JPH0980045A - Inspection method for biological reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which a biological reaction participating in immunity, inflammation and the like can be inspected by a more simplified measuring system while whole blood is used. SOLUTION: A polymer material (e.g. chitosan or a chitosan derivative) which comprises at least one chemical structure selected from a group composed of a hydroxyl group, of an amide skelton and of an ester skelton or a polymer material which comprises a cationic functional group is brought into contact with blood. Thereby, the generation of an interleukin-1, a TNF (tumor necrotizing factor) or of interleukin-6 is induced, and the generation amount of the interleukin-1, the TNF or the interleukin-6 is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an interleukin-1 (IL-IL) by contacting a material with blood.
1), TNF (TNF: Tumor Necrosis Factor) or interleukin 6 (IL-6) is induced and produced, and the production capacity is measured to examine a biological reaction relating to immunity, inflammation and the like.

[0002]

2. Description of the Related Art As is well known, there are leukocytes such as monocytes, macrophages, granulocytes, and lymphocytes, killer cells, NK cells, LAK cells, etc. as immune cells in the living body. In addition to performing immune surveillance in each organ, it plays various roles in various immune reactions.

It is generally known that in various diseases and malignant tumors, the functions of these cells are suppressed or enhanced by various immune reactions. Therefore, it is important to understand the immune function of the living body by examining the functions of these cells in order to understand various pathological conditions and to determine the timing of drug administration. Conventionally, such a method for measuring the immune function of the living body, for example,
Lymphocyte blast transformation test, granulocyte phagocytosis function test, granulocyte bactericidal ability (reactive oxygen production ability) test, etc. have been conducted. Recently, fluorescent cytochrome-labeled monoclonal antibody against flow cytometry device and various immunocompetent cell surface antigens. Surface antigen tests have been performed with. However, these tests require special techniques such as cell separation, cell culture, and microscopic measurement, are time-consuming, and require RI (radioisotope) facilities and expensive equipment.
A simpler, less dangerous, and more accurate inspection method is desired.

[0004] Various immune factors produced by these immune cells also play an important role in the immune system of the living body. In particular, with the recent development of cell engineering and genetic engineering techniques, the structure of many humoral immune factor (cytokine, etc.) genes has been elucidated using recombinant DNA technology. Up to now, various substances such as cytokines such as interleukin, interferon, TNF and the like have been reported. It has been found that these factors are closely related to each other to form a cytokine network and regulate the immune function of the living body. At present, it is considered very important to know the dynamics of these cytokines and the like when considering the immune system of the living body.

Therefore, it has been recently conducted to examine the blood levels of these cytokines in the blood of patients with various diseases. However, since these cytokines act in a very small amount in the living body, their blood concentration is low and they are rapidly decomposed. Therefore, it is usually difficult to measure these cytokines.

On the other hand, since cytokines are produced by the above immunocompetent cells, it is important to know the cytokine producing capacity of the immunocompetent cells of the patient. From such an idea, Japanese Patent Publication No. 63-502695 discloses a method of reacting a specific antigen such as a tuberculin purified protein derivative with blood to measure γ-interferon released from sensitized lymphocytes. ing. In addition, Japanese Unexamined Patent Application Publication No.
Japanese Patent Publication No. 196961 discloses a method in which lipopolysaccharide (LPS) or lectin is reacted with blood to measure production-induced lymphokines. However, since these methods use a biologically derived substance, a purification step from the bacterial cells or the like is required, and there are problems such as lot-to-lot differences,
There is a problem that measurement results with good reproducibility cannot be obtained. In addition, when stimulants such as lipopolysaccharide and lectin are used, when quantifying the amount of cytokines by bioassays such as cytotoxicity tests and cell proliferation tests, the effect of those stimulants on various cells is Measurement is difficult.

IL-1 is a cytokine produced mainly by cells such as monocytes / macrophages and neutrophils. IL-1 acts on various cells, exhibits various biological activities, and plays an important role in biological reactions such as immunity, inflammation, hematopoiesis, endocrine, and cranial nerves (Oppenheim, JJ et al .:
There is more than one interleukin 1. Immunol. Tod
ay, 7, 45-56, 1986). IL-1 is also present in normal individuals.
Is constantly produced and is considered to be an essential factor for maintaining the function of the living body. On the other hand, it has been suggested that when IL-1 is abnormally produced, it causes a disease associated with inflammation.

For example, in the synovial fluid of patients with rheumatoid arthritis,
L-1 activity has been detected, and a correlation between the activity and the degree of progression has been confirmed, suggesting that IL-1 is involved in the pathological condition (Eastgare, JA et al .: Corre
relation of plasma interleukin 1 levels with disease
activity in rheumatoid arthriitis. Lancet, 8913,7
06-709, 1988). In addition, IL-1 has been suggested to be involved in various diseases such as glomerulonephritis, granuloma, Alzheimer's disease, Down syndrome, Kawasaki disease, gout, and acute myelogenous leukemia. It has been found that IL-1 exhibits a wide variety of actions in various tissues and cells throughout the body and is involved in various reactions of the living body found in infectious diseases and inflammatory diseases. As described above, it has become extremely important to know the production and production ability of IL-1 and its kinetics during the pathological condition.

[0009] However, there is not much evidence that the relationship between the amount of IL-1 produced in blood and the disease has been clearly proved. This is because the action of IL-1 is very strong and pg
Since it works at a concentration of the order of 1 ml / ml, it is possible that changes in the amount of production that may cause functional abnormality in the living body cannot be accurately detected by the current measurement system.

On the other hand, TNF is a cytokine that causes tumor necrosis. Initially, it attracted attention as a factor that damages tumor cells, but it is now known that normal cells also exhibit various actions related to inflammation and metabolic disorders.
As described above, it has been revealed that TNF exhibits a wide variety of actions in various tissues and cells throughout the body and is involved in various reactions of the living body found in infectious diseases and inflammatory diseases. Therefore, it is becoming extremely important to know the production and production ability of TNF and its kinetics during the pathological condition.

For example, TNF is said to play an important role in the pathological conditions of many inflammatory diseases including rheumatoid arthritis, Kawasaki disease, Behcet's disease and infectious diseases such as meningitis and malaria (Hideo Wada). , Saito Reiko, Nagao Mimasa et al .: Blood Tumor necrosis factor in disseminated intravascular coagulation syndrome
Value and therapeutic effect of FOY administration. Modern medicine. 22: 265-2
69, 1990).

[0012] As described above, although TNF is presumed to be involved in the etiology of a very wide range of diseases, the relationship between the amount of TNF and its production has been clearly proved in actual patients. Is not so many. This is TNF
Is very strong and acts at a concentration of the order of pg / ml, so it is possible that changes in the amount of production that can cause dysfunction in the living body cannot be accurately detected by the current measurement system.

