JP2005350396A - Treating method for specific cell and treating apparatus for specific cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To relatively simply and inexpensively annihilate or decrease a specific cell by deactivating cell division performances of the specific cell such as cancer cells using magnetism. <P>SOLUTION: The subject treating apparatus comprises a container 10 to accommodate a matter to be treated containing the specific cell taken out from a human body, a variable magnetic field-generating part 12 for applying a variable magnetic field having a magnetic field strength of 0.01≤T(Tesla)≤3 on the matter to be treated in the container 10, a temperature-retaining part 15 for retaining the temperature (t) in the container 10 within a specified temperature range, and the variable magnetic field-generating part 12 is equipped with a coil 13 surrounding the container 10 and generating a pulsed magnetic field having a pulse frequency of 1-250 Hz. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a specific cell processing method and a specific cell processing apparatus for inactivating or dividing a specific cell such as a cancer cell to kill or reduce the specific cell.

  In general, for example, as an effective technique for purifying blood for blood transfusion, a technique for selectively killing or reducing specific cells such as a small amount of cancer cells in blood has been studied, but sufficient results have been obtained. Absent. In means using radiation irradiation or chemicals, serious side effects become a problem. In addition, in the treatment by extracorporeal circulation such as hemodialysis, the technology remains to filter out waste products.

  Therefore, the inventors of the present invention perform removal of specific cells such as cancer cells by using magnetism without using drugs or radiation, simplify the processing, reduce the burden, and reduce the cost. I have studied.

  By the way, conventionally, for example, the following Patent Document 1 (Japanese Patent Application Laid-Open No. 8-47528), which is not intended for cells, proposes a device that kills or reduces bacteria and viruses in human blood. Has been.

  As shown in FIG. 5, this apparatus takes out blood from a human body, and sends the extracted blood to an irradiation unit 1 that irradiates radiation, X-rays, ultrasonic waves, weight beams, ultraviolet rays, strong magnetism, and the like. The irradiation unit 1 processes blood and returns the processed blood to the human body again.

  However, in this device, there is a disclosure of using strong magnetism, but since the condition setting has not been established, when used for cell processing, not only specific cells of cancer cells but also good quality cells are adversely affected. There is a problem that it cannot be applied as it is.

Therefore, the inventors of the present application have previously proposed a treatment technique for specific cells described in Patent Document 2 (Japanese Patent Application Laid-Open No. 2004-141032).
This is a treatment method in which a magnetic field is applied to a specific cell to deactivate the division ability of the specific cell, and the magnetic field is applied by placing the cell in a static magnetic field space formed by a superconducting magnet. The strength is treated as 6 ≦ T (Tesla) ≦ 12.
As a result, if the specific cell is a malignant cell such as a cancer cell having a relatively high division rate, the division ability can be inactivated by the action of a magnetic field. For example, T lymphocytes, B lymphocytes, natural cells can be inactivated. In immune cells such as killer cells, there is almost no influence in the range of the set magnetic field, and therefore, specific cells can be effectively killed or reduced.

JP-A-8-47528 JP 2004-141032 A

  By the way, in the former conventional processing technique, processing is performed in a strong magnetic field, and even in the latter technique, cells are arranged in a static magnetic field space formed by a superconducting magnet and having a relatively large magnetic field strength. Since the processing is performed, the processing function is sufficiently ensured, but there is a disadvantage that the apparatus is complicated and the cost is significantly increased, resulting in poor versatility.

  The present invention has been made in view of the above problems, and kills specific cells by deactivating the division ability of specific cells such as cancer cells using magnetism so that they are relatively simple and not expensive. Alternatively, it is an object of the present invention to provide a specific cell processing method and a specific cell processing apparatus which can be reduced.

