JP2011240115A - Apoptosis inducing method using nanosecond pulsed electric field - Google Patents
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Abstract
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本発明は、がん細胞に効果的に高電界を印加し、アポトーシスを誘発させる方法に関する。 The present invention relates to a method for inducing apoptosis by effectively applying a high electric field to cancer cells.
がん細胞治療の一環として、交流波パルスやマイクロ秒からミリ秒のパルスを用いるものが多かった。 As part of cancer cell therapy, there are many that use AC wave pulses or microsecond to millisecond pulses.
近年ナノ秒パルス高電界を用いて、例えばメラノーマ（がん細胞）にアポトーシスを引き起こす技術が研究されてきた（例えば、特許文献1）。 In recent years, techniques for causing apoptosis in, for example, melanoma (cancer cells) using a nanosecond pulse high electric field have been studied (for example, Patent Document 1).
特許文献1によれば、持続時間約10〜300ナノ秒、電界強度10~350KV/cm、繰り返し周波数0.5Hz(pps)のナノ秒パルス電界を複数回印加することで細胞死や組織中の血流阻害を引き起こすことを開示している。 According to Patent Document 1, cell death and blood in tissues are applied by applying a nanosecond pulse electric field having a duration of about 10 to 300 nanoseconds, an electric field strength of 10 to 350 KV / cm, and a repetition frequency of 0.5 Hz (pps) multiple times. Discloses causing flow inhibition.
しかしながら、特許文献1によれば、繰り返し周波数が0.5pps(pulse per second)のみのデータで開示されているため、細胞によっては最適な繰り返し周波数ではなく、電界強度に頼った治療になるため目的の治療部位以外の周囲の正常な細胞を死なせたり、なんらかの異常を誘因したりなど好ましくない影響がでる虞がある。 However, according to Patent Document 1, since the repetition frequency is disclosed only with data of 0.5 pps (pulse per second), depending on the cell, it is not the optimal repetition frequency, but the treatment depends on the electric field strength. There is a risk that unfavorable effects may occur, such as the death of normal cells around the treatment site or the cause of some abnormalities.
そこで上述の課題に鑑み、本発明の目的はナノ秒パルス電界の繰り返し周波数を変化させ用いることにより、目的臓器および細胞、例えば、がん細胞を含有する臓器などを標的とし、高効率にアポトーシスを誘導する方法を提供することにある。 Accordingly, in view of the above-mentioned problems, the object of the present invention is to change the repetition frequency of the nanosecond pulse electric field to target and target organs and cells, for example, organs containing cancer cells, and to efficiently induce apoptosis. It is to provide a method of guiding.
請求項1の発明は、組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、繰り返し周波数は0.01pps超過0.5pps未満であることを特徴とするナノ秒パルス電界を用いたアポトーシスなど細胞死誘導方法を用いることを特徴とする。 The invention of claim 1 is a method for inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV / cm. Thus, it is characterized by using a cell death induction method such as apoptosis using a nanosecond pulse electric field characterized by having a pulse width of 1 to 300 ns and a repetition frequency exceeding 0.01 pps and less than 0.5 pps.
請求項2の発明は、組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、繰り返し周波数は50pps以上であることを特徴とするナノ秒パルス電界を用いたアポトーシスなど細胞死誘導方法を用いることを特徴とする。 The invention of claim 2 is a method for inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV / cm. Thus, it is characterized by using a cell death induction method such as apoptosis using a nanosecond pulse electric field characterized by a pulse width of 1 to 300 ns and a repetition frequency of 50 pps or more.
組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、前記ナノ秒パルス電界複数回を1回としたクラスターとして印加して、クラスターの繰り返し周波数が0.01回/秒超過0.5回/秒未満であることを特徴とするナノ秒パルス電界印加シークエンスを用いたアポトーシスなど細胞死誘導方法を用いることを特徴とする。 A method of inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV / cm and a pulse of 1 to 300 ns. The nanosecond pulse electric field application sequence is characterized in that the repetition frequency of the cluster is 0.01 times / second and less than 0.5 times / second by applying the nanosecond pulse electric field multiple times as a single cluster in width. The cell death induction method such as apoptosis used is used.
請求項1の発明によれば、組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、繰り返し周波数は0.0１pps超過0.5pps未満である構成であるから、2~100秒間隔の低繰り返し周波数を用いることによって、ナノ秒パルス電界による細胞膜上のイオンチャンネルの乱れを常に起きている状態を作り出し、電界にあまり依存せず、目的以外の周囲の細胞への影響をできるだけ抑制した細胞のメカニズムを用いた柔和な条件で高効率にアポトーシスなどの細胞死誘導する方法を提供する。 According to the invention of claim 1, a method of inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV / cm, with a pulse width of 1 to 300 ns and a repetition frequency of more than 0.01 pps and less than 0.5 pps. By using a low repetition frequency of 2 to 100 seconds, a nanosecond pulse electric field on the cell membrane Cells that are constantly disturbed by ion channels, that do not depend much on the electric field, and that have a high degree of efficiency under mild conditions using the cellular mechanism that suppresses the influence on surrounding cells other than the target as much as possible. Provide a way to induce death.
請求項2の発明によれば、組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、繰り返し周波数は50pps以上である構成であるから、0.2秒間隔以下の連続したパルスによりパルスによって標的細胞に蓄積される熱が積極的に上昇していくことから、電界と温熱効果を併用してアポトーシスなどの細胞死を誘導することができる。 According to the invention of claim 2, a method for inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV / cm, with a pulse width of 1 to 300 ns and a repetition frequency of 50 pps or more, the heat accumulated in the target cells is actively increased by the pulses with continuous pulses of 0.2 seconds or less. Therefore, cell death such as apoptosis can be induced by using both the electric field and the thermal effect.
