WO2005075004A1 - Procede et appareil pour la regeneration d'organes et procede de test de selection d'un medicament favorisant la regeneration d'organes - Google Patents

Procede et appareil pour la regeneration d'organes et procede de test de selection d'un medicament favorisant la regeneration d'organes Download PDF

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Publication number
WO2005075004A1
WO2005075004A1 PCT/JP2005/001696 JP2005001696W WO2005075004A1 WO 2005075004 A1 WO2005075004 A1 WO 2005075004A1 JP 2005001696 W JP2005001696 W JP 2005001696W WO 2005075004 A1 WO2005075004 A1 WO 2005075004A1
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Prior art keywords
organ
tissue
regeneration
inflammation
site
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PCT/JP2005/001696
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English (en)
Japanese (ja)
Inventor
Tetsuhiro Kitamura
Nobuyuki Asanuma
Haruhiko Kouhara
Ichiro Kawase
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Osaka Industrial Promotion Organization
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Publication of WO2005075004A1 publication Critical patent/WO2005075004A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/08—Wound clamps or clips, i.e. not or only partly penetrating the tissue ; Devices for bringing together the edges of a wound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a technique for continuously inducing desemination in a part of a living body to thereby regenerate and repair an organ or tissue.
  • the present invention also relates to a method for screening a drug that promotes regeneration or repair of organs and tissues, an evaluation method, and a non-human mammal that can be used for evaluation of a drug candidate compound.
  • Patent Document 1 describes that various sub-organs can be reconstructed and repaired using bladder submucosa, but a specific method other than regenerating part of the bladder is described. Is not disclosed.
  • Patent Document 1 Special Table 2000— 516503
  • An object of the present invention is to provide a technique necessary for regenerating a wide range of organs or tissues.
  • the present invention also relates to a screening method and an evaluation method for a candidate compound that regenerates an organ or tissue, and a non-human mammal suitable for such a method.
  • the present inventor locally induces inflammation using a regeneration reactor (Regeneration Reactor) in organs that require regeneration and repair.
  • a regeneration reactor Regeneration Reactor
  • organ regeneration is induced at the inflamed site.
  • the present invention provides the following organ or tissue regeneration method and device, and a drug screening method that promotes organ or tissue regeneration.
  • An organ or tissue regeneration method comprising the following steps (1) and (2):
  • a method for regenerating an organ or tissue comprising
  • An organ regeneration device having a force bar that secures a space for a regenerated organ to be generated.
  • Methods for evaluating drug candidates that regenerate or repair organs or tissues including the following steps:
  • a non-human mammal having a regeneration reactor having a structure in which a localized inflammatory site of an organ or tissue is covered with a cover that secures a space for the organ or tissue to regenerate the inflammatory site.
  • an organ or tissue
  • an organ or tissue
  • a drug useful for the regeneration or repair of organs or tissues can be obtained.
  • FIG. 1 schematically shows an organ regeneration inducing apparatus according to the present invention.
  • FIG. 2A Spleen regeneration method. “a”, “b”, and “c” indicate procedures in which a part of the spleen is tied with silk thread and excised. d and e show the state where the silk knot is covered with a silicon tube. F indicates the presence of acinar cells (green), transplanted cells (red), and FGF7 (blue). In “a”, the arrow is an FGF7 producing cell
  • FIG. 2B Effect of spleen regeneration.
  • FIG. 2C Quantification of spleen regeneration.
  • l and m indicate spleen cells in the presence or absence of FGF7 by color development using antigen-antibody reaction against PCNA protein.
  • Brown is the cell nucleus. Brown nucleus of acinar cells Z total number of nuclei (0), brown nucleus of splenic duct Z total number of nuclei (n), number of splenic duct (p).
  • FIG. 2D Effect of silicon cover.
  • the upper part a-d is the result of staining with anti-PCNA staining (a, b) and anti-pCK antibody (c, d) in the presence and absence of the cover.
  • the lower row shows PCNA labeling index (a) in the tubular complex after 4 weeks, PCNA labeling index (b) in the splenic acinar cells after 4 weeks, and the number of tubular complexes after 4 weeks ( c) is shown respectively.
  • FIG. 3A Regeneration Reactor 6 months old C57BL / 6J installed in the cut ureter C57BL / 6 Tgl4 (act-EGFP) OsbYOl mice were used for the experiments.
  • the left kidney is pulled out of the retroperitoneal cavity.
