WO2014208425A1 - Gene transfer device and gene transfer method - Google Patents
Gene transfer device and gene transfer method Download PDFInfo
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- WO2014208425A1 WO2014208425A1 PCT/JP2014/066185 JP2014066185W WO2014208425A1 WO 2014208425 A1 WO2014208425 A1 WO 2014208425A1 JP 2014066185 W JP2014066185 W JP 2014066185W WO 2014208425 A1 WO2014208425 A1 WO 2014208425A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- the other electrode is attached to the storage container.
- the one electrode has a cylindrical shape, and the plasma material supply means can supply the plasma material between the electrodes through the inside of the one electrode. It is preferable that it is comprised.
- the gene introduction method according to the present invention preferably includes a storage container to which the other electrode is attached, and the gene dispersion solution is stored and disposed in the storage container. In these cases, the plasma generated from the plasma raw material supplied from one electrode toward the other electrode can be electrically accelerated and efficiently applied to the gene dispersion solution, further increasing gene transfer efficiency. Can do.
- the length of the cylindrical electrode 33a is 20 mm
- the distance between the electrodes 33a and 33b is 13 mm
- the distance from the lower electrode 33b to the lower end of the glass tube 31 is 32 mm
- the lower end of the glass tube 31 to the surface of the gene dispersion solution 35 It was 5 mm.
- the apparatus used for the test is the apparatus shown in FIG. 2, in which the distance from the lower electrode 23b to the lower end of the glass tube 21 is 73 mm (hereinafter referred to as “long diffusion type”), and the lower electrode 23b to the glass tube.
- the test was performed on a sample having a length of 35 mm up to the lower end of 21 (hereinafter referred to as “short diffusion type”).
- the cells used for the test are 3T3L1 cells (floating state). In the test, two kinds of fluorescent substances, YOYO-1 and LIVE / DEAD Stain, were used instead of the gene.
Abstract
[Problem] To provide a gene transfer device and a gene transfer method, whereby it becomes possible to introduce a gene into a cell with higher introduction efficiency.
[Solution] A pair of electrodes (13a, 13b) are so arranged as to hold therebetween a storage container (12) in which a gene-dispersed solution (15) is to be placed, wherein the gene-dispersed solution contains a gene and a cell into which the gene is to be introduced. The other (13b) of the electrodes is attached to the storage container (12). A plasma raw material supply means is so adapted that a helium gas, which is a plasma raw material, can be supplied from one (13a) of the electrodes toward the other (13b) of the electrodes. A power supply unit (14) is so adapted as to apply a voltage between the electrodes (13a, 13b) so that low-temperature plasma can be generated between the electrodes (13a, 13b) to which the plasma raw material has been supplied. The storage container (12) is so arranged that the gene-dispersed solution (15) contained therein can be irradiated with the generated plasma directly.
Description
本発明は、プラズマを利用した遺伝子導入装置および遺伝子導入方法に関する。
The present invention relates to a gene introduction apparatus and a gene introduction method using plasma.
近年、遺伝子治療の研究やｉＰＳ細胞の作製などに、細胞に遺伝子を導入する遺伝子導入技術が用いられている。例えば、遺伝子治療の研究では、がんやＨＩＶ、単一遺伝子病、Ｃ型肝炎などを治療するために、患者から抽出した細胞に、正常な遺伝子や治療用の遺伝子を導入し、それをワクチンとして患者に投与する技術の開発が進められている。また、ｉＰＳ細胞については、患者から抽出した体細胞に、複数の遺伝子を導入することによりｉＰＳ細胞が作製されている。
In recent years, gene introduction techniques for introducing genes into cells have been used for gene therapy research and iPS cell production. For example, in gene therapy research, in order to treat cancer, HIV, single-gene disease, hepatitis C, etc., normal genes and therapeutic genes are introduced into cells extracted from patients and used as vaccines. As a result, development of technology for administration to patients is underway. As for iPS cells, iPS cells are prepared by introducing a plurality of genes into somatic cells extracted from a patient.
従来の遺伝子導入方法として、リポソーム法などの化学的手法や、エレクトロポレーション法などの物理学的手法、ウイルス法などの生物学的手法が用いられている。しかし、リポソーム法では、適用できる細胞が限られており、導入効率も低いという問題があった。また、エレクトロポレーション法では、導入効率は高いが、細胞が死にやすく、生存率が低いという問題があった。また、ウイルス法では、使用するウイルスにより、細胞が病気に感染する危険性があるという問題があった。
As a conventional gene introduction method, a chemical method such as a liposome method, a physical method such as an electroporation method, or a biological method such as a virus method is used. However, the liposome method has a problem that applicable cells are limited and introduction efficiency is low. In addition, the electroporation method has a problem that the introduction efficiency is high, but the cells are likely to die and the survival rate is low. In addition, the virus method has a problem in that there is a risk of infecting cells with a disease depending on the virus used.
そこで、これらの問題を解決するため、細胞を低温ガスプラズマで処理することにより、細胞の近傍に存在する遺伝子を細胞内に導入する方法が開発されている（例えば、特許文献１、２、非特許文献１参照）。この方法では、細胞にプラズマを照射することにより、プラズマ中に存在するラジカルの作用により、脂質過酸化反応で細胞膜に小孔を開けるとともに、細胞の表面に電界を発生させて、その電界により小孔から細胞内に遺伝子を押し込むものと考えられる。この方法によれば、病気感染の危険性がなく、導入効率も目視で最大６０～７０％、ＦＡＣＳ（ｆｌｕｏｒｅｓｃｅｎｃｅ ａｃｔｉｖａｔｅｄ ｃｅｌｌ ｓｏｒｔｉｎｇ）による検討で最大２５～３０％程度と高くなっている。
In order to solve these problems, a method has been developed in which cells are treated with low-temperature gas plasma to introduce genes existing in the vicinity of the cells into the cells (for example, Patent Documents 1 and 2). Patent Document 1). In this method, by irradiating a cell with plasma, a radical is present in the plasma, thereby opening a small hole in the cell membrane by a lipid peroxidation reaction and generating an electric field on the surface of the cell. It is thought that the gene is pushed into the cell from the hole. According to this method, there is no risk of disease infection, and the introduction efficiency is as high as 60 to 70% visually, and as high as 25 to 30% as a result of FACS (fluorescence activated cell sorting) studies.
しかし、特許文献１、２および非特許文献１に記載の低温ガスプラズマを利用した遺伝子導入方法によれば、比較的高い導入効率が得られているが、照射したプラズマにより細胞に障害が発生しやすく、細胞の生存率が高いとは言い難いという問題があった。そこで、低温ガスプラズマの照射による細胞の生存率を高めるために、プラズマを照射する液体培地に、細胞の障害を防止するためのタンパク質から成る障害防止成分を含有した遺伝子導入方法が開発されている（例えば、特許文献３参照）。この方法では、先端に向かってテーパー状に細くなるよう形成された細長いガラス管の側面に、所定の間隔をあけて１対の電極を設けたプラズマ発生装置を用い、ガラス管の上部からプラズマ原料のヘリウムガスを注入し、各電極間に電圧を印加して低温プラズマを発生させ、ガラス管の先端から、細胞および遺伝子が入った液体培地に向かってプラズマを噴射させることにより、細胞内に遺伝子を導入することができる。
However, according to the gene introduction methods using low-temperature gas plasma described in Patent Documents 1 and 2 and Non-Patent Document 1, a relatively high introduction efficiency is obtained, but cells are damaged by the irradiated plasma. There was a problem that it was easy and it was difficult to say that the survival rate of cells was high. Therefore, in order to increase the survival rate of cells by irradiation with low-temperature gas plasma, a gene introduction method has been developed in which a liquid medium that is irradiated with plasma contains a disorder-preventing component consisting of a protein for preventing cell damage. (For example, refer to Patent Document 3). In this method, a plasma material is used in which a pair of electrodes is provided on a side surface of an elongated glass tube formed so as to be tapered in a tapered shape toward the tip, and a pair of electrodes is provided at a predetermined interval. Helium gas is injected, a voltage is applied between the electrodes to generate low-temperature plasma, and plasma is injected from the tip of the glass tube toward the liquid medium containing the cells and genes. Can be introduced.
特許文献３に記載の低温ガスプラズマを利用した遺伝子導入方法によれば、比較的高い導入効率が得られるが、稀少遺伝子を利用する場合や体内細胞への導入を考慮した場合、さらに高い導入効率で、遺伝子を細胞に導入できる方法の開発が望まれている。
According to the gene introduction method using low-temperature gas plasma described in Patent Document 3, a relatively high introduction efficiency can be obtained. However, when a rare gene is used or introduction into a body cell is taken into consideration, the introduction efficiency is higher. Therefore, development of a method capable of introducing a gene into a cell is desired.
本発明は、このような課題に着目してなされたもので、より高い導入効率で遺伝子を細胞に導入することができる遺伝子導入装置および遺伝子導入方法を提供することを目的とする。
The present invention has been made paying attention to such a problem, and an object thereof is to provide a gene introduction apparatus and a gene introduction method capable of introducing a gene into cells with higher introduction efficiency.