IL-6 is a cytokine mainly produced by cells such as monocyte / macrophage cells and lymphocytes [edited by Shinpei Kasakura, published by The Nihon Igakukan, "Cytokine 94", 74-81 ( 1994)]. IL-6 acts on various cells, shows various biological activities, and plays an important role in biological reactions such as immunity, inflammation, and hematopoiesis. Moreover, IL-6 is constantly produced in normal individuals, and is considered to be an essential factor for maintaining the function of the living body. On the other hand, when IL-6 is abnormally produced,
It has also been suggested to cause diseases associated with inflammation.

For example, in patients with Castleman's disease, multiple myeloma, and mesangial proliferative nephritis, high IL-6 activity was detected in the blood, and a correlation was observed between the activity and the degree of progression. Therefore, the involvement of IL-6 in the pathological condition is suggested. It has been found that IL-6 exhibits a wide variety of actions in various tissues and cells throughout the body and is involved in various reactions of the living body found in infectious diseases and inflammatory diseases. As described above, it is becoming extremely important to know the production and production capacity of IL-6 and its kinetics during the pathological condition.

However, there is not much evidence that the relationship between the amount of IL-6 produced in blood and the disease has been clearly proved. This is because the action of IL-6 is very strong and pg
Since it works at a concentration of the order of 1 ml / ml, it is possible that changes in the amount of production that may cause functional abnormality in the living body cannot be accurately detected by the current measurement system.

Therefore, recently, peripheral blood monocytes and the like have been collected,
Studies have been conducted to examine the ability of IL-1, TNF, and IL-6 to be produced in vitro, and to clarify the relationship between diseases and these cytokines (Hidemaro Ono, Hide Kikuchi,
Nakamura S. et al .: Study of tumor necrosis factor production in cancer patients. Ayumi of Medicine. 150: 165-166,1989).

However, collection and preparation of monocytes and the like from blood requires complicated operations including aseptic processing,
The operation technique has a great influence on the cells. In addition, these tests are carried out in cells that have been separated and prepared from blood in an appropriate culture medium, and are accurate in that they do not contain various humoral factors or other cells contained in blood. It cannot be said that it reflects the biological reaction in the body.

[0018]

The object of the present invention is to solve the drawbacks of the conventional test methods and to produce IL-1, TNF and IL-6 by stimulants such as LPS and various drugs,
IL-1, in a more simplified assay system using whole blood,
It is to provide a new test method for carrying out a biological reaction test by measuring the TNF or IL-6 production ability.

[0019]

The present invention has been made to achieve the above object, and the inventions described in claims 1 to 4 respectively have the following configurations.

That is, the invention described in claim 1 is a polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton or a polymer material having a cationic functional group in the molecule. The method for inspecting biological reaction is characterized in that production of IL-1 is induced by contacting the blood with blood, and the production amount of the IL-1 is measured.

Further, the invention according to claim 2 is a polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton or a polymer material having a cationic functional group in the molecule. Contacting the blood with blood induces the production of TNF,
It is a biological reaction test method characterized by measuring the production amount of

According to the third aspect of the present invention, a polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton or a polymer material having a cationic functional group and blood in the molecule. By inducing the production of IL-6,
It is a biological reaction test method characterized by measuring the production amount of

Further, in the invention described in claims 1 to 3, preferably, as described in claim 4, the polymer material is brought into contact with blood in a temperature range of 15 ° C to 42 ° C. As described above, according to the inventions of claims 1 to 4, the cytokine (IL
-1, TNF or IL-6) is induced and the production amount of the cytokine is measured in common, and the living body involved in immunity, inflammation, etc. by measuring the production capacity of the cytokine. It is intended to provide a more accurate and simple biopsy method that makes it possible to test a reaction.

In the biological reaction test method of the present invention, whole blood or the like can be used as it is, and in that case, it is not necessary to separate monocytes, neutrophils or the like from the blood, and operations and Needless to say, the treatment time is not required, and there is no problem that the cell activity is lowered or unnecessary activation is caused by the operation, the treatment time and the like.

Further, according to the present invention, the amount of blood required for the test can be very small, and the burden on the blood sample can be reduced. Moreover, since whole blood or diluted blood thereof is used, the involvement of various humoral factors and cells contained in the blood works in the same manner as in the living body, and the defense function in the living body can be more accurately grasped.

The present invention will be described in detail below. In one example of an embodiment of the method of the present invention, blood is collected as a sample, and this blood is added to a tissue culture plate, a test tube or the like as it is or as whole blood. The bottom surface or the side surface of the inner wall of the plate well or the test tube is composed of a material for inducing the production of IL-1, TNF and IL-6 from cells, or is coated with these materials, or It is filled. By culturing blood using these reaction vessels, these materials interact with blood to induce the production of IL-1, TNF, and IL-6. This IL-1, TNF or IL-6
By measuring the amount of production, it is possible to grasp the biological reaction relating to immunity, inflammation and the like.

In the above, the blood sample can be collected by an arbitrary method according to a conventional method. For example, heparin blood sampling, citric acid blood sampling and the like can be mentioned. However, IL-1,
Since the induction of TNF and IL-6 production is the result of the biological reaction of cells, calcium ions in blood and medium,
Heparin blood collection that does not chelate magnesium ions and the like is more preferable.

When diluting blood, for example, phosphate buffer, Hanks buffer, MEM, RPMI-16
Any conventional medium such as 40 can be used. Culturing is usually carried out at a temperature of around 37 ° C.
It can be performed in the range of 2 ° C. Mixing plates and test tubes using a shaker or a rotary incubator during this culture is
It is preferable for inducing the production of -1, TNF and IL-6, and it becomes easy to understand the production capacity of each blood sample.

The production amount of IL-1, TNF or IL-6 after culturing can be measured by various immunoassay methods such as immunoenzyme antibody method. Moreover, since a stimulant such as lipopolysaccharide or lectin is not used in the method for inducing the production of IL-1, TNF and IL-6, IL-1, TNF or IL is determined by a bioassay such as a cytotoxicity test or a cell proliferation test. Even when the activity of -6 is measured, there is no effect of those stimulants on various cells, and accurate measurement is possible.

Next, the background of the present invention will be described. When leukocytes such as monocytes and neutrophils in the blood react with foreign substances such as microorganisms, they are activated through adhesion reaction and phagocytosis reaction, release of intracellular enzymes and activated oxygen, prostaglandins, cytokines, etc. It releases various mediators and plays an important role in the biological defense reaction. These reactions are likewise triggered when in contact with the material. For example, leukocytes such as monocytes and neutrophils also actively phagocytose synthetic polymeric materials such as latex particles. This occurs when the material is regarded as a foreign substance, and these reactions greatly vary depending on the properties of the polymer material such as surface properties and physicochemical composition. Therefore, it is considered that this reaction can be controlled by controlling the properties of the material.

