WO2010018764A1 - Anti-angiogenic agent, and screening method and screening kit for same - Google Patents

Anti-angiogenic agent, and screening method and screening kit for same Download PDF

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Publication number
WO2010018764A1
WO2010018764A1 PCT/JP2009/063753 JP2009063753W WO2010018764A1 WO 2010018764 A1 WO2010018764 A1 WO 2010018764A1 JP 2009063753 W JP2009063753 W JP 2009063753W WO 2010018764 A1 WO2010018764 A1 WO 2010018764A1
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bazf
angiogenesis
cbf
gene
screening
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PCT/JP2009/063753
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French (fr)
Japanese (ja)
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繁樹 東山
博文 井上
秀隆 大貫
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国立大学法人愛媛大学
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to an angiogenesis control agent, a screening method thereof, and a screening kit. More specifically, the present invention relates to an agent that inhibits or promotes angiogenesis involving BAZF, a screening method thereof, and a screening kit. Is.
  • angiogenesis is related to the growth of cancer, and this control of angiogenesis was considered to play an important role in the treatment of cancer.
  • Non-Patent Document 1 VEGF (Vascular endothelial growth factor)
  • Non-Patent Document 2 the inhibition of angiogenesis involves the Notch1 signal pathway
  • BAZF Bcl-6 associated Zinc finger protein
  • Bcl-6 Bcl-6 associated Zinc finger protein
  • This BAZF has the same transcriptional repression activity as Bcl-6, and functions by forming a heterodimer with Bcl-6, and is known to have some effect on the immune system such as T cells.
  • the relationship with angiogenesis was not known at all (Non-patent Document 3).
  • the present inventors have been able to temporarily block Notch1 signal by stimulating human umbilical vein endothelial cells (HUVEC) with VEGF (that is, blood vessels) BAZF, whose expression is increased by VEGF signaling, is involved in the blockade of neonatal accelerator (VEGF stimulation) and brake (Notch1 signaling)) CBF-1 by binding to CBF-1 downstream of the Notch1 signaling pathway and promoting the formation of a complex between CBF-1 and the protein “Cull3 (E-3 ligase)” that breaks CBF-1.
  • the present invention has been achieved by releasing the angiogenesis brake function of the present invention, and the object of the present invention is to provide a novel angiogenesis control agent, a screening method for an angiogenesis control agent, and a screening Provide kit There is.
  • the angiogenesis control agent characterized by including the substance which controls following (I) and / or (II).
  • the angiogenesis control agent according to the first invention wherein the substance that controls (I) and / or (II) is a substance that inhibits (I) and / or (II).
  • the agent for controlling angiogenesis according to the first invention wherein the substance that controls (I) and / or (II) is a substance that promotes (I) and / or (II).
  • a method for screening an angiogenesis control agent comprising the following steps (M-1) to (M-3): (M-1) Step of coexisting BAZF gene and test substance (M-2) Step of confirming effect of test substance on expression of BAZF (M-3) Selection of test substance that affects expression of BAZF Step to do
  • ⁇ Fifth invention> A method for screening an angiogenesis control agent, comprising the following steps (N-1) to (N-3): (N-1) A step of causing a test substance to coexist with the following (A) and (B): (A) BAZF and / or a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF (B) CBF-1 And / or the step of confirming the influence of the polypeptide (N-2) test substance comprising the amino acid sequence corresponding to positions 1 to 178 of CBF-1 on the interaction between (A) and (B) (N -3) selecting a test substance that affects the interaction between (A) and (B)
  • kits for screening an angiogenesis control agent comprising the following (X) or (Y): (X) BAZF gene (Y) (y-1) and (y-2) below (Y-1) one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1, One or more selected from the group consisting of a polypeptide comprising an amino acid sequence corresponding to positions 1 to 178 of CBF-1, and a gene encoding any of these
  • the angiogenesis controlling agent of the present invention can control (inhibit or promote) angiogenesis, it is caused by unnecessary angiogenesis, such as cancer, diabetic retinopathy, rheumatism, and concomitant lack of angiogenesis. It is useful as a preventive or therapeutic agent for angiogenic diseases such as ischemic heart disease (angina, myocardial infarction, etc.), cerebral infarction, obstructive arteriosclerosis and the like.
  • the screening method for an angiogenesis controlling agent of the present invention can obtain an angiogenesis controlling agent targeting a downstream substance compared to VEGF, which has been the target of an angiogenesis inhibitor, and thus has a more adverse effect. Less medication can be obtained. Furthermore, the angiogenesis control ability of a candidate compound can be easily tested with the screening kit for an angiogenesis controlling agent of the present invention.
  • Notch1 signal can be temporarily blocked by stimulating HUVEC with VEGF.
  • VEGF can block the Notch1 signal, which is known to suppress angiogenesis, that is, the Notch1 signal pathway (brake) that has been considered to be an independent control mechanism. This means that there was an association between the VEGF signaling pathway (accelerator).
  • the present inventors have found that the suppression of HEY2, HES1 gene expression by VEGF stimulation is due to BAZF.
  • BAZF plays a key role in controlling “angiogenesis promoting action by VEGF” and also plays a role in controlling “angiogenesis inhibitory action by Notch1 signal”.
  • BAZF binds to the intracellular domain of Notch1 (N1ICD). It was found that it can bind to “CBF-1 which is a nuclear transcriptional regulator” (see Test Examples 6, 7, FIGS. 9, 10 and the like).
  • CBF-1 is a type of nuclear transcriptional regulator that is known to promote the expression of HEY1, HEY2, and HES1 genes in cooperation with Notch1 signal.
  • Test Examples 6 and 7 suggest that the promotion of angiogenesis by BAZF may be due to the inhibition of the interaction between N1ICD and CBF-1.
  • the angiogenesis controlling agent of the present invention can be obtained by a substance that controls the above (I) and / or (II).
  • the present inventors succeeded in narrowing down the amino acid sites involved in the interaction between BAZF and CBF-1 (see Test Example 8, FIGS. 11 to 15). That is, it was confirmed that there was a binding site in the polypeptide corresponding to positions 330 to 465 in the case of BAZF and in the polypeptide corresponding to positions 1 to 178 in the case of CBF-1.
  • FIG. 3 is a schematic diagram showing various BAZF partial fragments used for examining the binding region of CBF-1 in BAZF. It is a figure which shows that CBF-1 couple
  • FIG. 3 is a view showing that CBF-1 binds to a polypeptide region corresponding to positions 330 to 465 in the ZnF region of BAZF.
  • FIG. 3 is a schematic diagram showing various partial fragments of CBF-1 used for examining the binding region of BAZF in CBF-1. It is a figure which shows that BAZF bind
  • FIG. 3 is a schematic diagram of a plasmid vector pME18s- FLAG-BAZF for expressing FLAG-BAZF. The schematic diagram of a “pAxCAwt-BAZF” vector for expressing AdBAZF is shown. The schematic diagram of plasmid vector “BAZF-pIRES2-AcGFP-1” for expressing BAZF simultaneously with GFP is shown.
  • the angiogenesis controlling agent of the present invention is an angiogenesis controlling agent characterized by containing a substance that controls the following (I) and / or (II).
  • the angiogenesis control agent of the present invention includes preventive or therapeutic agents for diseases related to angiogenesis, in addition to experimental and research reagents for controlling angiogenesis in an experimental system such as in vitro.
  • CBF-1 means inhibiting the function of CBF-1 as a protein. More specifically, for example, by reducing the stability of CBF-1 as a protein, CBF- 1 means to inhibit interaction with N1ICD.
  • control includes “inhibition (suppression)” and “promotion”. Therefore, “substances that control (I) and / or (II)” include “substances that inhibit (I) and / or (II)” and “(I) and / or (II), “Promoting substances”.
  • siRNA gene antisense DNA, antisense RNA, decoy DNA, decoy RNA for BAZF gene
  • BAZF antibody antibody having an amino acid sequence portion corresponding to positions 330-465 of BAZF as an antigenic determinant
  • iv An amino acid sequence portion corresponding to positions 1-178 of CBF-1 as an antigenic determinant antibody
  • siRNA genes for the BAZF gene in (i) for example, SEQ ID NO: 1 (sense: 5 'aguuuaucuguaaauauaaTT 3') and SEQ ID NO: 2 (antisense: 5 'uuauauuuacagauaaacuGA 3') (both lower case letters represent RNA and upper case letters represent DNA) )), but is not limited to this.
  • the siRNA genes for other BAZF genes can be appropriately designed based on the knowledge of known RNAi technology.
  • Antisense DNA and antisense RNA for BAZF gene in (i) are oligonucleotides (including modified nucleotides) complementary to DNA sense strand and RNA of BAZF, and specifically for transcription and translation of BAZF gene It inhibits and can be appropriately designed by a known gene recombination technique or the like.
  • the decoy DNA and decoy RNA of (i) has a sequence similar to the transcriptional regulatory factor binding site on BAZF DNA, and binds to the transcriptional regulatory factor and binds the transcriptional regulatory factor to the BAZF DNA. It is a decoy that inhibits transcription, and as a result, specifically inhibits transcription, and can be appropriately designed by a known gene recombination technique or the like.
  • the antibodies (ii) to (iv) are prepared according to a known method using an amino acid sequence portion corresponding to positions 330-465 of BAZF and BAZF, an amino acid sequence portion corresponding to positions 1 to 178 of CBF-1, and the like as antigens.
  • the antibody can be prepared by immunizing an appropriate host or the like, but can also be purchased as a known antibody.
  • an antibody of Bcl-6 which is a homologue (congener) of BAZF, can be used.
  • Bc-7388 Bcl-6 (D-8)
  • sc-858 Bcl-6 (N-3)
  • BCL6B mouse polyclonal antibody A01 ( Abnova, catalog ID H00255877-A01) etc.
  • the term “gene” includes purine bases such as adenine (A) and guanine (G), pyrimidine bases such as thymine (T), uracil (U), and cytosine (C), and their modified bases.
  • the gene can be used in the form of a plasmid, a viral vector, etc. In this case, it may be single-stranded or double-stranded.
  • genes are artificially synthesized using a DNA synthesizer or transformed cells according to conventional methods, extract naturally-occurring polynucleotides, or delete some of the bases of extracted polynucleotides from nature , Substitution, addition, insertion, use of a sequence complementary to the target sequence and synthesis of the target sequence by reverse transcriptase, DNA polymerase, RNA polymerase, etc. It can be produced by a method such as modifying the base.
  • the antibody may be any of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a phage antibody.
  • the angiogenesis control agent of the present invention can contain other components in addition to the above-mentioned “substances that control (I) and / or (II)” within a range not inhibiting the purpose of the present invention. For example, the following may be mentioned.
  • Excipients include organic excipients and inorganic excipients.
  • Examples of the dosage form of the angiogenesis controlling agent of the present invention include tablets, capsules, granules, powders, pills, troches, syrups, and injections.
  • composition of active ingredients The content of the active ingredient (“the substance that controls (I) and / or (II)”) in the angiogenesis controlling agent of the present invention varies depending on the dosage form, and cannot be generally limited.
  • Various dosage forms In the case of a liquid preparation, for example, it is preferably 0.0001 to 10 (w / v%), more preferably 0.001 to 5 (w / v).
  • v%) preferably 0.0002 to 0.2 (w / v%), more preferably 0.001 to 0.1 (w / v%), although it can be prepared as 0.01 to 50 (w / w%), more preferably 0.02 to 20 (w / w%), etc., it is not necessarily limited to this range.
  • the angiogenesis controlling agent of the present invention can be formulated by a known method using the above components.
  • angiogenesis controlling agent of the present invention can be used as a combination with known angiogenesis controlling agents.
  • known angiogenesis control agents include the following.
  • ⁇ Angiogenesis inhibitors > Notch1, Notch1 gene, CBF-1, CBF-1 gene, HEY1, HEY1 gene, HEY2, HEY2 gene, HES1, HES1 inheritance, etc.
  • Notch1 signal inhibitors product name DAPT: ⁇ -secretase inhibitors such as N- [N- (3,5-Difluorophenacetyl-L-alanyl) -S-phenylglycine t-Butyl Ester)], VEGF, VEGF gene, etc.
  • DAPT is a substance known to block Notch1 signal by inhibiting the generation of its intracellular domain (N1ICD) by inhibiting the function of ⁇ -secretase to cleave the Notch1 membrane.
  • the administration route of the angiogenesis control agent of the present invention includes systemic administration and local administration, and any of them may be used. Specifically, oral administration, intravenous administration such as intravenous injection, intramuscular administration such as intramuscular injection, transdermal Administration, nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intrarectal administration, mucosal administration, inhalation, intraarticular administration, etc., which should be selected appropriately according to the disease or symptom of the therapeutic purpose Can do.
  • angiogenesis control agent of the present invention is in the form of a gene therapy agent containing siRNA or the like, for example, when a plasmid is used, a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), liposome Method, lipofectin method, microinjection method, calcium phosphate method, electroporation method and the like, and DNA vaccine method and liposome method are particularly preferable.
  • a viral vector When a viral vector is used, it can be carried out by incorporating a target gene into a virus according to a known method.
  • viruses used for viral vectors include detoxified retrovirus, adenovirus, adeno-associated (associated) virus, herpes virus, Sendai virus, vaccinia virus, poxvirus, poliovirus, symbis virus, SV40, immunodeficiency. And various DNA viruses or RNA viruses such as HIV virus (HIV).
  • retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are preferable, and adenoviruses with particularly high infection efficiency are preferable.
  • the gene In order to actually make a gene act as a medicine, in addition to the “in vivo method” in which the gene is directly introduced into the body, the gene is introduced into a cell collected from a human, and then the gene-introduced cell is returned to the body. And “ex vivo method”.
  • the “in vivo method” is preferable in terms of simplicity and cost and labor, and the “ex vivo method” is preferable in terms of high efficiency of gene introduction into cells.
  • the “in vivo method” is considered preferable.
  • an appropriate administration route can be selected according to the disease, symptom and the like for the purpose of treatment.
  • the administration route include vein, artery, subcutaneous, intradermal, intramuscular and the like.
  • in vivo method in the case of administration by “in vivo method”, for example, it can be in the form of a preparation such as a liquid.
  • a form such as an injection containing a gene is preferable, and various components commonly used in injections and the like can be added as necessary.
  • a liposome containing a gene or a membrane-fused liposome can be used in the form of a liposome preparation such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent.
  • a liposome preparation such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent.
  • the dose of the angiogenesis control agent of the present invention varies depending on the administration route, symptoms, age, body weight, form of the angiogenesis control agent, etc.
  • the amount of the active ingredient in the angiogenesis control agent requires treatment.
  • the active ingredient in the angiogenesis controlling agent of the present invention is a gene, for example, 0.0001 to 100 mg, preferably 0.001 to 10 mg, etc. as a gene, about once every several days to several months, etc. Administration is preferred.
  • diseases to which the angiogenesis controlling agent of the present invention can be applied include cancer, diabetic retinopathy, rheumatism and the like when the angiogenesis controlling agent is an angiogenesis inhibitor.
  • examples include ischemic heart disease (angina, myocardial infarction, etc.), cerebral infarction, obstructive arteriosclerosis, and the like.
  • the screening method for an angiogenesis controlling agent of the present invention is a screening method for an angiogenesis controlling agent characterized by including the following steps (M-1) to (M-3).