上記目的を達成するために、本発明に係る遺伝子導入装置は、遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液を収納する収納容器と、前記収納容器を挟むよう配置された１対の電極と、一方の電極から他方の電極に向かって、プラズマ原料を供給するプラズマ原料供給手段と、前記プラズマ原料が供給された各電極間でプラズマを発生させるよう、各電極間に電圧を印加する電源部とを有し、前記収納容器は、収納された前記遺伝子分散溶液に、発生した前記プラズマが直接当たるよう配置されていることを特徴とする。
In order to achieve the above object, a gene transfer device according to the present invention includes a storage container that stores a gene dispersion solution containing a gene and a cell into which the gene is introduced, and a pair of the storage containers disposed between the storage container. A voltage is applied between the electrodes so as to generate plasma between the electrode, the plasma raw material supply means for supplying the plasma raw material from one electrode to the other electrode, and each electrode supplied with the plasma raw material And the storage container is arranged so that the generated plasma directly hits the stored gene dispersion solution.
また、本発明に係る遺伝子導入方法は、遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液を、１対の電極の間に配置し、一方の電極から他方の電極に向かってプラズマ原料を供給しつつ、各電極間に電圧を印加してプラズマを発生させ、前記遺伝子分散溶液に前記プラズマを直接当てることにより、前記細胞に前記遺伝子を導入することを特徴とする。
In the gene introduction method according to the present invention, a gene dispersion solution containing a gene and a cell into which the gene is introduced is disposed between a pair of electrodes, and the plasma raw material is directed from one electrode to the other electrode. While supplying, a voltage is applied between the electrodes to generate plasma, and the plasma is directly applied to the gene dispersion solution to introduce the gene into the cells.
本発明に係る遺伝子導入装置および遺伝子導入方法は、遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液にプラズマを直接当てることにより、その細胞に遺伝子を導入することができる。特に、遺伝子分散溶液を各電極の間に配置していることにより、遺伝子導入効率を高めることができる。このため、稀少遺伝子を導入するのに好適に使用することができる。
The gene introduction apparatus and gene introduction method according to the present invention can introduce a gene into a cell by directly applying plasma to a gene dispersion solution containing the gene and a cell into which the gene is introduced. In particular, the gene introduction efficiency can be increased by arranging the gene dispersion solution between the electrodes. For this reason, it can be suitably used for introducing a rare gene.
本発明に係る遺伝子導入装置および遺伝子導入方法は、プラズマを利用するため、エレクトロポレーション法と比べて細胞の生存率が高く、ウイルスによる病気の感染の危険性もない。また、本発明に係る遺伝子導入装置および遺伝子導入方法は、様々な細胞に遺伝子を導入することができる。遺伝子を導入する細胞は、いかなるものであってもよく、例えば、ＨｅＬａ細胞、繊維芽細胞、神経芽細胞、乳がん細胞などを利用することができる。
Since the gene introduction apparatus and the gene introduction method according to the present invention use plasma, the cell survival rate is higher than that of the electroporation method, and there is no risk of infection by a virus. Moreover, the gene introduction apparatus and the gene introduction method according to the present invention can introduce genes into various cells. The cell into which the gene is introduced may be any cell, and for example, HeLa cells, fibroblasts, neuroblasts, breast cancer cells and the like can be used.
使用するプラズマは、細胞を傷つけないよう低温プラズマから成ることが好ましい。また、プラズマを発生する装置は、密閉型であっても、開放型であってもよいが、体内細胞への導入を考慮すると、開放型であることが好ましい。プラズマ原料は、生成ガスとしてヘリウム、アルゴン、窒素、酸素、二酸化炭素、空気など、いかなるものを含んでいてもよい。
The plasma used is preferably composed of low temperature plasma so as not to damage the cells. The device for generating plasma may be a sealed type or an open type, but it is preferably an open type in consideration of introduction into cells in the body. The plasma raw material may contain any product gas such as helium, argon, nitrogen, oxygen, carbon dioxide, air.
本発明に係る遺伝子導入装置は、前記他方の電極が前記収納容器に取り付けられていることが好ましい。また、本発明に係る遺伝子導入装置で、前記一方の電極は筒状を成しており、前記プラズマ原料供給手段は、前記一方の電極の内部を通して、各電極の間に前記プラズマ原料を供給可能に構成されていることが好ましい。本発明に係る遺伝子導入方法は、前記他方の電極が取り付けられた収納容器を有し、前記遺伝子分散溶液を前記収納容器に収納して配置することが好ましい。これらの場合、一方の電極から他方の電極に向かって供給されるプラズマ原料から発生するプラズマを、電気的に加速して効率的に遺伝子分散溶液に当てることができ、遺伝子導入効率をさらに高めることができる。
In the gene introduction apparatus according to the present invention, it is preferable that the other electrode is attached to the storage container. Further, in the gene introduction apparatus according to the present invention, the one electrode has a cylindrical shape, and the plasma material supply means can supply the plasma material between the electrodes through the inside of the one electrode. It is preferable that it is comprised. The gene introduction method according to the present invention preferably includes a storage container to which the other electrode is attached, and the gene dispersion solution is stored and disposed in the storage container. In these cases, the plasma generated from the plasma raw material supplied from one electrode toward the other electrode can be electrically accelerated and efficiently applied to the gene dispersion solution, further increasing gene transfer efficiency. Can do.
本発明に係る遺伝子導入装置で、前記細胞は接着細胞から成り、前記収納容器の底面から、前記収納容器に収納された前記遺伝子分散溶液の表面までの高さを調整可能に構成されていてもよい。この場合、収納容器の底面から遺伝子分散溶液の表面までの高さを調整することにより、接着細胞（ｂｏｕｎｄ ｃｅｌｌ）に対して、遺伝子導入効率を高めることができる。特に高い遺伝子導入効率を得るために、本発明に係る遺伝子導入装置で、前記細胞は接着細胞から成り、前記収納容器は、底面から前記遺伝子分散溶液の表面までの高さが１．５ｍｍ以下で、前記底面に存在する前記細胞が前記遺伝子分散溶液の表面に露出しないよう、前記遺伝子分散溶液を収納可能に設けられていることが好ましい。また、本発明に係る遺伝子導入方法で、前記細胞は接着細胞から成り、前記収納容器の底面から前記遺伝子分散溶液の表面までの高さが１．５ｍｍ以下で、前記底面に存在する前記細胞が前記遺伝子分散溶液の表面に露出しないよう、前記遺伝子分散溶液を前記収納容器に収納することが好ましい。
In the gene introduction device according to the present invention, the cell is composed of an adherent cell, and the height from the bottom surface of the storage container to the surface of the gene dispersion solution stored in the storage container can be adjusted. Good. In this case, by adjusting the height from the bottom surface of the storage container to the surface of the gene dispersion solution, it is possible to increase the gene transfer efficiency for the bound cells. In order to obtain particularly high gene transfer efficiency, in the gene transfer device according to the present invention, the cells are composed of adherent cells, and the storage container has a height from the bottom surface to the surface of the gene dispersion solution of 1.5 mm or less. It is preferable that the gene dispersion solution can be stored so that the cells existing on the bottom surface are not exposed on the surface of the gene dispersion solution. Further, in the gene introduction method according to the present invention, the cells are composed of adherent cells, the height from the bottom surface of the storage container to the surface of the gene dispersion solution is 1.5 mm or less, and the cells present on the bottom surface are It is preferable to store the gene dispersion solution in the storage container so as not to be exposed on the surface of the gene dispersion solution.
本発明に係る遺伝子導入装置および遺伝子導入方法で、前記プラズマ原料は水を含んでいることが好ましい。この場合、プラズマ中にヒドロキシルラジカル（ＯＨラジカル）が生成されるため、そのヒドロキシルラジカルの作用により細胞への遺伝子の導入が促進され、高い遺伝子導入効率を得ることができる。
In the gene introduction apparatus and the gene introduction method according to the present invention, the plasma raw material preferably contains water. In this case, since hydroxyl radicals (OH radicals) are generated in the plasma, introduction of genes into cells is promoted by the action of the hydroxyl radicals, and high gene introduction efficiency can be obtained.
本発明によれば、より高い導入効率で遺伝子を細胞に導入することができる遺伝子導入装置および遺伝子導入方法を提供することができる。
According to the present invention, it is possible to provide a gene introduction apparatus and a gene introduction method capable of introducing a gene into cells with higher introduction efficiency.
以下、図面に基づき、本発明の実施の形態について説明する。
図１は、本発明の実施の形態の遺伝子導入装置の一例を示している。
図１に示すように、遺伝子導入装置１０は、ガラス管１１と収納容器１２と１対の電極１３ａ，１３ｂとプラズマ原料供給手段（図示せず）と電源部１４とを有している。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows an example of a gene introduction apparatus according to an embodiment of the present invention.
As shown in FIG. 1, thegene introduction apparatus 10 includes a glass tube 11, a storage container 12, a pair of electrodes 13 a and 13 b, plasma raw material supply means (not shown), and a power supply unit 14.
