JP2007332082A - Protein for cell adhesion/extension - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein for cell adhesion/extension, and to provide a method for cell adhesion/extension using the same. <P>SOLUTION: A composition for cell adhesion and/or extension is provided, comprising the protein, called as adrenal cortex zonal factor-1(AZ-1) or the like, mentioned below: (a) A protein comprising a specific amino acid sequence. (b) A protein comprising an amino acid sequence wherein one or several amino acids is(are) substituted, deleted, inserted or added in the above-mentioned amino acid sequence and having the activity of cell adhesion and/or extension. The method for cell adhesion/extension using the composition comprises culturing smooth muscle cells or vascular endothelial cells using the above composition and fibronectin. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel cell adhesion / extension protein.

  Cell adhesion / extension proteins play an important role in the adhesion, migration, proliferation, differentiation, morphology, metabolism, and the like of cells constituting tissues. Its role is involved in many biological phenomena such as reproduction, development, maturation, aging, inflammation, wound healing, immunity, and tumors.

 Cell adhesion / extension proteins are useful for studying the above-mentioned biological phenomena, and for diagnosis and examination or treatment of diseases or wounds in which these proteins are involved. Further, it can be applied to tissue regeneration in regenerative medicine and artificial tissue formation in vitro. Therefore, it is desired to develop a method for producing and utilizing a biological activity such as cell adhesion and spreading activity by producing an external environmental factor of cells constituting a tissue.

  By the way, adrenocortical zonation factor-1 (Adrenocortical zonation factor-1, hereinafter referred to as “AZ-1”) is a secreted protein having a 466 amino acid sequence, which is a secretory signal, a cysteine-rich sequence (EGF-like domain) and a proprotein. A 52 kD mouse protein consisting of cathepsin B-related regions. Its expression is closely linked to zonal differentiation of adrenocortical cells. The ortholog of this protein is interstitial tubular nephritis antigen (TIN-ag) -like protein (TIN-ag RP) (Wex, T. et al. (2001) Biochemistry 40: 1350-1357) in humans or rats. Or called lipocalin 7 (lcn7) and these homologs are known in mammals as TIN-ag (Nelson, TR at al. (1995) J. Biol. Chem. 270, 16265-16270 ). The cysteine-rich sequence has domains similar to somatomedin B (N-terminal fragment of vitronectin) and von Willebrand factor and laminin chain. Procathepsin B-related sequences are inactivated by proteolysis by substitution of a conserved catalytic cysteine residue in cathepsin B. As previously reported, in immunohistochemical analysis using anti-AZ-1 antibody, AZ-1 is localized in the basement membrane along the adrenal cortex and surrounds vascular smooth muscle cells of various organs. (Mukai, K. et al. (2005) Biochemistry 77, 970-970).

AZ-1 is identified by cloning of the gene (cDNA) encoding this protein, and acts to regulate signal transduction between cells, regulates cell adhesion / binding, protease activity or regulates it, cell migration or It was predicted to be a protein having an effect of regulating cancer metastasis (Patent Document 1, Non-Patent Document 1).
However, details regarding the action of AZ-1 are not clear.
JP 2001-211887 Mukai, K. et al. (2003) J. Biol. Chem. 278 (19), 17084-17092

  The present invention has been made in view of the above problems, and an object thereof is to provide a novel cell adhesion and / or extension protein.

As a result of intensive studies in view of the above problems, the present inventor has found a secreted protein derived from a mouse (Mukai, K. et al. (2003) J Biol Chem 278 (19), 17084-17092) already cloned by the inventor. The cDNA was used to express a recombinant protein, and its function was analyzed. As a result, it was found that this protein has a novel cell adhesion / extension activity, and the present invention was completed.