In order to solve such a problem, the inventors of the present invention have found out that the division ability of a specific cell having a high division rate is lost under the condition of a variable magnetic field.
The mechanism is that in the differentiation of cells accompanied by nuclear division, the nuclear matter mainly composed of ions undergoes a magnetic force repeatedly when moving in a variable magnetic field, and thus deviates from the normal division process. it is conceivable that.
As a result, the following is inferred.
1. A fluctuating magnetic field causes cytoskeletal protein sequence abnormalities, cell division ends abnormally, and cells die.
2. Induction of structural abnormality of the cell membrane is performed.
3. The magnetic field causes DNA synthesis failure.

  For example, in the case of cancer cells, as in the case of the above results, when the magnetic field is applied in the process of actively dividing, it becomes possible to inhibit the division process in the division process of the cancer cell, and the increase in the number of disordered division of the cancer cell Can be prevented.

  On the other hand, for example, it was also confirmed that immune cells such as T lymphocytes, B lymphocytes, and natural killer cells have almost no effect under conditions with a varying magnetic field.

  Further, a specific technical means of the present invention is a specific cell processing method in which a magnetic field is applied to a specific cell and the division ability of the specific cell is deactivated. Yes.

With this configuration, when a specific cell is a malignant cell such as a cancer cell having a relatively high division rate, the division ability is inactivated by the action of a varying magnetic field. In other words, when a malignant cell is stimulated to divide, the cell is differentiated with nuclear division, but when the nuclear substance mainly composed of ions moves in the magnetic field, it receives magnetic force from the magnetic field and deviates from the normal division process. As a result, cell damage occurs, and the division ability is deactivated. And since malignant cells have a high division rate and the opportunity of magnetic field stimulation at the time of division increases, the malignant cells die or decline because disordered increase in division is prevented.
On the other hand, for example, immune cells such as T lymphocytes, B lymphocytes, and natural killer cells have almost no effect in the set magnetic field range, that is, prevent immune function changes and cytotoxicity. Is done.
Further, since the process is performed in a varying magnetic field, the apparatus can be made relatively simple and not expensive, the process can be simplified and simplified, and versatility can be improved.

If necessary, the applied magnetic field strength is set to 0.01 ≦ T (Tesla) ≦ 3. Desirably, 0.05 ≦ T (Tesla) ≦ 2.
With a relatively simple device, it is possible to generate a fluctuating magnetic field that reliably exhibits the above-described functions and effects.

If necessary, the cells are arranged in a pulse magnetic field having a pulse frequency of 1 to 250 Hz. Desirably, it is 5 to 100 Hz.
With a relatively simple device, it is possible to generate a fluctuating magnetic field that reliably exhibits the above-described functions and effects.

Furthermore, if necessary, the temperature t of the specific cell is set to 4 ° C. ≦ t ≦ 37 ° C. Desirably, the temperature t of the specific cell was set to 25 ° C. ≦ t ≦ 37 ° C.
Thereby, when a specific cell is processed with the blood of a human body etc., the influence which it has on a good quality cell is reduced.
That is, when the temperature is lower than 4 ° C. or higher than 37 ° C., not only specific cells but also other cells are greatly affected.

Furthermore, the magnetic field is applied for 5 min to 240 min. Thereby, processing is easy and can be simplified, the burden is reduced, and the cost is reduced.
In this case, if the time is shorter than 5 minutes, a sufficient effect cannot be obtained. On the other hand, when the time is longer than 240 min, the influence on not only a specific cell but also other cells becomes large.

  And it is set as the structure which adds the division rate promoter which accelerates | stimulates the division rate of the said specific cell as needed. Thereby, specific cells are activated (cell division is stimulated), and abnormal cells can be removed more powerfully and effectively.

Examples of the mitogenic rate promoter include phytohemagglutinin (PHA), concanavalin A (ConA), lymphocyte-specific peptide, lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglyceol, tetradecanoyl phorbol acetate (TPA) It is effective to use at least one of the following substances. These substances are mainly substances that stimulate the cells of the immune system and usually do not always have a bad action. For example, lymphocyte-specific monoclonal antibodies are also used as therapeutic agents for leukemia.
In general, each immune cell (T cell or NK cell) is activated by a specific molecule (peptide, protein, antibody or sugar, lipid) but is hardly activated by other molecules. Therefore, by activating specific immune cells (specific cells) that have caused abnormalities and applying a variable magnetic field (giving division and growth stimulation), only abnormal immune cells can be selected without damaging normal immune cells Therefore, it can be removed without side effects.

