WO2015093402A1 - Myeloid stem cell attractant and myeloid stem cell-attracting method - Google Patents
Myeloid stem cell attractant and myeloid stem cell-attracting method Download PDFInfo
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- the present invention has been made in view of the above-mentioned problems and the like, and an object thereof is to provide an attractant for bone marrow stem cells having excellent attraction performance for bone marrow stem cells. It is another object of the present invention to provide a method for attracting bone marrow stem cells having excellent attraction performance for bone marrow stem cells.
- the bone marrow stem cell attractant may include a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer compound, and the like.
- the bone marrow stem cell attractant may include a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer compound, and the like.
- the concentration of kaempferitrin contained in the bone marrow stem cell attractant is not particularly limited, and examples thereof include 0.001 to 100% by weight.
- the above-mentioned attractant for bone marrow stem cells containing kaempferritrin can be produced by, for example, dissolving commercially available kaempferritrin in an appropriate solvent.
- the attractant for bone marrow stem cells containing quercetin-3,7-dirhamnoside can be produced by, for example, dissolving commercially available quercetin-3,7-dirhamnoside in an appropriate solvent.
- the method for attracting bone marrow stem cells according to the present embodiment is a method for attracting bone marrow stem cells using the bone marrow stem cell attractant.
- a method of applying a bone marrow stem cell attractant to a predetermined body tissue and attracting bone marrow stem cells to the body tissue can be performed.
- a method of attracting bone marrow stem cells to the epidermal tissue by applying an attractant to the skin can be performed.
- a method of attracting bone marrow mesenchymal stem cells present in blood to the epidermal tissue by incorporating the bone marrow stem cell attractant into the epidermal tissue (such as the stratum corneum) of the skin by application or the like. can be implemented.
- various tissues such as a muscle tissue, a cartilage tissue, and a liver tissue can be cited in addition to an epidermal tissue.
- the method for attracting bone marrow stem cells can be carried out in a human living body or a living body of a non-human animal.
- the method for attracting bone marrow stem cells is carried out non-therapeutically in a human body, specifically for cosmetic purposes, for example.
- Example 1 Borkensiflavone (isolated and purified from Garcinia vilersiana ) was dissolved in a solvent (Dulbecco's modified Eagle's medium DMEM) to a concentration of 500 ⁇ g / mL to produce an attractant containing Borkenciflavone.
- a solvent Dulbecco's modified Eagle's medium DMEM
- Example 3 An attractant was produced in the same manner as in Example 1 except that the concentration of Borkensiflavone was 100 ⁇ g / mL.
- Example 5 An attractant was produced in the same manner as in Example 4 except that the concentration of morelloflavone was 200 ⁇ g / mL.
- Example 9 An attractant was produced in the same manner as in Example 7 except that the concentration of kaempferitrin was 100 ⁇ g / mL.
- Example 11 An attractant was produced in the same manner as in Example 7 except that the concentration of quercetin-3,7-dirhamnoside was 200 ⁇ g / mL.
- DMEM “FBS ( ⁇ ), P / S ( ⁇ )” was prepared as a negative control sample.
- a DMEM solution having a concentration of 20 ng / mL PDGF-BB (platelet-derived growth factor PEPRO TECH) was prepared.
- mMSC mouse bone marrow mesenchymal stem cells
- the stained image is digitized and loaded into a computer, and the image is converted to a white portion of the blue-stained portion so that the image is binarized into black and white.
- the measurement was performed using the function of editing software (trade name “Photoshop”).
- the mMSC attracting activity of the bone marrow stem cell attractant was assessed by comparing the brightness in the negative and positive control samples with the brightness in each test sample.
- the measured value was each calculated by averaging two measurement results.
- the bone marrow stem cell attractant and bone marrow stem cell attracting method of the present invention are used, for example, to attract bone marrow stem cells generated in the bone marrow to a predetermined body tissue before the bone marrow stem cells are differentiated in the predetermined body tissue.
- the method for attracting bone marrow stem cells and the method for attracting bone marrow stem cells of the present invention includes, for example, bone marrow stem cells circulating in the body along the bloodstream by performing a treatment that includes the attractant in a predetermined body tissue. Is preferably used for the purpose of attracting and accumulating in the body tissue.
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Abstract
The present invention addresses the problem of providing a myeloid stem cell attractant with excellent myeloid stem cell-attracting ability, and a myeloid stem cell-attracting method. Provided are: a myeloid stem cell attractant comprising at least one of volkensii flavone, morelloflavone, kaempferitrin, and quercetin-3,7-dirhamnoside; and a myeloid stem cell-attracting method for attracting myeloid stem cells using said attractant.
Description
本願は、日本国特願２０１３－２６３０５８号の優先権を主張し、該出願が引用によって本願明細書の記載に組み込まれる。
This application claims the priority of Japanese Patent Application No. 2013-263058, which is incorporated herein by reference.
本発明は、骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法に関する。
The present invention relates to an attractant for bone marrow stem cells and a method for attracting bone marrow stem cells.
骨髄幹細胞は、骨髄において生じた未だ分化していない細胞であり、様々な体内組織の細胞へ分化し得るものとして知られている。また、分化を誘導する分化誘導剤の影響を受けて、機能が失われた組織においてその組織細胞に分化することにより組織の機能を回復させ得るものとして注目されている。
Bone marrow stem cells are undifferentiated cells generated in the bone marrow and are known to be able to differentiate into cells of various body tissues. In addition, it has been attracting attention as a tissue function that can be restored by differentiating into tissue cells in a tissue that has lost its function under the influence of a differentiation-inducing agent that induces differentiation.
具体的には、骨髄幹細胞は、例えば、炎症のある組織又は損傷を受けた組織へ血流にのって骨髄から移動した後に、分化誘導剤の影響を受けてその組織細胞へ分化し得るものとして注目されている。
Specifically, bone marrow stem cells can be differentiated into tissue cells under the influence of a differentiation-inducing agent after moving from the bone marrow to the inflamed tissue or damaged tissue through the bloodstream, for example. It is attracting attention as.
ところで、従来、骨髄幹細胞を各種の細胞に分化させ得る分化誘導剤としては、様々なものが知られており、例えば、骨髄幹細胞を心臓の筋肉細胞に分化させ得る線維芽細胞増殖因子（Fibroblast Growth Factor ＦＧＦ）や血小板由来成長因子（Platelet-Derived Growth Factor ＰＤＧＦ）などが知られている（特許文献１）。
By the way, conventionally, various agents are known as differentiation inducers capable of differentiating bone marrow stem cells into various cells. For example, fibroblast growth factor (Fibroblast Growth, which can differentiate bone marrow stem cells into cardiac muscle cells. Factor FGF) and platelet-derived growth factor (Platelet-Derived Growth Factor PDGF) are known (Patent Document 1).
この種の分化誘導剤は、骨髄幹細胞を所定の組織細胞へと分化させる性能を有するものの、骨髄幹細胞を誘引する性能、具体的には例えば、血流にのって体内を循環している骨髄幹細胞を所定の体内組織へ誘引する性能が、必ずしも十分なものではないという問題がある。
Although this type of differentiation inducer has the ability to differentiate bone marrow stem cells into predetermined tissue cells, it has the ability to attract bone marrow stem cells, specifically, for example, bone marrow circulating in the body along the bloodstream. There is a problem that the ability to attract stem cells to a predetermined body tissue is not always sufficient.
