JP5271632B2 - Type VI collagen with neuronal cell death inhibitory action - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a neurocyte death inhibiting factor and/or a neurocyte death inhibitor or a neurocyte death inhibiting method. <P>SOLUTION: Inclusion of type IV collagen having a neurocyte death inhibiting action inhibits neurocyte death. In addition, an extension accelerator of neurite containing type IV collagen is also provided. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

The present invention relates to type VI collagen having a nerve cell death inhibitory action.

When a mammalian peripheral nerve axon is damaged, the distal side of the injury site degenerates and disappears, and regeneration of the axon begins from the proximal stump. These biological reactions are expected to involve a large number of substances, but the whole picture has not been clarified. In order to suppress neuronal cell death and extend axons, it has been suggested that interaction between neurons and extracellular matrix (ECM) is required. It remains almost unknown.

Collagen, which is a major component of ECM, is a group of different types (types), and its existence from type I to type XIX has now been clarified. These are broadly classified into those involved in the structure and adhesion of the fiber structure and those having a network structure. Type I, type II, type III, type V and type XI collagen are fibrogenic, whereas type IV collagen forms a basement membrane, and type VI collagen forms microfibrils. It is known that type VII collagen forms anchoring fibrils, and type VIII and type X collagen form a single-chain sheet structure (Non-patent Document 1). Among these, type I collagen is the most abundant in the living body and is used for various purposes as a medical material. For example, by coating type I collagen on a culture vessel, cell adhesion is improved and differentiation is promoted (Patent Documents 1 and 2). It has also been reported that type I collagen has a process of promoting the extension of protrusions on cultured neurons (Non-patent Document 2). However, the interaction between other collagens and nerve cells is not necessarily the same as that of type I collagen. For example, it has been reported that type IV collagen contained in large amounts in scars of damaged nerves has a neurite outgrowth inhibitory effect (Non-patent Document 3). There is also a report that type V collagen suppresses neurite outgrowth (Non-patent Document 4). As described above, collagen may act to promote nerve regeneration depending on the type, or may act in a suppressive manner. The effect of collagen other than the type mentioned here on nerve regeneration has not been investigated.

Elucidation of pathological conditions associated with neuronal cell death and establishment of treatment methods are important issues in the central nervous system. In particular, there is a strong social demand for the development of treatments for neurodegenerative diseases represented by Alzheimer's disease and Parkinson's disease. For example, although neuronal cell death induced by Alzheimer's disease is thought to be due to apoptosis, although elucidation of the molecular mechanism has made significant progress in recent years, there are many unknown parts. Therefore, identification of new factors that regulate neuronal cell death including apoptosis is expected.

In recent years, research on regenerative medicine using cell differentiation / proliferation ability has become active with the aim of reconstructing damaged tissues, organs, or organs that have been damaged or degenerated. Nerve regeneration research is no exception, and the transplantation of nerve cells directly into the lesion is expected to solve side effects due to repeated administration of the drug, complicated drug delivery problems, and the like. In fact, nerve cell transplantation is performed in the treatment of Parkinson's disease, and a certain effect has been shown in clinical trials (Non-patent Document 5). However, in the method of transplanting the nerve tissue obtained from the aborted fetus, about 8 embryonic nerve tissues are required for one transplantation, so the lack of donor cells is a big problem. Nor does it solve ethical issues. Even if the patient's own sympathetic ganglion tissue piece is autotransplanted, there is a risk of damage to the dominant region of the collected nerve cells, so the amount of collection is limited. In any case, it is necessary to develop a method for efficiently engrafting cells by suppressing the death of nerve cells and a method for producing transplantable neurons. In neural stem cell transplantation, which has recently been in the limelight, it is expected that the time and economic burden of maintaining cells will be reduced by developing similar methods.
JP2002-142751 JP2007-014352 Collagen metabolism and disease, page 112, Kodansha (1982) Cell, vol. 68, no. 2, pp. 303-322, January 24, 1992 Journal of Neurotrauma, vol. 23, no. 3-4, pp. 422-436, April 1, 2006 Journal of Neuroscience, vol. 21, no. 16, pp. 6125-6135, August 15, 2001 Japanese Journal of Neurosurgery (Tokyo), vol. 14, no. 8, pp. 487-492, August 20, 2005

Therefore, identification of a factor that suppresses neuronal cell death and development of a neuronal cell death inhibitor or a method of inhibiting neuronal cell death are highly desired.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a neuronal cell death inhibitory factor and / or a neuronal cell death inhibitor or a neuronal cell death inhibitory method.

The regulatory mechanisms of neuronal cell death are still unclear. Therefore, in the present invention, a novel neuronal cell death inhibitor was identified.

That is, according to the present invention, a neuronal cell death inhibitor containing Type VI collagen is provided. Since the present neuronal cell death inhibitor contains type VI collagen having a neuronal cell death inhibitory action, it suppresses neuronal cell death.

The present invention also provides a neurite extension promoter containing type VI collagen. Since this neurite extension promoter contains type VI collagen having a neurite extension promoting action, it promotes neurite extension.

Moreover, according to this invention, the nerve regeneration promoter containing VI type collagen is provided. Since this nerve regeneration-promoting agent contains type VI collagen having a nerve regeneration-promoting action, it promotes nerve regeneration.

In addition, according to the present invention, there is provided a method for suppressing neuronal cell death outside the human body, the method comprising the step of bringing neuronal cells into contact with type VI collagen. According to this method, nerve cell death is suppressed because the type VI collagen having a nerve cell death inhibitory effect is brought into contact with the nerve cell.

In addition, according to the present invention, there is provided a method for promoting neurite outgrowth outside the human body, the method comprising the step of contacting a neuron with a type VI collagen to promote neurite outgrowth. According to this method, since the type VI collagen having the neurite extension promoting action is brought into contact with the nerve cell, the extension of the neurite is promoted.

In addition, according to the present invention, there is provided a method for promoting nerve regeneration outside the human body, which comprises a step of bringing nerve cells into contact with type VI collagen. According to this method, nerve regeneration is promoted because the type VI collagen having nerve regeneration promoting action is brought into contact with nerve cells.

