JP4869020B2 - Neutrophil function test system and neutrophil function test method - Google Patents

Description

  The present invention relates to a system for examining cell functions such as migration ability and active oxygen production ability in neutrophils of animals and humans.

The neutrophil function test system of the present invention quantitatively detects an increase or decrease in neutrophil function, and is useful for diagnosis and prevention of diseases of animals and humans.

  It can also be used to screen the effects of various drugs on neutrophil function.

Animal and human leukocytes play an important role in the defense of living organisms. Among leukocytes, neutrophils are the most numerous. When foreign substances such as bacteria enter the body, the neutrophils present in the blood migrate to the outside of the blood vessels toward the foreign substances, releasing active oxygen and killing the bacteria. To do. Therefore, when the neutrophil migration ability and active oxygen production ability decrease, the risk of falling into a pathological condition such as an infection increases.

It is also known that certain antibacterial drugs inhibit or promote the ability of neutrophils to migrate or react with active oxygen (for example, Tadatoshi Kuramata, Journal of Infectious Diseases, 52, 358- 363 (1978); Non-patent document 1, Hirohiko Akamatsu, Inflammation, 14, 59-60 (1994); see Non-patent document 2).

More recently, it has been pointed out that active oxygen produced by neutrophils that have migrated to abnormal sites in arteries is a factor in arteriosclerosis.

Although it is known that the evaluation of neutrophil function is important in this way, in general, only the neutrophil count is examined, and the neutrophil migration ability and active oxygen production ability are examined. Is not done. This is because neutrophil migration ability and active oxygen production ability cannot be easily evaluated.

As a device for evaluating cell migration ability, Boyden's membrane is used to measure the number of cells that pass through the membrane and move to the chemotactic factor by installing a microporous membrane between the cell suspension and the chemotactic factor solution. There are a chamber system, an array system using a large number of microchannels formed on a substrate instead of a microporous film. However, these methods require very laborious work, and are generally limited.

On the other hand, neutrophils can be tested for the ability to produce active oxygen using a flow cytometer, but the apparatus is large and the cost of the test must be high. In order to measure the ability of neutrophils to produce active oxygen without using a special machine such as a flow cytometer, it is necessary to separate only neutrophils from the blood, which is troublesome such as density gradient centrifugation. Needed work. Furthermore, during the operation of separating neutrophils from this whole blood, the activity of neutrophils decreased, making it difficult to measure the ability of normal neutrophils to produce active oxygen.
Kuratoshi Tadatoshi, Journal of Infectious Diseases, 52, 358-363 (1978) Akamatsu Hirohiko, Inflammation, 14, 59-60 (1994)

An object of the present invention is to provide a neutrophil function testing system that solves the problems of the conventional neutrophil function testing system described above. That is, to provide a neutrophil function test system that does not require separation of neutrophils from whole blood in advance and can simultaneously test neutrophil migration ability and active oxygen production ability with a simple operation. It is in.

As a result of intensive studies, the present inventors have used a hydrogel in which neutrophils are selectively adsorbed or invaded; the neutrophil that has invaded into the hydrogel is converted into the activity of the neutrophil in the hydrogel. The present inventors have found that detection by oxygen production ability is effective in solving the above problems, and have completed the present invention.

  The present inventors have found that when whole blood is brought into contact with a specific hydrogel, only neutrophils selectively enter the hydrogel and produce active oxygen. At this time, it was found that when a reagent that emits light or absorbs light in response to active oxygen coexists, the amount of light emitted or absorbed from the hydrogel corresponds to the ability to produce active oxygen of neutrophils, thereby completing the present invention.

According to the present invention, the ability of neutrophils to migrate in a physiological environment in which plasma components including complement and the like coexist without using a specific hydrogel to separate neutrophils from whole blood in advance. Can be evaluated.

Furthermore, according to the present invention, active oxygen produced by neutrophils migrated in the hydrogel is quantified, so that the ability of neutrophils to produce active oxygen can be evaluated simultaneously.

Furthermore, according to the present invention, the ability to produce active oxygen can be measured by measuring the amount of chemiluminescence, and the time-dependent change in the ability to produce active oxygen of neutrophils can be observed by continuously measuring the amount of luminescence. .

With the neutrophil function test system of the present invention, it becomes possible to simultaneously and easily test the neutrophil migration ability and active oxygen production ability under physiological conditions, which has been difficult to measure conventionally. Therefore, it is extremely useful for diagnosis, prevention, development of therapeutic agents, etc. for various pathological conditions related to neutrophil function.

(Detection of active oxygen by chemiluminescence)
Neutrophils produce various reactive oxygen species such as superoxide (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), and hypochlorous acid (HOCl / OCl ⁻ ). Therefore, by allowing a reagent that chemically reacts with these active oxygen molecular species to emit light, the ability of neutrophils to produce active oxygen can be quantified by the amount of light emitted.

Examples of the reagent that emits light by chemically reacting with the active oxygen molecular species used in the present invention include luminol and lucigenin.
Luminescent reagents such as (lucigenin), hydroxyphenylfluorescein
(hydroxyphenylfluorescein) and aminophenylfluorescein
A fluorescent reagent such as (aminophenylfluorescein) can be exemplified.

  In particular, neutrophils are cells that contain an enzyme called myeloperoxidase (MPO) in cytoplasmic granules and metabolize reactive oxygen species different from other leukocytes. That is, this MPO is an enzyme that exists specifically in neutrophils, and produces hypochlorous acid using hydrogen peroxide and chloride ions as substrates.

  Here, luminol, which is a chemiluminescent sensitizer, mainly detects hypochlorous acid produced via this MPO. (A) Since MPO exists only in neutrophils, it is possible to monitor a reaction specific to neutrophils. (A) If neutrophils are activated and MPO is not degranulated, the reaction does not occur. ) Because it detects powerful reactive oxygen species (hypochlorous acid) directly related to sterilization and tissue injury, it is useful for pathological significance. (D) Luminescence has low background noise, and it is useful for other chemiluminescent substances. Compared to other chemiluminescent materials, it has superior detection power (excellent S / N ratio) and (e) can be stored stably as a reagent.

