宿主細胞内で自律複製可能なエピゾーマルベクター等が挙げられる。該ベクターを間葉系幹細胞に導入する場合は、リポフェクション法、マイクロインジェクション法、ＤＥＡＥデキストラン法、遺伝子銃法、エレクトロポレーション法、リン酸カルシウム法等の方法を用いることができる。 Although it does not specifically limit as an expression vector, For example, retrovirus (including lentivirus), adenovirus, adeno-associated virus, Sendai virus, herpes virus, vaccinia virus, pox virus, poliovirus, silbis virus, rhabdovirus, Viral vectors such as paramyxovirus and orthomyxovirus; artificial chromosome vectors such as YAC (Year Artificial Chromome) vector, BAC (Bacterial Artificial Chromome) vector, PAC (P1-derived artificial chromosome) vector; Examples include an episomal vector capable of autonomous replication. When introducing the vector into mesenchymal stem cells, a lipofection method, a microinjection method, a DEAE dextran method, a gene gun method, an electroporation method, a calcium phosphate method, or the like can be used.
また、発現ベクターとしてウイルスベクターを用いる場合は、パッケージング細胞を利用することもできる。パッケージング細胞とは、ウイルスの構造タンパク質をコードする遺伝子を導入した細胞であって、この細胞に目的遺伝子を組み込んだ組換えウイルスＤＮＡを導入すると、該組換えウイルス粒子を産生するものをいう。それゆえパッケージング細胞としては、ウイルス粒子の構成に必要なタンパク質を組換えウイルスベクターに対して補給する細胞であればいかなるものも用いることができ、例えば、ヒト腎臓由来のＨＥＫ２９３細胞やマウス線維芽細胞由来のＮＩＨ３Ｔ３細胞をベースにしたパッケージング細胞；Ｅｃｏｔｒｏｐｉｃ ｖｉｒｕｓ由来エンベロープ糖タンパク質を発現するよう設計されているＰｌａｔ−Ｅ細胞、Ａｍｐｈｏｔｒｏｐｉｃ ｖｉｒｕｓ由来エンベロープ糖タンパク質を発現するよう設計されているＰｌａｔ−Ａ細胞、及び水疱性口内炎ウイルス由来エンベロープ糖タンパク質を発現するよう設計されているＰｌａｔ−ＧＰ細胞等を用いることができる（Ｐｌａｔ−Ｅ細胞、Ｐｌａｔ−Ａ細胞及びＰｌａｔ−ＧＰ細胞は、ＣＥＬＬ ＢＩＯＬＡＢＳ社より購入することができる）。該パッケージング細胞へのウイルスベクターの導入方法としては、特に限定されるものではなく、リポフェクション、エレクトロポレーション、リン酸カルシウム法等の従来公知の方法を利用することができる。 In addition, when a viral vector is used as an expression vector, a packaging cell can be used. A packaging cell refers to a cell into which a gene encoding a viral structural protein has been introduced, which produces the recombinant virus particle when a recombinant viral DNA incorporating the target gene is introduced into the cell. Therefore, any cell can be used as the packaging cell as long as it is a cell that replenishes the recombinant virus vector with a protein necessary for the construction of the virus particles. For example, HEK293 cells derived from human kidney or mouse fibroblasts can be used. Cell-derived NIH3T3 cell-based packaging cells; Plato-E cells designed to express Ecotropic virus-derived envelope glycoproteins, Plato-A cells designed to express Amphoteric virus-derived envelope glycoproteins , And Plato-GP cells designed to express vesicular stomatitis virus-derived envelope glycoproteins (Plat-E cells, Plat-A cells and Plat-GP cells are CEL It can be purchased from BIOLABS Co., Ltd.). The method for introducing the viral vector into the packaging cell is not particularly limited, and a conventionally known method such as lipofection, electroporation, or calcium phosphate method can be used.