  • the ureter and renal arteries and veins are observed (a). They were combined and ligated with silk thread at the renal hilar (b).
  • the kidney body was completely excised, and it was confirmed that no kidney tissue remained (c).
  • the ligature was covered with a silicone tube (d). The entire device was placed in the abdominal cavity and incision cage J was closed.
  • GFP green glomeruli
  • Dil red transplanted cells
  • WT-1 tea ⁇ 'glomera
  • LTA tea g proximal tubule
  • PNA tea nephron
  • FIG. 3D Dedifferentiation and redifferentiation of the ureter.
  • the remaining ureter (a, and the distal part (b, d) near the cut end was immunostained. Both Pax2 and Ret were expressed in the ureter, but the expression intensity was The tissue near the cut surface was analyzed at 1 week (e, f, g), 2 weeks (h, i, j), and 4 weeks (k, 1, m) after surgery.
  • FIG. 3E One week after the detection of both Pax2 and WT1 positive cells in the vicinity of the cut ureter, mesoderm cells expressed Pax2 (a) and WT1 (b). These cells proliferated near the cut ureter (c). Because these immature markers to analyze whether the simultaneous expression underwent immunofluorescent staining (d, e) 0 2 double stained the cells could be detected in the vicinity of the urinary tract (£).
  • FIG. 3F HE staining (a, b) and tissue staining (cj) of the tissue in the silicon cover 4 weeks after analysis of new nephron.
  • New ectopic glomeruli (arrows) and tubules (arrowheads) were detected between silk (stars) and spleen (a).
  • the glomeruli retained red blood cells (b), GSA lectin-positive endothelial cells (c), and WT1-positive podocytes. There are WT1-positive cells around the glomerulus (d).
  • tubules around the glomeruli were positive for LTA lectin binding to the proximal tubule (and also positive for PNA lectin binding to the proximal and distal tubules (£). Urine near the glomerulus. Since the tubules are positive for PNA lectin but negative for LTA lectin (f: arrowhead), they are considered to be distal tubules, and other tubular structures exist are collecting tubes that are positive for DBA lectin (g). Because of the presence of PNA and LTA positive cells in the vicinity of, it is considered to be a proximal tubule (h, i). All of these tubules, collecting ducts, and stromal cells were PCNA positive and proliferated ⁇ .
  • FIG. 3G Continuous cytokeratin immunostaining (ad) and DBA lectin binding (e) in the residual ureter and glomeruli were examined. The positional relationship between consecutive samples is shown in (£). The part in (a) was magnified 4 times in (. The cytokeratin-positive tube was traced between the glomeruli and the remaining ureter. (Ac) 0 The DBA-positive part was the junction between the ureter and the tubular structure. (E, ⁇ ) 0 Tubular structures at both ends are DBA positive (e, red line in 1) but negative at the center and extended to new glomeruli (a, d, Do
  • FIG. 3H Promotion of nephron formation by adult kidney tissue strips.
  • Schematic (a) The kidneys of wild-type mice were cut and fragmented. It was introduced into the Regeneration Reactor installed at the cut ureteral stump of the GFP mouse.
  • HE staining (b, c) and fluorescence staining (d, e) were performed 4 weeks after the operation. New GFP-positive glomeruli were present between necrotic tissue (b: asterisk) and spleen.
  • the frame in (b) was enlarged 4 times in (c).
  • the kidney tissue of the wild strain (D and GFP mice (g) is shown as a control for fluorescence staining.
  • the glomeruli are GFP negative (f: arrowhead) in the wild strain, but positive in the GFP mouse (g: arrowhead). These glomeruli were analyzed with a kidney-specific marker to confirm nephron, and the podocytes and surrounding tissues were stained with WT1 (h) Proximal tubules and glomeruli Anti-Pax2 antibody (stained with 0. Pax2 is a fetal marker and is not expressed in adult kidney.
  • FIG. 4 A schematic diagram of complete skin regeneration.
  • FIG. 5 shows a schematic diagram of endocrine gland regeneration.
  • FIG. 6 A schematic diagram of a skin regeneration assay device is shown.
  • organs or tissues that are regenerated by the present invention are targeted.
  • organs or tissues that are regenerated by the present invention include spleen, kidney, liver, heart, lung, limbs, bladder, adrenal gland, pituitary gland, thyroid gland, parathyroid gland, esophagus, stomach, intestine (small intestine, large intestine, rectum), genital organs, nerves, arteriovenous, skin, etc.