According to the present invention, there is also provided a method for culturing a nerve cell, the method comprising culturing a nerve cell, comprising a step of contacting the nerve cell with type VI collagen in a culture vessel. According to this method, since the type VI collagen having a nerve cell death inhibitory action, a neurite extension promoting action and / or a nerve regeneration promoting action is brought into contact with the nerve cell, the nerve cell death is suppressed, the neurite extension is promoted, and Promotion of nerve regeneration occurs.

Moreover, according to this invention, the nerve cell culture kit containing a type VI collagen and a culture container is provided. Since this kit contains type VI collagen having a neuronal cell death inhibitory effect, a neurite outgrowth promoting effect and / or a neural regeneration promoting effect, it suppresses neuronal cell death, promotes neurite outgrowth and / or promotes nerve regeneration Happens.

Moreover, according to this invention, the medical material for nerve regeneration containing a type VI collagen is provided. Since this medical material contains type VI collagen having a nerve cell death inhibitory action, a neurite extension promoting action and / or a nerve regeneration promoting action, it suppresses nerve cell death, promotes neurite extension and / or nerve regeneration. Promotion occurs.

According to the present invention, since it contains type VI collagen having a nerve cell death inhibitory action, a neurite extension promoting action and / or a nerve regeneration promoting action, it suppresses nerve cell death, promotes neurite extension and / or nerve regeneration. The promotion of

<Explanation of terms>
In this specification, the meaning of various terms shall be defined as follows.

(1) Type VI collagen The term “type VI collagen” as used herein includes native sequence type VI collagen and type VI collagen variants. The type VI collagen described herein may be prepared by recombinant or synthetic methods or isolated from various sources.

“Native sequence type VI collagen” is a polypeptide having the amino acid sequence of naturally occurring type VI collagen. Such native sequence type VI collagen can be produced by recombinant or synthetic means and can be isolated from various sources.

By “type VI collagen variant” is meant type VI collagen having 80% or more amino acid sequence identity with the full-length native sequence type VI collagen disclosed herein.

(Type VI collagen variant polynucleotide)
A “variant polynucleotide” is a nucleic acid molecule that encodes a polypeptide having the inherent activity of the polypeptide, and is 80% of the nucleic acid sequence encoding the full-length native sequence type VI collagen polypeptide disclosed herein. % Sequence identity.

(2) Suppression of neuronal cell death “suppressing neuronal cell death” is to inhibit neuronal cells from being killed by apoptosis or necrosis. A person skilled in the art can measure neuronal cell death using trypan blue staining or the TUNEL method (TUNEL: terminal deoxynucleotidyl transferase (TdT) -mediated dUTP-biotin nick end labeling). Moreover, it is not necessary to suppress the death of all the nerve cells, and 50%, 60%, 70%, 80%, 90%, 95% It means to suppress 100%.

(3) Extension of neurite “Extend neurite” means that dendrites or axons extend from nerve cells. One skilled in the art can measure neurite outgrowth using morphological techniques and measurement software. Also, the promotion of neurite extension means 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to the case where neurite extension is not promoted. Alternatively, it means that neurite extension increases by 100% or more.

(4) Nerve regeneration “Nerve regeneration” means suppression of nerve cell death, nerve cell proliferation, neurite extension, synapse (re) formation, neural circuit (re) formation and / or target organ ( (Re) control will take place. Further, the promotion of nerve regeneration means that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more compared to the case where nerve regeneration is not promoted. Means to induce nerve regeneration.

(5) Contact between nerve cells and type VI collagen “Contact between nerve cells and type VI collagen” refers to a state in which type VI collagen can act on neurons, and is not particularly limited as long as it acts. Absent. For example, type VI collagen may not adhere directly to nerve cells, may adhere to only part of nerve cells, or may adhere to cells surrounding the nerve cells.

(6) Culture “culture” refers to differentiation, proliferation, growth and / or development of nerve cells (in this case, not only differentiated nerve cells but also undifferentiated neural stem cells, neural progenitor cells, and neuroblasts). However, as for the culture conditions of nerve cells, appropriate conditions can be appropriately applied from the corresponding conventionally known culture conditions. A person skilled in the art can easily select culture conditions corresponding to nerve cells, and culture can be performed based on the selected culture conditions.

(7) The vector vector may be any vector that can be conveniently subjected to recombinant DNA procedures, and selection of the vector often depends on the host cell into which the vector is to be introduced. Thus, the vector may be a self-replicating vector, ie a vector that exists as extrachromosomal material whose replication is independent of chromosomal replication.

(8) Promoter As used herein, “promoter” refers to a sequence that controls the expression of a polypeptide present downstream of a promoter sequence. Here, a promoter that can be expressed in a region including nerve cells such as cerebrum, cerebellum, brain stem, spinal cord and peripheral somatic or autonomic ganglia is desirable, and those skilled in the art can select an appropriate promoter. it can.

Embodiments of the present invention will be described below.

<Type VI collagen>
The type VI collagen used in the present embodiment below is a native sequence type VI collagen and / or a type VI collagen variant. A type VI collagen variant is a type VI collagen in which one or more (for example, 2, 3,...) Amino acid residues are added, substituted, or deleted at the N- or C-terminus of the full-length natural amino acid sequence. included. Typically, a type VI collagen variant is 80% or more amino acid sequence identity, preferably 90% or more amino acid sequence identity with the full length natural amino acid sequence disclosed herein, or the full length natural sequence VI collagen sequence disclosed herein. More preferably 95% or more amino acid sequence identity. SEQ ID NO: 1 shown below shows the amino acid sequence of the full-length native sequence type VI collagen α1 subunit, and SEQ ID NO: 3 shows the amino acid sequence of the full-length native sequence type VI collagen α2 subunit. 5 shows the amino acid sequence of the full-length native sequence type VI collagen α3 subunit.

As defined herein, “percent (%) amino acid sequence identity” may align sequences and introduce gaps so that maximum percent sequence identity is obtained, and conservative substitutions may be considered part of sequence identity. Defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a type VI collagen sequence, not considered part. Selection of alignments for the purpose of determining percent amino acid sequence identity can be achieved using methods well known to those skilled in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR). Is possible by using. One skilled in the art can determine appropriate parameters to measure alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Examples of mutants of type VI collagen include solubilized type VI collagen and fragmented type VI collagen.

