JP3053764B2 - Sugar chain polymer, cell treating agent and treatment method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sugar chain polymer, and more particularly to a sugar chain polymer having a sugar chain selectively recognized by cells and having a component having a second action added thereto, and a sugar chain polymer thereof. The present invention relates to a cell treatment agent and a treatment method using a sugar chain polymer. Here, the cell treating agent in the present invention includes all agents for performing various treatments on cells, such as a cell culture substrate, a cell encapsulating agent for encapsulating cells, or a cell collecting agent.

[0002]

2. Description of the Related Art Recent advances in glycotechnology have been remarkable. For example, as a biopolymer having a sugar chain,
Proteoglycans in the cell wall of plant cells that contribute to cell stabilization; glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc .; and glycoproteins that are involved in cell-cell interactions and cell recognition. However, the mechanism by which these high-molecular sugar chains substitute, assist, amplify, regulate, or inhibit the functions of each other and control sophisticated and precise biological reactions is gradually being clarified.

[0003] Furthermore, if the relationship between such sugar chains and cell differentiation and proliferation, cell adhesion, immunity, and canceration of cells is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering Can be expected to be closely related to the development of new developments.

As an example, sugar chains on the cell surface and sugar chains
-Research on the occurrence of diseases due to abnormal interaction between receptors, and the role of sugar chains in viral infections such as AIDS, etc., has been active. Research on the involvement of sugar chains in cell-cell interaction and cell-matrix interaction has also become important for understanding biological reactions.

[0005] To illustrate the specificity of sugar chain recognition by various cells, hepatocytes preferentially recognize galactose (Gal) and mannose (Man), whereas nonhepatic cells par- ticulate Man, glucose (Glc). , N-acetylglucosamine (GlcNAc). Serum tissue was Man, GlcNAc, and N-acetylmannosamine (ManNAc), lymph tissue was Ma
It specifically recognizes nNAc and GlcNAc.

[0006] In addition, sugar chain recognition proteins such as asialoglycoprotein and lectin-like protein on macrophages present on the surface of various cells also selectively recognize sugar chains. For example, a protein with a molecular weight of 175K on alveolar macrophages is known to be Man, Fucose, and Gl.
cNAc, 180K molecular weight protein on peritoneal macrophages Man, Fuc, and GlcNAc, 30K molecular weight protein on rat Kupffer cells, 42K molecular weight protein on peritoneal macrophages, and 45-60K molecular weight on activated macrophages The proteins are Gal and N-acetylgalactosamine (GalN
Ac) are each specifically recognized.

The inventors of the present invention have been conducting intensive studies focusing on the specific affinity of sugar chains. For example, as a model of a ligand for asialoglycoprotein receptor, a polymer having galactose in the side chain has been used. A certain poly (Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 → 4) -D-gluconamide]) (abbreviated as PVLA) was designed and synthesized. For example, in a hepatocyte culture experiment on a petri dish on which this PVLA was immobilized, PVLA was used.
Hepatocytes are selectively adhered through specific affinity with the asialo-sugar receptor on the surface of hepatocytes and the specific form of the adhesion leads to a three-dimensional cell aggregate. . ("Glycotechnology and artificial organs", Toshihiro Akaike et al., Nikkei Science, 114-129, 1994.
Year)

[0008] In addition, living organisms can differentiate cells,
It has other factors that affect proliferation, adhesion, migration, etc., which act in combination to achieve precise biological control.
In addition, there are factors outside of the body that affect them. Therefore, the present inventors have conducted research to add a second action derived from a living body or ex vivo to a polymer containing a sugar chain that imparts cell selectivity as described above, and have led to the present invention. Was.

[0009]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide, in addition to cell selectivity utilizing sugar chains specifically recognized by cells, growth and differentiation of the specifically selected cells. Another object of the present invention is to provide a sugar chain polymer having a second effect, which is effective in performing various treatments on the cells.