Among the above reactions, the present inventors paid attention to the production of cytokines by leukocytes, and conducted intensive studies on induction of cytokine production by contacting blood with various natural and synthetic polymer materials, It has been discovered that various polymeric materials cause significant production of cytokines such as IL-1, TNF and IL-6 in blood. In particular, when a polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton in the molecule or a polymer material having a cationic functional group is brought into contact with blood, remarkable IL -1, TN
Induction of F and IL-6 production was observed, whereas in polymer materials such as polystyrene, polyethylene, polypropylene and polyvinyl chloride, IL-1 and TNF were mostly produced.
Induction of IL-6 production was not observed. Further, in a polymer material having only an anionic group such as a sulfone group or a carboxyl group, a low level of IL-1, TNF is obtained.
And the induction of IL-6 production was only observed.

The polymer material having at least one chemical structure selected from the group consisting of hydroxyl group, amide skeleton and ester skeleton in the molecule used in the present invention includes agarose, cellulose, starch, pullulan, dextran, glycogen, Mannan, glucomannoglycan,
Galactomannoglycan, pectin, alginic acid, hyaluronic acid, chondroitin sulfate, chitin, natural polysaccharides such as chitosan and their carboxymethylation, succinylation, sugar side chain graft, peptide side chain graft, glycolation, acylation, There are various derivatives modified by amination.

For example, according to the findings obtained by the present inventors, chitin and its N-deacetylated product, chitosan, and a primary amine, a secondary amine, a tertiary amine or a quaternary ammonium salt are introduced into the amino group of chitosan. Chitosan derivative, a derivative having an alkyl group introduced, a sulfone group in the hydroxyl group,
Derivatives and the like into which a carboxymethyl group was introduced showed large induction of IL-1, TNF and IL-6 upon contact with blood. Also, for agarose and agarose 2,
Cross-linked agarose with increased strength by allowing 3-dibromopropanol to act under strong alkaline conditions to cross-link,
By contacting blood with gel beads composed of an agarose derivative having an ion exchange group such as a diethylaminoethyl (DEAE) group ether-bonded thereto, or an agarose derivative modified with a quaternary ammonium salt, IL-1, TNF can also be obtained.
And significant induction of IL-6 production was observed. Also, by dissolving sodium alginate or the like in water, C and C
By contacting blood with alginic acid gel beads prepared by contacting with an aqueous solution containing a polyvalent metal ion such as a ²⁺ , Al ³⁺ , Ba ²⁺ and Cu ²⁺ , IL-1, T
A significant induction of NF and IL-6 production was seen.

Further, the polymeric material used in the present invention having at least one chemical structure selected from the group consisting of hydroxyl group, amide skeleton and ester skeleton in the molecule includes polyvinyl alcohol, polyhydroxyethyl acrylate, poly Synthetic polymeric materials such as hydroxymethyl acrylate, polyphenol, polyacrylamide, polylysine, polyvinylpyrrolidone, polyacrylic acid ester, and polymethacrylic acid ester, and various derivatives and crosslinked products thereof, and vinyl monomers such as styrene and methacrylic acid ester. Various copolymers of

For example, according to the findings obtained by the present inventors, the polyvinyl alcohol gel was treated with IL-IL by contact with blood.
1, showed a significant induction of TNF and IL-6 production. This polyvinyl alcohol gel introduces crosslinking by adding boric acid, or uses a photocrosslinkable polyvinyl alcohol having a styrylpyridinium group or a styrylquinolinium group which is a photosensitive functional group in a side chain, or as a gelling agent. It can be prepared by adding various organic and inorganic compounds to be referred to, or by repeating the freeze-drying or freeze-thawing of the aqueous solution.

Further, according to the knowledge obtained by the present inventors, by modifying a methacrylic acid ester monomer and copolymerizing it with various vinyl monomers and the like, a modified product introduced with a hydroxyl group or a material introduced with a phenyl group or the like. But IL-
1. A significant induction of TNF and IL-6 production was confirmed.

The polymeric material having a cationic functional group used in the present invention is a polymeric material having an amino group, an imino group, a nitrilo group, a quaternary ammonium group, a sulfonium group, a phosphonium group, and the like. , Polyvinyl pyridine and salts thereof, ionene polymer, N-trialkylaminomethyl polystyrene, amino acetalized polyvinyl alcohol, polyvinyl imidazole, polyethylene imine, polydialkyl diallyl ammonium salt, polydialkyl diallyl ammonium salt-SO ₂ copolymer, polyvinyl Examples thereof include benzylsulfonium salt and polyvinylbenzylphosphonium salt.

The polymeric material having the above-mentioned cationic functional group can be introduced into the above-mentioned cationic functional group or synthetic functional polymers such as polystyrene by using various chemical modification methods. It can be obtained by a copolymerization or a crosslinking reaction between the vinyl monomers. For example, by copolymerizing styrene and divinylbenzene, Friedel-
It is possible to introduce a cationic functional group into polystyrene by introducing a chloromethyl group into a benzene nucleus through a Krafts reaction, treating the chloromethyl group with an amine to aminate, and then performing alkyl substitution. it can.

As another method, for example, a compound having a chloromethyl group and an oxirane ring in the molecule such as epichlorohydrin is reacted with imidazoles to synthesize a modified imidazole, which is then treated with a polyfunctional epoxy compound. It can also be obtained by converting. Further, regarding the polymer material having a cationic functional group,
Various other synthetic methods are possible, but the present invention is not limited to these methods.

According to the findings of the present inventors, a polystyrene modified product in which a trimethylammonium group having a trialkyl-substituted nitrogen atom or a dimethylethanolammonium group which is a dialkylethanol is introduced as a cationic group is an unmodified polystyrene or Significantly higher IL compared to materials consisting of polystyrene modified with anionic groups
The production induction of -1, TNF and IL-6 was shown. Also,
Large production induction of IL-1, TNF, and IL-6 was confirmed even with a cationic group having an amino group, an imino group, or a nitrilo group. On the other hand, almost no induction of production of IL-1, TNF and IL-6 was observed in the polystyrene derivative having a sulfonic acid group as an anionic group introduced therein.