  • spleen, kidney, liver, lung and the like are preferably exemplified.
  • regeneration includes both a case where an organ or tissue is completely regenerated and a case where an organ or tissue is partially regenerated.
  • the “regenerative reactor” means a reactor capable of causing continuous inflammation.
  • the inflamed site is not an organ and a cover is provided so as to secure a space for tissue regeneration. It can be formed by covering with.
  • the “site that can be divided into organs or tissues” means, for example, the ureter for the kidney.
  • the regeneration reactor of the present invention is installed in the ureter, nephron, which is a functional unit of the kidney, is regenerated.
  • the ureter corresponds to “a part that can be divided into organs or tissues”.
  • Induction of inflammation may be performed by either physical means or chemical means. Specifically, it can be tied with thread, string, etc., or affixed or pressed with cloth (knitted fabric, woven fabric), tape or sheet, band, non-woven fabric, etc. Stoppers (such as fish hooks such as bone's horns, needle-like protrusions cut into a spider or cage, stables, sticks to prey, keeps them from coming off, bark hooks, etc.), ultraviolet light, laser Local irradiation with light, radiation, etc., local injury with scalpels, needles, scissors, etc., pinching with forceps, clips, etc., fibers, particles such as metal, grease, ceramic, Fix the plate etc.
  • Stoppers such as fish hooks such as bone's horns, needle-like protrusions cut into a spider or cage, stables, sticks to prey, keeps them from coming off, bark hooks, etc.
  • ultraviolet light laser Local irradiation with light, radiation, etc., local injury with scalp
  • the organ surface or the regeneration site site that causes inflammation
  • substances that cause inflammation eg carrageenan, liquid paraffin, medium chain fatty acid triglyceride, lipopolysaccharide, muramyl dipipe
  • a pathogenic agent eg carrageenan, liquid paraffin, medium chain fatty acid triglyceride, lipopolysaccharide, muramyl dipipe
  • it includes various methods such as a possible material releasing pathogenic substance fixed to the local surface of the organ.
  • nephron can be regenerated from the organ excision site by removing the whole kidney to cause inflammation.
  • the portion of the spleen including the spleen is tied with silk thread, and the upper side (tip side) is cut and the silk thread and the cut surface are covered with a cover.
  • regeneration of an organ or organ can be realized by excising part or all of the organ or organ to form a regeneration reactor.
  • the site that causes inflammation is preferably localized.
  • localized means that the site involved in inflammation is relatively small in the organ (or tissue) or site that can be separated into the organ (or tissue) to be regenerated. It means having a few inflammatory sites (especially one). In the case of a large organ where the inflamed site is distant, it is possible to regenerate the organ at many more inflamed sites.
  • the ends of the branching spleen are tied with a thread, the localized part where the tied spleen is concentrated is cut, and the cut part and the part tied with the thread are localized.
  • the spleen can be regenerated.
  • the present inventors have found for the first time that a plurality of functional nephrons having glomeruli, tubules (proximal and distal), collecting ducts, etc. are formed in the kidney after the kidney has been removed. .
  • the kidney can be regenerated by increasing the number of nephrons formed and increasing the nephrons.
  • the regeneration of the organ is performed with little or no formation of granulation tissue, and the proliferation of the organ tissue around the inflamed site is enhanced.
  • stimulation for the inflammation on the organ surface is continuously performed.
  • Part of the organ is tied with a thread (especially silk thread) or a clip or forceps is used. Local stimulation is desirable.
  • clipped with clips or forceps it is possible to apply a cloth made of silk (woven fabric or non-woven fabric) and then clamp the force.
  • Inflammation promotes the regeneration of the organ, and a relatively large regenerated organ is formed around the inflamed site as compared to the inflamed site. It is desirable that a space for exists. For example, when a part of an organ or the like is tied with a thread or pinched with a clip or forceps, it is preferable to cut the raised tissue that has been tightened by these to expose the inside of the tissue. In this way, regeneration of organs or tissues is promoted.
  • the space required on the surface of the inflammatory site includes not only the surface or the outside of the inflammatory site, but also the side of the inflammatory site.
  • the stem cells that can be regenerated to the inflamed site simply by covering the inflamed site with a cover. It is also possible to promote the regeneration of organs or tissues.
  • cells for example, other organs or tissues, or endocrine cells
  • the present inventor has confirmed that dedifferentiated or weakened cells accumulate at or near the site of inflammation, which contributes to regeneration or repair of organs or tissues.