図１は、本発明の実施の形態の遺伝子導入装置の一例を示している。
図１に示すように、遺伝子導入装置１０は、ガラス管１１と収納容器１２と１対の電極１３ａ，１３ｂとプラズマ原料供給手段（図示せず）と電源部１４とを有している。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows an example of a gene introduction apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the
ガラス管１１は、上下方向に伸びるよう配置され、下端部が徐々に細くなるようテーパー状に形成されている。収納容器１２は、ガラス管１１の下方に、ガラス管１１の下端から所定の間隔を開けて配置されている。収納容器１２は、ガラス管１１の側の上部が開口しており、内部に、遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液１５を収納するよう構成されている。
The glass tube 11 is disposed so as to extend in the vertical direction, and is formed in a tapered shape so that the lower end portion is gradually narrowed. The storage container 12 is disposed below the glass tube 11 with a predetermined interval from the lower end of the glass tube 11. The storage container 12 is open at the top of the glass tube 11 and is configured to store therein a gene dispersion solution 15 containing a gene and cells into which the gene is introduced.
１対の電極１３ａ，１３ｂのうち一方の電極１３ａは、ステンレス製で筒状を成し、ガラス管１１の内部上方に、上下方向に伸びるよう配置されている。他方の電極１３ｂは、銅線から成り、収納容器１２の下部外側面に巻き付けられている。
One electrode 13 a of the pair of electrodes 13 a and 13 b is made of stainless steel and has a cylindrical shape, and is disposed above the inside of the glass tube 11 so as to extend in the vertical direction. The other electrode 13 b is made of a copper wire and is wound around the lower outer surface of the storage container 12.
プラズマ原料供給手段は、筒状の一方の電極１３ａの内部を通して、各電極１３ａ，１３ｂの間にプラズマ原料を供給可能に構成されている。また、プラズマ原料供給手段は、一方の電極１３ａから他方の電極１３ｂに向かって、プラズマ原料を噴射して供給するようになっている。
The plasma raw material supply means is configured to be able to supply a plasma raw material between the electrodes 13a and 13b through the inside of one cylindrical electrode 13a. Further, the plasma raw material supply means jets and supplies the plasma raw material from one electrode 13a toward the other electrode 13b.
電源部１４は、各電極１３ａ，１３ｂに接続され、プラズマ原料が供給された各電極１３ａ，１３ｂの間で低温プラズマを発生させるよう、各電極１３ａ，１３ｂ間に電圧を印加可能に構成されている。これにより、遺伝子導入装置１０は、発生したプラズマが、収納容器１２に収納された遺伝子分散溶液１５に直接当たるようになっている。
The power supply unit 14 is connected to the electrodes 13a and 13b, and is configured to be able to apply a voltage between the electrodes 13a and 13b so as to generate a low temperature plasma between the electrodes 13a and 13b supplied with plasma raw materials. Yes. As a result, the gene transfer apparatus 10 is configured such that the generated plasma directly hits the gene dispersion solution 15 stored in the storage container 12.
なお、図１に示す具体的な一例では、ガラス管１１は、内径が８ｍｍ、下端の内径が２ｍｍ、一方の電極１３ａは、内径が２ｍｍ、長さが２０ｍｍ、一方の電極１３ａの下端からガラス管１１の下端までは、５８ｍｍである。また、ガラス管１１の下端から収納容器１２の底面までは、１２ｍｍである。
In the specific example shown in FIG. 1, the glass tube 11 has an inner diameter of 8 mm, the lower end has an inner diameter of 2 mm, and the one electrode 13a has an inner diameter of 2 mm and a length of 20 mm. The distance to the lower end of the tube 11 is 58 mm. Further, the distance from the lower end of the glass tube 11 to the bottom surface of the storage container 12 is 12 mm.
本発明の実施の形態の遺伝子導入方法は、遺伝子導入装置１０により好適に実施することができる。本発明の実施の形態の遺伝子導入方法では、まず、遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液１５を、他方の電極１３ｂが取り付けられた収納容器１２に収納する。プラズマ原料供給手段により、一方の電極１３ａから他方の電極１３ｂに向かってプラズマ原料を供給しつつ、電源部１４で各電極１３ａ，１３ｂ間に電圧を印加して低温プラズマを発生させ、遺伝子分散溶液１５にプラズマを直接当てる。
The gene introduction method according to the embodiment of the present invention can be preferably implemented by the gene introduction apparatus 10. In the gene introduction method according to the embodiment of the present invention, first, a gene dispersion solution 15 containing a gene and cells into which the gene is introduced is stored in a storage container 12 to which the other electrode 13b is attached. While supplying the plasma raw material from one electrode 13a to the other electrode 13b by the plasma raw material supply means, the power supply unit 14 applies a voltage between the electrodes 13a and 13b to generate a low temperature plasma, and the gene dispersion solution 15 is directly exposed to plasma.
本発明の実施の形態の遺伝子導入装置１０および遺伝子導入方法は、遺伝子分散溶液１５にプラズマを直接当てることにより、その細胞に遺伝子を導入することができる。特に、遺伝子分散溶液１５が各電極１３ａ，１３ｂの間に配置され、プラズマが電気的に加速され効率的に遺伝子分散溶液１５に当たるため、遺伝子導入効率をより高めることができる。このため、稀少遺伝子を導入するのに好適に使用することができる。
The gene introduction apparatus 10 and the gene introduction method of the embodiment of the present invention can introduce a gene into the cell by directly applying plasma to the gene dispersion solution 15. In particular, since the gene dispersion solution 15 is disposed between the electrodes 13a and 13b and the plasma is electrically accelerated to efficiently hit the gene dispersion solution 15, the gene introduction efficiency can be further increased. For this reason, it can be suitably used for introducing a rare gene.
本発明の実施の形態の遺伝子導入装置１０および遺伝子導入方法は、プラズマを利用するため、エレクトロポレーション法と比べて細胞の生存率が高く、ウイルスによる病気の感染の危険性もない。また、本発明の実施の形態の遺伝子導入装置１０および遺伝子導入方法は、様々な細胞に遺伝子を導入することができる。低温プラズマを使用するため、細胞を傷つけず、細胞の生存率を高めることができる。
Since the gene introduction apparatus 10 and the gene introduction method according to the embodiment of the present invention use plasma, the cell survival rate is higher than that of the electroporation method, and there is no risk of infection by a virus. Moreover, the gene introduction apparatus 10 and the gene introduction method of the embodiment of the present invention can introduce genes into various cells. Since low temperature plasma is used, the cell viability can be increased without damaging the cells.
以下に、比較例として、図２に示す試験装置を用いて、プラズマを利用した遺伝子導入技術についての試験を行った。なお、以下の比較例で使用する方法および装置は、特許文献３に記載のものとほぼ同じ方法および装置である。
Hereinafter, as a comparative example, a test for gene transfer technology using plasma was performed using the test apparatus shown in FIG. Note that the method and apparatus used in the following comparative examples are substantially the same method and apparatus as described in Patent Document 3.
［プラズマを利用した遺伝子導入試験－比較例］
プラズマを利用した遺伝子導入技術の導入効率等を調べるための試験を行った。図２に示すように、試験装置は、上下方向の伸びるよう配置されたガラス管２１の側面に、所定の間隔をあけて、銅製の１対の電極２３ａ，２３ｂを設けている。また、下端部がテーパー状に形成されたガラス管２１の下方に、遺伝子分散溶液２５を収納した容器２２を設置している。試験装置では、ガラス管２１の内径を８ｍｍ、ガラス管２１の下端の内径を２ｍｍとした。また、各電極２３ａ，２３ｂの間隔を３３ｍｍ、下方の電極２３ｂからガラス管２１の下端までを７３ｍｍ、ガラス管２１の下端から遺伝子分散溶液２５の表面までを５ｍｍとした。 [Plasma-based gene transfer test-comparative example]
A test was conducted to investigate the introduction efficiency of gene transfer technology using plasma. As shown in FIG. 2, the test apparatus is provided with a pair of copper electrodes 23 a and 23 b on a side surface of a glass tube 21 arranged to extend in the vertical direction with a predetermined interval. Further, a container 22 containing the gene dispersion solution 25 is installed below the glass tube 21 having a tapered lower end. In the test apparatus, the inner diameter of the glass tube 21 was 8 mm, and the inner diameter of the lower end of the glass tube 21 was 2 mm. The distance between the electrodes 23a and 23b was 33 mm, the distance from the lower electrode 23b to the lower end of the glass tube 21 was 73 mm, and the distance from the lower end of the glass tube 21 to the surface of the gene dispersion solution 25 was 5 mm.