[0010]

This problem can be solved by a sugar chain polymer comprising a copolymer of a sugar chain-containing styrene derivative and a vinyl monomer having at least one site having a second action. . That is, as shown in the following formula (1), the sugar chain polymer of the present invention has a second action with a sugar chain-containing monomer having a sugar chain (SUG) bonded to the benzyl position of a p-substituted styrene derivative. It is a copolymer with a vinyl monomer to which a site (X) having is linked.

[0011]

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[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The sugar chain-containing monomer constituting the sugar chain polymer of the present invention is a monomer having a sugar chain (SUG) bonded to the styrene derivative at the p-benzyl position. Examples of this sugar chain include glucose, galactose, lactose, mannose, N-acetylglucosamine, laminaribiose,
Uronic acid-related substances, monosaccharides such as sulfated sugars, oligosaccharides, etc. can be used, and specifically interact with cells in a state where these monomers are bonded as side chains of a sugar chain polymer obtained by polymerizing these monomers. There is no particular limitation as long as it can hold a sugar residue.

The method for bonding the styrene derivative and the sugar chain in these sugar chain-containing monomers is preferably a covalent bond such as an amide bond, an ether bond, or an ester bond.
These sugar chain-containing monomers can be synthesized, for example, by forming an amide bond between the amino group of p-aminomethylstyrene and the terminal carbonyl group of the lactonized sugar. Among such sugar chain-containing monomers, for example, sugar chain-containing monomers represented by the following formulas (2) to (11) are particularly preferably used.

[0014]

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[0015]

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[0016]

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[0017]

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[0018]

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[0019]

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[0020]

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[0021]

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[0022]

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[0023]

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Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 → 4) -D- represented by the above formula (2)
Gluconamide] (hereinafter abbreviated as VLA)
It is a monomer synthesized from aminomethylstyrene and lactose and has a β-galactose residue.

Np-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4) -D-gluconamide] (hereinafter abbreviated as VMA) represented by the above formula (3) -A monomer synthesized from aminomethylstyrene and maltose, having a glucose residue.

N-p-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4) -D-mannamide] (hereinafter abbreviated as VMan) represented by the above formula (4) is It is a monomer synthesized from aminomethylstyrene and mannobiose and has a mannose residue.

Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 → 6) -D- represented by the above formula (5)
Gluconamide] (hereinafter abbreviated as VMeA)
-Aminomethylstyrene and O-α-D-galactopyranosyl
A monomer synthesized from-(1 → 6) -D-glucose and having an α-galactose residue.

Np-vinylbenzyl- [O-6-carboxymethyl-β-D-galactopyranosyl- (1 → 4) -OD-6-carboxymethyl represented by the above formula (6) -Gluconamide] (hereinafter abbreviated as VLACOOH) is p-
It is obtained by carboxymethylating a monomer synthesized from aminomethylstyrene and lactose, and has a carboxymethylated-β-galactose residue.

3-O-4'-vinylbenzyl-D-glucose represented by the above formula (7) (hereinafter abbreviated as VG)
Is a monomer synthesized from p-chloromethylstyrene and glucose, and has a glucose residue.

Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 4) -OD-2-acetamide- represented by the above formula (8) 2-deoxy-β-D-glucopyranosyl- (1 → 4) -OD-2-
Acetamido-2-deoxy-β-D-gluconamide],
Np-vinylbenzyl- [OD] represented by the above formula (9)
-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 4) -OD-2-acetamido-2-deoxy-β
-D-gluconamide] or a mixture thereof (hereinafter, referred to as “
Both are abbreviated as VGlcNac), both of which have an N-acetylglucosamine residue.

Np-vinylbenzyl-D-gluconamide represented by the above formula (10) (hereinafter abbreviated as VGA)
Is a monomer obtained by ring-opening D-glucose and binding to p-aminomethylstyrene.

Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 → 3) -D- represented by the above formula (11)
Gluconamide] (hereinafter abbreviated as VLam)
-A monomer synthesized from aminomethylstyrene and laminaribiose, having β1 → 3 glucose residues.

