JP2008162983A - Method for constructing functional neural network using projector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology for providing a universal regenerative technique. <P>SOLUTION: A composition for tissue regeneration is provided, comprising a cell having such a property as to be variable in state by photoreaction. A system for conducting tissue regeneration at a desired place is also provided, comprising (A) the cell and (B) a light source providing light on such a desired place. This system contributes particularly to realizing the regenerative medicine for recovering the functions of patients who suffered tissue damage such as those who suffered neural damage. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a functional reproduction method using light. In another aspect, the present invention relates to a novel experimental system using light. The present invention also relates to a biological control method using light.

  For regenerative medicine to restore the function of a patient who has been damaged in a nerve (brain), (1) to create and transplant a nerve cell, and (2) to construct a transplanted functional nerve cell network. Two consecutive techniques are required. Even if one of them is missing, the function of these patients cannot be restored quickly and effectively.

However, research on technology (1) has been energetically promoted and results have been achieved, but technology (2) remains completely undeveloped, and the current state is that transplanted neurons have independently built a network. It remains passive, expecting to do. In order to quickly and effectively restore patient function, a technology that constructs a functional network by projecting nerve cell axons onto target cells, forming synapses, and connecting nerve cells together (2) is required. Non-Patent Document 1 does not describe such a technique.
Ishizuka et al. , Neurosci. Res. 54 (2006) 85-94

  An object of the present invention is to provide a technique for providing a free reproduction technique. In particular, it contributes to the realization of regenerative medicine for recovering the function of a patient who has suffered tissue damage such as a patient who has suffered nerve damage.

  In order to achieve this object, the present inventor has focused on “light” as a stimulus source for constructing a functional nerve cell network. The intensity, intensity width (pattern), type (that is, wavelength or color), and spatial arrangement of light can be freely controlled. By utilizing a projector whose output can be controlled by a computer, it is possible to create “a variety of strengths, patterns and colors, as well as fine spatio-temporal patterns of stimuli with temporal changes”. On the other hand, for the nerve cells on the side to be stimulated, the nerve cells in which a cation channel that responds to light is forcibly expressed (Non-Patent Document 1 = Ishizuka et al., Neurosci. Res. 54 (2006) 85-94) are used. use. As described above, by combining the “computer output projector and the cation channel forced expression nerve cell”, it is possible to reconstruct an arbitrary nerve network at the nerve damage site of the patient. Such fine stimulus control cannot be realized by a solution or electric stimulus that has been generally used.

  First, a nerve cell in which a protein that responds to light (for example, channel rhodopsin-2) is forcibly expressed is prepared (see Non-Patent Document 1 for a method). These neurons become excited in response to blue irradiation, and as a result, nerve axon extension and synapse formation are promoted. That is, by irradiating blue, a nerve cell can be connected and a functional nerve cell network can be constructed. Next, the above nerve cells are injected into a site necessary for restoring the nerve function of the patient. Finally, a computer-generated light pattern is irradiated onto a necessary site using a computer output projector to activate nerve cells and connect nerve cells to induce a functional nerve network.

  FIG. 1 illustrates the present invention. First, for example, a nerve cell in which ckmelrhodopsin · 2 that is a cation channel that is opened with blue light is forcibly expressed is created (see Non-Patent Document 1 for an exemplary method).

  These neurons become excited in response to blue irradiation, and as a result, nerve axon extension and synapse formation are promoted. That is, by irradiating blue, a nerve cell can be connected and a functional nerve cell network can be constructed. Next, the above nerve cells are injected into a site necessary for restoring the nerve function of the patient. Finally, a computer-generated light pattern is irradiated onto a necessary site using a computer output projector to activate nerve cells and connect nerve cells to induce a functional nerve network.

  An example of an optical system necessary to realize the present invention is shown in FIG.

Accordingly, the present invention provides the following.
(1) A composition for tissue regeneration comprising cells having a property of changing state by a photoreaction.
(2) The composition according to item 1, wherein the change in the state is a change appropriate for the tissue regeneration.
(3) The composition according to item 1, wherein the change in the state is selected from the group consisting of cell death, morphological change, migration, change in cell-cell interaction, differentiation and proliferation.
(4) The composition according to item 1, wherein the cell is a cell used for tissue regeneration.
(5) The composition according to item 1, wherein the cell is a nerve cell.
(6) The composition according to item 1, wherein the cell is a cell which has been converted to have the above-mentioned property by converting a cell having the above-mentioned property in nature or not having the above-mentioned property in nature.
(7) The composition according to item 1, wherein the cell is transiently transformed so as to have the above property.
(8) The composition according to item 6, wherein the conversion is achieved by transformation using a nucleic acid encoding a photoresponsive protein.
(9) The composition according to Item 8, wherein the photoresponsive protein is a G protein-coupled photoreceptor, a cation channel or an anion channel.
(10) The photoresponsive protein is a cation channel, and the cation channel is channel rhodopsin-1, channel rhodopsin-2, opsin, red opsin, green opsin, blue opsin, UV opsin or pinopsin. Item 9. The composition according to Item 8.
(11) A system for performing tissue regeneration at a desired location,
A system comprising: A) a cell having a property of changing its state by a light reaction; and B) a light source that provides light to the desired location.
(12) The system according to Item 11, wherein the change in the state is a change appropriate for the tissue regeneration.
(13) The system according to Item 11, wherein the change in the property is selected from the group consisting of cell death, morphological change, migration, change in cell-cell interaction, differentiation and proliferation.
(14) The system according to Item 11, wherein the cell is a cell used for tissue regeneration.
(15) The system according to Item 11, wherein the cell is a nerve cell.
(16) The system according to Item 11, wherein the cell is a cell that has been converted to have the above-mentioned property by converting a cell that has the above-mentioned property in nature or does not have the above-mentioned property in nature.
(17) The system according to Item 11, wherein the cell is transiently transformed so as to have the above property.
(18) The system according to Item 16, wherein the conversion is achieved by transformation using a nucleic acid encoding a light-responsive protein.
(19) The system according to Item 18, wherein the photoresponsive protein is a G protein-coupled photoreceptor, a cation channel or an anion channel.
(20) The photoresponsive protein is a cation channel, and the cation channel is channel rhodopsin-1, channel rhodopsin-2, opsin, red opsin, green opsin, blue opsin, UV opsin or pinopsin. Item 19. The system according to Item 18.
(21) The system according to Item 11, wherein the light source is a projector.
(22) The system according to Item 11, wherein the light source is a projector whose output can be controlled by a computer.
(23) The system according to item 11, wherein the light source can form a stimulus pattern having a parameter change selected from the group consisting of intensity, pattern, color, and time.
(24) The system according to Item 11, wherein the light source has a shape suitable for irradiation at the desired location.
(25) A system according to item 24, wherein the shape is a shape suitable for irradiation during surgery.
(26) The system according to Item 11, wherein the light source realizes irradiation specific to the desired place.
(27) A system according to item 26, wherein the light source includes means for realizing a multiphoton excitation method.
(28) The multiphoton excitation method is realized by exciting the reaction of the photoreactive protein by simultaneously irradiating two light beams having twice the wavelength, which is a photon having half the energy, The described system.
(29) A system according to item 27, wherein the means includes an infrared laser.
(30) A system according to item 29, wherein the means can irradiate light having a wavelength of 750 nm to 3000 nm.
(31) A system according to item 27, wherein the means is a pulsed laser that emits light intermittently.
(32) The system according to item 27, wherein the photoresponsive protein is channelrhodopsin-2, and the means is capable of irradiating a strong pulse laser near 920 nm.
(33) A system for performing tissue regeneration so as to have a desired pattern,
A system comprising: A) a cell having a property of changing its state by a photoreaction; B) a light source corresponding to the photoreaction; and C) means for processing the light source into a desired pattern.
(34) The system according to item 33, wherein the system has one or more features according to items 12 to 32.
(35) A system according to item 33, wherein the means for processing the light source into a desired pattern is selected from the group consisting of an infrared laser, a laser scan, a virtual mask, a real mask, a filter, and a projector.
(36) Item 33, wherein the tissue is a nerve, the cell is a nerve cell transformed with channelrhodopsin-2, the light source is a blue light source, and the means is a nervous system pattern. The described system.
(37) The system according to Item 33, wherein the nerve cell is activated to form a nerve network by irradiation with blue light.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  The present invention provides a method for constructing a functional nerve network by connecting regenerative medicine, particularly nerve cells, and is also effective for the following purposes. That is, it can be used for any other transplanted organ, for example, as a method of connecting the transplanted skin and the nerve of the patient.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