本発明は、上記の問題点等に鑑み、骨髄幹細胞の誘引性能に優れた骨髄幹細胞の誘引剤を提供することを課題とする。また、骨髄幹細胞の誘引性能に優れた骨髄幹細胞の誘引方法を提供することを課題とする。
The present invention has been made in view of the above-mentioned problems and the like, and an object thereof is to provide an attractant for bone marrow stem cells having excellent attraction performance for bone marrow stem cells. It is another object of the present invention to provide a method for attracting bone marrow stem cells having excellent attraction performance for bone marrow stem cells.
本発明に係る骨髄幹細胞の誘引剤は、ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのうちの少なくとも１種を含む。
The attractant for bone marrow stem cells according to the present invention includes at least one of borkenciflavone, moreroflavone, kaempferitrin, and quercetin-3,7-dirhamnoside.
本発明に係る骨髄幹細胞の誘引方法は、前記誘引剤により骨髄幹細胞を誘引するものである。
The method for attracting bone marrow stem cells according to the present invention involves attracting bone marrow stem cells with the attractant.
以下に、本発明に係る骨髄幹細胞の誘引剤の実施形態について説明する。
Hereinafter, embodiments of the attractant for bone marrow stem cells according to the present invention will be described.
本実施形態の骨髄幹細胞の誘引剤は、ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのうちの少なくとも１種を含むものである。
本実施形態の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、骨髄幹細胞の誘引性能に優れるという効果を奏する。 The attractant for bone marrow stem cells of the present embodiment includes at least one of borkenciflavone, moreroflavone, kaempferritrin, and quercetin-3,7-dirhamnoside.
The attractant for bone marrow stem cells and the method for attracting bone marrow stem cells according to the present embodiment have an effect that the attracting performance of bone marrow stem cells is excellent.
本実施形態の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、骨髄幹細胞の誘引性能に優れるという効果を奏する。 The attractant for bone marrow stem cells of the present embodiment includes at least one of borkenciflavone, moreroflavone, kaempferritrin, and quercetin-3,7-dirhamnoside.
The attractant for bone marrow stem cells and the method for attracting bone marrow stem cells according to the present embodiment have an effect that the attracting performance of bone marrow stem cells is excellent.
前記ボルケンシフラボン（タルボタフラボン）［Volkensiflavone（Talbotaflavone）］は、下記式（１）の分子構造を有する化合物である。
The Volkensiflavone (Talbotaflavone) is a compound having a molecular structure represented by the following formula (1).
前記骨髄幹細胞の誘引剤に含まれるボルケンシフラボンの濃度は、特に限定されず、該濃度としては、例えば、０．００１～１００重量％が挙げられる。
The concentration of Borkensiflavone contained in the bone marrow stem cell attractant is not particularly limited, and examples thereof include 0.001 to 100% by weight.
前記骨髄幹細胞の誘引剤は、ボルケンシフラボン以外に、ｐＨ緩衝剤、水などの溶媒、界面活性剤、防腐剤、油類、多価アルコール類、水溶性高分子化合物などを含み得る。
In addition to Borkensiflavone, the bone marrow stem cell attractant may include a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer compound, and the like.
前記ボルケンシフラボンを含む骨髄幹細胞の誘引剤は、例えば、市販されているボルケンシフラボンを適当な溶媒に溶解させることにより製造することができる。
The attractant for bone marrow stem cells containing the borkensiflavone can be produced, for example, by dissolving a commercially available borkensiflavone in an appropriate solvent.
また、前記ボルケンシフラボンを含む骨髄幹細胞の誘引剤は、例えば、Garcinia vilersianaなどの植物におけるボルケンシフラボンを含む部分を抽出処理することにより製造することができる。
In addition, the bone marrow stem cell attractant containing the borkensiflavone can be produced, for example, by subjecting a part containing a borkenciflavone in a plant such as Garcinia vilersiana to extraction treatment.
前記モレロフラボン「Morelloflavone」は、下記式（２）の分子構造を有する化合物である。
The Morelloflavone “Morelloflavone” is a compound having a molecular structure represented by the following formula (2).
前記骨髄幹細胞の誘引剤に含まれるモレロフラボンの濃度は、特に限定されず、該濃度としては、例えば、０．００１～１００重量％が挙げられる。
The concentration of moreroflavone contained in the bone marrow stem cell attractant is not particularly limited, and examples thereof include 0.001 to 100% by weight.
前記骨髄幹細胞の誘引剤は、モレロフラボン以外に、ｐＨ緩衝剤、水などの溶媒、界面活性剤、防腐剤、油類、多価アルコール類、水溶性高分子化合物などを含み得る。
In addition to moreroflavone, the bone marrow stem cell attractant may include a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer compound, and the like.
前記モレロフラボンを含む骨髄幹細胞の誘引剤は、例えば、市販されているモレロフラボンを適当な溶媒に溶解させることにより製造することができる。
The above-mentioned attractant for bone marrow stem cells containing moreroflavone can be produced, for example, by dissolving commercially available moreroflavone in an appropriate solvent.
また、前記モレロフラボンを含む骨髄幹細胞の誘引剤は、例えば、Garcinia vilersianaなどを抽出処理することにより製造することができる。
Further, the bone marrow stem cell attractant containing moreroflavone can be produced, for example, by extracting Garcinia vilersiana and the like.
前記ケンフェリトリン［Kaempferitrin］（又は、ケンフェロール－３，７－ジラムノシドともいう）は、下記式（３）の分子構造を有する化合物である。
The kaempferitrin (or kaempferol-3,7-dirhamnoside) is a compound having a molecular structure represented by the following formula (3).
前記骨髄幹細胞の誘引剤に含まれるケンフェリトリンの濃度は、特に限定されず、該濃度としては、例えば、０．００１～１００重量％が挙げられる。
The concentration of kaempferitrin contained in the bone marrow stem cell attractant is not particularly limited, and examples thereof include 0.001 to 100% by weight.
前記骨髄幹細胞の誘引剤は、ケンフェリトリン以外に、ｐＨ緩衝剤、水などの溶媒、界面活性剤、防腐剤、油類、多価アルコール類、水溶性高分子化合物などを含み得る。
The attractant for bone marrow stem cells may contain a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer compound, etc., in addition to kaempferitrin.
前記ケンフェリトリンを含む骨髄幹細胞の誘引剤は、例えば、市販されているケンフェリトリンを適当な溶媒に溶解させることにより製造することができる。
The above-mentioned attractant for bone marrow stem cells containing kaempferritrin can be produced by, for example, dissolving commercially available kaempferritrin in an appropriate solvent.
また、前記ケンフェリトリンを含む骨髄幹細胞の誘引剤は、例えば、ゲンノショウコ（Geranium thunbergii）、セイロンニッケイ（Cinnamomum osmophloeum）、キャッツクロー（Uncaria guianensis）、フユボダイジュ（Tilia cordata）などの植物におけるケンフェリトリンを含む部分を抽出処理することにより製造することができる。
In addition, the attractant for bone marrow stem cells containing kaempferritrin includes, for example, kaempfertrins in plants such as Geranium thunbergii , Cinnamomum osmophloeum , cats claw ( Uncaria guianensis ), and jutebo ( Tilia cordata ). It can manufacture by extracting the part containing.
