JP2005287979A - Immunoactivation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To clarify the most suitable light source means to activate an immune system and a construction method of the same by clarifying a mechanism of optical action affecting the immune system. <P>SOLUTION: The apparatus emits a pulsed laser beam with a wavelength of 1.4-2.5 μm to activate Langerhans cell which subcutaneously exists. Thereby, an internal immune system is activated and a tumor can be contracted so that the apparatus is effective for immunodeficiency and tumor reduction. The intensity of energy density and a laser output are selected on the basis of an interaction between the light and a biological cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus that brings about an immune activity of a living body by using optical and mechanical means or an immunoactive agent, an immunoactive functional food, and the like together.

  The therapeutic effect by the interaction between the living body and light has been known for a long time. In particular, in Japan, there is a known method of using the luminescence generated by passing an electric current through a carbon electrode known as a “phototherapy device” to treat the human body, and it is still used as a means of acupuncture. Has been.

  Phototherapy devices are said to be effective against a wide range of symptoms. Phototherapy devices are said to be effective for pain, itching, allergic diseases, ulcers, inflammatory diseases, and the like. (Non-Patent Document 1)

  On the other hand, new medical devices using lasers have been clinically used for more than 20 years. As is well known, lasers that have been put into practical use as medical lasers are of many types, such as carbon dioxide lasers, Nd: YAG lasers, ruby lasers, and semiconductor lasers, and their oscillation wavelengths range from the ultraviolet region to the far infrared region. . Examples of applications using these laser beams are incision, transpiration, coagulation, and hemostasis of a living body.

In addition to the above-described application examples, the effect of low output energy on the cell level is known for the interaction between light and a living body. Biological action by low output energy is known to be pain relief, wound healing promoting effect, etc., and its effectiveness including the mechanism of action has been extensively studied, and these devices have already spread in clinical practice.
As for the mechanism of interaction between light and living body, (1) collagen production mechanism, (2) cell activation mechanism, etc. are known. In any case, irradiated cells do not cause cell destruction by light irradiation. It is known to have a specific effect on.

It has been shown that when fibroblasts are irradiated with laser light, the intracellular cGMP (protein production circuit) is dependent on light stimulation, resulting in enhanced collagen production. Actually, in the cell experiment, a production of procollagen 1.5 times that of the control was obtained at an irradiation energy of 5.4 J / cm 2 (wavelength 632.8 nm). On the other hand, in the case of 15 J / cm 2, the production was slightly lower and 1.4 times.
In addition, it is known that pulse irradiation is more effective than continuous light for cell activation. The basis is the law of Arndt Rudolph and Schultz Hugo. “Any stimulus does not stimulate the body's response if it is a very weak stimulus. However, when it reaches a certain stimulation zone, it promotes the body's reaction and conversely suppresses a strong stimulus. A stronger stimulus stops the biological reaction. This law may be empirically verified.

Russian Academy of Sciences Karu TI has proposed a biological photoresponse model for visible red light. When flavin dehydrogenase, cytochrome, cytochrome oxidase, etc., which are respiratory chain compounds in the mitochondria in the cell, absorb light energy, the mitochondria are activated and NAD increases, resulting in a high metabolic state and increased ATP. Is released into the cytoplasm, changing the redox state of mitochondria and cytoplasm. A series of light-mediated phenomena occur inside the cell, affecting the calcium ions that flow inside and outside the cell membrane, amplifying the signal, affecting the nucleus, RNA,
It promotes DNA synthesis and affects cell differentiation (Non-patent Document 1). In addition, Smith KC of the United States has proposed a similar model, which says that cell differentiation is caused by an action similar to that of visible red light (Non-patent Document 2).
Young SR et al. Also examined the light irradiation effect of calcium intake by macrophages. This clarifies the biological effect of the low-power laser, and cell activity is caused by light irradiation, and in particular, the influence of the light wavelength, energy density, and frequency was investigated (Non-patent Document 3). As a result, cell activation occurred at an optical wavelength of 600-900 nm, an energy density of 4-8 J / cm 2 at maximum, and a frequency of 5-5000 Hz. This data is approximately equivalent to the clinical irradiation conditions related to related wound repair, pain, etc. obtained so far, and can therefore be regarded as the basis for determining the irradiation conditions.