In addition, the specific cell processing apparatus of the present invention for solving the above-described problems includes a container in which a processed material containing specific cells taken out from a human body is placed, and a magnetic field strength of 0.01 ≦ T on the processed material in the container. (Tesla) A variable magnetic field generation unit that applies a variable magnetic field of ≦ 3 and a temperature holding unit that holds the temperature t in the container in a predetermined temperature range are provided.
And the said fluctuation | variation magnetic field generation | occurrence | production part is provided with the coil which surrounds the said container and produces | generates the pulse magnetic field of pulse frequency 1-250Hz as needed.
Thereby, a variable magnetic field can be reliably applied to a specific cell to inactivate the division ability of the specific cell. Further, the apparatus can be made relatively simple and not expensive, the processing can be simplified and simplified, and the versatility can be improved.

As described above, according to the specific cell processing method and the specific cell processing apparatus of the present invention, if the specific cell is a malignant cell such as a cancer cell having a relatively high speed of division, it is divided by the action of a magnetic field. On the other hand, immune cells such as T lymphocytes, B lymphocytes, and natural killer cells have almost no effect in the set magnetic field range. Can be killed or reduced.
Further, since the process is performed in a varying magnetic field, the apparatus can be made relatively simple and not expensive, the process can be simplified and simplified, and versatility can be improved. Therefore, it can be expected to be applied to medical fields such as purification of blood for transfusion.

Hereinafter, a specific cell processing method and a specific cell processing apparatus according to an embodiment of the present invention will be described with reference to the accompanying drawings.
Since the specific cell processing method according to the embodiment is realized by the specific cell processing apparatus according to the embodiment, its operation will be described.

  The processing apparatus according to this embodiment is an apparatus that processes specific cells in blood collected from a human body as a processed object. Specific cells include various cancer cells such as leukemia and malignant cells such as virus-infected cells.

  As shown in FIG. 1, the basic configuration of the processing apparatus S includes a container 10 in which blood, which is taken out from a human body and contains processed cells containing specific cells, is placed. The container 10 is a plurality of elongate plastic containers that can be sealed, for example, and is fixed to the support 11.

  In addition, the apparatus S includes a variable magnetic field generation unit 12 that applies a variable magnetic field with a magnetic field intensity of 0.01 ≦ T (Tesla) ≦ 3 to a processed object in the container 10. The fluctuating magnetic field generating unit 12 includes a coil 13 that surrounds the container 10 and generates a pulse magnetic field having a pulse frequency of 1 to 250 Hz by a pulse power source. In the embodiment, for example, 50 Hz and 0.15T are set. FIG. 2 shows a pulse magnetic field current waveform according to the embodiment.

  Furthermore, this apparatus S is provided with the temperature holding part 15 which hold | maintains the temperature in the container 10 in a fixed range. The temperature holding unit 15 includes a constant temperature water tank 16 that houses the container 10 and the coil 13, a stirring blade 17 that stirs the water in the constant temperature water tank 16, and a heating and cooling unit 18 that heats and cools the water. And the temperature in the constant temperature water tank 16 is hold | maintained in a fixed range, and the temperature t in the container 10 is hold | maintained in a predetermined temperature range by the control part outside illustration. For example, the temperature is kept in the range of 4 ° C. ≦ t ≦ 37 ° C., preferably 25 ° C. ≦ t ≦ 37 ° C. In the embodiment, for example, t = 25 ° C. is set.

  And by this apparatus S, the container 10 is put into the constant temperature water tank 16, and after the predetermined time progresses, the container 10 is taken out and processed. Blood is kept in a varying magnetic field for a predetermined time. As the time, a fluctuating magnetic field is applied for 5 min to 240 min. In the embodiment, for example, it is set to 180 min (3 hours).