Further, the polymethacrylic acid ester material obtained by copolymerization of a methacrylic acid ester monomer having a cationic group such as a dimethylethanol ammonium group, a diethylamino group, and an amino group at the terminal has remarkable IL-1, The induction of TNF and IL-6 production was shown. On the other hand, in the case of a methacrylic acid ester-based material having a carboxyl group which is an anionic group, most of IL-1, T
No induction of NF and IL-6 production was seen.

In addition, chitin and its N-deacetylated product, chitosan, and a primary amine, 2 are added to the amino group of chitosan.
Chitosan derivative with primary amine, tertiary amine or quaternary ammonium salt, diethylaminoethyl (DEAE)
As described above, the agarose derivative in which an ion-exchange group such as a group is ether-bonded, and the agarose derivative modified with a quaternary ammonium salt also showed particularly high production induction of IL-1, TNF, and IL-6.

As the shape of the various polymeric materials used in the present invention, any known shape such as a particle shape, a fiber shape, a hollow fiber shape or a film shape can be used. For example, spherical particles of styrene / divinylbenzene copolymer can be easily obtained by adding divinylbenzene and a radical initiator benzoyl peroxide to a styrene monomer and continuing suspension stirring in water at 60 ° C. for 5 hours. The particle size can be controlled by the stirring speed, the type and concentration of the stabilizer added to water, the volume ratio of the monomer to water, and the like. Further, it is also easy to make it porous by carrying out suspension polymerization of a mixed liquid of a diluent and a monomer. Further, when the polymerization of various methacrylic acid ester monomers is carried out by suspension polymerization in a good solvent for the monomers and a poor solvent for the polymers, spherical particles of polymethacrylic acid ester can be easily obtained. The particle size can be controlled by the stirring speed, the type and concentration of the stabilizer added, and the like. The spherical particles thus synthesized are subjected to the chemical modification reaction as described above to obtain various IL-
1, TNF and IL-6 production inducers are obtained.

As for the method of contacting the blood with the material, any method can be used as long as the blood can be sufficiently contacted with the material. For example, a method in which the above fibrous material is packed in a column and blood is circulated in the column, or a method in which a bead-shaped material having a particle size of 50 μm to 5 mm is packed in the column and blood is circulated can be used. Further, the material may be brought into contact with the blood by suspending the material in various shapes in the blood.

The temperature at which the above material is brought into contact with blood is
IL-1, TNF and IL-6 in the range of 15 ° C to 42 ° C
It is preferable for enhancing the induction of production of. According to the findings obtained by the present inventors, as is clear from the examples described below, sufficient induction of production of IL-1, TNF and IL-6 was not observed when the contact temperature was lowered. Moreover, when the contact temperature was increased, sufficient induction of production of IL-1, TNF and IL-6 was not observed. When the contact temperature was higher than 45 ° C, denaturation of plasma protein, remarkable hemolysis, and leukocyte disintegration occurred, and the induction of IL-1, TNF and IL-6 production was drastically reduced.

In the present invention, the contact between the material and blood causes the interaction between the material and cells,
Induction of production of IL-1, TNF and IL-6.
The IL-1, TNF or IL-6 producing cells are not limited to cells in peripheral blood, but also include cells obtained from lymph vessels, lymph nodes, spleen and the like. The blood is rich in these cells that produce IL-1, TNF or IL-6. Also, these cells in the blood interact with the material,
IL-1, TNF and IL-6 are induced to be produced, but even if they are not directly acted on, IL-1, TNF is mediated by another factor induced by the action of the above material and some factor in blood.
Production of NF or IL-6 may be induced.

Using the above-mentioned materials, IL-1, TNF and IL-6 can be simply and conveniently obtained from blood of healthy persons or patients with various diseases.
Can be induced, and the ability to produce IL-1, TNF and IL-6 in an individual can be examined by measuring the degree of the induction. This can be an effective parameter that reflects the state of health and the pathology of various diseases.

(Operation) As described above, according to the present invention,
By contacting the specific polymeric material with blood,
IL-1, TNF and IL-6 are rapidly and conveniently induced. Therefore, since the production of IL-1, TNF and IL-6 can be easily and rapidly induced from the blood of the person being examined, the IL-of the person being examined can be measured by measuring the amount of production. 1, the ability to produce TNF and IL-6 can be examined.

[0049]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to examples and comparative examples of the present invention. However, the present invention is not limited to the following examples. First,
IL-1, TNF, and IL-6 production-inducing materials were produced by the methods described in Examples 1 to 44 and Comparative Examples 1 to 9 below, and the obtained materials were used to prepare IL-1, TN
The induction of F and IL-6 production was tested.

(Material for Inducing Production by Chitosan or Chitosan Derivative) Example 1 Chitosan gel particles in which the amino group of chitosan remains as it is (Fuji Spinning Co., Ltd., trade name "Chitopearl basic AL-0"
3 "and an average particle size of 0.3 mm) were filled in a polypropylene test tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml. Saline solution for injection (Otsuka Pharmaceutical Co., Ltd.) 12
After adding ml and stirring lightly, the mixture was centrifuged at 500 rpm for 5 minutes, and the supernatant was suctioned and discarded for washing. This washing operation was repeated twice more and then left overnight at 4 ° C. Then, the same washing operation was further performed 5 times, and finally, physiological saline for injection was removed as much as possible.
Polypropylene sample tube for 2 ml (eppendorf
Physiological saline for injection, and then at 120 ° C for 2
It was sterilized by autoclave for 0 minutes, the physiological saline for injection was discarded, and then the physiological saline for injection was further washed three times. This 2 ml polypropylene sample tube was filled with 500 μl of the chitosan gel particles obtained above in a bulk volume.

Example 2 Instead of the chitosan gel particles of Example 1 (manufactured by Fuji Spinning Co., Ltd., trade name "Chitopearl basic AL-03"), chitosan derivative gel particles in which the amino group of chitosan was acetylated (Fuji Spinning Co., Ltd. Manufactured by Chitopearl basic BL-03, having an average particle size of 0.3 mm) was used, and the same procedure as in Example 1 was carried out.
A polypropylene sample tube for ml was obtained.

Example 3 Instead of the chitosan gel particles of Example 1 (trade name "Chitopearl basic AL-03" manufactured by Fuji Spinning Co., Ltd.), a primary amine was introduced into the amino group of chitosan via a benzyl group. Chitosan derivative gel particles (Fuji Spinning Co., Ltd., trade name "Chitop
Earl BCW-3503 ", average particle size 0.3 mm) was used, and the same procedure as in Example 1 was carried out to obtain a 2 ml polypropylene sample tube filled with chitosan derivative gel particles.