請求項3の発明によれば、組織中の標的細胞に対して、ナノ秒パルス電界を印加して、アポトーシスなどの細胞死を誘導する方法であって、前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、前記ナノ秒パルス電界複数回を1回としたクラスターとして印加して、クラスターの繰り返し周波数が0.01回/秒超過0.5回/秒未満であるである構成であるから、
例えば250ppsのナノ秒パルスの40ms分、すなわち、10発分を1クラスターとし、前記クラスター
を0.01回/秒 超過0.5回/秒未満で繰り返すシークエンスを用いることにより、標的細胞の温度をほとんど上昇させることなくアポトーシスなどの細胞死を誘導させることができる。
According to the invention of claim 3, a method for inducing cell death such as apoptosis by applying a nanosecond pulse electric field to a target cell in a tissue, wherein the nanosecond pulse electric field is 0.5 to 250 KV A configuration in which the repetition frequency of the cluster is 0.01 times / second and less than 0.5 times / second by applying the nanosecond pulse electric field multiple times as a cluster at a pulse width of 1 to 300 ns at / cm Because
For example, 40 ms of 250 pps nanosecond pulse, that is, 10 shots as 1 cluster, and the sequence is repeated 0.01 times / second and less than 0.5 times / second, the temperature of the target cell is almost increased. Cell death such as apoptosis can be induced.
以下に、本発明の実施形態について図を用いて説明する。本実施形態のナノ秒パルス電界（nanosecond Pulsed Electric Fields以下「nsPEF」という）を用いたnsPEF印加システム12について図1を用いて説明する。本実施形態のnsPEF印加システム12は、nsPEF発生装置1・充電器2・制御装置3・外部繰り返し周波数制御装置4とパルス発生機より作製されたnsPEFを目的の組織もしくは細胞に印加するための電極5により構成される。 Embodiments of the present invention will be described below with reference to the drawings. An nsPEF application system 12 using nanosecond pulsed electric fields (hereinafter referred to as “nsPEF”) according to the present embodiment will be described with reference to FIG. The nsPEF application system 12 of the present embodiment includes an electrode for applying nsPEF produced by the nsPEF generator 1, the charger 2, the controller 3, the external repetition frequency controller 4 and the pulse generator to a target tissue or cell. Consists of 5.
nsPEF発生装置1は1〜300nsのパルス幅で0.5~250KV/cmの電界強度の単極パルスを出力できる装置である。また、充電器2は0.1〜5Jの充電が可能な装置である。さらに制御装置3は、電界強度と0.1〜2000ppsの連続印加型繰り返し周波数を制御する装置である。これらnsPEF発生装置1・充電器2・制御装置3は、例えば、磁気パルス圧縮回路方式（Magnetic Pulse Compression Circuit: MPC）を使用している末松電子社製の公知の機器類を使用することができる。これらの機器は性能的に同じならば他社製品でも良いものとする。 The nsPEF generator 1 is a device capable of outputting a unipolar pulse with an electric field strength of 0.5 to 250 KV / cm with a pulse width of 1 to 300 ns. The charger 2 is a device capable of charging 0.1 to 5J. Furthermore, the control device 3 is a device that controls the electric field strength and the continuous application type repetition frequency of 0.1 to 2000 pps. These nsPEF generator 1, charger 2, and controller 3 can use, for example, known devices manufactured by Suematsu Electronics Co., Ltd. that use a magnetic pulse compression circuit (MPC). . These devices may be products of other companies as long as they have the same performance.
外部繰り返し周波数制御装置4は、例えば公知のファンクションジェネレーターを用いることができる。外部繰り返し周波数制御装置4は、自己のトリガーパルスを有し、任意の繰り返し周波数を選択でき、さらに印加回数も設定できる。また、外部繰り返し周波数制御装置4により、例えば、図2に示すように250ppsの繰り返し周波数のnsPEF10回印加を１つのクラスター10として設定し、そのクラスターパルスを制御することができる。電界強度は制御装置3により制御される。 As the external repetition frequency control device 4, for example, a known function generator can be used. The external repetition frequency control device 4 has its own trigger pulse, can select an arbitrary repetition frequency, and can also set the number of times of application. Further, for example, as shown in FIG. 2, the external repetition frequency control device 4 can set application of nsPEF 10 times with a repetition frequency of 250 pps as one cluster 10 and control the cluster pulse. The electric field strength is controlled by the control device 3.
電極5は、細胞実験用のキュベットタイプ以外に上皮系組織に刺すニードルタイプ・組織を挟むパットタイプ外部から組織内部に電界を送り込めるアンテナタイプなど用途に適合したものを用いることができる。そして、電極5は、パルス発生装置1のパルス電界発生部6と接地電位7に接続される。 As the electrode 5, other than the cuvette type for cell experiments, a needle type that pierces epithelial tissue, a pad type that sandwiches the tissue, an antenna type that sends an electric field from the outside to the inside of the tissue, and the like can be used. The electrode 5 is connected to the pulse electric field generator 6 of the pulse generator 1 and the ground potential 7.