  Although there has been little research on the immune activity of the living body in light and biological action, it is predicted that it will naturally have some kind of action on the cells responsible for immunity in the action of light and cells. In relation to this, a “method and apparatus for stimulating the immune system and generating healing at the cellular level” has been disclosed (Patent Document 1). This method stimulates the immune system to promote healing by irradiating the skin of a disease to be treated as a method of stimulating the immune system with an infrared ray having a wavelength near 1917 nm through a tube or a guide, thereby stimulating the immune system. It is about a device that allows healing at the cellular level without harming the body.

  Regarding the light and biological action by the device, the immune system including T cells in the living body is stimulated, and the production of B cells in the bone marrow is promoted by selecting the irradiation site. The device that brings about such an action is non-coherent light, the wavelength range is 1800-2400 nm, and the irradiation light recommends pulse irradiation of 7.5 Hz. The irradiation energy is set to 10 to 200 mW with an average irradiation diameter of 10 mm.

  As explained in detail above, the stimulating action on the immune system is a non-destructive cellular action, but the details of the mechanism of action and why non-coherent light is effective are not clear at present. is there.

Surgery, anticancer drug administration, and radiation therapy inevitably cause a decline in the immune system of the living body. However, immunologically active drugs, immunologically active functional foods, or other means may be used alone or in combination as a means of immunization against this decline. Sometimes used in a complementary manner. The present invention can be expected to further improve the effect when used in combination with these means.
Special Table 2002-519162 "Visible Comprehensive Phototherapy" published by Ray Research Institute Karu TI, Biological action intensity visible monochromatic light and some of its medical applications.Laser ed.by Galletti G, p 381, Monduzzui Editore, Bologna, 1985 Smith KC, Thephotobilogical basis of low level laser therapy. Laser 3, 19, 1991 Young SR, DysonM and Bolton P, Effect of Light on Calcium Uptake by Macrophages, Laser Therapy, 2, 53-57, 1990

  The problem to be solved is to clarify the mechanism of light action on the immune system, and to provide an optimal light source means and a method for constructing it.

  The main object of the present invention is to clarify the light irradiation conditions exerted on the immune system by animal experiments, and to establish a laser device, light guide / irradiation means, and irradiation conditions as the optimum light source means.

  The immune activation device of the present invention reduces the effect of lowering the immune system after surgical treatment, anticancer drug treatment, and radiation treatment, which are currently the main cancer treatment methods, and is in addition to other immune activation methods. As a result, the cancer treatment method is safety and effectiveness synergistically and synergistically, and when used alone, there is an advantage that it has an effect of reducing tumor growth.

When irradiating light to living cells, it is necessary to consider the interaction between light and cells. It is well known that the interaction in this case is related to irradiation energy (J / cm 2), irradiation time, and irradiation power density (W / cm 2). As for the irradiation energy and output density to the cells, how much laser light is transmitted into the living body is defined by the difference in reflectance, absorption coefficient, scattering coefficient, etc. on the living body surface.
In general, in the visible light region, hemoglobin and melanin in blood are strongly absorbed, and scattering is generated in addition to the absorption of transmitted light, which complicates the irradiation light action. In general, light in this wavelength range is transmitted into the living body. On the other hand, infrared light of 1 μm or more has less absorption and scattering action of hemoglobin, melanin, etc., and on the contrary, absorption which seems to be due to OH acts strongly. As a result, the light transmission distance becomes very short and the action of light is limited to the skin surface.
Young SR et al. Have studied the interaction between cells and light, particularly cell activation. In general, effective conditions seem to exist in the following range.
Energy density: 4-8J / cm2
Irradiation conditions: 5-100Hz
Of course, this value is general and approximate. According to said patent on the method of stimulating the immune system, the optimal irradiation conditions are described below.
Wavelength: 1.917nm
Frequency: 7.5Hz
Irradiation energy density: 14 J / cm2 (output 200 mW, frequency 7.5 Hz, on
: 25ms, irradiation area 10mm circle, irradiation time 5 minutes)
This irradiation energy density is a maximum value, which is about one tenth of the minimum value. Which condition is selected depends on the treatment target. In any case, it can be assumed that the irradiation conditions for the cells are in the above range.