That is, the present invention is as follows.
(1) A composition for cell adhesion and / or extension comprising the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and a cell A protein having adhesion and / or spreading activity (2) A method of adhering and / or spreading cells comprising culturing smooth muscle cells or vascular endothelial cells in the presence of the composition according to (1) above .
(3) A smooth muscle cell or vascular endothelial cell is cultured in the presence of the composition described in (1) above, and the smooth muscle cell or vascular endothelial cell is collected from the obtained culture. Manufacturing method.
(4) A cell adhesion and / or extension kit comprising the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and a cell Protein having adhesion and / or extension activity (5) Pharmaceutical composition for treatment of at least one disease selected from the group consisting of inflammation, tumor, wound and immune disease, comprising the following protein (a) or (b) .
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and a cell Protein having adhesion and / or extension activity In the present invention, examples of the cell to be subjected to cell adhesion and / or extension include smooth muscle cells and vascular endothelial cells. The smooth muscle cell is preferably a vascular smooth muscle cell.

  In one embodiment of the present invention, smooth muscle cells can be adhered and / or extended in the presence of fibronectin or a fragment thereof.

 According to the present invention, a protein having cell adhesion and / or spreading activity is provided. Since AZ-1 has been shown to have adhesion and / or spreading activity to vascular smooth muscle cells and vascular endothelial cells, AZ-1 has the proliferation, differentiation, morphology, migration, metabolism, etc. of these cells. It can be said that it controls. Therefore, it is possible to develop technology using AZ-1 in many biological phenomena such as reproduction, development, maturation, aging, inflammation, wound healing, immunity, tumor formation / metastasis of living body. Furthermore, it can be applied to tissue regeneration in regenerative medicine and artificial tissue formation in vitro. Therefore, the AZ-1 protein used in the present invention can be used for the development of artificial organs, therapeutic agents for tissue damage and recovery including organ transplantation, and basic research for these developments.

   Hereinafter, the present invention will be described in detail.

  The present invention relates to a cell adhesion and / or spreading composition containing AZ-1 (also referred to as “angionectin” or “Angionectin”). Hereinafter referred to as “AZ-1 / angionectin” or “present protein”.

Recombinant AZ-1 / angionectin was isolated from conditioned medium of HEK293 cells stably transfected with the expression vector. By solid phase protein-protein interaction and cell adhesion assay using AZ-1 / angionectin, this protein interacts with major extracellular matrix (ECM) proteins such as fibronectin, collagen, laminin-1 and cell adhesion It became clear to promote. When the localization of AZ-1 / angionectin in the tissue is analyzed, it is detected in adrenal cortex-like vascular basement membrane, vascular smooth muscle basement membrane, renal glomerulus, etc., but tubules, skeletal muscle cells and cardiomyocytes It was found that it could not be detected in the basement membrane. Therefore, AZ-1 / angionectin is a novel member of the basement membrane protein and can be said to be a protein that is preferentially localized in the blood vessel wall.
The present invention produces and purifies novel cell adhesion / extension proteins, analyzes their functions, and proposes methods for using them.
1. AZ-1 / angionectin and gene AZ-1 / angionectin used in the present invention is the following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (b) comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and a cell A protein having adhesion and / or extension activity The AZ-1 / angionectin used in the present invention is at least a part of the base sequence of the full-length AZ-1 / angionectin gene (SEQ ID NO: 1), for example, at least 118- The amino acid sequence encoded by the region of the base sequence No. 1464 (SEQ ID NO: 3) (amino acid sequence Nos. 18 to 466 of the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 4)) is included. Moreover, the amino acid sequence (SEQ ID NO: 2: full-length coding region) encoded by Nos. 67 to 1467 in the base sequence shown in SEQ ID NO: 1 is also included. In the present invention, in addition to the full-length AZ-1 / angionectin gene sequence (SEQ ID NO: 1) and the amino acid sequence of full-length AZ-1 / angionectin (SEQ ID NO: 2), the above partial fragments (SEQ ID NOs: 3 and 4) should be used. However, it is preferable to use full length AZ-1 / angionectin.
Furthermore, in the present invention, “gene” encoding AZ-1 / angionectin includes RNA and DNA. RNA includes mRNA, and DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding strand serving as an antisense strand (the antisense strand may be used as a probe or an antisense strand). Available as a drug). Furthermore, the “gene” of the present invention includes sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences) in addition to the sequence encoding the protein (a) or (b). It may be a thing. If necessary, AZ-1 / angionectin can be provided with a histidine tag (His tag) or the like to form a fusion protein. Giving a His tag is preferable in that purification using a nickel column and detection using an anti-His tag antibody are possible.