また、発現ベクターを用いて遺伝子を導入する場合は、該遺伝子の導入を確認するため同時にマーカー遺伝子を利用することもできる。マーカー遺伝子とは、該マーカー遺伝子を細胞に導入することにより、細胞の選別や選択を可能とするような遺伝子全般をいい、例えば、薬剤耐性遺伝子、蛍光タンパク質遺伝子、発光酵素遺伝子、発色酵素遺伝子等が挙げられる。薬剤耐性遺伝子としては、ネオマイシン耐性遺伝子、テトラサイクリン耐性遺伝子、カナマイシン耐性遺伝子、ゼオシン耐性遺伝子、ハイグロマイシン耐性遺伝子等が挙げられ、蛍光タンパク質遺伝子としては、緑色蛍光タンパク質（ＧＦＰ）遺伝子、黄色蛍光タンパク質（ＹＦＰ）遺伝子、赤色蛍光タンパク質（ＲＦＰ）遺伝子等が挙げられる。また、発光酵素遺伝子としては、ルシフェラーゼ遺伝子等が挙げられ、発色酵素遺伝子としては、βガラクトシターゼ遺伝子、βグルクロニダーゼ遺伝子、アルカリフォスファターゼ遺伝子等が挙げられる。これらのマーカー遺伝子については一種又は二種以上を組み合わせて用いることができ、また、ネオマイシン耐性遺伝子とβガラクトシダーゼ遺伝子との融合遺伝子であるβｇｅｏ遺伝子等のような、二種以上のマーカー遺伝子を含む融合遺伝子も用いることができる。 In addition, when a gene is introduced using an expression vector, a marker gene can be used simultaneously to confirm introduction of the gene. The marker gene refers to all genes that enable selection and selection of cells by introducing the marker gene into cells, such as drug resistance genes, fluorescent protein genes, luminescent enzyme genes, chromogenic enzyme genes, etc. Is mentioned. Examples of drug resistance genes include neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene, zeocin resistance gene, hygromycin resistance gene and the like, and fluorescent protein genes include green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP). ) Gene, red fluorescent protein (RFP) gene and the like. Further, examples of the luminescent enzyme gene include a luciferase gene, and examples of the chromogenic enzyme gene include a β-galactosidase gene, a β-glucuronidase gene, and an alkaline phosphatase gene. These marker genes can be used singly or in combination of two or more, and also include a fusion containing two or more marker genes such as a βgeo gene that is a fusion gene of a neomycin resistance gene and a β-galactosidase gene. Genes can also be used.
発現ベクターは、所望によりエンハンサー、スプライシングシグナル、ポリＡ付加シグナル、ＳＶ４０複製オリジン（以下、ＳＶ４０ｏｒｉと略称する場合がある）などを、それぞれ機能可能な態様で含有していてもよい。 The expression vector may contain an enhancer, a splicing signal, a poly A addition signal, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like in a functional manner, if desired.
本発明は、このようにして得られたＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞をも提供する。当該間葉系幹細胞においては、導入された発現ベクターがゲノム内に組み込まれていてもよい。このような間葉系幹細胞は、ゲノム構造において、もとの間葉系幹細胞や、天然の哺乳動物から単離された間葉系幹細胞と明確に区別され得る。また、染色体外で自律複製可能なエピゾーマルベクターを用いた場合、ゲノム上には組み込まれないが、ＰＲＥＬＰは遺伝的に安定に間葉系幹細胞内に存在し、発現され得るので、このようなベクターを用いて得られる間葉系幹細胞もまた、本発明の範囲に包含される。 The present invention also provides mesenchymal stem cells into which the PRELP expression vector thus obtained has been introduced. In the mesenchymal stem cell, the introduced expression vector may be integrated into the genome. Such mesenchymal stem cells can be clearly distinguished from the original mesenchymal stem cells and mesenchymal stem cells isolated from natural mammals in the genome structure. In addition, when an episomal vector capable of autonomous replication outside the chromosome is used, PRELP is genetically stably present in mesenchymal stem cells and can be expressed. Mesenchymal stem cells obtained using such vectors are also encompassed within the scope of the present invention.