  • Pax2 and Ret which are specific markers for nephroblastic epithelium, are expressed on the cut end face (inflamed site) of the ureter, which means that substances that induce detachment at the inflamed site.
  • the cover that covers the inflamed site maintains the formation of organs or cells with the ability to form tissues.
  • a wound site caused by surgery or trauma is pressed with gauze, tape, or the like.
  • the granulation tissue grows at the wound site and the wound is healed, but at the same time, the growth of cells that have been devolatilized or weakened is suppressed, and the regeneration or repair of the tissue or organ is inhibited.
  • melting of existing tissue accumulation of dedifferentiated cells such as organ stem cells
  • the organ or tissue is continuously regenerated, so that a large tissue regeneration occurs, and the organ itself Can also be played.
  • a part of an organ is tied with silk thread, the tied part is cut, and the wound surface generated by the cutting is a material having shape retaining properties such as a silicone tube or a greave sheet. Covers the wound surface with the surrounding tissue and wound surface to release the cellular force that inhibits regeneration and exposes it to uncompressed space to prevent the surface from being compressed or adhered .
  • the wound (inflammation) site The tissue or organ is regenerated by sustaining the growth of the thawing tissue (group of undivided cells).
  • Undifferentiated cells have the ability to change differentiated cells into undifferentiated cells by various factors produced at the inflamed site. Undifferentiated cells (for example, various organ stem cells, mesenchymal stem cells) are supplied, or both accumulate at the inflamed site.
  • the present invention in order to differentiate from a mesoderm organ together with a young mesoderm organ to a mature organ, for example, when acting on an endocrine gland, unlike simply transplanting, it is different from an endocrine gland cell. Since the surrounding mesoderm stromal cells interact and mature in the same way as in development, it is possible to create the same environment as general endocrine glands.
  • factors supplied to the inflamed site include bFGF (FGF2), FGF7 (KGF), HGF, IG Fl, GDNF, TGF jS, LIF, FGFIO, PDGF, EGF, and the like.
  • Preferred growth factors include growth factors such as FGF7, HGF, GDNF, IGF-1 and FGFIO. They may isolate the growth factor protein and supply it to the inflamed site, or a factor that induces or stimulates the release of these growth factors may be administered to the inflamed site. Alternatively, cells that highly express these growth factors (for example, transformed cells transformed with a vector capable of expressing growth factors) may be provided at or near the inflammatory site.
  • These growth factors, or other drugs useful for tissue regeneration are drug delivery systems (DDS) such as beads, biocompatible gels, or gradual release by soaking into threads, fibers or fabrics. It is more preferable to continuously supply to the inflamed site using.
  • DDS drug delivery systems
  • the inflammatory site is preferably covered with a cover so as not to inhibit organ regeneration.
  • This force bar prevents the inflamed site from adhering to adjacent organs and the wall-side peritoneum (diaphragm, peritoneum, etc.), and preventing regeneration of the organ or tissue from being in contact with surrounding tissues.
  • a cover examples include an hill shape, a cylindrical shape, a spherical shape, and an ellipsoid shape as long as it has a space that can accommodate an organ or tissue to be regenerated on the outside. Is done.
  • the cover may be closed on the entire surface.
  • the hole or opening communicating with the outside can be used. May have a part.
  • an inlet for injecting a drug such as the growth factor can be provided in the cover.
  • the regenerative tissue has a part that is regenerated from the inflamed area toward the outside (upper side) and a part that is regenerated so as to rise from the lower side (inside) of the inflamed area. Therefore, it is desirable that there is a space around the inflammatory site (for example, the part in contact with the silk thread) where the upper part of the inflammatory site is not so large. Since the organ or tissue is regenerated while moving (or swelling) the original inflammatory site to the outside, a small capacity is sufficient for the space portion with respect to the regenerated organ Z tissue.
  • the material of the cover is not particularly limited as long as it is biocompatible, and is a biocompatible material such as silicon, lactic acid, glycolic acid, ⁇ ? Examples thereof include bioabsorbable polymers such as caprolatatone or copolymers thereof, synthetic polymers such as polyamide, polyester, polyolefin, polyurethane, etc.
  • the non-biocompatible material is preferably covered with a biocompatible material.
  • FIG. 1 A preferred embodiment of the present invention will be schematically described based on FIG.
  • the apparatus of the present invention is a regenerative reactor formed by inflammation-inducing means and a cover.