（実施例1）: nsPEFによるHeLa S3細胞アポトーシス誘導
材料および方法
ヒト子宮頸ガン由来のHeLa S3細胞をATCC(Manassas,VA)から入手し、培養を行い細胞を増殖させた後、上記本実施形態のnsPEF印加システム12を用いて実験を行った。本実施例1では、前記電極5はキュベットタイプ電極を用いた。0.5〜１×106cells/mlの濃度に調節した細胞含有培地を4mmキュベット電極に約800μl入れ、電極支持部8にキュベット電極を差し込み。nsPEFを印加した。オシロスコープ9などで電界値の観察を行い、実施例1では12.5〜25KV/cmの電界強度を使用した。
(Example 1): Material and method for inducing apoptosis of HeLa S3 cells by nsPEF After obtaining HeLa S3 cells derived from human cervical cancer from ATCC (Manassas, VA) and culturing the cells to proliferate, the above embodiment The experiment was conducted using the nsPEF application system 12 of the present invention. In Example 1, the electrode 5 was a cuvette type electrode. About 800 μl of cell-containing medium adjusted to a concentration of 0.5-1 × 10 6 cells / ml is put into a 4 mm cuvette electrode, and the cuvette electrode is inserted into the electrode support 8. nsPEF was applied. The electric field value was observed with an oscilloscope 9 or the like. In Example 1, an electric field strength of 12.5 to 25 KV / cm was used.
繰り返し周波数の影響測定
実施例1において、電界値を12.5KV/cm、印加回数を100回に固定し、繰り返し周波数を250pps,
10pps, 0.5ppsの条件で細胞死への影響を調べた（図3）。細胞死判定にはヨウ化プロピジウム(PI)染色法を行い、フローサイトメトリー法で解析した。その結果、2時間後、0.5ppsがもっとも細胞死を誘導することがわかった。さらに、低繰り返し周波数を詳細に調べるために電界強度25KV/cm,印加回数25回の条件で、0.01~250pps繰り返し周波数を用いて実験を行い、細胞死判定を行ったところ、0.33〜0.05ppsを中心に細胞死が強く誘導された(図4)。なお、図4では、同様な実験を３回繰り返し、標準誤差を付け加えている。しかし、0.01ppsではあまり細胞死が誘導されなかった。そこで、我々は0.01超過0.5pps未満で細胞死が強く誘導される可能性が示唆された。加えて、0.025ppsや0.01ppsの条件では印加実験中に適度にタッピングを行い、常に細胞が混和した状態を維持している。また、17度以下の条件では細胞死にむらができるので、実験中の室温は22度以上25度以下で行っている。
In Example 1 of measurement of influence of repetition frequency, the electric field value is 12.5 KV / cm, the number of times of application is fixed to 100 times, the repetition frequency is 250 pps,
The effect on cell death was examined under conditions of 10 pps and 0.5 pps (Fig. 3). Cell death was determined by propidium iodide (PI) staining and analyzed by flow cytometry. As a result, after 2 hours, 0.5 pps was found to induce cell death most. Furthermore, in order to investigate the low repetition frequency in detail, an experiment was performed using a repetition frequency of 0.01 to 250 pps under the conditions of an electric field strength of 25 KV / cm and an application frequency of 25 times. Cell death was strongly induced in the center (FIG. 4). In FIG. 4, the same experiment is repeated three times, and standard errors are added. However, cell death was not induced at 0.01 pps. Therefore, we suggested that cell death might be strongly induced at over 0.01 and under 0.5 pps. In addition, under the conditions of 0.025 pps and 0.01 pps, appropriate tapping is performed during the application experiment, and the cells are always mixed. In addition, since cell death can be uneven under the condition of 17 degrees or less, the room temperature during the experiment is 22 degrees or more and 25 degrees or less.
xCelligence測定
細胞増殖活性を継時測定可能なxCelligenceシステム（Roche）を用いて、15分置きに3日間測定した。電界条件12.5KV/cm、印加回数100回で、繰り返し周波数を250pps, 10pps, 1pps, 0.5pps, 0.2pps, 0.1ppsの条件で細胞増殖活性を測定したところ、250pps,10ppsは無処理の細胞と同じ増殖を示したが、1pps, 0.5pps, は数時間後に一時的に細胞増殖が止まり、0.2pps,
0.1ppsでは48時間は細胞増殖が停止していることが示唆された。
xCelligence Measurement Using a xCelligence system (Roche) capable of measuring cell proliferation activity over time, measurement was performed every 15 minutes for 3 days. When cell proliferation activity was measured under conditions of electric field conditions of 12.5 KV / cm, application frequency of 100 times, and repetition rates of 250 pps, 10 pps, 1 pps, 0.5 pps, 0.2 pps, and 0.1 pps, 250 pps and 10 pps were treated as untreated cells. The same growth, but 1pps, 0.5pps, temporarily stopped cell growth after a few hours, 0.2pps,
It was suggested that cell growth stopped for 48 hours at 0.1 pps.
（実施例2）50pps以上の繰り返しnsPEFによるHeLa S3細胞アポトーシス誘導
ヒト子宮頸ガン由来のHeLa
S3細胞をATCC(Manassas,VA)から入手し、培養を行い細胞を増殖させた後、上記本実施形態のnsPEF印加システム12を用いて実験を行った。本実施例2では、前記電極5はキュベットタイプ電極を用いた。0.5〜１×106cells/mlの濃度に調節した細胞含有培地を4mmキュベット電極に約800μl入れ、電極支持部8にキュベット電極を差し込み。nsPEFを印加した。実施例2では12.5KV/cmの電界強度を使用した。
(Example 2) HeLa S3 cell apoptosis induction HeLa from human cervical cancer induced by repeated nsPEF of 50 pps or more
After obtaining S3 cells from ATCC (Manassas, VA), culturing and growing the cells, experiments were performed using the nsPEF application system 12 of the present embodiment. In Example 2, the electrode 5 was a cuvette type electrode. About 800 μl of cell-containing medium adjusted to a concentration of 0.5-1 × 10 6 cells / ml is put into a 4 mm cuvette electrode, and the cuvette electrode is inserted into the electrode support 8. nsPEF was applied. In Example 2, an electric field strength of 12.5 KV / cm was used.