However, when whole blood is used as a test sample, chemiluminescence is attenuated by the quenching action of the coexisting red blood cells with hemoglobin dye, and antioxidants contained in plasma erase reactive oxygen species. There was a problem in detection. In the neutrophil function test system of the present invention, a hydrogel that selectively invades neutrophils is used, and only the neutrophils that have invaded into the hydrogel are suppressed while suppressing the influence of the above measurement inhibitory factors. It became possible to detect by oxygen production. On the other hand, neutrophils that have not entered the hydrogel are inhibited from emitting light by the overwhelming number of red blood cells that surround them even if reactive oxygen species are produced. The reaction of only the neutrophil will be detected.

  As the medium (cell culture medium) for swelling the hydrogel, Hanks' Balanced Salt Solution (HBSS) often used in leukocyte-related experiments, RPMI, DMEM and the like used for cell culture can be used. From the viewpoint of increasing the detection sensitivity of luminescence, those not containing a pigment such as phenol red are preferably used.

The luminescent reagent may be previously contained in the hydrogel of the present invention described later, or may be mixed with whole blood and coexisted in the solution on the hydrogel. Regarding luminol, the condition was set so that it was first mixed with whole blood and allowed to migrate into the hydrogel, but the detection power of luminol-dependent chemiluminescence of neutrophils was better than that previously diffused into the gel. This is considered to be because the neutrophils are exposed to luminol from the beginning, thereby increasing the detection power.

  Thus, by using the neutrophil function test system of the present invention, it is possible to easily evaluate neutrophil migration ability and active oxygen production ability simultaneously in a short time using whole blood. Compared with this measurement method, it is a measurement method that reflects the kinetics of neutrophil activity close to the living body, and is considered to be a useful novel inflammatory marker.

For detection of active oxygen produced by neutrophils by chemiluminescence, the following documents can be referred to.
Hasegawa, H., Suzuki, K., Nakaji, S., and Sugawara, K: Analysis and assessment of the capacity ofneutrophils to produce reactive oxygen species in a 96-well microplate formatusing lucigenin- and luminol-dependent chemiluminescence. Journal of Immunological Methods 210: 1-10, 1997.

(Detection of active oxygen by colorimetry)
Detection of active oxygen by chemiluminescence requires a measuring instrument such as a luminometer, and has a problem in that it cannot be easily spread to general medical institutions because of the measurement cost. The application of the color reaction with nitroblue tetrazolium (NBT), which is a redox reagent, has the advantage that neutrophil function can be easily monitored without a special measuring device.

Conventionally, this color reaction was used as a histochemical NBT reduction method for microscopic examination of neutrophils' ability to produce active oxygen, and diagnosed chronic granulomatosis lacking ability to produce active oxygen and enhanced ability to produce active oxygen. A micro blood test method using hematocrit capillaries has been devised so that it can be used to monitor inflammation such as severe infections and can be tested even in children who can only collect micro blood. However, the procedure is complicated, and it takes time to observe the cells with a microscope. Since the judgment is based on the subjectivity of the examiner, there is a problem in quantitativeness, and it has not been used as a clinical test.

  In the neutrophil function test system of the present invention, when a neutrophil infiltrated into a transparent gel generates active oxygen by using a hydrogel that selectively infiltrates neutrophils, NBT is reduced and blackened. It is possible to make semi-quantification because it is possible to visually distinguish and visually.

NBT may also be contained in advance in the hydrogel of the present invention described later, or may be mixed with whole blood and coexisted in a solution on the hydrogel.

Since NBT has an antithrombin action and can prevent blood clotting, if it is first mixed with whole blood and allowed to migrate into the gel, whole blood can be tested without using an anticoagulant such as heparin. There is an advantage that it can be used.

As described above, by using the neutrophil function testing system of the present invention, it is possible to easily evaluate the neutrophil migration ability and active oxygen production ability simultaneously in a short time using whole blood as it is.

In the future, in order to proceed with G-CSF therapy etc. at home, it is necessary to evaluate the neutrophil function and determine the right or wrong of drug administration without simply using a special device with a trace amount of blood. It is considered that the present invention using as a detection system is a useful test index.

The following literature can be referred to for colorimetric detection of active oxygen produced by neutrophils.
Suzuki Katsuhiko et al., Analysis and evaluation method of phagocyte active oxygen production ability by NBT reduction test and its applicability, Journal of Physical Fitness and Nutrition Immunology, 3, 31-39, 1993.

(Hydrogel)
The hydrogel used in the present invention can be used without particular limitation as long as it maintains a gel state at 37 ° C. and neutrophils selectively adsorb or invade. In order to evaluate the migration ability of neutrophils, it is preferable that the neutrophils can enter into the hydrogel, not simply adsorption. When blood cell components other than neutrophils enter the hydrogel, it becomes difficult to detect active oxygen produced by neutrophils. In addition, in a hydrogel in which blood cell components other than neutrophils can invade, neutrophils can invade into the hydrogel regardless of the migratory ability, and thus the neutrophil migratory ability cannot be evaluated.

A suitable hydrogel in the present invention can be screened by the following method.

Materials and equipment:
2mL cryotube (AXY, product code: MCT-200-LC)
Hank's balanced salt solution (HBSS, manufactured by Invitrogen, catalog No. 1425-076)
Luminol reagent: Luminol (manufactured by Sigma, catalog No.
A-8511) 17.72 mg is dissolved in 10 mL of 1N NaOH, pH is adjusted to 8.5 with 1N hydrochloric acid, 0.34 g of NaCl is added, and the total volume is made up to 40 mL with distilled water. This stock solution is diluted 5 times with HBSS, and the pH is adjusted to 7.4 with 0.2 N NaOH or hydrochloric acid (luminol concentration 0.5 mM).
Heparin-added peripheral blood luminometer; (Genlite GL55, manufactured by Microtech Nichion)

Method:
On the bottom of a 2 mL cryotube, 0.05 mL of hydrogel using HBSS as a solvent is formed without gaps. Luminol reagent (0.075 mL) and heparin-added peripheral blood (0.075 mL) are mixed and placed on the hydrogel. Let stand at 37 ° C. for 60 minutes, put the cryotube in a luminometer, and measure the amount of luminescence.