(Regenerative medicine)
As used herein, “regeneration” refers to a phenomenon in which a remaining tissue grows and is restored when a part of the tissue of an individual is lost. Depending on the tissue species between animal species or in the same individual, the degree and mode of regeneration varies. Many human tissues have limited regenerative ability, and complete regeneration cannot be expected if they are largely lost. In a large injury, a tissue with strong proliferative power different from the lost tissue proliferates, and incomplete regeneration may occur in which the tissue is regenerated incompletely and the function cannot be recovered. In this case, a structure made of a bioabsorbable material is used to prevent the invasion of a tissue having a strong proliferation ability into the tissue defect portion, thereby securing a space in which the original tissue can grow, and further, a cell growth factor. Regenerative medicine is performed to replenish the natural tissue and enhance the regenerative capacity of the original tissue. Examples of this include regenerative medicine of cartilage, bone, heart and peripheral nerves. It has previously been thought that cartilage, nerve cells and myocardium are incapable or remarkably incapable of regeneration. In recent years, the existence of tissue stem cells (somatic stem cells) having the ability to differentiate into these tissues and the ability to self-proliferate has been reported, and expectations for regenerative medicine using tissue stem cells are increasing. Embryonic stem cells (ES cells) are cells that have the ability to differentiate into all tissues. Regeneration of complex organs such as kidneys and livers using these cells has been attempted but has not been realized.

A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism, surrounded by a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. The number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is used as a tissue slice, and hematoxylin-eosin (HE) staining is performed, whereby the extracellular matrix (for example, elastin or collagen) and the nucleus derived from the cells are dyed with a dye. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μm × 200 μm) can be estimated and counted. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or such as cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line Examples include but are not limited to cell mixtures. As the cells, for example, primary cultured cells can be used, but subcultured cells can also be used. Preferably, cells that have passed through passage 3 to 8 are used. In the present specification, the cell density can be represented by the number of cells per unit area (for example, cm ² ).

  As used herein, “stem cell” refers to a cell having a self-renewal ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells can preferably be embryonic stem cells or tissue stem cells.

  When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, and nervous system. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells, mesenchymal stem cells (for example, fat-derived, cartilage-derived, bone-derived, bone marrow-derived). Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells.

  In the present specification, “somatic cells” refers to all cells that are cells other than germ cells such as eggs and sperm and that do not directly transfer the DNA to the next generation. Somatic cells usually have limited or disappeared pluripotency. The somatic cells used in the present specification may be naturally occurring or genetically modified.

  Cells can be classified into stem cells derived from ectoderm, mesoderm and endoderm by origin. Cells derived from ectoderm are mainly present in the brain and include neural stem cells. Cells derived from mesoderm are mainly present in bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like. Endoderm-derived cells are mainly present in organs, and include liver stem cells, pancreatic stem cells, and the like. As used herein, somatic cells may be derived from any mesenchyme. Preferably, a mesenchymal cell can be used as the somatic cell.

  As used herein, “isolated” means that the substances naturally associated with it in a normal environment are at least reduced, preferably substantially free. Thus, an isolated cell, tissue, etc. refers to a cell that is substantially free of other materials (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment. When referring to a tissue, an isolated tissue refers to a tissue that is substantially free of substances other than the tissue (eg, scaffolds, sheets, coatings, etc.).

  As used herein, “established” or “established” cells maintain certain properties (eg, pluripotency) and the cells continue to grow stably under culture conditions. State. Thus, established stem cells maintain pluripotency.

  As used herein, “non-embryonic” means not derived directly from an early embryo. Therefore, cells derived from body parts other than early embryos fall under this category, but cells obtained by modifying embryonic stem cells (eg, genetic modification, fusion, etc.) are also within the scope of non-embryonic cells. It is in.

  As used herein, “differentiated cells” refers to cells with specialized functions and morphology (eg, muscle cells, nerve cells, etc.), and unlike stem cells, there is no or almost no pluripotency. . Examples of differentiated cells include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, Examples include smooth muscle cells, fat cells, bone cells, chondrocytes.