Example 4 Instead of the chitosan gel particles of Example 1 (manufactured by Fuji Boshoku Co., Ltd., trade name "Chitopearl basic AL-03"), a primary amine was introduced into the amino group of chitosan via a linear alkyl group. Chitosan derivative gel particles (trade name "Ch
itopearl BCW-3003 ", average particle size 0.3 mm) was used, and the same procedure as in Example 1 was carried out to obtain a 2 ml polypropylene sample tube filled with chitosan derivative gel particles.

Example 5 Instead of the chitosan gel particles of Example 1 (trade name "Chitopearl basic AL-03" manufactured by Fuji Spinning Co., Ltd.), chitosan derivative gel particles in which a tertiary amine was introduced into the amino group of chitosan ( Fuji Spinning Co., Ltd. product name "Chitopearl BCW-2603",
Example 1 except that an average particle size of 0.3 mm) was used
The same procedure as in (1) was performed to obtain a polypropylene sample tube for 2 ml filled with chitosan derivative gel particles.

Example 6 Chitosan derivative gel particles in which a quaternary ammonium salt was introduced into the amino group of chitosan in place of the chitosan gel particles of Example 1 (trade name “Chitopearl basic AL-03” manufactured by Fuji Spinning Co., Ltd.) (Product name "Chitopearl BCW-2, manufactured by Fuji Spinning Co., Ltd.
503 ", average particle size 0.3 mm)
The same procedure as in Example 1 was carried out to obtain a 2 ml polypropylene sample tube filled with chitosan derivative gel particles.

Example 7 Instead of the chitosan gel particles of Example 1 (manufactured by Fuji Spinning Co., Ltd., trade name "Chitopearl basic AL-03"), 6 of chitosan was used.
Chitosan derivative gel particles in which a carboxymethyl group is introduced into the hydroxyl group at the position (Fuji Spinning Co., Ltd., trade name "Chitopearl C
M-03 ", average particle size 0.3mm)
The same procedure as in Example 1 was carried out to obtain a 2 ml polypropylene sample tube filled with chitosan derivative gel particles.

Example 8 Instead of the chitosan gel particles of Example 1 (manufactured by Fuji Boshoku Co., Ltd., trade name "Chitopearl basic AL-03"), 6 of chitosan was used.
Chitosan derivative gel particles in which a sulfone group has been introduced into the hydroxyl group at position (Fuji Spinning Co., Ltd., trade name "Chitopearl SU-03",
Example 1 except that an average particle size of 0.3 mm) was used
The same procedure as in (1) was performed to obtain a polypropylene sample tube for 2 ml filled with chitosan derivative gel particles.

Example 9 5 g of chitosan powder particles (the product name "CHITOSAN 10B" manufactured by Katokichi Co., Ltd.) in which the amino group of chitosan remains as it is
Was suspended in 30 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), centrifuged at 1500 rpm for 1 minute, and the supernatant was aspirated and discarded. This washing operation was repeated twice more and then left overnight at 4 ° C. Then, the same washing operation was further performed 5 times, and finally, physiological saline for injection was removed as much as possible. After adding physiological saline for injection to a polypropylene sample tube for 2 ml (made by Eppendorf), sterilized by autoclaving at 120 ° C for 20 minutes, discarding physiological saline for injection, and further using physiological saline for injection 3 times. Washed. This 2 ml polypropylene sample tube was filled with 500 μl of the chitosan powder obtained above in a bulk volume.

(Material for Inducing Production by Agarose and Agarose Derivatives) Example 10 Agarose (Nakarai Chemical Co., Ltd., special reagent for electrophoresis)
GP-36) was dissolved in distilled water at a concentration of 5% by weight, and
Autoclave was performed at 1 ° C for 20 minutes. 6 this solution
It was kept warm at 0 ° C. and dropped into cold distilled water (4 ° C.) using a microsyringe to prepare agarose gel beads (particle size 5 mm).

The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection.
A polypropylene sample tube for ml (manufactured by Eppendorf) was filled. The filling amount was 20 agarose gel beads.

Example 11 Sepharose 2B consisting of about 2% by weight agarose
3 ml suspension of (Pharmacia LKB Biotechnology)
15ml polypropylene test tube (made by Iwaki Glass Co., Ltd.)
I put it in. This was centrifuged at 1000 rpm for 5 minutes, the supernatant was aspirated and discarded, 12 ml of physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.) was added and stirred, and the mixture was centrifuged under the same conditions and the supernatant was aspirated and discarded. This washing operation was performed three times and left at 4 ° C. overnight.

As in Example 1, 500 μl (bulk volume) of this gel carrier was put into a polypropylene sample tube for 2 ml (made by Eppendorf) and washed with physiological saline for injection (made by Otsuka Pharmaceutical).

Example 12 Sepharos consisting of cross-linked agarose at a concentration of about 6% by weight
A gel-filled tube was obtained in the same manner as in Example 11 except that eCL-6B (Pharmacia LKB Biotechnology) was used.

Example 13 DEA in which diethylaminoethyl (DEAE) group was introduced by ether bond to crosslinked agarose having a concentration of about 6% by weight.
A gel-filled tube was obtained in the same manner as in Example 11 except that ESepharose CL-6B (Pharmacia LKB Biotechnology) was used.

Example 14 Phenyl Sepharose CL-4B (P was prepared by introducing a phenyl group into an agarose having a concentration of about 4% by weight through an ether bond.
A gel-filled tube was obtained in the same manner as in Example 11 except that harmacia LKB Biotechnology) was used.

Example 15 Q Sepharose FF (Pharmacia LKB Biotech) prepared by introducing a quaternary ammonium salt into cross-linked agarose having a concentration of about 6% by weight.
gel-filled tube was obtained in the same manner as in Example 11 except that (Nology) was used.

(Material for inducing production by alginic acid gel) Example 16 2% by weight of sodium alginate (AL-1, medium viscosity type, manufactured by Nitta Gelatin Co., Ltd.) was suspended in physiological saline, and the suspension was placed in an autoclave at 121 ° C. By performing the treatment for 20 minutes, the sodium alginate was dissolved simultaneously with the heat sterilization. Particle size of about 2.5 mm by dropping this into sterilized 1.5 wt% calcium chloride solution and gelling.
Calcium alginate gel beads were prepared. The gel beads were placed in a 15 ml polypyroprene test tube (manufactured by Iwaki Glass Co., Ltd.) in a bulk volume of 1 ml. To this, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred. Centrifuge at 500 rpm for 1 minute, aspirate and discard the supernatant, and inject saline (Otsuka Pharmaceutical Co., Ltd.) 12
ml was added, and the mixture was stirred, centrifuged, and the supernatant was aspirated and discarded.
This washing operation was performed 3 times and left overnight at 4 ° C. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.

2 ml polypropylene sample tube (eppendorf) filled with physiological saline for injection and sterilized and washed
(Manufactured by the company) were filled with 70 of these gel beads.