The hydrogel used in the present invention has a light emission amount (RLU) obtained by the above operation of 50 or more, preferably 100 or more, more preferably 500 or more. If the amount of luminescence is less than 50, neutrophils do not migrate and enter the hydrogel, or other blood cell components have entered the hydrogel at the same time. Since it is considered that chemiluminescence cannot be detected, it is not preferable as the hydrogel used in the present invention.

As a material for the hydrogel, a natural polymer material or a synthetic polymer material can be appropriately selected and used.

Natural polymer materials include proteins such as collagen and gelatin, acidic polysaccharides such as hyaluronic acid and alginic acid and their salts, neutral polysaccharides such as agar, agarose and cellulose, and derivatives thereof (low melting point agarose, methylcellulose, etc.) And basic polysaccharides such as chitin and chitosan.

Synthetic polymer materials include polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly N-methyl acrylamide, polyhydroxymethyl acrylate, polyacrylic acid, Polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and salts thereof, poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate, poly N, N-dimethylaminopropylacrylamide And salts thereof.

As the hydrogel used in the present invention, it exhibits a thermoreversible sol-gel transition phenomenon in which it is gelled at a low temperature and in a high temperature, and the gel is substantially insoluble at a temperature higher than the sol-gel transition temperature. The thermoreversible hydrogel which is can be preferably used. The thermoreversible hydrogel-forming polymer usable in the present invention is not particularly limited as long as it exhibits a thermoreversible sol-gel transition as described above (that is, has a sol-gel transition temperature).

Specific examples of the polymer in which the aqueous solution has a sol-gel transition temperature and reversibly shows a sol state at a temperature lower than the transition temperature include, for example, a block copolymer of polypropylene oxide and polyethylene oxide. Known polyalkylene oxide block copolymers; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (KRHolme. Et al. Macromolecules, 24, 3828 (1991)) and the like are known.

As a polyalkylene oxide block copolymer, Pluronic F-127 (trade name, BASF Wyandotte) in which polyethylene oxide is bonded to both ends of polypropylene oxide.
(Chemicals Co.) gel has been developed. It is known that this high-concentration aqueous solution of Pluronic F-127 becomes a hydrogel at about 20 ° C. or higher and becomes an aqueous solution at a lower temperature. However, in the case of this material, it becomes a gel state only at a high concentration of about 20% by mass or more, and even if it is kept at a temperature higher than about 20% by mass and higher than the gelation temperature, water is further added. And the gel will dissolve. Pluronic F-127 has a relatively small molecular weight and not only exhibits a very high osmotic pressure in a gel state of high concentration of about 20% by mass or more, but also easily permeates the cell membrane, thus adversely affecting neutrophils. there is a possibility.

  On the other hand, according to the study by the present inventors, a thermoreversible hydrogel-forming polymer having a sol-gel transition temperature that is preferably higher than 0 ° C. and lower than or equal to 42 ° C. (for example, a plurality of clouds having a cloud point) A polymer having a sol-gel transition temperature and a reversible sol state at a temperature lower than the sol-gel transition temperature). It has been found that the above problem can be solved when a ball function testing system is configured.

(Suitable hydrogel-forming polymer)
The thermoreversible hydrogel-forming polymer utilizing a hydrophobic bond that can be suitably used in the present invention is preferably formed by bonding a plurality of blocks having a cloud point and a hydrophilic block. The hydrophilic block is preferably present because the hydrogel becomes water-soluble at a temperature lower than the sol-gel transition temperature, and the plurality of blocks having a cloud point have a hydrogel having a sol-gel transition temperature. It is preferably present to change to a gel state at higher temperatures. In other words, a block having a cloud point dissolves in water at a temperature lower than the cloud point and changes to insoluble in water at a temperature higher than the cloud point, so that at a temperature higher than the cloud point, the block has a gel. It serves as a cross-linking point consisting of a hydrophobic bond to form.

  That is, the cloud point derived from the hydrophobic bond corresponds to the sol-gel transition temperature of the hydrogel. However, the cloud point and the sol-gel transition temperature do not necessarily match. This is because the cloud point of the “block having a cloud point” described above is generally affected by the bond between the block and the hydrophilic block.

The thermoreversible hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger as the temperature increases, but the change is reversible with respect to temperature. From the point that a plurality of crosslinking points are formed in one molecule and a gel having excellent stability is formed, the thermoreversible hydrogel-forming polymer may have a plurality of “blocks having cloud points”. preferable. On the other hand, the hydrophilic block in the hydrogel-forming polymer has the function of changing the hydrogel-forming polymer to water-soluble at a temperature lower than the sol-gel transition temperature, as described above. The hydrogel has a function of forming a hydrogel state while preventing the hydrogel from aggregating and precipitating at a temperature higher than the transition temperature.

From the point that it is easy to show a suitable sol-gel change at a physiological temperature (about 0 to 42 ° C.), for example, a plurality of blocks having cloud points in the thermoreversible hydrogel-forming polymer and hydrophilicity It is preferable to achieve this by adjusting the cloud point, the composition of both blocks and the hydrophobicity, hydrophilicity, and / or molecular weight of both blocks.

(Multiple blocks with cloud points)
The block having a cloud point is preferably a polymer block having a negative solubility-temperature coefficient in water, and more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, A polymer selected from the group consisting of poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. Can be preferably used. The above-mentioned polymer (block having a cloud point) has a cloud point higher than 4 ° C. and not higher than 40 ° C., and the thermoreversible hydrogel-forming polymer used in the present invention (multiple blocks having a cloud point and hydrophilicity) The sol-gel transition temperature of the compound to which the block is bonded is preferably from 4 ° C. to 40 ° C. Here, the cloud point is measured by, for example, cooling an aqueous solution of about 1% by mass of the above polymer (block having a cloud point) to form a transparent uniform solution, and then gradually increasing the temperature (temperature increase rate of about 1). C./min), and the point at which the solution becomes cloudy for the first time is taken as the cloud point.

  Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below. Poly-N-acroylpiperidine; poly-Nn-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.

The polymer may be a homopolymer or a copolymer of a monomer constituting the polymer and another monomer. As another monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. In general, copolymerization with a hydrophilic monomer raises the cloud point of the product and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Therefore, a polymer having a desired cloud point (for example, a cloud point higher than 4 ° C. and lower than 40 ° C.) can be obtained also by selecting the monomer to be copolymerized.