前記ケルセチン－３，７－ジラムノシド［Quercetin-3,7,-di-O-α-L-rhamnoside］は、下記式（４）の分子構造を有する化合物である。
The quercetin-3,7-dirhamnoside [Quercetin-3,7, -di-O-α-L-rhamnoside] is a compound having a molecular structure represented by the following formula (4).
前記骨髄幹細胞の誘引剤に含まれるケルセチン－３，７－ジラムノシドの濃度は、特に限定されず、該濃度としては、例えば、０．００１～１００重量％が挙げられる。
The concentration of quercetin-3,7-dirhamnoside contained in the bone marrow stem cell attractant is not particularly limited, and examples thereof include 0.001 to 100% by weight.
前記骨髄幹細胞の誘引剤は、ケルセチン－３，７－ジラムノシド以外に、ｐＨ緩衝剤、水などの溶媒、界面活性剤、防腐剤、油類、多価アルコール類、水溶性高分子化合物などを含み得る。
In addition to quercetin-3,7-dirhamnoside, the bone marrow stem cell attractant includes a pH buffer, a solvent such as water, a surfactant, an antiseptic, an oil, a polyhydric alcohol, a water-soluble polymer, and the like. obtain.
前記ケルセチン－３，７－ジラムノシドを含む骨髄幹細胞の誘引剤は、例えば、市販されているケルセチン－３，７－ジラムノシドを適当な溶媒に溶解させることにより製造することができる。
The attractant for bone marrow stem cells containing quercetin-3,7-dirhamnoside can be produced by, for example, dissolving commercially available quercetin-3,7-dirhamnoside in an appropriate solvent.
また、前記ケルセチン－３，７－ジラムノシドを含む骨髄幹細胞の誘引剤は、例えば、ハマカヅラ（Bauhinia forficata）、エジプトイチジク（Ficus sycomorus）、Siparuna guianensis、フユボダイジュ（Tilia cordata）などの植物におけるケルセチン－３，７－ジラムノシドを含む部分を抽出処理することにより製造することができる。
Examples of the bone marrow stem cell attractant containing quercetin-3,7-dirhamnoside include quercetin-3 in plants such as Bauhinia forficata , Egyptian fig ( Ficus sycomorus ), Siparuna guianensis , and Tilia cordata. , 7-Dirhamnoside can be produced by extraction treatment.
なお、前記骨髄幹細胞の誘引剤は、ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのうちのいずれか１つを含んでいてもよく、いずれか２つ又は３つを含んでいてもよい。前記骨髄幹細胞の誘引剤は、ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのいずれも含み得る。
The attractant for bone marrow stem cells may contain any one of vorkenciflavone, moreroflavone, kaempferitrin, and quercetin-3,7-dirhamnoside, and either two or three It may contain one. The bone marrow stem cell attractant may include any of borkenciflavone, moreroflavone, kaempferitrin, and quercetin-3,7-dirhamnoside.
続いて、本発明の骨髄幹細胞の誘引方法の実施形態について説明する。本実施形態の骨髄幹細胞の誘引方法は、前記骨髄幹細胞の誘引剤により骨髄幹細胞を誘引するものである。
Subsequently, an embodiment of the method for attracting bone marrow stem cells of the present invention will be described. The method for attracting bone marrow stem cells according to the present embodiment is a method for attracting bone marrow stem cells using the bone marrow stem cell attractant.
骨髄幹細胞は、骨髄組織に含まれており、さらに骨髄組織内で分化したり又は血流にのって骨髄組織から他の体内組織へ移動したりし得る。
前記骨髄幹細胞としては、軟骨細胞、脂肪細胞、筋肉細胞などの中胚葉性組織の細胞へ分化し得る骨髄間葉系幹細胞、赤血球や白血球などの血液細胞へ分化し得る造血幹細胞などが挙げられる。
前記骨髄幹細胞のなかでも骨髄間葉系幹細胞は、中胚葉性組織の細胞に分化するだけでなく、神経などの外胚葉性組織、肝臓などの内胚葉性組織の細胞へも分化し得る。本実施形態の誘引方法において誘引される骨髄幹細胞としては、誘引された後に、より多様な細胞に分化し得るという点で、骨髄間葉系幹細胞が好ましい。 Bone marrow stem cells are contained in the bone marrow tissue and can further differentiate within the bone marrow tissue or migrate from the bone marrow tissue to other body tissues along the bloodstream.
Examples of the bone marrow stem cells include bone marrow mesenchymal stem cells that can differentiate into cells of mesodermal tissue such as chondrocytes, adipocytes, and muscle cells, and hematopoietic stem cells that can differentiate into blood cells such as erythrocytes and leukocytes.
Among the bone marrow stem cells, bone marrow mesenchymal stem cells can differentiate not only into cells of mesodermal tissue but also into cells of endoderm tissue such as nerves and endoderm tissue such as liver. As the bone marrow stem cell attracted in the attracting method of this embodiment, a bone marrow mesenchymal stem cell is preferable in that it can be differentiated into more diverse cells after being attracted.
前記骨髄幹細胞としては、軟骨細胞、脂肪細胞、筋肉細胞などの中胚葉性組織の細胞へ分化し得る骨髄間葉系幹細胞、赤血球や白血球などの血液細胞へ分化し得る造血幹細胞などが挙げられる。
前記骨髄幹細胞のなかでも骨髄間葉系幹細胞は、中胚葉性組織の細胞に分化するだけでなく、神経などの外胚葉性組織、肝臓などの内胚葉性組織の細胞へも分化し得る。本実施形態の誘引方法において誘引される骨髄幹細胞としては、誘引された後に、より多様な細胞に分化し得るという点で、骨髄間葉系幹細胞が好ましい。 Bone marrow stem cells are contained in the bone marrow tissue and can further differentiate within the bone marrow tissue or migrate from the bone marrow tissue to other body tissues along the bloodstream.
Examples of the bone marrow stem cells include bone marrow mesenchymal stem cells that can differentiate into cells of mesodermal tissue such as chondrocytes, adipocytes, and muscle cells, and hematopoietic stem cells that can differentiate into blood cells such as erythrocytes and leukocytes.
Among the bone marrow stem cells, bone marrow mesenchymal stem cells can differentiate not only into cells of mesodermal tissue but also into cells of endoderm tissue such as nerves and endoderm tissue such as liver. As the bone marrow stem cell attracted in the attracting method of this embodiment, a bone marrow mesenchymal stem cell is preferable in that it can be differentiated into more diverse cells after being attracted.
前記骨髄幹細胞の誘引方法においては、例えば、生体外において又は生体において、前記骨髄幹細胞の誘引剤により骨髄幹細胞を誘引することができる。
In the method for attracting bone marrow stem cells, for example, bone marrow stem cells can be attracted by the bone marrow stem cell attractant in vitro or in vivo.
具体的には、生体外での骨髄幹細胞の誘引方法においては、例えば、厚み方向に貫通する微細孔を有するメンブレン（膜）を備えた装置を用い、メンブレンの一方側に骨髄幹細胞を配し、他方側に前記骨髄幹細胞の誘引剤を配し、所定時間をおいて骨髄幹細胞をメンブレンの他方側へ誘引する方法などを実施することができる。
また、生体外での骨髄幹細胞の誘引方法においては、例えば、スライドグラス上に播種した骨髄幹細胞の一部を掻き取り、掻き取った部分に骨髄幹細胞の誘引剤を含む培地を塗布して骨髄幹細胞の培養条件下におくことにより、掻き取った部分へ骨髄幹細胞を誘引する方法などを実施することができる。 Specifically, in the method of attracting bone marrow stem cells in vitro, for example, using a device having a membrane (membrane) having micropores penetrating in the thickness direction, the bone marrow stem cells are arranged on one side of the membrane, For example, a method for attracting bone marrow stem cells to the other side of the membrane by placing the attractant for the bone marrow stem cells on the other side and waiting for a predetermined time can be performed.