間葉系幹細胞の培養に用いられる培地は、動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地としては、例えば、ＢＭＥ培地、ＢＧＪｂ培地、ＣＭＲＬ １０６６培地、Ｇｌａｓｇｏｗ ＭＥＭ培地、Ｉｍｐｒｏｖｅｄ ＭＥＭ Ｚｉｎｃ Ｏｐｔｉｏｎ培地、ＩＭＤＭ培地、Ｍｅｄｉｕｍ １９９培地、Ｅａｇｌｅ ＭＥＭ培地、αＭＥＭ培地、ＤＭＥＭ培地、ハムＦ１２培地、ＲＰＭＩ １６４０培地、Ｆｉｓｃｈｅｒ’ｓ培地、及びこれらの混合培地等、動物細胞の培養に用いることのできる培地であれば特に限定されない。 A medium used for culturing mesenchymal stem cells can be prepared using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham F12 medium, RPMI 1640 The medium is not particularly limited as long as it can be used for culturing animal cells, such as a medium, Fischer's medium, and mixed medium thereof.
また、該培地は、血清含有培地又は無血清培地とすることができる。無血清培地とは、無調整又は未精製の血清を含まない培地を意味し、精製された血液由来成分や動物組織由来成分（例えば、増殖因子）が混入している培地や血清代替試薬（例えば、KSR（Invitrogen社）等）が添加されている培地などは無血清培地に該当するものとする。 The medium can be a serum-containing medium or a serum-free medium. A serum-free medium means a medium that does not contain unadjusted or unpurified serum, and is a medium or serum replacement reagent (for example, a purified blood-derived component or animal tissue-derived component (eg, growth factor)). , KSR (Invitrogen) etc.) is added to the serum-free medium.
該培地は、自体公知の添加物を含むことができる。添加物としては、特に限定されないが、例えば増殖因子（例えばインスリン等）、鉄源（例えばトランスフェリン等）、ミネラル（例えばセレン酸ナトリウム等）、糖類（例えばグルコース等）、有機酸（例えばピルビン酸、乳酸等）、血清蛋白質（例えばアルブミン等）、アミノ酸（例えば、非必須アミノ酸）、還元剤（例えば２−メルカプトエタノール等）、抗酸化剤、ビタミン類（例えばアスコルビン酸、ｄ−ビオチン等）、脂肪酸又は脂質、抗生物質（例えばストレプトマイシン、ペニシリン、ゲンタマイシン等）、緩衝剤（例えばＨＥＰＥＳ等）、無機塩類等が挙げられる。当該添加物は、それぞれ自体公知の濃度範囲内で含まれることが好ましい。 The medium can contain additives known per se. The additive is not particularly limited, but for example, growth factors (such as insulin), iron sources (such as transferrin), minerals (such as sodium selenate), saccharides (such as glucose), organic acids (such as pyruvic acid, Lactic acid etc.), serum proteins (eg albumin etc.), amino acids (eg nonessential amino acids), reducing agents (eg 2-mercaptoethanol etc.), antioxidants, vitamins (eg ascorbic acid, d-biotin etc.), fatty acids Alternatively, lipids, antibiotics (for example, streptomycin, penicillin, gentamicin, etc.), buffers (for example, HEPES, etc.), inorganic salts and the like can be mentioned. Each of the additives is preferably contained within a concentration range known per se.
一態様において、間葉系幹細胞は、シャーレ、プレート、ボトル等の培養容器中で、接着培養される。 In one embodiment, mesenchymal stem cells are adherently cultured in a culture container such as a petri dish, a plate, or a bottle.