  As used herein, “tissue” refers to a cell population having the same function and form in a cell organism. In multicellular organisms, the cells that make up it usually differentiate, function becomes specialized, and division of labor occurs. Therefore, it cannot be a mere assembly of cells, and an organization as an organic cell group or social cell group having a certain function and structure is formed. Examples of tissues include, but are not limited to, integument tissue, connective tissue, muscle tissue, nerve tissue, and the like. The tissue of the present invention may be any organ or tissue derived from an organism. In a preferred embodiment of the present invention, the tissue targeted by the present invention includes bone, cartilage, tendon, ligament, meniscus, intervertebral disc, periosteum, blood vessel, blood vessel-like tissue, heart, heart valve, pericardium, dura mater, etc. Examples include but are not limited to organizations.

  Sufficient biocompatibility in an implantable artificial tissue varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the degree of biocompatibility. Usually, the desired biocompatibility includes, but is not limited to, performing biological binding with surrounding tissues without causing inflammation or the like and without causing an immune reaction. In some cases, for example, in the cornea and the like, it is difficult to cause an immune reaction. Therefore, even if there is a possibility of causing an immune reaction in other organs, it can be considered biocompatible for the purpose of the present invention. Examples of the biocompatibility parameter include, but are not limited to, the presence / absence of an extracellular matrix, the presence / absence of an immune response, and the degree of inflammation. Such biocompatibility can be determined by checking the compatibility at the transplant site after transplantation (for example, confirming that the transplanted artificial tissue has not been destroyed) (human transplant organ rejection) Histopathological diagnosis criteria for differential diagnosis and biopsy specimen handling (diagrams) Kidney transplantation, liver transplantation and heart transplantation. See Japanese Society of Transplantation, Japanese Pathology Society, Kanehara Publishing Co., Ltd. (1998)). According to this document, Grade 0, 1A, 1B, 2, 3A, 3B, and 4 are classified into Grade 0 (no accurate rejection), and biopsy specimens have no evidence of acute rejection, cardiomyocyte damage, or the like. State. Grade 1A (focal, mild accurate rejection) is a state in which large lymphocytes infiltrate locally, around blood vessels or in the stroma, but without cardiomyocyte injury. This finding is observed in one or more biopsy specimens. Grade 1B (diffuse, mild accurate rejection) is a state in which large lymphocytes are more diffusely infiltrated around the blood vessel, stroma or both, but there is no cardiomyocyte injury. Grade 2 (focal, moderate rate rejection) is a state in which an inflammatory cell infiltrate clearly demarcated from the surrounding area is found in only one place. Inflammatory cells consist of large, activated lymphocytes that may be eosinophils. Cardiomyocyte injury with alteration of myocardial architecture is observed in the lesion. Grade 3A (multifocal, moderate accurate rejection) is a state in which inflammatory cell infiltrates composed of large activated lymphocytes are formed in a multiplicity, and may also cause eosinophils. Two or more of these multiple inflammatory cell infiltrates are accompanied by cardiomyocyte injury. Sometimes accompanied by coarse inflammatory cell infiltration into the endocardium. This invasive lesion is found in one or more biopsy specimens. Grade 3B (multifocal, borderline severe-acute-rejection) is a condition in which the inflammatory cell infiltrate seen in 3A becomes more confluent or diffuse and is found in more biopsy specimens. There is cardiomyocyte injury, with inflammatory cell infiltration involving large lymphocytes and eosinophils, and sometimes neutrophils. There is no bleeding. In Grade 4 (severe account rejection), various inflammatory cell infiltrations including activated lymphocytes, eosinophils, and neutrophils are diffusely observed. Cardiomyocyte injury and cardiomyocyte necrosis are always present. Edema, bleeding, and vasculitis are also usually observed. Inflammatory cell infiltration into the endocardium, which is different from the “Quilty” effect, is usually observed. Edema and hemorrhage can be more prominent than cell infiltration if treated with immunosuppressants for a significant period of time.

  Sufficient biofixability in a transplantable artificial tissue varies depending on the portion intended for transplantation, but those skilled in the art can appropriately set the degree of biofixability. Examples of the biocompatibility parameter include, but are not limited to, biological connectivity between the transplanted artificial tissue and the transplanted site. Such biostability can be determined by the presence of biological binding at the site of transplantation after transplantation. In the present specification, preferable bio-fixation property includes that the transplanted artificial tissue is arranged so as to exhibit the same function as the transplanted site.

  In this specification, “organ” and “organ” are used interchangeably, and a function of a living individual is localized and operated in a specific part of the individual, and the part is morphologically A structure with independence. In general, in a multicellular organism (eg, animal, plant), an organ is composed of several tissues having a specific spatial arrangement, and the tissue is composed of many cells. Such organs or organs include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, joint, bone, cartilage, extremity, retina, etc. It is not limited to them. Such organs or organs also include epidermis, pancreatic parenchymal system, pancreatic duct system, liver system, blood system, myocardial system, skeletal muscle system, osteoblast system, skeletal myoblast system, nervous system, vascular endothelial system, pigment system And organs such as, but not limited to, smooth muscle system, fat system, bone system, and cartilage system.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed. The present invention can be performed in vivo.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo. The present invention can be performed in vitro.

  In the present specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again. The present invention can be performed ex vivo.

  As used herein, a “recipient” (recipient) refers to an individual that receives a graft (tissue, cell, organ, etc.) or a transplant (tissue, cell, organ, etc.), and is also referred to as a “host”. In contrast, an individual that provides a graft (tissue, cell, organ, etc.) or transplant (tissue, cell, organ, etc.) is called a “donor” (donor).

  As used herein, “subject (also subject)” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

  The cells used in the present invention may be derived from the same line (derived from the self (autologous)), derived from the same species (derived from another individual (other family)), or derived from different species with respect to the target tissue. Self-derived cells are preferred because rejection is considered, but they may be allogeneic if rejection is not a problem. Moreover, what causes rejection reaction can also be utilized by performing the treatment which eliminates rejection reaction as needed. Procedures for avoiding rejection are well known in the art. For example, the New Surgery System, Vol. 12, Organ Transplantation (From Heart Transplantation / Lung Transplantation Technical and Ethical Development to Implementation) (Revised 3rd Edition) ), Listed in Nakayama Shoten. Examples of such methods include methods such as the use of immunosuppressants and steroids. Immunosuppressants that prevent rejection are currently "cyclosporine" (Sandimune / Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (Predonin, Methylpredonin), "T cells There are “antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs “cyclosporine, azathioprine, steroid hormone”. The immunosuppressive agent is desirably administered at the same time as the medicament of the present invention, but it is not always necessary. Therefore, as long as the immunosuppressive effect is achieved, the immunosuppressive agent can be administered before or after the regeneration / treatment method of the present invention.