Therefore, when a malignant cell in blood is processed by the processing apparatus S according to this embodiment, blood is first put in the container 10. Then, this container 10 is put into a constant temperature water tank 16, the coil 13 is operated, and is kept in a varying magnetic field for a predetermined time.
Thereby, when a fluctuating magnetic field acts on a malignant cell having a high division rate, the division ability is inactivated. In other words, when a malignant cell is stimulated to divide, the cell is differentiated with nuclear division, but when the nuclear substance mainly composed of ions moves in the magnetic field, it receives magnetic force from the magnetic field and deviates from the normal division process. As a result, cell damage occurs, and the division ability is deactivated. And since malignant cells have a high division rate and the opportunity of magnetic field stimulation at the time of division increases, the malignant cells die or decline because disordered increase in division is prevented.

  On the other hand, for example, immune cells such as T lymphocytes, B lymphocytes, and natural killer cells have almost no effect in the range of the set magnetic field strength, temperature, and retention time, that is, immune function change, cell damage. Effects such as sex are prevented.

  In the above-described embodiment, it is effective to add a mitotic rate accelerator that accelerates the mitotic rate of specific cells as necessary. For example, when there are specific immune cells (specific cells) that have caused abnormalities in the cells of the immune system and it is desired to remove them, phytohemagglutinin (PHA), concanavalin A (ConA), lymphocytes It is effective to use at least one substance such as a specific peptide, a lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglyceol, tetradecanoyl phorbol acetate (TPA).

  In general, each immune cell (T cell or NK cell) is activated by a specific molecule (peptide, protein, antibody or sugar, lipid) but is hardly activated by other molecules. Therefore, by activating specific immune cells (specific cells) that have caused abnormalities and applying a variable magnetic field (giving division and growth stimulation), only abnormal immune cells can be selected without damaging normal immune cells Therefore, it can be removed without side effects. Therefore, by adding this mitotic rate accelerator, specific cells are activated (stimulation of cell division), and abnormal cells can be removed more powerfully and effectively.

  Moreover, in the above embodiment, it is effective to add a deactivation auxiliary agent that deactivates specific cells. For example, in order to remove cancer cells, vitamin A, thyroid hormone, glucocorticoid, cyclophosphamide, azathioprine, adriamycin, bleomycin, antimetabolite, alkylating agent, plant alkaloid (bin alkaloid) It is effective to use at least one substance of cytoskeletal disorder drug. These are known as drugs used for the treatment of cancer, and the addition of these substances makes it possible to mainly inactivate cancer cells with high efficiency. That is, in addition to the inactivation of specific cells by a magnetic field, inactivation by an inactivation auxiliary agent is also performed, the effect becomes synergistic, and the removal efficiency of specific cells is further improved.

Experimental example

Next, an experimental example is shown. The effect of a varying magnetic field on human immune cells was tested.
The analysis method and analysis contents are that the immunocompetent cells (lymphocytes, natural killer cells) of peripheral blood (24 to 52 years old) of healthy subjects are separated by centrifugation and suspended in a culture solution (RPMI1640 / 10% FCS). It was.
Then, two groups, a lymphocyte activation stimulation group (PHA addition) and a non-stimulation group (PHA non-addition) were incubated with a varying magnetic field applied, and analyzed after 24 hours of culture.

Test conditions are
Pulse frequency: 50Hz
Magnetic field strength: 0.05T, 0.15T
Holding temperature: t = 25 ° C.
Magnetic field application time: 120 min (2 hours).

First, lymphocyte subsets (CD4, CD8, CD20, CD45RA) were measured by flow cytometry.
The results are shown in FIG. FIG. 3 is a graph showing the results of testing the effect of a magnetic field on T lymphocytes as immune cells, with the number of cells in the control group being 100%, showing the increase or decrease in the number of cells.
From this result, the following can be said.
1. Unstimulated lymphocytes Fluctuating magnetic field did not significantly affect lymphocyte count, CD4 T cell count, CD8 T cell count.
2. Lectin PHA-stimulated lymphocytes Due to cell division stimulation (PHA administration), the ratio of dead cells increased in the variable magnetic field loading group compared to the non-loading group.