  • Organ regeneration is performed by (Regeneration Reactor).
  • Inflammation-inducing means include thread, string, cloth (knitted fabric, woven fabric), tape, sheet, band, non-woven fabric, and locking tool (bone-shaped hooks, needle-like projections made by cutting into a spider or spear. Tables, prey sticks, etc., such as barbage hooks, light (ultraviolet light, laser light, radiation, etc.), scalpels, needles, scissors, forceps, clips, etc., preferably silk thread Is exemplified. For example, a part of an organ or tissue is bound with silk thread, and if necessary, the part that has been tied up and raised is excised to induce inflammation locally, leading to regeneration of the organ or tissue.
  • silk thread induces inflammation strongly because it is heterogeneous for mammals.
  • the regenerative reactor formed by the means for inducing inflammation and the cover not only grows the organ buds, but also provides a place for the grown organ buds to grow. It is also possible to transplant organ stem cells to form organ buds and develop the organ ectopically. Furthermore, it is preferable to secure a space for growing the organ buds thus grown with a silicon cover or the like.
  • a growth factor source is preferably arranged in the vicinity of the reactor.
  • nucleic acids peptide hormones, transferrin, etc.
  • intercellular matrix collagen, laminin, etc.
  • chemical substances vitamins, amino acids, steroid hormones, trace gold
  • Genus, ethanolamine, etc. can also be supplied to the organ bud and grown.
  • FGF2, FGF7, FGFIO, HGF, IGF-1 etc. kidney (HGF, FGF7, GDNF, IG Fl, LIF), liver (HGF, FGF8, IGF-1), lung (FGFIO, HGF, IGF-1) Etc. are shown as examples.
  • a step of inducing localized inflammation in the organ, tissue, or a site that can be separated from these, and (2) a cover for securing a space for the organ or tissue to regenerate the inflammation site force The step of attaching can be performed at the same time or first.
  • the epidermis regenerates, but the subdermis and the subcutaneous tissue do not regenerate.
  • tissue force below the dermis granulates grow and fill the gaps, forming scars.
  • the muscle is sutured with a surgical thread.
  • the skin tissue is then fixed linearly with a clip made of synthetic resin, such as a paper clip, lined with silk fiber or similar material (see Figure 4).
  • the inside of the synthetic resin has a structure that is filled with, for example, a water-retaining gel so that the silk fibers do not dry, and that the silk fibers are in close contact with the skin.
  • the unbroken mesoderm cell mass accumulates around the silk fibers and regenerates.
  • This partial force skin tissue is regenerated in a centrifugal manner.
  • silk fibers are excluded due to foreign substances, and no surgical wound remains. Since the skin proliferates in the horizontal direction, the partial force sandwiched between clips and the like also extends in the horizontal direction, so the skin may be covered so as not to be pressed because it is a cloth. Since this is a free space on the outside, the regenerated skin will sink under the cloth, causing skin regeneration.
  • the present invention can be applied to regeneration of endocrine glands.
  • the hormone-producing organ has the following characteristics:
  • hormone-producing cells at the inflamed site. Whether or not hormones are secreted from the transplanted cells is determined by measuring the hormone levels in the blood. Examples of hormone-producing cells transplanted or supplied to the inflamed site include Langerhans islet cells, thyroid cells, gonadal cells, and adrenal cells (cortex, medulla).
  • Endocrine glands are partially collected from self and organ donors. This amount can be obtained by collecting a part of the endocrine organ using a puncture needle that does not require surgery. This is broken down to the cellular level using proteolytic enzymes and strengthened with silk fibers. This is placed in a hollow silicon tube to form a regenerative reactor, which is then implanted into an appropriate tissue, such as subcutaneously or intramuscularly ( Figure 5). At this time, the tube size corresponding to the post-growth volume is required according to the endocrine gland. After transplantation, a specific growth factor is administered as necessary. Langernos cells are energetic to engraft in the liver.
  • Topical growth factors such as liver growth factors and blood sugar levels, and if necessary, factors such as HGF that proliferate Langernos and islands are administered locally. .
  • an antithyroid agent is administered and TSH is secreted by pituitary force to promote proliferation.
  • the gonadal cells are isolated from the ovary or testis, transplanted to an appropriate location, and FSH and LH are administered systemically. For postmenopausal women, these are high and do not need to be dared.