  The cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates). Preferably, cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. In the most preferred embodiment, human-derived cells are used. Usually, it is preferable to use cells of the same species as the host.

  The tissue targeted by the present invention may be any organ or organ of an organism, and the tissue targeted by the present invention may be derived from any type of organism. Examples of organisms targeted by the present invention include vertebrates and invertebrates. Preferably, the organism targeted by the present invention is a mammal (eg, primate, rodent, etc.). More preferably, the organism targeted by the present invention is a primate. Most preferably, the present invention is directed to humans.

  As used herein, the term “part” refers to any part, tissue, cell, organ in the body. Such portions, tissues, cells, organs include, but are not limited to, portions that can be treated with skeletal myoblasts, fibroblasts, synoviocytes, stem cells. As long as it is a specific marker, any parameters such as nucleic acid molecule (mRNA expression), protein, extracellular matrix, specific phenotype, and cell shape can be used. Therefore, even if the marker is not specifically described in the present specification, any marker can be used in the present invention as long as it can indicate that it is derived from the portion. An artificial tissue may be determined. Representative examples of such parts include, for example, parts of the heart other than the myocardium, parts containing mesenchymal stem cells or cells derived therefrom, tissues, organs, myoblasts (for example, skeletal myoblasts), fibroblasts, etc. Examples include, but are not limited to, cells and synovial cells.

  As used herein, the term “derived from” means that a certain cell has been separated, isolated or extracted from the cell mass, tissue, organ, etc. from which the cell originally resided. Or the cell was derived from a stem cell.

  The cells, systems and the like provided in the present invention can be provided as pharmaceuticals, or can be provided by known preparation methods as animal drugs, quasi drugs, marine drugs, cosmetics, and the like.

  Accordingly, the animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has an organ or an organ. Preferably, it is a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (for example, single holes, marsupials, Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents , Rabbit eyes, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, a primate (for example, chimpanzee, Japanese monkey, human) is used. Most preferably humans can be targeted. This is because there is a limit in transplantation treatment.

  When the present invention is used as a medicament, the medicament of the present invention may further contain a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. The amount of medicament (artificial tissue, combined pharmaceutical compound, etc.) used in the treatment method of the present invention includes, but is not limited to, an agent, delivery vehicle, diluent, excipient and / or pharmaceutical adjuvant. It can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, tissue form or type, and the like. The frequency with which the treatment method of the present invention is applied to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  In the present specification, “administering” means that the artificial tissue, three-dimensional structure or the like provided in the present invention or a medicament containing the same is administered alone or in combination with other therapeutic agents. . For the artificial tissue provided in the present invention, the following introduction method, introduction form, and introduction amount to a treatment site (for example, a damaged heart) can be used. That is, as an administration method of the artificial tissue and the three-dimensional structure provided in the present invention, for example, direct attachment to a damaged part of myocardial tissue that has been ischemic damaged due to myocardial infarction, angina, etc., and suture after the application And insertion methods. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes the presentation that the combined drugs are administered together as a therapeutic mixture, and the combined drugs are separate but at the same time (eg, artificial tissue or the like is provided by direct surgery, other drugs are intravenously injected) Also included are procedures administered (if given by). “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  In the present specification, the “instruction” refers to a method of administering or diagnosing the pharmaceutical of the present invention, such as a reagent handling, usage method, preparation method, artificial tissue preparation method, contraction method, etc. It is described for the person who performs the diagnosis and the person who diagnoses (which may be the patient himself). This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

  As used herein, “prophylaxis” or “prevention” refers to a certain disease or disorder that prevents or reduces such a condition before it is triggered. Treatment that occurs in a condition or delays the occurrence of the condition.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means that it is reduced. As used herein, “curative treatment” refers to treatment involving the eradication of the root or cause of a pathological process. Therefore, when a radical treatment is given, in principle, the disease will not recur.

  As used herein, “prognosis” is also referred to as prognosis treatment, and refers to diagnosing or treating a post-treatment condition for a certain disease or disorder.

  In the present specification, the “property whose state changes by photoreaction” refers to any property that can cause a change by light irradiation. Thus, such properties can include, for example, morphology, migration, cell-cell interactions, differentiation and proliferation.

  Such a property has a case where it has nature, and a case where it adds artificially. For example, natural cells may be used for cells that originally have the property of moving in response to light (phototaxis). Therefore, when such a natural cell is used, its movement can be controlled by light without intentionally expressing a photoreceptive channel unlike a nerve cell.

  In this specification, “light” is used in the broadest sense in the field, and an electromagnetic wave having a wavelength in the range of about 1 nm to 1 mm is referred to as light by combining ultraviolet rays and infrared rays. This is almost equivalent to what is emitted and absorbed by the transition between the energy levels of atoms and molecules. When emphasizing the nature of a wave, it is also called a light wave, but it sometimes occurs as a group of photons having energy corresponding to each wavelength. The energy propagation path is called a light beam, and the light may be represented by a light beam or a set of light beams.

  As used herein, “change in state” refers to any change in a cell when used with respect to the cell. It is understood that such state changes can include, for example, cell death, morphological changes, migration, changes in cell-cell interactions, differentiation and proliferation.

  In this specification, “transformation” is understood in a broad sense, and includes, for example, a process that causes an arbitrary change. It will be appreciated that such transformation can include any transformation, transduction, transfection, infection, mutagenesis, etc., for example.

  In this specification, transformation is understood to have the ordinary meaning in the field. When DNA extracted from a cell having a certain inheritance (donor cell) is given to another cell (recipient cell), the inheritance of that cell is obtained. Changes in the intrinsic properties of donor cells.

  As used herein, “photoresponsive protein” refers to any protein that responds to light. Examples of such a photoresponsive protein include a photoresponsive cation channel.

In the present specification, the “cation channel” means a cation channel through a cation (cation). Representative examples include Na ⁺ channels and K ⁺ channels that are responsible for propagation of nerve impulses. These are voltage-dependent channels. These opening and closing generate impulses that propagate through nerve axons. There is also a voltage-dependent Ca ²⁺ channel. The cation channels involved in excitatory synaptic transmission are ligand-operated channels, and typical ones include nicotinic acetylcholine receptors and glutamate receptors.