Further, in the method for attracting bone marrow stem cells in vitro, for example, a portion of bone marrow stem cells seeded on a slide glass is scraped, and a medium containing an attractant for bone marrow stem cells is applied to the scraped portion, and the bone marrow stem cells are applied. In this culture condition, a method for attracting bone marrow stem cells to the scraped portion can be carried out.
また、生体外での骨髄幹細胞の誘引方法においては、例えば、スライドグラス上に播種した骨髄幹細胞の一部を掻き取り、掻き取った部分に骨髄幹細胞の誘引剤を含む培地を塗布して骨髄幹細胞の培養条件下におくことにより、掻き取った部分へ骨髄幹細胞を誘引する方法などを実施することができる。 Specifically, in the method of attracting bone marrow stem cells in vitro, for example, using a device having a membrane (membrane) having micropores penetrating in the thickness direction, the bone marrow stem cells are arranged on one side of the membrane, For example, a method for attracting bone marrow stem cells to the other side of the membrane by placing the attractant for the bone marrow stem cells on the other side and waiting for a predetermined time can be performed.
Further, in the method for attracting bone marrow stem cells in vitro, for example, a portion of bone marrow stem cells seeded on a slide glass is scraped, and a medium containing an attractant for bone marrow stem cells is applied to the scraped portion, and the bone marrow stem cells are applied. In this culture condition, a method for attracting bone marrow stem cells to the scraped portion can be carried out.
このような生体外での骨髄幹細胞の誘引方法は、比較的簡便に実施できることから、例えば、後述する生体での骨髄幹細胞の誘引方法における誘引剤の最適濃度を決定する目的で予備実験的に実施することができる。
Since such an in vitro method of attracting bone marrow stem cells can be performed relatively easily, for example, a preliminary experiment was performed for the purpose of determining the optimum concentration of the attractant in the method of attracting bone marrow stem cells in vivo described below. can do.
なお、上記のような骨髄幹細胞の誘引方法における骨髄幹細胞の誘引の程度は、骨髄幹細胞を染色し、染色度を測定することにより評価できる。また、誘引された骨髄幹細胞を目視にて確認することにより評価できる。
In addition, the degree of attraction of bone marrow stem cells in the above method for attracting bone marrow stem cells can be evaluated by staining the bone marrow stem cells and measuring the degree of staining. Moreover, it can evaluate by confirming the attracted bone marrow stem cell visually.
一方、生体での骨髄幹細胞の誘引方法においては、例えば、前記骨髄幹細胞の誘引剤を含む水性ゲルを通常のマウスの皮下に埋め込み、マウスの骨髄幹細胞をこのマウスの静脈に注入し、所定期間マウスを飼育することにより、骨髄幹細胞を水性ゲルに誘引する方法などを実施することができる。
また、生体での骨髄幹細胞の誘引方法においては、例えば、マウスの骨髄幹細胞を、創傷モデルマウスの骨髄に移植するとともに、該マウスの創傷部に前記骨髄幹細胞の誘引剤を塗布することにより、創傷部に骨髄幹細胞を誘引する方法などを実施することができる。 On the other hand, in the method for attracting bone marrow stem cells in a living body, for example, an aqueous gel containing the above-mentioned bone marrow stem cell attractant is implanted subcutaneously into a normal mouse, and the mouse bone marrow stem cells are injected into the vein of the mouse, and the mouse is injected for a predetermined period. Can be performed to attract bone marrow stem cells to an aqueous gel.
In addition, in the method of attracting bone marrow stem cells in vivo, for example, a bone marrow stem cell of a mouse is transplanted into the bone marrow of a wound model mouse, and the bone marrow stem cell attractant is applied to the wound part of the mouse, thereby A method of attracting bone marrow stem cells to the part can be performed.
また、生体での骨髄幹細胞の誘引方法においては、例えば、マウスの骨髄幹細胞を、創傷モデルマウスの骨髄に移植するとともに、該マウスの創傷部に前記骨髄幹細胞の誘引剤を塗布することにより、創傷部に骨髄幹細胞を誘引する方法などを実施することができる。 On the other hand, in the method for attracting bone marrow stem cells in a living body, for example, an aqueous gel containing the above-mentioned bone marrow stem cell attractant is implanted subcutaneously into a normal mouse, and the mouse bone marrow stem cells are injected into the vein of the mouse, and the mouse is injected for a predetermined period. Can be performed to attract bone marrow stem cells to an aqueous gel.
In addition, in the method of attracting bone marrow stem cells in vivo, for example, a bone marrow stem cell of a mouse is transplanted into the bone marrow of a wound model mouse, and the bone marrow stem cell attractant is applied to the wound part of the mouse, thereby A method of attracting bone marrow stem cells to the part can be performed.
なお、上記のような骨髄幹細胞の誘引方法における骨髄幹細胞の誘引の程度は、骨髄幹細胞として、緑色蛍光タンパク質（Green Fluorescent Protein，以下ＧＦＰともいう）を発現させたマウスの骨髄幹細胞を用いて、誘引された骨髄幹細胞における蛍光の強度を測定することにより評価できる。
The degree of attraction of bone marrow stem cells in the method for attracting bone marrow stem cells as described above is determined by using bone marrow stem cells of mice expressing green fluorescent protein (Green Fluorescent Protein) (hereinafter also referred to as GFP) as bone marrow stem cells. It can be evaluated by measuring the intensity of fluorescence in the obtained bone marrow stem cells.
さらに、生体での骨髄幹細胞の誘引方法においては、例えば、所定の体内組織に骨髄幹細胞の誘引剤を含有させる処置を施し、その体内組織に骨髄幹細胞を誘引する方法などを実施することができる。
具体的には、誘引剤を皮膚に塗布することにより、骨髄幹細胞を表皮組織に誘引する方法などを実施することができる。より具体的には、例えば、皮膚の表皮組織（角質層など）に前記骨髄幹細胞の誘引剤を塗布などにより含有させて、血液中に存在する骨髄間葉系幹細胞を表皮組織に誘引する方法などを実施することができる。
なお、生体での誘引方法において骨髄幹細胞が誘引されて来る体内組織としては、表皮組織の他に、筋肉組織、軟骨組織、肝臓組織などの各種組織が挙げられる。 Furthermore, in the method for attracting bone marrow stem cells in a living body, for example, a method of applying a bone marrow stem cell attractant to a predetermined body tissue and attracting bone marrow stem cells to the body tissue can be performed.
Specifically, a method of attracting bone marrow stem cells to the epidermal tissue by applying an attractant to the skin can be performed. More specifically, for example, a method of attracting bone marrow mesenchymal stem cells present in blood to the epidermal tissue by incorporating the bone marrow stem cell attractant into the epidermal tissue (such as the stratum corneum) of the skin by application or the like. Can be implemented.