  • Another screening method for an angiogenesis controlling agent of the present invention is a screening method for an angiogenesis controlling agent characterized by including the following steps (N-1) to (N-3).
  • A Polypeptide containing amino acid sequence corresponding to positions 330-465 of BAZF and / or BAZF
  • B Polypeptide containing amino acid sequence corresponding to positions 1-178 of CBF-1 and / or CBF-1
  • Known methods include, for example, co-immunoprecipitation method, Western blot method, pull-down method using beads, method using mass spectrometer, imaging method using fluorescent or luminescent label, or yeast two-hybrid method. These can be used alone or in appropriate combination.
  • polypeptide containing an amino acid sequence corresponding to positions 330-465 of BAZF” in (A) is a polypeptide in which another amino acid is added before or after the amino acid sequence corresponding to positions 330-465 of BAZF. But it means good.
  • polypeptide containing an amino acid sequence corresponding to positions 1 to 178 of CBF-1 is also classified before and after the amino acid sequence corresponding to positions 1 to 178 of CBF-1. It means that a polypeptide to which is added an amino acid may be used.
  • polypeptide containing an amino acid sequence corresponding to positions 330-465 of BAZF As described above, the amino acid sites involved in the interaction with BAZF and CBF-1 can be used in the polypeptide corresponding to positions 330-465 of BAZF and positions 1-178 of CBF-1, respectively. This is because the present inventors have confirmed that the polypeptide is within the polypeptide corresponding to.
  • full-length (wild-type) BAZF and CBF-1, or a polypeptide close to this should be used. It is preferable to use it.
  • the screening kit for an angiogenesis controlling agent of the present invention comprises the following (X) or (Y).
  • (Y-1) one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1,
  • Y-2 one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1
  • the kit containing (X) can be used in the screening method including the steps (M-1) to (M-3).
  • kit containing (Y) can be used in the screening method including the steps (N-1) to (N-3).
  • (y-1) Is a full-length (wild-type) BAZF or a polypeptide encoding the same, or a gene encoding any of them
  • (y-2) is CBF-1, or a polypeptide close thereto, or any of these It is preferable to use genes encoding these in the kit.
  • the kit of the present invention includes various cells and media necessary for gene expression, various amino acids, and other methods for confirming protein interactions (co-immunoprecipitation, Western (Blot method, pull-down method using beads, method using mass spectrometer, imaging method using fluorescent or luminescent label, yeast two-hybrid method, etc.) Accordingly, it is preferable to include them appropriately.
  • the gene includes single-stranded or double-stranded DNA, cDNA, RNA, hybrids thereof, chimeras, and the like.
  • the gene expression level was measured using a quantitative-PCR method.
  • the HEY1 gene which is also the target gene for Notch signal, was not clearly suppressed by VEGF as compared to the HEY2 and HES1 genes, but the increase tended to be suppressed compared to the case where VEGF was not added. (Not shown).
  • VEGF-A 50 ng / ml is added to 2.6 x 10 4 (cells / cm 2 ) HUVEC, 37 ° C, CO 2 5% (fetal calf serum 10%, basic FGF 10 ng / ml, Heparin 18mU / MC, Hydrocortisone 1 ⁇ g / ml, L-Glutamine 10 mM, Amphotericin B 0.25 ⁇ g / ml, penicillin 60 ⁇ g / ml, MCDB131 medium containing streptomycin 100 ⁇ g / ml). The expression level was examined.
  • the BAZF expression level was measured by Western blotting using “Antibodies to BAZF” in Example 4 described later.
  • Example 3 Increase in the number of branch vessels by BAZF-1
  • the gene was introduced into HUVEC using the angiogenesis control agent (promoter) of Example 2 described later.
  • an angiogenesis control agent (promoter) (MOI 0 to 1000) of Example 2 described later is added to 2.6 ⁇ 10 4 (cells / cm 2 ) HUVEC, and the temperature is 37 ° C. and CO 2 5%.
  • MCDB131 medium containing fetal bovine serum 10%, basic FGF 10 ng / ml, Heparin 18 mU / ml, Hydrocortisone 1 ⁇ g / ml, L-Glutamine 10 mM, Amphotericin B 0.25 ⁇ g / ml, penicillin 60 ⁇ g / ml, streptomycin 100 ⁇ g / ml was cultured under the conditions of
  • the BAZF expression level was measured by Western blotting using “Antibodies to BAZF” in Example 4 described later.
  • FIGS. 3 and 4 show the relationship between the expression level of BAZF and the number of protrusions).
  • MOI multiple of infection
  • WB Anti-BAZF
  • FIG. 4 means Western blotting using an anti-BAZF antibody.
  • angiogenesis regulators BAZF gene and BAZF of the present invention are useful as angiogenesis promoters.
  • Example 4 Increase in the number of blood vessel branches by BAZF-2
  • the angiogenesis control agent (promoter) of Example 3 and Comparative Example 2 was introduced into HUVEC to which various concentrations (0, 0.1, 1, 10 ng / ml) of VEGF-A were added, respectively.
  • an angiogenesis control agent (inhibitor) of Example 3 (BAZF-pIRES2-AcGFP-1) or Comparative Example 2 (pIRES2-AcGFP-1) is applied to 2.6 ⁇ 10 4 (cells / cm 2 ) HUVEC.
  • the introduction was performed by a known lipofection method using a gene introduction reagent.
  • angiogenesis regulators BAZF gene and BAZF of the present invention are useful as angiogenesis promoters.
  • Example 5 Suppression of increase in the number of blood vessels by inhibiting BAZF expression
  • the angiogenesis control agent (inhibitor) of Example 1, Comparative Example 1 and Control Example 1 described later was introduced into HUVEC, and the influence on the formation of vascular endothelial network was examined.
  • Example 1 the angiogenesis control agent (inhibitor) of Example 1, Comparative Example 1, and Control Example 1 (each 50 ng / ml) was added to 2.6 ⁇ 10 4 (cells / cm 2 ) HUVEC, and the temperature was 37 ° C.
  • CO 2 5% (fetal bovine serum 10%, basic FGF 10 ng / ml, Heparin 18 mU / ml, Hydrocortisone 1 ⁇ g / ml, L-Glutamine 10 mM, Amphotericin B 0.25 ⁇ g / ml, penicillin 60 ⁇ g / ml, streptomycin 100 ⁇ g / ml
  • the culture was performed under the condition of MCDB131 medium containing ml).
  • angiogenesis regulator of the present invention acts as an angiogenesis inhibitor by inhibiting the expression of BAZF.
  • the upper and lower rows indicate the cell-cell adhesion (contact) portion.
  • angiogenesis controlling agent of the present invention can also be used as a Tip cell controlling agent.
  • the pME18s-FLAG-BAZF plasmid vector is represented in FIG.
  • IP in FIG. 9 means immunoprecipitation.
  • WB in FIG. 9 means Western blotting.
  • the anti-FLAG in FIG. 9 means an anti-FLAG antibody.
  • the anti-BAZF in FIG. 9 means an anti-BAZF antibody.
  • the anti-CBF-1 in FIG. 9 means an anti-CBF-1 antibody.
  • the detection band by the anti-BAZF antibody or the anti-CBF-1 antibody appeared at the position of the band corresponding to BAZF or CBF-1. This indicates that BAZF and CBF-1 are co-precipitated, that is, interact (coupled).
  • anti-FLAG mouse antibody (Monoclonal ANTI-FLAGR M2 antibody, Sigma-Aldrich Corporation, St. Louis, USA) was reacted with FLAG-BAZF, and then FITC-labeled secondary antibody (FITC-conjugated Affinipure Anti-mouse IgG (H + L) donkey antibody (catalog NO. 711-095-151, Jackson Immunoresearch Institute, Pennsylvania, USA) was used to confirm the location of BAZF.
  • FITC-labeled secondary antibody (FITC-conjugated Affinipure Anti-mouse IgG (H + L) donkey antibody (catalog NO. 711-095-151, Jackson Immunoresearch Institute, Pennsylvania, USA) was used to confirm the location of BAZF.
  • anti-CBF-1 rabbit antibody (SUH / CBF1 / RBP-J ⁇ rabbit polyclonal antibody, product code ab25949, Abcam, Cambridge, UK) and Cy3-labeled secondary antibody (Cy3-conjugated Affinipure (F (ab ') 2 Fragment® anti-rabbit IgG® (H + L) donkey antibody (catalog NO. 711-166-152, Jackson Immunoresearch Institute, Pennsylvania, USA) was used to confirm the location of CBF-1.
  • Cy3-conjugated Affinipure F (ab ') 2 Fragment® anti-rabbit IgG® (H + L) donkey antibody
  • FITC-labeled secondary antibody (detection of FLAG-BAZF): 488 nm (argon laser) / 505-530 nm (BP filter)
  • Cy3-labeled secondary antibody (detection of CBF-1): 543 nm (HeNe laser) / 560-615 nm
  • the rightmost “Hoechst33342” in FIG. 10 is a test result using Hoechst33342 (a cell membrane-permeable DNA-binding dye (Beckman Coulter, Tokyo, Japan)). It is a figure which shows that the localized part is a cell nucleus.
  • FIG. 12 shows that CBF-1 binds to the Zinc finger region, not the BTB region, of BAZF. Further, FIG. 13 shows that the Zinc finger region binds particularly to a region containing an amino acid sequence corresponding to positions 330-465 of BAZF.
  • FIG. 15 reveals that BAZF binds in the region of 178 amino acids on the amino terminal side, not in the repression domain having a repressor function of CBF-1 (the right diagram in FIG. 15).
  • anit-V5 in FIGS. 13 and 15 means the above-mentioned anti-V5 antibody.
  • BTB in FIGS. 11 and 12 means a BTB region in zinc finger protein (BAZF).
  • ZnF in FIGS. 11 to 13 means a Zn finger motif region in zinc (Zn) finger protein (BAZF).
  • ⁇ CT in FIGS. 11 and 13 means a fragment in which 15 amino acids have been deleted from the C-terminus.
  • V5-CBF-1 labeleled full length CBF-1).
  • Example 1 Angiogenesis controlling agent (inhibitor)
  • a double strand consisting of SEQ ID NO: 1 sense: 5 ′ aguuuaucuguaaauauaaTT 3 ′
  • SEQ ID NO: 2 antisense: 5 ′ uuauauuuacagauaaacuGA 3 ′
  • An angiogenesis control agent (inhibitor) containing the “siRNA gene for BAZF gene” was manufactured. Specifically, the procedure was as follows.
  • Comparative Example 1 also, in place of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 (sense: 5 'uucuccgaacgugucacguTT 3') and SEQ ID NO: 4 (antisense: 5 'acgugacacguucggagaaTT 3') (both lowercase letters represent RNA and uppercase letters represent DNA)
  • the angiogenesis control agent (inhibitor) of Comparative Example 1 was produced using a double-stranded “control siRNA gene” (trade name: Negative control siRNA 1027310, Qiagen, Dusseldorf, Germany).
  • Control Example 1 As Control Example 1 (Mock), 4 ⁇ l of CodeBreaker (registered trademark, Promega, Wisconsin, USA) and 625 ⁇ l of Opti MEM (Invitrogen Corporation, California, USA) were mixed, and only the reagent (CodeBreaker: registered trademark, Angiogenesis control agents (inhibitors) including Promega Corp., Wisconsin, USA) were manufactured and used in the above test examples.
  • CodeBreaker registered trademark, Angiogenesis control agents (inhibitors) including Promega Corp., Wisconsin, USA
  • Example 2 Angiogenesis controlling agent (promoter)
  • An adenovirus vector (AdBAZF) incorporating BAZF cDNA was prepared and used as an angiogenesis control agent (promoter) in Example 2.
  • pAxCAwt-BAZF in which a BAZF gene was incorporated was prepared in pAxCAwt (adenovirus expression vector, Takara Bio Inc., Japan) (FIG. 17), and this was used in HEK293 cells (human embryonic kidney cell-derived cell line).
  • AdBAZF was produced by expression in a recombinant adenovirus-producing host.
  • Example 3 Angiogenesis controlling agent (promoter)
  • the angiogenesis controlling agent (promoter) of Example 3 was used.
  • Example 4 BAZF antibody
  • New Zealand White Rabbit male was given 100 ⁇ g of the peptide fragment represented by SEQ ID NO: 6 (LSLPGGPEARGFAPLL: positions 78 to 93 of BAZF) corresponding to a part of the BTB domain of human-derived BAZF at a rate of once a week.
  • Immunization was performed for 5 weeks, blood was collected 1 week after the last immunization, serum was separated, and purified by affinity chromatography coupled with BAZF to obtain a polyclonal antibody against BAZF.
  • This antibody was used as a primary antibody for BAZF detection in the above test example.
  • This antibody can also be used as an angiogenesis controlling (inhibiting) agent of the present invention.
  • the present invention relates to an angiogenesis control agent, a screening method thereof, and a screening kit, and more particularly to an agent that inhibits or promotes angiogenesis involving BAZF, a screening method thereof, and a screening kit. It is.
  • SEQ ID NO: 1 BAZF siRNA sense: 5 'aguuuaucuguaaauauaaTT 3' Sequence number 2: BAZF siRNA antisense: 5 'uuauauuuacagauaaacuGA 3' Sequence number 3: Control siRNA sense: 5'uucuccgaac gugucacguT T3 ' Sequence number 4: Control siRNA antisense: 5'acgugacacg uucggagaaT T3 ' Sequence number 5: V5 Peptide Tag: GKPIPNPLLGLDST Sequence number 6: a part of BTB domain of human BAZF: LSLPGGPEARGFAPLL

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Abstract

Disclosed are: a novel anti-angiogenic agent; a method for screening for an anti-angiogenic agent; and a kit for screening for an anti-angiogenic agent. Specifically disclosed are: an anti-angiogenic agent comprising a substance capable of regulating (I) the expression of BAZF and/or (II) the inhibition of CBF-1 by BAZF; a method for screening for an anti-angiogenic agent, which utilizes the regulation of BAZF-related neovascularization; and a kit for screening for an anti-angiogenic agent.

Description

血管新生制御剤，及びそのスクリーニング方法，並びにスクリーニング用キットAngiogenesis control agent, screening method thereof, and screening kit
本発明は、血管新生制御剤，及びそのスクリーニング方法，並びにスクリーニング用キットに関するものであり、更に詳しくは、BAZFが関与する血管新生を阻害又は促進する薬剤，及びそのスクリーニング方法，並びにスクリーニング用キットに関するものである。 The present invention relates to an angiogenesis control agent, a screening method thereof, and a screening kit. More specifically, the present invention relates to an agent that inhibits or promotes angiogenesis involving BAZF, a screening method thereof, and a screening kit. Is.
従来から、癌の発育には、血管新生が関係していることが知られており、この血管新生の制御が、癌の治療にとって重要な役割を果たすと考えられていた。 Conventionally, it is known that angiogenesis is related to the growth of cancer, and this control of angiogenesis was considered to play an important role in the treatment of cancer.
血管新生の促進には、VEGF（Vascular endothelial growth factor：血管内皮細胞増殖因子）（非特許文献１）が関与していること知られていた。
また逆に、血管新生の阻害には、Notch１シグナル経路（非特許文献２）が関与していることが知られていた。 It has been known that VEGF (Vascular endothelial growth factor) (Non-Patent Document 1) is involved in promoting angiogenesis.