  • the adrenal gland after the cell transplantation, the adrenal cortex hormone is proliferated in order to promote the secretion of ACTH, which is a pituitary gland, by administering a normal maintenance dose or a slightly lower dose of corticosteroid. Screening method and evaluation method
  • the present invention also provides a screening method for a drug that promotes the regeneration or repair of an organ or tissue, and an evaluation method for evaluating a drug that promotes the regeneration or repair of an organ or tissue. Also related.
  • the drug candidate compound when persistent inflammation is caused in a part of an organ or tissue, the drug candidate compound is supplied to the inflamed site, and the effect of giving the candidate compound is affected.
  • a drug effective for regeneration of an organ or tissue can be screened. For example, how much regeneration of an organ or tissue is promoted by giving the candidate compound to a control that does not contain the candidate compound, the weight of the regenerated organ or tissue, the regeneration rate (unit days).
  • a drug useful for regeneration or repair of an organ or tissue can be obtained by comparing the function of the regenerated tissue or the like as an index.
  • the drug candidate compound may be impregnated into, for example, silk thread or silk fabric, bead, biocompatible gel, thread (particularly silk thread), fiber or cloth (particularly silk woven or non-woven cloth). It can be supplied continuously to the inflamed site using a drug delivery system (DDS) that is soaked and gradually released, or by oral or parenteral administration (injection or transdermal absorption preparation). You may administer to the animal which has this inflammatory site.
  • DDS drug delivery system
  • the screening method of the present invention can be used for screening therapeutic agents for all diseases in which regeneration of an organ or tissue is involved in the mechanism.
  • the step of inducing localized inflammation in the organ or tissue and (2) the inflammation site force is attached a cover that secures a space for the organ or tissue to be regenerated.
  • the steps can be done at the same time, or V, the deviation can be done first.
  • the present invention also relates to a drug screening method for complete regeneration of skin wounds.
  • the mouse skin is picked up together with its subcutaneous tissue.
  • This root is ligated with silk thread.
  • This step can be performed with a device that places the skin on the bottom of a cylindrical synthetic resin and drags it with medium forceps (the part that sandwiches the skin is covered with silk) (see Figure 6).
  • the raised skin is necrotized because the roots are ligated.
  • the silk thread in contact with this causes inflammation and induces a regenerative reaction.
  • the necrotic layer is formed so as to traverse the skin, and therefore the dermis layer, which is supposed to contain skin stem cells, has a structure that receives the inflammation of the silk thread over the entire longitudinal section, and the regenerative mechanism extending downward from there has a direction. Is possible.
  • the entire skin layer regenerated from stem cells extends in a centrifugal manner. This is a force mouse that is recognized as a new skin tissue that exists on a concentric circle. Since the body hair is dense, the regenerated skin can be visually discerned because the body hair is also thin. This area is the object of evaluation.
  • the basic principle of drug screening is whether the area expansion due to the same inflammatory substance can be promoted not only by the selection of the inflammatory substance mentioned above, but also by systemic administration of the drug or local administration to the installation device. is there.
  • the device itself can be the basic form of a medical device for wound healing. Skin healing was mainly based on methodologies that burned, punched out, and psychologically resistant. These are invasive and time consuming. This method consists of a simple methodology that only fits the kit on the skin, and the evaluation method consists of a thin measurement of the body hair and a simple measurement method that measures the area of the part.
  • the screening method of the present invention can be performed using a bandage having inflammation and a spatial effect.
  • the present invention has shown that actively utilizing inflammation promotes general wound healing. Applicable to screening of candidate compounds that stimulate skin stem cells and promote wound healing by mixing an appropriate amount of an appropriate inflammatory substance into an adhesive bandage that is widely used for wound treatment. Specifically, the adhesive bandage is closely attached to prevent entry of bacteria, moisture, etc., but the central part is structured to have a space, and a continuous inflammation is caused by an inflammable substance such as silk thread or tuberculin inside. Then, by adding the candidate compound to this, screening of candidate compounds that promote healing (regenerate normal skin tissue) can be performed.
  • Drugs obtained by such screening methods function not only in organ or tissue regeneration in vivo or in vitro but also in inflammation (eg, hepatitis, splenitis, nephritis, cholecystitis, thyroiditis, etc.) It may be useful for repairing or treating reduced organs.
  • the present invention also relates to a non-human mammal having a regenerative reactor embedded in a localized inflammatory site covered with a cover.
  • non-human mammals include rats, mice, guinea pigs, rats, musters, nu, monkeys, pigs, rushes, and rabbits.