好ましい態様において、間葉系幹細胞の培養は、３次元培養により行われる。３次元培養を行うことにより、平面での接着培養と比較して平行線維性結合組織の形成が促進される。本発明で「３次元培養」とは、ゾル−ゲル転移温度を有し、該転移温度より低い温度で可逆的にゾル状態を示すような高分子化合物を培養用の担体として使用し、必要に応じて、当該培養用担体に、細胞培養用生理活性物質を配合せしめて培養用の組成物として、それを用いて、当該培養用担体又は培養用組成物中の該高分子化合物をゲル状態にした培地中で、該ゲル物質に培養されるべき細胞及び／又は組織が包埋された状態で培養することを意味する。 In a preferred embodiment, the mesenchymal stem cells are cultured by three-dimensional culture. By performing the three-dimensional culture, the formation of parallel fibrous connective tissue is promoted as compared to the adhesion culture on a flat surface. In the present invention, “three-dimensional culture” means that a polymer compound having a sol-gel transition temperature and reversibly showing a sol state at a temperature lower than the transition temperature is used as a culture carrier. Accordingly, a physiologically active substance for cell culture is mixed with the culture support as a culture composition, and the polymer compound in the culture support or culture composition is gelled using the culture support. It means that the cells and / or tissues to be cultured in the gel material are embedded in the prepared medium.
３次元培養用の担体として使用する高分子化合物としては、例えば、ポリプロピレンオキサイドとポリエチレンオキサイドのブロック共重合体などに代表されるポリアルキレンオキサイドブロック共重合体；メチルセルロース、ヒドロキシプロピルセルロースなどのエーテル化セルロース；キトサン誘導体(K. R. Holme. et al. Macromolecules, 24, 3828(1991))；プルラン誘導体（出口茂ら、Polymer Preprints. Japan. 19, 936(1990))などの変性多糖類；ポリN-置換（メタ）アクリルアミド誘導体、ポリビニルアルコール部分酢化物、ポリビニルメチルエーテルなどに代表される温度感応性高分子と水溶性高分子化合物との結合体（例えば松田武久ら、Polymer Preprints. Japan. 39(8), 2559(1990))、生理的な条件下で自己重合し、微小な線維構造を有するハイドロゲルを形成するペプチドなどがあげられるが、これらに限定されるものではない。ＰｕｒａＭａｔｒｉｘ等の市販品を適宜使用することができる。 Examples of the polymer compound used as a carrier for three-dimensional culture include polyalkylene oxide block copolymers represented by block copolymers of polypropylene oxide and polyethylene oxide; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose Modified polysaccharides such as chitosan derivatives (KR Holme. Et al. Macromolecules, 24, 3828 (1991)); pullulan derivatives (Shigeru Deguchi, Polymer Preprints. Japan. 19, 936 (1990)); poly N-substituted (meta ) Conjugates of temperature-sensitive polymers and water-soluble polymer compounds represented by acrylamide derivatives, polyvinyl alcohol partial acetylates, polyvinyl methyl ether, etc. (for example, Takehisa Matsuda, Polymer Preprints. Japan. 39 (8), 2559 (1990)), a pepper that self-polymerizes under physiological conditions to form a hydrogel with a fine fibrous structure. Such as de the like, but not limited thereto. Commercial products such as PuraMatrix can be used as appropriate.
本発明においては、繊維組織を形成するのに十分な期間（通常１日以上、好ましくは２日以上）、ＰＲＥＬＰと接触させた状態で間葉系幹細胞が培養される。 In the present invention, mesenchymal stem cells are cultured in a state of being contacted with PRELP for a period sufficient for forming a fibrous tissue (usually 1 day or more, preferably 2 days or more).
本発明の工程（ｂ）における培養条件は、通常の動物細胞の培養条件に準じ適宜設定することができる。例えば、培養温度は、通常約３０〜４０℃、好ましくは約３７℃である。ＣＯ２濃度は、通常、約１〜１０％、好ましくは約２〜５％である。 The culture conditions in the step (b) of the present invention can be appropriately set according to normal animal cell culture conditions. For example, the culture temperature is usually about 30 to 40 ° C, preferably about 37 ° C. CO 2 concentration is typically about 1-10%, preferably about 2-5%.