Conversely, it has been known that the inhibition of angiogenesis involves the Notch1 signal pathway (Non-patent Document 2).
しかし、これらは、それぞれ別個に認識されているに過ぎず、その相関については全く知られていなかった。
血管新生その他の生理現象には、複数の制御システムが関与していることが普通であるが、それらの制御システムは、必ずしも相関しているとは限らないからである。 However, these were only recognized separately, and their correlation was completely unknown.
This is because a plurality of control systems are usually involved in angiogenesis and other physiological phenomena, but these control systems are not always correlated.
一方、血管内皮細胞等にも発現が見られるタンパク質として、BAZF（Bcl-6 associated Zinc finger protein）が知られている。
このBAZFは、Bcl-6と同様の転写抑制活性を有するほか、Bcl-6とヘテロ２量体を形成して機能し、T細胞等の免疫系にも何らかの影響を及ぼすこと等が知られているが、血管新生との関わりは、全く知られていなかった（非特許文献３）。 On the other hand, BAZF (Bcl-6 associated Zinc finger protein) is known as a protein that is also expressed in vascular endothelial cells and the like.
This BAZF has the same transcriptional repression activity as Bcl-6, and functions by forming a heterodimer with Bcl-6, and is known to have some effect on the immune system such as T cells. However, the relationship with angiogenesis was not known at all (Non-patent Document 3).
本発明者等は、上述の課題を解決するべく鋭意検討を行った結果、ヒト臍帯静脈内皮細胞(HUVEC)をVEGFで刺激することによって、Notch1シグナルを一時的に遮断し得ること（つまり、血管新生の、アクセル（VEGF刺激）とブレーキ（Notch1シグナル伝達）の間に、ある種の相関があること），更には、その遮断に、VEGFシグナルによって発現が増加するBAZFが関与しており、BAZFが、Notch1シグナル伝達経路の下流にあるCBF-1に結合し、CBF-1を壊すタンパク質“Cull3（E-3リガーゼ）”とCBF-1との複合体形成を促進することによって、CBF-1の血管新生のブレーキ機能を解除すること等を見いだし、本発明を達成したものであって、その目的とするところは、新規な血管新生制御剤，及び血管新生制御剤のスクリーニング方法，並びにスクリーニング用キットを提供するにある。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have been able to temporarily block Notch1 signal by stimulating human umbilical vein endothelial cells (HUVEC) with VEGF (that is, blood vessels) BAZF, whose expression is increased by VEGF signaling, is involved in the blockade of neonatal accelerator (VEGF stimulation) and brake (Notch1 signaling)) CBF-1 by binding to CBF-1 downstream of the Notch1 signaling pathway and promoting the formation of a complex between CBF-1 and the protein “Cull3 (E-3 ligase)” that breaks CBF-1. The present invention has been achieved by releasing the angiogenesis brake function of the present invention, and the object of the present invention is to provide a novel angiogenesis control agent, a screening method for an angiogenesis control agent, and a screening Provide kit There is.
上述の目的は、下記の第一～第六の発明によって達成される。 The above object is achieved by the following first to sixth inventions.
〈第一の発明〉
下記（Ｉ）及び／又は（II）を、制御する物質を含むことを特徴とする、血管新生制御剤。
（Ｉ）BAZFの発現
（II）BAZFによるCBF-1の阻害 <First invention>
The angiogenesis control agent characterized by including the substance which controls following (I) and / or (II).
(I) Expression of BAZF (II) Inhibition of CBF-1 by BAZF
〈第二の発明〉
（Ｉ）及び／又は（II）を制御する物質が、（Ｉ）及び／又は（II）を、阻害する物質であることを特徴とする、第一の発明記載の、血管新生制御剤。 <Second invention>
The angiogenesis control agent according to the first invention, wherein the substance that controls (I) and / or (II) is a substance that inhibits (I) and / or (II).
〈第三の発明〉
（Ｉ）及び／又は（II）を制御する物質が、（Ｉ）及び／又は（II）を、促進する物質であることを特徴とする、第一の発明記載の、血管新生制御剤。 <Third invention>
The agent for controlling angiogenesis according to the first invention, wherein the substance that controls (I) and / or (II) is a substance that promotes (I) and / or (II).
〈第四の発明〉
下記（Ｍ－１）乃至（Ｍ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法。
（Ｍ－１）BAZF遺伝子と、被験物を共存させるステップ
（Ｍ－２）被験物が、BAZFの発現に及ぼす影響を確認するステップ
（Ｍ－３）BAZFの発現に影響を及ぼす被験物を選択するステップ <Fourth Invention>
A method for screening an angiogenesis control agent comprising the following steps (M-1) to (M-3):
(M-1) Step of coexisting BAZF gene and test substance (M-2) Step of confirming effect of test substance on expression of BAZF (M-3) Selection of test substance that affects expression of BAZF Step to do
〈第五の発明〉
下記（Ｎ－１）乃至（Ｎ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法。
（Ｎ－１）下記（Ａ）及び（Ｂ）と、被験物を共存させるステップ
　（Ａ）BAZF及び／又は、BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド
　（Ｂ）CBF-1及び／又は、CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド
（Ｎ－２）被験物が、（Ａ），（Ｂ）間の相互作用に及ぼす影響を確認するステップ
（Ｎ－３）（Ａ），（Ｂ）間の相互作用に影響を及ぼす被験物を選択するステップ <Fifth invention>
A method for screening an angiogenesis control agent, comprising the following steps (N-1) to (N-3):
(N-1) A step of causing a test substance to coexist with the following (A) and (B): (A) BAZF and / or a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF (B) CBF-1 And / or the step of confirming the influence of the polypeptide (N-2) test substance comprising the amino acid sequence corresponding to positions 1 to 178 of CBF-1 on the interaction between (A) and (B) (N -3) selecting a test substance that affects the interaction between (A) and (B)
〈第六の発明〉
下記（Ｘ）又は（Ｙ）を含むことを特徴とする、血管新生制御剤のスクリーニング用キット。
（Ｘ）BAZF遺伝子
（Ｙ）下記の（ｙ－１）及び（ｙ－２）
　（ｙ－１）BAZF，BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上
　（ｙ－２）CBF-1，CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上 <Sixth Invention>
A kit for screening an angiogenesis control agent, comprising the following (X) or (Y):
(X) BAZF gene (Y) (y-1) and (y-2) below
(Y-1) one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1, One or more selected from the group consisting of a polypeptide comprising an amino acid sequence corresponding to positions 1 to 178 of CBF-1, and a gene encoding any of these
本発明の血管新生制御剤は、血管新生を制御（阻害又は促進）できるため、不必要な血管新生が一因となる癌，糖尿病性網膜症，リューマチや、逆に血管新生の不足が原因となる虚血性心疾患（狭心症，心筋梗塞等），脳梗塞，閉塞性動脈硬化症等，血管新生に関わる疾患の予防又は治療剤として有用である。
また本発明の血管新生制御剤のスクリーニング方法は、従来、血管新生阻害剤の標的であったVEGFよりも、より下流の物質を標的とした血管新生制御剤を得ることができるため、より副作用の少ない薬剤を得ることができる。
更に、本発明の血管新生制御剤のスクリーニング用キットによって、候補化合物の血管新生制御能が、簡便に検査できる。 Since the angiogenesis controlling agent of the present invention can control (inhibit or promote) angiogenesis, it is caused by unnecessary angiogenesis, such as cancer, diabetic retinopathy, rheumatism, and concomitant lack of angiogenesis. It is useful as a preventive or therapeutic agent for angiogenic diseases such as ischemic heart disease (angina, myocardial infarction, etc.), cerebral infarction, obstructive arteriosclerosis and the like.
In addition, the screening method for an angiogenesis controlling agent of the present invention can obtain an angiogenesis controlling agent targeting a downstream substance compared to VEGF, which has been the target of an angiogenesis inhibitor, and thus has a more adverse effect. Less medication can be obtained.
Furthermore, the angiogenesis control ability of a candidate compound can be easily tested with the screening kit for an angiogenesis controlling agent of the present invention.
作用Action
本発明者等は、HUVECをVEGFで刺激することによって、Notch1シグナルを一時的に遮断し得ることを見いだした。 The present inventors have found that Notch1 signal can be temporarily blocked by stimulating HUVEC with VEGF.
すなわち、Notch1シグナルによって発現が促進される筈のHEY2遺伝子, HES1遺伝子の、発現抑制が認められた（試験例１，図１等参照）。 That is, the suppression of expression of moth HEY2 gene and moth HES1 gene whose expression is promoted by Notch1 signal was observed (see Test Example 1, FIG. 1, etc.).
つまり、このことは、血管新生の抑制をすることが知られているNotch1シグナルを、VEGFが遮断し得ること，つまり、これまで独立の制御機構と考えられて来たNotch1シグナル経路（ブレーキ）と、VEGFシグナル伝達経路（アクセル）の間に、関連があったこと意味している。 In other words, this means that VEGF can block the Notch1 signal, which is known to suppress angiogenesis, that is, the Notch1 signal pathway (brake) that has been considered to be an independent control mechanism. This means that there was an association between the VEGF signaling pathway (accelerator).
更に、本発明者等は、このVEGF刺激によるHEY2, HES1 遺伝子の発現抑制が、BAZFによるものであることを見いだした。 Furthermore, the present inventors have found that the suppression of HEY2, HES1 gene expression by VEGF stimulation is due to BAZF.
すなわち、
１）VEGFによる刺激によって、HUVEC におけるBAZFの発現量が増加すること（試験例２，図２等参照）
２）HUVEC へのBAZF遺伝子の導入によって、血管新生の一つの指標である「血管の分岐数」が上昇すること（試験例３，４，及び図３，４，５等参照）
３）VEGFを添加しても、BAZFの発現を阻害すれば、HUVECにおける血管分岐数は、増加しないこと（試験例５，図６，７，８等参照）
等が確認された。 That is,
1) Increased expression level of BAZF in HUVEC by stimulation with VEGF (see Test Example 2, Fig. 2 etc.)
2) By introducing the BAZF gene into HUVEC, the “number of branches of blood vessels”, which is an index of angiogenesis, is increased (see Test Examples 3, 4, and FIGS. 3, 4, 5, etc.)
3) Even if VEGF is added, if the expression of BAZF is inhibited, the number of blood vessels in HUVEC does not increase (see Test Example 5, FIGS. 6, 7, 8, etc.)
Etc. were confirmed.
つまり、これらは、BAZFが、「VEGFによる血管新生促進作用」の制御の鍵となると同時に、「Notch1シグナルによる血管新生阻害作用」の制御の役割をも担っていることを意味する。 That is, these mean that BAZF plays a key role in controlling “angiogenesis promoting action by VEGF” and also plays a role in controlling “angiogenesis inhibitory action by Notch1 signal”.
更に本発明者等は、BAZFが、どのようにNotch1シグナルを制御しているかについて検討を進めた結果、BAZFは、「Notch1の細胞内ドメイン（N1ICD）」との相互作用（結合）が知られている「核内転写調節因子であるCBF-1」と、結合し得ることを見いだした（試験例６，７，図９，１０等参照）。 Furthermore, as a result of investigations on how BAZF regulates Notch1 signal, the present inventors have found that BAZF interacts with (binding to) the intracellular domain of Notch1 (N1ICD). It was found that it can bind to “CBF-1 which is a nuclear transcriptional regulator” (see Test Examples 6, 7, FIGS. 9, 10 and the like).
CBF-1とは、Notch1シグナルと協調して、HEY1，HEY2, HES1 遺伝子の発現を促進していることが知られている、核内転写調節因子の一種である。 CBF-1 is a type of nuclear transcriptional regulator that is known to promote the expression of HEY1, HEY2, and HES1 genes in cooperation with Notch1 signal.
つまり、試験例６，７の結果は、BAZFによる血管新生の促進が、N1ICDとCBF-1の相互作用を阻害することによるものである可能性を示唆している。 That is, the results of Test Examples 6 and 7 suggest that the promotion of angiogenesis by BAZF may be due to the inhibition of the interaction between N1ICD and CBF-1.
従って、上記（Ｉ）及び／又は（II）を、制御する物質によって、本発明の血管新生制御剤を得ることが可能となる。 Therefore, the angiogenesis controlling agent of the present invention can be obtained by a substance that controls the above (I) and / or (II).
また、本発明者等は、BAZFとCBF-1との相互作用に関わるアミノ酸部位をそれぞれ絞り込むことに成功した（試験例８,図１１～１５等参照）
すなわち、BAZFの場合は、330-465位に相当するポリペプチド内，CBF-1の場合は、１-178位に相当するポリペプチド内に、それぞれ結合部位があることが確認できた。 In addition, the present inventors succeeded in narrowing down the amino acid sites involved in the interaction between BAZF and CBF-1 (see Test Example 8, FIGS. 11 to 15).
That is, it was confirmed that there was a binding site in the polypeptide corresponding to positions 330 to 465 in the case of BAZF and in the polypeptide corresponding to positions 1 to 178 in the case of CBF-1.
従って、全長の「BAZF」あるいは「BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド」と、「CBF-1」あるいは「CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド」との間の、相互作用を制御し得る物質であれば、血管新生を制御することができると考えられる。 Therefore, full-length “BAZF” or “polypeptide containing an amino acid sequence corresponding to positions 330-465 of BAZF” and “CBF-1” or “polypeptide containing an amino acid sequence corresponding to positions 1 to 178 of CBF-1” Any substance that can control the interaction with the “peptide” is considered to be able to control angiogenesis.