  The relationship between light absorption and scattering on the skin is an important parameter when considering the interaction between light and a living body. In the general visible light range, oxygenated hemoglobin, reduced hemoglobin, and melanin are known as main absorbents in vivo. When considering the light irradiation effect on the immune system, stimulation of immune cells by transmitting light to the inside of the living body and stimulation of immune cells directly under the surface of the living body can be considered. However, since immune cells in the living body mainly exist in blood, lymphatic vessels, digestive tracts, lungs, other organs, and surrounding lymphoid tissues, it is necessary to transmit light to the inside. Visible light is useful for penetrating deeply into the living body. In this case, it is absorbed by hemoglobin, oxygenated hemoglobin, and melanin pigment in the blood, so that the target immune cells can be irradiated with light. There is doubt whether there is.

  On the other hand, Langerhans cells (dendritic cells) are recently attracting attention as cells involved in immunity. The Langerhans cells are present directly under the skin and are widely distributed on the body surface. The functions of the Langerhans cells have been revealed to play an important role in the immune system in the body, such as antigen presentation by capturing bacteria, viruses, etc. that have entered from outside the skin, induction of immune responses, and induction of T cells. is there. In the present invention, the Langerhans cells are activated by light irradiation or increase the Langerhans cells by cell differentiation, and as a result, the immune system in the body is activated.

  Dendritic cells are a type of immune cell with many long arms and are found in various tissues. Dendritic cells that are particularly abundant in the skin and mucous membranes and distributed in the epidermis layer of the skin are called Langerhans cells. These dendritic cells find very few antigens, capture them efficiently, and present them. The dendritic cells presenting the antigen ride on the blood to the spleen and to the lymph nodes together with the lymph, mature, and present the antigen-bearing MHC molecule to naive T cells. Dendritic cells that have become immunostimulatory cells present antigen to undifferentiated T cells and activate helper T cells. Activated helper T cells produce lymphokines, induce antibody production in B cells, activate macrophages, or turn cytotoxic T progenitor cells into cytotoxic lymphocytes.

  The distribution of dendritic cells in human skin has already been investigated in studies of delayed hypersensitivity reactions, and usually skin Langerhans cells present on the surface of the cortex migrate to the epithelium of the epidermis during the course of the reaction. It is said that it will be stripped and detached from the surface of the epidermis. Within 24 hours after sensitization, helper T cells, monocytes, and Langerhans cells gather around the skin blood vessels, and antigen presentation is performed. Helper T cells presented with antigen interact with immune cells such as antibody-producing B cells and cytotoxic T cells that attack infected cells. As described above, Langerhans cells have a mechanism for collecting antigens around helper T cells after gathering around a subcutaneous blood vessel after receiving some kind of stimulation. Therefore, it is hypothesized that Langerhans cells are stimulated by light irradiation, and as a result, produce the same action as antigen presentation.

  Although it has been clarified that cells are activated by light irradiation as described in the above-mentioned 0008, it is also valid that Langerhans cells are similarly activated by light irradiation.

  When light is irradiated on a living body, the relationship between the irradiation light wavelength and the bioabsorbable substance is important in order to absorb light energy within a certain range directly under the skin. The requirements are (1) no absorption by blood and (2) no absorption by melanin. With such absorption, the energy of irradiation light to the surface of the living body necessary for acting on immune cells increases, and problems such as enlargement of the device and skin damage arise. Therefore, in order to act on the Langerhans cells, it is necessary to select the irradiation light wavelength so that the light irradiation effect is obtained at least 1 mm or less from the skin surface. As the light wavelength for producing such an effect, infrared light of 1 μm or more is selected.

  In a generally known Nd: YAG laser beam, a skin attenuation coefficient of 5.7 cm-1 is obtained. Substituting this value into the Lambert-Beer equation to obtain the distance at which the irradiation energy drops to 10% of the incident light is approximately 4 mm, and the light transmission intensity is too high in this wavelength region. On the other hand, the absorption coefficient at a wavelength of 1.9 μm is about 150 cm−1, but according to a separate experimental result, it is 75 cm−1. When light transmission is calculated in the same manner as described above based on an absorption coefficient of 150 cm−1, it is about 0.15 mm. Since the thickness of the epidermis is about 0.1 mm, it satisfies the condition that it acts in the epidermis where Langerhans cells are present but does not act on the deeper subcutaneous tissue. In addition, the biological action due to this wavelength is characterized in that there is almost no light scattering in the skin and that it has an absorption characteristic of transmitting in a direction perpendicular to the irradiation surface. It is known that the light wavelength having a large scattering coefficient in the vicinity of the irradiated region is generally 1 μm or less, but it is pointed out that the light intensity in the vicinity of the irradiated region increases when the scattering coefficient is large. Therefore, there is a risk of damaging the cell, not the cell activity. Therefore, in order to activate immune cells safely, it is necessary to select a wavelength that does not cause scattering in the body tissue by irradiation light. From this point of view, light in the 1.9 μm band is suitable.