The variant polynucleotide used in the present embodiment below has a nucleic acid sequence identity of 80% or more, preferably 90% of the nucleic acid sequence encoding the full-length native sequence type VI collagen polypeptide disclosed herein. It has identity, more preferably 95% nucleic acid sequence identity. Variants do not contain natural nucleic acid sequences. SEQ ID NO: 2 shown below contains a full-length sequence encoding human type VI collagen α1 subunit, and SEQ ID NO: 4 contains a full-length sequence encoding human type VI collagen α2 subunit. Number: 6 contains the full length sequence encoding the human type VI collagen α3 subunit.

“Percent (%) nucleic acid sequence identity” relative to the type VI collagen-encoding nucleic acid sequence identified herein may align the sequences and introduce gaps to obtain maximum percent sequence identity. Defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides of a type I collagen polypeptide-encoding nucleic acid sequence. Selection of alignments for the purpose of determining percent nucleic acid sequence identity can be accomplished using methods well known to those skilled in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR). This is possible by using wear. One skilled in the art can determine appropriate parameters to measure alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The “type VI collagen variant polynucleotide” encodes a type VI collagen variant polypeptide, and preferably a nucleic acid encoding the polypeptide shown in SEQ ID NO: 1 under stringent hybridization and washing conditions. It may be a nucleic acid molecule that hybridizes to a sequence. The type VI collagen variant polypeptide may be encoded by a type VI collagen variant polynucleotide.

In type VI collagen, there are different subunits of α1, α2, and α3. Two α subunits that are associated in the opposite direction gather to form a tetramer. Outside the cell, it further polymerizes to form microfibrils. The type VI collagen used in this embodiment may not be a tetramer but a dimer. The structural unit of the α subunit may also be a monomer or a dimer of the α1 subunit, the α2 subunit and / or the α3 subunit.

<Embodiment 1: Neuronal cell death inhibitor>
This embodiment is a neuronal cell death inhibitor containing type VI collagen. As shown in Examples described later, since type VI collagen has a nerve cell death inhibitory action, a nerve cell death inhibitor containing it also suppresses nerve cell death. Further, in the neuronal cell death inhibitor of this embodiment, 100% of the collagen contained is preferably type VI collagen, but it is not an essential condition that it is 100%, but 50%, 60% 70%, 80%, 90%, or 95% or more of type VI collagen.

<Embodiment 2: Neurite extension promoter>
This embodiment is a neurite extension promoter containing type VI collagen. As shown in the Examples described later, since type VI collagen has a neurite extension promoting action, a neuronal cell death inhibitor containing the same also promotes neurite extension. Further, in the neurite extension promoter of this embodiment, 100% of the collagen contained is preferably type VI collagen, but it is not an essential condition that it is 100%. %, 70%, 80%, 90%, or 95% or more of type VI collagen may be contained.

<Embodiment 3: Nerve regeneration promoter>
This embodiment is a nerve regeneration promoter containing type VI collagen. As shown in Examples described later, since type VI collagen has a nerve regeneration promoting action, a nerve cell death inhibitor containing the same promotes nerve regeneration. Further, in the nerve regeneration promoting agent of the present embodiment, 100% of the contained collagen is preferably type VI collagen, but it is not an essential condition that it is 100%, 50%, 60%, 70%, 80%, 90%, or 95% or more of type VI collagen may be contained.

The agent in the present embodiment can be made into a medicine by adding a carrier that is alone or pharmaceutically acceptable. Pharmaceutically acceptable carriers include, for example, excipients, diluents, additives, disintegrants, binders, coating agents, wetting agents, lubricants, lubricants, flavoring agents, sweeteners, solubilizers, etc. Specifically, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, gelatin, starch, cellulose and cellulose derivatives, animal oils and vegetable oils, polyethylene glycol, water, monovalent or polyvalent Mention may be made of alcohol.

The dosage form of the agent in the present embodiment is not particularly limited, and examples thereof include capsules, tablets, pills, solutions, syrups, troches, powders, granules, infusions, injections, and the like. Is a tablet, granule, powder or capsule that is easy to formulate.

Moreover, the agent of this embodiment can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms, such as powders, granules, capsules, syrups, suspensions, etc., or, for example, in dosage forms such as solutions, emulsions, suspensions, etc. In addition to the topical administration parenterally in the form of injection, it can also be administered in a liquid, sheet or tube form so as to contact the site of nerve injury.

Furthermore, the agent in the present embodiment may be an agent including a vector having a nucleic acid sequence encoding a desired protein. Examples of the vector of this embodiment include a plasmid, phage, cosmid, minichromosome, or virus. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the host cell genome and replicated with one or more chromosomes integrated therein. Examples of suitable vectors are bacterial expression vectors and yeast expression vectors. Examples of the viral vector include, but are not limited to, vectors derived from viruses such as adenovirus, retrovirus, papoba virus such as SV40, vaccinia virus, fowlpox virus, and pseudorabies virus. By using an agent containing a vector prepared by selecting a suitable promoter, a desired amount of type VI collagen can be expressed at a desired site.

Moreover, the type VI collagen used in the present embodiment promotes the adhesion of nerve cells as shown in Examples described later. By having an action of promoting the adhesion of nerve cells, it can be used, for example, as a fixing accelerator for transplanted nerve cells during nerve transplantation.

Moreover, the type VI collagen used in this embodiment forms a scaffold for nerve cells in nerve regeneration, as shown in the examples described later. By having a nerve cell scaffold-forming action, it can be used, for example, as a drug that induces axonal regeneration in damaged nerves that have undergone cutting, crushing, and the like.

Moreover, the type VI collagen used in the present embodiment promotes the adhesion of nerve cells as shown in Examples described later. By having a nerve cell adhesion promoting action, for example, when culturing nerve cells, it can also be used as a nerve cell fixing promoter.

Moreover, the type VI collagen used in this embodiment forms a scaffold for nerve cells in nerve regeneration, as shown in the examples described later. By having a nerve cell scaffolding action, it can be used, for example, as an axon inducing agent when axons are elongated outside the human body.

<Embodiment 4: Method for suppressing neuronal cell death>
This embodiment is a method for suppressing neuronal cell death in vitro, and includes a step of bringing neuronal cells into contact with type VI collagen. As shown in the Examples described later, since type VI collagen has a nerve cell death inhibitory action, it is brought into contact with nerve cells to suppress nerve cell death.