プラズマを利用した遺伝子導入技術の導入効率等を調べるための試験を行った。図２に示すように、試験装置は、上下方向の伸びるよう配置されたガラス管２１の側面に、所定の間隔をあけて、銅製の１対の電極２３ａ，２３ｂを設けている。また、下端部がテーパー状に形成されたガラス管２１の下方に、遺伝子分散溶液２５を収納した容器２２を設置している。試験装置では、ガラス管２１の内径を８ｍｍ、ガラス管２１の下端の内径を２ｍｍとした。また、各電極２３ａ，２３ｂの間隔を３３ｍｍ、下方の電極２３ｂからガラス管２１の下端までを７３ｍｍ、ガラス管２１の下端から遺伝子分散溶液２５の表面までを５ｍｍとした。 [Plasma-based gene transfer test-comparative example]
A test was conducted to investigate the introduction efficiency of gene transfer technology using plasma. As shown in FIG. 2, the test apparatus is provided with a pair of
試験に使用した細胞は、マウス繊維芽細胞の３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、蛍光物質を使用した。使用した蛍光物質は、細胞中のＤＮＡに付着すると発光するＹＯＹＯ－１と、細胞が死ぬと発光するＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類である。
The cells used in the test are mouse fibroblast 3T3L1 cells (floating state). In the test, a fluorescent substance was used instead of the gene. There are two types of fluorescent substances used: YOYO-1, which emits light when attached to DNA in cells, and LIVE / DEAD Stein, which emits light when cells die.
試験では、ガラス管２１の上部からプラズマ原料のヘリウムガスを注入し、電源２４により各電極２３ａ，２３ｂ間に１０ｋＨｚの電圧を印加して、各電極２３ａ，２３ｂの間で低温プラズマを発生させ、遺伝子分散溶液２５に向かってプラズマを噴射させた。プラズマ噴射後の細胞の蛍光を観察し、遺伝子の導入効率および細胞の生存率を求めた。なお、印加電圧は６．８～１２．５ｋＶ、プラズマ照射時間は１～６０秒、ヘリウムガスの流量は３．０ｓｌｍ、遺伝子分散溶液２５の量は１０～１５０μＬである。
In the test, helium gas as a plasma material is injected from the upper part of the glass tube 21, and a voltage of 10 kHz is applied between the electrodes 23a and 23b by the power source 24 to generate low temperature plasma between the electrodes 23a and 23b. Plasma was sprayed toward the gene dispersion solution 25. The fluorescence of the cells after plasma injection was observed, and the gene transfer efficiency and cell viability were determined. The applied voltage is 6.8 to 12.5 kV, the plasma irradiation time is 1 to 60 seconds, the flow rate of helium gas is 3.0 slm, and the amount of the gene dispersion solution 25 is 10 to 150 μL.
蛍光物質として、ＹＯＹＯ－１を使用した結果の一例を図３に、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎを使用した結果の一例を図４に示す。図３および図４は、印加電圧が７．８ｋＶ、プラズマ照射時間が５秒、遺伝子分散溶液２５の量が１００μＬのときの結果である。なお、図３および図４中には、比較のため、同じ条件で、プラズマを照射しないときの結果（以下、「コントロール」という）および、エレクトロポレーション法による結果も示す。
FIG. 3 shows an example of the result of using YOYO-1 as the fluorescent material, and FIG. 4 shows an example of the result of using LIVE / DEAD Stain. 3 and 4 show the results when the applied voltage is 7.8 kV, the plasma irradiation time is 5 seconds, and the amount of the gene dispersion solution 25 is 100 μL. 3 and 4 also show the results when the plasma is not irradiated under the same conditions (hereinafter referred to as “control”) and the results by the electroporation method for comparison.
図３に示すように、コントロールでは、蛍光がほとんど認められず、ほとんど遺伝子が導入されていないのに対し、プラズマ照射によるもの、およびエレクトロポレーション法によるものは、多くの細胞に蛍光が認められ、遺伝子が導入されていることが確認された。また、図４に示すように、プラズマ照射によるものは、蛍光がほとんど認められないため、遺伝子が導入された細胞がほとんど死んでおらず、生存率が高いことが確認された。これに対し、エレクトロポレーション法によるものは、多くの細胞に蛍光が認められ、図３と比較することにより、遺伝子が導入された細胞のほとんどが死んでいることが確認された。また、コントロールでは、蛍光がほとんど認められないが、これは図３にしめすように、遺伝子がほとんど導入されていないためであると考えられる。
As shown in FIG. 3, in the control, almost no fluorescence was observed, and almost no gene was introduced. On the other hand, fluorescence was observed in many cells by plasma irradiation and by electroporation. It was confirmed that the gene was introduced. Moreover, as shown in FIG. 4, since the fluorescence by plasma irradiation was hardly observed, it was confirmed that the cells into which the gene was introduced were hardly dead and the survival rate was high. On the other hand, in the case of the electroporation method, fluorescence was observed in many cells, and it was confirmed that most of the cells into which the gene was introduced were dead by comparing with FIG. In the control, almost no fluorescence was observed, but this is considered to be because almost no gene was introduced as shown in FIG.
図３および図４の結果から、生存率を考慮した導入効率は、コントロールが２．５６％、プラズマ照射によるものが２１．１％、エレクトロポレーション法によるものがほぼ０％であった。以上より、プラズマ照射を利用した遺伝子導入方法は、生存率および導入効率が高いといえる。
3 and 4, the introduction efficiency considering the survival rate was 2.56% for the control, 21.1% for the plasma irradiation, and almost 0% for the electroporation method. From the above, it can be said that the gene introduction method using plasma irradiation has high survival rate and introduction efficiency.
次に、導入効率（η）および生存率（Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ）と、プラズマ照射時間（ｔｄ）との関係を調べ、その結果を図５に示す。なお、図５は、印加電圧が７．８ｋＶ、遺伝子分散溶液２５の量（図５中、Ｉと表示）が１００μＬおよび１５０μＬのときの結果である。図５（ａ）に示すように、導入効率は、照射時間が３～５秒のときと、２０秒以上のときに、３０％以上と高くなることが確認された。また、図５（ｂ）に示すように、生存率は、照射時間にかかわらずほぼ９０％以上と、高い値を示すことが確認された。これらの結果から、プラズマ照射を利用した遺伝子導入方法は、常に生存率が高く、照射時間を調整することにより、導入効率を高めることができるといえる。
Next, the relationship between introduction efficiency (η) and survival rate (Cell Viability) and plasma irradiation time (t d ) was examined, and the results are shown in FIG. FIG. 5 shows the results when the applied voltage is 7.8 kV and the amount of the gene dispersion solution 25 (indicated as I in FIG. 5) is 100 μL and 150 μL. As shown in FIG. 5 (a), it was confirmed that the introduction efficiency was as high as 30% when the irradiation time was 3 to 5 seconds and when it was 20 seconds or more. Moreover, as shown in FIG.5 (b), it was confirmed that a survival rate shows a high value with about 90% or more irrespective of irradiation time. From these results, it can be said that the gene introduction method using plasma irradiation always has a high survival rate, and the introduction efficiency can be improved by adjusting the irradiation time.
［プラズマ原料に水を含んだときの遺伝子導入試験－比較例］
プラズマ原料に水を含んだときの導入効率等を調べるための試験を行った。試験に使用した装置を、図６に示す。図６に示すように、試験装置は、上下方向の伸びるよう配置されたガラス管３１の内部上方に、ステンレス製の円筒状の電極３３ａを設け、その電極３３ａの下方のガラス管３１の側面に銅線の電極３３ｂが巻き付けられている。また、下端部がテーパー状に形成されたガラス管３１の下方に、遺伝子分散溶液３５を収納した容器３２を設置している。試験装置では、ガラス管３１の内径を８ｍｍ、ガラス管３１の下端の内径を２ｍｍとした。また、円筒状の電極３３ａの長さを２０ｍｍ、各電極３３ａ，３３ｂ間隔を１３ｍｍ、下方の電極３３ｂからガラス管３１の下端までを３２ｍｍ、ガラス管３１の下端から遺伝子分散溶液３５の表面までを５ｍｍとした。 [Gene transfer test when plasma raw material contains water-comparative example]
A test was conducted to investigate the introduction efficiency when water was included in the plasma raw material. The apparatus used for the test is shown in FIG. As shown in FIG. 6, the test apparatus is provided with a stainlesssteel cylindrical electrode 33a above the inside of the glass tube 31 arranged to extend in the vertical direction, and on the side surface of the glass tube 31 below the electrode 33a. A copper wire electrode 33b is wound. In addition, a container 32 containing the gene dispersion solution 35 is installed below the glass tube 31 having a tapered lower end. In the test apparatus, the inner diameter of the glass tube 31 was 8 mm, and the inner diameter of the lower end of the glass tube 31 was 2 mm. Further, the length of the cylindrical electrode 33a is 20 mm, the distance between the electrodes 33a and 33b is 13 mm, the distance from the lower electrode 33b to the lower end of the glass tube 31 is 32 mm, and the lower end of the glass tube 31 to the surface of the gene dispersion solution 35 It was 5 mm.