  As described in 0015 above, the irradiation energy density that brings about cell activation is generally 4-10 J / cm 2. Although it is unclear whether this irradiation energy density is applied to all cell activations, it is an index for setting conditions. As an example for this, when the irradiation site area is 5.5 mm in diameter and the laser light intensity is 100 mW, the irradiation energy value at 0.3 mm from the surface is 1.4 J / cm 2, and the cell action range is 0. It can be seen that a sufficiently possible condition is satisfied within 3 mm.

On the other hand, absorption in a red light region such as He—Ne laser light is about 10 cm −1, and absorption mainly by melanin pigment is dominant. On the other hand, the absorption coefficient of oxyhemoglobin and deoxyhemoglobin is almost an order of magnitude higher than that of melanin. As a result, it can be determined that the light action distance reaches 5 mm or more. In addition, in this wavelength range, reflection from the inside of the living body is large, and even if it varies depending on the skin color, it is about 50%. As a result, the light irradiation energy required for the action increases, and at the same time, it penetrates deeply into the living body. As a result, it seems difficult to bring about interaction with the target immune cells.
From the above schematic explanation, wavelength selection is an important condition for the action distance transmitted by laser irradiation in order to act on Langerhans cells immediately below the surface. The light wavelength range having such a light working distance is 1.4 to 2.5 μm.

  In order to effectively carry out the interaction with cells by light irradiation, it is necessary to perform light irradiation on Langerhans cells distributed almost in the subcutaneous epidermis layer. In general, in the case of non-coherent light, the incident angle of the irradiation light varies depending on the location in the irradiation site, and the transmission angle is dispersed inside the living body according to the incident angle. For this reason, the action with cells differs from cell to cell even if the incident light intensity is the same. For this purpose, it is important to use coherent light in order to keep the action and effect constant, but it is obvious that this does not mean that non-coherent light cannot be used.

  In order to realize a predetermined action, the light irradiation area at the irradiation site is an important condition. When laser light acts on Langerhans cells present under the skin, it depends on how many cells are distributed within the area of the irradiated site. Even if the Langerhans cell distribution exists spatially in the epidermis, it is considered to exist in parallel rather than in multiple layers when viewed functionally. Accordingly, the effect depends on the irradiation site area. As a result, it is necessary to satisfy a condition that a predetermined irradiation energy density can be ensured and thermal damage to the skin does not occur. It is important to select a 3-15 mm diameter as the irradiation area for this purpose.

  Pulsed light irradiation is known as a means for improving the efficiency of cell activation by light irradiation. In order to pulse the irradiation light, generally, a method of pulsing the light source itself or a chopper capable of intermittently shielding light between the light source emitting end and the light guide unit is known. ing. In order to reduce the size of the apparatus, a method of pulsing on the light source side is advantageous. In the case of pulsed light, it may be necessary to change the pulse duty. In particular, in order to efficiently perform the cell action, it is clinically necessary to optimize each irradiation site targeted for the irradiation condition. For this purpose, it is important that the laser output setting is varied by pulse width modulation.

  As described in the paragraph 0015, irradiation time, irradiation energy, and irradiation intensity are known as parameters that define light and biological action. One condition that brings about cell activation is an energy density of 4-8 J / cm 2, but the irradiation time is not sufficiently known. Under pulse irradiation conditions, the pulse width modulation method can change the average irradiation energy without changing the peak irradiation value when the pulse duty is changed, but in the case of mechanical pulsing, the duty is Fixed. Therefore, in the case of the mechanical pulsing method (chopper method), the variable output needs to continuously change the excitation energy. In this case, both the peak irradiation intensity and the irradiation energy will fluctuate.