As used herein, “anion channel” refers to an ion channel through which an anion (anion) passes. Channels that play an important role in nerve excitation transmission and the like are mostly those that allow Cl ⁻ to pass through (chlorine (ion) channels), but there are also channels with low selectivity. As the ligand-operated anion channel, a γ-aminobutyric acid (GABA) receptor, a glycine receptor, etc., which are integrated with an inhibitory neurotransmitter receptor, are known. VDACs in bacteria and mitochondrial membranes are known as voltage-dependent ones.

  As used herein, “G protein-coupled receptor” is a guanine nucleotide-binding regulatory protein that specifically binds to GTP (guanosine 5-triphosphate) or GDP (guanosine 5-diphosphate), A GTP-binding protein that shows enzymatic activity to break down bound GTP into GDP and phosphate. A factor that transduces and transmits information through intracellular signaling pathways via receptors such as hormones and neurotransmitters. Among the proteins that function as a receptor, it refers to a receptor that transmits extracellular information into the cell. It exists in the cell membrane of eukaryotes from yeast to humans. It includes many hormones, neurotransmitter receptors, visual, olfactory, and taste receptors.

  In addition, it is known that there are photoreceptors inside the living body other than the cone visual substance (red opsin, green opsin, blue opsin, UV opsin) and eyes. For example, the chicken pineal gland has a photoreceptor called pinopsin, which is known to be related to circadian rhythm (see http://www.kaorin.net/casa/misc/phototransduction.html thing).

  In this specification, “channelrhodopsin-2” is a kind of opsin and is a unique photogated ion channel possessed by algae. Ishizuka et al. , Neurosci. Res. 54 (2006) 85-94.

When channelrhodopsin-2 is used, it can be carried out as follows. CDNA (nucleic acid) encoding protein channel opsin-2 is artificially introduced into cells. Intracellularly produced chanelopsin-2 forms a complex with all-trans retinal and forms a cation channel channelrhodopsin-2 on the cell membrane. That is, (channel rhodopsin = channel opsin-2 + all-trans retinal). This channel rhodopsin-2 is a cation channel that opens in response to light. There are other channel rhodopsin-1 and the like, but at present, channel rhodopsin-2 is the best in terms of performance.
In the normal eye retina, rhodopsin is contained in cells. Rhodopsin is a complex of opsin and retinal (a type of vitamin A (retinoid)) that responds to light. Channel rhodopsin is similar to rhodopsin but is itself an ion channel. The image is (channel rhodopsin = rhodopsin + ion channel).

  In this specification, the “projector” is also called a projector or a projector, and refers to an optical system that forms an image so that a real image of an appropriately illuminated object can be seen, photographed, or observed by other methods.

  Among the techniques that are currently in practical use, in a preferred embodiment, the present invention can be implemented using a method of irradiating channelrhodopsin with a multiphoton excitation method using an infrared laser.

  As is clear from the fact that brain measurement machines (optical topography) that use infrared rays are actually commercialized, infrared rays can pass through the skull.

  The multi-photon excitation method is a method that is often used recently in the field of fluorescence observation. Taking the two-photon excitation method as an example, two photons with half energy (double wavelength) are applied almost simultaneously. This is a method for exciting a fluorescent dye or the like. A high-power laser is often used, and by focusing the laser, the light becomes dense only at the focal point and two photons can be applied almost simultaneously. The short wavelength side in infrared (wavelength 750 nm to 1,000,000 nm) is called near infrared (or near infrared light, wavelength 750 nm to 3000 nm) and is used in various industries including wireless communication such as remote control. It is often used in the field of biological measurement. The good point of infrared rays is that it is good for measuring the inside of a living body because it is less absorbed and scattered by the living body. The shortest wavelength of near-infrared light (wavelength 750 nm to 1200 nm, SWNIR: also called Short Wave Near Infrared) is particularly low in absorption by the living body and is commonly referred to as the “light window in the living body”. It is used for living body measurement. Biological measurement is a method for taking out information inside the living body outside the living body (output, diagnosis), and the present invention is a method for sending information into the living body from outside the living body (input, treatment). Although the directionality is different, the same medium of infrared light can be used. Concerning safety, near infrared rays having a wavelength of around 1000 nm among infrared rays are said to be highly safe because they are less absorbed by the living body. Further, it is known that if a pulse laser that emits light intermittently is used, it is minimally invasive even at high output.

  Therefore, specifically, the embodiment can be assumed as follows. Since channel rhodopsin-2 has a maximum absorption near 460 nm, channel rhodopsin in the brain can be activated from outside the skull by scanning with a strong pulsed laser near the double wavelength (920 nm).

  Laser scanning is different from a projector, but it is a kind of “machine that projects an image of light”. It is understood that by operating a multicolor laser, it can be used in the same way as a projector.

  Moreover, it is not always necessary to apply from the outside, and it is possible to irradiate on the spot during the operation. In such a case, a shape or irradiation method suitable for surgery is used. It is understood that such irradiation methods can use arable land techniques in the art.

  In this specification, “control” of an organism refers to various harmonious adaptations exhibited by the organism in biology. Includes a wide range of concepts across cells, tissues, organs, individuals, populations, and their mutual levels.

(Description of Preferred Embodiment)
The best mode of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Organizational regeneration technology)
In one aspect, the present invention provides a composition for tissue regeneration comprising cells having a property that changes state by a photoreaction. This technique can achieve a flexible regeneration method by freely controlling the transplanted cells, rather than a passive regeneration method in which the transplanted cells are expected to autonomously construct a network. It is also understood that meaningful functional networks can be achieved by this active regeneration method in nerve cells and the like.

  As a representative example of the change in properties that can be caused by the cells of the present invention, as shown in FIG. 1, taking a nerve cell as an example, one extends axon by photoreaction, and the other is light. The formation of synapses between cells by reaction (see FIG. 3). An axon is an elongated process that extends from one nerve cell to another (including another nerve cell, muscle, etc.), and acts as a conductor that transmits information. A synapse is a special contact that can be made between a nerve cell and another cell. By making this contact, information can be transmitted. Therefore, by applying light, axons are extended from one neuron to another, and further synapses are formed. If you compare two neurons to two CPUs, it is an image where a lead wire extends from one CPU and can be soldered to a specific leg of the other CPU by applying light.

  Even in muscles or skin, not only simply increase, but if the shape changes, moves, or the interaction between cells is strengthened, there is a situation that can be used. It will be appreciated that any cell may be used, if any.

  It is understood that the state change in the cells used in the tissue regeneration technique of the present invention may be any change as long as it is appropriate for tissue regeneration.