Example 17 A bead-filled tube was obtained in the same manner as in Example 16 except that 2% by weight of sodium alginate was changed to a concentration of 5% by weight.

Example 18 A bead-filled tube was obtained in the same manner as in Example 16 except that 2% by weight of sodium alginate was changed to a concentration of 10% by weight.

Example 19 A bead-filled tube was obtained in the same manner as in Example 16 except that the 1.5% by weight calcium chloride solution was changed to a concentration of 3% by weight.

Example 20 A bead-filled tube was obtained in the same manner as in Example 16 except that the 1.5 wt% calcium chloride solution was changed to the 1.5 wt% barium chloride solution.

Example 21 All performed except that the 1.5 wt% calcium chloride solution was changed to a 1.5 wt% barium chloride solution and the 5 wt% sodium alginate was changed to a 3 wt% concentration. The beads-filled tube was obtained in the same manner as in Example 16.

Example 22 Sodium alginate was replaced with sodium alginate (100-
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 150 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

Example 23 Sodium alginate was replaced with sodium alginate (300-
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 400 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

Example 24 Sodium alginate was replaced with sodium alginate (500-
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 600 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

(Material for Inducing Production by Polyvinyl Alcohol Gel) Example 25 A 10% by weight aqueous solution of polyvinyl alcohol (manufactured by Kishida Chemical Co., Ltd.) having a polymerization degree of about 1400 and a saponification degree of 99 mol% was prepared. This aqueous solution was defined as R = [polyvinyl alcohol monomer unit] / [metal ion], R =
Fe ³⁺ is included so as to be 10. This viscous solution was added dropwise to a sterilized 1M NaOH aqueous solution using a syringe, and left for about 30 minutes. As a result, polyvinyl alcohol gel beads having a particle size of about 2.5 mm were prepared. [H. Yokoi: Complex gel of PVA and PAA, polymer processing, Vol. 40, No. 11, 1991].

The above gel beads were placed in a polypropylene test tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml. 12m of physiological saline for injection (made by Otsuka Pharmaceutical Co., Ltd.)
1 and added and stirred lightly. Then, the mixture was centrifuged at 500 rpm for 1 minute, and the supernatant was aspirated and discarded. Similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added, and the mixture was stirred, centrifuged under the same conditions as above, and the supernatant was aspirated. Threw away. This washing operation was performed three times and left at 4 ° C. overnight. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible. 2 ml that was filled with physiological saline for injection and sterilized and washed
Polypropylene sample tube (made by Eppendorf)
Then, 70 gel beads thus obtained were filled.

Example 26 The same procedure as in Example 25 was carried out except that the polyvinyl alcohol used was changed to a polymerization degree of about 2000 and a saponification degree of 98.5 to 99.4 mol% of polyvinyl alcohol (manufactured by Kishida Chemical Co., Ltd.). Then, a tube filled with gel beads was obtained.

Example 27 A gel bead-filled tube was obtained in the same manner as in Example 25 except that the concentration of polyvinyl alcohol used was changed to 15% by weight.

Example 28 A gel bead-filled tube was obtained in the same manner as in Example 26 except that the concentration of polyvinyl alcohol used was changed to 15% by weight.

Example 29 A gel bead-filled tube was obtained in the same manner as in Example 25 except that the amount of metal ions used was changed to R = 20.

Example 30 A gel bead-filled tube was obtained in the same manner as in Example 26 except that the amount of metal ions used was changed to R = 20.

Example 31 A gel bead-filled tube was obtained in the same manner as in Example 25 except that the metal ion used was changed to Cu ²⁺ .

Example 32 A gel bead-filled tube was obtained in the same manner as in Example 26 except that the metal ion used was changed to Cu ²⁺ .

Example 33 Photocrosslinkable polyvinyl alcohol (degree of polymerization: 1700, degree of saponification: 88 mol% and ratio of styrylpyridinium substituents: 1.3%) was added to physiological saline for injection to be 5% by weight. In addition, 121 ℃, 20 by autoclave
The photocrosslinkable polyvinyl alcohol was dissolved at the same time as the heat sterilization by treating with minutes to obtain a photocrosslinkable polyvinyl alcohol solution.

The obtained solution was added to liquid paraffin and stirred sufficiently to suspend the solution. After that, using a slide projector equipped with a 300 W halogen lamp, by irradiating light while stirring for 30 minutes,
Photo-crosslinkable polyvinyl alcohol gelled, particle size 2.5
mm gel beads were prepared. The gel beads were thoroughly washed with physiological saline for injection to remove the attached liquid paraffin. A gel bead-filled tube was obtained in the same manner as in Example 25 except for the above method for producing gel beads.

Example 34 The concentration of the photocrosslinkable polyvinyl alcohol used was 10% by weight.
A gel bead-filled tube was obtained in the same manner as in Example 33 except that the procedure was changed to.

Example 35 The concentration of the photocrosslinkable polyvinyl alcohol used was 15% by weight.
A gel bead-filled tube was obtained in the same manner as in Example 33 except that the procedure was changed to.

(Material for Inducing Production by Modified Polystyrene) Example 36 Diaion SA11A, which is a styrene-divinylbenzene copolymer having a trimethylammonium group.
(Mitsubishi Chemical Co., Ltd.) was placed in a 50 ml polypropylene test tube (Iwaki Glass Co., Ltd.) with a bulk volume of 3 ml. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was aspirated and removed. After this was repeated 3 times, 40 ml of methanol was added in the same manner, and the mixture was allowed to stand overnight at room temperature.

Next, the methanol was removed by suction, and the same washing with methanol was performed twice. To this, 40 ml of sterilized distilled water was added and gently stirred. The mixture was centrifuged at 500 rpm for 1 minute, the upper portion was removed by suction, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours.

Next, the same washing operation was performed three times, and finally distilled water was removed as much as possible. To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred.
Centrifuge at 500 rpm for 1 minute, aspirate the upper part,
Similarly, physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was washed three times, and finally 40 ml of physiological saline for injection was added, and the mixture was allowed to stand overnight at room temperature. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible. A polystyrene sample tube for 2 ml (manufactured by Eppendorf) filled with physiological saline for injection and sterilized was filled with 500 μl of the polystyrene particles in a bulk volume.

Example 37 The same procedure as in Example 36 was carried out except that the polystyrene particles used were changed to Diaion SA21A (manufactured by Mitsubishi Kasei Co.) having a dimethylethanol ammonium group to obtain a particle-filled tube.