Here, in the present invention, as long as the AZ-1 / angionectin or a partial fragment thereof has cell adhesion and / or spreading activity, at least one, preferably one or several amino acids in the amino acid sequence of the protein. Mutations such as deletion, substitution and addition may occur.
For example, one or several amino acids of the amino acid sequence shown in SEQ ID NO: 2 or 4 may be deleted, for example, 1 to 10, preferably 1 to 5 amino acids may be deleted. 1 or several, for example 1 to 10, preferably 1 to 5 amino acids may be added, or one or several of the amino acid sequence shown in SEQ ID NO: 2 or 4, for example 1 to 10 , Preferably 1 to 5 amino acids may be substituted with other amino acids. Therefore, a gene encoding a protein containing an amino acid sequence into which the mutation is introduced is also included in the gene that can be used in the present invention as long as the protein has cell adhesion and / or spreading activity.

  “Cell adhesion and / or spreading activity” means the activity of cell adhesion activity and / or cell spreading activity, and “cell adhesion activity” means that a substance coated on the surface of the incubator is used. The term “cell spreading activity” refers to the activity of adhering cells in a form in which a substance coated on the surface of the incubator extends its temporary foot. These activities can be measured by staining the attached cells with a fluorescent dye such as Hoechst33342, and the criteria for determining the activity are bovine serum albumin (BSA) having no adhesion / extension activity as a negative control. The difference in the fluorescence intensity with the coating is significant. The determination of whether the activity is adhesion activity or extension activity is based on microscopic observation, depending on whether the form of each attached cell is substantially spherical and does not have a temporary foot, or a shape in which the temporary foot is extended.

The introduction of mutations such as the above-mentioned deletion, substitution, addition, etc. is carried out using a mutation-inducing kit using site-directed mutagenesis, such as GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan- K, Mutan-Super Express Km, etc. (Takara Bio)) and the like.

  Furthermore, in the present invention, a DNA capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA encoding AZ-1 / angionectin (SEQ ID NO: 1). DNA encoding a protein having cell adhesion and / or spreading activity can also be used in the present invention. “Stringent conditions” are conditions at the time of washing after hybridization, wherein the salt concentration is 15 to 900 mM, the temperature is 40 to 75 ° C., preferably the salt concentration is 15 to 300 mM, and the temperature is 65 ° C. means. For example, conditions such as 65 ° C. with 2 × SSC can be mentioned. Those skilled in the art can set hybridization conditions in consideration of various conditions such as reaction time in addition to such conditions as salt concentration and temperature of the buffer. For the detailed procedure of the hybridization method, Sambrook J and Russel D. Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001, etc. can be referred to.

  As shown in the examples below, AZ-1 / angionectin used in the present invention has binding properties with other extracellular matrix factors as a result of functional analysis, in particular, fibronectin and a part of fibronectin. It has been experimentally confirmed that it has binding ability with III1-C (anasterin), type 4 and type 1 collagen.

  The recombinant expression vector used for expressing AZ-1 / angionectin in the present invention contains a gene encoding the protein (a) or (b), the full-length protein, or a partial fragment thereof. For example, a recombinant expression vector into which a base sequence shown in SEQ ID NO: 1 or 3 or a gene containing a base sequence of Nos. 67 to 1467 in the base sequence shown in SEQ ID NO: 1 is inserted can be mentioned. For the production of the recombinant expression vector, a plasmid, phage, cosmid, or viral vector can be used, but it is not particularly limited, and a known method can be employed (for example, Sambrook J and Russel D. See Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001).