<Embodiment 5: Method for Promoting Neurite Extension>
The present embodiment is a method for promoting neurite outgrowth outside the human body, and includes a step of bringing a nerve cell into contact with type VI collagen and promoting neurite outgrowth. As shown in Examples described later, type VI collagen has a neurite outgrowth promoting action, so that it is brought into contact with nerve cells to promote neurite outgrowth.

<Embodiment 6: Method for promoting nerve regeneration>
The present embodiment is a method for promoting nerve regeneration in vitro, and includes a step of bringing nerve cells into contact with type VI collagen. As shown in the Examples described later, since type VI collagen has a nerve regeneration promoting action, nerve regeneration is promoted by bringing it into contact with nerve cells.

<Embodiment 7: Nerve cell culture method>
In addition, the present embodiment is a method for culturing nerve cells, and includes a step of contacting nerve cells with type VI collagen in a culture container. As shown in Examples described later, since type VI collagen has a nerve cell death inhibitory action, it is used for culturing nerve cells and brought into contact with nerve cells to suppress nerve cell death. Further, in Examples described later, neuronal cell death is suppressed by culturing using a plate coated with type VI collagen, but the method of contacting type VI collagen and neurons is not limited thereto. . For example, neuronal cell death can be suppressed by adding type VI collagen or solubilized type VI collagen to the medium or by introducing a gene.

Moreover, the type VI collagen used in the present embodiment suppresses the aggregation of nerve cells as shown in Examples described later. By having an action of suppressing aggregation of nerve cells, the nerve cells can be cultured efficiently.

The culture solution used in the culturing method of the present embodiment may be of a conventionally known composition that is suitable for culturing nerve cells. For example, a commercially available culture solution for culturing nerve cells can be used.

The incubator used for the culture of the present embodiment is not particularly limited as long as it can culture nerve cells, but a CO ₂ incubator similar to that generally used for culture can be used. Usually, the CO ₂ incubator is often set to a CO ₂ concentration of 5 to 10%, a temperature of 37 ° C., and a relative humidity of 80% or more.

The culture container used in the culture method of the present embodiment may be a conventionally known culture container that is suitable for culturing nerve cells, and for example, a commercially available culture container can be used. In the examples described later, a 96-hole polystyrene plate is used, but a plate with 4 holes, 6 holes, 12 holes, 24 holes, and 48 holes can be used, and a dish or the like can also be used. In addition, the material of the culture vessel is not limited to polystyrene, and a conventionally known material suitable for culture such as methylpentene resin and polycarbonate can be used.

In addition, the culture vessel used in the culture method of the present embodiment is processed so that nerve cells and type VI collagen are in contact. In the examples described later, the culture vessel is coated with type VI collagen, but the invention is not limited to this, and it is sufficient that the nerve cells and type VI collagen are in contact with each other. For example, the entire inside of the culture vessel may be coated, or only the inner bottom surface or the inner side surface may be coated. In the present embodiment, the term “coated with type VI collagen” means that nerve cells and type VI collagen can be contacted even if the culture vessel material is not completely covered. You may coat | cover in mesh shape and filament shape. In addition, the type VI collagen may be coated in multiple layers such as single, double, triple, etc., and the thickness thereof should be selected by those skilled in the art to be suitable for culture. Can do.

Moreover, this embodiment is a nerve cell culture kit including type VI collagen and a culture vessel. The kit of this embodiment may be a kit further containing reagents used for culture of nerve cells. Although it does not specifically limit as a reagent contained in the kit of this embodiment, For example, the culture solution for commercially available nerve cells can be used suitably. For example, Dulbecco's modified Eagle's medium (DMEM), Hanks'F-12 medium, or a mixture of them at 1: 1 can be suitably used as a reagent.

<Eighth embodiment: medical material>
The type VI collagen used in the present embodiment can be formed into a sheet for use as a medical material or a culture material without being used as a medicine. In addition to being able to cover the culture container in the form of a sheet, it can act as a scaffold for nerve regeneration by administering it so as to contact nerve cells at the nerve injury site, for example, covering the injury site it can. Moreover, the agent of this embodiment made into the sheet form can be administered also as a composition containing a nerve cell. By adopting a form containing nerve cells, it can be used for nerve transplantation and the like, and nerve regeneration can be promoted.

Further, the type VI collagen used in the present embodiment can be formed into a tube shape for use as a medical material or a culture material without being used as a medicine. By making it into a tube shape, proliferation of nerve cells and / or extension of neurites can be efficiently induced in the hollow portion, and nerve regeneration at the nerve cutting portion can be promoted as an artificial neural tube. Furthermore, the agent of this embodiment can also be used by filling a biocompatible material. Examples of biocompatible materials that can be used include aliphatic polyester resins such as silicon, polylactic acid, glycolic acid, polydioxanic acid, lactic acid-glycolic acid copolymer, and glycolic acid-polydioxane-trimethylene carbonate. Moreover, these sheet-form or tube-form VI type collagen can take forms, such as a mesh form, a filament form, and sponge form, and those skilled in the art can select according to a condition.

<Combination with other ingredients>
In this embodiment, it can also be used in combination with other substances having a nerve cell death inhibitory effect, a neurite extension promoting effect and / or a nerve regeneration promoting effect. Examples of other substances include neurotrophic factors such as nerve growth factor (NGF), cell adhesion molecules such as laminin, and hormones such as erythropoietin. As long as it has an extension promoting action and / or nerve regeneration promoting action, it is not limited thereto.

As mentioned above, although embodiment of this invention was described, these are only illustrations of this invention.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

Example 1 Identification of genes involved in nerve regeneration

(Materials and methods)
Surgical treatment and tissue preparation Wistar adult rats (body weight 200-300 grams) were anesthetized with diethyl ether and chloral hydrate (intraperitoneal administration, 100 mg / kg), then the left sciatic nerve was cut, and the right sciatic nerve was Left untreated. 3, 5 and 7 days after cutting, the proximal and distal cut ends of the left sciatic nerve and the right sciatic nerve trunk were removed and stored at −80 ° C. until use. Chronic strangulation model rats were prepared by the method of Bennett and Xie (1988). All treatments were performed according to the Kanazawa University animal experiment regulations.