プラズマ原料に水を含んだときの導入効率等を調べるための試験を行った。試験に使用した装置を、図６に示す。図６に示すように、試験装置は、上下方向の伸びるよう配置されたガラス管３１の内部上方に、ステンレス製の円筒状の電極３３ａを設け、その電極３３ａの下方のガラス管３１の側面に銅線の電極３３ｂが巻き付けられている。また、下端部がテーパー状に形成されたガラス管３１の下方に、遺伝子分散溶液３５を収納した容器３２を設置している。試験装置では、ガラス管３１の内径を８ｍｍ、ガラス管３１の下端の内径を２ｍｍとした。また、円筒状の電極３３ａの長さを２０ｍｍ、各電極３３ａ，３３ｂ間隔を１３ｍｍ、下方の電極３３ｂからガラス管３１の下端までを３２ｍｍ、ガラス管３１の下端から遺伝子分散溶液３５の表面までを５ｍｍとした。 [Gene transfer test when plasma raw material contains water-comparative example]
A test was conducted to investigate the introduction efficiency when water was included in the plasma raw material. The apparatus used for the test is shown in FIG. As shown in FIG. 6, the test apparatus is provided with a stainless
試験に使用した細胞は、３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、ＹＯＹＯ－１と、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類の蛍光物質を使用した。試験では、上方の電極３３ａの内部を通して、プラズマ原料のヘリウムガスと水とを注入し、電源３４により各電極３３ａ，３３ｂ間に１０ｋＨｚの電圧を印加して、各電極３３ａ，３３ｂの間に低温プラズマを発生させ、遺伝子分散溶液３５に向かってプラズマを噴射させた。プラズマ噴射後の細胞の蛍光を観察し、遺伝子の導入効率および細胞の生存率を求めた。なお、印加電圧は７．８ｋＶ、プラズマ照射時間は５秒、ヘリウムガスの流量は１．０ｓｌｍ、遺伝子分散溶液３５の量は１００μＬ、水の導入量は２．１１μＬ／ｍｉｎである。
The cells used in the test are 3T3L1 cells (floating state). In the test, two kinds of fluorescent substances, YOYO-1 and LIVE / DEAD Stein, were used instead of the gene. In the test, helium gas and water as a plasma raw material are injected through the inside of the upper electrode 33a, a voltage of 10 kHz is applied between the electrodes 33a and 33b by the power source 34, and a low temperature is applied between the electrodes 33a and 33b. Plasma was generated and sprayed toward the gene dispersion solution 35. The fluorescence of the cells after plasma injection was observed, and the gene transfer efficiency and cell viability were determined. The applied voltage is 7.8 kV, the plasma irradiation time is 5 seconds, the flow rate of helium gas is 1.0 slm, the amount of the gene dispersion solution 35 is 100 μL, and the amount of water introduced is 2.11 μL / min.
試験結果を、図７に示す。図７（ａ）に示すように、ＹＯＹＯ－１の場合には、多くの細胞に蛍光が認められ、遺伝子が導入されていることが確認された。また、図７（ｂ）に示すように、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの場合には、蛍光がほとんど認められないため、遺伝子が導入された細胞がほとんど死んでおらず、生存率が高いことが確認された。このときの生存率は９６．６％、導入効率は１２．９％であった。このように、プラズマ原料中に水を含むときには、プラズマ中にヒドロキシルラジカル（ＯＨラジカル）が生成されるため、そのヒドロキシルラジカルの作用により細胞への遺伝子の導入が促進され、高い遺伝子導入効率を得ることができると考えられる。
The test results are shown in FIG. As shown in FIG. 7 (a), in the case of YOYO-1, fluorescence was observed in many cells, confirming that the gene was introduced. Further, as shown in FIG. 7 (b), in the case of LIVE / DEAD Stain, since almost no fluorescence was observed, it was confirmed that the cells into which the gene was introduced were hardly dead and the survival rate was high. It was. The survival rate at this time was 96.6%, and the introduction efficiency was 12.9%. Thus, when water is contained in the plasma raw material, hydroxyl radicals (OH radicals) are generated in the plasma, so that the introduction of genes into cells is promoted by the action of the hydroxyl radicals, and high gene introduction efficiency is obtained. It is considered possible.
［プラズマ発生域からの距離と導入効率との関係－比較例］
プラズマ発生域から遺伝子分散溶液までの距離と導入効率との関係を調べるための試験を行った。試験に使用した装置は、図２に示す装置であり、下方の電極２３ｂからガラス管２１の下端までを７３ｍｍとしたもの（以下、「長拡散型」という）と、下方の電極２３ｂからガラス管２１の下端までを３５ｍｍとしたもの（以下、「短拡散型」という）について試験を行った。試験に使用した細胞は、３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、ＹＯＹＯ－１と、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類の蛍光物質を使用した。 [Relationship between distance from plasma generation area and introduction efficiency-comparative example]
A test was conducted to investigate the relationship between the distance from the plasma generation area to the gene dispersion solution and the introduction efficiency. The apparatus used for the test is the apparatus shown in FIG. 2, in which the distance from thelower electrode 23b to the lower end of the glass tube 21 is 73 mm (hereinafter referred to as “long diffusion type”), and the lower electrode 23b to the glass tube. The test was performed on a sample having a length of 35 mm up to the lower end of 21 (hereinafter referred to as “short diffusion type”). The cells used for the test are 3T3L1 cells (floating state). In the test, two kinds of fluorescent substances, YOYO-1 and LIVE / DEAD Stain, were used instead of the gene.
プラズマ発生域から遺伝子分散溶液までの距離と導入効率との関係を調べるための試験を行った。試験に使用した装置は、図２に示す装置であり、下方の電極２３ｂからガラス管２１の下端までを７３ｍｍとしたもの（以下、「長拡散型」という）と、下方の電極２３ｂからガラス管２１の下端までを３５ｍｍとしたもの（以下、「短拡散型」という）について試験を行った。試験に使用した細胞は、３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、ＹＯＹＯ－１と、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類の蛍光物質を使用した。 [Relationship between distance from plasma generation area and introduction efficiency-comparative example]
A test was conducted to investigate the relationship between the distance from the plasma generation area to the gene dispersion solution and the introduction efficiency. The apparatus used for the test is the apparatus shown in FIG. 2, in which the distance from the
試験では、ガラス管２１の上部からプラズマ原料のヘリウムガスを注入し、各電極２３ａ，２３ｂ間に１０ｋＨｚの電圧を印加して、各電極２３ａ，２３ｂの間に低温プラズマを発生させ、遺伝子分散溶液２５に向かってプラズマを噴射させた。プラズマ噴射後の細胞の蛍光を観察し、遺伝子の導入効率および細胞の生存率を求めた。なお、印加電圧は６．０～１２．５ｋＶ、プラズマ照射時間は１～１５秒、ヘリウムガスの流量は３．０ｓｌｍ、遺伝子分散溶液１５の量は１００μＬである。
In the test, a plasma raw material helium gas is injected from the upper part of the glass tube 21, a voltage of 10 kHz is applied between the electrodes 23a and 23b, a low temperature plasma is generated between the electrodes 23a and 23b, and a gene dispersion solution. Plasma was injected toward 25. The fluorescence of the cells after plasma injection was observed, and the gene transfer efficiency and cell viability were determined. The applied voltage is 6.0 to 12.5 kV, the plasma irradiation time is 1 to 15 seconds, the flow rate of helium gas is 3.0 slm, and the amount of the gene dispersion solution 15 is 100 μL.
印加電圧（Ｖｐ－ｐ）を変化させたときの導入効率（η）および生存率（Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ）の変化を、図８に示す。なお、このときのプラズマ照射時間は５秒である。また、プラズマ照射時間（ｔｄ）を変化させたときの導入効率（η）の変化を、図９に示す。なお、このときの印加電圧は、７．８ｋＶである。また、図９には、比較のため、プラズマを水に照射してプラズマ処理水を作製し、そのプラズマ処理水を遺伝子分散溶液に注ぐことで遺伝子導入を行ったときの結果（図９中の「処理水」）も示している。
FIG. 8 shows changes in introduction efficiency (η) and survival rate (Cell Viability) when the applied voltage (V p−p ) is changed. The plasma irradiation time at this time is 5 seconds. Further, FIG. 9 shows a change in introduction efficiency (η) when the plasma irradiation time (t d ) is changed. The applied voltage at this time is 7.8 kV. For comparison, FIG. 9 shows the results of gene introduction by irradiating plasma with water to produce plasma-treated water and pouring the plasma-treated water into the gene dispersion solution (in FIG. 9). “Treated water”).
図８（ａ）に示すように、導入効率は、印加電圧が８ｋＶ付近で最も高くなることが確認された。また、図８（ｂ）に示すように、生存率は、長拡散型でも短拡散型でも常に９０％以上と高くなっていることが確認された。また、図９に示すように、導入効率は、長拡散型でプラズマ照射時間が１～５秒のとき、短拡散型でプラズマ照射時間が３～１０秒のとき高くなることが確認された。また、図８（ａ）および図９に示すように、印加電圧およびプラズマ照射時間にかかわらず、短拡散型の方が、長拡散型よりも常に導入効率が高いことも確認された。これらの結果から、プラズマ発生域から遺伝子分散溶液２５までの距離を短くするほど、導入効率が高くなるものと推定される。
As shown in FIG. 8A, it was confirmed that the introduction efficiency was highest when the applied voltage was around 8 kV. Further, as shown in FIG. 8B, it was confirmed that the survival rate was always as high as 90% or more in both the long diffusion type and the short diffusion type. Further, as shown in FIG. 9, it was confirmed that the introduction efficiency was high when the long diffusion type plasma irradiation time was 1 to 5 seconds and when the short diffusion type plasma irradiation time was 3 to 10 seconds. Further, as shown in FIGS. 8A and 9, it was confirmed that the short diffusion type always has higher introduction efficiency than the long diffusion type regardless of the applied voltage and the plasma irradiation time. From these results, it is estimated that the introduction efficiency increases as the distance from the plasma generation region to the gene dispersion solution 25 is shortened.
