JP4446711B2 - Cell capable of inducing differentiation into osteoblast and method of forming osteoblast - Google Patents
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本発明は、骨芽細胞へ分化誘導可能な細胞及び骨芽細胞の形成方法に関する。本発明は、骨の修復等に有用である。 The present invention relates to a cell capable of inducing differentiation into an osteoblast and a method for forming an osteoblast. The present invention is useful for bone repair and the like.
従来より、外傷や骨腫瘍の除去等により骨の修復が必要な場合には、患者本人の大腿骨等の自家骨を採取し、これを移植することが行われている。しかしながら、この方法は、患者の負担が大変大きい。一方、再生医学や組織工学の分野では、骨細胞に分化し得る幹細胞(骨幹細胞）を移植することにより骨の修復を行うことが研究されている。従来より、骨幹細胞は、骨髄中や脂肪細胞層中に見出されている。しかしながら、これらは安定供給に難がある。 Conventionally, when bone repair is required due to removal of trauma, bone tumor, etc., autologous bone such as the patient's own femur is collected and transplanted. However, this method is very burdensome for the patient. On the other hand, in the fields of regenerative medicine and tissue engineering, research has been conducted on bone repair by transplanting stem cells that can differentiate into bone cells (bone stem cells). Conventionally, bone stem cells have been found in bone marrow and adipocyte layers. However, these are difficult to supply stably.
本発明の目的は、安定供給が可能であり、骨芽細胞への誘導が可能な細胞及びそれを利用した、骨芽細胞の形成方法を提供することである。 An object of the present invention is to provide a cell that can be stably supplied and can be induced into an osteoblast, and a method for forming an osteoblast using the cell.
本願発明者は、鋭意研究の結果、ヒト羊膜上皮細胞層中に、骨芽細胞分化培地で培養することにより骨芽細胞に分化誘導可能な細胞が含まれていることを見出し、本発明を完成した。 As a result of diligent research, the present inventor has found that human amnion epithelial cell layers contain cells that can be induced to differentiate into osteoblasts by culturing in an osteoblast differentiation medium, and completed the present invention. did.
すなわち、本発明は、ヒト羊膜上皮細胞層中に存在する、骨芽細胞へ分化誘導可能な細胞を含む、骨芽細胞形成用細胞を提供する。さらに、本発明は、ヒト羊膜上皮細胞層に含まれる細胞を、骨芽細胞分化培地で培養し、骨芽細胞を誘導することを含む、骨芽細胞の形成方法を提供する。
That is, the present invention is present in human amniotic epithelial cell layer, including differentiation inducible cells into osteoblasts, providing osteoblasts forming cells. Furthermore, the present invention provides an osteoblast formation method comprising culturing cells contained in a human amniotic epithelial cell layer in an osteoblast differentiation medium and inducing osteoblasts.
本発明により、ヒト羊膜上皮細胞層中に存在し、骨芽細胞へ分化誘導可能な細胞が初めて提供された。本発明の細胞は、ヒト羊膜由来であり、ヒト羊膜は大きな組織でしかも出産後に医療廃棄物として処理されるものであるから安定供給が可能である。本発明の細胞又はこれから誘導された骨芽細胞は、骨の欠損部に移植することにより、骨の修復を行うことができる。 The present invention provides for the first time cells that are present in the human amniotic epithelial cell layer and can be induced to differentiate into osteoblasts. The cells of the present invention are derived from human amniotic membrane, and human amniotic membrane is a large tissue and is treated as medical waste after giving birth, and thus can be stably supplied. The cells of the present invention or osteoblasts derived therefrom can be repaired by transplanting them into a bone defect.
上記の通り、本発明の細胞は、ヒト羊膜上皮細胞層から分離されるものである。羊膜は、胎児由来の組織であるが、母体由来の胎盤に付着した状態で採取することができ、しかも、子宮内壁の全体を被覆する大きな組織であるので、大量に採取することができる。さらに、胎盤やそれに付着している羊膜は、医療廃棄物として処理されるものであるので、採取に倫理上の問題も生じない。 As described above, the cell of the present invention is isolated from the human amniotic epithelial cell layer. The amniotic membrane is a tissue derived from a fetus, but can be collected while attached to the placenta derived from the mother's body, and since it is a large tissue covering the entire inner wall of the uterus, it can be collected in large quantities. Furthermore, since the placenta and the amniotic membrane adhering to it are treated as medical waste, there is no ethical problem in collection.
本発明の細胞は、ヒト羊膜の羊膜上皮細胞層＋間葉細胞層を絨毛膜層から剥離し、これをトリプシン処理することにより、トリプシン溶液中に分離、回収することができる。このようにして分離、回収される細胞には、骨芽細胞に分化誘導可能な細胞以外の細胞も含まれるが、それらの細胞をそのまま後述の方法で培養することにより、骨芽細胞を形成することができる。従って、トリプシン溶液中に分離、回収することができる上記細胞は、さらに分離することなく骨芽細胞形成用細胞としての用途を有する。 The cells of the present invention can be separated and recovered in a trypsin solution by detaching the amniotic epithelial cell layer + mesenchymal cell layer of human amniotic membrane from the chorionic membrane layer and treating it with trypsin. The cells separated and collected in this manner include cells other than cells that can be induced to differentiate into osteoblasts. By culturing these cells as they are by the method described later, osteoblasts are formed. be able to. Therefore, the cells that can be separated and collected in a trypsin solution have applications as osteoblast-forming cells without further separation.