VEGF刺激による、HUVECにおける「Notch1シグナルの遮断」を示す図である。It is a figure which shows "blocking of Notch1 signal" in HUVEC by VEGF stimulation. VEGF刺激による、HUVEC における「BAZFの発現量の増加」を示す図である。It is a figure which shows "the increase in the expression level of BAZF" in HUVEC (s) by VEGF stimulation. アデノウイルスベクターを用いた、HUVEC へのBAZF遺伝子の導入による、「血管の分岐数」の上昇を、微分干渉顕微鏡写真によって示す図である。尚、図中の白いバーは、１００μｍを表す。It is a figure which shows the raise of the "branch number of the blood vessel" by introduction | transduction of the BAZF gene into HUVEC * using an adenovirus vector by a differential interference micrograph. In addition, the white bar in a figure represents 100 micrometers. 図３の「血管の分岐数」の上昇を数値によって示す図である。It is a figure which shows the raise of the "blood vessel branch number" of FIG. 外来VEGFの添加量毎（0，0.1，1，10ng/ml）の、HUVEC へのBAZF遺伝子導入による「血管の分岐数」の上昇の差を、微分干渉顕微鏡写真によって示す図である。It is a figure which shows the difference of the increase in "the number of branching of blood vessels" by BAZF gene introduction | transduction to HUVEC * for every addition amount (0, 0.1, 1, 10 ng / ml) of foreign VEGF by a differential interference micrograph. VEGF存在下での、BAZFの発現阻害による、HUVECにおける血管分岐数の増加防止効果を、微分干渉顕微鏡写真によって示す図である。尚、図中の白いバーは、５００μｍを表す。It is a figure which shows the increase prevention effect of the vascular branch number in HUVEC by the expression inhibition of BAZF in presence of VEGF by a differential interference micrograph. In addition, the white bar in a figure represents 500 micrometers. 図６の「HUVECにおける血管分岐数の増加防止効果」を、数値によって示す図である。尚、図中の＊＊＊は、「有意差検定：p＜0.001」を表す。It is a figure which shows the "inhibition effect of the increase in the number of blood vessel branches in HUVEC" of FIG. In the figure, *** represents “significant difference test: p <0.001”. VEGF存在下での、BAZFの発現阻害による、HUVECにおける血管分岐数の増加防止（細胞間ネットワークの形成阻害）効果の経時的な変化を、微分干渉顕微鏡下でのタイムラプスイメージによって示す図である。It is a figure which shows the time-dependent change by the time-lapse image under a differential interference microscope of the increase prevention of the number of blood vessels branching in HUVEC (inhibition of formation of an intercellular network) by BAZF expression inhibition in presence of VEGF. BAZFが、CBF-1に結合すことを示す図である。It is a figure which shows that BAZF couple | bonds with CBF-1. BAZFが、実際に細胞内で、CBF-1に結合し、共局在していることを、蛍光顕微鏡写真によって示す図である。It is a figure which shows that BAZF actually couple | bonded with CBF-1 and colocalized in a cell with a fluorescence micrograph. BAZF中の、CBF-1の結合領域を調べるために用いた、各種のBAZFの部分フラグメントを示す模式図である。FIG. 3 is a schematic diagram showing various BAZF partial fragments used for examining the binding region of CBF-1 in BAZF. CBF-1が、BAZFの、BTB領域では無く、ZnF領域に結合することを示す図である。It is a figure which shows that CBF-1 couple | bonds not with the BTB area | region but with a ZnF area | region of BAZF. CBF-1が、BAZFのZnF領域の中でも特に、330-465位に相当するポリペプチド領域内に結合することを示す図である。FIG. 3 is a view showing that CBF-1 binds to a polypeptide region corresponding to positions 330 to 465 in the ZnF region of BAZF. CBF-1中の、BAZFの結合領域を調べるために用いた、各種のCBF-1の部分フラグメントを示す模式図である。FIG. 3 is a schematic diagram showing various partial fragments of CBF-1 used for examining the binding region of BAZF in CBF-1. BAZFが、CBF-1の１-178位に相当するポリペプチド領域内に結合することを示す図である。It is a figure which shows that BAZF bind | bond | couples in the polypeptide area | region corresponding to 1-178 position of CBF-1. FLAG-BAZFを発現させるための、プラスミドベクターpME18s- FLAG-BAZFの模式図である。FIG. 3 is a schematic diagram of a plasmid vector pME18s- FLAG-BAZF for expressing FLAG-BAZF. AdBAZF を発現させるための、「pAxCAwt-BAZF」ベクターの模式図を表す。The schematic diagram of a “pAxCAwt-BAZF” vector for expressing AdBAZF is shown. BAZF をGFPと同時に発現させるための、プラスミドベクター「BAZF-pIRES2-AcGFP-1」の模式図を表す。The schematic diagram of plasmid vector “BAZF-pIRES2-AcGFP-1” for expressing BAZF simultaneously with GFP is shown.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
［血管新生制御剤］
本発明の血管新生制御剤は、下記（Ｉ）及び／又は（II）を、制御する物質を含むことを特徴とする、血管新生制御剤である。
本発明の血管新生制御剤には、試験管内等の実験系において血管新生を制御するための、実験・研究用試薬の他、血管新生に関わる疾患の予防又は治療薬も含まれる。 [Angiogenesis control agent]
The angiogenesis controlling agent of the present invention is an angiogenesis controlling agent characterized by containing a substance that controls the following (I) and / or (II).
The angiogenesis control agent of the present invention includes preventive or therapeutic agents for diseases related to angiogenesis, in addition to experimental and research reagents for controlling angiogenesis in an experimental system such as in vitro.
（Ｉ）BAZFの発現
（II）BAZFによるCBF-1の阻害 (I) Expression of BAZF (II) Inhibition of CBF-1 by BAZF
CBF-1の阻害とは、CBF-1のタンパク質としての機能を阻害することを意味するが、より具体的には、例えば、CBF-1のタンパク質としての安定性を低下させることによって、CBF-1が、N1ICDと相互作用することを阻害することを意味する。 The inhibition of CBF-1 means inhibiting the function of CBF-1 as a protein. More specifically, for example, by reducing the stability of CBF-1 as a protein, CBF- 1 means to inhibit interaction with N1ICD.
本発明において、「制御」には、「阻害（抑制）」及び「促進」が含まれる。
従って、「（Ｉ）及び／又は（II）を、制御する物質」としては、「（Ｉ）及び／又は（II）を、阻害する物質」及び「（Ｉ）及び／又は（II）を、促進する物質」が挙げられる。 In the present invention, “control” includes “inhibition (suppression)” and “promotion”.
Therefore, “substances that control (I) and / or (II)” include “substances that inhibit (I) and / or (II)” and “(I) and / or (II), “Promoting substances”.
（（Ｉ）及び／又は（II）を、阻害する物質）
（Ｉ）及び／又は（II）を、阻害する物質としては、具体的には、下記のようなものが挙げられるが、必ずしもこれらに限られるものでは無い。 (Substance that inhibits (I) and / or (II))
Specific examples of substances that inhibit (I) and / or (II) include, but are not limited to, the following.
（ｉ）BAZF遺伝子に対するsiRNA遺伝子，アンチセンスDNA，アンチセンスRNA，デコイDNA，デコイRNA
（ｉｉ）BAZF抗体
（ｉｉｉ）BAZFの330-465位に相当するアミノ酸配列部分を抗原決定基とする抗体
（ｉｖ）CBF-1の１-178位に相当するアミノ酸配列部分を抗原決定基とする抗体 (I) siRNA gene, antisense DNA, antisense RNA, decoy DNA, decoy RNA for BAZF gene
(Ii) BAZF antibody (iii) Antibody having an amino acid sequence portion corresponding to positions 330-465 of BAZF as an antigenic determinant (iv) An amino acid sequence portion corresponding to positions 1-178 of CBF-1 as an antigenic determinant antibody
（ｉ）のBAZF遺伝子に対するsiRNA遺伝子としては、例えば配列番号１（sense：5' aguuuaucuguaaauauaaTT 3'）及び配列番号２（antisense:5' uuauauuuacagauaaacuGA 3'）（いずれも小文字はRNA，大文字はDNAを表す。）からなる二本鎖の遺伝子等が挙げられるが、これに限られるものでは無い。
その他のBAZF遺伝子に対するsiRNA遺伝子は、公知のRNAi技術の知見に基づいて、適宜設計することが可能である。
（ｉ）のBAZF遺伝子に対するアンチセンスDNA，アンチセンスRNAとは、BAZFのDNAセンス鎖やRNAに相補的なオリゴヌクレオチド（修飾ヌクレオチドを含む）であって、BAZF遺伝子の転写や翻訳を特異的に阻害するものであり、公知の遺伝子組換技術等によって適宜設計することができる。
（ｉ）のデコイDNA，デコイRNAとは、BAZF DNA上の転写調節因子結合部位と類似の配列を有しており、転写調節因子と結合して転写調節因子がBAZFのDNAに結合するのを、おとりとなって阻害し、その結果、転写を特異的に阻害するものであり、公知の遺伝子組換技術等によって適宜設計することができる。 As siRNA genes for the BAZF gene in (i), for example, SEQ ID NO: 1 (sense: 5 'aguuuaucuguaaauauaaTT 3') and SEQ ID NO: 2 (antisense: 5 'uuauauuuacagauaaacuGA 3') (both lower case letters represent RNA and upper case letters represent DNA) )), But is not limited to this.
The siRNA genes for other BAZF genes can be appropriately designed based on the knowledge of known RNAi technology.
Antisense DNA and antisense RNA for BAZF gene in (i) are oligonucleotides (including modified nucleotides) complementary to DNA sense strand and RNA of BAZF, and specifically for transcription and translation of BAZF gene It inhibits and can be appropriately designed by a known gene recombination technique or the like.
The decoy DNA and decoy RNA of (i) has a sequence similar to the transcriptional regulatory factor binding site on BAZF DNA, and binds to the transcriptional regulatory factor and binds the transcriptional regulatory factor to the BAZF DNA. It is a decoy that inhibits transcription, and as a result, specifically inhibits transcription, and can be appropriately designed by a known gene recombination technique or the like.
（ｉｉ）～（ｉｖ）の抗体は、公知の方法に従い、BAZF，BAZFの330-465位に相当するアミノ酸配列部分，CBF-1の１-178位に相当するアミノ酸配列部分等を抗原として、適当な宿主に免疫すること等によって、作製することができるが、公知の抗体として購入することもできる。 The antibodies (ii) to (iv) are prepared according to a known method using an amino acid sequence portion corresponding to positions 330-465 of BAZF and BAZF, an amino acid sequence portion corresponding to positions 1 to 178 of CBF-1, and the like as antigens. The antibody can be prepared by immunizing an appropriate host or the like, but can also be purchased as a known antibody.
例えば、（ｉｉ）又は（ｉｉｉ）のBAZF抗体としては、BAZFのホモログ（同族体）であるBcl-6の抗体等も使用でき、例えば、「sc-7388（Bcl-6（D-8）」，（Santa Cruz Biotechnology,Inc.，カリフォルニア州，USA），「sc-858（Bcl-6（N-3）」（Santa Cruz Biotechnology,Inc.，カリフォルニア州，USA），BCL6B mouse polyclonal antibody A01, （Abnova社、catalog ID H00255877-A01）等として購入することができる。 For example, as the BAZF antibody of (ii) or (iii), an antibody of Bcl-6, which is a homologue (congener) of BAZF, can be used. For example, “sc-7388 (Bcl-6 (D-8)” , (Santa Cruz Biotechnology, Inc., California, USA), "sc-858 (Bcl-6 (N-3)" (Santa Cruz Biotechnology, Inc., California, USA), BCL6B mouse polyclonal antibody A01, ( Abnova, catalog ID H00255877-A01) etc.
（（Ｉ）及び／又は（II）を、促進する物質）
（Ｉ）及び／又は（II）を、促進する物質としては、具体的には、下記のようなものが挙げられるが、必ずしもこれらに限られるものでは無い。 (Substance that promotes (I) and / or (II))
Specific examples of substances that promote (I) and / or (II) include, but are not limited to, the following.
（ｉ）BAZF遺伝子
（ｉｉ）BAZFタンパク質 (I) BAZF gene (ii) BAZF protein
本発明において「遺伝子」とは、アデニン（A），グアニン（G）等のプリン塩基や、チミン（T），ウラシル（U），シトシン（C）等のピリミジン塩基やそれらの修飾塩基を構成要素として含むポリヌクレオチドであり、一本鎖又は二本鎖のDNA，cDNA，一本鎖又は二本鎖のRNA，一本鎖DNAと一本鎖RNAからなるハイブリッド体，RNAとDNAが結合して一本鎖となったキメラ体をも含むものである。 In the present invention, the term “gene” includes purine bases such as adenine (A) and guanine (G), pyrimidine bases such as thymine (T), uracil (U), and cytosine (C), and their modified bases. A single-stranded or double-stranded DNA, cDNA, single-stranded or double-stranded RNA, a hybrid composed of single-stranded DNA and single-stranded RNA, and RNA and DNA It also includes a single-stranded chimera.
尚、ヒトBAZFの、遺伝子及びアミノ酸の配列は、「Cloning and characterization of the human BAZF gene, a homologue of the BCL6 oncogene.（Biochem. Biophys. Res. Commun. 291 (3), 567-573 (2002)）」等に記載されており、また、「NCBI GENE accession number; NM 181844」として、NCBI（メリーランド州，米国）等から入手可能である。
  The gene and amino acid sequences of human BAZF are described in "Cloning and characterization of the human BAZF gene, a homologue of the BCL6 oncogene. (Biochem. Biophys. Res. Commun. 291 (3), 567-573 (2002). ) ", Etc., and is available from NCBI (Maryland, USA) as" NCBI GENE accession number; NM 181844 ".
遺伝子は、プラスミド，ウイルスベクター等の形態で用いることができ、その際には、一本鎖であっても二本鎖であっても構わない。 The gene can be used in the form of a plasmid, a viral vector, etc. In this case, it may be single-stranded or double-stranded.
これらの遺伝子は、常法に従い、DNA合成装置や形質転換細胞等を用いて人工的に合成する，天然に存在するポリヌクレオチドを抽出する，天然からの抽出ポリヌクレオチドの塩基の一部を欠失，置換，付加, 挿入する，目的とする配列と相補的な配列を用い、逆転写酵素やDNAポリメラーゼ，RNAポリメラーゼ等によって目的の配列のものを合成させる，これらの方法で得られたポリヌクレオチドの塩基を修飾する等の方法によって製造することができる。 These genes are artificially synthesized using a DNA synthesizer or transformed cells according to conventional methods, extract naturally-occurring polynucleotides, or delete some of the bases of extracted polynucleotides from nature , Substitution, addition, insertion, use of a sequence complementary to the target sequence and synthesis of the target sequence by reverse transcriptase, DNA polymerase, RNA polymerase, etc. It can be produced by a method such as modifying the base.
尚、本発明において抗体とは、モノクローナル、ポリクローナル、もしくはキメラ抗体やヒト化抗体のいずれであっても良く、ファージ抗体であっても良い。 In the present invention, the antibody may be any of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a phage antibody.
（その他の成分）
本発明の血管新生制御剤には、上記の、「（Ｉ）及び／又は（II）を制御する物質」の他、本発明の目的を阻害しない範囲で、他の成分を含有させることができ、例えば、以下のようなものが挙げられる。 (Other ingredients)
The angiogenesis control agent of the present invention can contain other components in addition to the above-mentioned “substances that control (I) and / or (II)” within a range not inhibiting the purpose of the present invention. For example, the following may be mentioned.
賦形剤，滑沢剤，結合剤，崩壊剤，安定剤，矯味矯臭剤，希釈剤，界面活性剤，乳化剤，可溶化剤，吸収促進剤，保湿剤，吸着剤，充填剤，増量剤，付湿剤，防腐剤等。
賦形剤としては、有機系賦形剤及び無機系賦形剤等が挙げられる。 Excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, humectants, adsorbents, fillers, extenders, Wetting agent, preservative, etc.
Examples of excipients include organic excipients and inorganic excipients.
（剤形）
本発明の血管新生制御剤の剤形は、例えば錠剤，カプセル剤，顆粒剤，散剤，丸剤，トローチ，もしくはシロップ剤，注射剤等の形態が挙げられる。 (Dosage form)
Examples of the dosage form of the angiogenesis controlling agent of the present invention include tablets, capsules, granules, powders, pills, troches, syrups, and injections.
（有効成分の含有量）
本発明の血管新生制御剤中の、有効成分（「（Ｉ）及び／又は（II）を制御する物質」）の含有量は、剤形によって様々であり、一概に限定できず、各種剤形化が可能な範囲で、投与量との関係で適宜選択すれば良いが、例えば液剤の場合、好ましくは０．０００１～１０（ｗ／ｖ％），より好ましくは０．００１～５（ｗ／ｖ％），特に注射剤の場合、好ましくは０．０００２～０．２（ｗ／ｖ％），より好ましくは０．００１～０．１（ｗ／ｖ％），固形剤の場合、好ましくは０．０１～５０（ｗ／ｗ％），より好ましくは０．０２～２０（ｗ／ｗ％）等として調製できるが、必ずしもこの範囲に限定されるものでは無い。 (Content of active ingredients)
The content of the active ingredient (“the substance that controls (I) and / or (II)”) in the angiogenesis controlling agent of the present invention varies depending on the dosage form, and cannot be generally limited. Various dosage forms In the case of a liquid preparation, for example, it is preferably 0.0001 to 10 (w / v%), more preferably 0.001 to 5 (w / v). v%), particularly in the case of injections, preferably 0.0002 to 0.2 (w / v%), more preferably 0.001 to 0.1 (w / v%), Although it can be prepared as 0.01 to 50 (w / w%), more preferably 0.02 to 20 (w / w%), etc., it is not necessarily limited to this range.