  The transformant used for expressing AZ-1 / angionectin in the present invention is a transformant in which a gene encoding the protein (a) or (b), the full-length protein or a partial fragment thereof is introduced. It is. Here, “gene introduced” means that the gene is introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). The “transformant” means not only cells / tissues / organs but also individual animals (transformed animals such as mice, rats, rabbits, pigs, monkeys). In particular, mice and rats are widely used as experimental animals and pathological model animals, and knockout mice and the like are used for functional analysis of extracellular matrix factors, pathological analysis of diseases associated with the same factors, diagnosis methods and diagnosis of the diseases. It is useful for the development of drugs, treatment methods, and therapeutic drugs.

  A recombinant vector for protein expression can be obtained by ligating the above-mentioned polynucleotide to an appropriate vector, and a transformant introduces the recombinant vector of the present invention into a host so that the target gene can be expressed. (Sambrook J and Russel D. Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001).

As the vector, a phage or a plasmid capable of autonomously growing in a host microorganism is used. Examples of plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis or yeast, and examples of phage DNA include λ phage. Furthermore, animal virus and insect virus vectors can also be used.
For the production of a recombinant vector, the purified DNA may be cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of an appropriate vector DNA, etc. and ligated to the vector.
The host used for transformation is not particularly limited as long as it can express the target gene. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.) and insect cells.
Methods for introducing a recombinant vector into a host are known and include any method (for example, a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, a calcium phosphate method, a lipofection method, etc.).

  In the present invention, the peptide of the present invention can also be obtained by culturing the transformant and collecting it from the culture. The “culture product” may be a culture supernatant, a cultured cell, a cultured microbial cell, or a disrupted product thereof. Culture methods are well known in the art (see Sambrook et al., Molecular Cloning, supra).

  After culturing, when AZ-1 / angionectin is produced in cells or cells, proteins are extracted by disrupting the cells or cells. When the protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, or the like, is used alone or in combination as appropriate. It can be separated and purified.

In the present invention, protein synthesis by in vitro translation can be employed to obtain AZ-1 / angionectin. As the in vitro translation system, a commercially available system such as Expressway ^™ system (Invitrogen), PURESYSTEM (registered trademark; Post Genome Research Institute), TNT system (registered trademark; Promega), etc. can be used.
2. Composition for cell adhesion and / or extension and method thereof AZ-1 / angionectin obtained as described above can be used as a composition for cell adhesion and / or extension.

  The type of cell to which the composition of the present invention is applied is not particularly limited, and examples thereof include vascular endothelial cells, vascular smooth muscle cells, adrenal cortex cells, fibroblasts, and tumor-derived cells. Vascular endothelial cells and vascular smooth muscle cells are preferred.

By culturing the cells in the presence of the composition of the present invention, the target cells can be adhered and / or extended. The phrase “in the presence of the composition of the present invention” means that the cells can come into contact with the composition of the present invention during culture, and the culture medium contains the composition of the present invention. Or coating a culture plate with the composition of the present invention. For example, the composition of the present invention is coated on the surface of the incubator, or the composition of the present invention is mixed with an appropriate cell culture medium (RPMI-1640, DMEM, etc.), and the target cells are subjected to predetermined conditions. Incubate with The cell concentration during the culture is 1 x 10 ^{3 to} 5 x 10 ⁵ cells / cm ² , preferably 1 x 10 ^{4 to} 3 x 10 ⁵ cells / cm ² , more preferably 3 x 10 ^{4 to} 1.5 x 10 ^5. cells / cm ² . The culture temperature and time are 25 to 37 ° C, preferably 37 ° C, and 0.2 to 48 hours, preferably 0.5 to 18 hours. During the culture, the CO ₂ concentration is preferably 5%. The culture can be performed in a serum-free medium, but when the serum such as FCS or FBS is contained in the culture solution, the content ratio is 1 to 20%.

  When cultured under the above conditions, cells proliferate while adhering and spreading. When the cells become confluent, the cells can be collected by treating with trypsin and / or EDTA to obtain the target cells. Therefore, the present invention provides a method for adhering and / or spreading these cells by culturing smooth muscle cells or vascular endothelial cells in the presence of the composition of the present invention. Provided is a method for producing a cell, which comprises collecting smooth muscle cells or vascular endothelial cells.