Example 38 The polystyrene particles used contain an amino group and an imino group.
[-CH₂NH (CH ₂CH₂NH) n H (n = 1 to
3)] with Diaion WA21 (Mitsubishi Kasei
Manufactured in the same manner as in Example 36 except that
Then, a particle-filled tube was obtained.

Example 39 The polystyrene particles used contained a nitrilo group [-(C
H₂) N N (CH_Three) ₂Di with (n = 1 to 3)]
aion WA30 (manufactured by Mitsubishi Kasei Co., Ltd.)
Particle-filled tube, all carried out as in Example 36.
I got

(Material for Inducing Production by Polymethacrylic Acid Ester Derivative) Example 40 SEPABEADS, which is a polymethacrylic acid ester-based material modified with dimethylethanolamine
FP-QA13 (manufactured by Mitsubishi Kasei Co., Ltd.) was placed in a polypropylene test tube for 50 ml (manufactured by Iwaki Glass Co., Ltd.) in a bulk volume of 3 ml. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was aspirated and removed. After this was repeated 3 times, 40 ml of methanol was added in the same manner, and the mixture was allowed to stand overnight at room temperature.

Then, the methanol was removed by suction, and the same washing with methanol was carried out twice. To this, 40 ml of sterilized distilled water was added and gently stirred. 500 rpm
The mixture was centrifuged at 1 minute for 1 minute, the supernatant was aspirated and removed, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours.

Next, the same washing operation was performed three times, and finally distilled water was removed as much as possible. To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred.
Centrifuge at 500 rpm for 1 minute, remove the supernatant by aspiration,
Similarly, physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was washed three times, and finally 40 ml of physiological saline for injection was added, and the mixture was allowed to stand overnight at room temperature. After that, the same washing device was performed 5 times, and finally the physiological saline was removed as much as possible.

2 ml polypropylene sample tube (eppendorf) filled with physiological saline for injection and sterilized and washed
(Manufactured by the company) was filled with 500 μl of particles of the polymethacrylic acid ester material in a bulk volume.

Example 41 A particle-filled tube was obtained in the same manner as in Example 40 except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-DA13 having a diethylamino group (manufactured by Mitsubishi Kasei). It was

Example 42 The same procedure as in Example 40 was carried out except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-HA13 (manufactured by Mitsubishi Kasei) having an amino group at the terminal, and a particle-filled tube was used. Got

Example 43 The polymethacrylic acid ester-based material used was SEPABEA SFP-HG13 (manufactured by Mitsubishi Kasei) having a hydroxyl group.
Except that the procedure is the same as in Example 40, except that
A particle-filled tube was obtained.

Example 44 The polymethacrylic acid ester material used is SEPABEADS PH-1 having a hydrophobic group [-OC ₆ H ₅ ].
Example 40 except that the number was changed to 3 (manufactured by Mitsubishi Kasei)
The same procedure as in (1) was performed to obtain a particle-filled tube.

Comparative Example 1 A polypropylene sample tube for 2 ml (manufactured by Eppendorf) washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.).

Comparative Example 2 The same procedure as in Example 36 was carried out except that the polystyrene particles used were changed to techpolymer: SB-100S (manufactured by Sekisui Plastics Co., Ltd.) which was an unmodified polystyrene particle, and the particle filling was carried out. A tube was obtained.

Comparative Example 3 The polystyrene particles used were Diaion S, which is a styrene / divinylbenzene copolymer having a sulfonic acid group.
A particle-filled tube was obtained in the same manner as in Example 36 except that K1B (manufactured by Mitsubishi Kasei) was used.

Comparative Example 4 A particle-filled tube was obtained in the same manner as in Example 40 except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-CM13 (manufactured by Mitsubishi Kasei) having a carboxyl group. It was

Comparative Example 5 Polyethylene beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection to 2 m.
It was filled in a polypropylene sample tube for l (made by Eppendorf). The filling amount was 70 beads.

Comparative Example 6 Polystyrene beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection to 2 m.
It was filled in a polypropylene sample tube for l (made by Eppendorf). The filling amount was 70 beads.

Comparative Example 7 Polyvinyl chloride beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection 2
A polypropylene sample tube for ml (manufactured by Eppendorf) was filled. The filling amount was 70 beads.

Comparative Example 8 Polypropylene beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection 2
A polypropylene sample tube for ml (manufactured by Eppendorf) was filled. The filling amount was 70 beads.

Comparative Example 9 Polytetrafluoroethylene propylene copolymer beads (particle size 2.5 mm) were prepared by injection molding. Next, the beads were washed with methanol and dried. Next, after washing the beads with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.),
A polypropylene sample tube for 2 ml (manufactured by Eppendorf) which was similarly washed with physiological saline for injection was filled.
The filling amount was 70 beads.

IL-1, TNF and IL-6 production induction test Using the materials obtained in Examples 1-44 and Comparative Examples 1-9, an IL-1, TNF and IL-6 production induction test was conducted. went. The production induction test for IL-1, TNF and IL-6 and the measuring method thereof were performed as follows. (1) IL-1, TNF and IL-6 production induction test To each tube obtained in Examples 1 to 44 and Comparative Examples 1 to 9 was added 1.6 ml of fresh blood of a healthy person from which heparin was collected, and the tube was attached to a rotating disk. At 37 ° C for 2 hours, rotation speed 26r
Mix by tumbling at pm. Thereafter, the blood was collected and the concentrations of IL-1, TNF and IL-6 in plasma were measured by the following method.

(2) Plasma IL-1, TNF and IL-
6 Concentration measurement method IL-1, TNF, and blood after the IL-6 production induction test were centrifuged to collect plasma, and IL-1 and TNF in plasma were collected.
And the concentration of IL-6 were measured as follows. IL-
The concentration of 1 was determined by immunoenzymatic antibody method ((Medgenix IL-1β
EASIA). The detection limit concentration of this measuring method was 40 pg / ml. The concentration of TNF is TNF
Using a monoclonal antibody, the immunoenzymatic antibody method (R & D
It was measured with Quantikine TNF-α manufactured by System. The detection limit concentration of this measuring method was 25 pg / ml. The concentration of IL-6 was measured by an immunoenzymatic antibody method (manufactured by R & D System, trade name: Quantikine IL-6 ^R ) using an IL-6 monoclonal antibody. The detection limit concentration of this measuring method was 2.8 pg / ml.

Immediately after the blood collection, the blood was centrifuged to collect plasma, and the concentrations of IL-1, TNF and IL-6 in the plasma were measured in the same manner. Plasma I in blood immediately after blood collection
The L-1, TNF, and IL-6 concentrations were all below the detection limit. Tables 1 to 3 show the results of the IL-1, TNF, and IL-6 production induction tests of each Example and Comparative Example.