（製造方法）
本発明の血管新生制御剤は、上記の成分を用いて、周知の方法で製剤化することができる。 (Production method)
The angiogenesis controlling agent of the present invention can be formulated by a known method using the above components.
（合剤）
また、本発明の血管新生制御剤は、公知の血管新生制御剤との合剤とすることもできる。
公知の血管新生制御剤としては、例えば下記のようなものが挙げられる。 (Mixture)
In addition, the angiogenesis controlling agent of the present invention can be used as a combination with known angiogenesis controlling agents.
Examples of known angiogenesis control agents include the following.
〈血管新生阻害剤〉
Notch1，Notch1遺伝子，CBF-1，CBF-1遺伝子，HEY1，HEY1遺伝子，HEY2,HEY2遺伝子，HES1，HES1 遺伝等 <Angiogenesis inhibitors>
Notch1, Notch1 gene, CBF-1, CBF-1 gene, HEY1, HEY1 gene, HEY2, HEY2 gene, HES1, HES1 inheritance, etc.
〈血管新生促進剤〉
Notch1シグナル阻害剤（製品名DAPT：N-[N-（3,5-Difluorophenacetyl-L-alanyl）-S-phenylglycine t -Butyl Ester）等のγ－セクレターゼ阻害剤等]，VEGF，VEGF遺伝子等 <Angiogenesis promoter>
Notch1 signal inhibitors (product name DAPT: γ-secretase inhibitors such as N- [N- (3,5-Difluorophenacetyl-L-alanyl) -S-phenylglycine t-Butyl Ester)], VEGF, VEGF gene, etc.
DAPTは、γ－セクレターゼによるNotch1膜内部位切断機能を阻害することで、その細胞内ドメイン（N1ICD）の発生を抑制し、Notch1シグナルを遮断することが知られている物質である。 DAPT is a substance known to block Notch1 signal by inhibiting the generation of its intracellular domain (N1ICD) by inhibiting the function of γ-secretase to cleave the Notch1 membrane.
（投与経路）
本発明の血管新生制御剤の投与経路としては、全身投与と局所投与があり、いずれでも良く、具体的には、経口投与，静注等の静脈投与，筋注等の筋肉内投与,  経皮投与，経鼻投与，皮内投与，皮下投与，腹腔内投与，直腸内投与，粘膜投与，吸入，関節腔内投与等が挙げられ、治療目的の疾患，症状等に応じて、適宜選択することができる。 (Administration route)
The administration route of the angiogenesis control agent of the present invention includes systemic administration and local administration, and any of them may be used. Specifically, oral administration, intravenous administration such as intravenous injection, intramuscular administration such as intramuscular injection, transdermal Administration, nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intrarectal administration, mucosal administration, inhalation, intraarticular administration, etc., which should be selected appropriately according to the disease or symptom of the therapeutic purpose Can do.
（投与方法）
本発明の血管新生制御剤が、siRNA等を含む遺伝子治療剤の形態を取っている場合には、例えば、プラスミドを用いる場合、発現プラスミドを直接筋肉内に投与する方法（ＤＮＡワクチン法）, リポソーム法, リポフェクチン法, マイクロインジェクション法, リン酸カルシウム法, エレクトロポレーション法等が挙げられ、特にＤＮＡワクチン法, リポソーム法が好ましい。 (Method of administration)
When the angiogenesis control agent of the present invention is in the form of a gene therapy agent containing siRNA or the like, for example, when a plasmid is used, a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), liposome Method, lipofectin method, microinjection method, calcium phosphate method, electroporation method and the like, and DNA vaccine method and liposome method are particularly preferable.
ウイルスベクターを用いる場合、公知の方法に従って、ウイルスに、目的とする遺伝子を組み込むことによって行うことができる。 When a viral vector is used, it can be carried out by incorporating a target gene into a virus according to a known method.
ウイルスベクターに用いるウイルスとしては、例えば、無毒化した、レトロウイルス，アデノウイルス，アデノ関連（随伴）ウイルス，ヘルペスウイルス，センダイウイルス，ワクシニアウイルス，ポックスウイルス，ポリオウイルス，シンビスウイルス，ＳＶ４０，免疫不全症ウイルス（ＨＩＶ）等の、各種ＤＮＡウイルス又はＲＮＡウイルスが挙げられる。
ウイルスの中では、レトロウイルス，アデノウイルス，アデノ関連ウイルス，ワクシニアウイルス等が好ましく、特に感染効率が高いアデノウイルスが好ましい。 Examples of viruses used for viral vectors include detoxified retrovirus, adenovirus, adeno-associated (associated) virus, herpes virus, Sendai virus, vaccinia virus, poxvirus, poliovirus, symbis virus, SV40, immunodeficiency. And various DNA viruses or RNA viruses such as HIV virus (HIV).
Among viruses, retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are preferable, and adenoviruses with particularly high infection efficiency are preferable.
遺伝子を実際に医薬として作用させるには、当該遺伝子を直接体内に導入する「ｉｎ　ｖｉｖｏ法」の他、ヒトかから採集した細胞に当該遺伝子を導入し、その後、遺伝子導入細胞を体内に戻すという、「ｅｘ　ｖｉｖｏ法」等がある。
「ｉｎ　ｖｉｖｏ法」は費用や手間が少なく、簡便である点で好ましく、「ｅｘ　ｖｉｖｏ法」は、遺伝子の細胞内への導入効率が良いという点で好ましいが、血管新生を制御する際には、「ｉｎ　ｖｉｖｏ法」が好ましと考えられる。 In order to actually make a gene act as a medicine, in addition to the “in vivo method” in which the gene is directly introduced into the body, the gene is introduced into a cell collected from a human, and then the gene-introduced cell is returned to the body. And “ex vivo method”.
The “in vivo method” is preferable in terms of simplicity and cost and labor, and the “ex vivo method” is preferable in terms of high efficiency of gene introduction into cells. However, when controlling angiogenesis, The “in vivo method” is considered preferable.
「ｉｎ　ｖｉｖｏ法」により投与する場合は、治療目的の疾患，症状等に応じた適当な投与経路を選択することができる。投与経路としては、例えば、静脈，動脈，皮下，皮内，筋肉内等が挙げられる。 In the case of administration by “in vivo method”, an appropriate administration route can be selected according to the disease, symptom and the like for the purpose of treatment. Examples of the administration route include vein, artery, subcutaneous, intradermal, intramuscular and the like.
「ｉｎ　ｖｉｖｏ法」によって投与する場合は、例えば、液剤等の製剤形態をとることができる。一般的には遺伝子を含有する注射剤等の形態が好ましく、必要に応じて、注射剤等に常用されている各種の成分等を加えることもできる。 In the case of administration by “in vivo method”, for example, it can be in the form of a preparation such as a liquid. In general, a form such as an injection containing a gene is preferable, and various components commonly used in injections and the like can be added as necessary.
また、遺伝子を含有するリポソームまたは膜融合リポソーム（センダイウイルス（ＨＶＪ）－リポソーム等）においては、懸濁剤，凍結剤，遠心分離濃縮凍結剤等のリポソーム製剤の形態として用いることができる。 In addition, a liposome containing a gene or a membrane-fused liposome (such as Sendai virus (HVJ) -liposome) can be used in the form of a liposome preparation such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent.
（投与量）
本発明の血管新生制御剤の投与量は、投与経路，症状，年齢，体重，血管新生制御剤の形態等によって異なるが、例えば、血管新生制御剤中の有効成分の量が、処置を必要としている対象体重１ｋｇ当たり好ましくは０．００５～５００ｍｇ，より好ましくは、０．１～１００ｍｇ，但し、成人に対して１日あたり、下限として好ましくは０．０１ｍｇ（より好ましくは０．１ｍｇ），上限として、好ましくは２０ｇ（より好ましくは２０００ｍｇ，更に好ましくは５００ｍｇ，特に好ましくは１００ｍｇ）となるように、１回又は数回に分けて、症状に応じて投与することが望ましい。 (Dose)
The dose of the angiogenesis control agent of the present invention varies depending on the administration route, symptoms, age, body weight, form of the angiogenesis control agent, etc. For example, the amount of the active ingredient in the angiogenesis control agent requires treatment. Preferably 0.005 to 500 mg per kg of subject body weight, more preferably 0.1 to 100 mg, provided that the lower limit is preferably 0.01 mg (more preferably 0.1 mg) per day for an adult, upper limit As mentioned above, it is desirable to administer according to the symptom in a single dose or in several doses so that the dose is preferably 20 g (more preferably 2000 mg, still more preferably 500 mg, particularly preferably 100 mg).
尚、本発明の血管新生制御剤中の有効成分が、遺伝子の場合、例えば、遺伝子として０．０００１～１００ｍｇ，好ましくは、０．００１～１０ｍｇ等を、数日乃至数ヶ月に１回程度等投与するのが好ましい。 When the active ingredient in the angiogenesis controlling agent of the present invention is a gene, for example, 0.0001 to 100 mg, preferably 0.001 to 10 mg, etc. as a gene, about once every several days to several months, etc. Administration is preferred.
本発明の血管新生制御剤を適用し得る疾病としては、血管新生制御剤が、血管新生阻害剤の場合には、癌，糖尿病性網膜症，リューマチ等が挙げられる。
また、本発明の血管新生制御剤が、血管新生促進剤である場合には、虚血性心疾患（狭心症，心筋梗塞等），脳梗塞，閉塞性動脈硬化症等が挙げられる。 Examples of diseases to which the angiogenesis controlling agent of the present invention can be applied include cancer, diabetic retinopathy, rheumatism and the like when the angiogenesis controlling agent is an angiogenesis inhibitor.
In addition, when the angiogenesis controlling agent of the present invention is an angiogenesis promoter, examples include ischemic heart disease (angina, myocardial infarction, etc.), cerebral infarction, obstructive arteriosclerosis, and the like.
［血管新生制御剤のスクリーニング方法］
本発明の血管新生制御剤のスクリーニング方法は、下記（Ｍ－１）乃至（Ｍ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法である。 [Screening method for angiogenesis control agent]
The screening method for an angiogenesis controlling agent of the present invention is a screening method for an angiogenesis controlling agent characterized by including the following steps (M-1) to (M-3).
（Ｍ－１）BAZF遺伝子と、被験物を共存させるステップ
（Ｍ－２）被験物が、BAZFの発現に及ぼす影響を確認するステップ
（Ｍ－３）BAZFの発現に影響を及ぼす被験物を選択するステップ (M-1) Step of coexisting BAZF gene and test substance (M-2) Step of confirming effect of test substance on expression of BAZF (M-3) Selection of test substance that affects expression of BAZF Step to do
また、本発明の血管新生制御剤の他のスクリーニング方法は、下記（Ｎ－１）乃至（Ｎ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法である。 Another screening method for an angiogenesis controlling agent of the present invention is a screening method for an angiogenesis controlling agent characterized by including the following steps (N-1) to (N-3).
（Ｎ－１）下記（Ａ）及び（Ｂ）と、被験物を共存させるステップ (N-1) A step of causing the test object to coexist with the following (A) and (B)
　（Ａ）BAZF及び／又は、BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド
　（Ｂ）CBF-1及び／又は、CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド (A) Polypeptide containing amino acid sequence corresponding to positions 330-465 of BAZF and / or BAZF (B) Polypeptide containing amino acid sequence corresponding to positions 1-178 of CBF-1 and / or CBF-1
（Ｎ－２）被験物が、（Ａ），（Ｂ）間の相互作用に及ぼす影響を確認するステップ (N-2) Step of confirming the influence of the test substance on the interaction between (A) and (B)
（Ｎ－３）（Ａ），（Ｂ）間の相互作用に影響を及ぼす被験物を選択するステップ (N-3) selecting a test substance that affects the interaction between (A) and (B)
（Ｎ－２）において、被験物が、（Ａ），（Ｂ）間の相互作用に及ぼす影響を確認するには、（Ａ），（Ｂ）間の「相互作用」の有無を確認する事が必要となる。この「相互作用」の有無の確認は、例えば、（Ａ），（Ｂ）間（タンパク質間）の「結合」を確認することによって行うことができる。
タンパク質間の結合の確認は、公知の方法によって実施することができる。 In (N-2), in order to confirm the effect of the test substance on the interaction between (A) and (B), confirm the existence of “interaction” between (A) and (B). Is required. The presence / absence of this “interaction” can be confirmed, for example, by confirming the “binding” between (A) and (B) (between proteins).
Confirmation of binding between proteins can be performed by a known method.
公知の方法としては、例えば、共免疫沈降法，ウエスタンブロット法，ビーズを用いたプルダウン法，質量分析計を用いた方法，蛍光または発光標識を用いたイメージング方法，又は酵母ツーハイブリッド法等が挙げられ、これらを、それぞれ単独で，あるいは適宜組み合わせて用いることもできる。 Known methods include, for example, co-immunoprecipitation method, Western blot method, pull-down method using beads, method using mass spectrometer, imaging method using fluorescent or luminescent label, or yeast two-hybrid method. These can be used alone or in appropriate combination.
（Ａ）の「BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド」とは、BAZFの330-465位に相当するアミノ酸配列の前後に、更に別のアミノ酸が付加されているポリペプチドでも良いことを意味している。 The “polypeptide containing an amino acid sequence corresponding to positions 330-465 of BAZF” in (A) is a polypeptide in which another amino acid is added before or after the amino acid sequence corresponding to positions 330-465 of BAZF. But it means good.
また、（Ｂ）の「CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド」とは、同様に、CBF-1の１-178位に相当するアミノ酸配列の前後に、更に別のアミノ酸が付加されているポリペプチドでも良いことを意味している。 In addition, the “polypeptide containing an amino acid sequence corresponding to positions 1 to 178 of CBF-1” in (B) is also classified before and after the amino acid sequence corresponding to positions 1 to 178 of CBF-1. It means that a polypeptide to which is added an amino acid may be used.
尚、（Ａ）として、「BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド」を，（Ｂ）として、「CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド」を用いることができるのは、上述した通り、BAZF及びCBF-1との相互作用に関わるアミノ酸部位が、それぞれ、BAZFの330-465位に相当するポリペプチド内及びCBF-1の１-178位に相当するポリペプチド内にあることが本発明者等によって、確認されたからである。 As (A), “a polypeptide containing an amino acid sequence corresponding to positions 330-465 of BAZF”, and (B), “a polypeptide containing an amino acid sequence corresponding to positions 1 to 178 of CBF-1”. As described above, the amino acid sites involved in the interaction with BAZF and CBF-1 can be used in the polypeptide corresponding to positions 330-465 of BAZF and positions 1-178 of CBF-1, respectively. This is because the present inventors have confirmed that the polypeptide is within the polypeptide corresponding to.