  For example, in one embodiment, the change in state can be a change in any parameter in the cell, such as cell death, morphological change, migration, changes in cell-cell interactions, differentiation and proliferation. Here, the morphological change means that the shape of the cell changes. Movement means that the position of the cell fluctuates. The cell-cell interaction refers to an interaction between cells, and includes tangible objects and intangible objects and concepts. Therefore, it is understood that informational differences or relative position variations are also included in this change in cell-cell interaction. Here, for example, differentiation refers to specialization of function and form when referring to the state of a cell. Differentiated cells reduce or eliminate pluripotency.

  In one embodiment, the cell used in the present invention may be a cell used for tissue regeneration. It is understood that such a cell may be any cell as long as it is a cell used for tissue regeneration. Examples of such cells include, but are not limited to, cells having differentiation ability.

  In a preferred embodiment, the cell used in the present invention is a nerve cell. Preferably, the cell is a cell that has a property that is naturally changed by a photoreaction or a property that changes a state by a photoreaction by converting a cell that does not have the property in nature. It is. Such conversion can be realized by any method known in the art, and examples thereof include transformation, transduction, transfection, and mutagenesis.

  In a preferred embodiment, the cells used in the present invention are transiently transformed so that they have the property of changing state by a light reaction. Such transient transformation can be accomplished using kits such as Clontech's Retro-X System, J. Org. Virol. , Dec 1997, 9557-9562, Vol 71, no. This can be realized by considering 12 or the like.

  In one embodiment, the conversion is achieved by transformation with a nucleic acid encoding a light responsive protein. In another embodiment, anything other than protein can be used as long as it is a biological material that responds to light. Examples of biological substances other than such proteins include lipids and carbohydrates. Being able to perform biological measurements using infrared rays or the like has some interaction with light, so if it is exposed to light, it can have some effect on the living body.

  In one embodiment, the light-responsive protein can include a G protein-coupled photoreceptor, a cation channel, or an anion channel.

  In one embodiment, the cation channel may include channel rhodopsin-1, channel rhodopsin-2, opsin, red opsin, green opsin, blue opsin, UV opsin, pinopsin and the like.

(system)
In another aspect, the present invention provides a system for performing tissue regeneration at a desired location. This system includes A) a cell having a property that changes its state by a light reaction, and B) a light source that provides light to the desired location. This technique can achieve a flexible regeneration method by freely controlling the transplanted cells, rather than a passive regeneration method in which the transplanted cells are expected to autonomously construct a network. It is also understood that meaningful functional networks can be achieved by this active regeneration method in nerve cells and the like. A system that realizes such a method is expected to be useful as a regenerative treatment device.

  In one embodiment, the change in state is a change appropriate for tissue regeneration. Such changes in the state of the cell can include cell death, morphological change, migration, change in cell-cell interaction, differentiation and proliferation. For example, in the case of nerve cells, a desired neural network can be constructed by appropriate cell death, differentiation and proliferation.

  In another embodiment, the cell used in the present invention is a cell used for tissue regeneration. It is understood that such a cell may be any cell as long as it is a cell used for tissue regeneration. Examples of such cells include, but are not limited to, cells having differentiation ability. In one specific embodiment, the cell is a neuronal cell.

  Preferably, the cell used in the system of the present invention has a property that is naturally changed by a photoreaction, or a property that changes a state by a photoreaction by converting a cell that does not have the property in nature. Cells that have been converted to have Such conversion can be realized by any method known in the art, and examples thereof include transformation, transduction, transfection, and mutagenesis. The system of the present invention may be provided with natural cells and means for realizing such conversion (for example, a kit containing a reagent for carrying out transformation).

  In a preferred embodiment, the cells used in the system of the invention are transiently transformed so that they have the property of changing state by a light reaction. Such transient transformation can be accomplished using kits such as Clontech's Retro-X System, J. Org. Virol. , Dec 1997, 9557-9562, Vol 71, no. This can be realized by considering 12 or the like.

  In one embodiment, the conversion is achieved by transformation with a nucleic acid encoding a light responsive protein.

  As the light-responsive protein used in the present invention, any protein described in the present specification can be used. Alternatively, in another embodiment, anything other than protein can be used as long as it is a biological substance that responds to light. Examples of biological substances other than such proteins include lipids and carbohydrates.

  As the light source used in the present invention, any light source can be used as long as it can provide light to a desired place. Examples of such a light source include a projector, an infrared laser, and an infrared pulse laser. An example of such a projector is a projector whose output can be controlled by a computer, but is not limited thereto.

  The light source used in the present invention may be a light source capable of forming a stimulus pattern having changes in parameters such as intensity, intensity width or pattern, color and time.

  Therefore, the light source used in the present invention can be used as long as it can realize specific irradiation at a desired location, and has a shape suitable for irradiation at the desired location. It will be understood that any shape can be used.

  In one embodiment, the shape of the light source of the present invention can include a shape suitable for irradiation during surgery. For example, examples of the shape include a catheter, an endoscope, and the like, but are not limited thereto.

  In one embodiment, the light source used in the present invention includes means for implementing multiphoton excitation. Multi-photon excitation is a phenomenon in which a large number of photons are used simultaneously, and these photons are absorbed to cause excitation. In the two cases, two photons are simultaneously absorbed into a molecule to cause excitation. For the two examples, two-photon excitation does not use two lasers, but femtosecond laser light compresses the light into very short pulses (about 100 femtoseconds), and the duration of the pulse When extremely strong light is emitted, if it is condensed with a lens, the light density is unusually strong at the focal point, and two-photon absorption and excitation that do not normally occur occur. Since the light density is not sufficient outside the focal point, two-photon absorption does not occur and the laser light passes through the specimen. Thus, absorption of light not related to observation or reaction can be eliminated. Further, since near-infrared light is used for two-photon excitation, it penetrates deeper into the tissue, so that observation or reaction can be performed in the deep part. Thus, multiphoton microscopy, such as two-photon excitation, allows to see or react with molecular and cellular mechanisms at slightly deeper parts of biological tissue.

  In one embodiment, the multi-photon excitation method used in the present invention is realized by exciting a photoreactive protein by simultaneously irradiating two double-wavelength light that is a photon of half energy. The

  Thus, in one embodiment, the means includes an infrared laser.

