JP2007284410A - Promoter of neurogenesis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicament for promoting neurogenesis, effective for treatment of neurologic disease. <P>SOLUTION: The promoter of the neurogenesis contains (1) (i) an HGF protein or (ii) a peptide being a partial peptide of the HGF protein, and having activities substantially equivalent to those of the HGF protein, or (2) (i) a DNA encoding the HGF protein, (ii) a DNA encoding the peptide being the partial peptide of the HGF protein, and having the activities substantially homogeneous to those of the HGF protein, or (iii) a DNA hybridizing with the DNA in a stringent condition, and encoding the protein or peptide having the activities substantially equivalent to those of the HGF protein as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an agent for promoting neurogenesis, and more particularly to an agent for promoting neurogenesis comprising as an active ingredient a DNA containing hepatocyte growth factor (hereinafter abbreviated as HGF) or a DNA encoding it.

  Once a neuron is differentiated and matured, it cannot divide, and neurodegeneration directly leads to a decrease in the number of neurons. For example, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or Huntington's disease are known as neurodegenerative diseases in which selective neuronal cell death occurs progressively. Currently, replacement therapies and symptomatic treatments that compensate for the depletion of neurotransmitters associated with neurodegeneration are used for the treatment of these diseases. However, these replacement therapy agents and symptomatic therapy agents do not suppress the progression of neurodegeneration, and their effects are gradually lost as the disease progresses. Therefore, development of a drug that suppresses the progression of neurodegeneration and promotes the regeneration of the remaining nerve is desired, but no drug having such an action has been found at present. In addition, it has been considered that neurogenesis is impossible when neurodegeneration occurs in the central nervous system for a long time. However, only recently it has been revealed that there are neural stem cells and neural progenitor cells capable of neurogenesis in the central nervous system of mature mammals including humans, activating endogenous neural stem cells. The possibility of regenerating the damaged nerve tissue and function has begun to be examined (for example, Non-Patent Documents 1 and 2).

  On the other hand, HGF was first identified as a powerful mitogen for mature hepatocytes, and its gene cloning was performed in 1989 (see Non-Patent Documents 3 and 4). Administration of HGF prevents endotoxin-induced lethal liver injury in mice with fulminant hepatitis by anti-apoptotic activity, and HGF gene therapy can improve the survival rate of rats with fatal cirrhosis (Non-Patent Document 5, 6). In addition, many recent studies in expression and functional analysis including the knockout / knock-in mouse technique have revealed that HGF is a novel neurotrophic factor (see Non-Patent Documents 7 and 8). In particular, HGF is known as one of the most powerful survival promoting factors for motor neurons comparable to glial cell line-derived neurotrophic factor (GDNF) in vitro (see Non-Patent Document 9).

In addition, Patent Document 1 discloses that a rat mesencephalic dopaminergic neuron cell death-induced rat that specifically kills mesocerebral substantia nigra dopamine neuron degenerated by Parkinson's disease is used to induce neurodegenerative disease of the HGF gene. Examples in which the effects are examined behaviorally and histologically are shown. Specifically, pre-administration of the HGF gene protected the midbrain substantia nigra dopamine neurons from the neurotoxin 6-hydroxydopamine (6-OHDA), and suppressed the symptoms of the midbrain nigra dopamine neuron cell death-induced rats It is shown. And in this patent document 1, based on such an experimental result, it describes that the HGF gene has a protective effect with respect to a dopamine neuron. Patent Document 2 describes that HGF has two actions, a direct neurotrophic factor activity for motor neurons and an indirect improvement action (neuroprotective action) of glutamate cytotoxicity for motor neurons. However, it cannot be predicted at all that HGF promotes the neurogenesis of nerve cells just because HGF exhibits a neuroprotective action against the neurotoxins 6-OHDA and glutamine.
Furthermore, Patent Document 3 describes a therapeutic agent for cranial neuropathy containing HGF as an active ingredient, and HGF prolongs the survival of nerve cells, but the DNA of rat pheochromocytoma PC12 cells, which are cells derived from nerve cells. It states that it does not promote synthesis. If reduced, HGF does not promote PC12 cell neogenesis. From this, too, it cannot be predicted that HGF promotes the neurogenesis of nerve cells.
International Publication No. 2003/045439 Pamphlet JP 2002-87983 A Japanese Patent No. 3680114 Nature Medicine, 1998, Vol. 4, 1313-1317. Nature Medicine, 2000, Vol. 6, pp. 271-277 Nakamura et al., Biochemical and Biophysical and Research Communications (Biochem. Biophys. Res. Commun.), 1984, Vol. 122, p. 1450-1459 Nakamura et al., Nature, 1989, 342, p. 440-443 Kosai et al., Hepatology, 1999, 30th, p. 151-159 Ueki et al., Nature Medicine, 1999, Vol. 5, p. 226-230. Matsumoto et al., Ciba Found. Symp., 1997, Vol. 212, p. 198-211; Discussion 211-194 Funakoshi et al., Clin. Chim. Acta., 2003, Vol. 327, p. 1-23 Neuron, 1996, Vol. 17, p. 1157-1172

  An object of the present invention is to provide a drug effective for neurogenesis.

  As a result of various studies to solve the above-mentioned problems, the present inventors have found that the HGF protein or a partial peptide of the HGF protein that has substantially the same activity as the HGF protein (hereinafter referred to as HGF protein or the like). Or a DNA that hybridizes under stringent conditions with a DNA that encodes them or a DNA comprising a base sequence complementary to the DNA, and has substantially the same activity as the HGF protein. It has been found that DNA containing DNA encoding a protein (hereinafter sometimes referred to as HGF gene) has a neurogenesis effect, and has been further studied to complete the present invention.

The present inventors first examined the involvement of HGF protein or the like or HGF gene in neurogenesis using R6 / 2-transformed mice as model mice for inducing neuronal cell death.
The present inventors introduced the rat HGF gene into the striatum in R6 / 2 transformed mice using a vector (type I herpes simplex virus (HSV-1) vector) that has neurotropism and lacks replication ability. Then, R6 / 2-transformed mice that express rat HGF protein in the striatum were prepared, and whether or not the HGF gene actually showed an effect on neurogenesis was examined using the mice. For neurogenesis, new neurons are born and cell division must be performed. During cell division, DNA is synthesized to copy genetic information. There is BrdU (bromodeoxyuridine) as a material for knowing whether or not DNA has been synthesized. When BrdU is injected into the body, this newly-born nerve (cell) is taken up by BrdU. Therefore, the presence or absence of neurogenesis can be determined using BrdU as an index. Thus, when BrdU was administered to R6 / 2-transformed mice expressing the rat HGF protein and BrdU incorporated into the striatum nerve cells was measured, it was found that at least the nerves were born in the striatum. It was. Further, in a differentiation assay using cultured neural stem cells (neurosphere), it has been found that HGF receptors are present in neural stem cells, neural cells differentiated from the neural stem cells, and astrocyte progenitor cells. Further, in a differentiation assay using cultured neural stem cells (neurosphere), HGF promotes differentiation from neural stem cells to neoplastic neurons (increase of neoplastic neurons), and nerve fiber elongation and neurites in differentiated neurons. Found to promote the branching.
Such a neurogenic action of the HGF protein or HGF gene was found for the first time in the present invention, and the inventors have further studied based on these findings and completed the present invention.

That is, the present invention
[1] (1) (a) HGF protein or (b) a peptide having a substantially the same activity as HGF protein or a salt thereof, or (2) (a) HGF protein DNA encoding (b) a partial peptide of HGF protein and encoding a peptide having substantially the same activity as HGF protein, or (c) hybridizing with DNA under stringent conditions, and HGF A neurogenesis-promoting agent comprising, as an active ingredient, a DNA comprising a DNA encoding a protein or peptide having substantially the same activity as a protein,
[2] The active ingredient is (b) a DNA encoding an HGF protein or (b) a DNA encoding a peptide that is a partial peptide of the HGF protein and has substantially the same activity as the HGF protein, or (c) the DNA. [1] characterized in that it is a DNA encoding a protein or peptide that hybridizes under stringent conditions with a DNA comprising a complementary base sequence and has substantially the same activity as the HGF protein. The described neurogenesis-promoting agent,
[3] DNA encoding HGF protein is (a) DNA consisting of the base sequence represented by SEQ ID NO: 1, 2, or 5 or (b) DNA consisting of a base sequence complementary to the base sequence of (a) above The neurogenesis promoting agent according to the above-mentioned [2], which comprises a DNA that encodes a protein that hybridizes under stringent conditions with and encodes a protein having substantially the same quality of activity as the HGF protein,
[4] The neurogenesis promoting agent according to [2] or [3] above, wherein the DNA is incorporated into a type I herpes simplex virus (HSV-1) vector, an adenovirus vector or an adeno-associated virus vector ,
[5] The above-mentioned [1], wherein the active ingredient is (a) an HGF protein or (b) a peptide having partial activity of the HGF protein and having substantially the same activity as the HGF protein, or a salt thereof. ] The neurogenesis-promoting agent,
[6] The HGF protein is a protein comprising (a) an amino acid sequence represented by SEQ ID NO: 3, 4 or 6, or (b) a protein comprising substantially the same amino acid sequence as the amino acid sequence. The neurogenesis promoting agent according to the above [5],
[7] The neurogenesis promoting agent according to any one of [1] to [6], wherein the neurogenesis promoting agent is for topical administration,
[8] The neurogenesis promoting agent according to the above [7], wherein the local administration is intrathecal administration,
[9] The neurogenesis promoting agent according to any one of the above [1] to [8], which is used in combination with a neurotrophic factor, and [10] The neurotrophic factor is NT-3, NT -4, neurogenesis promoting agent according to [9] above, which is at least one selected from CNTF, GDNF and BDNF,
About.