  • the regeneration reactor has a structure in which an inflammatory site (for example, it can be formed by tying an organ or tissue with silk thread and cutting the tied part) is covered with a cover. It may be formed in an organ or tissue, or may be formed by forming a regenerative reactor having an inflammatory site and a cover and implanting it under the skin or muscle of a non-human mammal.
  • a part of the spleen of a mouse is tied up with silk thread to which bone marrow cells that secrete FGF7 (growth factor, KGF) are attached. Cut the upper part of the tie (a, b, c) 0 Cover this knot with a silicone tube (d, e) 0 Then the cells attached to the thread will contact the spleen below, where Growth factors are supplied to the spleen (£).
  • FGF7 growth factor 7
  • PCNA which is expressed when cells divide, and what percentage of protein is expressed in what type of cells, cut the tissue around the device and fix it on the glass slide.
  • the cell nuclei shining in brown are examined by measuring with a microscope using a coloring method that utilizes an antigen-antibody reaction (l, m). Two types of slides were compared, with and without FGF7 (1) and without (m). The calculation was performed using the ratio of the total number of brown nuclei Z in the acinar cells (0) and the total number of splenic brown nuclei Z (n).
  • FGF7 FGF7
  • FGF7 has been shown to promote the proliferation of the spleen itself (0) and the proliferation of the spleen itself (n), which has the ability to divide into the spleen that has appeared due to inflammation.
  • the splenic duct was confirmed to contain the receptor for FGF7, and FGF7 secreted from bone marrow cells was accurately assayed. It has also been shown that FGF7 acts as a knee regeneration factor in the organ regeneration device, and is particularly preferred as a factor during spleen construction.
  • the silk thread was covered with a silicon cover (b, d) and the cocoon group (a, c), and the tissue specimens were evaluated 4 weeks after surgery.
  • the cells were stained with anti-PCNA antibody (a, b) for the purpose of examining proliferation, and anti-pCK antibody (c, d) for examining the number of splenic ducts.
  • the arrow indicates the position where the silk thread was present.
  • the group with a silicone cover has a high number of knee tubes with a high growth rate.
  • the lower graph in Fig. 2D is a statistical analysis of several locations of these samples.
  • A The labeling index (LI) of PCNA in the tubular complex did not change much with or without the cover, but (b) the labeling index in the splenic acinar cells was 1.7 times more likely to have a cover.
  • C shows the number of tubular complexes. In the presence of a force cover, the number of splenic ducts increased 5.4 times.
  • the “C” in (a) and (b) is a force that indicates the PCNA labeling index of normal splenic and acinar cells proximal to the covered silk. This indicates that the silicone cover does not affect splenocyte proliferation.
  • the present inventor reconstructed nephrons in adult mice using a novel methodology named Regeneration Reactor.
  • a silk thread covered with a silicone cover was placed on the ureteric section after nephrectomy. After this, the cut ureter expressed Pax2 and Ret, which are specific markers for the ureteric bud epithelium, and WT1 and Pax2-positive fibroblasts specific for the metanephric mesoderm proliferated around it. .
  • a month later several nephrons were induced and connected to the cut ureter.
  • a glomerulus is a filter that turns blood into urine.
  • the glomerulus is green, and this is because the organ regeneration device was installed in the green mouse, and the reactor that does not change the non-green transplanted cells into the glomerulus induced the glomerulus derived from the green mouse in the green mouse ureter. Means that helped. In fact, a green glomerulus was made only with silk without transplanting cells. In order to confirm whether it was really a glomerulus, we examined multi-directional forces.
  • WT-1 protein peculiar to glomeruli was confirmed by an immunoantibody method (£).
  • the glomerular force is specific to the proximal tubule (which can be stained with LTA) (g), which is a tube that passes after urine has been filtered, and nephron (which is a basic structure of the kidney, and many of these form a kidney) It was connected with the glomerulus when we examined the binding substance (which can be stained with PNA) (h). When this tube is followed, it leads to a collecting tube (which can be stained with DBA) (0, finally connected to the ureter!).
  • the glomeruli are part of the nephron induced by the cut ureter.
  • Pax2 belongs to a group of transcription factors involved in morphogenesis. It begins to express in the pronephros from the intermediate mesoderm. However, its expression is suppressed by WT1 in the renal epithelium in adults. This Pax2 was strongly expressed in the cut ureteral stump (a). Pax2 was expressed weaker at the distal end of the ureter than at the proximal end (b). The ureter near the cut end was still Pax2 positive after 2 weeks (h), but its expression disappeared after 4 weeks (k). Pax2 is not detected in normal adult mouse ureters (data not shown).