Preparation of Complementary DNA (cDNA) Library Total cellular RNA was extracted from the proximal cut end of the left sciatic nerve and the right sciatic nerve trunk by the method of Chirwin et al. (1979), and poly (A) ⁺ RNA was obtained from Promega. , Madison, Wis.) Kit (PolyATract mRNA Isolation System III). Furthermore, the suppression subtractive hybridization polymerase chain reaction method of Diatchenko et al. (1996). (Hybridization PCR) method was performed using a Clontech kit (PCR Select; Clontech Laboratories). At this time, the proximal cut end of the left sciatic nerve and cDNA derived from the right sciatic nerve trunk were used as a driver and a tester, respectively. After obtaining a pool of cDNA fragments of the gene whose expression is increased at the proximal cut end of the left sciatic nerve and digesting with restriction enzymes Sau3AI and NotI, pGEM-3zf (+) (Promega) was digested with BamHI and NotI. It was incorporated into a vector prepared by digestion and transformed into E. coli competent cells (TOP10F ′, Invitrogen).

Determination of cDNA base sequence and database analysis The base sequence of cloned cDNA was determined by the dideoxy method using a 370A DNA sequencer from Applied Biosystems, Foster City, CA. Identification of the protein encoded by the obtained sequence was performed using the BLAST program while collating with the database of the National Center for Biotechnology Information (NCBI).

RNA blot hybridization analysis Total cellular RNA (2 μg each) extracted from the proximal and distal cut ends of the left sciatic nerve and the right sciatic nerve trunk by the method of Chomczynski and Sacchi (1987) was added to 1.0% agarose. After separation by electrophoresis on a -2.2M formamide gel, it was transferred to a nylon membrane (Zeta-Probe; Bio-Rad Laboratories, Hercules, CA) and hybridized with radioisotope labeled cDNA. The composition of the hybridization solution was 50% formamide, 50 mM sodium phosphate buffer (pH 7.0), 5 × SSC (1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate), 250 μg / ml salmon. Sperm DNA, 0.5% sodium dodecyl sulfate (SDS), 0.1% bovine serum albumin (BSA), 0.1% ficoll and 0.1% polyvinylpyrrolidone. After hybridizing overnight at 42 ° C., the nylon membrane was finally rinsed with 0.1 × SSC containing 0.1% SDS at 65 ° C. Autoradiography was performed at −30 ° C. and an intensifying screen was used. The scion image program (Scion Image program; Scion Corp., Frederick, MD) was used for quantification of the degree of blackening of the band.

(Results and discussion)
In order to identify genes involved in nerve regeneration from the extracellular matrix, a gene whose expression was elevated at the cut end of the proximal rat sciatic nerve was cloned. 135 cDNA clones were isolated, of which the genes encoding the ECM genes were type III collagen and type VI collagen α1 chain, lumican, and laminin. Further, when RNA blot hybridization analysis was performed, it was detected that type VI collagen, which is a 4.2 kb mRNA, was expressed at the proximal and distal cuts of the sciatic nerve. In particular, it was confirmed that the expression at the proximal cut portion was significantly increased as compared with the untreated sciatic nerve trunk (FIGS. 1A and 1B).

Although redundantly described, FIG. 1A shows expression of α1 subunit of type VI collagen and 18S ribosomal RNA. Each lane was loaded with 2 μg of total cellular RNA extracted from the proximal cut of the sciatic nerve (Prox), the distal cut (Dist) and the sciatic nerve trunk (Contra). The position of the molecular weight marker is shown on the left side. FIG. 1B shows the result of quantitative analysis of α1 subunit expression of type VI collagen using 18S ribosomal RNA as an internal standard and standardized based on the value of nerve trunk. Bars indicate mean ± standard error (n = 3). An asterisk (*) indicates a significant difference when compared with the value of the nerve trunk (P <0.05). From the above, it was revealed that the expression of type VI collagen was elevated in the cut rat sciatic nerve.

Example 2 Analysis of the activity of type VI collagen

(Materials and methods)
Cell Culture Adult rat rat dorsal root ganglion (DRG) neurons were cultured according to the method of Rizzo et al. (1994). After removing D4 of L4 and L5 to remove connective tissue, enzyme treatment was performed in a collagenase-containing solution and a papain-containing solution. Further, a mixture of Dulbecco's modified Eagle's medium (DMEM) and Hanks'F-12 medium 1: 1 was mixed with 10% fetal bovine serum, 1.5 mg / ml trypsin inhibitor, 1.5 mg / ml. DRG was transferred to a solution containing mlBSA, 100 U / ml penicillin and 0.1 mg / ml streptomycin, the cells were dispersed by pipetting, and then plated on a 96-well polystyrene plate (269620; Nunc, Roskilde, Denmark). . Polystyrene plates prepared in advance and not coated with type VI collagen were prepared.

The culture of superior cervical ganglion (SCG) neurons followed the method of Fernandez-Fernandez et al. (1999). SCG was removed from adult rats, first incubated in a collagenase (2 mg / ml) solution at 4 ° C. for 30 minutes, then in a trypsin (0.5 mg / ml) solution at 37 ° C. for 20 minutes, and a tapered path The cells were dispersed by pipetting with a tool pipette, and then centrifuged. The collected cells were dispersed in L-15 medium containing fetal bovine serum, 2 mM glutamic acid, 24 mM sodium bicarbonate, 38 mM glucose, 100 U / ml penicillin, 0.1 mg / ml streptomycin and 100 ng / ml NGF, and 96-well polystyrene. Plated (269620; Nunc).

Rat pheochromocytoma-derived PC12 cells (Greene et al., 1998) were maintained in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and 5% horse serum in an incubator at 37 ° C., 10% CO ₂ , humidified conditions. . This was dispersed in the same culture solution plus 250 ng / ml NGF, and seeded in the same 96-well polystyrene plate as described above at a density of 2500 cells per well.

Neuroblastoma × spinal neuron hybrid cell line NSC34 (Cashman et al., 1992) was maintained in Dulbecco's modified Eagle medium containing 10% fetal calf serum and then plated on polystyrene plates in the same manner as described above.