  The present invention also relates to (1) (a) an HGF protein or (b) a peptide having partial activity of the HGF protein and having substantially the same activity as the HGF protein, or a salt thereof, or (2) (a) HGF DNA encoding a protein or (b) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) hybridizing with the DNA under stringent conditions, The present invention also relates to a method for promoting neurogenesis, which comprises administering to a mammal a DNA comprising a DNA encoding a protein or peptide having substantially the same activity as that of an HGF protein. Furthermore, the present invention provides (1) (a) an HGF protein or (b) a peptide having a substantially the same activity as the HGF protein, or (b) an HGF protein partial peptide for producing a medicament for promoting neurogenesis These salts, or (2) (a) DNA encoding an HGF protein or (b) DNA encoding a peptide that is a partial peptide of the HGF protein and has substantially the same activity as the HGF protein, or (c) The present invention relates to the use of DNA containing DNA encoding a protein or peptide that hybridizes with DNA under stringent conditions and has substantially the same activity as HGF protein.

  The neurogenesis-promoting agent of the present invention can promote the neurogenesis of a new nerve at a site where neuronal degeneration or neuronal cell death is induced in a neurodegenerative disease or neurological disorder associated with neuronal degeneration or neuronal cell death.

  In the present invention, “DNA encoding HGF protein” refers to DNA capable of expressing HGF protein. Examples of DNA containing DNA encoding HGF protein include Nature, 342, 440 (1989); Japanese Patent No. 2777678; Biochem. Biophys, Res. Commun. 1989, 163, p. 967-973; Proc. Natl. Acad. Sci. U. S. A. 1991, 88 (16), p. 7001-7005 etc., for example, Accession No. in GeneBank / EMBL / DDBJ. Preferred examples include DNA encoding a human-derived HGF protein registered as M60718, M73240, AC004960, AY246560, M29145 or M73240. The DNA encoding the HGF protein of the present invention is DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA, and has substantially the same activity as the HGF protein. For example, DNA encoding a protein having mitogenic activity, motogenic activity, etc. is included.

  Specific examples of DNA encoding the HGF protein include, for example, a DNA having a base sequence represented by SEQ ID NO: 1 or 2 or a base sequence complementary to a DNA having a base sequence represented by SEQ ID NO: 1 or 2 Preferable examples include DNA that hybridizes with DNA under stringent conditions and encodes a protein having substantially the same quality of activity as HGF protein, such as mitogenic activity and motogenic activity. Here, the base sequence represented by SEQ ID NO: 1 is Accession No. It corresponds to the nucleotide sequence Nos. 73 to 2259 of M60718, and this DNA corresponds to DNA encoding the HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 3. The base sequence represented by SEQ ID NO: 2 is Accession No. It corresponds to the nucleotide sequence Nos. 66 to 2237 of M73240, and the DNA corresponds to DNA encoding the HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 4.

  The DNA hybridized under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2 is, for example, a colony hybridization method, plaque high using the partial sequence of the DNA as a probe. It means DNA obtained by using the hybridization method or Southern blot hybridization method. Specifically, hybridization was performed at about 65 ° C. in the presence of about 0.7 to 1.0 M sodium chloride using a filter on which DNA derived from colonies or plaques was immobilized, and then about 0.1 to DNA that can be identified by washing the filter under a condition of about 65 ° C. using a SSC solution of a double concentration (the composition of the SSC solution of a single concentration consists of 150 mM sodium chloride and 15 mM sodium citrate). Can be mentioned. Stringent conditions are the same in the following.

  Specifically, as a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2, the base sequence represented by SEQ ID NO: 1 or 2 Examples include DNA having a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more. Hybridization is performed by a known method such as molecular cloning (Molecular Cloning, A laboratory Manual, Third Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001: hereinafter abbreviated as Molecular Cloning 3rd Edition). )), Etc. When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual.

  Furthermore, the DNA encoding the HGF protein of the present invention is not limited to the above. As long as the expressed protein is a DNA encoding a protein having substantially the same action as the HGF protein, the DNA encoding the HGF protein of the present invention Can be used as For example, DNA encoding a partial peptide of the HGF protein and encoding a peptide having substantially the same activity as the HGF protein can be preferably used.

  The DNA encoding the partial peptide of the HGF protein may be any DNA as long as it has a base sequence encoding the partial peptide and encodes a peptide having substantially the same activity as the HGF protein. Good. Specific examples of the DNA encoding the partial peptide of the present invention include (a) a DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 1 or 2, and substantially equivalent to the HGF protein. A DNA encoding a peptide having the same activity as the above, (b) a DNA comprising a base sequence complementary to a DNA having a partial base sequence of the DNA represented by SEQ ID NO: 1 or 2, and a stringent condition And a DNA encoding a protein that has a substantially the same activity as the HGF protein. More specifically, such DNA includes, for example, the 94th to 630th base sequences of the human HGF base sequence represented by SEQ ID NO: 1 (from the N-terminal hairpin loop of HGF to the first kringle. DNA having a peptide encoding a peptide up to the domain) and the 94th to 864th base sequences of the human HGF base sequence represented by SEQ ID NO: 1 (from the N-terminal hairpin loop of HGF to the second kringle domain) Preferred examples include DNA having a DNA encoding the above peptide).

  DNA encoding HGF protein or a partial peptide of HGF protein and encoding a peptide having substantially the same activity as HGF protein can be easily obtained by, for example, a normal hybridization method or PCR method. Specifically, the DNA can be obtained with reference to basic documents such as the Molecular Cloning Third Edition.

The DNA used in the present invention includes a DNA encoding a HGF protein or a partial peptide of the HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein. Rally, cell / tissue-derived cDNA, cell / tissue-derived cDNA library, or synthetic DNA is preferable. Examples of the vector into which the genomic DNA fragment is cloned in the library include bacteriophage, plasmid, cosmid, or phagemid.
In addition, RNA encoding the HGF protein used in the present invention or RNA encoding a partial peptide of the HGF protein and encoding a peptide having substantially the same activity as that of the HGF protein can also be obtained by reverse transcriptase. Any substance capable of expressing HGF protein or partial peptide can be used in the present invention. Examples of the RNA include RNA obtained by preparing an mRNA fraction from a cell or tissue and amplified by RT-PCR, and are within the scope of the present invention. The RNA can also be obtained by known means.

The HGF protein used in the present invention is a known substance, and any protein prepared by various methods can be used as long as it is purified to the extent that it can be used as a medicine.
The HGF protein can be obtained, for example, by culturing primary cultured cells or cell lines that produce HGF protein, and separating and purifying from the culture supernatant or the like. Alternatively, the gene encoding the HGF protein is incorporated into an appropriate vector by genetic engineering techniques, and this is inserted into an appropriate host cell for transformation, and the desired recombinant HGF protein is obtained from the culture supernatant of the transformant. It can also be obtained by separating the. (See, for example, JP-A No. 5-111382, Biochem. Biophys. Res. Commun. 1989, Vol. 163, p. 967). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast or animal cells can be used. As long as the HGF protein thus obtained has substantially the same action as that of the natural HGF protein, one or more of the amino acid sequences (for example, 1 to about 20, preferably 1 to about 10). The same shall apply hereinafter)) may be substituted, deleted or added, and the sugar chain may be similarly substituted, deleted or added. Examples of such an HGF protein include the 5-amino acid deficient HGF protein described below. Here, with respect to the amino acid sequence, “one or a plurality of amino acids are deleted, substituted or added” refers to genetic engineering techniques, well-known technical means such as site-directed mutagenesis, or the like. It means that a sufficient number (one to plural) is deleted, substituted or added. An HGF protein in which a sugar chain is substituted, deleted or added is, for example, an HGF protein in which a sugar chain added to a natural HGF protein is treated with an enzyme or the like to delete the sugar chain, or a sugar chain is not added. The amino acid sequence of the glycosylation site is mutated, or the amino acid sequence is mutated so that the sugar chain is added to a site different from the natural glycosylation site. Specifically, for example, the Accession No. registered in the NCBI database. For the human HGF of NP_001010932, substitution of the glycosylation site at position 289 Asn to Gln, position 397 Asn to Gln, position 471 Thr to Gly, position 561 Asn to Gln, position 648 Asn to Gln HGF [Fukuta K et al. , Biochemical Journal, 388, 555-562 (2005)].
A protein having at least about 80% homology with the amino acid sequence of the HGF protein, preferably a protein having about 90% homology, more preferably a protein having about 95% homology, A protein having substantially the same activity as the HGF protein is also included in the HGF protein used in the present invention. “Homology” in the above amino acid sequences means the degree of coincidence of amino acid residues constituting each sequence by comparing the primary structures of proteins.