In the method of the present invention, when smooth muscle cells are cultured in the presence of fibronectin or a fragment thereof, the smooth muscle cells can be efficiently attached and / or extended.
“In the presence of fibronectin or a fragment thereof” means that the cells can be in contact with fibronectin or a fragment thereof during culture, and the culture medium contains fibronectin or a fragment thereof, or is cultured. It means either coating the plate with fibronectin or a fragment thereof.

  For example, in the case of culturing vascular smooth muscle cells, fibronectin or a fragment thereof is first coated on the culture plate (first coating) and then coated with AZ-1 / angionectin (second coating), and then the blood vessel When smooth muscle cells are cultured, cell adhesion and / or extension is observed depending on the concentration of AZ-1 / angionectin.

  The fibronectin used here is known, and commercially available products (eg, III1-C (Sigma)) can be used. The amount or concentration of III1-C used is 0-100 μM.

  Confirmation that the cells have adhered may be determined by adhering to the culture plate regardless of the form of the cells. The confirmation that the cells have extended may be determined by the fact that the cell attached to the culture plate is not spherical but has a temporary leg extended.

3. Kit AZ-1 / angionectin used in the present invention can be packaged in advance and supplied as a cell adhesion and / or extension kit. Specifically, the kit of the present invention contains the AZ-1 / angionectin or mutant protein or a fragment thereof, but in addition, a cell culture medium, antibiotics added to the medium, serum (fetal bovine serum, etc.) ), Sodium bicarbonate, and the like, if necessary, may include reagents necessary for carrying out positive control and / or negative control of cell adhesion and / or extension, or blocking of an incubator and fluorescent staining of cells. .

4). Pharmaceutical Composition In the present invention, AZ-1 / angionectin can be used as a pharmaceutical composition (therapeutic agent) for treating inflammation, tumor, wound or immune disease. These diseases can be applied alone or in two or more diseases, and can be applied even when diseases other than the above-mentioned diseases occur together.
Inflammation is a defensive reaction for the living body to maintain its homeostasis when an invasion causing some organic change is applied to the living tissue. Inflammation includes purulent inflammation caused by bacterial infection of tissues, serous inflammation where exudates mainly composed of liquid components ooze into tissues, fibrinous inflammation, necrotic inflammation, etc., but are limited to these inflammations Is not to be done.

The following are mentioned as a tumor.
(a) Oral cavity or pharynx: tongue cancer, gingival cancer, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer
(b) Brain tumor: glioma, meningioma, astrocytoma
(c) Respiratory organ: Lung cancer (squamous cell carcinoma, adenocarcinoma, alveolar epithelial cancer, large cell undifferentiated cancer, small cell undifferentiated cancer), bronchiole cancer
(d) Breast: Breast cancer, breast sarcoma, breast sarcoma
(e) Gastrointestinal: Esophageal cancer, stomach cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer
(f) Female genitals: endometrial cancer, ovarian cancer, cervical cancer
(g) Urinary organs: prostate cancer, renal cancer, bladder cancer, testicular tumor, urethral cancer, renal pelvic and ureteral cancer
(h) Motor organs: rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, Ewing sarcoma, leiomyosarcoma
(i) Skin: skin cancer, skin Paget's disease, malignant melanoma
(j) Blood: Acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Adult T-cell leukemia, malignant lymphoma (lymphosarcoma, reticulosarcoma, Hodgkin's disease), multiple myeloma, primary macroglobulinemia Wound means damage to body tissue, especially caused by physical external forces Cut wounds, laceration wounds, stab wounds, bite wounds, gun wounds, wound wounds, and the like.

  An immune disease refers to a disease caused by loss or destruction of tissue function resulting from the action of humoral or cellular immunity against body components, and includes autoimmune diseases. These diseases include, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Hashimoto's disease, systemic scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease, Graves' disease, etc. Can be mentioned.