In addition, as a body tissue from which bone marrow stem cells are attracted in the attracting method in a living body, various tissues such as a muscle tissue, a cartilage tissue, and a liver tissue can be cited in addition to an epidermal tissue.
具体的には、誘引剤を皮膚に塗布することにより、骨髄幹細胞を表皮組織に誘引する方法などを実施することができる。より具体的には、例えば、皮膚の表皮組織（角質層など）に前記骨髄幹細胞の誘引剤を塗布などにより含有させて、血液中に存在する骨髄間葉系幹細胞を表皮組織に誘引する方法などを実施することができる。
なお、生体での誘引方法において骨髄幹細胞が誘引されて来る体内組織としては、表皮組織の他に、筋肉組織、軟骨組織、肝臓組織などの各種組織が挙げられる。 Furthermore, in the method for attracting bone marrow stem cells in a living body, for example, a method of applying a bone marrow stem cell attractant to a predetermined body tissue and attracting bone marrow stem cells to the body tissue can be performed.
Specifically, a method of attracting bone marrow stem cells to the epidermal tissue by applying an attractant to the skin can be performed. More specifically, for example, a method of attracting bone marrow mesenchymal stem cells present in blood to the epidermal tissue by incorporating the bone marrow stem cell attractant into the epidermal tissue (such as the stratum corneum) of the skin by application or the like. Can be implemented.
In addition, as a body tissue from which bone marrow stem cells are attracted in the attracting method in a living body, various tissues such as a muscle tissue, a cartilage tissue, and a liver tissue can be cited in addition to an epidermal tissue.
生体での骨髄幹細胞の誘引方法は、例えば、骨髄幹細胞を各種の組織細胞へ分化させる前に、骨髄幹細胞をその組織へ集積させる目的で実施することができる。
The method for attracting bone marrow stem cells in a living body can be performed, for example, for the purpose of accumulating bone marrow stem cells in various tissues before differentiation into bone marrow stem cells.
前記骨髄幹細胞の誘引方法は、ヒトの生体又はヒト以外の動物の生体において実施することができる。好ましくは、前記骨髄幹細胞の誘引方法は、ヒトの生体において、具体的には例えば美容上の目的で、非治療的に実施する。
The method for attracting bone marrow stem cells can be carried out in a human living body or a living body of a non-human animal. Preferably, the method for attracting bone marrow stem cells is carried out non-therapeutically in a human body, specifically for cosmetic purposes, for example.
前記骨髄幹細胞の誘引方法においては、ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのうちの少なくとも１種が適当な濃度に希釈された誘引剤を用いることができる。希釈するための液としては、特に限定されるものではなく、例えば、水、生理食塩水、骨髄幹細胞の培養液などを用いることができる。
例えば、ボルケンシフラボンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのボルケンシフラボン濃度、より好ましくは、１００～５００μｇ／ｍＬのボルケンシフラボン濃度にて使用される。
また、例えば、モレロフラボンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのモレロフラボン濃度、より好ましくは、１００～５００μｇ／ｍＬのモレロフラボン濃度にて使用される。
また、例えば、ケンフェリトリンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのケンフェリトリン濃度、より好ましくは、１００～４００μｇ／ｍＬのケンフェリトリン濃度にて使用される。
また、例えば、ケルセチン－３，７－ジラムノシドを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのケルセチン－３，７－ジラムノシド濃度、より好ましくは、１００～４００μｇ／ｍＬのケルセチン－３，７－ジラムノシド濃度にて使用される。 In the method for attracting bone marrow stem cells, an attractant in which at least one of borkenciflavone, moreroflavone, kaempferriline, and quercetin-3,7-dirhamnoside is diluted to an appropriate concentration can be used. . The liquid for dilution is not particularly limited, and for example, water, physiological saline, bone marrow stem cell culture medium, and the like can be used.
For example, the bone marrow stem cell attractant containing borkenciflavone is preferably used at a borkenciflavone concentration of 10 to 1,000 μg / mL, more preferably at a borkenciflavone concentration of 100 to 500 μg / mL.
In addition, for example, a bone marrow stem cell attractant containing moreroflavone is preferably used at a concentration of more than 10 to 1,000 μg / mL, more preferably more than 100 to 500 μg / mL.
In addition, for example, an attractant for bone marrow stem cells containing kaempferritrin is preferably used at a kaempferitrin concentration of 10 to 1,000 μg / mL, more preferably at a kaempferritrin concentration of 100 to 400 μg / mL. The
In addition, for example, the bone marrow stem cell attractant containing quercetin-3,7-dirhamnoside preferably has a quercetin-3,7-dirhamnoside concentration of 10 to 1,000 μg / mL, more preferably 100 to 400 μg / mL. Used at quercetin-3,7-dirhamnoside concentration.
例えば、ボルケンシフラボンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのボルケンシフラボン濃度、より好ましくは、１００～５００μｇ／ｍＬのボルケンシフラボン濃度にて使用される。
また、例えば、モレロフラボンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのモレロフラボン濃度、より好ましくは、１００～５００μｇ／ｍＬのモレロフラボン濃度にて使用される。
また、例えば、ケンフェリトリンを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのケンフェリトリン濃度、より好ましくは、１００～４００μｇ／ｍＬのケンフェリトリン濃度にて使用される。
また、例えば、ケルセチン－３，７－ジラムノシドを含む骨髄幹細胞の誘引剤は、好ましくは、１０～１，０００μｇ／ｍＬのケルセチン－３，７－ジラムノシド濃度、より好ましくは、１００～４００μｇ／ｍＬのケルセチン－３，７－ジラムノシド濃度にて使用される。 In the method for attracting bone marrow stem cells, an attractant in which at least one of borkenciflavone, moreroflavone, kaempferriline, and quercetin-3,7-dirhamnoside is diluted to an appropriate concentration can be used. . The liquid for dilution is not particularly limited, and for example, water, physiological saline, bone marrow stem cell culture medium, and the like can be used.
For example, the bone marrow stem cell attractant containing borkenciflavone is preferably used at a borkenciflavone concentration of 10 to 1,000 μg / mL, more preferably at a borkenciflavone concentration of 100 to 500 μg / mL.
In addition, for example, a bone marrow stem cell attractant containing moreroflavone is preferably used at a concentration of more than 10 to 1,000 μg / mL, more preferably more than 100 to 500 μg / mL.
In addition, for example, an attractant for bone marrow stem cells containing kaempferritrin is preferably used at a kaempferitrin concentration of 10 to 1,000 μg / mL, more preferably at a kaempferritrin concentration of 100 to 400 μg / mL. The
In addition, for example, the bone marrow stem cell attractant containing quercetin-3,7-dirhamnoside preferably has a quercetin-3,7-dirhamnoside concentration of 10 to 1,000 μg / mL, more preferably 100 to 400 μg / mL. Used at quercetin-3,7-dirhamnoside concentration.
本実施形態の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、上記例示の通りであるが、本発明は、上記例示の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法に限定されるものではない。また、本発明の作用効果は、上述した作用効果に限定されない。また、本発明の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、本発明の要旨を逸脱しない範囲で種々の変更が可能である。
The bone marrow stem cell attractant and bone marrow stem cell attractant of the present embodiment are as exemplified above, but the present invention is not limited to the above exemplified bone marrow stem cell attractant and bone marrow stem cell attractant. . Moreover, the effect of this invention is not limited to the effect mentioned above. Further, the bone marrow stem cell attractant and bone marrow stem cell attractant of the present invention can be variously modified without departing from the gist of the present invention.