Examples of the HGF protein include Accession No. registered in the NCBI database. P14210 (SEQ ID NO: 3) or Accession No. A human-derived protein represented by the amino acid sequence represented by NP_001010932 (SEQ ID NO: 4) is preferred. The HGF protein represented by SEQ ID NO: 4 is a 5-amino acid deficient HGF protein in which the 5th amino acid residues from 161 to 165th of the amino acid sequence represented by SEQ ID NO: 3 have been deleted. Both of the proteins having the amino acid sequence represented by SEQ ID NO: 3 or 4 are human-derived natural HGF proteins, and have mitogenic activity, motogenic activity, etc. as HGF.
The protein comprising the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 or 4 is at least about 80% or more, preferably about 90% or more, with the amino acid sequence represented by SEQ ID NO: 3 or 4. More preferably, a protein comprising an amino acid sequence having about 95% or more identity, and having substantially the same activity as the HGF protein, such as the amino acid sequence represented by SEQ ID NO: 3 or 4, -Amino acid sequence in which a plurality of amino acid residues are inserted or deleted, amino acid sequence in which one to a plurality of amino acid residues are replaced with another amino acid residue, or one to a plurality of amino acid residues are modified A protein containing an amino acid sequence or the like, preferably a protein having substantially the same activity as the HGF protein. The amino acid to be inserted or substituted may be an unnatural amino acid other than the 20 types of amino acids encoded by the gene. The non-natural amino acid may be any compound as long as it has an amino group and a carboxyl group, and examples thereof include γ-aminobutyric acid.
These proteins may be used alone or as a mixed protein thereof. Examples of a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 or 4 include Accession No. registered in the NCBI database. Although human origin HGF, such as BAA14348 or AAC71655, is mentioned, it is not limited to these.

  The HGF protein used in the present invention or the DNA encoding the same is preferably used from humans as described above when applied to humans, but mammals other than humans (for example, monkeys, cows, horses, pigs) HGF protein derived from sheep, dog, cat, rat, mouse, rabbit, hamster, guinea pig, chimpanzee, etc.) or DNA encoding it. Examples of such HGF include mouse-derived HGF (for example, Accession No. AAB31855, NP_034557, BAA01065, BAA01064, etc.) and rat-derived HGF [for example, Accession No. NP — 58713 (protein consisting of the amino acid sequence represented by SEQ ID NO: 6) and the like, bovine-derived HGF (eg, Accession No. NP — 0010269921, XP874740, BAD02475, etc.), cat-derived HGF (eg, Accession No. NP — 001009830, BAC10545, BAB21499, etc.), Examples include, but are not limited to, dog-derived HGF (for example, Accession No. NP — 001002964, BAC57560, etc.) or chimpanzee-derived HGF (for example, Accession No. XP519174, etc.).

In the HGF protein used in the present invention, the C-terminus is any of a carboxyl group (—COOH), a carboxylate [—COOM (M represents a metal)], an amide (—CONH ₂ ), or an ester (—COOR). Also good. Here, R in the ester is, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α -C6-12 aryl groups such as naphthyl, for example, C7-14 aralkyl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, C2-6 alkanoylmethyl groups such as acetyloxymethyl and pivaloyloxymethyl are used. When the HGF protein used in the present invention has a carboxyl group or a carboxylate other than the C-terminal, those in which the carboxyl group or carboxylate is amidated or esterified are also included in the HGF protein of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used. Further, in the HGF protein used in the present invention, the amino group of the N-terminal methionine residue is a protecting group (for example, a C1-6 acyl group such as a C2-6 alkanoyl group such as formyl group and acetyl). Etc.), the N-terminal side cleaved in vivo and the glutamyl group produced by pyroglutamine oxidation, the reactive group on the side chain of the amino acid in the molecule (for example, —OH, —SH, amino Group, imidazolyl group, indolyl group, guanidino group, etc.) protected with an appropriate protecting group (for example, C1-6 acyl group such as C2-6 alkanoyl group such as formyl group, acetyl, etc.), or sugar chain Also included are complex proteins such as so-called glycoproteins to which is bound.

The HGF protein partial peptide used in the present invention having substantially the same activity as the HGF protein (hereinafter sometimes abbreviated as HGF partial peptide) is the above-mentioned partial peptide of HGF protein. Any one can be used as long as it has substantially the same activity as the HGF protein. In the present invention, the number of amino acids of the HGF partial peptide is at least about 20 or more, preferably about 50 or more, more preferably about 100 or more of the amino acid sequences constituting the HGF protein described above. Etc. are preferred. Specifically, for example, the amino acid sequence from the 32nd amino acid to the 210th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 3 (from the N-terminal hairpin loop of HGF to the first kringle domain) Amino acid sequence from the 32nd amino acid to the 288th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 3 (from the N-terminal hairpin loop of HGF to the second kringle domain) Peptides represented by the sequence are preferred.
In the HGF partial peptide of the present invention, the C-terminus is carboxyl group (—COOH), carboxylate [—COOM (M is as defined above)], amide (—CONH ₂ ) or ester (—COOR; R is as described above. Any of the above may be used. Furthermore, in the HGF partial peptide, the amino group of the N-terminal methionine residue is protected with a protecting group, and the Gln produced by cleaving the N-terminal side in vivo is pyroglutamine oxidized, as in the HGF protein described above. And those in which a substituent on the side chain of an amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.

  Examples of the salt of the HGF protein or partial peptide thereof used in the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

  The partial peptide of HGF protein or a salt thereof used in the present invention can be produced according to a known peptide synthesis method or by cleaving the HGF protein with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that may have a protecting group capable of constituting an HGF protein is condensed with a remaining part that may have a protecting group, and when the product has a protecting group, The desired peptide can be produced by removing. Known condensation methods and protecting group removal include, for example, M.M. Bodanszky and M.C. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966), Schroeder and Luebke, The Peptide, Academic Press, New York (1965), etc. It is done. After the reaction, a partial peptide of HGF protein can be purified and isolated by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, crystallization or recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. Can do.

In the present invention, “neurogenesis” refers to differentiation of a young undifferentiated cell (for example, a neural stem cell) into a new nerve (nerve cell) via a neuroblast, a neuroblast that differentiates into a nerve cell, Includes proliferation of undifferentiated cells and the like. Neurogenesis also includes regeneration of damaged nerves and nerve repair.
“Nerve” includes central nerves (eg, brain, spinal cord, etc.) and peripheral nerves (eg, ulnar nerve, radial nerve, radial nerve, etc.).

  The neurogenesis promoting agent of the present invention can be applied not only to humans but also to mammals other than humans (for example, monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). Applicable.

When administering the neurogenesis-promoting agent of the present invention to a patient, the administration form, administration method, dosage and the like may be slightly different from the case where the active ingredient is HGF protein and the DNA encoding HGF protein.
For example, when the active ingredient is HGF protein, the preparation of the present invention can take various preparation forms, for example, liquids, solids, etc., but in general, only HGF protein or an injection with a conventional carrier is used. In addition, it is preferably formulated into a propellant, sustained-release preparation (for example, depot) and the like. The injection may be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to a pharmaceutically acceptable additive such as an isotonic agent (sodium chloride, potassium chloride, glycerin, mannitol, Sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon) Aminocaproic acid buffer, etc.), preservatives (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boro Acid, borax, etc.), thickener (hydroxyethylcellulose) Hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH adjusters (hydrochloric acid, sodium hydroxide) , Phosphoric acid, acetic acid, etc.) can be prepared by dissolving the HGF protein in a solution to which it has been added as appropriate, and then sterilizing it by filtration through a filter or the like, and then filling it into an aseptic container. Further, an appropriate solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.) may be further blended. Good. In the case of an oily injection, for example, sesame oil or soybean oil is used as the oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection solution is usually filled in an appropriate ampoule or vial. The HGF protein content in the injection can be adjusted to usually about 0.0002 to 0.2 w / v%, preferably about 0.001 to 0.1 w / v%. In addition, it is desirable to store liquid preparations such as injections after removing moisture by freezing or lyophilization. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.

  Propellants can also be prepared by routine pharmaceutical methods. In the case of producing as a propellant, the additive added to the propellant may be any additive as long as it is generally used for inhalation preparations. A solvent, a preservative, a stabilizer, an isotonic agent, a pH adjuster, and the like can be blended. Examples of the propellant include a liquefied gas propellant or a compressed gas. Examples of the liquefied gas propellant include fluorinated hydrocarbons (alternative chlorofluorocarbons such as HCFC22, HCFC-123, HCFC-134a, and HCFC142), liquefied petroleum, dimethyl ether, and the like. Examples of the compressed gas include soluble gas (carbon dioxide gas, nitrous oxide gas, etc.) or insoluble gas (nitrogen gas, etc.).