なお、図９に示すように、プラズマを直接遺伝子分散溶液に照射する場合と比べると導入効率は低くなっているが、プラズマ処理水を利用しても、遺伝子導入可能であることが確認された。プラズマ処理水で遺伝子導入できるのは、プラズマ処理水の中に生成された活性酸素種の作用によるものと考えられる。
In addition, as shown in FIG. 9, the introduction efficiency is low compared with the case of directly irradiating the gene dispersion solution with plasma, but it was confirmed that gene introduction was possible even using plasma treated water. . It is considered that the gene can be introduced with the plasma-treated water due to the action of reactive oxygen species generated in the plasma-treated water.
以上の比較例の試験結果を参考にして、以下に本発明の実施の形態の遺伝子導入装置１０による試験を行った。
With reference to the test results of the comparative examples described above, the following tests were performed using the gene introduction apparatus 10 according to the embodiment of the present invention.
図１に示す本発明の実施の形態の遺伝子導入装置１０を使用して、遺伝子導入試験を行った。使用した細胞は、マウス繊維芽細胞の３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、蛍光物質を使用した。使用した蛍光物質は、細胞中のＤＮＡに付着すると発光するＹＯＹＯ－１と、細胞が死ぬと発光するＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類である。また、電極１３ａにはタングステン製のものを用い、電極１３ｂには銅製のものを用いた。
A gene introduction test was performed using the gene introduction apparatus 10 of the embodiment of the present invention shown in FIG. The cells used were mouse fibroblast 3T3L1 cells (floating state). In the test, a fluorescent substance was used instead of the gene. There are two types of fluorescent substances used: YOYO-1, which emits light when attached to DNA in cells, and LIVE / DEAD Stein, which emits light when cells die. The electrode 13a was made of tungsten, and the electrode 13b was made of copper.
試験では、上方の電極１３ａの内部を通して、プラズマ原料のヘリウムガスを注入し、各電極１３ａ，１３ｂ間に１０ｋＨｚの電圧を印加して、各電極１３ａ，１３ｂの間に低温プラズマを発生させ、遺伝子分散溶液１５に向かってプラズマを噴射させた。プラズマ噴射後、細胞の蛍光を観察した。なお、印加電圧は７．８ｋＶ、プラズマ照射時間は５秒、ヘリウムガスの流量は３．０ｓｌｍ、遺伝子分散溶液１５の量は１００μＬである。これらの試験条件は、図３および図４のものと同じである。
In the test, helium gas as a plasma material is injected through the inside of the upper electrode 13a, a voltage of 10 kHz is applied between the electrodes 13a and 13b, and a low-temperature plasma is generated between the electrodes 13a and 13b. Plasma was sprayed toward the dispersion solution 15. After plasma injection, the fluorescence of the cells was observed. The applied voltage is 7.8 kV, the plasma irradiation time is 5 seconds, the flow rate of helium gas is 3.0 slm, and the amount of the gene dispersion solution 15 is 100 μL. These test conditions are the same as those in FIGS.
蛍光物質として、ＹＯＹＯ－１を使用した結果、および、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎを使用した結果を、それぞれ図１０（ａ）および（ｂ）に示す。試験の結果、図１０（ａ）に示すように、多くの細胞にＹＯＹＯ－１の蛍光が認められ、遺伝子が導入されていることが確認された。また、図１０（ｂ）に示すように、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの蛍光がほとんど認められないため、遺伝子が導入された細胞がほとんど死んでおらず、生存率が高いことが確認された。これらの結果を、図２の装置を用いてほぼ同じ条件で行われた、図３（ｃ）および図４（ｃ）の試験結果と比較すると、導入された細胞の生存率はほとんど同じであるが、図１の装置を利用した方が、より高い導入効率で遺伝子を細胞に導入できることがわかる。
10A and 10B show the results of using YOYO-1 as the fluorescent material and the results of using LIVE / DEAD Stein, respectively. As a result of the test, as shown in FIG. 10 (a), YOYO-1 fluorescence was observed in many cells, confirming that the gene was introduced. Further, as shown in FIG. 10 (b), since the fluorescence of LIVE / DEAD Stein was hardly observed, it was confirmed that the cells into which the gene was introduced were hardly dead and the survival rate was high. Comparing these results with the test results of FIG. 3 (c) and FIG. 4 (c), which were performed under the same conditions using the apparatus of FIG. 2, the viability of the introduced cells is almost the same. However, it can be seen that the gene can be introduced into cells with higher introduction efficiency by using the apparatus of FIG.
図８および図９の結果から、プラズマ発生域から遺伝子分散溶液までの距離を短くすればするほど、導入効率が高くなると考えられる。このことを調べるため、図１に示す本発明の実施の形態の遺伝子導入装置１０による試験を行った。遺伝子導入装置１０は、プラズマ発生域から遺伝子分散溶液１５までの距離を、実質的にゼロにしたものと考えられる。試験に使用した細胞は、３Ｔ３Ｌ１細胞（浮遊状態）である。また、試験では、遺伝子の代わりに、ＹＯＹＯ－１と、ＬＩＶＥ／ＤＥＡＤ Ｓｔａｉｎの２種類の蛍光物質を使用した。
8 and 9, it is considered that the introduction efficiency increases as the distance from the plasma generation region to the gene dispersion solution is shortened. In order to investigate this, a test was conducted using the gene introduction apparatus 10 according to the embodiment of the present invention shown in FIG. The gene introduction apparatus 10 is considered to have substantially reduced the distance from the plasma generation area to the gene dispersion solution 15 to zero. The cells used for the test are 3T3L1 cells (floating state). In the test, two kinds of fluorescent substances, YOYO-1 and LIVE / DEAD Stein, were used instead of the gene.
試験では、上方の電極１３ａの内部を通して、プラズマ原料のヘリウムガスを注入し、各電極１３ａ，１３ｂ間に１０ｋＨｚの電圧を印加して、各電極１３ａ，１３ｂの間に低温プラズマを発生させ、遺伝子分散溶液１５に向かってプラズマを噴射させた。プラズマ噴射後の細胞の蛍光を観察し、遺伝子の導入効率および細胞の生存率を求めた。なお、印加電圧は７．８ｋＶ、８．７ｋＶ、１１．６ｋＶ、プラズマ照射時間は０～３０秒、ヘリウムガスの流量は３．０ｓｌｍ、遺伝子分散溶液１５の量は１００μＬである。
In the test, helium gas as a plasma material is injected through the inside of the upper electrode 13a, a voltage of 10 kHz is applied between the electrodes 13a and 13b, and a low-temperature plasma is generated between the electrodes 13a and 13b. Plasma was sprayed toward the dispersion solution 15. The fluorescence of the cells after plasma injection was observed, and the gene transfer efficiency and cell viability were determined. The applied voltage is 7.8 kV, 8.7 kV, 11.6 kV, the plasma irradiation time is 0 to 30 seconds, the flow rate of helium gas is 3.0 slm, and the amount of the gene dispersion solution 15 is 100 μL.
プラズマ照射時間（ｔｄ）を変化させたときの導入効率（η）および生存率（Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ）の変化を調べ、図１１に示す。図１１（ａ）に示すように、プラズマ照射時間が５秒以上のとき、導入効率が高くなることが確認された。特に、印加電圧が１１．６ｋＶのとき、５５％以上の非常に高い導入効率が得られていることが確認された。また、図１１（ｂ）に示すように、生存率は、プラズマ照射時間および印加電圧にかかわらず、常に９０％程度より高くなっていることが確認された。この結果から、遺伝子導入装置１０によれば、より高い導入効率で遺伝子を細胞に導入することができるといえる。
Changes in introduction efficiency (η) and survival rate (Cell Viability) when the plasma irradiation time (t d ) is changed are shown in FIG. As shown in FIG. 11A, it was confirmed that the introduction efficiency was increased when the plasma irradiation time was 5 seconds or more. In particular, it was confirmed that a very high introduction efficiency of 55% or more was obtained when the applied voltage was 11.6 kV. Further, as shown in FIG. 11B, it was confirmed that the survival rate was always higher than about 90% regardless of the plasma irradiation time and the applied voltage. From this result, it can be said that the gene introduction apparatus 10 can introduce genes into cells with higher introduction efficiency.
図１に示す本発明の実施の形態の遺伝子導入装置１０を使用して、細胞に遺伝子を導入する試験を行った。使用した細胞は、ヒトの乳癌細胞由来のＭＣＦ－７細胞（浮遊状態）である。また、使用した遺伝子は、蛍光色素Ｃｙ５で標識されたｄｓ－ｓｉＲＮＡである。また、電極１３ａにはタングステン製のものを用い、電極１３ｂには銅製のものを用いた。
Using the gene introduction apparatus 10 of the embodiment of the present invention shown in FIG. 1, a test for introducing a gene into a cell was performed. The cells used are MCF-7 cells (floating state) derived from human breast cancer cells. The gene used is ds-siRNA labeled with the fluorescent dye Cy5. The electrode 13a was made of tungsten, and the electrode 13b was made of copper.