印加回数の影響測定
HeLa S3細胞などの培養液は抵抗が約10Ωと導電性が高いため、50pps以上の繰り返し周波数で積算的に加熱されることが、レーザー干渉法から割り出された。そこで、繰り返し周波数250ppsで印加回数を変えて調べたところ、2時間後に200〜300回の印加条件領域で死亡率が高くなり始め、その後、印加回数に伴い細胞死率は上昇する(図5)。このときの温度上昇率は印加回数300回でおよそ27ケルビンで、実際の細胞の温度は一時的に約50度前後となる。このときの温度上昇に使用された時間は約1秒で、その後、外気によってすばやく冷却される。このことから、温熱単体だけではなく、温熱と電界の併用効果により細胞死が誘導されると考えられる。この結果より、細胞死を短時間の処理で引き起こせることがわかった、しかし、熱や印加回数が必要となり、総エネルギー投入率の割合が多くなるため、目標領域（患部）周辺の細胞にも損傷を引き起こす虞がある。この方法で細胞死を引き起こす部分は再生力の高い臓器や死亡率を優先したいときなど限定して使用する方が良いと考えられる。
Effect measurement of the number of applied times
Since the culture fluid such as HeLa S3 cells has a high conductivity of about 10Ω, it was determined from laser interferometry that it can be heated cumulatively at a repetition rate of 50 pps or more. Therefore, when the number of times of application was changed at a repetition frequency of 250 pps and examined, mortality began to increase in the application condition region of 200 to 300 times after 2 hours, and then the cell death rate increased with the number of times of application (Fig. 5). . At this time, the rate of temperature increase is approximately 27 Kelvin after 300 applications, and the actual cell temperature temporarily becomes approximately 50 degrees. The time used for the temperature rise at this time is about 1 second, and then it is quickly cooled by the outside air. From this, it is considered that cell death is induced not only by the heat alone but also by the combined effect of the heat and the electric field. From this result, it was found that cell death can be caused by short-time treatment. However, heat and the number of times of application are required, and the ratio of the total energy input rate increases, so the cells around the target area (affected area) are also affected. May cause damage. The part that causes cell death by this method may be better used only when organs with high regenerative power or mortality is prioritized.
（実施例3）シークエンスnsPEFと繰り返し周波数を組み合わせたアポトーシスなど細胞死誘導方法
ヒト子宮頸ガン由来のHeLa
S3細胞をATCC(Manassas,VA)から入手し、培養を行い細胞を増殖させた後、上記本実施形態のnsPEF印加システム12を用いて実験を行った。本実施例3では、前記電極5はキュベットタイプ電極を用いた。0.5〜１×106cells/mlの濃度に調節した細胞含有培地を4mmキュベット電極に約800μl入れ、電極支持部8にキュベット電極を差し込み。nsPEFを印加した。実施例3では12.5KV/cmと25KV/cmの電界強度を使用した。
(Example 3) Cell death induction method such as apoptosis combining sequence nsPEF and repetition frequency HeLa derived from human cervical cancer
After obtaining S3 cells from ATCC (Manassas, VA), culturing and growing the cells, experiments were performed using the nsPEF application system 12 of the present embodiment. In Example 3, the electrode 5 was a cuvette type electrode. About 800 μl of cell-containing medium adjusted to a concentration of 0.5-1 × 10 6 cells / ml is put into a 4 mm cuvette electrode, and the cuvette electrode is inserted into the electrode support 8. nsPEF was applied. In Example 3, electric field strengths of 12.5 KV / cm and 25 KV / cm were used.
例えば、約100ナノ秒のパルス幅とパルス間隔約4ミリ秒の10回計40ミリ秒のパルス集団を1つのクラスター10とし、このときクラスター10をクラスター（250:10）と表記する（図2）。このクラスター（250:10）の印加繰り返し周波数を調べたところ、クラスター（250:10）の印加数10回で、12.5KV/cmのクラスターパルスの繰り返し周波数が0.025回/秒~0.2回/秒の範囲で細胞死誘導効果が強く現れた（図6）。 For example, a cluster of 10 milliseconds with a pulse width of about 100 nanoseconds and a pulse interval of about 4 milliseconds for a total of 40 milliseconds is defined as one cluster 10, and cluster 10 is represented as a cluster (250: 10) (Fig. 2). ). When the application repetition frequency of this cluster (250: 10) was examined, the repetition frequency of the 12.5 KV / cm cluster pulse was 0.025 times / second to 0.2 times / second when the number of times of application of the cluster (250: 10) was 10. The cell death induction effect was strong in the range (Fig. 6).
加えて、パルス幅約60nsの25KV/cmのクラスターパルス（250:5）を5回印加したところ、0.025回/秒をピークとする細胞死誘導効果が得られた（図7）。 In addition, when a 25 KV / cm cluster pulse (250: 5) with a pulse width of about 60 ns was applied 5 times, a cell death inducing effect peaking at 0.025 times / second was obtained (FIG. 7).