  The HGF protein used in the present invention can be used as a sustained-release preparation (for example, a depot) together with a biodegradable polymer. By using HGF protein as a depot, effects such as a reduction in the number of administrations, sustained action and reduction of side effects can be expected. The sustained-release preparation can be produced according to a known method. The biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers. For example, polysaccharides such as starch, dextran or chitosan; proteins such as collagen or gelatin Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine; polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer; polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyanhydride Or polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer; polyalkyl cyanoacrylic acid such as polyorthoester or polymethyl-α-cyanoacrylic acid; polycarbonate such as polyethylene carbonate or polypropylene carbonate. Polyester is preferable, and polylactic acid or lactic acid-glycolic acid copolymer is more preferable. When a lactic acid-glycolic acid copolymer is used, the composition ratio (lactic acid / glycolic acid) (mol%) varies depending on the sustained release period. For example, the sustained release period is about 2 to 3 months, preferably about 2 weeks. In the case of 1 month, about 100/0 to 50/50 is preferable. The weight average molecular weight of the polylactic acid or lactic acid-glycolic acid copolymer is generally preferably about 5,000 to 20,000. Polylactic acid or lactic acid-glycolic acid copolymer can be produced according to a known production method, for example, a production method described in JP-A No. 61-28521. The mixing ratio of the biodegradable polymer and the HGF protein is not particularly limited. For example, the HGF protein is preferably about 0.01 to 30 w / w% with respect to the biodegradable polymer.

  As an administration method, an injection or a spray is directly injected into a site where neurodegeneration or neuronal cell death is induced (intrathecal or spinal parenchyma, intrathecal continuous administration by a sustained-release pump, etc.) or spray. Alternatively, it is preferable to embed a sustained-release preparation (depot agent) in a site close to a tissue having a site where neurodegeneration or neuronal cell death is induced. The dosage is appropriately selected according to the dosage form, the degree of disease or age, but is usually 1 μg to 500 mg, preferably 10 μg to 50 mg, more preferably 1 to 25 mg per dose. In addition, the number of administrations is appropriately selected according to the dosage form, the degree of disease, age, or the like, and can be administered once or continuously at a certain interval. In the case of continuous administration, the administration interval may be from once a day to once every several months. For example, in the case of administration by a sustained release preparation (depot) or intrathecal continuous administration by a sustained release pump, several months It may be once.

On the other hand, in order to administer the HGF gene to a patient, a conventional method, for example, separate experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate experimental medicine, gene transfer & expression analysis experimental method, Yodosha, 1997, It is preferably performed according to the method described in the Gene Therapy Research Handbook edited by the Japanese Society for Gene Therapy, NTS, 1999, and the like.
Specific administration methods include, for example, a method of locally injecting a recombinant expression vector incorporating an HGF gene into a tissue (for example, spinal nerve, brain, etc.) where neurodegeneration or neuronal cell death has been induced, or Then, after removing cells from the tissue of the lesion site of the patient or the spinal cord and the like, introducing a recombinant expression vector incorporating the HGF gene into the cells and transforming the cells, the transformed cells are Examples include a method of transplanting into a lesion site or spinal cord.

Expression vectors include naked plasmids, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpesviruses (type I herpes simplex virus, etc.), vaccinia viruses, poxviruses, polioviruses, symbis viruses, Sendai viruses, SV40 or Examples include, but are not limited to, DNA viruses such as immunodeficiency virus (HIV) or RNA viruses. By introducing a gene of interest into the expression vector and infecting the cell with a recombinant virus, DNA encoding the HGF protein can be introduced into the cell. Among them, type I herpes simplex virus (HSV-1) vector, adenovirus vector, adeno-associated virus (AAV) vector, and the like are preferable.
The HSV-1 vector is a neurotropic vector. HSV-1 vectors are preferably those that have a large (152 kb) genome that incorporates multiple genes (up to 30 kb) and that have the ability to potentially establish infection in neurons over life. A specific HSV-1 vector is a non-replicating HSV-1 in a severely impaired state due to the deletion of three genes encoding ICR4, ICP34.5 and VP16 (vmw65) for viral replication. (HSV1764 / 4- / pR19) vector (Coffin RS, et al., J. Gen. Virol. 1998, Vol. 79, p. 3019-3026; Palmer JA, et al., J. Virol., 2000 74, p. 5604-5618; Lilley CE, et al., J. Virol., 2001, vol. 75, p. 4343-4356). AAV vectors belong to non-pathogenic viruses, are highly safe, and can efficiently introduce genes into non-dividing cells such as nerve cells. AAV-2, AAV-4, AAV-5 etc. are mentioned as an AAV vector. These HSV-1 vectors and AAV vectors can express the target gene in nerve cells or the like for a long time.
In order to evaluate the effect of HGF on neurogenesis, for example, an HSV-1 vector or an AAV vector is used to change the HGF gene to a site where neurodegeneration or neuronal cell death is induced, such as the striatum or intrathecal space It is preferred to transduce.

As the dosage form, various known dosage forms [for example, injections, sprays, sustained-release preparations (depots), microcapsules, etc.] suitable for the respective administration forms can be taken. Injections, sprays and sustained-release preparations (depots) can be prepared in the same manner as for HGF protein. In the case of producing a microcapsule, for example, a host cell into which an expression plasmid containing an HGF gene has been introduced is used as a core substance, and this is performed according to a known method (for example, coacervation method, interfacial polymerization method or double nozzle method). By covering with a coating substance, it can be produced as fine particles having a diameter of about 1 to 500, preferably about 100 to 400 μm. Coating materials include carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, alginic acid or salts thereof, gelatin, gelatin gum arabic, nitrocellulose, polyvinyl alcohol or hydroxypropyl cellulose, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer And film-forming polymers such as chitosan-alginate, cellulose sulfate-poly (dimethyldiallyl) ammonium chloride, hydroxyethyl methacrylate-methyl methacrylate, chitosan-carboxymethylcellulose, alginate-polylysine-alginate, and the like.
The content and dosage of DNA in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. The dose varies depending on the type of HGF gene transfer vector, but is usually 1 × 10 ⁶ pfu to 1 × 10 ¹² pfu, preferably 1 × 10 ⁷ pfu to 2 × 10 ¹¹ pfu in terms of HGF gene transfer vector. More preferably, 1.5 × 10 ⁷ pfu to 1.5 × 10 ¹¹ pfu is preferably administered once every several days to several months.

The neurogenesis-promoting agent of the present invention may be administered as a combination agent in combination with other neurotrophic factors for complementation and / or enhancement of the HGF protein or the like or the neurogenesis-promoting effect of the HGF gene.
The combination of HGF protein or the like or HGF gene and other neurotrophic factors may be administered in the form of a combination preparation in which both components are combined in one preparation, or may be administered in separate preparations. . In the case of administration as separate preparations, simultaneous administration and administration by time difference are included. In addition, administration by time difference may be such that a preparation containing HGF protein or the like or HGF gene is administered first, and a preparation containing other neurotrophic factor may be administered later, or a preparation containing other neurotrophic factor May be administered first, and a preparation containing HGF protein or the like or the HGF gene may be administered later. Each administration method may be the same or different. Neurotrophic factors include factors that promote the proliferation and differentiation of undifferentiated neuroblasts or the maintenance or promotion of survival and function of mature neurons. In addition, neurotrophic factors include those that exhibit properties as a lesion factor that is associated with the prevention of tissue degeneration and functional repair, where synthetic secretion is temporarily increased when nerve tissue is damaged. The neurotrophic factor is not particularly limited. For example, NT-3 (Neurotrophin 3), NT-4 (Neurotrophin 4), CNTF (Ciliary Neurotrophic Factor), GDNF (Glial-Derived Neurotrophic Factor) or BDNF (Brain-derived neurotrophic factor).

Since the neurogenesis-promoting agent of the present invention can promote the neurogenesis of a new nerve at a site where nerve degeneration or neuronal cell death is induced, it can be used in neurodegenerative diseases or neurological disorders associated with neurodegeneration or neuronal cell death. For example, it can be used for nerve regeneration, nerve repair or nerve function improvement.
Neurodegenerative diseases and neurological disorders include all neurodegenerative diseases or neurological disorders that induce neuronal degeneration and cell death. Factors that induce neurodegenerative diseases or neurological disorders are not particularly limited, and include, for example, genetics, physical injuries (for example, intense exercise, accidents, exposure to cold or hot air for a long time, repeated compression, etc.), radiation Irradiation, infectious diseases, toxic substances and drugs, cancer nerve invasion, complications of diseases such as diabetes, etc. are included.
Further, neurodegenerative diseases and neurological disorders include central neurological disorders, central neurological disorders, peripheral neurological disorders and peripheral neuropathies.
Examples of central neurological diseases or disorders include aphasia, apraxia, agnosia, amnesia (Wernicke encephalopathy, Korsakoff symptoms, transient global amnesia, false (psychogenic) amnesia), stupor and coma, Delirium and dementia, Alzheimer's disease, convulsive disorders, sleep disorders, insomnia, hypersomnia [Narcolepsy], sleep apnea syndrome, parasomnia, etc .; depression, manic depression Psychiatric disorders such as schizophrenia; ischemic syndrome [transient cerebral ischemic attack (TIA), ischemic attack, hemorrhagic syndrome, intracranial hemorrhage, subarachnoid hemorrhage], cerebrovascular disease such as cerebral arteriovenous malformation Head trauma; acute bacterial meningitis, acute viral encephalitis and aseptic meningitis, subacute and chronic meningitis, brain abscess, subdural pus, central nervous system infections; nervous system radiation Injury; cerebral cortex basal ganglia degeneration; spinal stenosis; spine Schizophrenia, posterior longitudinal ligament ossification; Horner syndrome, internuclear ocular palsy, gaze paralysis, retinopathy and other optic nerve disorders; 3rd cranial nerve palsy, 4th cranial nerve palsy, 6th cranial nerve palsy, trigeminal neuralgia, face Cranial nerve diseases such as neurological disease (bell paralysis); tremor, dyskinesia (myoclonus, tic, Tourette syndrome, athetosis (dystonia); Huntington's chorea, bulbar spinal atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia Polyglutamine diseases such as type 2, spinocerebellar ataxia 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, spinal cerebellar ataxia Ryukyu atrophy, etc .; Movement disorders such as induced movement disorders, progressive supranuclear palsy; spinal cord compression, subdural and epidural abscesses and hematomas, cavitation, vascular disease, hereditary spastic paraplegia, acute transverse myelitis Spinal cord disorders such as spinal cord injury; , Diabetic retinopathy and the like.
Peripheral neuropathy or peripheral neuropathy includes, for example, trauma involving nerve cutting, etc .; lower and upper motor neuron disease (hereditary spinal muscular atrophy), nerve root disease (nuclear nucleus herniation, cervical spondylosis), plexus Diseases of the peripheral nervous system such as diseases, chest outlet compression syndrome, peripheral neuropathy (Gillan-Barre syndrome, hereditary neuropathy, neurofibromatosis, Proteus syndrome), neuromuscular transmission disease (myasthenia gravis); muscular dystrophy, Myopathy, such as myopathy, channelopathy (muscular tonic disease, family periodic paralysis), etc .; or diabetic neuropathy, etc.