(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acrylic having an acid group. Acid, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid and the like, and N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl having a basic group Examples include, but are not limited to, acrylamide and salts thereof.

(Hydrophobic monomer)
On the other hand, as the hydrophobic monomer, acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, butyl methacrylate and glycidyl methacrylate, N-substituted alkylmethacrylamide derivatives such as Nn-butylmethacrylamide, vinyl chloride, Examples include, but are not limited to, acrylonitrile, styrene, vinyl acetate, and the like.

(Hydrophilic block)
On the other hand, as the hydrophilic block to be combined with the above-described block having a cloud point, specifically, methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide , Poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and their salts; poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate , Poly N, N-dimethylaminopropylacrylamide and salts thereof.

The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited. For example, a polymerizable functional group (for example, acryloyl group) is introduced into one of the blocks to give the other block. This can be done by copolymerizing monomers. Further, the combined product of a block having a cloud point and the hydrophilic block may be obtained by block copolymerization of a monomer that gives a block having a cloud point and a monomer that gives a hydrophilic block. Is possible.

In addition, the block having a cloud point and the hydrophilic block are bonded to each other by introducing a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) in advance and bonding them together by a chemical reaction. Can also be done. At this time, usually a plurality of reactive functional groups are introduced into the hydrophilic block.

In addition, the bond between the polypropylene oxide having a cloud point and the hydrophilic block is repeated, for example, by anionic polymerization or cationic polymerization by repeatedly repeating propylene oxide and a monomer (for example, ethylene oxide) constituting “another hydrophilic block”. By polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bonded can be obtained. Such a block copolymer can also be obtained by copolymerizing monomers constituting a hydrophilic block after introducing a polymerizable group (for example, acryloyl group) to the terminal of polypropylene oxide.

Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of binding reaction with a functional group (for example, hydroxyl group) at the end of polypropylene oxide into the hydrophilic block and reacting both. .

Moreover, the hydrogel-forming polymer used in the present invention can also be obtained by linking materials such as Pluronic F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.) in which polyethylene glycol is bonded to both ends of polypropylene glycol. Can be obtained.

The polymer of the present invention in a mode including a block having a cloud point is water-soluble together with the hydrophilic block at the temperature lower than the cloud point, because the above-mentioned “block having a cloud point” is present in the molecule. It is completely dissolved in water and shows a sol state. However, when the temperature of the polymer aqueous solution is raised to a temperature higher than the above cloud point, the “block having a cloud point” present in the molecule becomes hydrophobic, and is associated between separate molecules by hydrophobic interaction. To do. On the other hand, since the hydrophilic block is still water-soluble at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention is a hydrophobic association part between blocks having a cloud point in water. A hydrogel having a three-dimensional network structure with a cross-linking point as a cross-linking point is generated.

When the temperature of this hydrogel is cooled again to a temperature lower than the cloud point of the “block having cloud point” existing in the molecule, the block having the cloud point becomes water-soluble and the crosslinking point due to hydrophobic association is released. The hydrogel structure disappears and the polymer of the present invention becomes a complete aqueous solution again. Thus, the sol-gel transition of the polymer of the present invention in a preferred embodiment is based on reversible changes in hydrophilicity and hydrophobicity at the cloud point of the block having the cloud point present in the molecule. Therefore, it has complete reversibility in response to temperature changes.

(Gel solubility)
As described above, the hydrogel-forming polymer of the present invention comprising at least a polymer having a sol-gel transition temperature in an aqueous solution is substantially insoluble in water at a temperature (d ° C.) higher than the sol-gel transition temperature. And is reversibly water soluble at a temperature (e ° C.) lower than the sol-gel transition temperature. The high temperature (d ° C.) is preferably a temperature that is 1 ° C. or more higher than the sol-gel transition temperature, and more preferably 2 ° C. or more (particularly 5 ° C. or more).

The “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (d ° C.) is 5.0 g or less (more preferably 0.5 g or less, particularly 0.1 g or less). ) Is preferable. On the other hand, the above-mentioned low temperature (e ° C.) is preferably 1 ° C. or more lower than the sol-gel transition temperature (in absolute value), more preferably 2 ° C. or higher (particularly 5 ° C. or higher). preferable. The “water-soluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (e ° C.) is preferably 0.5 g or more (more preferably 1.0 g or more).

Further, “reversibly water-soluble” means that the aqueous solution of the thermoreversible hydrogel-forming polymer is once gelled (at a temperature higher than the sol-gel transition temperature). At a temperature lower than the transition temperature, it means to exhibit the above-mentioned water solubility.

The “thermo-reversible hydrogel-forming polymer” usable in the present invention becomes a hydrogel at a temperature higher than a specific temperature in a temperature range of 0 ° C. or higher and 42 ° C. or lower when the aqueous solution or aqueous dispersion is used. In addition, it has a characteristic of becoming sol or liquid at a temperature lower than the above temperature.

(Sol-gel transition temperature)
In the present invention, the measurement of the sol-gel transition temperature of a sample is carried out using literature (H. Yoshioka et al., Journal of
Macromolecular Science, A31 (1), 113 (1994)).

That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured by gradually changing the temperature (from the low temperature side to the high temperature side or from the high temperature side to the low temperature side by 1 ° C./1 minute), and the storage elastic modulus of the sample is measured. The temperature at the point where (G ′, elastic term) and the loss elastic modulus (G ″, viscosity term) intersect is defined as the sol-gel transition temperature. Generally, the state of G ″> G ′ is sol, G ″ <G The state of 'is defined as a gel. If the sol-gel transition temperature differs between the temperature rise and the temperature fall (for example, 2 ° C or more in absolute value), the intermediate temperature is taken as the sol-gel transition temperature. In measuring the sol-gel transition temperature, the following measurement conditions can be preferably used.

<Measuring conditions of dynamic elastic modulus>
Measuring instrument: Product name = Stress control type rheometer AR500, manufactured by TA Instruments, concentration of sample solution (or dispersion) (however, as concentration of “thermo-reversible hydrogel-forming composition”): 10 (weight) %, Amount of sample solution: about 0.8
g Shape and dimensions of measurement cell: acrylic parallel disk (diameter 4.0 cm), gap 600 μm. Applied stress: in the linear region. Observation frequency: 1 Hz.