この結果、実施例1の単発パルスと同様のメカニズムで0.01回/秒超過0.5回/秒未満のときに細胞死が強く誘導されると考えられた。また、総エネルギー量から鑑みると、クラスターパルスを適切な繰り返し周波数で用いる方が効率的にもよく、また、総印加時間的にも単発パルス印加時よりも早く処理が終わる。このような前記クラスター10を複数回印加するシークエンスを用いることで、単発パルスよりも有効にアポトーシスなどの細胞死を誘導できる。 As a result, it was considered that cell death was strongly induced by the same mechanism as that of the single pulse of Example 1 when it exceeded 0.01 times / second and less than 0.5 times / second. In view of the total energy amount, it is more efficient to use the cluster pulse at an appropriate repetition frequency, and the processing is completed earlier than the time of single pulse application in terms of total application time. By using such a sequence in which the cluster 10 is applied a plurality of times, cell death such as apoptosis can be induced more effectively than a single pulse.
クラスターは約2〜100発の0.5pps以上の繰り返し周波数のパルスで形成され、クラスター自体の繰り返し周波数でクラスター間隔11の時間を調整し、温熱効果を含むか、非加熱にするかを決められるので、細胞や臓器の特性に対して適応した処理が可能となり、有効なアポトーシス誘導効果を得ることができると推測される。 The cluster is formed with about 2 to 100 pulses with a repetition frequency of 0.5 pps or more, and the time of the cluster interval 11 is adjusted by the repetition frequency of the cluster itself, so that it can be determined whether to include the thermal effect or not to heat it It is presumed that treatment adapted to the characteristics of cells and organs becomes possible, and an effective apoptosis-inducing effect can be obtained.
（細胞死分類）
実施例1〜3の実験に関して、電界印加後のHeLa
S3細胞の細胞死がアポトーシスかネクローシスか、もしくは異なる細胞死かを調べた。その結果、12.5KV/cm, 0.5pps, 100回印加した細胞では、24時間培養後、核の断片化を調べることができるフローサイトメトリー用TUNEL染色（TACS）により、核の断片化が示唆された。図8で示すように灰色・黒実線部分は非処理群（TUNEL染色）13を示し、右側に移動している白色・黒実線部分はnsPEF処理群（TUNEL染色）14を示す。また、Y軸のEventは細胞数を示し、X軸のFL-1Pは蛍光強度を示す。蛍光強度は発現量や染色度合いを示す。図8の場合はTUNEL染色の強さを示し、nsPEF処理群（TUNEL染色）14が染色が強いこと、つまり核の断片化が多いことを示す。加えて、この核の断片化は追試により２時間では始まらず、６時間で開始することがわかった。
(Cell death classification)
For the experiments of Examples 1-3, the HeLa after application of the electric field
We examined whether S3 cell death was apoptotic, necrotic, or different. As a result, in cells applied at 12.5 KV / cm, 0.5 pps, 100 times, nuclear fragmentation was suggested by TUNEL staining (TACS) for flow cytometry, which can examine nuclear fragmentation after 24 hours of culture. It was. As shown in FIG. 8, the gray / black solid line portion indicates the non-treated group (TUNEL staining) 13 and the white / black solid line portion moving to the right side indicates the nsPEF-treated group (TUNEL staining) 14. Y-axis Event indicates the number of cells, and X-axis FL-1P indicates fluorescence intensity. The fluorescence intensity indicates the expression level and the degree of staining. In the case of FIG. 8, the intensity of TUNEL staining is shown, and the nsPEF-treated group (TUNEL staining) 14 shows strong staining, that is, a lot of nuclear fragmentation. In addition, it was found that fragmentation of this nucleus did not start in 2 hours but started in 6 hours in a follow-up test.
(Caspase3測定)
アポトーシス時に活性が高くなる活性型Caspase3をActive-Caspase抗体（Abcam）を用いてフローサイトメトリー法で測定した。電界強度12.5KV/cm, 印加回数100回で、0.5ppsで行ったところ、（図9）に示すように、nsPEF印加２時間後では、非処理群(活性型Caspase3)15よりnsPEF処理群(活性型Caspase3)16が活性型Caspase3の発現が多いことが確認された。この結果、nsPEFで誘導される細胞死はアポトーシスのような細胞死である推測できる。このことより、DNA断片化は2時間から24時間のあたりで行われていると予想される。
(Caspase3 measurement)
Active caspase3, which increases its activity during apoptosis, was measured by flow cytometry using an Active-Caspase antibody (Abcam). When the electric field strength was 12.5 KV / cm, the number of times of application was 100, and 0.5 pps, as shown in FIG. 9, after 2 hours of nsPEF application, the nsPEF treatment group (active caspase 3) 15 It was confirmed that active caspase3) 16 has a high expression of active caspase3. As a result, it can be inferred that cell death induced by nsPEF is cell death like apoptosis. From this, DNA fragmentation is expected to take place from 2 to 24 hours.