培養の結果、培養物中に平行線維性結合組織が形成される。本明細書中、「平行線維性結合組織」とは、膠原線維が一方向に並列配列した密性結合組織をいう。密性結合組織とは、膠原線維が線維束を作って蜜に配列することにより形成された結合組織をいう。膠原線維は、通常Ｉ型コラーゲンを含有する。平行線維性結合組織は靭帯組織であり得る。 As a result of the culture, parallel fibrous connective tissue is formed in the culture. In the present specification, “parallel fibrous connective tissue” refers to a dense connective tissue in which collagen fibers are arranged in parallel in one direction. Close connective tissue refers to connective tissue formed by collagen fibers forming fiber bundles and arranging them in nectar. Collagen fibers usually contain type I collagen. The parallel fibrous connective tissue can be ligament tissue.
培養物とは、細胞を培養することにより得られる結果物をいい、細胞、培地、細胞分泌性成分等が含まれる。 The culture refers to a result obtained by culturing cells, and includes cells, culture media, cell secretory components, and the like.
肉眼観察や顕微鏡観察等によって、平行線維性結合組織の形成を確認した後、培養物から平行線維性結合組織を単離することにより、平行線維性結合組織を得ることができる。 A parallel fibrous connective tissue can be obtained by confirming the formation of the parallel fibrous connective tissue by macroscopic observation, microscopic observation or the like and then isolating the parallel fibrous connective tissue from the culture.
こうして得られる平行線維性結合組織は、人工靱帯として、スポーツ選手などの靭帯断裂による運動障害などの治療に有用である。 The parallel fibrous connective tissue thus obtained is useful as an artificial ligament for the treatment of movement disorders caused by ligament rupture in athletes.
（組み合わせ物、キット）
また、本発明は、ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞、及び３次元培養用の担体を含む組み合わせ物（組み合わせ物Ｉ）を提供する。
(Combination, kit)
The present invention also provides a combination (combination I) comprising mesenchymal stem cells into which a PRELP expression vector has been introduced, and a carrier for three-dimensional culture.
更に、本発明は、
（１）ＰＲＥＬＰ、又はその発現ベクター；及び
（２）間葉系幹細胞
を含む組み合わせ物（組み合わせ物ＩＩ）を提供する。
Furthermore, the present invention provides
Provided is a combination (Combination II) comprising (1) PRELP or an expression vector thereof; and (2) a mesenchymal stem cell.
組み合わせ物ＩＩは、更に３次元培養用の担体を含むことができる。 The combination II can further contain a carrier for three-dimensional culture.
ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞、組み合わせ物Ｉ及びＩＩを用いれば、上記本発明の製造方法により容易に平行線維性結合組織を製造することができる。従って、ＰＲＥＬＰ発現ベクターが導入された間葉系幹細胞、組み合わせ物Ｉ及びＩＩのそれぞれを、平行線維性結合組織製造用キットとすることもできる。 By using the mesenchymal stem cells and the combinations I and II into which the PRELP expression vector has been introduced, parallel fibrous connective tissue can be easily produced by the production method of the present invention. Therefore, each of the mesenchymal stem cells and the combinations I and II into which the PRELP expression vector has been introduced can be used as a kit for producing a parallel fibrous connective tissue.
本発明のキットに含まれる各構成要素は、各々別個に（あるいは可能であれば混合した状態で）水もしくは適当な緩衝液（例：TEバッファー、PBSなど）中に適当な濃度となるように溶解又は懸濁し、約−２０℃〜４℃で保存することができる。 Each component contained in the kit of the present invention is adjusted to an appropriate concentration in water or an appropriate buffer (eg, TE buffer, PBS, etc.) separately (or in a mixed state if possible). It can be dissolved or suspended and stored at about -20 ° C to 4 ° C.
本発明のキットには、上記本発明の製造方法に用いた種々の試薬（培地、遺伝子導入用試薬等）、培養容器、試験プロトコールを記載した指示書等を含めることができる。 The kit of the present invention can include various reagents (medium, reagent for gene introduction, etc.) used in the production method of the present invention, culture vessels, instructions describing the test protocol, and the like.
本発明の組み合わせ物及びキットに関する各用語の定義は、上述の「平行線維性結合組織の製造方法」の項における各用語の定義と同一である。 The definition of each term regarding the combination and kit of this invention is the same as the definition of each term in the above-mentioned "Method for producing parallel fibrous connective tissue" section.