A sugar chain polymer obtained by polymerizing a sugar chain-containing monomer having a sugar chain bonded to such a polystyrene derivative has a polystyrene derivative as a main chain, and a sugar chain is bonded to the main chain as a side chain. It has a shape. Therefore, this sugar chain polymer dissolves in water by forming stable micelles having a polyhydrophobic styrene main chain as a core and a hydrophilic sugar chain surrounding the core.

On the other hand, the vinyl monomer constituting the sugar chain polymer of the present invention represented by the formula (1) has a site (X) having a second action in its side chain. The second action includes an action that directly affects the function of the cell that specifically recognizes the sugar chain, such as adhesion, growth, or differentiation of the cell. The site having is preferably composed of at least one selected from vitamins, drugs, cytokines, growth factors, flavonoids, nucleic acids, peptides, and hormones, and is bonded to a vinyl monomer while maintaining their action. I have.

Here, vitamins include vitamin A,
Fat-soluble vitamins such as D, E and K, water-soluble vitamins such as vitamin B group and vitamin C, all vitamins such as riboflavin (vitamin G) or biotin (vitamin H), and ubiquinone and lipoic acid It should include vitamin-like agents.

For example, VL which is a sugar chain-containing monomer of the above formula (2) having a galactose residue specific to hepatocytes
A and a sugar-chain polymer obtained by copolymerizing a vinyl monomer having biotin, which is a kind of vitamin, in the side chain thereof, is a sugar-chain polymer consisting of only VLA with respect to HepG2 which is a hepatocyte or a liver cancer cell It has been confirmed that the sugar chain polymer has a larger interaction, and the sugar chain polymer into which biotin having a second action is introduced can be used, for example, as a diagnostic reagent for identifying canceration of hepatocytes.

The drug is not particularly limited as long as it acts on functions such as cell adhesion, proliferation and differentiation. For example, a sugar chain obtained by copolymerizing, with the VB-LA, a vinyl monomer having a sulfonylurea structure, which is a fat-soluble drug controlling a K ⁺ channel involved in insulin secretion from β cells of the pancreatic islets of Langerhans, with the VB-LA. The macromolecule is insulinoma M derived from a transgenic mouse having high insulin secretion ability derived from pancreatic β cells.
It has been confirmed to affect the insulin release of IN6 cells. That is, this sugar chain polymer can be used, for example, as an insulin release regulator.

The growth factors include all growth factors such as epidermal growth factor, fibroblast growth factor, somatomedin, and T cell growth factor. Flavonoids include flavone, flavonol, flavanone, catechin, chalcone and the like. The nucleic acid includes all flavonoids, and the nucleic acid also includes DNA and RNA, or a macromolecular substance having a similar structure.

Further, the above-mentioned V having a galactose residue specific to an asialoglycoprotein receptor on the surface of a hepatocyte
It was obtained by copolymerizing LA with a vinyl monomer having an RGDSG (arginine / glycine / aspartic acid / serine / glycine) peptide in the side chain that is also specifically recognized by the integrin family, which is also a receptor on the surface of hepatocytes. It has been confirmed that sugar chain macromolecules are specifically recognized by each receptor on hepatocytes, and such multi-point recognition sugar chain macromolecules are highly accurate and highly sensitive cell discriminating agents. Can be applied.

In the present invention, the second action may act non-specifically on cells that specifically recognize the sugar chain. Here, “non-specifically acting” means that the site having the second action does not directly and specifically act on cell adhesion, proliferation, differentiation, etc. For example, it means indirectly affecting the cell by changing the environment or configuration around the cell, or changing the physicochemical properties of the glycan macromolecule itself. I do.