トリプシン溶液中に分離、回収された細胞を次いで、骨芽細胞分化培地で培養することにより、骨芽細胞を誘導することができる。本発明の細胞の骨芽細胞への分化に用いられる骨芽細胞分化培地としては、公知のいずれの骨芽細胞分化培地をも採用することができる。このような骨芽細胞分化培地の好ましい例としては、100 nMデキサメタゾン、10 mMβ−グリセロールリン酸、0.25 mMアスコルビン酸塩(ascorbate)及び10% FBS (ウシ胎児血清)をDMEM (Dulbecco's modified Eagle's medium) 中に含有する培地(非特許文献１)を挙げることができる。培養条件は、特に限定されないが、ヒトの体温である３７℃で２〜４週間程度培養することが好ましい。また、培養は、5% CO2ガス雰囲気下で行うことが好ましい。なお、骨芽細胞分化培地での培養に先立ち、細胞を通常の動物細胞培養用培地（例えば牛胎児血清含有DMEM培地 (Dulbecco's modified Eagle's medium)で４日〜２週間程度、さらに好ましくは約１週間予備培養することが好ましい。この予備培養の培養条件も、特に限定されないが、5% CO2ガス雰囲気下、３７℃が好ましい。 The cells separated and collected in the trypsin solution are then cultured in an osteoblast differentiation medium, whereby osteoblasts can be induced. Any known osteoblast differentiation medium can be employed as the osteoblast differentiation medium used for differentiation of the cells of the present invention into osteoblasts. Preferred examples of such osteoblast differentiation medium include 100 nM dexamethasone, 10 mM β-glycerol phosphate, 0.25 mM ascorbate and 10% FBS (fetal bovine serum) in DMEM (Dulbecco's modified Eagle's medium). Examples thereof include a medium (Non-patent Document 1) contained therein. The culture conditions are not particularly limited, but it is preferable to culture at 37 ° C., which is the human body temperature, for about 2 to 4 weeks. The culture is preferably performed in a 5% CO 2 gas atmosphere. Prior to culture in osteoblast differentiation medium, the cells are cultured in a normal animal cell culture medium (eg, DMEM medium containing fetal bovine serum (Dulbecco's modified Eagle's medium) for about 4 days to 2 weeks, more preferably about 1 week. The pre-culture is preferably performed, and the culture conditions for the pre-culture are not particularly limited, but are preferably 37 ° C. in a 5% CO 2 gas atmosphere.
なお、上記本発明の細胞を一次培養又は継代培養をして得られる培養細胞であって、骨芽細胞に分化誘導可能な細胞も本発明の範囲に含まれる。 In addition, cells that are obtained by primary culture or subculture of the above-described cells of the present invention and that can be induced to differentiate into osteoblasts are also included in the scope of the present invention.
骨芽細胞に誘導されたか否かは、アルカリフォスファターゼ及びカルシウムの産生を調べることにより判定することができる（下記実施例参照）。細胞が、アルカリフォスファターゼ及びカルシウムを産生するようになった場合には、骨芽細胞に分化誘導されたと判定される。 Whether or not induced by osteoblasts can be determined by examining the production of alkaline phosphatase and calcium (see Examples below). When the cells produce alkaline phosphatase and calcium, it is determined that differentiation has been induced in osteoblasts.
本発明の細胞はヒト羊膜由来であり、羊膜は胎児由来であり、免疫寛容を示す。すなわち免疫組織染色ではHLA Class I には陽性反応を呈し、HLA Class II の染色性はない。またFas ligand 陽性細胞が存在している。最近、羊膜組織が拒絶反応を惹起しにくい理由は、HLA Class Ib (HLA-G)の発現とFas ligand 陽性細胞の存在が拒絶抑制に貢献していると考えられている（眼科42:257-269, 2000）。従って、HLAの適合性を問題にすることなく移植することが可能である。 The cells of the present invention are derived from human amniotic membrane, which is derived from fetus and exhibits immune tolerance. In other words, immunohistochemical staining shows a positive reaction with HLA Class I and no staining with HLA Class II. Fas ligand positive cells are also present. Recently, the reason why amniotic tissue is less likely to cause rejection is thought to be due to the expression of HLA Class Ib (HLA-G) and the presence of Fas ligand positive cells (Ophthalmology 42: 257- 269, 2000). Therefore, it is possible to transplant without questioning the compatibility of HLA.