以下に実施例を挙げて本発明をさらに具体的に説明するが、これらは単なる例示であって、本発明の範囲を何ら限定するものではない。 The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.
ヒトＰＲＥＬＰ ｃＤＮＡをクローニングした。クローニングしたＰＲＥＬＰ ｃＤＮＡをＸｈｏｌ処理し、ｐＣＡＧＧＳ発現ベクターのＸｈｏｌサイトに挿入した。発現ベクターがデザイン通りに構築されているか確認するために、構築後のベクターを制限酵素で消化し、電気泳動パターンを確認した。その結果、予想されたパターンの泳動図が確認された。また、挿入されたｃＤＮＡ領域のシーケンス解析を行ったところ、目的とする配列（GENBANKアクセッション番号：EAW91481.1）と一致することを確認した。 Human PRELP cDNA was cloned. The cloned PRELP cDNA was treated with Xhol and inserted into the Xhol site of the pCAGGS expression vector. In order to confirm whether the expression vector was constructed as designed, the constructed vector was digested with restriction enzymes, and the electrophoresis pattern was confirmed. As a result, an electrophoretogram of the expected pattern was confirmed. Moreover, when the sequence analysis of the inserted cDNA area | region was conducted, it confirmed that it corresponded with the target arrangement | sequence (GENBANK accession number: EAW91481.1).
Ｃ３Ｈ／１０Ｔ１／２−ｃｌｏｎｅ８マウス間葉系幹細胞（ＪＣＲＢ０００３）を１０％ＦＢＳ含むＢａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ（Ｗａｋｏ）にて培養後、０．２５％ Ｔｒｙｐｓｉｎ ａｎｄ ０．０２％ ＥＤＴＡにて剥離することを繰り返すことにより、継代増殖培養をおこなった。Ｃ３Ｈ／１０Ｔ１／２−ｃｌｏｎｅ８マウス間葉系幹細胞を３．５ｍｍシャーレ（ＢＤ）に７０％コンフルエントとなるまで培養後、培養液を５００μＬに調整した。２０ｍＭＴｒｉｓ−ＨＣＬ（ｐＨ７．４）バッファー（ＤＡＫＯ）１００μＬに、ｐＣＡＧＧＳ−ＰＲＥＬＰベクター２．０μｇを加え、Ｍｕｌｔｉ Ｆｅｃｔａｍ（Ｐｒｏｍｅｇａ）５０．０μＬを添加、混合後、室温にて３０分間静置させ、Ｏｐｔｉ−ＭＥＭ培養液（ｉｎｖｉｔｒｏｇｅｎ）５０μＬを加え、混合後、室温にて５分静置させた。３．５ｍｍウェル１枚に２００μＬのＭｕｌｔｉ Ｆｅｃｔａｍ、ｐＣＡＧＧＳ−ＰＲＥＬＰプラスミドＤＮＡ複合体を加え、均一となるようにプレートを揺らし、３７℃、５％ＣＯ２にて４時間培養した。培地を、新しい培養液（１０％ＦＢＳ含むＢａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ）に交換し、更に９６時間培養後、上清中にＰＲＥＬＰタンパク質が発現していることをウエスタンブロットにて確認した（図１）。２日に１度の頻度で、培養液を交換した。ＰｕｒａＭａｔｒｉｘ培養開始前のｐＣＡＧＧＳ−ＰＲＥＬＰトランスフェクションしたＣ３Ｈ／１０Ｔ１／２−ｃｌｏｎｅ８マウス間葉系幹細胞を図２に示す。 C3H / 10T1 / 2-clone8 mouse mesenchymal stem cells (JCRB0003) are cultured in Basal Medium Eagle (Wako) containing 10% FBS and then repeatedly detached with 0.25% Trypsin and 0.02% EDTA. By subcultivation culture was performed. After culturing C3H / 10T1 / 2-clone8 mouse mesenchymal stem cells in a 3.5 mm petri dish (BD) until 70% confluent, the culture solution was adjusted to 500 μL. To 100 μL of 20 mM Tris-HCL (pH 7.4) buffer (DAKO), 2.0 μg of pCAGGS-PRELP vector is added, and 50.0 μL of Multi Fectam (Promega) is added, mixed, and then allowed to stand at room temperature for 30 minutes. MEM culture solution (invitrogen) 50 μL was added, mixed, and allowed to stand at room temperature for 5 minutes. 200 μL of Multi Fectam and pCAGGS-PRELP plasmid DNA complex were added to one 3.5 mm well, and the plate was shaken so as to be uniform, and cultured at 37 ° C., 5% CO 2 for 4 hours. The medium was replaced with a new culture solution (Basal Medium Eagle containing 10% FBS), and after further culturing for 96 hours, it was confirmed by Western blot that the PRELP protein was expressed in the supernatant (FIG. 1). The culture medium was changed once every two days. FIG. 2 shows C3H / 10T1 / 2-clone8 mouse mesenchymal stem cells transfected with pCAGGS-PRELP before the start of PuraMatrix culture.