Next, it was examined from the expression of CD56 whether the magnetic field can affect the subclass frequency of natural killer cells.
Natural killer cells can be defined as CD3-negative and CD16-positive cells, but CD56 is a further subcategory of natural killer cells, and it is possible to infer differences in the function of natural killer cells based on CD56 expression. Is done.
The results are shown in FIG. As a result, no significant effect of the varying magnetic field on unstimulated natural killer cells was observed.

Experiments on natural killer cell (NK cell) function and neutrophil function were also conducted. As a result, the fluctuation magnetic field of 0.15T did not affect the NK cell function (killer activity) and neutrophil function (neutrophil bactericidal ability, neutrophil phagocytic ability).
From the above results, it was revealed that the fluctuating magnetic field has no significant effect on unstimulated leukocytes. On the other hand, in the case of stimulated lymphocytes, cell viability was affected.

  That is, under the above experimental conditions, it is presumed that malignant cells such as cancer cells having a relatively high division rate can inactivate division ability by the action of a magnetic field. It was found that immune cells such as spheres, B lymphocytes, and natural killer cells have almost no effect in the set magnetic field range.

  In the above embodiment, the present invention is applied to specific cells in blood. However, the present invention is not necessarily limited thereto, and may include specific cells other than blood or specific cells themselves, and may be changed as appropriate. There is no problem. Further, the specific cell may be any cell.

It is a figure which shows the processing apparatus of the specific cell which concerns on embodiment of this invention. It is a figure which shows the pulse magnetic field current waveform which concerns on embodiment of this invention. It is a graph which shows the result of having tested the influence of the magnetic field which gives to T lymphocyte as an immune cell, and shows the increase / decrease in the number of cells by making the number of cells of a control group into 100%. It is a graph which shows the influence which a magnetic field has on the subclass frequency of a natural killer cell. It is a figure which shows an example of the conventional blood processing apparatus.

Explanation of symbols

S processing device 10 container 11 support 12 fluctuating magnetic field generating unit 13 coil 15 temperature holding unit 16 constant temperature water tank 17 stirring blade 18 heating and cooling unit

Claims (9)

  A method for treating a specific cell, wherein a magnetic field is applied to the specific cell to deactivate the division ability of the specific cell, wherein the magnetic field to be applied is a variable magnetic field.   2. The method for treating specific cells according to claim 1, wherein the applied magnetic field intensity is 0.01 ≦ T (Tesla) ≦ 3.   The method for treating a specific cell according to claim 1 or 2, wherein the cell is placed in a pulse magnetic field having a pulse frequency of 1 to 250 Hz.   4. The method for treating specific cells according to claim 1, wherein the temperature t of the specific cells is 4 ° C. ≦ t ≦ 37 ° C.   The method for treating specific cells according to claim 1, 2, 3 or 4, wherein a magnetic field is applied for 5 min to 240 min.   6. The method for treating a specific cell according to claim 1, 2, 3, 4 or 5, wherein a mitotic rate accelerator for accelerating the division rate of the specific cell is added.   Examples of the mitogenic rate promoter include phytohemagglutinin (PHA), concanavalin A (ConA), lymphocyte-specific peptide, lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglyceol, tetradecanoyl phorbol acetate (TPA) 7. The method for treating specific cells according to claim 6, wherein at least one substance is used.   A container in which a processed product containing specific cells taken out from the human body is placed; a variable magnetic field generating unit that applies a variable magnetic field of magnetic field intensity 0.01 ≦ T (Tesla) ≦ 3 to the processed material in the container; And a temperature holding unit that holds the temperature t within a predetermined temperature range.   9. The specific cell processing apparatus according to claim 8, wherein the fluctuating magnetic field generating unit includes a coil that surrounds the container and generates a pulsed magnetic field having a pulse frequency of 1 to 250 Hz.

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