The hydrogel preferably used in the present invention has a sol-gel transition temperature obtained by the above dynamic viscoelasticity measurement in a range of 0 ° C. or higher and 42 ° C. or lower.

The hydrogel preferably used in the present invention has a storage elastic modulus (G ′) at 37 ° C. obtained by the dynamic viscoelasticity measurement of 10 Pa to 1000 Pa, more preferably 50 Pa to 500 Pa, and still more preferably 100 Pa. The range is 200 Pa or less. If G 'falls below this range, the strength of the hydrogel is low at the temperature at which neutrophils migrate (37 ° C), and cells other than neutrophils are liable to enter the hydrogel, which is not preferable. On the other hand, if it exceeds this range, the strength of the hydrogel is too high, and it becomes difficult for neutrophils to enter the hydrogel, which is not preferable.

In order to adjust the strength (G ′) of the hydrogel to the above preferable range, the concentration of the hydrogel-forming polymer is adjusted. The preferred concentration range of the hydrogel-forming polymer varies depending on the type of the hydrogel-forming polymer, but is usually 0.2% by mass or more and 20% by mass or less, preferably 1% by mass or more and 12% by mass or less, more preferably 5% by mass. % Or more and 10% by mass or less.

(Cell adhesion factor)
Furthermore, the present inventors have found that inclusion of a cell adhesion factor in the hydrogel of the present invention has an effect of promoting migration of neutrophils in the gel. As this cell adhesion factor, extracellular matrix molecules such as collagen, fibronectin, laminin, vitronectin, thrombospondin, tenascin, and osteonectin can be used.

  It is also useful to use gelatin as a cell adhesion factor, but an aqueous gelatin solution has a property of gelling at a low temperature. When a thermoreversible hydrogel that gels at a high temperature is used as the hydrogel of the present invention, it is thermoreversible. There are cases where an aqueous solution or aqueous dispersion of a hydrogel-forming polymer is prevented from becoming a fluid aqueous solution at a low temperature. In this case, it is preferable to use a low molecular weight collagen peptide or water-soluble gelatin obtained by degrading collagen or gelatin with an enzyme or the like. When the molecular weight of collagen or gelatin is large, the tendency to gel at low temperatures becomes strong. Therefore, the molecular weight of collagen or gelatin used in the present invention is 30,000 or less, preferably 10,000 or less, more preferably 5,000 or less.

A synthetic peptide can also be used as a cell adhesion factor used in the present invention. That is, an amino acid sequence synthesized in an extracellular matrix molecule such as collagen, fibronectin, laminin, vitronectin, thrombospondin, tenascin, or osteonectin as a basic structure related to cell adhesion is synthesized and reconstructed.

Specifically, RGD (Arg-Gly-Asp, arginine-glycine-aspartic acid) and RGDS (Arg-Gly-Asp-Ser, arginine-glycine-aspartic acid-serine), YIGSR, PDGSR, RYVVLPR, RNAEIIKDA, REDV, LRGDN, LRE, IKVAV, EILDV, IDAPS, etc. are known.

As a method of incorporating the cell adhesion factor into the hydrogel, the cell adhesion factor can be mixed and gelled as it is in an aqueous polymer solution that forms the hydrogel. It is also possible to directly bind the cell adhesion factor to the polymer that forms the hydrogel.

When the cell adhesion factor is water-soluble, it may not function as a cell adhesion factor just by mixing in a hydrogel. Therefore, as another method, the cell adhesion factor can be bound to a polymer compound having a cloud point, and the cell adhesion factor can be insolubilized and phase-separated at a temperature at which neutrophils migrate. The polymer compound having a cloud point used here may be the same as the block having a cloud point in the thermoreversible hydrogel-forming polymer described above.

  For the binding of a polymer compound having a cloud point and a cell adhesion factor, a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) is previously introduced into the two, and the two are bound by a chemical reaction. Can be done by. Alternatively, a polymer compound having a cloud point can also be obtained by introducing a polymerizable functional group into a cell adhesion factor and copolymerizing the polymerizable cell adhesion factor and a monomer that gives a polymer compound having a cloud point. And cell adhesion factor can be bound.

(Neutrophil chemotactic factor)
Furthermore, the present inventors have found that inclusion of a neutrophil chemotactic factor in the hydrogel of the present invention has an effect of promoting migration of neutrophils in the gel. Examples of neutrophil chemotactic factors include complement-derived factors C3a, C5a, N-formyl-Met-Leu-Phe (fMLP), Interleukin 8, and the like. As a method for containing the neutrophil chemotactic factor in the hydrogel of the present invention, the above cell adhesion factor can be mixed and gelled as it is in an aqueous polymer solution that forms the hydrogel. It is also possible to directly bind the cell adhesion factor to the polymer that forms the hydrogel.

(Mode of causing neutrophils to migrate)
In the neutrophil function testing system of the present invention, the container containing the hydrogel of the present invention is preferably a transparent container that can directly detect neutrophils by luminescence, fluorescence, and colorimetry. If the detection of neutrophils by luminescence, fluorescence, and colorimetry is not hindered, the container need not be completely transparent, and may be a clouded container. Examples of containers that are preferably used include plastic containers such as cryotubes and multiwell plates and glass containers such as test tubes.

In the neutrophil function testing system of the present invention, the neutrophils migrate as described above. The hydrogel of the present invention is spread on the bottom surface of the container, and whole blood is placed on the hydrogel. The thickness of the hydrogel is 100 μm to 5 mm, preferably 300 μm to 3 mm, more preferably 500 μm to 2 mm. The temperature at which neutrophils migrate is in the range of 20 to 40 ° C, preferably 35 to 38 ° C. As described above, the detection reagent may be dissolved in the hydrogel, may coexist in the whole blood, or may coexist in both the gel and the whole blood.