An example of such a non-specifically acting second-site-containing vinyl monomer is a monomer having a temperature-sensitive group such as isopropylacrylamide. A polymer obtained by polymerizing the polymer has a temperature sensitivity of dehydrating and acting as a hydrophobic polymer at about 32 ° C. or higher, and hydrating and behaving as a hydrophilic polymer below that temperature. Therefore, a sugar chain polymer obtained by copolymerizing the sugar chain-containing monomer and the temperature-sensitive group-containing monomer has a hydrophobic property at a certain temperature or higher and a hydrophilic property at a lower temperature.

For example, when the sugar chain polymer is added while maintaining an aqueous dispersion in which cells specific to the sugar chain are kept at a certain temperature or lower, the hydrophilic sugar chain polymer is dissolved and dispersed. Cells specifically bind to sugar chains. Next, when the water temperature is raised to a certain temperature or higher, the sugar chain polymer bound to the cells becomes hydrophobic and precipitates. Therefore, the sugar chain polymer of the present invention can be used as a cell collection agent capable of selectively collecting target cells.

A vinyl monomer having a basic group such as an amino group is also an example of the above-mentioned non-specifically acting second-site-containing vinyl monomer. A basic group such as an amino group easily forms a complex with an acidic polysaccharide such as alginic acid having gel forming ability. Therefore, by forming a gel with a complex of a sugar chain polymer obtained by copolymerizing such a basic group-containing monomer and a sugar chain-containing monomer and an acidic polysaccharide, the sugar chain is contained,
A cell culture substrate having a three-dimensional structure can be obtained.

The “three-dimensional structure” is different from a conventional two-dimensional structure in which the surface of a cell culture substrate is coated with a cell adhesive substance such as collagen, and is a sugar chain having a specific adhesive function of cells. Means a three-dimensionally distributed structure. By culturing cells specific to the sugar chain on a gel-like cell culture substrate having such a three-dimensional structure, it becomes possible to control the three-dimensional structure of the formed cell aggregate.

Further, by granulating with a gel formed of the above-mentioned sugar chain polymer and acidic polysaccharide, cells can be cultured in a closed environment of particles, and excess cells can be cultured. Aggregate formation can be prevented and moderately controlled aggregate formation can be achieved. Such cell culture in which the three-dimensional structure is controlled is particularly important in hepatocyte culture, and can be applied to a hybrid artificial liver in which hepatocytes derived from a living body are cultured and used.

In the above, particularly, examples using the specific affinity between the galactose residue of VLA and hepatocytes have been described, but the present invention is not limited to these, and includes various applications. That is, the sugar chain polymer of the present invention is obtained by copolymerizing the sugar chain-containing monomer described above and the second functional site-containing vinyl monomer, Gives specific recognizability of cells, and various second effects are added depending on the site of action of the vinyl monomer.

In the sugar chain polymer of the present invention, the combination of the sugar chain and the second active site is free, and can be appropriately selected according to the cells to be selectively treated and the treatment to be performed. In addition, their composition ratio may be appropriately selected depending on the strength of selectivity and purpose, and is not particularly limited. Furthermore, it goes without saying that a sugar chain polymer obtained by copolymerizing two or more kinds of sugar chain-containing monomers and / or two or more kinds of active site-containing vinyl monomers is also within the scope of the present invention.

[0048]

EXAMPLES The sugar chain polymer of the present invention will be described more specifically with reference to the following examples. (Example 1) 1.92 g (7.9 mmol) of hydroxysuccinimide ester of biotin and vinylbenzylamine were dissolved in 20 ml of DMF, and this was dissolved in a nitrogen stream.
The reaction was performed at 7 ° C. for 24 hours. After completion of the reaction, the reaction mixture was filtered, 200 ml of ether was added to the filtrate, and the mixture was allowed to stand. The resulting precipitate was separated by filtration. The filtrate was concentrated and then purified by a silica gel column to obtain 2.1 g of vinylbenzylbiotinamide (VBA), which is a target vinyl monomer containing a second action site (yield: 82%). Further, VLA of the above formula (2) was synthesized as a sugar chain-containing monomer.