Measurement of neurite extension Measurement of neurite extension was according to the method of Turner et al. (1989). PC12 cells were detached with Hanks buffered saline containing 0.125% (w / v) trypsin and 1 mM EDTA, washed with DMEM without serum, and then seeded on a 96-well polystyrene plate (269620; Nunc). The hole remains untreated, laminin (50 μg / ml), poly-D-ornithine (100 μg / ml), poly-L-lysine (50 μg / ml), type I collagen (10 μg / ml), or type VI collagen (10 μg / ml) solution coated overnight at 4 ° C. was used. Cells were seeded at a density of 2500 per well and NGF was added to 250 ng / ml. Cells having neurites with a length of three times the diameter of cell bodies were made positive, and the numbers were compared 3 hours, 3, 6, 9, and 14 days after the start of culture.

Measurement of cell death Cell death was measured by trypan blue (0.4%) staining and the TUNEL method. The TUNEL method was carried out using a Roche kit (In Situ Cell Death Detection Kit, POD; Roche, Penzberg, Germany) according to the description of Osamura et al. (2005). PC12 cells cultured on polystyrene plates coated with different substrates as described above were fixed with 4% paraformamide and treated with proteinase K (20 μg / ml) dissolved in 10 mM Tris buffer (pH 7.5) for 30 minutes. . Subsequently, after washing with phosphate-buffered saline (PBS), the mixture was immersed in methanol containing 3% hydrogen peroxide for 30 minutes at room temperature in order to inactivate endogenous peroxidase. After washing again with PBS, 0.1% Triton X-100 diluted with 0.1% sodium citrate was added to increase membrane permeability and allowed to react on ice for 2 minutes. Further, the reaction was carried out at 37 ° C. for 1 hour with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluororesin-labeled UTP. In order to detect the incorporated fluororesin, after reacting with an anti-fluororesin antibody labeled with HRP (horse-radish peroxidase), 3,3′-diaminobenzidine was added as a chromogenic substrate. The specimen was observed under an optical microscope and visualized with a camera connected to a computer.

Immunoperoxidase-stained tissue sections were prepared according to the method described by Felts et al. (1997). Three days after the sciatic nerve was cut, the rats were deeply anesthetized with diethyl ether and chloral hydrate, and perfused in the order of PBS and fixative (4% paraformamide) from the heart. The proximal and distal cut ends of the left sciatic nerve and the right sciatic nerve trunk were removed and immersed in the same fixative solution (4% paraformamide) at 4 ° C. overnight for postfixation. Furthermore, it was immersed in a 30% sucrose solution at 4 ° C. for 2 nights to improve the freezing resistance (cryoprotection). The tissue specimen was cut to a thickness of 2-16 μm and placed on a glass slide coated with poly-L-lysine. Sections were blocked with PBS containing Triton X-100, 1% BSA and 1% normal goat serum for 3 hours, and then at 4 ° C. overnight rabbit polyclonal anti-type VI collagen antibody (H-200; 1: 200 dilution, Santa Cruz) Biotechnology, Santa Cruz, CA). To detect immunoreactivity, a Nichirei kit (Histofine Simple Stain MAX PO (M); Nichirei, Tsukiji, Japan) was used according to the attached instructions. After color development, the sections were dehydrated and transparent, covered with a cover glass, and observed under an optical microscope.

From a tissue 3 days after the fluorescence double-staining sciatic nerve was cut, a 20 μm-thick section was prepared in the same manner as described above, and a rabbit anti-type VI collagen antibody (H-200; 1: 200 dilution) overnight at 4 ° C .; (Santa Cruz Biotechnology). At the same time, as mouse monoclonal antibodies, S-100 protein antibody (1: 200 dilution; Sigma), anti-glial fibrillary acidic protein (GFAP) antibody (GA-5; 1: 400 dilution; Sigma), Anti-signal regulatory protein (SIRP) antibody (OX-41; 1:50 dilution; Chemicon International, Temecula, CA), anti-Thy1 antibody (MRC OX-7; 1:50 dilution; Chemicon International), anti-neuro Double staining with filament antibody (RT97; 1: 100 dilution; Roche, Mannheim, Germany) or anti-tyrosine hydroxylase antibody (Clone45; 1: 500 dilution; BD Biosciences, Pharmingen, San Diego, CA) It was. Secondary antibodies include fluorescein isothiocyanate (FITC) labeled anti-rabbit IgG antibody (1: 500 dilution; Molecular Probes, Eugene, OR) and Texas red labeled anti-mouse IgG antibody (1: 500 dilution; (Molecular Probes) and reacted at 4 ° C. for 3 hours. Sections were observed using a confocal laser microscope (LSM5 PASCAL; Carl Zeiss, Gottingen, Germany), and the resulting images were corrected using Adobe Photoshop version 5.5 (Adobe Systems, San Jose, CA). did.

(result)
(Effect of type VI collagen on neurite outgrowth)
The effect of type VI collagen on neurite outgrowth was tested using DRG neurons, SCG neurons, PC12 cells and NSC34 cells. As shown in FIG. 2, it was observed that DRG neurons adhered to a plate coated with type VI collagen and the neurites were extended. The cell adhesion was finally observed. Similar trends were observed in SCG neurons, PC12 cells and NSC34 cells. In addition, the following experiment was performed using PC12 cells having excellent cell stability.

FIG. 2 is a diagram showing the extension of neurites observed when various neurons were cultured on a plate coated with type VI collagen (upper) or an uncoated plate (lower). It is. From the left, the results of DRG neurons, SCG neurons, neuroblastoma x spinal cord neuron hybrid cell line NSC34 cells, and pheochromocytoma-derived PC12 cells are shown. When cultured on uncoated plates, neurite outgrowth was not observed in all cell types and aggregated. The scale bar represents 50 μm.