  For neurogenesis, new neurons are born and cell division must be performed. During cell division, DNA is synthesized to copy genetic information. Examples of the substance that can be used as a clue to know whether or not DNA has been synthesized include BrdU (bromodeoxyuridine). For example, when BrdU is injected into the body, newly born cells take in BrdU, so that the newborn of a neuron can be determined using BrdU as an index. Therefore, the neurogenesis effect can be measured using a method of measuring BrdU taken up by nerve cells as an index. Moreover, it measures using the method etc. which are described in the below-mentioned test example including the method of measuring the differentiation from a cultured neural stem cell to a glial cell (astrocyte, oligodendrocyte) or a neuron in vitro. Can do. The above-described nerve cells include cell bodies, dendrites and axons. Glial cells include astrocytes, oligodendrocytes and microglial cells. Astrocytes can further differentiate into neurons. In the present invention, astrocytes include all GFAP positive cells.

  Hereinafter, the present invention will be described using test examples, but the present invention is not limited thereto. In Examples,% represents mass% unless otherwise specified.

[Test Example 1]
Effect of HGF on the formation of brain neurons Experimental animals B6CBA-TgN (HD exon 1) 62 Gpb / J female mice (Mangiarini L et al., Cell, 1996, Vol. 87, p.493-506) Obtained from Jackson Laboratory (Bar Harbor, ME), bred and mated with B6CBAF1 / J male mice.
The mated offspring determines the gene system by PCR analysis of genomic DNA extracted from its tail tissue, and a mouse having a TgN62Gpb gene is a Huntington's disease model mouse R6 / 2 mouse (hereinafter abbreviated as R6 / 2 mouse). It was. In addition, a mouse that had the same litter as the R6 / 2 mouse and did not have the TgN (HD exon 1) 62Gpb gene was used as a wild-type littermate mouse.
All experiments were conducted according to the guidelines of the Osaka University Animal Ethics Committee. All efforts made the animal burden as small as possible and used as few animals as possible.

2. Construction, production and purification of vector GFP (green fluorescent protein) gene of pR19GFPWPRE (Lilley CE et al., J. Virol, 2001, Vol. 75, p. 4343-4356) is encoded by rat HGF. CDNA (Sun W et al., J. Neurosci, 2002, Vol. 22, p. 6537-6548) with a KT3 tag (3′-CCGCCCGCAGCCAGAGACT-5 ′; SEQ ID NO: 7) added to the full length (rat HGF; SEQ ID NO: 5). ) To construct pR19ratHGFKT3WPRE. The sequence of this vector (pR19ratHGFKT3WPRE) was confirmed by analysis using an ABI 310 capillary sequencer. Subsequently, homologous recombination was carried out by co-transfection of plasmid pR19ratHGFKT3WPRE DNA with HSV1764 / -4 / pR19LacZ viral DNA using M49 cells. White plaques were selected, purified three times, and viruses with no replication capacity were propagated according to the method of Palmer JA et al. (J. Virol, 2000, 74, p. 5604-5618). Rat HGF expression was confirmed by immunostaining. The expression was further confirmed by Western blotting and enzyme-linked immunosorbent assay (ELISA) of rat HGF. HSV1764 / -4 / pR19 HGF viral vector (HGF expression vector; hereinafter referred to as HSV-HGF) having a titer of 1-2 × 10 ⁹ pfu (plaque forming unit) / mL for use in this test (Abbreviated) and HSV1764 / -4 / pR19LacZ viral vector (HGF non-expression vector; hereinafter abbreviated as HSV-LacZ) having a titer of 1 to 1.5 × 10 ⁹ pfu / mL.

3. HSV insertion into the brain (In vivo)
Four-week-old R6 / 2 mice were deeply anesthetized with 50 mg / kg intravenous injection of pentobarbital. The mice were placed in a Kopf brain stereotaxic apparatus and fixed for injection into the striatum (around −0.4 mm, ± 1.8 mm medial / lateral direction, and −3.5 mm dorsoventral direction). The mice were injected with 5 μL of HSV-LacZ (5 × 10 ⁶ pfu) or HSV-HGF (3 × 10 ⁵ pfu). The injection was performed at a rate of 0.3 μL / min using a 10 μL Hamilton syringe on the striatum of the mouse. Hereinafter, mice injected with HSV-LacZ are referred to as R6 / 2 (HSV-LacZ) mice, and mice injected with HSV-HGF are referred to as R6 / 2 (HSV-HGF) mice.

4). Immunohistochemical analysis Mice were deeply anesthetized, and ice-cold phosphate buffered saline (PBS) was flushed through the heart to perfuse the entire body of the mice, and then contained 4% (w / w%, and so on) paraformaldehyde Perfusion was fixed with PBS. The brain was cryo-protected with 10% and 20% sucrose stepwise, then frozen and then frozen brain was made into 20 μm serial sections. The obtained frozen section was stained with cresyl violet in order to stain the niss substance.
Immunohistochemical staining was performed by washing frozen sections with PBS, immersing in PBS containing 10% goat or donkey serum for 1 hour and then incubating with antibody overnight at 4 ° C.

(1) Effect of HGF on Ki-67 cells The proliferation of neurons in the subventricular zone (SVZ) and striatum was examined. Ki-67 was selected as a marker for proliferating cells, Ki-67 immunostaining was performed, and the number of Ki-67 positive cells in the subventricular zone and striatum was measured. In the striatum, the number of Ki-67 positive cells in R6 / 2 (HSV-HGF) mice was significantly increased compared to that in R6 / 2 and R6 / 2 (HSV-LacZ) mice. (FIG. 1).

(2) Effect of HGF on BrdU incorporation Five-week-old mice were administered BrdU (4 × 75 mg / kg ip, dissolved in physiological saline for a total of 4 times every 2 hours), and 28 days after BrdU injection (9 Slaughtered at weekly age). Mice were anesthetized and PBS was perfused from the heart to perfuse the whole body of the mice, and then fixed by perfusion with PBS containing 4% paraformaldehyde. The brain was cryo-protected with 10% and 20% sucrose stepwise, then frozen and then frozen brain was made into 20 μm serial sections.
For BrdU immunohistochemical staining, frozen sections were incubated with 1N hydrochloric acid at 60 ° C. for 30 minutes and immersed in PBS containing 10% goat serum for 1 hour. Thereafter, the frozen sections were incubated with anti-BrdU (rat monoclonal antibody; manufactured by Oxford Biotechnology; catalog number OBT0030) at 4 ° C. for 36 hours. Double staining was visualized with a secondary antibody (Molecular Probes) conjugated with fluorescent dye Alexa 488 and fluorescent dye Alexa 546, and sections were counterstained with Topro-3 (Molecular Probes). Fluorescence images were detected with a Zeiss LSM-510 confocal fluorescence microscope.
As a result of measuring the number of BrdU positive cells in the subventricular zone and the striatum, no significant difference in the number of BrdU positive cells was observed between the groups in the subventricular zone. However, in the striatum, the number of BrdU positive cells in R6 / 2 (HSV-HGF) mice was significantly increased compared to R6 / 2 and R6 / 2 (HSV-LacZ) mice (FIG. 2). These data indicate that HSV-HGF treatment enhances neuronal cell proliferation.

(3) Action of HGF on cells positive for both nestin and BrdU Nestin is a marker for neural stem cells. Nestin was stained according to the above immunohistochemical staining using a nestin antibody [mouse polyclonal antibody (manufactured by BD Biosciences; catalog number 556309) diluted 100-fold] as an antibody.
The number of cells in which both nestin and BrdU were positive was measured. In the subventricular zone and striatum, cells that are positive for both nestin and BrdU in R6 / 2 (HSV-HGF) mice are compared to those in R6 / 2 and R6 / 2 (HSV-LacZ) mice. Significantly increased (FIG. 3).