The above-mentioned VLA and VBA were mixed at a charging molar ratio of 9
Copolymerized at 0:10. That is, 1.77 g of VLA
(3.7 mmol) and 0.15 g (0.42 mm) of VBA
ol) in 2 ml of DMSO and the initiator (AIB)
N) was added and polymerized in a glass ampoule for polymerization to obtain a sugar chain polymer. Similarly, a sugar chain polymer having a charge molar ratio of 80:20 (abbreviated as P (VLA-co-VBA)),
And a sugar chain polymer (P (VMA-) having a molar ratio of 80:20 using VMA of the above formula (3) instead of VLA.
co-VBA). For comparison, PVLA and PV, which are homopolymers of the VLA and VMA, were used.
MA was synthesized, and VLA or VMA120 was added to each polymer.
FITC was introduced at a rate of one per unit for fluorescent labeling.

The concentration of each polymer was 0.1%, 0.0%
A polymer solution dissolved in a biotin-free medium was prepared so as to be 1% and 0.001% (W / V). However, a mixture of a fluorescently labeled polymer and a non-fluorescently labeled polymer was prepared so that there was no difference in the amount of fluorescence between the polymer solutions at each concentration.

The cancer cells (Hep G2) were treated with 10% fetal calf serum (FCS), 0.07 g / l potassium penicillin G,
In a Williams medium E (buffer A) supplemented with 0.01 g / l streptomycin sulfate, a hydrophilic bottle (Falc
on3045). Subculture third day, after the culture medium was discarded, the cell surface PBS (-) was washed with a solution of trypsin solution 8 ml (0.25% triflate ° Singh, Hank's solution was dissolved 0.02% EDTA) was added, CO ₂ under 2 at 37 ° C
Incubated for minutes. After adding FCS-supplemented medium and pipetting, the cells were collected and centrifuged at 800 rpm for 1 minute. This process was repeated several times to wash the cells, and then dispensed into tubes so that the number of cells reached 1,000,000.

The above-mentioned tubes were filled with 10% of the polymer solution of each concentration.
After stirring, the mixture was treated at 37 ° C. and 4 ° C. for 45 minutes. During the reaction, stirring was performed every 15 minutes, and finally PBS (+) containing NaN ₃ was added. After washing twice with the same solvent, analysis by flow cytometry was performed.
FIG. 1 shows the results when the polymer concentration was 0.01%.

Hep G2 is a VMA having a glucose residue.
VLA having a galactose residue rather than a sugar chain polymer
The recognizability of the system sugar chain polymer was high, and the recognizability of the sugar chain polymer coexisting with biotin was further enhanced. That is, Hep G2 recognizes and interacts with both galactose and biotin, and from this, it becomes cancerous by using a sugar chain polymer containing biotin as the second component in addition to VLA. It became clear that Hep G2, a cell, could be more clearly identified.

Example 2 As a sugar chain-containing monomer, VLA of the above formula (2) and VMA of the formula (3) were synthesized.
A vinyl monomer having a sulfonylurea structure represented by the following formula (12) was synthesized as a vinyl monomer having an action site of (1).

[0055]

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The above vinyl monomer having a sulfonylurea structure was synthesized as follows. First, p-aminoethylbenzenesulfonamide (1 g, 5 mmol) was dissolved in a mixed solution of 5 ml of acetone and 5 ml of a 1N aqueous solution of NaOH, and 0.54 g of acroyl chloride was dissolved.
(6 mmol) was added and reacted at room temperature for 3 hours. After the solvent was distilled off, the residue was dissolved in methanol, and the insoluble matter was separated by filtration. This was recrystallized three times from methanol to give 4-
[(2 ′)-Acrylamidoethyl] benzenesulfonylamide (AEBSA) was obtained (80% yield).