When whole blood or a mixture of whole blood and detection reagent is placed on the hydrogel and allowed to stand, blood cell components in the whole blood settle on the top surface of the hydrogel, and only neutrophils with migratory activity migrate into the hydrogel. invade. When neutrophils that have entered the hydrogel produce active oxygen, a reactive oxygen detection reagent (such as a luminescent reagent, a fluorescent reagent, or a color reagent) reacts to develop or develop color.

When detecting luminescence or fluorescence, an embodiment in which light is captured from the bottom surface of the hydrogel is preferably used. That is, it is preferable to place an optical sensor such as a photomultiplier tube or a photodiode at the bottom of a container containing a hydrogel such as a cryotube or a multiwell plate, and the light receiving surface is parallel at a position close to the bottom of the hydrogel (see FIG. 1).

The time for allowing the neutrophils to migrate is suitably in the range of 10 minutes to 90 minutes. Usually, the longer the migration time, the greater the number of neutrophils that migrate in the hydrogel. However, if the migration time becomes too long, the activity of neutrophils decreases and the amount of active oxygen produced tends to decrease. According to the present invention, this process can be observed over time by continuously measuring luminescence and coloration correlated with the amount of active oxygen produced.

(Measurement of the number of migrating cells)
In the neutrophil function testing system of the present invention, when the thermoreversible hydrogel is used as a hydrogel on which neutrophils are selectively adsorbed or invaded, the neutrophil is at a temperature equal to or higher than its sol-gel transition temperature. After migration into the gel, the components outside the gel (red blood cells, platelets, leukocytes that did not migrate, etc.) are washed away at a temperature above the sol-gel transition temperature, and the remaining gel is below the sol-gel transition temperature. By dissolving at temperature, neutrophils that have migrated into the gel can be recovered.

By measuring the number of neutrophils collected as described above, it is possible to know the number of neutrophils that have migrated into the gel, so it is possible to independently evaluate the ability of neutrophils to produce active oxygen and the ability to migrate. is there. Here, the neutrophil count can be measured in the same manner as the white blood cell count or neutrophil count in whole blood using an automatic blood cell counter or the like. Alternatively, cell activity correlated with the neutrophil count can be used as an index of the neutrophil count.

As a method for measuring cell activity correlated with the neutrophil count, various conventionally known methods can be appropriately employed. For example, absorbance measurement typified by MTT assay, fluorescence intensity measurement method using Alamar Blue, and emission intensity measurement method based on ATP activity can be used. In particular, the ATP measurement method using the luciferin / luciferase reaction measures luminescence intensity in the same manner as the active oxygen measurement by the luminol reaction, and is advantageous in terms of ease of operation, sensitivity, and wide measurement dynamic range. Yes, it is preferably used as an index of neutrophil count.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

Example 1 (Synthesis of thermoreversible hydrogel-forming polymer)
602 g of N-isopropylacrylamide (Kojin Co., Ltd.) and 37.1 g of n-butyl methacrylate (Wako Pure Chemical Industries, Ltd.) were dissolved in 9470 g of ethanol, and polyethylene glycol dimethacrylate (PDE-6000, Nippon Oil & Fats ( Ltd.) An aqueous solution prepared by dissolving 192.9 g in 1093 g of distilled water and 5466 g of distilled water were added, and 68 mL of a 10% by mass ammonium persulfate (APS) aqueous solution and N, N, N ′, N′-tetramethylethylenediamine were added at 70 ° C. under a nitrogen stream. (TEMED) 6.8mL was added and it was made to react for 30 minutes, hold | maintaining at 70 degreeC under nitrogen stream. The operation of adding 68 mL of the APS aqueous solution and 6.8 mL of TEMED while being maintained at 70 ° C. under a nitrogen stream was repeated 4 times every 30 times.

The reaction solution was diluted with 30 L of distilled water at 4 ° C., and the aqueous solution was concentrated to 10 L at 4 ° C. using an ultrafiltration membrane (Biomax 100, P2B100V20, manufactured by Millipore) having a fractional molecular weight of 100,000. Diluted water (35 L) was added to the concentrate to dilute, and ultrafiltration was concentrated again to 10 L at 4 ° C. This dilution and concentration operation was further repeated 8 times (9 times in total) to remove unreacted substances and low molecular weight substances.

The final concentrated liquid obtained was lyophilized to obtain 496 g of a thermoreversible hydrogel-forming polymer. Distilled water was added to 1.0 g of the obtained hydrogel-forming polymer to make 10 g as a whole, and dissolved under cooling (4 ° C.) to obtain a uniform aqueous solution having a concentration of 10% by mass. When this aqueous solution was heated, it became a hydrogel at body temperature, and the sol-gel transition temperature was determined by measuring dynamic viscoelasticity. The aqueous solution was always liquid at a temperature of 0 ° C. or higher and lower than 15 ° C., and did not gel.

Example 2 (Binding of high molecular compound having cloud point and cell adhesion factor)
24 g of acid-treated pig skin-derived collagen enzyme degradation product (collagen peptide SCP-5100, molecular weight 5,000, manufactured by Nitta Gelatin Co., Ltd.) was dissolved in 96 g of distilled water at 37 ° C., and N-acryloylsuccinimide (Kokusan Chemical Co., Ltd.) 3.26 g was added and reacted at 37 ° C. for 4 days to obtain an aqueous solution of a polymerizable collagen peptide obtained by introducing a polymerizable group into the collagen peptide.

108.5 g of N-isopropylacrylamide (Kojin Co., Ltd.) and 4.26 g of n-butyl methacrylate (Wako Pure Chemical Industries, Ltd.) are dissolved in 600 g of ethanol, 123.3 g of an aqueous solution of the above-described polymerizable collagen peptide and distilled water. Add 400 g, add 10 mL of 10 mass% ammonium persulfate (APS) aqueous solution and 1 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) at 37 ° C. under a nitrogen stream, and keep at 37 ° C. under a nitrogen stream The reaction was allowed for 5 hours.

The reaction solution was diluted with 30 L of distilled water at 4 ° C., and the aqueous solution was concentrated to 3 L at 4 ° C. using an ultrafiltration membrane (Biomax 100, P2B100V20, manufactured by Millipore) having a molecular weight cut off of 100,000. Diluted water (30 L) was added to the concentrate to dilute, and ultrafiltration concentration was again performed at 4 ° C to 3 L. This dilution and concentration operation was further repeated 4 times (5 times in total) to remove unreacted substances and low molecular weight substances.