AZ-1 / angionectin can be administered to a mammal in need of cell adhesion and / or extension. Examples of mammals to be administered include humans, domestic animals such as cattle, horses, sheep and goats, pets such as dogs and cats, and laboratory animals such as mice, rats, guinea pigs and rabbits. It is not limited to these animals.
The dosage form of the pharmaceutical composition of the present invention can be either oral or parenteral.

  In the case of oral administration, for example, administration by tablet, capsule, granule, powder or syrup is possible. In the case of parenteral administration, injection, suppository or eye drop, etc., pulmonary dosage form (for example, nephriser etc.), nasal dosage form, transdermal dosage form (eg ointment, cream), etc. Is mentioned. In the case of an injection type, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. These preparations are produced by well-known methods using pharmaceutically acceptable additives such as excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.

  Examples of the excipient include starch such as potato starch and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like. Examples of the lubricant (coating agent) include ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, paraffin and the like. Examples of the binder include polyvinyl pyrrolidone, macrogol and the same compound as the excipient. Examples of the disintegrant include the same compounds as the above excipients and chemically modified starch / celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone. Examples of the stabilizer include paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; and phenols such as phenol and cresol; Mention may be made of thimerosal; dehydroacetic acid; and sorbic acid. Examples of the flavoring agent include sweeteners, acidulants, and fragrances that are commonly used. Moreover, ethanol, phenol, chlorocresol, purified water, distilled water, or the like can be used as a solvent for producing the liquid agent. Examples of the surfactant or emulsifier include polysorbate 80, polyoxyl 40 stearate, lauromacrogol and the like.

  The above additives and the like are selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical composition of the present invention. For example, when used as an injectable preparation, purified AZ-1 / angionectin is dissolved in a solvent (for example, physiological saline, buffer solution, glucose solution, etc.), and then Tween 80, Tween 20, gelatin, human serum albumin The thing which added etc. can be used. Alternatively, the pharmaceutical composition of the present invention may be lyophilized to obtain a dosage form that dissolves before use. As the freeze-drying excipient, for example, sugar alcohols and saccharides such as mannitol and glucose can be used.

  The dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptom, administration route, administration frequency, and dosage form. The administration method is appropriately selected depending on the age and symptoms of the patient. The effective dose is, for example, 1 μg to 200 mg, preferably 200 μg to 60 mg per kg body weight at a time. However, the pharmaceutical composition is not limited to these dosages.

  The pharmaceutical composition of the present invention can be used in combination with known cell adhesion and / or spreading agents. Cell adhesion and / or spreading agents that can be used in combination include, but are not limited to, fibronectin, collagen, laminin, vitronectin and the like.

  Furthermore, in the present invention, by expressing (for example, gene expression) AZ-1 / angionectin in a mammal that requires cell adhesion and / or extension, cells can be attached and / or extended in the mammal. . “Expression” refers to production of AZ-1 / angionectin in a mammal. When gene therapy is performed, the AZ-1 / angionectin gene is administered to a mammal alone or supported on a suitable carrier.

  In the case of gene therapy, in addition to a method in which each gene is directly administered by injection, a method in which a vector in which a nucleic acid is incorporated is administered. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using these virus vectors.

  It is also possible to introduce the gene into a phospholipid vesicle such as a liposome and administer the vesicle. For example, an endoplasmic reticulum holding the above gene is introduced into a predetermined cell by a lipofection method. Then, the obtained cells are systemically administered, for example, intravenously or intraarterially. Cells can also be administered locally to the brain or the like.

The dosage of the pharmaceutical composition of the present invention in the case of gene therapy varies depending on age, sex, symptoms, administration route, number of administrations, dosage form, but in the case of gene therapy, for example, the dosage for adenovirus is 1 About 10 ⁶ -10 ¹¹ per day.
In order to introduce the AZ-1 / angionectin gene used for gene therapy into a target tissue or organ, a commercially available gene introduction kit (for example, Adeno Express: Clontech) can also be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. In the following examples, a method for producing AZ-1 / angionectin and purifying it from the culture solution, and the results of examining the properties of AZ-1 / angionectin will be described.