  In another embodiment, the means can irradiate light having a wavelength of about 750 nm to 3000 nm, for example, about 1000 nm. The short wavelength side in infrared (wavelength 750 nm to 1,000,000 nm) is called near infrared (or near infrared light, wavelength 750 nm to 3000 nm) and is used in various industries including wireless communication such as remote control. It is often used in the field of biological measurement. The good point of infrared rays is good for measuring the inside of a living body because it is less absorbed and scattered by the living body.

  In a preferred embodiment, the means is a pulsed laser that shines intermittently. It is because the influence on a living body is reduced by intermittent light irradiation.

  In another specific embodiment, the photoresponsive protein is channelrhodopsin-2, and the means is a means capable of irradiating a strong pulsed laser near 920 nm. As a result, a system using channel rhodopsin-2 is realized.

  In another aspect, the present invention is a system for performing tissue regeneration so as to have a desired pattern, and A) a cell having a property that changes its state by a photoreaction, and B) a light source corresponding to the photoreaction. And C) means for processing the light source into a desired pattern. It will be understood that this system may also have any one or more features as described herein above.

  In one embodiment, means for processing the light source used in the present invention into a desired pattern can include, but are not limited to, infrared lasers, virtual masks, real masks, filters, projector screens, and the like.

  In one embodiment, the target tissue in the present invention is a nerve, the cell is a nerve cell transformed with channelrhodopsin-2, the light source is a blue light source, and the means is a nervous system pattern. is there. Alternatively, this means may be a means capable of irradiating a strong pulse laser near 920 nm.

  In one embodiment, the nerve cell may be activated to form a nerve network by irradiation with blue light.

  In another aspect, the present invention provides a method for controlling a light-responsive organism. This method includes irradiating the photoresponsive organism with light of a wavelength to which the photoresponsive organism responds in a manner suitable for the control. It is understood that such a method can be implemented in any form.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the embodiments, but only by the claims.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. With the exception of exceptions, reagents and supports used in the following examples may be those commercially available from Sigma (St. Louis, USA), Wako Pure Chemicals, and the like.

(Example 1: Experiment showing that channelrhodopsin-2 can be opened with blue light)
Sample: Cell irradiation with forced expression of channelrhodopsin-2: Irradiation with blue light (near 460 nm) under a microscope Observation: Observation of cell morphological changes under a microscope or calcium imaging using a fluorescent calcium indicator Observe whether changes can be detected by irradiation.

(Preparation of cells forcibly expressing channelrhodopsin-2)
Cells forcibly expressing channelrhodopsin-2 are described in Ishizuka et al. , Neurosci. Res. 54 (2006) 85-94. Specifically, it is cloned into an appropriate vector (for example, pDsRed2-N1 (Clontech)) using channelrhodopsin-2 (SEQ ID NO: 1) described in SEQ ID NO: 1. This is transfected into, for example, desired nerve cells or PC12 cells to prepare cells in which channelrhodopsin-2 is forcibly expressed.

The multi-photon excitation method can be adapted using techniques similar to those in the world of fluorescence images.
B) It shows that nerve regeneration is possible with blue light.

FIG. 2 shows an example of an optical system for neural networking used in this embodiment. Using the computer output projector illustrated in FIG. 2, it is possible to create a light stimulus having “a fine spatiotemporal pattern of stimulus having various intensities, patterns, and colors”. Its light characteristics are
1) Wavelength: Any visible light (300-700nm)
2) Strength range: optional
3) Spatial layout: Any
4) Spatial resolution: ≤ 5 μm
5) Time change: Arbitrary
6) Time resolution: ~ 10 ms
It is.

  Examination of conditions: The animal's skull and tissue sections are sandwiched between the laser and the sample, and the same experiment as above is performed. Examine the relationship between laser output, bone and tissue type, thickness, etc., and check how transparent the current technology is. Alternatively, it can be performed during surgery. According to Ishizuka et al's paper, it reacts with an exponential function with a time constant of about 2 ms when light is applied, and it returns with an exponential function with a time constant of about 6 ms when light is turned off. Therefore, it can be considered that the response is made in about 10 ms.

  In this way, it is demonstrated that the present invention can arbitrarily regenerate the nervous system in the brain.

(Example 2: Experiment showing that channelrhodopsin-2 can be opened by the multiphoton excitation method)
Sample: Cell irradiation with forced expression of channelrhodopsin-2: Irradiation using an infrared pulsed laser under a microscope and multiphoton excitation observation: Observation of cell morphological changes with a microscope and calcium imaging using a fluorescent calcium indicator Observe whether changes can be detected by laser irradiation.

(Preparation of cells forcibly expressing channelrhodopsin-2)
Cells forcibly expressing channelrhodopsin-2 are described in Ishizuka et al. , Neurosci. Res. 54 (2006) 85-94. Specifically, it is cloned into an appropriate vector (for example, pDsRed2-N1 (Clontech)) using channelrhodopsin-2 (SEQ ID NO: 1) described in SEQ ID NO: 1. This is transfected into, for example, desired nerve cells or PC12 cells to prepare cells in which channelrhodopsin-2 is forcibly expressed.

The multi-photon excitation method can be adapted using techniques similar to those in the world of fluorescence images.
B) It shows that the skull can be transmitted through the multi-photon excitation method.

FIG. 2 shows an example of an optical system for neural networking used in this embodiment. Using the computer output projector illustrated in FIG. 2, it is possible to create a light stimulus having “a fine spatiotemporal pattern of stimulus having various intensities, patterns, and colors”. Its light characteristics are
1) Wavelength: Any infrared light (750-3000nm)
2) Strength range: optional
3) Spatial layout: Any
4) Spatial resolution: ≤ 5 μm
5) Time change: Arbitrary
6) Time resolution: ~ 13 ms
It is.

  Examination of conditions: The animal's skull and tissue sections are sandwiched between the laser and the sample, and the same experiment as above is performed. Examine the relationship between laser output, bone and tissue type, thickness, etc., and check how transparent the current technology is.

  In this way, it is demonstrated that the present invention can arbitrarily regenerate the nervous system in the brain.

(Example 3: nerves other than brain)
Next, the present invention shows that adaptation is possible not only in the brain but also in nerves (peripheral nerves) in various tissues.

  Try with tissues that have several nerves (such as extremities). Consider which tissue can be permeated to what thickness. Various reproduction methods can be realized by using the examined results.

  In addition, as long as the nerve exists on the surface, the blue light method can be used, and the multiphoton excitation method may be used to reach the deep part of the living body. As for the detailed conditions, any realization condition can be selected by changing the transmittance according to fine conditions such as laser output, wavelength, pulse width (pattern), and focusing.