但し、生体内と同じ血管新生制御機構を、より正確に再現し、より有効な血管新生制御剤をスクリーニングするには、全長（野生型）のBAZF及びCBF-1，もしくはこれに近いポリペプチドを用いることが好ましい。 However, in order to more accurately reproduce the same angiogenesis control mechanism as in vivo and screen for more effective angiogenesis control agents, full-length (wild-type) BAZF and CBF-1, or a polypeptide close to this, should be used. It is preferable to use it.
本発明で言うスクリーニングには、複数候補の中から、目的の血管新生制御剤を選択するための、いわゆる一次スクリーニングの他、公知の血管新生制御剤の、血管新生制御効果を確認するための、二次スクリーニング（再評価又は確認試験）も、含まれる。 In the screening referred to in the present invention, in addition to so-called primary screening for selecting a target angiogenesis controlling agent from among a plurality of candidates, for confirming the angiogenesis controlling effect of a known angiogenesis controlling agent, Secondary screening (re-evaluation or confirmation testing) is also included.
［血管新生制御剤のスクリーニング用キット］
本発明の血管新生制御剤のスクリーニング用キットは、下記（Ｘ）又は（Ｙ）を含むことを特徴とするものである。 [Angiogenesis control agent screening kit]
The screening kit for an angiogenesis controlling agent of the present invention comprises the following (X) or (Y).
（Ｘ）BAZF遺伝子
（Ｙ）下記の（ｙ－１）及び（ｙ－２） (X) BAZF gene (Y) (y-1) and (y-2) below
（ｙ－１）BAZF，BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上
（ｙ－２）CBF-1，CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上 (Y-1) one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1, One or more selected from the group consisting of a polypeptide comprising an amino acid sequence corresponding to positions 1 to 178 of CBF-1, and a gene encoding any of these
尚、（Ｘ）を含むキットは、（Ｍ－１）乃至（Ｍ－３）のステップを含む上記スクリーニング方法において用いることが出来る。 The kit containing (X) can be used in the screening method including the steps (M-1) to (M-3).
また、（Ｙ）を含むキットは、（Ｎ－１）乃至（Ｎ－３）のステップを含む上記スクリーニング方法において用いることが出来る。 Further, the kit containing (Y) can be used in the screening method including the steps (N-1) to (N-3).
但し、本発明のスクリーニング方法の項でも記載した通り、生体内と同じ血管新生制御機構を、より正確に再現し、より有効な血管新生制御剤をスクリーニングするためには、（ｙ－１）としては、全長（野生型）のBAZFもしくはこれに近いポリペプチド，あるいはこれらのいずれかをコードする遺伝子を、（ｙ－２）としては、CBF-1，もしくはこれに近いポリペプチド，あるいはこれらのいずれかをコードする遺伝子を、それぞれキットに用いることが好ましい。 However, as described in the section of the screening method of the present invention, in order to more accurately reproduce the same angiogenesis control mechanism as in vivo and to screen for a more effective angiogenesis control agent, (y-1) Is a full-length (wild-type) BAZF or a polypeptide encoding the same, or a gene encoding any of them, and (y-2) is CBF-1, or a polypeptide close thereto, or any of these It is preferable to use genes encoding these in the kit.
本発明のキットには、上記の遺伝子やタンパク質の他、遺伝子の発現に必要な各種の細胞や培地，各種のアミノ酸等の他、タンパク質相互作用の確認のための方法（共免疫沈降法，ウエスタンブロット法，ビーズを用いたプルダウン法，質量分析計を用いた方法，蛍光または発光標識を用いたイメージング方法，又は酵母ツーハイブリッド法等）において、一般的に必要とされる試薬等を、必要に応じて、適宜含ませることが好ましい。 In addition to the genes and proteins described above, the kit of the present invention includes various cells and media necessary for gene expression, various amino acids, and other methods for confirming protein interactions (co-immunoprecipitation, Western (Blot method, pull-down method using beads, method using mass spectrometer, imaging method using fluorescent or luminescent label, yeast two-hybrid method, etc.) Accordingly, it is preferable to include them appropriately.
尚、上記のスクリーニング方法やスクリーニング用キットにおいても、遺伝子とは、一本鎖又は二本鎖のDNA，cDNA，RNA，これらのハイブリッド体，キメラ体等を含むものである。 In the screening method and screening kit described above, the gene includes single-stranded or double-stranded DNA, cDNA, RNA, hybrids thereof, chimeras, and the like.
本発明の実施例を挙げるに先だって、本発明者等が確認した、VEGF刺激とNotch1シグナルの関係や、それとBAZFとの関わり等に関する試験結果を示す。 Prior to giving examples of the present invention, test results regarding the relationship between VEGF stimulation and Notch1 signal, the relationship with BAZF, etc., which were confirmed by the present inventors, are shown.
［試験例１：VEGFによる、Notch1シグナル伝達系の阻害］
2.6 x 104（cells／cm2）のHUVECに、VEGF-A（50 ng/ml）を添加し、37℃，CO2 5％(牛胎児血清 10％，basic FGF 10 ng/ml, Heparin 18mU/ml，Hydrocortisone 1μg/ml，L-Glutamine 10 mM，Amphotericin B 0.25μg/ml，ペニシリン60μg/ml，ストレプトマイシン 100μg/mlを含むMCDB131培地（インビトロジェン　コーポレーション，カリフォルニア州，米国）))という条件下、培養を行うことよって、Notch1 シグナルが、その発現を促進するHEY2遺伝子，HES1遺伝子の発現の経時的変化について検討した。
尚、VEGF-Aとは、A, B, C, D, Eと５つあるVEGFアイソフォームの一つである。 [Test Example 1: Inhibition of Notch1 signaling system by VEGF]
Add VEGF-A (50 ng / ml) to 2.6 x 10 4 (cells / cm 2 ) HUVEC, 37 ° C, CO 2 5% (fetal calf serum 10%, basic FGF 10 ng / ml, Heparin 18mU MCDB131 medium (Invitrogen Corporation, California, USA))) / ml, Hydrocortisone 1 μg / ml, L-Glutamine 10 mM, Amphotericin B 0.25 μg / ml, Penicillin 60 μg / ml, Streptomycin 100 μg / ml) As a result, we examined the temporal changes in the expression of the HEY2 gene and the HES1 gene, in which Notch1 signal promotes its expression.
VEGF-A is one of five VEGF isoforms, A, B, C, D, and E.
尚、遺伝子発現量は、定量-PCR法を用いて測定した。 The gene expression level was measured using a quantitative-PCR method.
その結果、添加後２－６時間といった比較的早い時間においてHEY2, HES1遺伝子の発現阻害が認められた（図１）。このことはVEGF 刺激が、一時的にNotch1 シグナルを遮断しうることを示している。 As a result, inhibition of HEY2 and HES1 gene expression was observed at a relatively early time such as 2-6 hours after addition (FIG. 1). This indicates that VEGF stimulation can temporarily block Notch1 signaling.
尚、同じくNotch シグナルの標的遺伝子であるHEY1遺伝子については、HEY2, HES1遺伝子ほど明確には、VEGFによる発現抑制は見られなかったが、VEGF未添加に比べると、増加が抑制される傾向にあった（図示せず）。 The HEY1 gene, which is also the target gene for Notch signal, was not clearly suppressed by VEGF as compared to the HEY2 and HES1 genes, but the increase tended to be suppressed compared to the case where VEGF was not added. (Not shown).
［試験例２：VEGF刺激によるBAZFの発現誘導］
2.6 x 104（cells／cm2）のHUVECに、VEGF-A（50 ng/ml）を添加し、37℃，CO2 5％ (牛胎児血清 10％、basic FGF 10 ng/ml, Heparin 18mU/ml，Hydrocortisone 1μg/ml，L-Glutamine 10 mM，Amphotericin B 0.25μg/ml，ペニシリン60μg/ml, ストレプトマイシン 100μg/mlを含むMCDB131培地)という条件下、培養を行うことよって、経時的なBAZFの発現量を調べた。 [Test Example 2: BAZF expression induction by VEGF stimulation]
VEGF-A (50 ng / ml) is added to 2.6 x 10 4 (cells / cm 2 ) HUVEC, 37 ° C, CO 2 5% (fetal calf serum 10%, basic FGF 10 ng / ml, Heparin 18mU / MC, Hydrocortisone 1 μg / ml, L-Glutamine 10 mM, Amphotericin B 0.25 μg / ml, penicillin 60 μg / ml, MCDB131 medium containing streptomycin 100 μg / ml). The expression level was examined.
BAZF発現量の測定は、後述する実施例４の「BAZFに対する抗体」を用いた、ウエスタンブロット法によって行った。 The BAZF expression level was measured by Western blotting using “Antibodies to BAZF” in Example 4 described later.
その結果、VEGF 添加後２時間くらいから、BAZFの発現量が上昇し、４時間をピークとする発現パターンが示された（図２）。 As a result, the expression level of BAZF increased from about 2 hours after the addition of VEGF and the expression pattern peaked at 4 hours was shown (FIG. 2).
尚、コントロールとして、βアクチンを用いた。 Note that β-actin was used as a control.
［試験例３：BAZFによる血管の分岐数の上昇－１］
後述する実施例２の血管新生制御剤（促進剤）を用いてHUVEC に遺伝子導入を行った。 [Test Example 3: Increase in the number of branch vessels by BAZF-1]
The gene was introduced into HUVEC using the angiogenesis control agent (promoter) of Example 2 described later.
具体的には、2.6 x 104（cells／cm2）のHUVECに、後述する実施例２の血管新生制御剤（促進剤）（MOI 0～1000）を添加し、37℃，CO2 5％ (牛胎児血清 10％，basic FGF 10 ng/ml，Heparin 18mU/ml，Hydrocortisone 1μg/ml，L-Glutamine 10 mM，Amphotericin B 0.25μg/ml，ペニシリン60μg/ml，ストレプトマイシン 100μg/mlを含むMCDB131培地)という条件下、培養を行った。 Specifically, an angiogenesis control agent (promoter) (MOI 0 to 1000) of Example 2 described later is added to 2.6 × 10 4 (cells / cm 2 ) HUVEC, and the temperature is 37 ° C. and CO 2 5%. (MCDB131 medium containing fetal bovine serum 10%, basic FGF 10 ng / ml, Heparin 18 mU / ml, Hydrocortisone 1 μg / ml, L-Glutamine 10 mM, Amphotericin B 0.25 μg / ml, penicillin 60 μg / ml, streptomycin 100 μg / ml ) Was cultured under the conditions of
BAZF発現量の測定は、後述する実施例４の「BAZFに対する抗体」を用いた、ウエスタンブロット法によって行った。 The BAZF expression level was measured by Western blotting using “Antibodies to BAZF” in Example 4 described later.
その結果、BAZFの過剰発現によって、外部からVEGF を添加していない状態でも、血管内皮細胞の突起形成伸長が認められた（図３，４はBAZF の発現量と突起数との関係を示す）。 As a result, overexpression of BAZF resulted in the formation of vascular endothelial cell protrusions even when VEGF was not added externally (FIGS. 3 and 4 show the relationship between the expression level of BAZF and the number of protrusions). .
尚、図３中のMOI（multiplicity of infection）とは、ベクター感染価（１細胞に感染が成立して１ヒット）を示す。
また、図４中の「ＷＢ：Anti-BAZF」とは、抗BAZF抗体を用いた、ウエスタンブロット法を意味する。 In addition, MOI (multiplicity of infection) in FIG. 3 shows a vector infectivity value (one hit when infection is established in one cell).
Further, “WB: Anti-BAZF” in FIG. 4 means Western blotting using an anti-BAZF antibody.
このことは、本発明の血管新生制御剤（BAZF遺伝子及びBAZF）が、血管新生促進剤として有用であることを示すものである。 This indicates that the angiogenesis regulators (BAZF gene and BAZF) of the present invention are useful as angiogenesis promoters.
［試験例４：BAZFによる血管の分岐数の上昇－２］
実施例３及び比較例２の血管新生制御剤（促進剤）を、それぞれ、各種濃度（0，0.1，1，10ng/ml）のVEGF-Aを添加したHUVECに導入した。 [Test Example 4: Increase in the number of blood vessel branches by BAZF-2]
The angiogenesis control agent (promoter) of Example 3 and Comparative Example 2 was introduced into HUVEC to which various concentrations (0, 0.1, 1, 10 ng / ml) of VEGF-A were added, respectively.
具体的には、2.6 x 104（cells／cm2）のHUVECに、実施例３（BAZF-pIRES2-AcGFP-1）又は比較例２（pIRES2-AcGFP-1）の血管新生制御剤（抑制剤）（30nM）を、各々添加し、37℃，CO2 5％ (牛胎児血清 10％，basic FGF 10 ng/ml，Heparin 18mU/ml，Hydrocortisone 1μg/ml，L-Glutamine 10 mM，Amphotericin B 0.25μg/ml，ペニシリン60μg/ml，ストレプトマイシン 100μg/mlを含むMCDB131培地)という条件下、培養を行った。 Specifically, an angiogenesis control agent (inhibitor) of Example 3 (BAZF-pIRES2-AcGFP-1) or Comparative Example 2 (pIRES2-AcGFP-1) is applied to 2.6 × 10 4 (cells / cm 2 ) HUVEC. ) (30 nM) each, 37 ° C, CO 2 5% (fetal bovine serum 10%, basic FGF 10 ng / ml, Heparin 18 mU / ml, Hydrocortisone 1 μg / ml, L-Glutamine 10 mM, Amphotericin B 0.25 The culture was performed under the conditions of μDB / ml, penicillin 60 μg / ml, and streptomycin 100 μg / ml MCDB131 medium).
導入は、遺伝子導入試薬を用いた、公知のリポフェクション法により行った。 The introduction was performed by a known lipofection method using a gene introduction reagent.
その結果、比較例２の血管新生制御剤（促進剤）を導入したHUVECと違って、実施例３の血管新生制御剤（促進剤）を導入し、BAZF を過剰発現したHUVEC では、通常なら反応しない低濃度VEGFにおいても、HUVEC間のネットワーク形成を認めた（図５）。 As a result, unlike HUVEC in which the angiogenesis control agent (promoter) of Comparative Example 2 was introduced, the angiogenesis control agent (promoter) in Example 3 was introduced, and HUVEC that overexpressed BAZF normally reacted. Even at low concentrations of VEGF, network formation between HUVECs was observed (FIG. 5).
このこともまた、本発明の血管新生制御剤（BAZF遺伝子及びBAZF）が、血管新生促進剤として有用であることを示すものである。 This also indicates that the angiogenesis regulators (BAZF gene and BAZF) of the present invention are useful as angiogenesis promoters.
［試験例５：BAZF発現阻害による血管分岐数の上昇抑制］
後述する実施例１，比較例１，対照例１の血管新生制御剤（阻害剤）をHUVEC に導入し、血管内皮ネットワーク形成への影響について検討した。 [Test Example 5: Suppression of increase in the number of blood vessels by inhibiting BAZF expression]
The angiogenesis control agent (inhibitor) of Example 1, Comparative Example 1 and Control Example 1 described later was introduced into HUVEC, and the influence on the formation of vascular endothelial network was examined.