Expression and purification of recombinant AZ-1 / angionectin (1) Expression of AZ-1 / angionectin AZ-1 / angionectin expressed in the present invention has a hexahistidine tag at the C-terminus. The expression vector pR / C11.13His of this fusion protein is the expression vector pR / C11.13FD1 (literature name: Mukai, K. et al. (2003) J. Biol. Chem. 278 (19 ), 17084-17092). For the modification, the following primers (i) and (ii) were used.
(I) 5′-TG GGGCCC ATGGTGGGGTGA-3 ′ (SEQ ID NO: 5)
(Ii) 5'-AA GGGCCC TC TAGATTAATC ACTTAGTGAT GGTGATGGTG GTGCTCGAGG TGGTGCCCCA TGTCCTCC-3 '(SEQ ID NO: 6)
Using the oligonucleotide primer described above, pR / C11.13FD1 was used as a template to amplify a fragment containing the C-terminal portion of the protein and having a restriction enzyme ApaI site (underlined) at both ends, and pR / C11.13FD1 According to the procedure to replace the corresponding fragment in.
PCR was performed in the following reaction cycle using the following reaction composition solution.

Composition liquid:
Template DNA (0.1μg / μl): 1μL
Pyrobest DNA polymerase: 2.5 units
(i) Primer (5μM): 20μL
(ii) Primer (5μM): 20μL
dNTP (each 2.5mM): 8μL
10 × Buffer: 10μL
Sterile water: appropriate amount total: 100μL

Number of cycles: 94 ° C x 0.5 min, 56 ° C x 0.5 min and 72 ° C x 1 min 24 cycles, 72 ° C x 7 min 1 cycle.

  The obtained pR / C11.13His was stably introduced into human HEK293 cells, and cell clones secreting AZ-1 / angionectin into the medium were selected. Clones were selected by Western blotting using anti-AZ-1 / angionectin antibody.

(2) Purification of AZ-1 / angionectin
HEK293 cells expressing AZ-1 / angionectin were cultured using a serum-free culture solution, and the culture solution was collected every one or two days and replaced with a new culture solution. The collected culture was centrifuged at 150 xg for 10 minutes at room temperature, and the supernatant was subsequently centrifuged at 48,400 xg for 20 minutes at 4 ° C to obtain a supernatant.

  For the purification, the low solubility of AZ-1 / angionectin was used. That is, 29.2 g of NaCl per liter was dissolved in the supernatant obtained by centrifugation, and concentrated 200 times with an ultrafilter (Amicon Ultra PL-30, Millipore) using centrifugal force. Most AZ-1 / angionectin became insoluble and accumulated in the ultrafiltration membrane, but phosphate-buffered saline containing an extra 0.5 M NaCl was used to increase the recovery of soluble AZ-1 / angionectin remaining in the concentrate. The concentrated solution was diluted 25 times with (PBS) and concentrated with the same ultrafiltration membrane. This dilution and concentration was further performed twice. The concentrate was discarded and the filter membrane was washed 3 times with the same buffer. AZ-1 / angionectin was solubilized and recovered by incubating for 15 minutes at room temperature using PBS containing 2 M Urea and 0.5 M NaCl. The purity was found to be about 90% by SDS-PAGE and silver staining.

Cell Preparation, Adhesion / Extension Assay (1) Cell Preparation Human coronary artery-derived vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC) (Cambrex Bio Science) were respectively used as SmGM-2 and EGM-2- Culture was performed using MV (manufactured by the same company). These cells were dispersed with trypsin and suspended in serum-free DMEM / F-12.
(2) Adhesion / extension assay 50 μL of AZ-1 / angionectin, III1-C (Sigma) diluted with PBS to a concentration of 0 to 30 μg / ml, or fibronectin (Sigma) as a positive control ) At 37 ° C. for 18 hours. After blocking the wells with PBS containing 1% BSA, VSMC (1 × 10 ⁴ ) or VEC (2 × 10 ⁴ ) suspended in serum-free medium was added and incubated at 37 ° C. for 1 hour. After shaking the microplate at a certain strength, non-adherent cells were removed by aspiration and fixed with 5% glutaraldehyde for 20 minutes. The fluorescence intensity was measured by staining with Hoechst 33342. In the VSMC assay, the following experiment was performed in addition to the above method. Microplate wells were first incubated with 50 μL of III1-C (30 μg / ml, Sigma) at 37 ° C. for 18 hours. After blocking with PBS containing 1% BSA for 1 hour, AZ-1 / angionectin (0 to 0.4 μM, 50 μL) was allowed to bind to III1-C for 18 hours at room temperature. The adhesion of VSMC to the wells subjected to these treatments was measured by the method described above. The morphology of the adhered cells was stained with Coomassie Blue R250 after fixation and photographed with transmitted light.