(Example 4: Tissues other than brain)
Next, the present invention shows that it can be applied not only to the brain but also to various tissues.

  Try with some tissues (bone, brain, skin, etc.). Consider which tissue can be permeated to what thickness. Various reproduction methods can be realized by using the examined results.

  In the multiphoton excitation method, reaching to the deep part of the living body is established theoretically and experimentally, and therefore, it can be irradiated to some extent with the current technology. As for the detailed conditions, any realization condition can be selected by changing the transmittance according to fine conditions such as laser output, wavelength, pulse width (pattern), and focusing.

    As mentioned above, although this invention was illustrated using the preferable embodiment and Example of this invention, it is understood that the scope of this invention should be interpreted only by a claim. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention can be used not only for regenerative medicine of the nerve (brain) but also as a method for connecting other transplanted organs, for example, transplanted skin and nerves of the nerve patient.

FIG. 1 illustrates the present invention. For example, the nerve cell which forcedly expressed channel rhodopsin * 2 which is a cation channel opened with blue light is produced (refer nonpatent literature 1 for an exemplary method). These neurons become excited in response to blue irradiation, and as a result, nerve axon extension and synapse formation are promoted. That is, by irradiating blue, a nerve cell can be connected and a functional nerve cell network can be constructed. Next, the above nerve cells are injected into a site necessary for restoring the nerve function of the patient. Finally, a computer-generated light pattern is irradiated onto a necessary site using a computer output projector to activate nerve cells and connect nerve cells to induce a functional nerve network. FIG. 2 shows an example of an optical system for neural networking. Using the computer output projector illustrated in FIG. 2, it is possible to create a light stimulus having “a fine spatiotemporal pattern of stimulus having various intensities, pattern widths, and colors”. Its optical characteristics are: 1) Wavelength: Arbitrary visible light (300-700nm) 2) Intensity range: Arbitrary 3) Spatial arrangement: Arbitrary 4) Spatial resolution: ≤ 5 μm 5) Time change: Arbitrary 6) Time resolution: ~ 10 ms. FIG. 3 shows a schematic diagram of axon guidance and synapse formation according to the present invention.

Sequence of channelrhodopsin-2 (AF461397) DNA and amino acid sequence

Claims (37)

A composition for tissue regeneration comprising cells having a property of changing state by a light reaction. The composition of claim 1, wherein the change in state is a change appropriate for the tissue regeneration. The composition of claim 1, wherein the change in state is selected from the group consisting of cell death, morphological change, migration, change in cell-cell interaction, differentiation and proliferation. The composition according to claim 1, wherein the cell is a cell used for tissue regeneration. The composition according to claim 1, wherein the cell is a nerve cell. 2. The composition of claim 1, wherein the cell is a cell that has been converted to have the property by converting a cell that has the property in nature or does not have the property in nature. The composition of claim 1, wherein the cell is transiently transformed to have the property. The composition according to claim 6, wherein the conversion is achieved by transformation with a nucleic acid encoding a light-responsive protein. 9. The composition of claim 8, wherein the photoresponsive protein is a G protein-coupled photoreceptor, a cation channel or an anion channel. The photoresponsive protein is a cation channel, and the cation channel is channel rhodopsin-1, channel rhodopsin-2, opsin, red opsin, green opsin, blue opsin, UV opsin or pinopsin. A composition according to 1. A system for tissue regeneration at a desired location,
A system comprising: A) a cell having a property of changing its state by a light reaction; and B) a light source that provides light to the desired location. The system of claim 11, wherein the change in state is a change appropriate to the tissue regeneration. 12. The system of claim 11, wherein the property change is selected from the group consisting of cell death, morphological change, migration, change in cell-cell interaction, differentiation and proliferation. The system according to claim 11, wherein the cell is a cell used for tissue regeneration. The system according to claim 11, wherein the cell is a nerve cell. 12. The system according to claim 11, wherein the cell is a cell that has been converted to have the property by converting a cell that has the property in nature or does not have the property in nature. 12. The system of claim 11, wherein the cell is transiently transformed to have the property. 17. The system of claim 16, wherein the conversion is achieved by transformation with a nucleic acid encoding a light responsive protein. 19. The system of claim 18, wherein the photoresponsive protein is a G protein coupled photoreceptor, a cation channel or an anion channel. 19. The photoresponsive protein is a cation channel, and the cation channel is channel rhodopsin-1, channel rhodopsin-2, opsin, red opsin, green opsin, blue opsin, UV opsin or pinopsin. The system described in. The system of claim 11, wherein the light source is a projector. The system according to claim 11, wherein the light source is a projector whose output can be controlled by a computer. The system of claim 11, wherein the light source is capable of forming a pattern of stimuli having a change in a parameter selected from the group consisting of intensity, pattern, color and time. The system of claim 11, wherein the light source has a shape suitable for illumination at the desired location. The system according to claim 24, wherein the shape is a shape suitable for irradiation during surgery. The system of claim 11, wherein the light source provides illumination specific to the desired location. 27. The system of claim 26, wherein the light source includes means for implementing a multiphoton excitation method. 28. The multi-photon excitation method according to claim 27, wherein the multi-photon excitation method is realized by exciting the reaction of the photoreactive protein by simultaneously irradiating two double-wavelength light having half energy. system. 28. The system of claim 27, wherein the means comprises an infrared laser. The system according to claim 29, wherein the means is capable of irradiating light having a wavelength of 750 nm to 3000 nm. 28. The system of claim 27, wherein the means is a pulsed laser that shines intermittently. 28. The system according to claim 27, wherein the photoresponsive protein is channelrhodopsin-2, and the means is a means capable of irradiating a strong pulsed laser near 920 nm. A system for performing tissue regeneration so as to have a desired pattern,
A system comprising: A) a cell having a property of changing its state by a photoreaction; B) a light source corresponding to the photoreaction; and C) means for processing the light source into a desired pattern. 34. The system of claim 33, having one or more features of claims 12-32. 34. The system of claim 33, wherein the means for processing the light source into a desired pattern is selected from the group consisting of an infrared laser, a laser scan, a virtual mask, a real mask, a filter, and a projector. 34. The tissue of claim 33, wherein the tissue is a nerve, the cell is a nerve cell transformed with channelrhodopsin-2, the light source is a blue light source, and the means is a nervous system pattern. system. 34. The system of claim 33, wherein the neural cell is activated to create a neural network upon irradiation with blue light.