  The difference between the above-described pulsing methods will be described in detail. Experimentally, if the optimal conditions for cell activity are 10 J / cm 2 as the irradiation energy density and the irradiation intensity is 0.127 W / cm 2 (laser output 100 mW, irradiation beam diameter 10 mm), the irradiation time is 79 seconds. This condition requires that the irradiation time is doubled or the output is doubled when a mechanical chopper (duty 50%) is used. However, in this case, if the output is changed, the irradiation intensity will deviate from the optimum condition. Further, when the irradiation energy density is changed, there is a drawback that the irradiation intensity is inevitably changed. On the other hand, in the case of the pulse width modulation method, the laser irradiation value is constant even when the output condition is changed. Therefore, to obtain a predetermined irradiation energy, only the irradiation time is changed, and the deviation from the optimum condition does not occur.

  As a device that can oscillate coherent light with a wavelength range of 1.4-2.5μm, it is generally a colored glass laser (oscillation wavelength: 1.4-1.8μm wavelength variable) with NaCl (F2 +) H, CO2 +: MgF2 laser (oscillation wavelength) 1.63-2.50 μm wavelength tunable) and Er3 +: glass “fiber laser” (oscillation wavelength: 1.522-1.575 μm, 1.590-1.603 μm wavelength tunable) laser excited by semiconductor laser ing.

A laser device with a wavelength of 1.9 μm is preferably a fiber laser that is small in size, has good coupling efficiency to a light guiding fiber, and excites a semiconductor laser with excellent operability. A laser oscillation can be obtained by exciting a Tm-doped fluoride fiber using a semiconductor laser having a wavelength of 800 ± 10 nm as an excitation laser. The fiber laser is generally continuous wave light. In the present invention, the excitation semiconductor laser drive circuit is provided with a pulse width modulation circuit for pulse oscillation.

FIG. 1 shows the configuration of the present invention. In FIG. 1, 1 is an excitation semiconductor laser, 2 is a semiconductor laser drive circuit, 3 is 800 nm transmission, 1900-2000 nm reflection mirror, 4 is Tm-doped fluoride fiber, 5 is a wavelength selection device, total reflection mirror, grating, Consists of lenses. 6 is an optical system for transmitting laser light from the Tm-doped fluoride fiber to the light guide fiber, 7 is a light guide fiber, and 8 is an irradiation handpiece. In the semiconductor laser drive circuit, a pulse width modulation circuit is provided, and the following relationship is established among the laser beam output peak value Pmax, the laser beam average output value Pave, the pulse ON time Ton, and the pulse OFF time Toff. .

Pave = Pmax × Ton / (Ton + Toff)

The pulse ON time can be set to be variable from 1 ms to 250 ms, and the pulse stop time can be varied from 1 ms to 250 ms. By oscillating the semiconductor laser by such a drive circuit and exciting the Tm-doped fluoride fiber, a pulsed laser beam having a wavelength of 1.95 μm was obtained. In addition, the oscillation wavelength could be adjusted in the range of 1.85 to 2.10 μm by adjusting the length of the Tm-doped fluoride fiber. In the case of an excitation semiconductor laser output of 0.7 W, a 1.95 μm laser output of 200 mW is obtained (maximum value). When the pulse repetition rate can be set to 10Hz and the duty is set to 50%, Ton and Toff are set to 50ms respectively, a laser beam with a pulse repetition rate of 10Hz, a laser output of 100mW, and a wavelength of 1.9μm can be obtained. It was.
In the confirmation of immune activation by animal experiments of this device, the laser was operated under the above set conditions. Further, in order to confirm the change in only the laser light output value, an output variable mechanism was used to oscillate an output of 55 mW without changing the pulse condition, and an animal experiment was conducted under this set condition. When the irradiation area is set to a diameter of 5.5 mm and the irradiation time is 10 minutes, the incident energy density to the skin is 63 J / cm 2 and 126 J / cm 2, respectively. This value is reasonable considering reflection on the surface of the skin and absorption in the skin, but it has not been confirmed whether it is the optimum value. Since the irradiation area may vary depending on the target site rather than being fixed, the irradiation site may be varied. Such a case can be dealt with by varying the distance from the handpiece emitting end to the skin irradiation surface or by providing the handpiece with a zoom optical system.