具体的には、2.6x104（cells／cm2）のHUVECに実施例１，比較例１，対照例１の血管新生制御剤（阻害剤）（各50 ng/ml）を添加し、37℃，CO2 5％ (牛胎児血清 10％，basic FGF 10 ng/ml，Heparin 18mU/ml，Hydrocortisone 1μg/ml，L-Glutamine 10 mM，Amphotericin B 0.25μg/ml，ペニシリン60μg/ml, ストレプトマイシン 100μg/mlを含むMCDB131培地)という条件下、培養を行った。 Specifically, the angiogenesis control agent (inhibitor) of Example 1, Comparative Example 1, and Control Example 1 (each 50 ng / ml) was added to 2.6 × 10 4 (cells / cm 2 ) HUVEC, and the temperature was 37 ° C. , CO 2 5% (fetal bovine serum 10%, basic FGF 10 ng / ml, Heparin 18 mU / ml, Hydrocortisone 1 μg / ml, L-Glutamine 10 mM, Amphotericin B 0.25 μg / ml, penicillin 60 μg / ml, streptomycin 100 μg / ml The culture was performed under the condition of MCDB131 medium containing ml).
その結果、通常VEGF 刺激によってマトリゲル（登録商標，ベクトン・ディッキンソン・アンド・カンパニー，ニュージャージー州，米国）（コラーゲンを含むマトリックス）上でネットワーク形成を示す血管内皮は、実施例１によって、完全に阻害されることを確認した（図６，７）。 As a result, vascular endothelium showing network formation on Matrigel (registered trademark, Becton Dickinson & Company, NJ, USA) (matrix containing collagen) by normal VEGF stimulation was completely inhibited by Example 1. (Figs. 6 and 7).
つまり、このことは、本発明の血管新生制御剤（BAZFのsiRNA遺伝子）が、BAZF の発現阻害によって、血管新生阻害剤として作用することを示すものである。 That is, this indicates that the angiogenesis regulator of the present invention (BAZF siRNA gene) acts as an angiogenesis inhibitor by inhibiting the expression of BAZF.
更にその変化を経時的に観察したところ、比較例１を導入したコントロール細胞では、細胞同士が集積した後、突起を形成して近くの細胞とネットワークを形成した（図８上段）。
これに対して、実施例１を導入したBAZF 発現阻害細胞では、細胞集積後の突起形成が認められないままでいた（図８下段）。 Further, when the change was observed over time, in the control cells into which Comparative Example 1 was introduced, after the cells accumulated, protrusions were formed to form a network with nearby cells (upper part of FIG. 8).
In contrast, in the BAZF expression-inhibited cells into which Example 1 was introduced, no protrusion formation was observed after cell accumulation (the lower part of FIG. 8).
尚、図８上下段の、▲印は、細胞-細胞接着（コンタクト）部分を示す。 In FIG. 8, the upper and lower rows indicate the cell-cell adhesion (contact) portion.
このことは、血管新生開始時に突起を伸ばすTip cellの抑制を示唆するものであり、本発明の血管新生制御剤は、Tip cell制御剤としても用いることができることを示している。 This suggests the suppression of Tip cell that extends the protrusion at the start of angiogenesis, and the angiogenesis controlling agent of the present invention can also be used as a Tip cell controlling agent.
［試験例６：BAZFとCBF-1 との分子間相互作用の確認］
2.6x104（cells／cm2）のHUVECに、FLAGタグ（登録商標，シグマ－アルドリッチ・コーポレイション，セントルイス州，米国）のついたBAZF （FLAG-BAZF）を発現させるための、「pME18s-FLAG-BAZFプラスミドベクター」2.0μgを導入後、抗FLAG 抗体（Monoclonal ANTI-FLAGR M2 antibody，シグマ－アルドリッチ・コーポレイション，セントルイス州，米国）で免疫沈降を行った。 [Test Example 6: Confirmation of intermolecular interaction between BAZF and CBF-1]
To express BAZF (FLAG-BAZF) with a FLAG tag (registered trademark, Sigma-Aldrich Corporation, St. Louis, USA) in 2.6 × 10 4 (cells / cm 2 ) HUVEC, “pME18s-FLAG- After introducing 2.0 μg of “BAZF plasmid vector”, immunoprecipitation was performed with an anti-FLAG antibody (Monoclonal ANTI-FLAG® M2 antibody, Sigma-Aldrich Corporation, St. Louis, USA).
pME18s-FLAG-BAZFプラスミドベクターは、図１６で表される。 The pME18s-FLAG-BAZF plasmid vector is represented in FIG.
次に、細胞を磨り潰し、沈降してきたタンパク質を、後述する実施例４の抗BAZF抗体，あるいは抗CBF-1抗体（SUH/CBF1/RBP-Jκ Rabbit polyclonal antibody，製品コードab25949，Abcam社，ケンブリッジ，英国）を用いてウエスタンブロット法で分析した。結果を、図９に示す。 Next, the cells that were crushed and sedimented were treated with the anti-BAZF antibody or anti-CBF-1 antibody (SUH / CBF1 / RBP-Jκ Rabbit polyclonal antibody, product code ab25949, Abcam, Cambridge, Inc., described later). , UK) and Western blot analysis. The results are shown in FIG.
図９中のIPとは、免疫沈降を意味する。
図９中のWBとは、ウエスタンブロット法を意味する。
図９中のanti-FLAGとは、抗FLAG抗体を意味する。
図９中のanti-BAZFとは、抗BAZF抗体を意味する。
図９中のanti-CBF-1とは、抗CBF-1抗体を意味する。 IP in FIG. 9 means immunoprecipitation.
WB in FIG. 9 means Western blotting.
The anti-FLAG in FIG. 9 means an anti-FLAG antibody.
The anti-BAZF in FIG. 9 means an anti-BAZF antibody.
The anti-CBF-1 in FIG. 9 means an anti-CBF-1 antibody.
図９では、抗BAZF抗体あるいは抗CBF-1抗体による検出バンドが、それぞれBAZFあるいはCBF-1に相当するバンドの位置に現れていた。このことは、BAZFとCBF-1とが、共沈している，つまり相互作用（結合）していることを示している。 In FIG. 9, the detection band by the anti-BAZF antibody or the anti-CBF-1 antibody appeared at the position of the band corresponding to BAZF or CBF-1. This indicates that BAZF and CBF-1 are co-precipitated, that is, interact (coupled).
［試験例７：HUVEC におけるBAZF 及びCBF-1 の局在性の確認］
2.6x104（cells／cm2）のHUVECに、FLAGタグ（登録商標，シグマ－アルドリッチ・コーポレイション，セントルイス州，米国）のついたBAZF （FLAG-BAZF）を発現させるための、「pME18s-FLAG-BAZFプラスミドベクター」（図１６）2.0μｇを導入後、HUVEC におけるBAZF 及びCBF-1 の存在位置を、細胞免疫染色法にて検討したところ、細胞核内で点状に共局在することが確認された（図１０）。 [Test Example 7: Confirmation of localization of BAZF and CBF-1 in HUVEC]
To express BAZF (FLAG-BAZF) with a FLAG tag (registered trademark, Sigma-Aldrich Corporation, St. Louis, USA) in 2.6 × 10 4 (cells / cm 2 ) HUVEC, “pME18s-FLAG- After introducing 2.0 μg of “BAZF plasmid vector” (FIG. 16), the location of BAZF and CBF-1 in HUVEC was examined by cell immunostaining, and was confirmed to be co-localized in the form of dots in the cell nucleus. (FIG. 10).
具体的には、抗FLAG マウス抗体（Monoclonal ANTI-FLAGR M2 antibody，シグマ－アルドリッチ・コーポレイション，セントルイス州，米国）を、FLAG-BAZFに反応させ、次いで、FITC標識した二次抗体（FITC-conjugated Affinipure 抗マウスIgG (H+L)ロバ抗体 (catalog NO. 711-095-151，ジャクソン・イムノリサーチ研究所，ペンシルバニア州，米国,)）で、BAZFの存在位置を確認した。 Specifically, anti-FLAG mouse antibody (Monoclonal ANTI-FLAGR M2 antibody, Sigma-Aldrich Corporation, St. Louis, USA) was reacted with FLAG-BAZF, and then FITC-labeled secondary antibody (FITC-conjugated Affinipure Anti-mouse IgG (H + L) donkey antibody (catalog NO. 711-095-151, Jackson Immunoresearch Institute, Pennsylvania, USA) was used to confirm the location of BAZF.
また、抗CBF-1ウサギ抗体（SUH/CBF1/RBP-Jκ ウサギポリクローナル抗体，製品コードab25949，Abcam社，ケンブリッジ，英国）及びCy3標識した二次抗体(Cy3-conjugated Affinipure (F(ab’)2 Fragment 抗ウサギIgG (H+L)ロバ抗体 (catalog NO. 711-166-152，ジャクソン・イムノリサーチ研究所，ペンシルバニア州，米国)))を用いて、CBF-1の存在位置を確認した。 In addition, anti-CBF-1 rabbit antibody (SUH / CBF1 / RBP-Jκ rabbit polyclonal antibody, product code ab25949, Abcam, Cambridge, UK) and Cy3-labeled secondary antibody (Cy3-conjugated Affinipure (F (ab ') 2 Fragment® anti-rabbit IgG® (H + L) donkey antibody (catalog NO. 711-166-152, Jackson Immunoresearch Institute, Pennsylvania, USA)) was used to confirm the location of CBF-1.
FITC標識した二次抗体と、Cy3標識した二次抗体の検出は、それぞれ、次の波長条件で行った。
FITC標識した二次抗体（FLAG-BAZFの検出）：488 nm(アルゴンレーザー)/505-530 nm（BPフィルター）
Cy3標識した二次抗体（CBF-1の検出）：543nm(HeNeレーザー)/560-615 nm  The FITC-labeled secondary antibody and the Cy3-labeled secondary antibody were detected under the following wavelength conditions, respectively.
FITC-labeled secondary antibody (detection of FLAG-BAZF): 488 nm (argon laser) / 505-530 nm (BP filter)
Cy3-labeled secondary antibody (detection of CBF-1): 543 nm (HeNe laser) / 560-615 nm
尚、図１０中の、最右図の「Hoechst33342」とは、Hoechst33342（細胞膜浸透性のDNA結合色素（Beckman Coulter，東京，日本））を用いた試験結果であり、色素によって着色された染色体の局在する部分が、細胞核であることを示す図である。 The rightmost “Hoechst33342” in FIG. 10 is a test result using Hoechst33342 (a cell membrane-permeable DNA-binding dye (Beckman Coulter, Tokyo, Japan)). It is a figure which shows that the localized part is a cell nucleus.
Hoechstについては、以下の条件で検出した。
405 nm(レーザーダイオード)/420-480 nm Hoechst was detected under the following conditions.
405 nm (laser diode) / 420-480 nm
その結果、BAZFがCBF-1と、分子内で、共局在していることが確認された（図１０）。 As a result, it was confirmed that BAZF colocalized with CBF-1 in the molecule (FIG. 10).
［試験例８：BAZFとCBF-1の結合部位の確認］
BAZFとCBF-1の、一部アミノ酸配列を除いた各種の改良フラグメントを用い（図１１，図１４）、BAZFとCBF-1の結合領域を、ＧＳＴプルダウン法によって検討した。 [Test Example 8: Confirmation of binding site between BAZF and CBF-1]
Various improved fragments of BAZF and CBF-1 excluding some amino acid sequences were used (FIGS. 11 and 14), and the binding region of BAZF and CBF-1 was examined by the GST pull-down method.
（BAZFにおけるCBF-1の結合領域の検討）
具体的には、GST（グルタチオンＳトランスフェラーゼ）とCBF-1の融合タンパク質を大腸菌中で発現させ、この大腸菌を磨り潰したものをグルタチオンビーズ（Glutathione-Sepharose 4B beads（GE Healthcare，英国））と混合し、洗浄する。
そして、公知のペプチドタグであるV5タグ（GKPIPNPLLGLDST（配列番号５））で標識した各種のBAZFフラグメント（図１１）と混合後、洗浄し、電気泳動を行い、下記の抗体及び基質等を用いて検出した結果を、図１２（右図）及び図１３（右図）に示す。 (Examination of binding region of CBF-1 in BAZF)
Specifically, GST (glutathione S transferase) and CBF-1 fusion protein is expressed in E. coli, and then ground E. coli is mixed with glutathione beads (Glutathione-Sepharose 4B beads (GE Healthcare, UK)). And wash.
And after mixing with various BAZF fragments (FIG. 11) labeled with a V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 5)) which is a known peptide tag, washing, electrophoresis, and using the following antibodies and substrates, etc. The detection results are shown in FIG. 12 (right diagram) and FIG. 13 (right diagram).
［一次抗体］
抗V5マウスモノクローナル抗体（カタログ番号R960-25，インビトロジェン　コーポレーション，カリフォルニア州，米国）
［二次抗体］
抗マウスIgGウサギ抗体－HRP(西洋ワサビペルオキシダーゼ) －コンジュゲート (カタログ番号 W402B, プロメガ, Madison, WI, USA)
［HRP基質］
ECL Plus Western Blotting Detection Reagents（製品コードRPN2132, GE ヘルスケアバイオサイエンス(株)，英国） [Primary antibody]
Anti-V5 mouse monoclonal antibody (Cat. No. R960-25, Invitrogen Corporation, California, USA)
[Secondary antibody]
Anti-mouse IgG rabbit antibody-HRP (horseradish peroxidase) -conjugate (Cat. # W402B, Promega, Madison, WI, USA)
[HRP substrate]
ECL Plus Western Blotting Detection Reagents (product code RPN2132, GE Healthcare Biosciences, UK)
尚、比較のため、細胞中で、各フラグメント遺伝子を発現させた後、細胞を磨り潰して、その溶解物中のタンパク質を検出する、Cell lysates分析も行った（図１２左図，図１３左図）。 For comparison, Cell lysates analysis was also performed in which each fragment gene was expressed in the cell, and then the cell was ground to detect the protein in the lysate (FIG. 12 left diagram, FIG. 13 left). Figure).
図１２によって、CBF-1は、BAZFの、BTB領域では無く、Zinc フィンガー領域に結合することが分かった。
また、図１３によって、Zinc フィンガー領域の中でも、特にBAZFの330-465位に相当するアミノ酸配列を含む領域に結合することが分かった。 From FIG. 12, it was found that CBF-1 binds to the Zinc finger region, not the BTB region, of BAZF.
Further, FIG. 13 shows that the Zinc finger region binds particularly to a region containing an amino acid sequence corresponding to positions 330-465 of BAZF.
（CBF-1におけるBAZFの結合領域の検討）
上述のGSTとCBF-1の融合タンパク質に変えて、GSTとBAZFとの融合タンパク質を大腸菌中で発現させ、この大腸菌を磨り潰したものをグルタチオンビーズ（Glutathione-Sepharose 4B beads（GE Healthcare，英国））と混合し、洗浄する。
そして、V5タグで標識した各種のCBF-1フラグメント（図１４）を用いて、上記と同様の実験を行い、その結果を図１５に示した。 (Examination of BAZF binding region in CBF-1)
Instead of the GST and CBF-1 fusion protein described above, a GST and BAZF fusion protein is expressed in E. coli, and this E. coli is crushed with glutathione beads (Glutathione-Sepharose 4B beads (GE Healthcare, UK) ) And wash.
Then, an experiment similar to the above was performed using various CBF-1 fragments labeled with a V5 tag (FIG. 14), and the results are shown in FIG.
尚、ここでも、比較のため、Cell lysates分析を行った（図１５左図）。 In this case, Celllysates analysis was also performed for comparison (the left figure in FIG. 15).