(4) Action of HGF on cells positive for both doublecortin and BrdU Doublecortin (DCX) is a marker for migrating neuroblasts. DCX was stained according to the above immunohistochemical staining using a DCX antibody [goat polyclonal antibody (manufactured by Santa Cruz Biotechnology; catalog number sc-8066) diluted 100-fold] as an antibody.
The number of cells positive for both DCX and BrdU was measured. Cells positive for both DCX and BrdU in R6 / 2 (HSV-HGF) mice are compared to those of R6 / 2 and R6 / 2 (HSV-LacZ) mice in the subventricular zone and striatum. Significantly increased (FIG. 4).

(5) Action of HGF on cells positive for both PSA-NCAM and BrdU PSA-NCAM is a marker for migrating neuroblasts. PSA-NCAM was stained according to the above immunohistochemical staining using PSA-NCAM antibody [mouse monoclonal antibody (AbCys S.A .; Catalog No. AbC0019) diluted 800 times] as an antibody.
The number of cells positive for both PSA-NCAM and BrdU was measured. Cells positive for both PSA-NCAM and BrdU in R6 / 2 (HSV-HGF) mice are compared to those in R6 / 2 and R6 / 2 (HSV-LacZ) mice in the subventricular zone and striatum. Significantly increased (FIG. 5).

(6) Action of HGF on cells positive for both βIII tubulin and BrdU βIII tubulin is an early to differentiated marker for neurons. βIII tubulin was stained according to the immunohistochemical staining using βIII tubulin antibody [TuJ1, mouse monoclonal antibody (manufactured by R & D Systems; catalog number MAB1195) diluted 200-fold] as an antibody.
The number of cells positive for both βIII tubulin and BrdU was measured. Cells positive for both βIII tubulin and BrdU in R6 / 2 (HSV-HGF) mice were found in the subventricular zone and striatum with those of R6 / 2 and R6 / 2 (HSV-LacZ) mice. There was a significant increase in comparison (FIG. 6).

(7) Action of HGF on cells positive for both NeuN and BrdU NeuN is a marker for differentiated neurons. NeuN was stained according to the above immunohistochemical staining using NeuN antibody [Mouse monoclonal antibody (Chemicon International; catalog number MAB377) diluted 500-fold] as an antibody.
The number of cells positive for both NeuN and BrdU was measured. Cells positive for both NeuNn and BrdU in R6 / 2 (HSV-HGF) mice are compared to those in R6 / 2 and R6 / 2 (HSV-LacZ) mice in the subventricular zone and striatum. Significantly increased (FIG. 7).

(8) Action of HGF on cells positive for both phosphorylated c-Met and nestin To examine the role of HGF in neurogenesis, HGF is involved in the tyrosine phosphorylation of c-Met in nestin-positive cells. We examined whether to give change. Phosphorylated c-Met and nestin are phosphorylated c-Met antibodies [rabbit polyclonal antibody (manufactured by Biosource; catalog number 44-888G) diluted 100-fold] and nestin antibody [mouse polyclonal antibody (BD Using Biosciences; catalog number 556309) diluted 100-fold] was used according to the above immunohistochemical staining.
In R6 / 2 (HSV-HGF) mice, cells positive for both nestin and phosphorylated c-Met were significantly increased compared to the other groups (FIG. 8).

(9) Action of HGF on cells positive for both phosphorylated c-Met and DCX In order to examine the role of HGF in neurogenesis, HGF is involved in tyrosine phosphorylation of c-Met in DCX positive cells. We examined whether to give change. Phosphorylated c-Met and DCX were immunohistochemically stained as described above.
In R6 / 2 (HSV-HGF) mice, cells positive for both phosphorylated c-Met and DCX were significantly increased compared to the other groups (FIG. 9).
[Test Example 2]
Expression of HGF in the spinal cord by spinal administration of a DNA-containing vector encoding the HGF protein Construction, manufacture and purification of vector (1) DNA-inserted HSV-1 vector encoding HGF protein As a type I herpes simplex virus (HSV-1) vector into which the HGF gene has been inserted, HSV 1764 / -4 prepared in Test Example 1 / PR19HGF viral vector was used. Hereinafter, this vector is abbreviated as HSV-HGF.
(2) DNA Insertion AAV-2 Vector and AAV-4 Vector Encoding HGF Protein Rat HGF- is added to the multicloning site of pCMV-MCS included in the AAV Helper-Free System (Stratagene, USA; catalog number # 240071) kit. KT3 [Rat HGF-encoding DNA (SEQ ID NO: 5) added with a KT3 tag sequence (3′-CCGCCCGCAGCCAGAGACT-5 ′; SEQ ID NO: 7) at the C-terminus; Sun, Funakoshi et al., J. Biol. Neurosci. 2002, Vol. 22, p. 6537-6548] was inserted. The sequence was confirmed to be correct. The vector was cleaved into two fragments at the NotI site, and the pAAV-rat HGF-KT3 for the production of AAV2-HGF was prepared by replacing the fragment containing rat HGF-KT3 with the one opened in the same manner as pAAV-MCS. . In order to prepare AAV4-HGF, pAAV ⁴ -rat HGF-KT3 was prepared in the same manner using pAAV-MCS modified for AAV4 (Proc. Natl. Acad. Sci. USA, 2000). 97, p. 3428-3432). Subsequently, pAAV-MCS into which rat HGF-KT3 was inserted was introduced into HEK193 cells contained in the kit according to the instruction manual of the kit. The expression and activity of rat HGF-KT3 in the cells were confirmed by ELISA and scatter analysis on MDCK cells. The resulting vectors are abbreviated as AAV2-HGF and AAV4-HGF.

2. Expression of HGF by administration to spinal cord parenchyma The spinal cord parenchyma of adult female SD rats was subjected to vector suspension (HSV-HGF: 3 × 10 ⁷ pfu, 3 × 10 ⁷ pfu; AAV2-HGF) using a minipump. : 3 × 10 ¹¹ pfu; or AAV4-HGF: 3 × 10 ¹¹ pfu) 5 μL was stereotaxically injected. Five days later, the animals were sacrificed under pentobarbiter deep anesthesia. Immediately remove the spinal cord and divide it into three regions: upper spinal (rostral side; U), middle (middle spinal; middle part; M) and lower part (lower spinal; caudal side; L). Fragments were homogenized as described above. The amount of HGF protein was measured by enzyme antibody immunoassay (ELISA). The ELISA method uses an anti-HGF polyclonal antibody (manufactured by Tokushu Meneki), Sun W et al., Brain Res Mol Brain Res, 2002, Vol. 103, p. Performed as described in 36-48.
The result is shown in FIG. When the vector was injected into the spinal cord parenchyma of the lumbar region, HGF expression was observed not only at the injection site but also at the rostral and central portions of the spinal cord. The intensity of expression was caudal (including lumbar spinal cord)> = middle part> rostral side. In HSV-HGF, the expression level of HGF increased in a concentration-dependent manner.

3. Expression of HGF by administration to the spinal cavity A vector suspension (HSV-HGF: 3 × 10 ⁷ pfu, 3 × 10 ⁷ pfu; AAV2-HGF) was used in the spinal cavity of the lumbar spinal cord of adult female SD rats using a minipump. : 3 × 10 ¹¹ pfu; or AAV4-HGF: 3 × 10 ¹¹ pfu) 5 μL was stereotaxically injected. Five days after that, the spinal cord was taken out in the same manner as in the administration to the spinal cord parenchyma, and the amount of HGF protein on the rostral, middle and caudal sides of the spinal cord was measured by ELISA as described above.
The result is shown in FIG. When the vector was injected into the spinal cavity of the lumbar region, HGF expression was observed not only at the injection site but also at the rostral and central portions of the spinal cord. Although the expression level of HGF was lower than that of injection into the spinal cord parenchyma, the expression intensity was almost the same in any part of the spinal cord [caudal side (including lumbar spinal cord), central part, rostral side]. That is, when administered to the spinal cavity, HGF could be supplied to a wide range of nerve cells. This was thought to be because the vector was diffused throughout the spinal cord by spinal fluid, compared to when the vector was administered to the spinal cord parenchyma. In HSV-HGF, the expression level of HGF increased in a concentration-dependent manner.

[Test Example 3]
Effects of HGF on neural stem cell differentiation (1) Neural stem cell culture method E15 C57BL6 E14 striatum is removed from the mouse fetus into the gas anesthesiology department, the cells are disassembled by mechanical pipetting, NeuroCult Basal Medium (nerve) EGF (epidermal growth factor) to a medium supplemented with 1/10 volume of NeuroCult Proliferation Supplements (supplement for neuronal cell growth; Stem cell technologies Inc.) to a basic culture medium for cell culture (Stem cell technologies Inc.) ) Was added to a medium (mouse 1/20 mL) to a concentration of 20 ng / mL, followed by suspension culture, and the cultured cells were used as primary cultured mouse neurospheres (neural stem cells).
After 2 weeks of suspension culture, a medium in which the primary cultured mouse neurosphere was added to NeuroCult Differentiation medium (medium for differentiation of neuronal cells; manufactured by Stem cell technologies Inc.) in a basal medium for neuronal culture (medium not added with EGF) ) It was resuspended in 20 mL and cultured in a culture dish coated with poly-L-lysine (PLL) (24-well plate; 1 mL / well of resuspended primary mouse neurosphere) for 2 days. The culture was performed at 37 ° C. using a CO ₂ incubator [5% (V / V) CO ₂ -95% (V / V) air]. The obtained cells were used as mouse neurostriatum-derived secondary neurosphere.

