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WO2014115571A1 - Procédé de visualisation de cellule d - Google Patents

Procédé de visualisation de cellule d Download PDF

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Publication number
WO2014115571A1
WO2014115571A1 PCT/JP2014/000430 JP2014000430W WO2014115571A1 WO 2014115571 A1 WO2014115571 A1 WO 2014115571A1 JP 2014000430 W JP2014000430 W JP 2014000430W WO 2014115571 A1 WO2014115571 A1 WO 2014115571A1
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brain tissue
reacting
biotin
primary antibody
visualizing
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PCT/JP2014/000430
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English (en)
Japanese (ja)
Inventor
桂子 池本
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Ikemoto Keiko
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Priority to JP2014558515A priority Critical patent/JPWO2014115571A1/ja
Publication of WO2014115571A1 publication Critical patent/WO2014115571A1/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • the present invention relates to a method for visualizing D-cells, and more particularly to a method for visualizing D-cells, which is considered to be one of cells that produce a neuroactive substance that acts on a trace amine-related receptor.
  • trace amine-related receptor type 1 trace amine associated with ventral tegmental dopamine neurons
  • TAAR1 trace amine-related receptor type1
  • the trace amine-related receptor type 1 is positioned as a target receptor for developing a novel antipsychotic drug that improves the pathology of neuropsychiatric disorders such as schizophrenia and sleep disorders.
  • Compounds such as 2-azetidinemethanamine and 2-pyrrolidinemethanamine that actually act on the traceamine-related receptor type 1 are depression, anxiety disorder, bipolar disorder, attention deficit hyperactivity disorder (ADHD), Stress-related disorders, schizophrenia, neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, diabetic complications, obesity, lipid metabolism It has been reported that it can be used to improve symptoms of abnormalities, energy consumption and assimilation, body temperature homeostasis and dysfunction, sleep and circadian rhythm disorders and cardiovascular disorders (Patent Document 1).
  • D-cells which are one of the cells that produce neuroactive substances that act on the trace amine-related receptors, were identified by Jaeger et al. In 1983 as “aromatic L-amino acid decarboxylase: AADC”.
  • AADC aromatic L-amino acid decarboxylase
  • FIG. 1 is a block diagram for explaining the “D-cell hypothesis”. The outline of the “D-cell hypothesis” will be described with reference to the block diagram of FIG. Trace amine present in the vicinity of the nucleus accumbens D-neuron due to a decrease in the number of nucleus accumbens D-neurons in the human brain due to a decrease in the function of neural stem cells in the subventricular zone of the lateral ventricle in the human brain The concentration of.
  • the D-cell may increase or decrease depending on the part of the human brain where the D-cell exists and the progression of the pathology of schizophrenia. Until now, it has been difficult to accurately grasp the basic information.
  • Non-patent Document 3 A technique for visualizing dopamine neurons in the ventral tegmental area has also been studied (Non-patent Document 3). However, this prior art also does not have a detailed reference to a method for visualizing D-cells.
  • An object of the present invention is to provide a method for visualizing D-cells contained in brain tissue obtained after human death and after autopsy.
  • the present invention [1] (1) fixing a brain tissue obtained after autopsy after human death; (2) slicing the fixed brain tissue; (3) inactivating peroxidase contained in the sliced brain tissue; (4) reacting a primary antibody obtained by reacting an animal with an antigen against brain tissue inactivated by the peroxidase; (5) reacting a brain antibody reacted with the primary antibody with a secondary antibody having the primary antibody as an antigen; (6) coloring the brain tissue reacted with the secondary antibody; including, A method for visualizing D-cells is provided.
  • the step (1) of slicing the fixed brain tissue is a step of slicing the fixed brain tissue to 30 to 100 ⁇ m
  • the step (4) of reacting the primary antibody obtained by reacting an antigen with an animal to the brain tissue inactivated with the peroxidase is an antigen with respect to the brain tissue inactivated with the peroxidase.
  • One of the present invention is [3]
  • the brain tissue reacted with the primary antibody is labeled with biotin.
  • Reacting with a secondary antibody anti-animal serum Reacting brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which a number of avidin and biotinylated peroxidase are bound;
  • the step (6) of coloring the brain tissue reacted with the secondary antibody is a step of coloring the brain tissue reacted with the avidin-biotin complex.
  • the method for visualizing D-cells described in [3] above is provided.
  • the step (1) of fixing the brain tissue includes: Slicing brain tissue obtained after autopsy to a thickness of 5 mm to 3 cm and immersing in a phosphate buffer solution containing paraformaldehyde for 1 to 3 days (1-1); Dipping in a phosphate buffer solution containing sucrose and sodium azide in a range of 3 days or more and less than 20 years (1-2);
  • the step (3) of inactivating the peroxidase comprises: The method for visualizing D-cells according to [1] above, comprising the step (3-1) of immersing the sliced brain tissue in a phosphate buffer solution containing methanol and aqueous hydrogen peroxide for 5 to 120 minutes. It is to provide.