Furthermore, using PC12 cells, the frequency of cells in which the neurites extended 3 times or more the cell diameter was measured. As shown in FIG. 3, it was observed that the neurites were extended more than 3 times the cell diameter in the most cells in the group using the plate coated with type VI collagen (type VI group). At 3 hours after culturing, neurite outgrowth was observed in 12.9 ± 2.6% (n = 5; total 257 cells) cells. In the sections using laminin-coated plates and type I collagen-coated plates (laminin sections and type I sections, respectively), neurite outgrowth was 9.6 ± 2.9% and 3.9 ± 1. 2% (3 hours after culture, n = 5) was observed. On day 3 of culture, there was not much difference between type VI and laminin groups, but on day 6 of culture, there were significantly more cells with neurites extending more than 3 times the cell diameter in type VI. It was. Furthermore, on the 14th day of culture, the type VI section was 4.7 ± 1.9% (n = 5), whereas the laminin section was substantially zero. Cells showing neurite outgrowth were also observed in the type I section, but not after day 6 of culture. In addition, no neurite outgrowth was observed in the section using an uncoated plate. The length of the neurite is 66.0 ± 2.5 μm (n = 37) 3 hours after the culture of the VI type section, and is almost the same length (70.3 ± 5.4 μm) until the 14th day of culture thereafter. , N = 14). A similar neurite length (55.7 ± 3.3 μm, n = 31, 3 hours after culture) was also observed in the laminin group, but in the type I group, neurites shorter than the other two groups were observed. Observed (45.9 ± 2.5 μm, n = 19, 3 hours after culture).

FIG. 3 is a diagram comparing the extension of neurites when using type VI collagen and other extracellular matrix proteins. FIG. 3A is a diagram showing changes in neurites over time. In the presence of NGF, PC12 cells were cultured using a plate coated with type VI collagen, laminin or type I collagen, or a plate not coated. The photo was taken after the time indicated in the figure. To determine the position, the bottom of the plate was marked with a needle and indicated with an asterisk (*). FIG. 3B is a diagram showing a change over time in the ratio of nerve cells in which neurites are extended. Cells in which the neurites extended more than 3 times the cell diameter were counted. FIG. 3C shows the measurement of neurite length in different extracellular matrix. The length of the longest neurite extending from each cell was measured using ScionImage software. Bars show mean ± standard error. The asterisk (*) is used to indicate the group where a significant difference was seen compared to the group using the uncoated plate (P <0.01).

(Effect of type VI collagen on neuronal cell death)
In the process of conducting a test on neurite outgrowth, the present inventor has developed a nerve cell cultured using a plate coated with type VI collagen, or a neuron cultured using a plate coated with another substance or It was discovered that cell death is less likely than that of neurons cultured on uncoated plates. Therefore, nerve cells were cultured on plates coated with various substances in the presence of NGF. FIG. 4A shows the result of quantitative analysis of neuronal cell viability using the trypan blue staining method. In the group in which nerve cells were cultured on the uncoated plate (None group), only 46.0 ± 8.1% (n = 5) survived, whereas in the plate coated with type VI collagen In the cultured group and the group cultured on the laminin-coated plate (ColVI group and Laminin group, respectively), 88.5 ± 3.1% (n = 6, P <0.001) and 88.1 ± 3.3. The survival rate was significantly increased to 7% (n = 6, P <0.001) (FIG. 4A). Sections cultured on plates coated with type I collagen, sections cultured on plates coated with poly-L-lysine, and sections cultured on plates coated with poly-D-ornithine (ColI section, PLL section and PORN section, respectively) Then, the survival rate of nerve cells is 62.7 ± 3.0% (n = 6), 48.5 ± 3.8% (n = 6) and 42.8 ± 7.7% (n = 6), respectively. And no significant difference was observed with respect to None.

Furthermore, the survival rate of nerve cells was analyzed using the TUNEL method (FIG. 4B). In the None ward, only 47.1 ± 7.6% (n = 3) survived, whereas in the ColVI ward and Laminin ward, 78.2 ± 3.2% (n = 3, P < 0.01) and 77.8 ± 2.1% (n = 3, P <0.01), and the survival rate was significantly increased (FIG. 4C). In the ColI, PLL and PORN groups, the survival rate of nerve cells was 68.2 ± 8.8% (n = 3), 39.1 ± 2.0% (n = 3) and 31.2 ± 7, respectively. It was 0.7% (n = 6), and no significant difference was observed with respect to None.

FIG. 4 shows that type VI collagen (ColVI), type I collagen (ColI), laminin, poly-L-lysine (PLL), or poly-D-ornithine (PORN) is a neuron. It is a figure which shows the influence which it has on survival. FIG. 4A is a diagram showing the results of a trypan blue dye exclusion test. PC12 cells were cultured using a plate coated with ColI, ColVI, Laminin, PLL or PORN, or a plate not coated (None), tested, and the survival rate was shown. Bars show mean ± standard error. The asterisks (*) are used to indicate the wards that showed a significant difference compared to the None ward (P <0.01). FIG. 4B is an image of neuronal cell death visualized using TUNEL staining. It has been shown that the number of TUNEL-positive darkly stained cells is small in the ColVI and Laminin groups. FIG. 4C is a diagram showing the cell survival rate calculated based on the TUNEL-stained image. Bars show mean ± standard error. The asterisks (*) are used to indicate the wards with significant differences compared to the None ward (*: P <0.01, **: P <0.001).

(Expression of type VI collagen)
To examine the localization of type VI collagen, immunoperoxidase staining was performed. At the proximal cut of the sciatic nerve, type VI collagen expression was detected around the axon (Figs. 5a and b), round cells at the proximal cut end (Fig. 5c), and around the blood vessel (Fig. 5d). It was. In addition, expression of type VI collagen was detected in cells in a region where a degenerative change was observed at the distal cut (FIG. 5e). Further, almost no expression of type VI collagen was detected in healthy sciatic nerve trunks (FIG. 5f).

FIG. 5 is a diagram showing the result of immunoperoxidase staining of type VI collagen. The left side (ae) and healthy right side (f) nerves 3 days after left sciatic nerve cut were stained with anti-type VI collagen antibody. At the left sciatic nerve proximal cut, a very strong response was observed in cells surrounding the axon (a and b, indicated by arrows), and was strongly stained with a small circular cell at the proximal cut end ( c, arrowhead). Furthermore, it was also observed that type VI collagen was accumulated around the blood vessel at the proximal cut (d, arrow). At the left sciatic nerve distal cut point, it was also observed in cells remaining after regression (e, arrow). In the healthy right sciatic nerve trunk, almost no type VI collagen was detected (f). The scale represents 20 μm.