(2) Immunostaining method The medium in which the cells in (1) above were cultured was removed, and 4% paraformaldehyde (PFA) -added phosphate buffered saline (PBS) was added thereto at room temperature for 15 minutes. The cells were fixed by standing. Thereafter, the fixed cells were washed with PBS three times, and blocking buffer (5% goat serum / PBS / 0.3% Triton X-100) was added for blocking (room temperature 15 minutes). The blocked cells were washed 3 times with PBS, and then incubated at 4 ° C. overnight in a blocking buffer containing a primary antibody. After washing with PBS three times, PBS added with anti-rabbit IgG (Invitrogen; diluted 600) conjugated with Alexa 488 was added and incubated at room temperature for 20 minutes. After incubation, the cells were washed 3 times with PBS and observed with an LSM510 confocal fluorescence microscope (Zeiss).
In addition, in order to confirm the specificity of the following c-Met antibody, an absorption test was performed. The absorption test was carried out using c-Met antibody previously treated with an excess amount (100-fold molar ratio to c-Met antibody) of c-Met antibody blocking peptide (sc162P) (manufactured by Santa Cruz Biotechnology). Similarly, immunostaining was performed. If the stained image is negative in the absorption test, a positive image by the antibody means that only c-Met is specifically detected.
The following was used as the primary antibody.
Nestin antibody: Mouse polyclonal antibody (BD Biosciences; catalog number 556309) was used after being diluted 100 times.
MAP2 antibody: A mouse monoclonal antibody (manufactured by Sigma; catalog number M4403) was diluted 500 times and used.
NeuN antibody: A mouse monoclonal antibody (Chemicon; catalog number MAB377) was diluted 500 times and used.
A2B5: A mouse monoclonal antibody (manufactured by Chemicon International; catalog number MAB312R) was diluted 100 times and used.
c-Met: SP260, rabbit polyclonal antibody (manufactured by Santa Cruz Biotechnology; catalog number sc-162) was diluted 50 times and used.
GFAP antibody: A mouse monoclonal antibody (Chemicon International) was diluted 600 times and used.

(3) Differentiation assay of primary cultured mouse neurosphere FIG. 12 shows the differentiation assay of primary cultured mouse neurosphere. Neural stem cells are called neurosphere because they grow in a spherical mass in suspension culture. In FIG. 12, nestin (NESTIN) indicates a neural stem cell marker, MAP2 indicates a neural cell marker, and GFAP indicates an astrocyte marker. Heochst shows that the nucleus is stained with Heochst33342. When the nestin image, MAP2 image or GFAP image and the nuclear staining image are superimposed (MERGE in the figure), the position of each cell and nucleus coincides, and the primary cultured mouse neurosphere has the ability to create neurons and astrocytes You can see that

(4) Differentiation assay for striatum-derived secondary neurosphere Next, in the differentiation assay for striatum-derived secondary neurosphere, co-staining with c-Met and other cell markers (nestin, GFAP, Heochst, A2B5) is performed. Thus, HGF target cells were identified in cell groups in various differentiation processes from the neurosphere to the neurogenesis process.
The results are shown in FIGS. Nestin-positive neural stem cells can be confirmed to be positive for c-Met, which is an HGF receptor (FIGS. 13 and 14). In addition, it can be confirmed that c-Met is expressed in GFAP-positive cells that are astrocyte markers (FIG. 15). In addition, it was confirmed that the expression of c-Met was very weak in the GFAP positive cells that had just differentiated from the neurosphere. In addition, c-Met expression was confirmed in A2B5-positive oligodendrocyte progenitor cells (FIG. 16). On the other hand, c-Met positive cells were also observed in the cell group not stained with A2B5. In addition, since the immunostaining property of c-Met disappeared by the absorption test, the specificity of the c-Met antibody in the immunostaining of c-Met was confirmed.
As described above, it was confirmed that c-Met is expressed in neural stem cells, glial cells (astrocytes, oligodendrocytes) and neurons differentiated from neural stem cells. This result indicates that cells expressing these c-Met can be HGF target cells.

(5) Neurosphere neurogenesis differentiation assay in the presence of various trophic factors The outline of this test is shown in FIG.
A mouse embryo-derived neurosphere was cultured in suspension in the presence of EGF (20 ng / mL) for 2 weeks or more, and then cultured for 2 days on a plate coated with PLL in a differentiation medium in the absence of EGF. After adhesion culture for 2 days, the cells were further cultured for 5 days under the conditions of addition of HGF and / or trophic factor (NT-3, CNTF) (hereinafter referred to as 7 days after EGF removal). Thereafter, the cells were fixed with 4% PFA and then stained by immunostaining. The stained cells were observed with an LSM510 confocal fluorescence microscope (Zeiss) to identify the cell type. HGF was added to the medium so that the final concentration was 3 ng / mL. Also, CNTF was added to the medium so that the final concentration was 3 ng / mL, and NT-3 was added so that the final concentration was 50 ng / mL. Immunostaining was performed according to (2) above. The nuclei were counterstained using Topro3 (Molecular Probes).

  The results are shown in FIGS. 18 and 19 show stained images of the control neurosphere 7 days after EGF removal. Neurogenesis was observed in many neurospheres even under control conditions. However, neurogenesis with low maturity, characterized by a small number of new neurons and short nerve fibers, was observed.

  20 and 21 show stained images of CNTF treated neurosphere 7 days after EGF removal. It is clear that the number of MAP2-positive neurons increases with the addition of CNTF compared to the control. Nerve cells obtained by the addition of CNTF showed a feature that the length of nerve fibers is very long although there are few branches of nerve fibers.

  22 and 23 show stained images of NT-3 treated neurosphere 7 days after EGF removal. Compared to the control, an increase in new nerve cells was recognized by the addition of NT-3. However, the degree was slightly weaker than when CNTF was added. The characteristics of new neurons by the addition of NT-3 were not so prominent in the effect of promoting elongation, although the nerve fiber length was slightly longer than that of the control. On the other hand, regarding neurite branching, a marked increase in the number of branches was observed, confirming that neuronal differentiation (more mature neurogenesis) different from control was induced.

  24 and 25 show stained images of HGF-treated neurosphere 7 days after EGF removal. Compared with the control, the increase of new nerve cells was recognized by the addition of HGF. The degree is remarkable compared with the addition of NT-3 as well as the control, and can be said to be stronger than when adding CNTF. The characteristics of new neurons by addition of HGF were slightly longer than the control, but the extent of the effect of promoting elongation was weaker than when CNTF was added. On the other hand, neurite branching was remarkable, and a marked increase in the number of branches was observed with the addition of HGF. Furthermore, new nerve cells with addition of HGF are characterized by having thicker nerve fibers than those with the addition of NT-3, which is clearly different from the control, CNTF, or NT-3 addition group (more mature neurogenesis). ) Was confirmed.

  FIGS. 26 and 27 show stained images of HGF and NT-3 treated neurosphere 7 days after EGF removal. A significant increase in the number of new neurons was observed by the simultaneous addition of HGF and NT-3 as compared to the control. The number of newborn neurons was larger than that when NT-3 alone was added. In addition, new nerves formed by the simultaneous addition of HGF and NT-3 showed morphological characteristics in which nerve fibers were longer than controls and had many branches. Furthermore, the thickness of the nerve fiber was also thick, and when NGF-3 and HGF were added simultaneously, the new nerve showed a different form compared to the control, CNTF, NT-3 and HGF added groups alone, that is, a more mature form.

  28 and 29 show stained images of HGF and CNTF treated neurosphere 7 days after EGF removal. A significant increase in the number of new neurons was observed by simultaneous addition of HGF and CNTF compared to the control. The number of newborn nerve cells was larger than that when CNTF alone was added. The new nerve showed morphological features in which nerve fibers were much longer than controls and there were also many branches. Furthermore, the simultaneous addition of HGF and CNTF showed characteristics such as the thickness of new nerve fibers being thick. From the above, it showed a different form compared to the control, CNTF and NT-3 single addition group, and it was confirmed that the formation of many more mature neurogenesis occurs by simultaneous addition of HGF and CNTF.

(6) Comparison of neurosphere formation ability with and without HGF
When HGF was used in place of EGF, which was known for neurosphere formation, neurospheres could be formed in the same manner as when EGF was added (whether an increase in neural stem cells causing neurogenesis was observed). In the section of [1) Neural stem cell (neurosphere) culture method of Test Example 3, HGF (10 ng / mL) was added in place of EGF (20 ng / mL) to prepare primary cultured mouse neurosphere. As a control, a mouse embryo-derived neurosphere was similarly cultured in suspension without addition of EGF or HGF.
The results are shown in FIG. A significant increase in the size of the neurosphere was determined as well as an increase in the number of neurospheres formed compared to the control without addition of EGF by HGF. From this, it was found that HGF has a prominent promoting effect on the division and / or survival of neural stem cells.