(細胞死誘導メカニズム)
図10で示すように、nsPEF印加後、HeLa S3細胞は正常細胞17とは異なり、nsPEF印加処理細胞18は5〜10分で細胞膜上に小胞（Bleb）19を形成し始める。この小胞の中や傷ついた細胞膜上でCeramide20という成分が産生されることがわかった。図11で示すように、Ceramide20は印加5分後の段階ですでに発現が上昇している。図11では、灰色・実線部分が非処理群（Ceramide発現）21、白色・実線部分が12.5KV/cm,0.5pps,100回のnsPEF処理群（Ceramide発現）21を示し、nsPEF処理群（Ceramide発現）21の方がCeramide20の発現をnsPEF印加５分後に多く発現していることを示唆している。他の研究でUV・過酸化水素・放射線・熱の刺激により細胞のCeramide20の量が上がることが示唆されている（Subham B et al., 1998, Oncogene, 17, 3277 -3285）。また、このCeramide20が小胞体由来のSAPK/JNK経路を刺激しアポトーシスを誘導することが知られている（Verheij M et al.,
1996, Nature, 380, 75-79）。
(Cell death induction mechanism)
As shown in FIG. 10, after application of nsPEF, HeLa S3 cells differ from normal cells 17, and nsPEF application-treated cells 18 start to form vesicles (Bleb) 19 on the cell membrane in 5 to 10 minutes. It was found that a component called Ceramide20 was produced in these vesicles and on damaged cell membranes. As shown in FIG. 11, the expression of Ceramide20 has already increased at the stage 5 minutes after application. In FIG. 11, the gray / solid line part represents the untreated group (Ceramide expression) 21, and the white / solid line part represents the 12.5 KV / cm, 0.5 pps, 100 nsPEF treatment group (Ceramide expression) 21, and the nsPEF treatment group (Ceramide expression) The expression (21) suggests that Ceramide20 is expressed more frequently after 5 minutes of nsPEF application. Other studies have suggested that the amount of Ceramide 20 in cells is increased by stimulation of UV, hydrogen peroxide, radiation, and heat (Subham B et al., 1998, Oncogene, 17, 3277-3285). Ceramide20 is also known to stimulate the SAPK / JNK pathway derived from the endoplasmic reticulum and induce apoptosis (Verheij M et al.,
1996, Nature, 380, 75-79).
同様に、上記と同じ12.5KV/cm,0.5pps,100回の条件でnsPEFを印加したものでは、JNK下流シグナルにあたるc-JunのmRNAの発現量が図12で示すように印加後30分で上昇し、さらに図13で示すようにタンパク質c-Junのリン酸化は２時間後に非処理群（リン酸化c-Jun発現）23に比べ、nsPEF処理群（リン酸化c-Jun発現）24が多くリン酸化していることが示唆された。加えて、図12は３回の実験を行い、標準誤差を加えている。c-Junはアポトーシスを引き起こす因子として知られており、c-Junによるアポトーシス誘導がp53正常細胞に比べ、p53変異性のガン細胞に対し、有意であることが示唆されている。（Shile H et al.,
2003, Molecular Cell, 11, 1491-1501）。p53変異性のがんはヒトのがんで50％以上を占めるため、nsPEFによるアポトーシス誘導方法はがん治療に有効であると推察される。
Similarly, in the case where nsPEF was applied under the same conditions of 12.5 KV / cm, 0.5 pps, 100 times as described above, the expression level of c-Jun mRNA corresponding to the JNK downstream signal was 30 minutes after application as shown in FIG. Furthermore, as shown in FIG. 13, the phosphorylation of protein c-Jun is more nsPEF-treated (phosphorylated c-Jun expression) 24 than untreated group (phosphorylated c-Jun expression) 23 after 2 hours. It was suggested that it was phosphorylated. In addition, FIG. 12 performs three experiments and adds standard error. c-Jun is known as a factor causing apoptosis, and it has been suggested that apoptosis induction by c-Jun is more significant for p53-mutant cancer cells than for p53 normal cells. (Shile H et al.,
2003, Molecular Cell, 11, 1491-1501). Since p53-mutant cancer accounts for more than 50% of human cancers, it is speculated that apoptosis induction by nsPEF is effective for cancer treatment.
実際にp53変異がん組織にnsPEFを印加した際、図14に示すように組織内で十分に印加された細胞からCeramide20が産生され、がん組織内にCeramide20が浸潤し、パラクリン作用によりさらに長時間周囲の境界部残存がん細胞25をアポトーシスに誘導することが予想される。また、Ceramide20は血管新生阻害作用もあり（Madhavi C et al., 2009, J. Agric. Food Chem.,
57, 5201-5210）、血管新生因子を産生するガン細胞を兵糧攻めすることができる。そのため、nsPEF誘導性Ceramide20により血管新生抑制による長期的兵糧攻め型がん治療も効果があると期待できる。先の特許文献1中でもnsPEFによる血管新生阻害が示唆されており、我々の発見したCeramide20による働きが大きいと考えられる。
When nsPEF was actually applied to p53 mutant cancer tissue, as shown in Fig. 14, Ceramide20 was produced from cells that were sufficiently applied in the tissue, and Ceramide20 infiltrated into the cancer tissue, which was further prolonged by paracrine action. It is expected that the cancer cells 25 remaining at the border around the time are induced to apoptosis. Ceramide 20 also has an angiogenesis inhibitory effect (Madhavi C et al., 2009, J. Agric. Food Chem.,
57, 5201-5210), can attack cancer cells that produce angiogenic factors. Therefore, it can be expected that nsPEF-inducible Ceramide20 will be effective for long-term cancer-based cancer treatment by inhibiting angiogenesis. The previous patent document 1 also suggests that nsPEF inhibits angiogenesis, and it is thought that the action of Ceramide 20 that we discovered is significant.
加えて、nsPEF印加時に細胞膜上にナノポア（ナノサイズの孔）の形成と同時に2価のカルシウムイオンなどのion 流入が起きる。図15に示すようにion 流入をEDTA・EGTA・カルシウムマグネシウムイオンフリーリン酸緩衝液(PBS(−))で遮断すると、12.5KV/cm条件下で引き起きる細胞死が抑制されることがわかった。加えて、小胞19の形成やCeramide20の産生を抑制した。図15に示す実験は３回繰り返し行った。図15には標準誤差を付加している。この結果、主にカルシウムイオンの流入により細胞死が引き起こされることが推察できた。 In addition, when nsPEF is applied, ion inflow such as divalent calcium ions occurs simultaneously with the formation of nanopores (nano-sized pores) on the cell membrane. As shown in Fig. 15, it was found that cell death caused under 12.5 KV / cm conditions was suppressed when ion inflow was blocked with EDTA / EGTA / calcium magnesium ion free phosphate buffer (PBS (-)). . In addition, vesicle 19 formation and Ceramide20 production were suppressed. The experiment shown in FIG. 15 was repeated three times. A standard error is added to FIG. As a result, it was inferred that cell death was caused mainly by the inflow of calcium ions.