The obtained final concentrated solution was freeze-dried to obtain 105 g of a polymer compound having a cloud point to which a collagen peptide was bound. 1 g of the obtained polymer was dissolved in 99 g of physiological saline at 4 ° C. to prepare a transparent 1% by mass aqueous solution. When the temperature of the aqueous solution was gradually increased, the solution became cloudy at 20 ° C. The cloudiness became even stronger when the temperature was raised to 37 ° C. When the cloudy aqueous solution was cooled to 4 ° C., it returned to a transparent aqueous solution again.

Example 3 (Hydrogel containing cell adhesion factor)
10 g of the thermoreversible hydrogel-forming polymer obtained in Example 1 and 10 g of the polymer compound having a cloud point obtained by binding the collagen peptide obtained in Example 2 were mixed with 313 g of Hanks balanced salt solution (HBSS, manufactured by Invitrogen). Was dissolved at 4 ° C. As a result of measuring the dynamic viscoelasticity of the solution at an observation frequency of 1 Hz using a stress-controlled rheometer AR500, the sol-gel transition temperature was 17 ° C., and the storage elastic modulus G ′ at 37 ° C. was 149 Pa. It was.

Example 4 (Preparation of chemiluminescent reagent)
Luminol (manufactured by Sigma, Catalog No.
A-8511) 17.72 mg was dissolved in 10 mL of 1N NaOH, pH was adjusted to 8.5 with 1N hydrochloric acid, 0.34 g of NaCl was added, and the total volume was adjusted to 40 mL with distilled water. This stock solution was diluted 5-fold with HBSS, and the pH was adjusted to 7.4 with 0.2N NaOH or hydrochloric acid to obtain a luminol reagent (luminol concentration 0.5 mM).

Example 5 (neutrophil function test)
0.05 mL of the polymer aqueous solution obtained in Example 3 was injected into a 2 mL cryotube (manufactured by AXY, product code: MCT-200-LC) at 4 ° C., heated to 37 ° C. and hydrogel on the bottom of the cryotube. Was uniformly formed. Luminol reagent (0.075 mL) heated to 37 ° C. and heparin-added peripheral blood (0.075 mL) were mixed and placed on the hydrogel at the bottom of the cryotube at 37 ° C. The result of measuring the amount of luminescence by luminol emission (RLU) with a luminometer (Genlite GL55, manufactured by Microtech Nichion) while keeping the temperature at 37 ° C. (average ± SD of n = 6), 21 ± 9 at 0 minutes , 741 ± 46 at 30 minutes, 838 ± 46 at 60 minutes, and 2072 ± 234 at 90 minutes.

Comparative Example 1
0.05 mL of a polymer aqueous solution obtained by dissolving the thermoreversible hydrogel-forming polymer obtained in Example 1 at a concentration of 10% by mass in HBSS at 4 ° C. is used in place of the polymer aqueous solution obtained in Example 3. Then, the same experiment as in Example 5 was performed, and the light emission amount (RLU) by luminol luminescence was measured (average of SD = n = 6), 6 ± 5 at 0 minutes, 28 ± 8 at 30 minutes, 60 minutes 102 ± 48 and 90 minutes 143 ± 76.

Example 6 (Measurement of the number of cells invading into the gel)
In Example 5, neutrophils migrated for 90 minutes, and then the luminol reagent and heparin-added peripheral blood (0.15 mL) on the hydrogel on the bottom of the cryotube were removed by suction with a pipette. 0.9 mL of physiological saline heated to 37 ° C. was injected onto the hydrogel on the bottom of the cryotube and stirred with a vortex mixer for 3 seconds. 0.9 mL of the physiological saline was removed by suction with a pipette, and 0.9 mL of physiological saline newly heated to 37 ° C. was injected onto the hydrogel on the bottom of the cryotube and stirred with a vortex mixer for 3 seconds. 0.9 mL of the physiological saline was removed by suction with a pipette, and 0.9 mL of physiological saline cooled to 4 ° C. was injected onto the hydrogel on the bottom of the cryotube. The cryotube was cooled to 4 ° C. to completely dissolve the hydrogel.

Next, Luciferin reagent (CellTiter-Glo,
Promega) 0.15 mL was added and allowed to stand at room temperature for 10 minutes, and then the luminescence (RLU) by luciferin luminescence was measured with a luminometer (Genlite GL55, manufactured by Microtech Nichion) (average of n = 6 ± SD), 51000 ± 5900. This amount of luciferin luminescence was based on the ATP activity of the cells and was confirmed separately to correlate with the number of cells that entered the gel (measured with an automated blood cell counter, Sysmex, pocH-100i). .

Example 7
(State of cells that have migrated into the hydrogel)
Cells collected from the gel by the method of Example 6 were centrifuged at 750 rpm for 5 minutes using a cell collection centrifuge (CytoSpin3 Cat.No.730 SHANDON), and a dedicated slide (CYTOSLIDE
Cat.No.59910051), and then stained with Merigrunwald solution (May-Grunwald's solution modified, manufactured by MERK) and Giemsa solution (Giemsa's solution, manufactured by MERK). The microscope observation image is shown in FIG. The stained image in FIG. 2 shows that the cells migrated into the hydrogel by the method of the present invention are neutrophils.

Comparative Example 2
After allowing neutrophils to migrate for 90 minutes in Comparative Example 1, the same operation as in Example 6 was performed, and the amount of luminescence (RLU) due to luciferin luminescence was measured with a luminometer (mean of SD = n = 6 ± SD). It was 4659 ± 5812. This light emission amount was significantly smaller than that of Example 6, and the variation was large.

Since the hydrogel used in Comparative Example 1 does not contain a cell adhesion factor, it is considered that neutrophil infiltration hardly occurs and the luminol luminescence amount is reduced. In addition, the hydrogel used in Comparative Example 1 has low selectivity for cell invasion, and the cells indiscriminately entered the hydrogel simply by sedimentation. Therefore, it is considered that the variation in the amount of luciferin luminescence based on the ATP activity is large.