Using the device according to Example 1, an animal experiment was conducted to confirm whether this device brings about immune activation, and its effect was confirmed.
In this confirmation experiment, the behavior of immune cells when tumor cells are transplanted under normal immunity is experimentally clarified. Under the condition that the tumor was naturally formed in its body. It should be noted that the behavior of tumor cells by light irradiation was not examined. Therefore, the following points are problems in this experiment.
1) What is the target immune cell behavior when cells that are foreign to the body are transplanted? 2) What are the immune cells that cause changes in tumor cells, and how these cell behaviors change with light irradiation? 3) How does the actual transplanted tumor cell change due to light irradiation? 4) Observation of the morphological change

As experimental animals, C3H / HeNCrj mice (8-12 weeks old, female) were used for both the abdominal cavity passage mice and tumor bearing mice. Tumor cell transplantation in the production of tumor-bearing mice was performed by collecting cells from the abdominal cavity of the mouse, washing with Ringer's solution several times, and preparing a cell suspension of 106 tumor cells / 0.1 ml. The transplanted site was subcutaneously shaved mouse buttocks, and 0.1 ml of cell suspension was injected into the site.
Laser beam irradiation is performed before tumor cell transplantation group (Group 2), post-transplant irradiation group (Group 3), and pre-transplant irradiation group (Group 4).
The tumor cells were transplanted, and a comparative study was conducted in the four experimental groups of the non-irradiated group of laser light and the control (Group 1). Pre-transplantation irradiation was performed for 3 days and post-transplantation irradiation for 7 days. All experimental groups were sacrificed on the 8th day after transplantation. The laser light irradiation site was a comparative study of two methods: a tumor transplant site and a non-tumor site of the shaved buttocks. The irradiation output to the non-tumor part was 100 mW.

  When the temperature of the tumor part increases to 43 degrees or more, the tumor cells are selectively killed. Therefore, in order to monitor the rise of the skin temperature at the irradiated site by laser irradiation, the temperature rise behavior at a laser output of 100 mW is observed. Measurement was performed by infrared thermography (FAIRIS, Nikon).

  Tumor tissues were collected from sacrificed mice, fixed with about 4% paraformaldehyde solution, and hematoxylin-eosin (HE) -stained tissue specimens were prepared, and tumor tissues were observed. HE staining is a method for staining cell nuclei and cytoplasm in order to observe tissue morphology, and is the most standard tissue staining method and is suitable for tissue analysis.

In order to clarify the behavior of immune cells by light irradiation, analysis using a cell analyzer was performed. There are two types of dyes used. One of them is FITC (Fluorescein Isothiocyanate), which absorbs 488 nm excitation light and mainly emits fluorescence at 530 nm. If FITC is labeled in advance on the antibody, the amount of antibody to be bound varies depending on the amount of antigen present on the cell surface. As a result, the fluorescence intensity of FITC varies, so that it exists on the surface of the cell. The amount of antigen can be estimated. The other one uses PE (Phycoerythrin), which mainly emits fluorescence at 585 nm. The collected spleen cells are fluorescently labeled with these two antibodies, and a cell analyzer (
EPICS Elite, BECKMAN COULTER). The antibodies used are anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD28 and anti-CD80 antibodies. CD3 is part of a large complex containing the T cell receptor (TCR), one of the immune cells, and expresses mature T cells and thymocytes. CD4 is a co-receptor in MHC class II-restricted antigen stimulation activation. CD8 molecules are found in T cell subsets of peripheral blood lymphocytes and CD antigens are also found in subsets of NK cells. As a co-receptor, CD8 is involved in MHC class I-restricted antigen recognition of the T cell receptor and is used as a cytotoxic T cell marker. CD25 is present on activated T and B cells and is also found on activated macrophages and T cell clones. CD28 is expressed on all T cells, mostly on CD3 positive T cells in humans, and CD28 antigen is frequently expressed on CD4 positive T cells. This antigen provides a major costimulatory signal in T cell activation. CD80 produces a costimulatory signal for T cell activation upon binding to CD28. These antibodies can be used to experimentally confirm immune activation by laser irradiation. In the experiment, cell adjustment was required, but the following method was used. For cell preparation, a spleen cell suspension was prepared from the spleen collected from C3H / HeNCrj mice (8-12 weeks old, female), and the number of cells collected was counted with a counter. A 10 μm fluorescently labeled monoclonal antibody was added to 1 × 10 6 cells and left for 40 minutes while cooling in the dark with ice. After the reaction, cell expression was analyzed with a cell analyzer.

  The measurement of the skin temperature of the irradiated part at the time of laser beam irradiation with 100 mW output was performed at the start of irradiation, 2, 4, 6, 8, and 10 minutes after the start. As a result, the temperature immediately after irradiation was 34.4 ° C., and the maximum temperature was 39.0 ° C. at 10 minutes after the start of irradiation.