図１５によって、BAZFは、CBF-1の、リプレッサー機能を有するリプレッションドメインでは無く、アミノ末端側の178アミノ酸の領域で結合することが明らかとなった（図１５右図）。 FIG. 15 reveals that BAZF binds in the region of 178 amino acids on the amino terminal side, not in the repression domain having a repressor function of CBF-1 (the right diagram in FIG. 15).
尚、図１３，１５中のanit-V5とは、上記の抗V5抗体を意味する。 In addition, anit-V5 in FIGS. 13 and 15 means the above-mentioned anti-V5 antibody.
図１１，１２中のBTBとは、ジンクフィンガータンパク質（BAZF）中の、BTB領域を意味する。 BTB in FIGS. 11 and 12 means a BTB region in zinc finger protein (BAZF).
図１１～１３中のZnFとは、ジンク(Zn)フィンガータンパク質（BAZF）中の、Znフィンガーモチーフ領域を意味する。 ZnF in FIGS. 11 to 13 means a Zn finger motif region in zinc (Zn) finger protein (BAZF).
図１１，１３中のΔCTとは、C末端から15個のアミノ酸を欠失させたフラグメントを意味する。 ΔCT in FIGS. 11 and 13 means a fragment in which 15 amino acids have been deleted from the C-terminus.
図１５中のFullとは、V5-CBF-1（標識した全長CBF-1）を意味する。 Full in FIG. 15 means V5-CBF-1 (labeled full length CBF-1).
［実施例１：血管新生制御剤（阻害剤）］
実施例１として、配列番号１（sense：5' aguuuaucuguaaauauaaTT 3'），及び配列番号２（antisense: 5' uuauauuuacagauaaacuGA 3'）（いずれも小文字はRNA，大文字はDNAを表す。）からなる二本鎖の「BAZF遺伝子に対するsiRNA遺伝子」を含有する、血管新生制御剤（阻害剤）を製造した。
具体的には、以下の様に行った。 [Example 1: Angiogenesis controlling agent (inhibitor)]
As Example 1, a double strand consisting of SEQ ID NO: 1 (sense: 5 ′ aguuuaucuguaaauauaaTT 3 ′) and SEQ ID NO: 2 (antisense: 5 ′ uuauauuuacagauaaacuGA 3 ′) (both lowercase letters represent RNA and uppercase letters represent DNA). An angiogenesis control agent (inhibitor) containing the “siRNA gene for BAZF gene” was manufactured.
Specifically, the procedure was as follows.
siRNAオリゴ専用のトランスフェクション試薬CodeBreaker（登録商標，プロメガ社，ウィスコンシン州，米国）4μlと、Opti MEM (登録商標，インビトロジェン　コーポレーション，カリフォルニア州，米国) 625μlを混合し、15分間室温でインキュベートした。これに、最終濃度が30 nM になるように配列番号１及び配列番号２からなる二本鎖siRNA遺伝子を添加し、ピペッティングで混合し、15分間室温でインキュベートすることによって、siRNA-CodeBreaker complexからなる、実施例１の血管新生制御剤（阻害剤）を製造した。 4 μl of transfection reagent CodeBreaker (registered trademark, Promega, Wisconsin, USA) dedicated to siRNA oligos and 625 μl of Opti MEM (registered trademark, Invitrogen Corporation, California, USA) were mixed and incubated at room temperature for 15 minutes. To this, add the double-stranded siRNA gene consisting of SEQ ID NO: 1 and SEQ ID NO: 2 to a final concentration of 30 nM, mix by pipetting, and incubate at room temperature for 15 minutes from the siRNA-CodeBreaker® complex. An angiogenesis controlling agent (inhibitor) of Example 1 was produced.
［比較例１］
また、配列番号１及び配列番号２に変えて、配列番号３（sense: 5' uucuccgaacgugucacguTT 3'）及び配列番号４（antisense: 5' acgugacacguucggagaaTT 3'）（いずれも小文字はRNA，大文字はDNAを表す。）で示される二本鎖の「コントロールsiRNA遺伝子」（商品名：Negative control siRNA 1027310，キアゲン社，デュッセルドルフ，ドイツ）を用いて、比較例１の血管新生制御剤（阻害剤）を製造した。 [Comparative Example 1]
Also, in place of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 (sense: 5 'uucuccgaacgugucacguTT 3') and SEQ ID NO: 4 (antisense: 5 'acgugacacguucggagaaTT 3') (both lowercase letters represent RNA and uppercase letters represent DNA) The angiogenesis control agent (inhibitor) of Comparative Example 1 was produced using a double-stranded “control siRNA gene” (trade name: Negative control siRNA 1027310, Qiagen, Dusseldorf, Germany).
［対照例１］
尚、対照例１（Mock）として、CodeBreaker（登録商標，プロメガ社，ウィスコンシン州，米国）4μlと、Opti MEM (インビトロジェン　コーポレーション，カリフォルニア州，米国) 625μlを混合し、試薬のみ（CodeBreaker：登録商標，プロメガ社，ウィスコンシン州，米国）を含む血管新生制御剤（阻害剤）を製造し、上述の試験例に用いた。 [Control Example 1]
As Control Example 1 (Mock), 4 μl of CodeBreaker (registered trademark, Promega, Wisconsin, USA) and 625 μl of Opti MEM (Invitrogen Corporation, California, USA) were mixed, and only the reagent (CodeBreaker: registered trademark, Angiogenesis control agents (inhibitors) including Promega Corp., Wisconsin, USA) were manufactured and used in the above test examples.
［実施例２：血管新生制御剤（促進剤）］
BAZF のcDNA を組み込んだアデノウイルスベクター（AdBAZF）を作製し、実施例２の血管新生制御剤（促進剤）とした。 [Example 2: Angiogenesis controlling agent (promoter)]
An adenovirus vector (AdBAZF) incorporating BAZF cDNA was prepared and used as an angiogenesis control agent (promoter) in Example 2.
具体的には、pAxCAwt（アデノウイスル発現ベクター，タカラバイオ株式会社，日本）に、BAZF遺伝子を組み込んだpAxCAwt-BAZFを作製し（図１７）、これをHEK293細胞（ヒト胎児腎細胞由来細胞株の、組換アデノウイスル作製用宿主。）中で発現させることによって、AdBAZFを作製した。 Specifically, pAxCAwt-BAZF in which a BAZF gene was incorporated was prepared in pAxCAwt (adenovirus expression vector, Takara Bio Inc., Japan) (FIG. 17), and this was used in HEK293 cells (human embryonic kidney cell-derived cell line). AdBAZF was produced by expression in a recombinant adenovirus-producing host.
［実施例３：血管新生制御剤（促進剤）］
CMVプロモーター等を含むプラスミドベクターである、pIRES2-AcGFP-1（タカラバイオ株式会社製，日本）に、BAZF のcDNA を組み込んだプラスミドベクター（BAZF-pIRES2-AcGFP-1）を作製し（図１８）、実施例３の血管新生制御剤（促進剤）とした。 [Example 3: Angiogenesis controlling agent (promoter)]
A plasmid vector (BAZF-pIRES2-AcGFP-1) in which BAZF cDNA is incorporated into pIRES2-AcGFP-1 (Takara Bio Inc., Japan), which is a plasmid vector containing a CMV promoter, is prepared (FIG. 18). The angiogenesis controlling agent (promoter) of Example 3 was used.
［比較例２］
また、BAZFのcDNAを導入しない、pIRES2-AcGFP-1プラスミドベクターを、比較例２とした。 [Comparative Example 2]
Further, a pIRES2-AcGFP-1 plasmid vector into which no BAZF cDNA was introduced was used as Comparative Example 2.
［実施例４：BAZF抗体］
ニュージーランドホワイトラビット（雄）に、ヒト由来BAZFのBTBドメインの一部に相当する配列番号６（LSLPGGPEARGFAPLL ：BAZFの78～93位）で表されるペプチドフラグメント１００μｇを、１週間に１回の割合で５週間免疫し、最後の免疫から１週間後に採血し血清を分離し、BAZFを結合させたアフィニティークロマトグラフィーで精製することによって、BAZFに対するポリクローナル抗体を得た。
この抗体を、上記試験例の、BAZF検出用一次抗体として用いた。
尚、この抗体は、本発明の血管新生制御（阻害）剤としても使用可能である。 [Example 4: BAZF antibody]
New Zealand White Rabbit (male) was given 100 μg of the peptide fragment represented by SEQ ID NO: 6 (LSLPGGPEARGFAPLL: positions 78 to 93 of BAZF) corresponding to a part of the BTB domain of human-derived BAZF at a rate of once a week. Immunization was performed for 5 weeks, blood was collected 1 week after the last immunization, serum was separated, and purified by affinity chromatography coupled with BAZF to obtain a polyclonal antibody against BAZF.
This antibody was used as a primary antibody for BAZF detection in the above test example.
This antibody can also be used as an angiogenesis controlling (inhibiting) agent of the present invention.
本発明は、血管新生制御剤，及びそのスクリーニング方法，並びにスクリーニング用キットに関するものであり、更に詳しくは、BAZFが関与する血管新生を、阻害又は促進する薬剤及びそのスクリーニング方法，スクリーニング用キットに関するものである。 The present invention relates to an angiogenesis control agent, a screening method thereof, and a screening kit, and more particularly to an agent that inhibits or promotes angiogenesis involving BAZF, a screening method thereof, and a screening kit. It is.
（小文字はRNA，大文字は、DNAを表す。）
配列番号１：BAZF siRNA sense：5' aguuuaucuguaaauauaaTT 3' 
配列番号２：BAZF siRNA antisense: 5' uuauauuuacagauaaacuGA 3'
配列番号３：Control siRNA sense: 5'uucuccgaac gugucacguT T3'
配列番号４：Control siRNA antisense: 5'acgugacacg uucggagaaT T3'
配列番号５：V5 Peptide Tag：GKPIPNPLLGLDST
配列番号６：a part of BTB domain of human BAZF：LSLPGGPEARGFAPLL (Lower case represents RNA, upper case represents DNA.)
SEQ ID NO: 1: BAZF siRNA sense: 5 'aguuuaucuguaaauauaaTT 3'
Sequence number 2: BAZF siRNA antisense: 5 'uuauauuuacagauaaacuGA 3'
Sequence number 3: Control siRNA sense: 5'uucuccgaac gugucacguT T3 '
Sequence number 4: Control siRNA antisense: 5'acgugacacg uucggagaaT T3 '
Sequence number 5: V5 Peptide Tag: GKPIPNPLLGLDST
Sequence number 6: a part of BTB domain of human BAZF: LSLPGGPEARGFAPLL

Claims (6)

  1. 下記（Ｉ）及び／又は（II）を、制御する物質を含むことを特徴とする、血管新生制御剤。
    （Ｉ）BAZFの発現
    （II）BAZFによるCBF-1の阻害     The angiogenesis control agent characterized by including the substance which controls following (I) and / or (II).
    (I) Expression of BAZF (II) Inhibition of CBF-1 by BAZF
  2. （Ｉ）及び／又は（II）を制御する物質が、（Ｉ）及び／又は（II）を、阻害する物質であることを特徴とする、請求項１記載の、血管新生制御剤。 The angiogenesis control agent according to claim 1, wherein the substance that controls (I) and / or (II) is a substance that inhibits (I) and / or (II).
  3. （Ｉ）及び／又は（II）を制御する物質が、（Ｉ）及び／又は（II）を、促進する物質であることを特徴とする、請求項１記載の、血管新生制御剤。 The angiogenesis control agent according to claim 1, wherein the substance that controls (I) and / or (II) is a substance that promotes (I) and / or (II).
  4. 下記（Ｍ－１）乃至（Ｍ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法。
    （Ｍ－１）BAZF遺伝子と、被験物を共存させるステップ
    （Ｍ－２）被験物が、BAZFの発現に及ぼす影響を確認するステップ
    （Ｍ－３）BAZFの発現に影響を及ぼす被験物を選択するステップ     A method for screening an angiogenesis control agent comprising the following steps (M-1) to (M-3):
    (M-1) Step of coexisting BAZF gene and test substance (M-2) Step of confirming effect of test substance on expression of BAZF (M-3) Selection of test substance that affects expression of BAZF Step to do
  5. 下記（Ｎ－１）乃至（Ｎ－３）のステップを含むことを特徴とする、血管新生制御剤のスクリーニング方法。
    （Ｎ－１）下記（Ａ）及び（Ｂ）と、被験物を共存させるステップ
    　（Ａ）BAZF及び／又は、BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド
    　（Ｂ）CBF-1及び／又は、CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド
    （Ｎ－２）被験物が、（Ａ），（Ｂ）間の相互作用に及ぼす影響を確認するステップ
    （Ｎ－３）（Ａ），（Ｂ）間の相互作用に影響を及ぼす被験物を選択するステップ     A method for screening an angiogenesis control agent, comprising the following steps (N-1) to (N-3):
    (N-1) A step of causing a test substance to coexist with the following (A) and (B): (A) BAZF and / or a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF (B) CBF-1 And / or the step of confirming the influence of the polypeptide (N-2) test substance comprising the amino acid sequence corresponding to positions 1 to 178 of CBF-1 on the interaction between (A) and (B) (N -3) selecting a test substance that affects the interaction between (A) and (B)
  6. 下記（Ｘ）又は（Ｙ）を含むことを特徴とする、血管新生制御剤のスクリーニング用キット。
    （Ｘ）BAZF遺伝子
    （Ｙ）下記の（ｙ－１）及び（ｙ－２）
    　（ｙ－１）BAZF，BAZFの330-465位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上
    　（ｙ－２）CBF-1，CBF-1の１-178位に相当するアミノ酸配列を含むポリペプチド，及びこれらのいずれかをコードする遺伝子からなる群から選択される一種以上     A kit for screening an angiogenesis control agent, comprising the following (X) or (Y):
    (X) BAZF gene (Y) (y-1) and (y-2) below
    (Y-1) one or more selected from the group consisting of BAZF, a polypeptide comprising an amino acid sequence corresponding to positions 330-465 of BAZF, and a gene encoding any of these (y-2) CBF-1, One or more selected from the group consisting of a polypeptide comprising an amino acid sequence corresponding to positions 1 to 178 of CBF-1, and a gene encoding any of these
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JP2015126722A (en) * 2013-12-28 2015-07-09 国立大学法人愛媛大学 New ubiquitin ligase and method of use thereof
WO2022092279A1 (en) * 2020-11-02 2022-05-05 カルナバイオサイエンス株式会社 siRNA AND PHARMACEUTICAL COMPOSITION, AND PROPHYLACTIC AND/OR THERAPEUTIC AGENT

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JP2004514404A (en) * 2000-03-22 2004-05-20 キュラゲン　コーポレイション Angiogenesis-related protein and nucleic acid encoding the same

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JP2004514404A (en) * 2000-03-22 2004-05-20 キュラゲン　コーポレイション Angiogenesis-related protein and nucleic acid encoding the same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015126722A (en) * 2013-12-28 2015-07-09 国立大学法人愛媛大学 New ubiquitin ligase and method of use thereof
WO2022092279A1 (en) * 2020-11-02 2022-05-05 カルナバイオサイエンス株式会社 siRNA AND PHARMACEUTICAL COMPOSITION, AND PROPHYLACTIC AND/OR THERAPEUTIC AGENT

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