1.7 g (7 mmol) of AEBSA was dispersed in 3.5 ml of acetone, and then 1N NaOH
7 ml of an aqueous solution was added and dissolved. To this, cyclohexyl isocyanate (0.8 ml) dissolved in 3.5 ml acetone was added.
76 g, 7 mmol) were added. After reacting for 16 hours, 7 ml of 1N HCl aqueous solution was added, the precipitate was collected by filtration, washed sufficiently with water, and recrystallized from methanol.
Cyclohexyl 4-[(2 ′)-acrylamidoethyl] benzenesulfonylurea (CA) represented by the above formula (12)
EBSU) was obtained (75% yield).

The sugar chain polymer having a charged molar ratio of 9: 1 between VLA and CAEBSU was 1.7 g of VLA and 0.13 g of VLA and CAEBSU.
g CAEBSU in 2 ml DMSO,
After adding 2 mg of AIBN, it was obtained by reacting at 60 ° C. for 6 hours.

Similarly, VLA or VMA and CA
EBSU was copolymerized at a charged molar ratio of 7: 3 to obtain a sugar chain polymer (P (VLA-co-SU) and P (V
MA-co-SU). 1.5 ml of an aqueous solution (0.1%) of each of these sugar chain polymers was added to a polystyrene Petri dish and left standing for coating. For comparison, petri dishes coated with PVLA and PVMA were also prepared.

The insulinoma MIN6 cells derived from the transgenic mouse were adjusted to 2 × 10 ⁵ cells / ml,
1.5 ml of each was seeded on a petri dish coated with each of the above polymers. After culturing at 37 ° C. under 5% CO ₂ for a predetermined time, the medium was collected, and the amount of insulin contained in the medium was measured by enzyme immunoassay (EIA). MIN
FIG. 2 shows the amount of insulin production for 24 hours after seeding of 6 cells. In FIG. 2, “control” is the result when cultured in an uncoated polystyrene dish.

As compared with the control, P having a glucose residue
In the system of VMA and P (VMA-co-SU), an increase in insulin production was observed, and in particular, P (VMA-co-SU)
The highest yield was obtained with the system. From this, MIN
By recognizing glucose and also sulfonylurea by the six cells, it was clearly suggested that interaction with them increased insulin production.

Example 3 A VLA represented by the above formula (2) was synthesized as a sugar chain-containing monomer. The RGDSG peptide is
Biotech Peptide Synthesizer with commercially available compounds or modified Sheppard's Fmoc
nthesizer).

4 g (27 mmol) of P-vinylbenzoic acid
Was dissolved in 10 ml of chloroform together with 3.5 g (30 mmol) of N-hydroxysuccinimide and 6 g (30 mmol) of N, N'-dicyclohexylcarbodiimide, and reacted at room temperature for 24 hours. After distilling off the solvent, the obtained precipitate was recrystallized from chlorophorom ether (yield 2.7 g). The obtained active ester (1.5 g (6 m
mol)) in 0.8 g of 6-amino-N-caproic acid (6 m
mol) together with THF and reacted at room temperature for 4 days. After evaporating the solvent, recrystallization from chloroform gave 6
-(P-Vinylbenzamide) -hexanoic acid was obtained.

1 g (2.1 mmol) of VLA and 6- (p-
61 mg of vinylbenzamide) -hexanoic acid (0.21
mmol) in 3 ml of DMSO and 2 mg of AI
BN was added and copolymerized at 60 ° C. for 24 hours. 100 mg of the obtained copolymer was added to 10 ml of a tetramethylethylenediamine buffer solution (TEMED, 50 mM, pH 4.7).
, And 0.063 g (2.2 mmol) of 1-ethyl-3-dimethylaminopropylcarbodiimide was added, and the mixture was reacted at room temperature for 24 hours. The activated polymer thus obtained has R
0.117 g (2.2 mmol) of GDSG peptide was added and reacted at room temperature for 3 days, and RG was used as a second action site.
A sugar chain polymer (P (VL) comprising a copolymer of VLA and a monomer represented by the following formula (13) having a DSG peptide:
A-co-RGDSG) was synthesized.