Furthermore, fluorescent double staining was performed to analyze the characteristics of type VI collagen positive cells. As shown in FIG. 6A, type VI collagen was observed in S-100 protein positive cells. In the proximal cut part, it was selectively localized around S-100 protein positive cells. In contrast, S-100 protein positive cells were uniformly observed at the distal cut. In addition, the same tendency was observed in co-staining with GFAP, which is a marker of myelinating Schwann cells (FIG. 6B). Furthermore, type VI collagen expression was also detected in OX-41 positive macrophages and Thy-1 positive fibroblasts (FIGS. 7A and B). However, no co-localization of RT97 positive neurofilament and type VI collagen in axons was observed (FIG. 7C).

Although overlapping description, FIG. 6 is an image obtained by confocal laser scanning with fluorescent double staining. Three days after cutting the left sciatic nerve, left (ac) and healthy right (Contralateral, d1-d3) nerves were collected. Sections were prepared and stained with rabbit anti-type VI collagen antibody and anti-mouse anti-S-100 protein antibody overnight at 4 ° C., and FITC-labeled anti-rabbit IgG antibody (a1-d1, green) or Texas red-labeled anti-mouse IgG Visualization with antibody (a2-d2, red). In the merged image (Merge), yellow indicates that type VI collagen and S-100 protein are co-expressed (a3-d3) (FIG. 6A). FIG. 6B shows staining using an anti-type VI collagen antibody (e1-h1, green) and an anti-glial fibrillary acidic protein (GFAP) antibody (e2-h2, red). e3-h3 represents a merged image. The scale represents 10 μm.

FIG. 7 is an image obtained by a confocal laser microscope using fluorescent double staining. Three days after the left sciatic nerve was cut, left (ae) and healthy right (f) nerves were collected. Sections were prepared and stained with rabbit anti-type VI collagen antibody and OX-41 mouse anti-signal control protein (SRIP) antibody overnight at 4 ° C., FITC-labeled anti-rabbit IgG antibody (a1-d1, green) or Texas Red Visualization with labeled anti-mouse IgG antibody (a2-d2, red). In the merged image, yellow indicates that type VI collagen and OX-41 antigen are co-expressed (a3-d3) (FIG. 7A). FIG. 7B shows staining using an anti-type VI collagen antibody (e1-g1, green) and an anti-Thy-1 antibody (e2-g2, red). e3-g3 represents a merged image. FIG. 7C shows staining using anti-type VI collagen antibody (h1, green) and RT97 mouse anti-neurofilament antibody (h2, red). h3 shows a merged image, but no co-expression was observed. The scale represents 50 μm for a, b, and d, and 10 μm for c, e, f, g, and h.

(Expression of type VI collagen in sympathetic nerve fibers)
Since type VI collagen strongly promotes neurite outgrowth and suppresses neuronal cell death, it was confirmed whether type VI collagen was localized in sympathetic nerve fibers at the damaged site. As shown in FIG. 8, it was observed that type VI collagen was accumulated close to the sympathetic nerve fibers.

FIG. 8 is an image showing that type VI collagen accumulates in the damaged sciatic nerve in the vicinity of tyrosine hydroxylase-positive nerve fibers. Tissues 3 weeks after ligation of the left sciatic nerve trunk were collected and sliced, and then stained with rabbit anti-type VI collagen antibody and mouse anti-tyrosine hydroxylase antibody, and FITC-labeled anti-rabbit IgG antibody (A, (Green) or Texas Red labeled anti-mouse IgG antibody (B, red). The scale represents 20 μm.

(Discussion)
From the above, it has been clarified that type VI collagen has neurite outgrowth promoting action, nerve cell death inhibiting action, and nerve regeneration promoting action. It is suggested that type VI collagen acts as a scaffold for germination and axonal extension in actual nerve tissue, and is involved in the regeneration process after injury.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

FIG. 1 is a diagram showing the results of RNA blot hybridization analysis. FIG. 2 is a diagram showing neurite outgrowth observed by culturing various neurons on a plate coated with type VI collagen (upper) or uncoated (lower). FIG. 3 is a diagram comparing neurite outgrowth when using type VI collagen and other extracellular matrix proteins. FIG. 4 shows the effect of type VI collagen (ColVI), type I collagen (ColI), laminin, poly-L-lysine (PLL) or poly-D-ornithine (PORN) on neuronal survival. FIG. FIG. 5 shows the results of immunoperoxidase staining of type VI collagen. FIG. 6 is an image obtained by a confocal laser scanning microscope using fluorescent double staining. FIG. 7 is a confocal laser microscope image of fluorescent double staining. FIG. 8 is an image showing that type VI collagen accumulates in the vicinity of nerve fibers that are tyrosine hydroxylase positive in the damaged sciatic nerve. FIG. 9 shows the amino acid sequence of type VI collagen α1 subunit. FIG. 10 is a view showing a nucleic acid sequence encoding a type VI collagen α1 subunit. FIG. 11 shows the amino acid sequence of type VI collagen α2 subunit. FIG. 12 is a view showing a nucleic acid sequence encoding a type VI collagen α2 subunit. FIG. 13 shows the amino acid sequence of type VI collagen α3 subunit. FIG. 14 is a view showing a nucleic acid sequence encoding a type VI collagen α3 subunit.

Claims (14)

  A neuronal cell death inhibitor comprising type VI collagen.   Neurite extension promoter containing type VI collagen.   A nerve regeneration promoter containing type VI collagen.   A method for suppressing neuronal cell death in vitro, comprising a step of contacting neuronal cells with type VI collagen.   A method of promoting neurite outgrowth in a human in vitro, the method comprising a step of contacting a neuron with a type VI collagen.   A method for promoting nerve regeneration in vitro, comprising the step of contacting nerve cells with type VI collagen.   A method for culturing a nerve cell, comprising the step of contacting the nerve cell with type VI collagen in a culture vessel.   The culture method according to claim 5, wherein the culture vessel is coated with type VI collagen.   A neural cell culture kit comprising type VI collagen and a culture vessel.   The neuronal cell culture kit according to claim 9, wherein the culture vessel is coated with type VI collagen.   A medical material for nerve regeneration containing type VI collagen.   The medical material according to claim 11, wherein the type VI collagen is in a sheet form.   The medical material according to claim 11, wherein the type VI collagen is tubular.   The medical material according to claim 11, wherein the type VI collagen is filled with a biocompatible material.