次に、実施例を挙げて本発明をさらに詳しく説明するが、本発明はこれらに限定されるものではない。
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
（実施例１）
ボルケンシフラボン（Garcinia vilersianaから単離精製したもの）を５００μｇ／ｍＬ濃度となるように溶媒（ダルベッコ変法イーグル培地 ＤＭＥＭ）に溶解させ、ボルケンシフラボンを含む誘引剤を製造した。 Example 1
Borkensiflavone (isolated and purified from Garcinia vilersiana ) was dissolved in a solvent (Dulbecco's modified Eagle's medium DMEM) to a concentration of 500 μg / mL to produce an attractant containing Borkenciflavone.
ボルケンシフラボン（Garcinia vilersianaから単離精製したもの）を５００μｇ／ｍＬ濃度となるように溶媒（ダルベッコ変法イーグル培地 ＤＭＥＭ）に溶解させ、ボルケンシフラボンを含む誘引剤を製造した。 Example 1
Borkensiflavone (isolated and purified from Garcinia vilersiana ) was dissolved in a solvent (Dulbecco's modified Eagle's medium DMEM) to a concentration of 500 μg / mL to produce an attractant containing Borkenciflavone.
（実施例２）
ボルケンシフラボンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例１と同様にして誘引剤を製造した。 (Example 2)
An attractant was produced in the same manner as in Example 1 except that the concentration of Borkensiflavone was 200 μg / mL.
ボルケンシフラボンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例１と同様にして誘引剤を製造した。 (Example 2)
An attractant was produced in the same manner as in Example 1 except that the concentration of Borkensiflavone was 200 μg / mL.
（実施例３）
ボルケンシフラボンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例１と同様にして誘引剤を製造した。 Example 3
An attractant was produced in the same manner as in Example 1 except that the concentration of Borkensiflavone was 100 μg / mL.
ボルケンシフラボンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例１と同様にして誘引剤を製造した。 Example 3
An attractant was produced in the same manner as in Example 1 except that the concentration of Borkensiflavone was 100 μg / mL.
（実施例４）
モレロフラボン（Garcinia vilersianaから単離精製したもの）を５００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、モレロフラボンを含む誘引剤を製造した。 Example 4
Moreroflavone (isolated and purified from Garcinia vilersiana ) was dissolved in a solvent (DMEM) to a concentration of 500 μg / mL to produce an attractant containing moreroflavone.
モレロフラボン（Garcinia vilersianaから単離精製したもの）を５００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、モレロフラボンを含む誘引剤を製造した。 Example 4
Moreroflavone (isolated and purified from Garcinia vilersiana ) was dissolved in a solvent (DMEM) to a concentration of 500 μg / mL to produce an attractant containing moreroflavone.
（実施例５）
モレロフラボンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例４と同様にして誘引剤を製造した。 (Example 5)
An attractant was produced in the same manner as in Example 4 except that the concentration of morelloflavone was 200 μg / mL.
モレロフラボンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例４と同様にして誘引剤を製造した。 (Example 5)
An attractant was produced in the same manner as in Example 4 except that the concentration of morelloflavone was 200 μg / mL.
（実施例６）
モレロフラボンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例４と同様にして誘引剤を製造した。 (Example 6)
An attractant was produced in the same manner as in Example 4 except that the concentration of Morelloflavone was adjusted to 100 μg / mL.
モレロフラボンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例４と同様にして誘引剤を製造した。 (Example 6)
An attractant was produced in the same manner as in Example 4 except that the concentration of Morelloflavone was adjusted to 100 μg / mL.
（実施例７）
ケンフェリトリン（フユボダイジュ（Tilia cordata）から単離精製したもの）を４００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、ケンフェリトリンを含む誘引剤を製造した。 (Example 7)
Kaempferitrin (isolated and purified from Tilia cordata ) was dissolved in a solvent (DMEM) to a concentration of 400 μg / mL to produce an attractant containing kaempferritrin.
ケンフェリトリン（フユボダイジュ（Tilia cordata）から単離精製したもの）を４００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、ケンフェリトリンを含む誘引剤を製造した。 (Example 7)
Kaempferitrin (isolated and purified from Tilia cordata ) was dissolved in a solvent (DMEM) to a concentration of 400 μg / mL to produce an attractant containing kaempferritrin.
（実施例８）
ケンフェリトリンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 (Example 8)
An attractant was produced in the same manner as in Example 7 except that the concentration of kaempferitrin was 200 μg / mL.
ケンフェリトリンの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 (Example 8)
An attractant was produced in the same manner as in Example 7 except that the concentration of kaempferitrin was 200 μg / mL.
（実施例９）
ケンフェリトリンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 Example 9
An attractant was produced in the same manner as in Example 7 except that the concentration of kaempferitrin was 100 μg / mL.
ケンフェリトリンの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 Example 9
An attractant was produced in the same manner as in Example 7 except that the concentration of kaempferitrin was 100 μg / mL.
（実施例１０）
ケルセチン－３，７－ジラムノシド（フユボダイジュ（Tilia cordata）から単離精製したもの）を４００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、ケルセチン－３，７－ジラムノシドを含む誘引剤を製造した。 (Example 10)
Quercetin-3,7-dirhamnoside (isolated and purified from Tilia cordata ) was dissolved in a solvent (DMEM) to a concentration of 400 μg / mL, and an attractant containing quercetin-3,7-dirhamnoside was added. Manufactured.
ケルセチン－３，７－ジラムノシド（フユボダイジュ（Tilia cordata）から単離精製したもの）を４００μｇ／ｍＬ濃度となるように溶媒（ＤＭＥＭ）に溶解させ、ケルセチン－３，７－ジラムノシドを含む誘引剤を製造した。 (Example 10)
Quercetin-3,7-dirhamnoside (isolated and purified from Tilia cordata ) was dissolved in a solvent (DMEM) to a concentration of 400 μg / mL, and an attractant containing quercetin-3,7-dirhamnoside was added. Manufactured.
（実施例１１）
ケルセチン－３，７－ジラムノシドの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 (Example 11)
An attractant was produced in the same manner as in Example 7 except that the concentration of quercetin-3,7-dirhamnoside was 200 μg / mL.
ケルセチン－３，７－ジラムノシドの濃度が２００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 (Example 11)
An attractant was produced in the same manner as in Example 7 except that the concentration of quercetin-3,7-dirhamnoside was 200 μg / mL.
（実施例１２）
ケルセチン－３，７－ジラムノシドの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 Example 12
An attractant was produced in the same manner as in Example 7 except that the concentration of quercetin-3,7-dirhamnoside was adjusted to 100 μg / mL.
ケルセチン－３，７－ジラムノシドの濃度が１００μｇ／ｍＬ濃度となるようにした点以外は、実施例７と同様にして誘引剤を製造した。 Example 12
An attractant was produced in the same manner as in Example 7 except that the concentration of quercetin-3,7-dirhamnoside was adjusted to 100 μg / mL.
製造した各実施例の骨髄幹細胞の誘引剤を使用し、生体外における細胞誘引試験による評価を行った。図１は、該評価方法の様子を模式的に示したものである。以下、図１を参照しながら評価方法の詳細を説明する。
Using the produced bone marrow stem cell attractant of each Example, evaluation was performed by an in vitro cell attraction test. FIG. 1 schematically shows the state of the evaluation method. The details of the evaluation method will be described below with reference to FIG.