[0065]

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P (VLA-co-RGDSG) and PVL
A 0.01% aqueous solution (w / v) of A
008), left for 24 hours, and washed with PBS to prepare a sugar chain polymer-coated petri dish. A suspension of rat hepatocytes isolated by the Seqlen method was dispensed into the petri dishes coated with the sugar chain polymer at a rate of 3.5 × 10 ⁵ cells / dish and cultured for 60 minutes. Cells not adhering to the petri dish were collected and counted by a Coulter counter to calculate the cell adhesion rate. At the same time as the start of culture,
VLA, RGDSG, or P (VLA-co-RGDS
An aqueous solution (0.01% (W / V)) of G) was added, and the inhibitory effect on cell adhesion was also measured. P (VLA-co-RG
DSG) The results of the uncoated Petri dish are shown in FIG.
FIG. 3B shows the results of the LA-coated petri dish.

When no inhibitor was added, both petri dishes showed an adhesion rate of 70% or more. As a result of the inhibition experiment, in the Petri dish coated with P (VLA-co-RGDSG),
In contrast to P (VLA-co-RGDSG), the inhibition was also inhibited by PVLA in the petri dish coated with PVLA. That is, P (VLA-co-RGDS
G) It was shown that hepatocyte adhesion in the coated petri dish was recognized not only through recognition of the galactose residue of VLA, but also through recognition of the second action site, RGDSG.

Example 4 As a sugar chain-containing monomer, VLA of the above formula (2) was synthesized. As the vinyl monomer, isopropylacrylamide (IPAAM) having a temperature-sensitive group as shown in the following formula (14) was used. When the composition ratio of IPAAM and VLA is 9: 1, 8: 2,
7: 3, 4: 6, and 2: 8 (the sum of the moles of both is 0.
01 mol), and potassium granulate was added so as to be 1 mol% with respect to each monomer.
These were made into a 2 ml aqueous solution, and then copolymerized at 60 ° C. for 24 hours to obtain a sugar chain polymer.

[0069]

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Lower Critica of the above sugar chain polymer aqueous solution
l Solution Temperature (LCST) was measured. The method was performed by measuring the change in the UV (wavelength 400 nm) transmittance of the aqueous sugar chain polymer solution under various temperature conditions of 16 ° C to 44 ° C. As a result, IPAAM: VLA = 9:
LCST is 3 only for sugar chain polymers copolymerized at a composition ratio of 1.
Observed at 4 ° C. This 9: 1 copolymer is referred to as the sugar chain polymer of this example (abbreviated as P (IPAAM-co-VLA)).
And

The sugar chain polymer (P (IPAAM-
Petri dishes were coated at various concentrations (co-VLA)). Seg
Rat hepatocytes isolated according len's method 4 × 10 ⁴
It was adjusted to cells / ml and dispensed into the coated petri dish at 37 ° C. The cells were cultured in a WE medium at 37 ° C. under 5% CO ₂ for 2 hours. Unadhered cells were collected at 37 ° C., and the cell adhesion rate to each coated petri dish was calculated. Next, a medium cooled to 4 ° C. was added to the cells adhered to each coated petri dish, the detached cells were collected, and the number of detached cells was counted using a Coulter counter. FIG. 4 shows the results.

At concentrations of 10 and 30 mg / cm ² , P (I
PAAM-co-VLA) coated petri dishes
% Was obtained. In addition, 30mg / c
In the Petri dish coated with m ² , almost all of the attached cells were detached, whereas in the Petri dish coated with 10 mg / cm ² , only about half of the attached cells were detached.
On the other hand, in the petri dish coated with 90 mg / cm ² , both the adhesion rate and the desorption rate were about 40%.

From the above results, rat hepatocytes were found to contain P (I) having a galactose residue and also having a temperature-sensitive group.
PAAM-co-VLA), indicating that the adhered cells can be easily detached only by adding the medium at 4 ° C. However, it was found that these adhesion-desorption also depend on the coating concentration of P (IPAAM-co-VLA).