  • the step (4) of reacting the primary antibody comprises: Anti-animal serum, which is a primary antibody obtained by reacting the peroxidase-inactivated brain tissue with an aromatic L-amino acid decarboxylase protein that is an antigen in an animal, in the range of 1 to 10 ° C. Including a step (4-1) of reacting for 10 days, The method for visualizing D-cells described in [2] above is provided.
  • One of the present invention is [8] The group consisting of rabbit, mouse, rat, chicken, goat, sheep and guinea pig, wherein the anti-animal serum is a primary antibody obtained by reacting the animal with an aromatic L-amino acid decarboxylase protein as an antigen.
  • One of the present invention is [9] The step (5) of reacting the brain tissue reacted with the primary antibody with a secondary antibody using the primary antibody as an antigen labels the brain tissue reacted with the primary antibody with the biotin. Reacting the obtained anti-animal serum in the range of 1 to 10 ° C. for 12 to 48 hours (5-1), The method for visualizing D-cells described in [3] above is provided.
  • the anti-animal serum labeled with biotin is anti-rabbit serum labeled with biotin, anti-mouse serum labeled with biotin, anti-rat serum labeled with biotin, anti-chicken serum labeled with biotin, anti-biotin labeled
  • the method for visualizing D-cells according to [9] above which is at least one selected from the group consisting of goat serum and anti-sheep serum labeled with biotin.
  • One of the present invention is [11] The step of reacting an avidin-biotin complex in which a large number of the avidin and biotinylated peroxidase are bound, A step of reacting brain tissue reacted with the biotin-labeled anti-animal serum with an avidin-biotin complex in which a large number of avidin and biotinylated peroxidase are bound (0.5-2 hours). )including, The method for visualizing D-cells described in [3] above is provided.
  • the step of coloring the brain tissue reacted with the avidin-biotin complex comprises: Carrying out in a solution containing tris (hydroxymethyl) aminomethane, diaminobenzidine tetrahydrochloride and aqueous hydrogen peroxide (6-1), The method for visualizing D-cells according to [4] above is provided.
  • D-cells contained in brain tissue obtained from a human autopsy brain can be visualized easily and clearly.
  • D-cell is “a cell that is positive for aromatic L-amino acid decarboxylase immunity but does not contain DA or serotonin”.
  • DA and serotonin biosynthetic enzymes, ie tyrosine hydroxylase and tryptophan hydroxylase, are not expressed. Therefore, immunostaining was performed by the above method using primary antibodies against tyrosine hydroxylase and tryptophan hydroxylase, and the cells were positive for aromatic L-amino acid decarboxylase immunity.
  • the enzyme cannot be visualized, it can be proved to be a D-cell by definition.
  • This step is performed by applying immunodouble staining with the same and serial sections. Since DA and serotonin are rapidly oxidatively decomposed in the human postmortem brain, it is difficult to visualize DA and serotonin itself, and it is necessary to label and visualize biosynthetic enzymes that are more stable biological materials. This is because the. Using this method, localization, morphology observation, and quantification of D-cells contained in the brain tissue can be performed.
  • the relationship between the pathology of a neuropsychiatric disorder such as schizophrenia and D-cells is elucidated by the D-cell visualization method of the present invention, and the relationship between the pathology and the increase or decrease in D-cells before and after administration of a novel antipsychotic drug. Can be compared.
  • the “D-cell hypothesis” shown in FIG. 1 proposed by the present inventor can be specifically verified.
  • the D-cell visualization method of the present invention further elucidating the molecular level of schizophrenia, analyzing D-cell signaling, and elucidating the molecular level of other neuropsychiatric disorders Can be dramatically accelerated.
  • FIG. 1 is a block diagram for explaining the D-cell hypothesis.
  • Step of fixing brain tissue obtained after autopsy after human death (1) After human death, brain tissue obtained after necropsy is removed, sliced to a thickness of 1 cm, and fixed with 0.1 M phosphate buffer solution (pH 7.4) containing 4% paraformaldehyde for 1 to 3 days. During this fixation, the 0.1M phosphate buffer solution containing 4% paraformaldehyde is exchanged once or twice with a fresh 0.1M phosphate buffer solution containing 4% paraformaldehyde.
  • the fixed brain tissue is transferred to a 0.1 M phosphate buffer solution (pH 7.4) containing 15% sucrose and 0.1% sodium azide. 1 to 2 times with 0.1 M phosphate buffer solution containing 15% sucrose and 0.1% sodium azide and 0.1M phosphate buffer solution containing 15% sucrose and 0.1% sodium azide Change and store in 0.1 M phosphate buffer solution containing 15% sucrose, 0.1% sodium azide for a period of not less than 3 days and less than 20 years.
  • Step of slicing fixed brain tissue to 30 to 100 ⁇ m (2) a block including a region predicted to contain D-cells is prepared, and sliced into a thickness of 50 ⁇ m with a cryostat or a microtome to prepare a sliced section. The obtained sliced sections are stored in a storage solution.