＜生体外における細胞誘引試験＞
各実施例の骨髄幹細胞の誘引剤を試験用サンプルとして用意した。
一方、陰性対照サンプルとして、ＤＭＥＭ「ＦＢＳ（－）、Ｐ／Ｓ（－）」を用意した。
また、陽性対照サンプルとして、２０ｎｇ／ｍＬ ＰＤＧＦ－ＢＢ（血小板由来成長因子 ＰＥＰＲＯ ＴＥＣＨ社製）濃度のＤＭＥＭ溶液を用意した。
また、マウス骨髄間葉系幹細胞（以下、ｍＭＳＣともいう）をコンフルエントまで培養して回収し、１０％ ＦＢＳ／ＤＭＥＭ「Ｐ／Ｓ（－）」で１×１０７ｃｅｌｌｓ／ｍｌとなるように懸濁し、細胞懸濁液を用意した。
なお、ＦＢＳは、１０％ウシ胎児血清を示し、Ｐ／Ｓは、１００ユニットペニシリン及び０．１ｍｇ／ｍＬストレプトマイシンを示している。また（－）の記号は、配合していないことを示している。
次に、複数の独立したウェルを備え、図１（ａ）に示すようにメンブレンＭによりウェルが上部ウェルＰと下部ウェルＱとに仕切られているボイデンチャンバーＢ（Ｎｅｕｒｏ Ｐｒｏｂｅ社製）を用意した。各試験用サンプルのいずれか１種と陰性対照サンプル及び陽性対照サンプルとが同一のボイデンチャンバーＢにおいて試験できるように設定し、該チャンバーＢの各下部ウェルに各サンプルをそれぞれ２８μＬずつアプライした。なお、ボイデンチャンバーＢのメンブレンＭとしては、商品名「Ｐｏｌｙｃａｒｂｏｎａｔｅ Ｍｅｍｂｒａｎｅｓ」（Ｎｅｕｒｏ Ｐｒｏｂｅ社製 細孔サイズ８μｍ）を用いた。
続いて、上部ウェルＰに上記細胞懸濁液を５０μＬずつ播種し、３７℃、５％ＣＯ２の条件下で４時間培養した（図１（ｂ）を参照）。
４時間培養後、図１（ｃ）に示すように、移動していないｍＭＳＣを付属のフィルターワイパーＲではぎ取り、メンブレンＭの下側へ移動したｍＭＳＣのみをディフ・クイック染色（Ｓｙｓｍｅｘ社製キットを使用）により染色した。
そして、染色像をデジタル化してコンピュータに取り込み、画像を黒と白に２値化すべく青色に染色された部分が白色になるように変換したうえで、各ウェル範囲内の輝度の平均値を画像編集ソフトウェア（商品名「フォトショップ」）の機能を用いて測定した。陰性対照サンプルおよび陽性対照サンプルにおける輝度を各試験用サンプルにおける輝度と比較することにより、骨髄幹細胞の誘引剤のｍＭＳＣ誘引活性を評価した。
なお、各試験サンプル、陽性対照サンプル、陰性対照サンプルについては、２回の測定結果を平均することにより、それぞれ測定値を算出した。 <In vitro cell attraction test>
The attractant for bone marrow stem cells of each Example was prepared as a test sample.
On the other hand, DMEM “FBS (−), P / S (−)” was prepared as a negative control sample.
As a positive control sample, a DMEM solution having a concentration of 20 ng / mL PDGF-BB (platelet-derived growth factor PEPRO TECH) was prepared.
In addition, mouse bone marrow mesenchymal stem cells (hereinafter also referred to as mMSC) were cultured and collected until confluent, and suspended with 10% FBS / DMEM “P / S (−)” to 1 × 10 7 cells / ml. It became cloudy and a cell suspension was prepared.
FBS represents 10% fetal bovine serum, and P / S represents 100 unit penicillin and 0.1 mg / mL streptomycin. The symbol (-) indicates that no blending is performed.
Next, a Boyden chamber B (manufactured by Neuro Probe) having a plurality of independent wells, in which the well is partitioned into an upper well P and a lower well Q by a membrane M as shown in FIG. did. One kind of each test sample was set so that a negative control sample and a positive control sample could be tested in the same Boyden chamber B, and 28 μL of each sample was applied to each lower well of the chamber B. In addition, as the membrane M of the Boyden chamber B, a trade name “Polycarbonate Membranes” (pore size: 8 μm, manufactured by Neuro Probe) was used.
Subsequently, 50 μL of the cell suspension was seeded in the upper well P, and cultured for 4 hours under conditions of 37 ° C. and 5% CO 2 (see FIG. 1B).
After culturing for 4 hours, as shown in FIG. 1 (c), unmigrated mMSC was removed with the attached filter wiper R, and only the mMSC that had moved to the lower side of the membrane M was subjected to Diff-Quick staining (Sysmex kit). Used).
Then, the stained image is digitized and loaded into a computer, and the image is converted to a white portion of the blue-stained portion so that the image is binarized into black and white. The measurement was performed using the function of editing software (trade name “Photoshop”). The mMSC attracting activity of the bone marrow stem cell attractant was assessed by comparing the brightness in the negative and positive control samples with the brightness in each test sample.
In addition, about each test sample, the positive control sample, and the negative control sample, the measured value was each calculated by averaging two measurement results.
各実施例の骨髄幹細胞の誘引剤を試験用サンプルとして用意した。
一方、陰性対照サンプルとして、ＤＭＥＭ「ＦＢＳ（－）、Ｐ／Ｓ（－）」を用意した。
また、陽性対照サンプルとして、２０ｎｇ／ｍＬ ＰＤＧＦ－ＢＢ（血小板由来成長因子 ＰＥＰＲＯ ＴＥＣＨ社製）濃度のＤＭＥＭ溶液を用意した。
また、マウス骨髄間葉系幹細胞（以下、ｍＭＳＣともいう）をコンフルエントまで培養して回収し、１０％ ＦＢＳ／ＤＭＥＭ「Ｐ／Ｓ（－）」で１×１０７ｃｅｌｌｓ／ｍｌとなるように懸濁し、細胞懸濁液を用意した。
なお、ＦＢＳは、１０％ウシ胎児血清を示し、Ｐ／Ｓは、１００ユニットペニシリン及び０．１ｍｇ／ｍＬストレプトマイシンを示している。また（－）の記号は、配合していないことを示している。
次に、複数の独立したウェルを備え、図１（ａ）に示すようにメンブレンＭによりウェルが上部ウェルＰと下部ウェルＱとに仕切られているボイデンチャンバーＢ（Ｎｅｕｒｏ Ｐｒｏｂｅ社製）を用意した。各試験用サンプルのいずれか１種と陰性対照サンプル及び陽性対照サンプルとが同一のボイデンチャンバーＢにおいて試験できるように設定し、該チャンバーＢの各下部ウェルに各サンプルをそれぞれ２８μＬずつアプライした。なお、ボイデンチャンバーＢのメンブレンＭとしては、商品名「Ｐｏｌｙｃａｒｂｏｎａｔｅ Ｍｅｍｂｒａｎｅｓ」（Ｎｅｕｒｏ Ｐｒｏｂｅ社製 細孔サイズ８μｍ）を用いた。
続いて、上部ウェルＰに上記細胞懸濁液を５０μＬずつ播種し、３７℃、５％ＣＯ２の条件下で４時間培養した（図１（ｂ）を参照）。
４時間培養後、図１（ｃ）に示すように、移動していないｍＭＳＣを付属のフィルターワイパーＲではぎ取り、メンブレンＭの下側へ移動したｍＭＳＣのみをディフ・クイック染色（Ｓｙｓｍｅｘ社製キットを使用）により染色した。
そして、染色像をデジタル化してコンピュータに取り込み、画像を黒と白に２値化すべく青色に染色された部分が白色になるように変換したうえで、各ウェル範囲内の輝度の平均値を画像編集ソフトウェア（商品名「フォトショップ」）の機能を用いて測定した。陰性対照サンプルおよび陽性対照サンプルにおける輝度を各試験用サンプルにおける輝度と比較することにより、骨髄幹細胞の誘引剤のｍＭＳＣ誘引活性を評価した。
なお、各試験サンプル、陽性対照サンプル、陰性対照サンプルについては、２回の測定結果を平均することにより、それぞれ測定値を算出した。 <In vitro cell attraction test>
The attractant for bone marrow stem cells of each Example was prepared as a test sample.