That is, the sugar chain polymer of this example specifically adheres cells with galactose residues, and physically changes the polymer by IPAAM, which is a second action site nonspecific for hepatocytes. To allow the cells to detach. Therefore, it was suggested that this sugar chain polymer can be effectively used as a selective recovery agent for cells and the like.

Example 5 As a sugar chain-containing monomer, VLA of the above formula (2) was synthesized. Examples of the vinyl monomer include p having an amino group as shown in the following formula (15).
-Aminomethylstyrene. These monomers are
The mixture was mixed at a molar ratio of 80:20 and copolymerized to obtain a sugar chain polymer of this example.

[0076]

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The sugar chain polymer of the present invention and sodium alginate were dissolved in water to prepare a gel-like cell culture substrate. The cell culture substrate was mixed with a hepatocyte suspension (30 × 10 ⁴ cells / ml) and processed into particles using a granulator.

The particulate cell culture substrate containing hepatocytes was dispersed in a polystyrene dish so that they did not come into contact with each other, and kept at 37 ° C. for 60 minutes in a humidified air / CO ₂ (95/5% by volume) incubator. . According to microscopic observation, the cultured liver parenchymal cells have an appropriate size (about 100 μm).
It was confirmed that a three-dimensional aggregate was formed.

[0079]

The sugar chain polymer of the present invention has cell selectivity given by the sugar chain, and has a second action added by the vinyl monomer. For example, when a vinyl-based monomer having a temperature-sensitive group is combined, a cell collection agent for selectively collecting target cells can be obtained.

When a vinyl-based monomer having a basic group is combined, a gel-like complex can be formed with an acidic polysaccharide, and a cell culture substrate having a three-dimensional structure can be obtained. If hepatocytes are cultured with the cell culture substrate having the three-dimensional structure, three-dimensional aggregates of hepatocytes can be easily obtained. The cell culture substrate can be formed into particles, and by culturing hepatocytes with the particle-formed cell culture substrate, a three-dimensionally controlled closed environment can be cultured. Hepatocyte aggregates cultured in such a stereocontrolled closed environment can be suitably incorporated into a hybrid artificial liver in its original form.

[Brief description of the drawings]

FIG. 1 is a graph showing the recognizability of the sugar chain polymer of Example 1 for cancer cells.

FIG. 2 is a graph showing the effect of the sugar chain polymer of Example 2 on the amount of insulin produced by MIN6 cells.

FIG. 3 shows a sugar chain polymer (A) and PVLA of Example 3.
It is a graph showing the adhesiveness of hepatocytes to (B).

FIG. 4 is a graph showing an adhesion rate and a desorption rate of hepatocytes to a petri dish coated with each concentration of the sugar chain polymer of Example 4.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-3-47802 (JP, A) JP-A-6-153927 (JP, A) JP-A-5-276923 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) C08F 212/14 C08F 12/14-12/28 C12N 5/00

Claims (7)

(57) [Claims] (1) The following formula (1): (Wherein, SUG represents a sugar chain, and X has a second action)
The second portion) .
The sites that have an effect are vitamins, cell adhesion, proliferation,
Drugs, cytokines, growth factors,
At least selected from flavonoids, nucleic acids and peptides
A sugar chain polymer characterized in that there is only one . 2. The method according to claim 1, wherein the site having the second action is RGD.
Cell recognition agent characterized by comprising a sugar polymer of claim 1 wherein SG peptide. 3. The method according to claim 1, wherein the site having the second action contained in the vinyl monomer acts non-specifically on cells that specifically recognize the sugar chain.
The sugar chain polymer according to the above. 4. The sugar chain polymer according to claim 3, wherein the site having the second action is a basic group that forms a complex with an acidic polysaccharide. 5. A gel cell culture substrate comprising a complex of the sugar chain polymer of claim 4 and an acidic polysaccharide. 6. A cell culture method comprising processing the cell culture substrate according to claim 5 into particles, and culturing cells in the particles. 7. The cell collection agent according to claim 3 , wherein the site having the second action is a temperature-sensitive group.