  • Step (3) for inactivating peroxidase contained in sliced brain tissue In order to suppress endogenous peroxidase contained in the brain tissue of the sliced slices obtained, it was treated with a 0.1 M phosphate buffer solution containing 40% methanol and 1% hydrogen peroxide at room temperature for 20 minutes, Rinse 10 minutes ⁇ 3 times with 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl.
  • Step (4) of reacting a primary antibody obtained by reacting an aromatic L-amino acid decarboxylase protein with an animal on brain tissue inactivated with peroxidase Although there is no limitation in particular in the animal used for this invention, if an example is given, 1 type or 2 types or more, such as a rabbit, a mouse
  • a primary antibody (autospecific antibody) obtained by reacting an aromatic L-amino acid decarboxylase protein with a rabbit is treated with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl. Dilute 1 to 70,000 times, place in a container, immerse the section in it, and allow to react for 1 week to 10 days with a shaker at 4 ° C. with gentle shaking.
  • “slowly shake” means that the shaker is preferably shaken at a rotation speed of 0.2 to 2 rpm. The same applies to the following.
  • Step (5) of reacting anti-animal serum labeled with biotin to the brain tissue reacted with the primary antibody After rinsing with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl for 10 minutes ⁇ 3 times, anti-rabbit serum labeled with biotin as a secondary antibody was treated with 0.3% Triton. Dilute 1000-fold with 0.01 phosphate buffer solution containing X-100 and 0.9% NaCl, in which the reaction is similarly carried out overnight at 4 ° C. with gentle shaking.
  • Step of reacting brain tissue reacted with biotin-labeled anti-animal serum with avidin-biotin complex in which many avidins and biotinylated peroxidase are bound After rinsing with a 0.01 M phosphate buffer solution containing 0.3% Triton X-100 and 0.9% NaCl for 10 minutes ⁇ 3 times, the brain tissue reacted with the biotin-labeled anti-animal serum, The reaction is carried out for 2 hours with gentle shaking in a 0.01 M phosphate buffer solution containing 0.3% Triton-X and 0.9% NaCl containing an avidin-biotin complex in which many avidins and biotinylated peroxidases are bound.
  • Step of developing color of brain tissue reacted with avidin-biotin complex (6) After rinsing with 0.01 M phosphate buffer solution containing 0.3% Triton X-100 for 10 minutes ⁇ 3 times, the brain tissue is developed with the following reaction solution.
  • Reaction solution In 50 ml of 0.05 M Tris-hydrochloric acid buffer (tris (hydroxymethyl) aminomethane and hydrochloric acid-containing buffer, pH 7.6), 0.5 g of ammonium nickel sulfate was dissolved while stirring with a stirrer. After dissolution, 5 mg of diaminobenzidine sodium tetrahydrochloride (DAB) is completely dissolved.
  • DAB diaminobenzidine sodium tetrahydrochloride
  • This solution is put into a 10 ml petri dish, and the above section is placed. Then, 3 ⁇ l of 1% hydrogen peroxide solution is dropped, and the color reaction is observed with occasional shaking under a stereomicroscope. When the color has developed sufficiently, transfer to 0.05 M Tris-HCl buffer (pH 7.6) to stop the reaction. If the color reaction is insufficient, add 1% hydrogen peroxide to the reaction solution. If the reaction is insufficient even after observing for more than 15 minutes, transfer to a new reaction solution and try to develop color in the same way. Thereafter, it is mounted on a slide glass coated with gelatin, dried, dehydrated and transparent, and encapsulated using enteran and cover glass. After drying, the visualized D-cells can be observed by observing under an optical microscope.
  • Example 1 In the case of Example 1, when a similar experiment was performed using tyrosine hydroxylase protein instead of the aromatic L-amino acid decarboxylase protein, no tyrosine hydroxylase protein was present in D-cells. Therefore, D-cells cannot be visualized.
  • Example 2 In the case of Example 1, when a similar experiment was performed using tryptophan hydroxylase protein instead of the aromatic L-amino acid decarboxylase protein, no tryptophan hydroxylase protein was present in D-cells. Therefore, D-cells cannot be visualized.
  • D-cells considered to be one of cells that produce a neuroactive substance that acts on a trace amine-related receptor Another possible method for visualizing D-cells is in situ hybridization, which visualizes a labeling substance as particles.
  • in situ hybridization which visualizes a labeling substance as particles.
  • Trace amine is an unstable biological material contained in trace amounts in the human brain and cannot be quantified in the autopsy brain after human death.
  • aromatic L-amino acid decarboxylase which is one of the synthases and a relatively stable biological substance in the postmortem brain. At present, this is the only way to visualize trace amine neurons. In the future, if other pathways related to biosynthesis and metabolism of trace amines in humans are elucidated, it will be necessary to label biological substances other than aromatic L-amino acid decarboxylase and try the same visualization method.
  • the mechanism of action of mesolimbic dopamine system overactivity in the pathogenesis of schizophrenia and the role of D-cells in the pathogenesis of other neuropsychiatric disorders will be elucidated. It can provide information essential for developing new antipsychotic drugs.
  • the molecular level pathogenesis of schizophrenia is further elucidated by using the D-cell visualization method of the present invention, the signal transduction of D-cells is analyzed, and the pathological level of other psychoneurological diseases at the molecular level. Elucidation can also be accelerated dramatically.