上記の現象を利用すれば、カルシウムイオンを多く含む緩衝液を細胞死を起こしたい患部に充填し、その後、nsPEFを印加することにより、患部のみを効率的に細胞死誘導を引き起こさせることが可能になると考えられる。もしくは印加しながらカルシウムイオン緩衝液を患部に充填する方法がよい。そのためにインジェクション針と電極が併用できる治療器具などがあれば効率的である。例えば、陽極をインジェクション針とする電極を用いることで、陰極の方へとカルシウムイオンを展開させやすいので、中心部にインジェクション針電極部（陽極）、周囲に陰極電極部を配置した構造のインジェクション可能電極を用いれば効果的だと考えられる。 By using the above phenomenon, it is possible to efficiently induce cell death only in the affected area by filling the affected area where cell death is desired with a buffer solution rich in calcium ions and then applying nsPEF. It is thought that it becomes. Alternatively, a method of filling the affected part with a calcium ion buffer solution while applying is preferable. Therefore, it is efficient if there is a therapeutic instrument that can be used in combination with an injection needle and an electrode. For example, by using an electrode with an anode as an injection needle, it is easy to spread calcium ions toward the cathode, so injection with an injection needle electrode (anode) at the center and a cathode electrode at the periphery is possible Use of electrodes is considered effective.
図16は、細胞死誘導メカニズムを時間軸でまとめたものを示す。 FIG. 16 shows a summary of cell death induction mechanisms on a time axis.
（実施例4）nsPEFによる成熟脂肪細胞脂肪球減少
マウス3T3L-1細胞をヒューマンサイエンス研究資源バンクより入手し、分化誘導剤（DEX, IBMX, Insullin）で分化誘導し、分化開始から10日目の成熟脂肪細胞を材料として使用した。100回印加条件で、電界強度、繰り返し周波数を変えて実験を行った結果、繰り返し周波数にはあまり依存せず、電界強度に依存し、脂肪球の減少が確認された。このようなアポトーシス以外の現象のいくつかには,250pps以上の高繰り返し周波数かつ高電界の使用が最適なものが存在すると考えられる。皮、筋肉、脂肪などでそれぞれに最適な印加パラメーターを使い分けることが望ましい。
(Example 4) Reduction of mature adipocyte fat globule by nsPEF Mouse 3T3L-1 cells were obtained from Human Science Research Resource Bank, induced to differentiate with differentiation inducers (DEX, IBMX, Insullin), and 10 days after the start of differentiation Mature adipocytes were used as material. As a result of experiment with changing the electric field strength and the repetition frequency under the application condition of 100 times, it was confirmed that the fat globule decreased depending on the electric field strength without depending on the repetition frequency. Some of these phenomena other than apoptosis may be best suited for the use of a high repetition frequency of 250 pps or higher and a high electric field. It is desirable to use the optimum applied parameters for skin, muscle, fat and so on.
以上のように、繰り返し周波数に対して反応する細胞活動の場合、ppsを0.01pps超過〜0.5pps未満の範囲の低繰り返し周波数の方が細胞死誘導効果が高く、ppsに対して反応しない細胞活動の場合は50pps以上の早いppsを使用して300発程度印加することが細胞死誘導に対して効率的である。また、電界強度を高くすると、細胞が焼け、皮膚などに行うとネクローシスやケロイドなどの痕ができるため、2時間後のPI染色率最高50％程度の電界強度でアポトーシス誘導を何日かに分けて複数回行うことが望ましいと考えられる。さらに、50〜500ppsを2〜30回を１つのクラスターとし、低繰り返し周波数の間欠暴露も細胞死誘導効果がある。 As described above, in the case of cellular activity that reacts to the repetition frequency, the cell repetition activity that has a higher cell death-inducing effect at low repetition frequencies in the range of more than 0.01 pps to less than 0.5 pps and does not react to pps In this case, applying about 300 shots using a fast pps of 50 pps or more is effective for cell death induction. In addition, when the electric field strength is increased, the cells are burned, and when it is applied to the skin, marks such as necrosis and keloid are formed. It is considered desirable to do this multiple times. Further, 50 to 500 pps is made 2 to 30 times as one cluster, and intermittent exposure at a low repetition frequency has an effect of inducing cell death.
さらに図17のように１つのクラスター10の中に電界強度やパルス幅が異なるnsPEFを複数入れることで、より強力な細胞死の誘導を可能にすると予測される。もしくは、ドラッグデリバリーや細胞分化制御などへの応用も期待できる。 Furthermore, as shown in FIG. 17, it is predicted that by introducing a plurality of nsPEFs having different electric field strengths and pulse widths in one cluster 10, it is possible to induce stronger cell death. Alternatively, application to drug delivery and cell differentiation control can be expected.