本発明の細胞は、そのまま、あるいは、アルカリフォスファターゼ及びカルシウムを産生する骨芽細胞にまで分化させた後、移植することにより骨の修復や再構成に用いることができる。移植する部位は、特に限定されないが、通常、骨の修復や再構成が望まれる、外傷や骨腫瘍の除去等に起因する骨欠損部である。移植は、公知の骨幹細胞移植と同様に行うことができる。移植する細胞の数は、骨欠損部の大きさや症状等に応じて適宜選択されるが、通常、１０３〜１０７個程度が適当である。 The cells of the present invention can be used for bone repair or reconstitution as they are or after being differentiated into osteoblasts producing alkaline phosphatase and calcium, and then transplanted. The site to be transplanted is not particularly limited, but is usually a bone defect caused by trauma, bone tumor removal, or the like for which bone repair or reconstruction is desired. Transplantation can be performed in the same manner as known bone stem cell transplantation. The number of cells to be transplanted is appropriately selected according to the size and symptoms of the bone defect portion, but usually about 10 3 to 10 7 cells are appropriate.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１． 細胞の分離及び培養
１）胎盤より、羊膜を絨毛層から剥離して分離した。
２）0.25％トリプシン溶液/1.3mM EDTAで37℃、15分間処理を行った。これを5回繰り返し、各トリプシン溶液分画を遠心処理して細胞を集め、10％牛胎児血清（FBS）含有RPMI1640培養液で洗浄した。
３）遠心処理して細胞を集め、10％牛胎児血清（FBS）含有DMEM培養液で懸濁し、培養ディッシュ上に播種し、37℃、5％CO２インキュベーター中で7日間程度初代培養した。
1. Separation and culture of cells 1) From the placenta, the amniotic membrane was separated from the chorionic layer and separated.
2) Treated with 0.25% trypsin solution / 1.3 mM EDTA at 37 ° C. for 15 minutes. This was repeated 5 times, and each trypsin solution fraction was centrifuged to collect cells and washed with RPMI1640 culture medium containing 10% fetal bovine serum (FBS).
3) The cells were collected by centrifugation, suspended in a DMEM culture solution containing 10% fetal bovine serum (FBS), seeded on a culture dish, and subjected to primary culture for about 7 days in a 37 ° C., 5% CO 2 incubator.
２． 骨芽細胞への分化誘導
１）羊膜上皮細胞を4.0×10４細胞/ｃｍ２で播種し、10％牛胎児血清（FBS）含有DMEM培養液で1週間程度培養を行った。
２）培養液を骨芽細胞分化培地（三光純薬社製: 100 nMデキサメタゾン、10 mMβ−グリセロールリン酸、0.25 mMアスコルビン酸塩及び10% FBSをDMEM中に含有)に置換し、2週間培養を行った。比較対照細胞は、10％牛胎児血清（FBS）含有DMEM培養液で2週間培養を行った。
2. Induction of differentiation into osteoblasts 1) Amnion epithelial cells were seeded at 4.0 × 10 4 cells / cm 2 and cultured in a DMEM culture solution containing 10% fetal bovine serum (FBS) for about one week.
2) The culture solution was replaced with osteoblast differentiation medium (manufactured by Sanko Junyaku Co., Ltd .: 100 nM dexamethasone, 10 mM β-glycerol phosphate, 0.25 mM ascorbate and 10% FBS in DMEM), and cultured for 2 weeks Went. Comparative control cells were cultured in a DMEM culture solution containing 10% fetal bovine serum (FBS) for 2 weeks.
３． 細胞化学染色
１）培養細胞を4％パラホルムアルデヒドで10分間固定し、アルカリフォスファターゼ染色キット（シグマ社製）を用いた免疫組織化学染色、およびアリザリンレッドＳ（シグマ社製）を用いてカルシウム染色を行った。操作は、各製品の添付文書に従った。
２）染色した細胞は、光学顕微鏡で観察し分析した。
3. Cytochemical staining 1) Fix cultured cells with 4% paraformaldehyde for 10 minutes, immunohistochemical staining using alkaline phosphatase staining kit (manufactured by Sigma), and calcium staining using alizarin red S (manufactured by Sigma). went. The operation followed the package insert for each product.
2) The stained cells were observed and analyzed with an optical microscope.
４． 結果
１）アルカリフォスファターゼ染色の結果、コントロールでは陰性であり、２週間分化誘導を施行した羊膜上皮細胞では陽性を示した。
２）また、アリザリンレッドＳ染色の結果、コントロールでは陰性であり、２週間分化誘導を施行した羊膜上皮細胞では陽性を示した。
4). Results 1) As a result of alkaline phosphatase staining, the control was negative and the amniotic epithelial cells subjected to differentiation induction for 2 weeks were positive.
2) As a result of staining with alizarin red S, it was negative in the control and positive in the amniotic epithelial cells subjected to differentiation induction for 2 weeks.
以上のように、羊膜上皮細胞の骨分化誘導により、アルカリフォスファターゼ活性およびカルシウム産生が認められ、骨芽細胞への分化誘導が可能であった。
As described above, alkaline phosphatase activity and calcium production were observed by induction of bone differentiation of amnion epithelial cells, and differentiation into osteoblasts was possible.
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