試験では、上方の電極１３ａの内部を通して、プラズマ原料のヘリウムガスを注入し、各電極１３ａ，１３ｂ間に１０ｋＨｚの電圧を印加して、各電極１３ａ，１３ｂの間に低温プラズマを発生させ、遺伝子分散溶液１５に向かってプラズマを噴射させた。プラズマ噴射後の細胞の蛍光を共焦点レーザー顕微鏡で観察し、同じ条件でプラズマを照射しないときの結果（以下、「コントロール」という）との比較を行った。なお、印加電圧は１１．６ｋＶ、プラズマ照射時間は１秒、ヘリウムガスの流量は１．５ｓｌｍ、遺伝子分散溶液１５の量は１００μＬ、遺伝子分散溶液１５中の遺伝子の濃度は１０μＭである。
In the test, helium gas as a plasma material is injected through the inside of the upper electrode 13a, a voltage of 10 kHz is applied between the electrodes 13a and 13b, and a low-temperature plasma is generated between the electrodes 13a and 13b. Plasma was sprayed toward the dispersion solution 15. The fluorescence of the cells after plasma injection was observed with a confocal laser microscope and compared with the results when the plasma was not irradiated under the same conditions (hereinafter referred to as “control”). The applied voltage is 11.6 kV, the plasma irradiation time is 1 second, the flow rate of helium gas is 1.5 slm, the amount of the gene dispersion solution 15 is 100 μL, and the concentration of the gene in the gene dispersion solution 15 is 10 μM.
試験結果を、図１２に示す。図１２（ｂ）に示すように、コントロールでは、細胞中に蛍光が認められず、遺伝子が導入されていないのに対し、図１２（ｄ）に示すように、プラズマ照射を行ったものは、細胞中に蛍光が認められ、遺伝子が導入されていることが確認された。
The test results are shown in FIG. As shown in FIG. 12 (b), in the control, no fluorescence was observed in the cells and no gene was introduced. On the other hand, as shown in FIG. Fluorescence was observed in the cells, confirming that the gene was introduced.
［接着細胞に対する遺伝子導入装置および遺伝子導入方法］
浮遊状態ではなく接着状態の細胞を使用する場合、遺伝子分散溶液１５を収納容器１２に入れると、遺伝子分散溶液１５中の接着細胞が収納容器１２の底面に存在する。このため、図１３に示すように、遺伝子導入装置１０は、収納容器１２が所定の深さを有しており、収納容器１２の底面から、収納容器１２に収納された遺伝子分散溶液１５の表面までの高さを調整可能になっていることが好ましい。図１３に示す一例では、収納容器１２は底を有する円柱状で、深さによらず同じ内径を有し、電極１３ｂは環状を成し、収納容器１２の外側面を円周方向に沿って１周するよう取り付けられている。 [Gene transfer device and gene transfer method for adherent cells]
When cells that are in an adherence state rather than a floating state are used, when thegene dispersion solution 15 is placed in the storage container 12, the adherent cells in the gene dispersion solution 15 are present on the bottom surface of the storage container 12. For this reason, as shown in FIG. 13, in the gene transfer device 10, the storage container 12 has a predetermined depth, and the surface of the gene dispersion solution 15 stored in the storage container 12 from the bottom surface of the storage container 12. It is preferable that the height can be adjusted. In the example shown in FIG. 13, the storage container 12 has a cylindrical shape with a bottom, the same inner diameter regardless of the depth, the electrode 13 b has an annular shape, and the outer surface of the storage container 12 extends along the circumferential direction. It is attached to make one round.
浮遊状態ではなく接着状態の細胞を使用する場合、遺伝子分散溶液１５を収納容器１２に入れると、遺伝子分散溶液１５中の接着細胞が収納容器１２の底面に存在する。このため、図１３に示すように、遺伝子導入装置１０は、収納容器１２が所定の深さを有しており、収納容器１２の底面から、収納容器１２に収納された遺伝子分散溶液１５の表面までの高さを調整可能になっていることが好ましい。図１３に示す一例では、収納容器１２は底を有する円柱状で、深さによらず同じ内径を有し、電極１３ｂは環状を成し、収納容器１２の外側面を円周方向に沿って１周するよう取り付けられている。 [Gene transfer device and gene transfer method for adherent cells]
When cells that are in an adherence state rather than a floating state are used, when the
接着細胞を使用する場合、照射されたプラズマにより遺伝子分散溶液１５の表面に形成された電界が、収納容器１２の底面に存在する接着細胞まで届く必要がある。また、遺伝子分散溶液１５の表面から接着細胞が露出すると、プラズマが接着細胞に直接当たるため、接着細胞が死んでしまう。このため、これらを考慮して、収納容器１２の底面から遺伝子分散溶液１５の表面までの高さを調整する必要がある。そこで、図１３に示す遺伝子導入装置１０を使用して、収納容器１２の底面から遺伝子分散溶液１５の表面までの高さｈを変化させて、導入効率を調べる試験を行った。
When using adherent cells, the electric field formed on the surface of the gene dispersion solution 15 by the irradiated plasma needs to reach the adherent cells present on the bottom surface of the storage container 12. Further, when the adherent cells are exposed from the surface of the gene dispersion solution 15, the plasma directly hits the adherent cells, so that the adherent cells die. For this reason, it is necessary to adjust the height from the bottom surface of the storage container 12 to the surface of the gene dispersion solution 15 in consideration of these. Therefore, using the gene introduction apparatus 10 shown in FIG. 13, a test for examining the introduction efficiency was performed by changing the height h from the bottom surface of the storage container 12 to the surface of the gene dispersion solution 15.
試験に使用した細胞は、マウス繊維芽細胞由来の３Ｔ３Ｌ１細胞（接着状態）である。また、試験では、遺伝子の代わりに、蛍光物質のＹＯＹＯ－１を使用した。試験では、上方の電極１３ａの内部を通して、プラズマ原料のヘリウムガスを注入し、各電極１３ａ，１３ｂ間に１０ｋＨｚの電圧を印加して、各電極１３ａ，１３ｂの間に低温プラズマを発生させ、遺伝子分散溶液１５に向かってプラズマを噴射させた。ｈ＝１．２８ｍｍ、３．８４ｍｍ、６．４０ｍｍとしたときの、プラズマ噴射後の細胞の蛍光をそれぞれ共焦点レーザー顕微鏡で観察した。なお、印加電圧は８．７ｋＶ、プラズマ照射時間は１秒、ヘリウムガスの流量は１．０ｓｌｍ、遺伝子分散溶液１５中のＹＯＹＯ－１の濃度は５μＭ、試験前日の細胞密度は１×１０５ｃｅｌｌｓ／ｄｉｓｈである。
The cells used for the test are 3T3L1 cells (adhered state) derived from mouse fibroblasts. In the test, the fluorescent substance YOYO-1 was used instead of the gene. In the test, helium gas as a plasma material is injected through the inside of the upper electrode 13a, a voltage of 10 kHz is applied between the electrodes 13a and 13b, and a low-temperature plasma is generated between the electrodes 13a and 13b. Plasma was sprayed toward the dispersion solution 15. When h = 1.28 mm, 3.84 mm, and 6.40 mm, the fluorescence of the cells after plasma injection was observed with a confocal laser microscope. The applied voltage was 8.7 kV, the plasma irradiation time was 1 second, the flow rate of helium gas was 1.0 slm, the concentration of YOYO-1 in the gene dispersion solution 15 was 5 μM, and the cell density on the day before the test was 1 × 10 5 cells. / Dish.
試験結果を、図１４に示す。図１４（ａ）および（ｂ）に示すように、ｈ＝１．２８ｍｍのとき、多くの細胞に蛍光が認められ、ＹＯＹＯ－１が導入されていることが確認された。これに対し、図１４（ｃ）乃至（ｆ）に示すように、ｈ＝３．８４ｍｍおよび６．４０ｍｍのときには、蛍光がほとんど認められず、ほとんどＹＯＹＯ－１が導入されていないことが確認された。これは、ｈ＝３．８４ｍｍおよび６．４０ｍｍのときには、照射されたプラズマにより遺伝子分散溶液１５の表面に形成された電界が、収納容器１２の底面に存在する接着細胞まで届いていないためであると考えられる。
The test results are shown in FIG. As shown in FIGS. 14A and 14B, when h = 1.28 mm, fluorescence was observed in many cells, confirming that YOYO-1 had been introduced. On the other hand, as shown in FIGS. 14C to 14F, when h = 3.84 mm and 6.40 mm, almost no fluorescence was observed, and it was confirmed that almost no YOYO-1 was introduced. It was. This is because when h = 3.84 mm and 6.40 mm, the electric field formed on the surface of the gene dispersion solution 15 by the irradiated plasma does not reach the adherent cells existing on the bottom surface of the storage container 12. it is conceivable that.