アンテナ型電極などでは、使用時に繰り返し周波数を低くすることで目的の細胞や臓器に効果的な影響を与える。また、アンテナ型電極で広範囲にアポトーシスを誘導する際には、繰り返し周波数が高い状態で印加する場所を移動させながら、印加起点と印加終点までを10秒ほどの時間をかけて複数回印加することで、同じ部位に対し0.1ppsの低繰り返しシークエンスを印加したときと同じ条件になり、効果的にアポトーシスを誘導できると考えられる。 In antenna-type electrodes and the like, effective frequency is affected to target cells and organs by reducing the repetition frequency during use. In addition, when inducing apoptosis over a wide range with an antenna-type electrode, apply multiple times over a period of about 10 seconds until the application start point and the application end point are moved while moving the place where the application frequency is high. Thus, it is considered that the same conditions as when applying a low repetitive sequence of 0.1 pps to the same site can induce apoptosis effectively.
本発明を使用して、ガンやメタボリックシンドロームなどの医療機器に利用できる。 The present invention can be used for medical devices such as cancer and metabolic syndrome.
1・・・nsPEF発生装置
2・・・充電器
3・・・制御装置
4・・・外部繰り返し周波数制御装置
5・・・電極
6・・・パルス電界発生部
7・・・接地電位
8・・・電極支持部
9・・・オシロスコープ
10・・・クラスター
11・・・クラスター間隔
12・・・nsPEF印加システム
13・・・非処理群（TUNEL染色）
14・・・nsPEF処理群（TUNEL染色）
15・・・非処理群(活性型Caspase3)
16・・・nsPEF処理群(活性型Caspase3)
17・・・正常細胞
18・・・nsPEF印加処理細胞
19・・・小胞
20・・・Ceramide
21・・・非処理群（Ceramide発現）
22・・・nsPEF処理群（Ceramide発現）
23・・・非処理群（リン酸化c-Jun）
24・・・nsPEF処理群（リン酸化c-Jun）
25・・・境界部残存がん細胞
1 ... nsPEF generator
2 ... Charger
3 ... Control device
4 ... External repetition frequency controller
5 ... Electrodes
6 ... Pulse electric field generator
7 ... Ground potential
8 ... Electrode support
9 ... Oscilloscope
10 ... Cluster
11 ... Cluster interval
12 ... nsPEF application system
13 ... Untreated group (TUNEL staining)
14 ... nsPEF treatment group (TUNEL staining)
15 ... Untreated group (active caspase 3)
16 ... nsPEF treatment group (active caspase 3)
17 ... Normal cells
18 ... nsPEF applied cells
19 ... Vesicles
20 ... Ceramide
21 ・ ・ ・ Non-treatment group (Ceramide expression)
22 ・ ・ ・ nsPEF treatment group (Ceramide expression)
23 ... Untreated group (phosphorylated c-Jun)
24 ・ ・ ・ nsPEF treatment group (phosphorylated c-Jun)
25 ... Residual cancer cells at the boundary
Claims (3)
アポトーシスなどの細胞死を誘導する方法であって、
前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、
繰り返し周波数は0.0１pps超過0.5pps未満であることを特徴とする
ナノ秒パルス電界を用いたアポトーシスなど細胞死誘導方法。
Apply a nanosecond pulse electric field to target cells in the tissue,
A method of inducing cell death such as apoptosis,
The nanosecond pulse electric field is 0.5 to 250 KV / cm, with a pulse width of 1 to 300 ns,
A cell death induction method such as apoptosis using a nanosecond pulse electric field, wherein the repetition frequency is more than 0.01 pps and less than 0.5 pps.
アポトーシスなどの細胞死を誘導する方法であって、
前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、
繰り返し周波数は50pps以上であることを特徴とする
ナノ秒パルス電界を用いたアポトーシスなど細胞死誘導方法。
Apply a nanosecond pulse electric field to target cells in the tissue,
A method of inducing cell death such as apoptosis,
The nanosecond pulse electric field is 0.5 to 250 KV / cm, with a pulse width of 1 to 300 ns,
A cell death induction method such as apoptosis using a nanosecond pulse electric field characterized by a repetition frequency of 50 pps or more.
アポトーシスなどの細胞死を誘導する方法であって、
前記ナノ秒パルス電界は、0.5~250KV/cmで、1~300nsのパルス幅で、
前記ナノ秒パルス電界複数回を1回としたクラスターとして印加して、
クラスターの繰り返し周波数が0.01回/秒超過0.5回/秒未満であることを特徴とする
ナノ秒パルス電界印加シークエンスを用いたアポトーシスなど細胞死誘導方法。 Apply a nanosecond pulse electric field to target cells in the tissue,
A method of inducing cell death such as apoptosis,
The nanosecond pulse electric field is 0.5 to 250 KV / cm, with a pulse width of 1 to 300 ns,
Applying the nanosecond pulse electric field multiple times as a single cluster,
A method for inducing cell death such as apoptosis using a nanosecond pulse electric field application sequence characterized in that the repetition frequency of the cluster is 0.01 times / second and less than 0.5 times / second.
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| JP2011047162A JP2011240115A (en) | 2010-04-19 | 2011-03-04 | Apoptosis inducing method using nanosecond pulsed electric field |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018094078A (en) * | 2016-12-13 | 2018-06-21 | 之一 市川 | Subcutaneous fat reduction device using high electric field |
| CN112694974A (en) * | 2020-11-26 | 2021-04-23 | 中国石油大学(华东) | Construction and monitoring method for nanosecond pulsed electric field ablation dynamic monitoring system |
-
2011
- 2011-03-04 JP JP2011047162A patent/JP2011240115A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018094078A (en) * | 2016-12-13 | 2018-06-21 | 之一 市川 | Subcutaneous fat reduction device using high electric field |
| CN112694974A (en) * | 2020-11-26 | 2021-04-23 | 中国石油大学(华东) | Construction and monitoring method for nanosecond pulsed electric field ablation dynamic monitoring system |
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