[0116]

[Table 1]

[0117]

[Table 2]

[0118]

[Table 3]

The following can be seen from the results of Examples 1-44 and Comparative Examples 1-9. IL-1 in plasma immediately after blood collection,
The concentrations of TNF and IL-6 were below the detection limit, and in a 2 ml tube washed with physiological saline for injection, which was only filled with blood (Comparative Example 1), IL-1 in plasma was
The concentrations of TNF and IL-6 were below the detection limit. From the results of Tables 1 to 3, the polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton or a polymer material having a cationic functional group in the molecule was found to be in contact with blood. Especially high IL-
It is clear that it induces the production of 1, TNF and IL-6. For hydrophobic polymer materials such as unmodified polystyrene, polyethylene and polyvinyl chloride and polymer materials having only anionic functional groups, IL-1, TNF and IL
It hardly induced the production of -6.

(Effect of Contact Temperature on TNF Production Induction) Example 45 Chitosan Gel Particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disc, and the rotation speed was 26 rpm at 37 ° C. for 2 hours. It fell and mixed.

Example 46 Chitosan gel particles Chitopear as in Example 1
1.6 ml of normal human fresh blood collected from heparin was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disk, and the rotation speed was 26 rpm at 15 ° C. for 2 hours. It fell and mixed.

Example 47 Chitosan gel particles Chitopear as in Example 1
1.6 ml of normal human fresh blood collected from heparin was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disc, and the rotation speed was 26 rpm at 30 ° C. for 2 hours. It fell and mixed.

Example 48 Chitosan gel particles Chitopear as in Example 1
l basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) was added to a polypropylene sample tube containing 1.6 ml of heparinized fresh blood of a healthy person and attached to a rotating disk, and the mixture was attached at 40 ° C. for 2 hours at a rotation speed of 26 rpm. It fell and mixed.

Example 49 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disc, and the rotation speed was 26 rpm at 42 ° C. for 2 hours. It fell and mixed.

Comparative Example 10 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disc, and the rotation speed was 26 rpm at 10 ° C. for 2 hours. It fell and mixed.

Comparative Example 11 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparinized fresh blood from a healthy person was added to a polypropylene sample tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.) and attached to a rotating disc, and the rotation speed was 26 rpm at 45 ° C. for 2 hours. It fell and mixed.

After mixing, each blood was centrifuged to collect plasma, and the plasma I was analyzed in the same manner as in the evaluation methods of Examples 1 to 44.
The concentrations of L-1, TNF and IL-6 were measured. Also,
The blood immediately after the blood collection was centrifuged to collect plasma, and the concentrations of IL-1 and TNF in the plasma were measured in the same manner. Immediately after blood collection, blood plasma levels of IL-1, TNF, and IL-6 were all below the detection limit. The results are shown in Table 4.

[0128]

[Table 4]

As is clear from the results in Table 4, when the contact temperature of blood with the above IL-1, TNF and IL-6 inducing materials is in the range of 15 to 42 ° C., IL-1, TNF and I
Induction of L-6 production was observed. Also, less than 15 ° C and 4
At temperatures above 2 ° C., IL-1, TNF and I
Almost no induction of L-6 production was observed.

(Measurement of TNF Production Inducing Ability in Drug-Administered Mouse Blood) Example 50 After acclimation for 1 week, ICR mice (Japan SLC,
Male, 9 weeks old, 0.4 mg / ml dexamethasone (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved or suspended in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) at 2 mg / kg and 0.4 mg / ml lentinan (Ajinomoto Co., Inc.) 2 mg / kg) was intraperitoneally administered for 3 days. As a control, physiological saline was administered.
One hour after the final administration, 1.5 ml of blood was collected from the mice of each administration group by a cardiac puncture needle (final heparin concentration 8 units / ml). Each blood sample was collected in groups. Next, Chitopearl B in which a primary amine was introduced into the amino group of chitosan through a linear alkyl group, which was washed in the same manner as in Example 1
A chitosan derivative of CW-3010 (trade name, manufactured by Fuji Spinning Co., Ltd.) was placed in each hole of a 24-well microplate (manufactured by Nunc Co.) at 0.1 g (wet weight) / hole. Next, 0.5 ml of mouse blood from each group was added to each hole of this plate, and the mixture was cultured with shaking at 37 ° C. for 2 hours.

Comparative Example 12 In the same manner as in Example 50, 0.5 mouse blood of each group was placed in an empty hole of a plate in which no Chitopearl BCW-3010 (trade name, manufactured by Fuji Spinning Co., Ltd.) was placed.
Each of the cells was added in an amount of ml and shake-cultured at 37 ° C. for 2 hours.

The blood after the reaction in Example 50 and Comparative Example 12 was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a monoclonal antibody by an immunoenzymatic antibody method (Genz.
ime company make, brand name Factor-Test-X M
Use TNF-α ELISA kit). The result is shown in FIG. As is clear from the results shown in FIG. 1, the dexamethasone-administered group had a predominantly decreased TNF-inducing ability and the lentinan-administered group had a predominantly increased TNF-inducing ability as compared with the physiological saline-administered group. That is, in the case of dexamethasone, which is an immunosuppressant originally, T
The ability to produce NF was suppressed, and lentinan, which is an immunopotentiator, had an enhanced ability to produce TNF.

[0133]

INDUSTRIAL APPLICABILITY IL-from the blood of the person being examined
By measuring the amount of 1, TNF or IL-6 production inducing amount, it becomes possible to detect a biological reaction related to immunity and inflammation of a person to be examined, prevent various diseases, and grasp the pathological condition in various diseases. , Helps to determine the dosage of drugs for treatment and treatment method.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measurement of TNF production-inducing ability in blood of mice administered with a drug.

Claims (4)

[Claims] 1. A polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton in the molecule or a polymer material having a cationic functional group is brought into contact with blood, Induces the production of interleukin-1 (IL-1),
A method for inspecting biological reaction, which comprises measuring the production amount of. 2. A polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton in the molecule or a polymer material having a cationic functional group is brought into contact with blood, TNF (T
NF: Tumor Necrosis Factor) production is induced, and the amount of TNF produced is measured, and the biological reaction test method is characterized. 3. A polymer material having at least one chemical structure selected from the group consisting of a hydroxyl group, an amide skeleton and an ester skeleton in the molecule or a polymer material having a cationic functional group is brought into contact with blood, The production of interleukin-6 (IL-6) is induced, and the IL-6
A method for inspecting biological reaction, which comprises measuring the production amount of. 4. The polymer material and blood are mixed at 15 ° C. to 42 ° C.
The biological reaction test method according to any one of claims 1 to 3, wherein the biological reaction test method is performed in the range of ° C.