  The results are shown in FIGS. In FIG. 1, (A) and (B) are photomicrographs showing that adhesion of vascular smooth muscle cells is weak and does not extend when AZ-1 / angionectin and III1-C are coated alone. (C) is a photomicrograph showing that vascular smooth muscle cells adhere and extend when III1-C is first coated alone and then AZ-1 / angionectin is bound to III1-C. (D) is a histogram showing that the adhesion / extension shown in (C) is capacity-dependent for AZ-1 / angionectin.

  As shown in FIGS. 1A and B, VSMC adhered weakly to the AZ-1 / angionectin and III1-C fragments coated directly on the wells and did not extend. However, when the well was first coated with III1-C and AZ-1 / angionectin was bound thereto, VSMC adhered and extended (FIG. 1C). This adhesion / extension depended on the concentration of AZ-1 / angionectin (FIG. 1D).

  In FIG. 2, (A) is a graph showing that the adhesion of vascular endothelial cells is dose-dependent when AZ-1 / angionectin or fibronectin is coated alone. (B) and (C) are photomicrographs showing that vascular endothelial cells adhere and extend to AZ-1 / angionectin and fibronectin, respectively.

  As shown in FIG. 2A, when AZ-1 / angionectin or fibronectin was directly coated, each VEC adhered in a dose-dependent manner. Adhesion to AZ-1 / angionectin was about 60% compared to adhesion to fibronectin. However, as shown in FIG. 2B, adhesion to AZ-1 / angionectin was accompanied by extension, as seen in adhesion and extension to fibronectin (FIG. 2C).

  From these results, AZ-1 / angionectin is a protein that shows almost no adhesion / extension activity to VSMC, but shows adhesion / extension activity when complexed with III1-C of fibronectin fragment. It has been found. On the other hand, for VEC, AZ-1 / angionectin was found to show adhesion and extension activity with a single coat.

It is a figure which shows adhesion and / or extension of vascular smooth muscle cells. It is a figure which shows adhesion | attachment and / or extension of a vascular endothelial cell.

Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA

Claims (9)

A cell adhesion and / or spreading composition comprising the following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 (b) containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4, and a cell Protein with adhesion and / or extension activity   The composition according to claim 1, wherein the cells are smooth muscle cells or vascular endothelial cells.   The composition according to claim 1, wherein smooth muscle cells can adhere and / or extend in the presence of fibronectin or a fragment thereof.   A method for adhering and / or spreading cells comprising culturing smooth muscle cells or vascular endothelial cells in the presence of the composition according to claim 1.   5. The method according to claim 4, wherein the smooth muscle cells are capable of adhering and / or spreading in the presence of fibronectin or a fragment thereof.   A method for producing a cell, comprising culturing smooth muscle cells or vascular endothelial cells in the presence of the composition according to claim 1, and collecting the smooth muscle cells or vascular endothelial cells from the obtained culture.   The method according to claim 6, wherein the smooth muscle cells are capable of adhering and / or spreading in the presence of fibronectin or a fragment thereof. A cell adhesion and / or extension kit comprising the following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 (b) containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4, and a cell Protein with adhesion and / or extension activity A pharmaceutical composition for treating at least one disease selected from the group consisting of inflammation, tumor, wound and immune disease, comprising the following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 (b) containing an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4, and a cell Protein with adhesion and / or extension activity

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