  The tumor shape after tumor cell transplantation as described in the above item 0034 was measured, and the weight was calculated by the method of converting to the weight. The results are shown in FIG. 2a for a laser irradiation output of 55 mW, in FIG. 2b for a laser output of 100 mW, and in FIG. 2c for a tumor growth curve when the non-tumor part is irradiated with a laser output of 100 mW. Although the tumor of the irradiation group tended to not grow greatly visually, the result of T test showed no significant difference from the analysis of the tumor weight. The reason for this was found to be due to tumor reduction factors that could not be determined by apparent shape measurement.

The tumor tissue observation result by HE staining is the non-irradiated group
FIG. 3 shows tumor tissues of 1), a tumor part irradiation group (Group 3) (output 100 mW), and a non-tumor irradiation group (Group 3) (output 100 mW). FIG. 3 b and FIG. 3 c are tumor-bearing mouse tumors that were irradiated with laser light before and after tumor cell transplantation. The epidermis side is shown from the right side of the figure, and the abdominal side appears as it goes to the left side. Observation with a microscope was set at an objective lens magnification of 2 times and an eyepiece lens magnification of 2.5 times. From the specimen observation result, in the tumor of the tumor irradiation group, a wide light red area appears on the abdominal side of the tumor area, the tumor cells are damaged, the nucleus disappears, and it becomes a connective tissue. On the other hand, even in the tumor tissue, the part close to the site of connective tissue shows a state leading to the disappearance of the nucleus. The same was observed in the tumor tissue with non-tumor irradiation.
When calculating the ratio of necrosis or connective tissue to the size of the entire tumor observed on the tissue specimen, it was almost 0% for non-irradiated tumor (a in FIG. 3), and about 59.7% for tumor irradiation ( In FIG. 3 b), irradiation to the non-tumor part was about 61.8% (FIG. 3 c), and the tumor cells (tissue) were substantially reduced.

The result of having analyzed CD expression of the immune system cell contained in the spleen cell collect | recovered from the tumor bearing mouse | mouth in the laser beam irradiation to a tumor part is shown. The results at an output of 55 mW and 100 mW are shown in FIGS. 4a and 4b, respectively. The result of CD expression of immune system cells of the tumor-bearing mouse in the irradiation output to the non-irradiated part is shown in FIG. From the results of this experiment, the analysis result at an irradiation output of 55 mW shows a difference in the expression of anti-CD3 antibody depending on the presence or absence of tumor cell transplantation (P <0.01 *), and the expression of anti-CD antibody is low in the presence of tumor. Met. In Groups 1, 2, 3 and 4, there was no difference in the expression of mature T cells, helper T cells and cytotoxic T cells. From the analysis result at an irradiation power of 100 mW, a significant difference is similarly observed in the expression of anti-CD3 antibody depending on the presence or absence of tumor cell transplantation, and the expression of anti-CD3 antibody is low in the presence of a tumor.
There was a significant difference in anti-CD3 antibody expression between 1 and Group 3 (P <0.01 *). In anti-CD4 and 28 antibodies, Group 2 and Group 4
There was a significant difference in comparison with 1 (P <0.01 *, P <0.03 **), and both were high (P <0.03 **).
Similar to the analysis result of the irradiation to the tumor part from the analysis result at the irradiation power of 100 mW in which the non-tumor part was irradiated, the anti-CD3 antibody was compared to Group 3 (P <0. 01 *), Group 2 for anti-CD8 antibody (P <0.03 *), Group for anti-CD28 antibody
There was a significant difference between 2 and Group 4 (P <0.01 *). However, in the anti-CD4 antibody, no significant difference was observed in Groups 2, 3, and 4.

The above experimental results can be summarized as follows.
1. Laser irradiation before tumor cell transplantation was effective in increasing helper T cells and cytotoxic T cells.
2. The Langerhans cells were stimulated by laser light irradiation, activated, transmitted signals to helper T cells, and stimulated to cytotoxic T cells.
3. The expression of mature T cells was significantly increased by laser light irradiation after transplantation.
4). Helper T cells and cytotoxic T cells were enhanced more effectively with higher irradiation power.
5). Even when the non-tumor part was irradiated with laser, the same result as that of the tumor part was obtained.
6). Under all irradiation conditions. There was a slight increase in the activation of B cells, T cells and macrophages between the tumor cell non-transplanted group and the tumor cell transplanted group.
7). In contrast to this, in the comparative experiment in which the He—Ne laser light was irradiated, the light irradiation did not cause a significant difference with respect to the immune system, unlike the experimental result obtained at 1.9 μm. In addition, about this experimental result, a detail is abbreviate | omitted and only a conclusion shall be shown.