On the other hand, DMEM “FBS (−), P / S (−)” was prepared as a negative control sample.
As a positive control sample, a DMEM solution having a concentration of 20 ng / mL PDGF-BB (platelet-derived growth factor PEPRO TECH) was prepared.
In addition, mouse bone marrow mesenchymal stem cells (hereinafter also referred to as mMSC) were cultured and collected until confluent, and suspended with 10% FBS / DMEM “P / S (−)” to 1 × 10 7 cells / ml. It became cloudy and a cell suspension was prepared.
FBS represents 10% fetal bovine serum, and P / S represents 100 unit penicillin and 0.1 mg / mL streptomycin. The symbol (-) indicates that no blending is performed.
Next, a Boyden chamber B (manufactured by Neuro Probe) having a plurality of independent wells, in which the well is partitioned into an upper well P and a lower well Q by a membrane M as shown in FIG. did. One kind of each test sample was set so that a negative control sample and a positive control sample could be tested in the same Boyden chamber B, and 28 μL of each sample was applied to each lower well of the chamber B. In addition, as the membrane M of the Boyden chamber B, a trade name “Polycarbonate Membranes” (pore size: 8 μm, manufactured by Neuro Probe) was used.
Subsequently, 50 μL of the cell suspension was seeded in the upper well P, and cultured for 4 hours under conditions of 37 ° C. and 5% CO 2 (see FIG. 1B).
After culturing for 4 hours, as shown in FIG. 1 (c), unmigrated mMSC was removed with the attached filter wiper R, and only the mMSC that had moved to the lower side of the membrane M was subjected to Diff-Quick staining (Sysmex kit). Used).
Then, the stained image is digitized and loaded into a computer, and the image is converted to a white portion of the blue-stained portion so that the image is binarized into black and white. The measurement was performed using the function of editing software (trade name “Photoshop”). The mMSC attracting activity of the bone marrow stem cell attractant was assessed by comparing the brightness in the negative and positive control samples with the brightness in each test sample.
In addition, about each test sample, the positive control sample, and the negative control sample, the measured value was each calculated by averaging two measurement results.
実施例１～６の誘引剤における評価結果を輝度のグラフによって図２に示す。また、実施例７～９の誘引剤における評価結果を同様に図３に示す。また、実施例１０～１２の誘引剤における評価結果を同様に図４に示す。
図２～図４から把握できるように、実施例の骨髄幹細胞の誘引剤は、骨髄幹細胞を十分に誘引することができる。 The evaluation results for the attractants of Examples 1 to 6 are shown in FIG. Similarly, the evaluation results of the attractants of Examples 7 to 9 are shown in FIG. The evaluation results for the attractants of Examples 10 to 12 are also shown in FIG.
As can be understood from FIG. 2 to FIG. 4, the attractant for bone marrow stem cells of the example can sufficiently attract bone marrow stem cells.
図２～図４から把握できるように、実施例の骨髄幹細胞の誘引剤は、骨髄幹細胞を十分に誘引することができる。 The evaluation results for the attractants of Examples 1 to 6 are shown in FIG. Similarly, the evaluation results of the attractants of Examples 7 to 9 are shown in FIG. The evaluation results for the attractants of Examples 10 to 12 are also shown in FIG.
As can be understood from FIG. 2 to FIG. 4, the attractant for bone marrow stem cells of the example can sufficiently attract bone marrow stem cells.
本発明の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、例えば、所定の体内組織において骨髄幹細胞を分化させる前に、骨髄で生じた骨髄幹細胞を所定の体内組織に誘引するために使用される。即ち、本発明の骨髄幹細胞の誘引剤及び骨髄幹細胞の誘引方法は、例えば、所定の体内組織に誘引剤を含ませる処置をすることにより、血流にのって体内を循環している骨髄幹細胞をその体内組織に誘引して集積させる目的で好適に使用される。
The bone marrow stem cell attractant and bone marrow stem cell attracting method of the present invention are used, for example, to attract bone marrow stem cells generated in the bone marrow to a predetermined body tissue before the bone marrow stem cells are differentiated in the predetermined body tissue. . That is, the method for attracting bone marrow stem cells and the method for attracting bone marrow stem cells of the present invention includes, for example, bone marrow stem cells circulating in the body along the bloodstream by performing a treatment that includes the attractant in a predetermined body tissue. Is preferably used for the purpose of attracting and accumulating in the body tissue.
Ｂ：ボイデンチャンバー、 Ｐ：上部ウェル、 Ｑ：下部ウェル、 Ｍ：メンブレン、 Ｒ：フィルターワイパー。
B: Boyden chamber, P: upper well, Q: lower well, M: membrane, R: filter wiper.
B: Boyden chamber, P: upper well, Q: lower well, M: membrane, R: filter wiper.
Claims (2)
- ボルケンシフラボン、モレロフラボン、ケンフェリトリン、及び、ケルセチン－３，７－ジラムノシドのうちの少なくとも１種を含む、骨髄幹細胞の誘引剤。 An attractant for bone marrow stem cells, comprising at least one of borkenciflavone, moreroflavone, kaempferitrin, and quercetin-3,7-dirhamnoside.
- 請求項１記載の骨髄幹細胞の誘引剤により骨髄幹細胞を誘引する、骨髄幹細胞の誘引方法。
A method for attracting bone marrow stem cells, wherein the bone marrow stem cells are attracted by the bone marrow stem cell attractant according to claim 1.
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| JP2011251925A (en) * | 2010-06-01 | 2011-12-15 | Pias Arise Kk | Mesenchymal stem cell inducer, and method for induction of mesenchymal stem cell |
| WO2012063514A1 (en) * | 2010-11-08 | 2012-05-18 | ピアス株式会社 | Attractant for bone marrow stem cells, and method for attracting bone marrow stem cells |
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| JP2011251925A (en) * | 2010-06-01 | 2011-12-15 | Pias Arise Kk | Mesenchymal stem cell inducer, and method for induction of mesenchymal stem cell |
| WO2012063514A1 (en) * | 2010-11-08 | 2012-05-18 | ピアス株式会社 | Attractant for bone marrow stem cells, and method for attracting bone marrow stem cells |
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| JP2015116169A (en) | 2015-06-25 |
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