  • Pax2 is an early renal marker, and its expression indicates that the cut ureter has been dedifferentiated with the Regeneration Reactor.
  • Ret a marker of the tip of the metanephric ureteric bud located downstream of Pax2.
  • This protein is a receptor for glial cell line-derived neurotrophic factor (GDNF) secreted from the metanephric mesoderm. It is expressed at the tip of the separated ureteric bud.
  • GDNF glial cell line-derived neurotrophic factor
  • Ret was expressed in the vicinity of the cut end of the ureter in the same manner as it was expressed at the tip of the ureter bud.
  • the ureter was still positive after 2 weeks (0, expression had disappeared by 4 weeks (1).
  • Ret expression was similar to Pax2.
  • Ret was found in normal ureteral cells. Detected only in the proximal tubule of the adult mouse, the above results indicate that in the Regeneration Reactor, the cut ureteral stump is dissected to the same state as the ureteric bud.
  • Ki67 whose expression correlates with proliferation, and some ureteral cells were Ki67-positive at 1 week after surgery (g), but at 2 and 4 weeks, there were no positive cells. (J, m)
  • fibroblasts around the remaining ureter were examined.
  • the elongation of the ureteroblast epithelium occurs near the metanephric mesoderm.
  • WT1 is one of the markers of metanephric mesoderm.
  • these cells accumulate around the kidney buds and express WT1 and Pax2.
  • fibroblasts around the remaining ureter were positive for Pax2, WT1, and Ki67 (a, b, c).
  • the tissue in the silicone cover was carefully examined and a new ectopic glomerulus was found between the silk thread and spleen (a).
  • the glomeruli contained red blood cells inside (b), and GSA lectin positive cells that bind to vascular endothelial cells were also found (c). This indicates that a blood vessel has invaginated into the glomerulus! Some cells in the glomerulus were WT1-positive, suggesting podocytes (d). In addition, nephrons were placed around the glomerulus. There were LTA lectin-positive tubules that bound to the proximal tubules.
  • the DBA binding site is present only in the collecting duct in mature kidneys, and as our results indicate, it is impossible for adults that have completed development to bind directly to the ureter, and this is strongly This indicates that the part was newly induced in the ureter, and the tracked tube showed DBA binding at both ends but not at the center (data not shown). Rather than in the collecting duct in adults, it appears to be more similar to the early branching of the ureteroblast epithelium That. These results, we are connected glomeruli and cutting ureter, it was concluded that those derived after the occurrence.

Abstract

Procédé de régénération d'un organe, caractérisé en ce qu'on induit une inflammation localisée dans un organe ou un site capable de se différencier en un organe.
PCT/JP2005/001696 2004-02-06 2005-02-04 Procede et appareil pour la regeneration d'organes et procede de test de selection d'un medicament favorisant la regeneration d'organes WO2005075004A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001003609A1 (fr) * 1999-07-07 2001-01-18 Tapic International Co., Ltd. Tube neural artificiel
JP2001316285A (ja) * 2000-05-01 2001-11-13 Yasuhiko Tabata 細胞と細胞増殖因子とからなる組織器官の再生のための材料
WO2003007784A2 (fr) * 2001-07-16 2003-01-30 Depuy Products, Inc. Dispositif et procede de regeneration du menisque
JP2003518517A (ja) * 1999-12-29 2003-06-10 チルドレンズ メディカル センター コーポレーション 無細胞化生体材料スカフォールドからの臓器再構築

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001003609A1 (fr) * 1999-07-07 2001-01-18 Tapic International Co., Ltd. Tube neural artificiel
JP2003518517A (ja) * 1999-12-29 2003-06-10 チルドレンズ メディカル センター コーポレーション 無細胞化生体材料スカフォールドからの臓器再構築
JP2001316285A (ja) * 2000-05-01 2001-11-13 Yasuhiko Tabata 細胞と細胞増殖因子とからなる組織器官の再生のための材料
WO2003007784A2 (fr) * 2001-07-16 2003-01-30 Depuy Products, Inc. Dispositif et procede de regeneration du menisque

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KITAMURA T. ET AL: "Regeneration of Tubular Complex is Promoted by a Free Space", PANCREAS, vol. 30, no. 2, March 2005 (2005-03-01), pages 174 - 179, XP008051657 *

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