From the detailed experimental results described above, it was shown that the immune system was activated by 1.9 μm band laser light irradiation. It is clear that the same result can be obtained if the oscillation wavelength of the laser beam is such that the incident energy intensity can be reduced to about 10% at 1 mm or less from the skin surface of the irradiation site.
In addition, it is clear from the distance of the irradiation light that the Langerhans cells present in the skin epidermis are directly involved in the activation of immune cells, but how the Langerhans cells are activated. Regarding the mechanism of action, it is currently assumed that the same effect as that of Langerhans cells captured antigens is caused by light action, but the possibility of increase in the number of Langerhans cells due to differentiation of monocytes as precursor cells Cannot be denied. Regardless of the mechanism of action, the usefulness of the present device is clear from the fact that Langerhans cells are substantially activated, the in vivo immune system is activated, and the immunity is improved.

  The present invention has the advantage that, when used alone, it has the effect of reducing tumor growth, and at the same time, the combined use of other immunoactive agents, etc., additively and synergistically brings the safety and effectiveness of cancer treatment methods. is there.

It is explanatory drawing which shows the whole structure of this apparatus. (Example 1) (A) is explanatory drawing which shows the tumor growth curve at the time of output 55mW irradiation to a tumor part. (Example 2) b is explanatory drawing which shows the tumor growth curve at the time of the output of 100 mW to a tumor part. (Example 2) c is explanatory drawing which shows the tumor growth curve at the time of the output of 100 mW to a non-tumor part. (Example 2) (A) is explanatory drawing which shows the tumor tissue sample in a non-irradiation group. (Example 2) b is explanatory drawing which shows the tissue sample at the time of the output of 100 mW to a tumor part. (Example 2) c is an explanatory view showing a tissue specimen at the time of irradiation with an output of 100 mW to a non-tumor part. (Example 2) (A) is explanatory drawing which shows the immune system cell CD expression of the tumor bearing mouse | mouth which is 55mW output to a tumor part. (Example 2) b is explanatory drawing which shows the immune system cell CD expression of the tumor bearing mouse | mouth with the output of 100 mW to a tumor part. (Example 2) c is an explanatory view showing immune system cell CD expression in a cancer-bearing mouse with 100 mW output to a non-tumor part. (Example 2)

Explanation of symbols

The code | symbol description of drawing in Example 1 is shown.
DESCRIPTION OF SYMBOLS 1 Semiconductor laser for excitation 2 Semiconductor laser drive circuit 3 800 nm transmission, 1900-2000 nm reflection mirror 4 Rare earth element doped fluoride fiber 5 Wavelength selection device 6 Coupling optical system 7 Light guide fiber 8 Irradiation handpiece

Claims (7)

  Immune activity characterized by having laser light oscillation means having an oscillation wavelength in the wavelength range of 1.4 to 2.5 μm, pulse oscillation means, and light guide and irradiation means that can irradiate the skin surface of a living body Device   2. The immune activation device according to claim 1, wherein the oscillation wavelength is variable in a range of 1.4 to 2.05 μm.   3. The immune activation device according to claim 1, wherein the oscillation wavelength is 1.85 to 2.05 μm, and the laser oscillation means is a fiber laser.   2. The laser oscillation means for oscillating an oscillation wavelength of 1.85 to 2.05 μm, wherein the laser excitation means is a semiconductor laser, and the means for pulsing the laser oscillation is constituted by a pulse width modulation system. The immune activation device according to 1 to 3   5. The immune activity according to claim 1, wherein the laser light pulsing means is a pulse width modulation system, and the pulse ON time is 1 to 250 ms and the pulse OFF time can be arbitrarily set to 1 to 250 ms. Device   The immunity according to any one of claims 1 to 5, wherein the irradiation energy density at the irradiation site surface is 10-200 J / cm2, the pulse repetition rate is 2-500 Hz, and the diameter of the irradiation part is 3-15 mm. Activation device The immune activation device according to any one of claims 1 to 6, which can be used in combination with an immunoactive drug, an immunoactive functional food, and the like.

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