以上の試験の結果から、収納容器１２の底面から遺伝子分散溶液１５の表面までの高さを調整することにより、接着細胞に対して、遺伝子導入効率を高めることができるといえる。特に、底面に存在する細胞が遺伝子分散溶液１５の表面に露出せず、収納容器１２の底面から遺伝子分散溶液１５の表面までの高さが１．５ｍｍ以下のとき、高い遺伝子導入効率が得られるといえる。
From the results of the above tests, it can be said that the gene transfer efficiency can be increased for the adherent cells by adjusting the height from the bottom surface of the storage container 12 to the surface of the gene dispersion solution 15. In particular, when the cell existing on the bottom surface is not exposed on the surface of the gene dispersion solution 15 and the height from the bottom surface of the storage container 12 to the surface of the gene dispersion solution 15 is 1.5 mm or less, high gene transfer efficiency is obtained. It can be said.
図１３に示す本発明の実施の形態の遺伝子導入装置１０を使用して、接着細胞に遺伝子を導入する試験を行った。使用した細胞は、マウス繊維芽細胞由来の３Ｔ３Ｌ１細胞（接着状態）である。また、使用した遺伝子は、緑色蛍光タンパク質ＧＦＰをコードする遺伝子を含むプラスミドＤＮＡ（ｐＡｃＧＦＰ１－Ｃ１）である。
Using the gene introduction apparatus 10 of the embodiment of the present invention shown in FIG. 13, a test for introducing genes into adherent cells was performed. The cells used are 3T3L1 cells (adhered state) derived from mouse fibroblasts. The gene used was plasmid DNA (pAcGFP1-C1) containing a gene encoding green fluorescent protein GFP.
試験では、上方の電極１３ａの内部を通して、プラズマ原料のヘリウムガスを注入し、各電極１３ａ，１３ｂ間に１０ｋＨｚの電圧を印加して、各電極１３ａ，１３ｂの間に低温プラズマを発生させ、遺伝子分散溶液１５に向かってプラズマを噴射させた。プラズマ照射中の装置周辺の温度を３７℃、ｈ＝１．２８ｍｍとし、プラズマ噴射後、４８時間の培養を経た細胞の蛍光を共焦点レーザー顕微鏡で観察した。なお、印加電圧は５．０ｋＶ、プラズマ照射時間は１秒、ヘリウムガスの流量は３．０ｓｌｍ、遺伝子分散溶液１５の量は１００μＬ、遺伝子分散溶液１５中の遺伝子の量は５０μｇ、試験前日の細胞密度は２×１０５ｃｅｌｌｓ／ｄｉｓｈである。
In the test, helium gas as a plasma material is injected through the inside of the upper electrode 13a, a voltage of 10 kHz is applied between the electrodes 13a and 13b, and a low-temperature plasma is generated between the electrodes 13a and 13b. Plasma was sprayed toward the dispersion solution 15. The temperature around the apparatus during plasma irradiation was 37 ° C. and h = 1.28 mm, and the fluorescence of the cells that had been cultured for 48 hours after plasma injection was observed with a confocal laser microscope. The applied voltage is 5.0 kV, the plasma irradiation time is 1 second, the flow rate of helium gas is 3.0 slm, the amount of the gene dispersion solution 15 is 100 μL, the amount of the gene in the gene dispersion solution 15 is 50 μg, and the cell the day before the test. The density is 2 × 10 5 cells / dish.
試験結果を、図１５に示す。図１５に示すように、プラズマ照射により、細胞中に蛍光が認められ、遺伝子が導入され、さらにその遺伝子が機能していることが確認された。
The test results are shown in FIG. As shown in FIG. 15, fluorescence was observed in the cells by plasma irradiation, the gene was introduced, and it was confirmed that the gene was functioning.
１０ 遺伝子導入装置
１１ ガラス管
１２ 収納容器
１３ａ，１３ｂ 電極
１４ 電源部
１５ 遺伝子分散溶液
DESCRIPTION OFSYMBOLS 10 Gene transfer apparatus 11 Glass tube 12 Storage container 13a, 13b Electrode 14 Power supply part 15 Gene dispersion solution
１１ ガラス管
１２ 収納容器
１３ａ，１３ｂ 電極
１４ 電源部
１５ 遺伝子分散溶液
DESCRIPTION OF
Claims (10)
- 遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液を収納する収納容器と、
前記収納容器を挟むよう配置された１対の電極と、
一方の電極から他方の電極に向かって、プラズマ原料を供給するプラズマ原料供給手段と、
前記プラズマ原料が供給された各電極間でプラズマを発生させるよう、各電極間に電圧を印加する電源部とを有し、
前記収納容器は、収納された前記遺伝子分散溶液に、発生した前記プラズマが直接当たるよう配置されていることを
特徴とする遺伝子導入装置。 A storage container for storing a gene dispersion solution containing a gene and a cell into which the gene is introduced;
A pair of electrodes arranged to sandwich the storage container;
Plasma raw material supply means for supplying a plasma raw material from one electrode to the other electrode;
A power source for applying a voltage between the electrodes so as to generate plasma between the electrodes supplied with the plasma raw material,
The gene transfer apparatus, wherein the storage container is arranged so that the generated plasma directly hits the stored gene dispersion solution. - 前記他方の電極が前記収納容器に取り付けられていることを特徴とする請求項１記載の遺伝子導入装置。 The gene transfer apparatus according to claim 1, wherein the other electrode is attached to the storage container.
- 前記一方の電極は筒状を成しており、
前記プラズマ原料供給手段は、前記一方の電極の内部を通して、各電極の間に前記プラズマ原料を供給可能に構成されていることを
特徴とする請求項１または２記載の遺伝子導入装置。 The one electrode has a cylindrical shape,
The gene introduction apparatus according to claim 1 or 2, wherein the plasma raw material supply means is configured to be able to supply the plasma raw material between the electrodes through the inside of the one electrode. - 前記細胞は接着細胞から成り、
前記収納容器の底面から、前記収納容器に収納された前記遺伝子分散溶液の表面までの高さを調整可能に構成されていることを
特徴とする請求項１乃至３のいずれか１項に記載の遺伝子導入装置。 The cells consist of adherent cells;
The height from the bottom face of the said storage container to the surface of the said gene dispersion solution accommodated in the said storage container is comprised so that adjustment is possible, The Claim 1 thru | or 3 characterized by the above-mentioned. Gene transfer device. - 前記細胞は接着細胞から成り、
前記収納容器は、底面から前記遺伝子分散溶液の表面までの高さが１．５ｍｍ以下で、前記底面に存在する前記細胞が前記遺伝子分散溶液の表面に露出しないよう、前記遺伝子分散溶液を収納可能に設けられていることを
特徴とする請求項１乃至３のいずれか１項に記載の遺伝子導入装置。 The cells consist of adherent cells;
The storage container has a height of 1.5 mm or less from the bottom surface to the surface of the gene dispersion solution, and can store the gene dispersion solution so that the cells existing on the bottom surface are not exposed on the surface of the gene dispersion solution. The gene introduction device according to any one of claims 1 to 3, wherein the gene introduction device is provided. - 前記プラズマ原料は水を含んでいることを特徴とする請求項１乃至５のいずれか１項に記載の遺伝子導入装置。 The gene introduction apparatus according to any one of claims 1 to 5, wherein the plasma raw material contains water.
- 遺伝子とその遺伝子を導入する細胞とを含む遺伝子分散溶液を、１対の電極の間に配置し、一方の電極から他方の電極に向かってプラズマ原料を供給しつつ、各電極間に電圧を印加してプラズマを発生させ、前記遺伝子分散溶液に前記プラズマを直接当てることにより、前記細胞に前記遺伝子を導入することを特徴とする遺伝子導入方法。 A gene dispersion solution containing a gene and a cell into which the gene is introduced is placed between a pair of electrodes, and a voltage is applied between the electrodes while supplying a plasma raw material from one electrode to the other electrode. Then, the gene is introduced into the cells by generating plasma and directly applying the plasma to the gene dispersion solution.
- 前記他方の電極が取り付けられた収納容器を有し、前記遺伝子分散溶液を前記収納容器に収納して配置することを特徴とする請求項５記載の遺伝子導入方法。 6. The gene introduction method according to claim 5, further comprising a storage container to which the other electrode is attached, wherein the gene dispersion solution is stored in the storage container.
- 前記細胞は接着細胞から成り、
前記収納容器の底面から前記遺伝子分散溶液の表面までの高さが１．５ｍｍ以下で、前記底面に存在する前記細胞が前記遺伝子分散溶液の表面に露出しないよう、前記遺伝子分散溶液を前記収納容器に収納することを
特徴とする請求項８記載の遺伝子導入方法。 The cells consist of adherent cells;
The height from the bottom surface of the storage container to the surface of the gene dispersion solution is 1.5 mm or less, and the gene dispersion solution is stored in the storage container so that the cells existing on the bottom surface are not exposed on the surface of the gene dispersion solution. The gene introduction method according to claim 8, wherein the gene introduction method is stored in a container. - 前記プラズマ原料は水を含んでいることを特徴とする請求項７乃至９のいずれか１項に記載の遺伝子導入方法。 The gene introduction method according to any one of claims 7 to 9, wherein the plasma raw material contains water.
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