１週間後、シャーレに張り付いたＣ３Ｈ／１０Ｔ１／２−ｃｌｏｎｅ８マウス間葉系幹細胞を０．２５％ Ｔｒｙｐｓｉｎ ａｎｄ ０．０２％ ＥＤＴＡにて剥離し、遠心回収し、細胞ペレットを５．０×１０４個に調整し、２０％スクロース溶液１ｍＬにて洗浄し、超音波処理したＰｕｒａＭａｔｒｉｘ（ＢＤ）１ｍＬを加え、素早く混合し、新たなウェル中で培養した。 One week later, the C3H / 10T1 / 2-clone8 mouse mesenchymal stem cells attached to the petri dish were detached with 0.25% Trypsin and 0.02% EDTA, and centrifuged to collect the cell pellet at 5.0 × 10. 4 to adjust, washed with a 20% sucrose solution 1 mL, sonicated PuraMatrix (BD) 1 mL was added, rapidly mixed, and cultured in fresh wells.
ＰｕｒａＭａｔｒｉｘ中で培養を開始してから１日後において、線維状の形態を示した（図３）。ＰｕｒａＭａｔｒｉｘ中で培養を開始してから２日後には、ウェル全体が線維状の形態を示した（図４）。 One day after the start of culture in PuraMatrix, it showed a fibrous morphology (FIG. 3). Two days after the start of culture in PuraMatrix, the whole well showed a fibrous morphology (FIG. 4).
顕微鏡観察の結果、膠原線維が一方向に並列配列した構造が確認された（図３及び４）。電子顕微鏡下での観察でも同じ構造が確認された（図５）。形成された線維組織をＩ型コラーゲンに対する抗体を用いて免疫組織染色した結果、Ｉ型コラーゲンの発現が確認された。以上の結果から、得られた線維状の組織は、靭帯等の平行線維性結合組織であることが示唆された。 As a result of microscopic observation, a structure in which collagen fibers were arranged in parallel in one direction was confirmed (FIGS. 3 and 4). The same structure was confirmed by observation under an electron microscope (FIG. 5). As a result of immunohistochemical staining of the formed fibrous tissue with an antibody against type I collagen, expression of type I collagen was confirmed. From the above results, it was suggested that the obtained fibrous tissue is a parallel fibrous connective tissue such as a ligament.
本発明によれば、インビトロの系で靱帯様の平行線維性結合組織を製造することができる。これにより、人工靱帯の作製が可能となり、スポーツ選手などの靭帯断裂による運動障害などの治療に有用である。 According to the present invention, a ligament-like parallel fibrous connective tissue can be produced in an in vitro system. This makes it possible to produce an artificial ligament, which is useful for treating movement disorders caused by ligament rupture in athletes and the like.
Claims (8)
（２）間葉系幹細胞
を含む組み合わせ物。 (1) PRELP, or an expression vector thereof; and (2) a combination comprising mesenchymal stem cells.
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