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Abstract

Le problème décrit par la présente invention est de fournir un procédé de visualisation de cellules D contenues dans des tissus de cerveau humain, obtenus après un décès ou une autopsie. La solution de l'invention porte sur un procédé de visualisation de cellules D, qui comprend : une étape de fixation de tissus de cerveau humain, obtenus après un décès ou une autopsie ; une étape de découpage des tissus de cerveau humain ; une étape de désactivation de la peroxydase contenue dans les tissus de cerveau découpés ; une étape consistant à faire réagir un anticorps principal, obtenu par réaction d'un antigène avec un animal, avec les tissus de cerveau, dans lesquels la peroxydase a été désactivée ; une étape consistant à faire réagir un anticorps secondaire, pour lequel l'anticorps principal sert d'antigène, avec les tissus de cerveau avec lesquels l'anticorps principal a été amené à réagir ; et une étape de coloration des tissus de cerveau avec lesquels l'anticorps secondaire a été amené à réagir.
PCT/JP2014/000430 2013-01-28 2014-01-28 Procédé de visualisation de cellule d WO2014115571A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008506380A (ja) * 2004-07-16 2008-03-06 シーエックスアール・バイオサイエンシズ・リミテッド 細胞のストレスの検出
JP2010538658A (ja) * 2007-09-14 2010-12-16 ベンタナ・メデイカル・システムズ・インコーポレーテツド 前立腺癌バイオマーカー

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008506380A (ja) * 2004-07-16 2008-03-06 シーエックスアール・バイオサイエンシズ・リミテッド 細胞のストレスの検出
JP2010538658A (ja) * 2007-09-14 2010-12-16 ベンタナ・メデイカル・システムズ・インコーポレーテツド 前立腺癌バイオマーカー

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KEIKO IKEMOTO: "Do all ot human midbrain tyrosine hydroxylase neurons synthesize dopamine?", BRAIN RESEARCH, vol. 805, no. 1-2, 14 September 1998 (1998-09-14), pages 255 - 258 *

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