(Statistical analysis)
Data are shown as mean ± standard deviation (SD), and statistical significance tests were evaluated by analysis of variance using Fisher's least significant difference method (PLSD).
Data of each group was analyzed with analysis software Statview 5.0 (SAS Institute, Inc.). Differences in probability values of p <0.05 were considered statistically significant.

  The neurogenesis-promoting agent of the present invention is useful as a drug for nerve regeneration or nerve repair in neurological diseases or neurological disorders associated with neurodegeneration or neuronal cell death.

FIG. 1 shows immunostained images of Ki-67 positive cells in the striatum of R6 / 2 (HSV-HGF) mice and wild-type littermates. In the figure, Str represents the striatum, LV represents the ventricle, and SVZ represents the subventricular zone. FIG. 2 is a diagram showing the number of BrdU positive cells in the mouse subventricular zone and the striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. * Indicates a significant difference (p <0.05) relative to wild-type littermates, and ** indicates a significant difference (p <0.05) relative to R6 / 2 (HSV-LacZ) mice. FIG. 3 is a diagram showing the number of cells in which both nestin and BrdU are positive in the mouse subventricular zone and striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. ** indicates a significant difference (p <0.05) from R6 / 2 (HSV-LacZ) mice. FIG. 4 is a diagram showing the number of cells in which both DCX and BrdU are positive in the mouse subventricular zone and the striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. ** Significant difference (p <0.05) relative to R6 / 2 (HSV-LacZ) mice. FIG. 5 shows the number of cells positive for both PSA-NCAM and BrdU in the mouse subventricular zone and striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. * Indicates a significant difference (p <0.05) relative to wild-type littermates, and ** indicates a significant difference (p <0.05) relative to R6 / 2 (HSV-LacZ) mice. FIG. 6 shows the number of cells positive for both βIII tubulin and BrdU in the mouse subventricular zone and striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. ** indicates a significant difference (p <0.05) from R6 / 2 (HSV-LacZ) mice. FIG. 7 is a diagram showing the number of cells positive for both NeuN and BrdU in the mouse subventricular zone and striatum. In the figure, A shows wild type littermates, B shows R6 / 2 mice, C shows R6 / 2 (HSV-LacZ) mice, and D shows R6 / 2 (HSV-HGF) mice. ** indicates a significant difference (p <0.05) from R6 / 2 (HSV-LacZ) mice. FIG. 8 is a diagram showing an immunostained image of cells in which both nestin and phosphorylated c-Met are positive in the mouse striatum. FIG. 9 shows immunostained images of cells positive for both DCX and phosphorylated c-Met in the mouse striatum. FIG. 10 shows that three vectors of HSV-HGF, AAV2-HGF and AAV4-HGF inserted with DNA encoding HGF protein were injected into the spinal cord parenchyma of rat lumbar spinal cord, and the expression level of HGF in the spinal cord after 5 days was determined. FIG. In the figure, U indicates the rostral side of the spinal cord, M indicates the central portion, and L indicates the caudal side. * Indicates a significant difference (p <0.05) from the control. FIG. 11 shows that three vectors of HSV-HGF, AAV2-HGF and AAV4-HGF inserted with DNA encoding HGF protein were injected into the spinal cavity of the rat lumbar spinal cord, and the expression level of HGF in the spinal cord after 5 days was determined. FIG. In the figure, U indicates the rostral side of the spinal cord, M indicates the central portion, and L indicates the caudal side. * Indicates a significant difference (p <0.05) from the control. FIG. 12 is a diagram showing immunostained images of nestin, MAP2, GFAP, and nucleus (Hoechst) in a differentiation assay of primary cultured mouse neurosphere. Merge: Overlaid images of immunostaining; Upper: Nestin and nucleus; Middle: MAP2 and nucleus; Lower: GFAP and nucleus. FIG. 13 is a diagram showing immunostained images of nestin, c-Met, and nucleus (Hoechst) in a mouse neurostriatum-derived secondary neurosphere differentiation assay. Merge: nestin, c-Met and nuclear immunostained images superimposed. FIG. 14 is a diagram showing immunostained images of nestin, c-Met, and nucleus (Hoechst) in a mouse neurostriatum-derived secondary neurosphere differentiation assay. Merge: nestin, c-Met and nuclear immunostained images superimposed. FIG. 15 is a view showing immunostained images of GFAP, c-Met and nucleus (Hoechst) in a mouse neurostriatum-derived secondary neurosphere differentiation assay. Merge: GFAP, c-Met and nuclear immunostained images superimposed. FIG. 16 shows immunostained images of oligodendrocyte progenitor cells (A2B5), c-Met, and nucleus (Hoechst) in a mouse neurostriate-derived secondary neurosphere differentiation assay (after 4 days). c-Met-Absorption: c-Met stained image after absorption test; Merge: A2B5, c-Met and nuclear immunostained images superimposed. FIG. 17 is a schematic diagram showing a neurosphere neurogenesis differentiation assay protocol in the presence of various trophic factors. FIG. 18 is an enlarged view of a typical example showing an immunostained image of MAP2 of the control neurosphere 7 days after EGF removal. FIG. 19 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of control neurosphere 7 days after EGF removal. FIG. 20 is an enlarged view of a typical example showing a MAP2 immunostained image of CNTF-treated neurosphere 7 days after EGF removal. FIG. 21 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of CNTF-treated neurosphere 7 days after EGF removal. FIG. 22 is an enlarged view of a typical example showing an immunostained image of MAP2 of NT-3 treated neurosphere 7 days after EGF removal. FIG. 23 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of NT-3 treated neurosphere 7 days after EGF removal. FIG. 24 is an enlarged view of a typical example showing an immunostained image of MAP2 of the HGF-treated neurosphere 7 days after EGF removal. FIG. 25 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of HGF-treated neurosphere 7 days after EGF removal. FIG. 26 is an enlarged view of a typical example showing an immunostained image of MAP2 of HGF and NT-3 treated neurosphere 7 days after EGF removal. In the figure, A shows a high degree of differentiation and B shows an increased number. FIG. 27 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of HGF and NT-3 treated neurosphere 7 days after EGF removal. FIG. 28 is an enlarged view of a typical example showing a MAP2 immunostained image of HGF and CNTF treated neurosphere 7 days after EGF removal. FIG. 29 is a diagram showing an immunostained image of MAP2 and nucleus (Topro3) of HGF and CNTF treated neurosphere 7 days after EGF removal. FIG. 30 is a diagram showing a comparison of the ability to form a neurosphere with and without HGF.

Claims (10)

  (1) (a) HGF protein or (b) a peptide that is a partial peptide of HGF protein and has substantially the same activity as HGF protein or a salt thereof, or (2) (a) DNA encoding HGF protein Or (b) DNA encoding a peptide that is a partial peptide of the HGF protein and having substantially the same activity as that of the HGF protein, or (c) hybridizes with the DNA under stringent conditions and is substantially the same as the HGF protein. A neurogenesis promoting agent comprising, as an active ingredient, a DNA comprising a DNA encoding a protein or peptide having the same activity.   The active ingredient is (b) a DNA encoding an HGF protein or (b) a partial peptide of the HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) complementary to the DNA. 2. The neurogenesis according to claim 1, which is a DNA encoding a protein or peptide that hybridizes under stringent conditions with a DNA comprising a simple nucleotide sequence and has substantially the same quality of activity as an HGF protein. Accelerator.   DNA encoding HGF protein is (a) DNA consisting of the base sequence represented by SEQ ID NO: 1, 2 or 5 or (b) DNA consisting of a base sequence complementary to the base sequence of (a) and stringent 3. The neurogenesis promoting agent according to claim 2, which is a DNA comprising a DNA that hybridizes under various conditions and encodes a protein having substantially the same activity as the HGF protein.   The neurogenesis promoting agent according to claim 2 or 3, wherein the DNA is incorporated into a type I herpes simplex virus (HSV-1) vector, an adenovirus vector, or an adeno-associated virus vector.   2. The nerve according to claim 1, wherein the active ingredient is (a) an HGF protein or (b) a partial peptide of the HGF protein and a peptide having substantially the same activity as the HGF protein or a salt thereof. Neonatal promoter.   The HGF protein is a protein comprising (a) an amino acid sequence represented by SEQ ID NO: 3, 4 or 6, or (b) a protein comprising substantially the same amino acid sequence as the amino acid sequence. The neurogenesis promoter as described.   The neurogenesis promoting agent according to any one of claims 1 to 6, wherein the neurogenesis promoting agent is for local administration.   The neurogenesis-promoting agent according to claim 7, wherein the local administration is intrathecal administration.   The neurogenesis promoting agent according to any one of claims 1 to 8, which is used in combination with a neurotrophic factor. 10. The neurogenesis promoting agent according to claim 9, wherein the neurotrophic factor is at least one selected from NT-3, NT-4, CNTF, GDNF and BDNF.