Example 8
The results of the measurements of Examples 5 and 6 performed on 43 volunteers are summarized in FIG. 3 (the horizontal axis is the luminol luminescence value after 90 minutes of migration, and the vertical axis is the luciferin luminescence value after 90 minutes of migration).
It is considered that there is an individual difference in the neutrophil migration ability and active oxygen production ability, and as shown in FIG. 2, the individual difference can be effectively evaluated by the neutrophil function test system of the present invention. As compared with the elderly group and the younger group, both the luminol luminescence value and the luciferin luminescence value tend to be higher in the elderly group. This may suggest that older people are often in mild inflammatory conditions.

Example 9
(Preparation of LPS reagent)
Lipopolysaccharide (LIPOPOLYSACCHARIDE (LPS): manufactured by SIGMA, Cat. No. L-2630) was dissolved in HBSS to obtain a 10 ug / mL LPS solution.

(Effect of LPS)
0.05 mL of the polymer aqueous solution obtained in Example 3 was injected into a 2 mL cryotube (manufactured by AXY, product code: MCT-200-LC) at 4 ° C., heated to 37 ° C. and hydrogel on the bottom of the cryotube. Was uniformly formed. 0.075 mL of luminol reagent heated to 37 ° C, 0.075 mL of heparinized peripheral blood, and 0.015 mL of LPS solution were mixed and placed on the hydrogel on the bottom of the cryotube at 37 ° C. While keeping the temperature at 37 ° C., the light emission amount (RLU) by luminol luminescence was measured with a luminometer. As a control, 0.015 mL of HBSS was mixed instead of the LPS solution, and the measurement was performed.

62 volunteers (each individual n = 2, 37 elderly people, 25 young people) measured the amount of luminescence by luminol luminescence (RLU) is shown in Table 1 (LPS and control measurement each individual n = 2, The average of the ratio of the results (± SD).

Although there are individual differences in the ability of neutrophils to migrate and react with active oxygen by LPS stimulation, LPS effects can be detected up to 60 minutes as shown in Table 2 (Percentage of people in time showing maximum LPS / HBSS). did it. There was no person who did not respond to LPS (LPS / HBSS ≦ 1) in the elderly group and the young group. When the elderly group and the younger group are compared by the neutrophil function test system of the present invention, the time for responding to LPS and higher than HBSS tends to be delayed in the elderly group. This suggests that there is a difference in response to LPS between the elderly and the young.

Example 10
(Preparation of lycopene reagent)
A solution containing 2% tomato lycopene (Tomat-O-Red 2% SG: manufactured by LycoRed Ltd.) was dissolved in HBSS to prepare a 0.002% solution of tomato lycopene.

(Evaluation of antioxidant power of lycopene)
0.05 mL of the polymer aqueous solution obtained in Example 3 was injected into a 2 mL cryotube (manufactured by AXY, product code: MCT-200-LC) at 4 ° C., heated to 37 ° C. and hydrogel on the bottom of the cryotube. Was uniformly formed. A mixture of 0.0675 mL of luminol reagent heated to 37 ° C, 0.0675 mL of heparinized peripheral blood, and 0.015 mL of a tomato lycopene 0.002% solution was placed on the hydrogel at the bottom of the cryotube at 37 ° C (lycopene final) Concentration 0.0002%). While keeping the temperature at 37 ° C., the amount of luminescence by luminol-dependent chemiluminescence (RLU) was measured with a luminometer (Genlite GL55, manufactured by Microtec Nithion) (each sample n = 2).

As shown in FIG. 4, compared to the chemiluminescence amount measured from the sample mixed with HBSS, the sample added with tomato lycopene 0.002% solution was 33% at 30 minutes, 24% at 60 minutes, 19 at 90 minutes. Decreased to%. This result may indicate that the neutrophil migration is suppressed by the antioxidant power of lycopene.

Example 11
(Preparation of pectin reagent)
Pectin (manufactured by Sansho Co., Ltd., GENU pectin type USP) was dissolved in distilled water to prepare a 1% pectin solution.

(Evaluation of antioxidant activity of pectin)
0.05 mL of the polymer aqueous solution obtained in Example 3 was injected into a 2 mL cryotube (manufactured by AXY, product code: MCT-200-LC) at 4 ° C., heated to 37 ° C. and hydrogel on the bottom of the cryotube. Was uniformly formed. A mixture of 0.0675 mL of luminol reagent warmed to 37 ° C, 0.0675 mL of heparinized peripheral blood, and 0.015 mL of 1% pectin solution (final concentration pectin 0.1%) at 37 ° C on the hydrogel at the bottom of the cryotube. I put it on. While keeping the temperature at 37 ° C., the amount of luminescence by luminol-dependent chemiluminescence (RLU) was measured with a luminometer (Genlite GL55, manufactured by Microtec Nithion) (each sample n = 2).

As shown in FIG. 5, the chemiluminescence amount of the sample to which the pectin 0.1% solution was added was 50% at 30 minutes, 58% at 60 minutes, compared to the chemiluminescence amount measured from the sample mixed with only HBSS. In 90 minutes, it decreased to 72%.

With the neutrophil function test system of the present invention, it becomes possible to simultaneously and easily test the neutrophil migration ability and active oxygen production ability under physiological conditions, which has been difficult to measure conventionally. Therefore, it is extremely useful for diagnosis, prevention, development of therapeutic agents, etc. for various pathological conditions related to neutrophil function.

These show the conceptual diagram of the neutrophil function test | inspection system of this invention. These are the dyeing | staining images which show that the blood cell which migrated in the hydrogel in the neutrophil function test | inspection system of this invention is a neutrophil. These are graphs of luminol luminescence values and luciferin luminescence values of volunteers examined by the neutrophil function testing method of the present invention. The black circles show the measured values for elderly people, and the white circles show the measured values for young people. These show that luminol-dependent chemiluminescence is reduced by adding tomato lycopene in the neutrophil function testing system of the present invention. These show that luminol-dependent chemiluminescence is reduced by adding pectin in the neutrophil function testing system of the present invention.

Claims (8)

Use of a hydrogel containing a cell adhesion factor insolubilized at a temperature at which neutrophils migrate; neutrophils that have entered the hydrogel are converted into active oxygen of the neutrophils in the hydrogel A neutrophil function testing system characterized by detecting by production ability. The neutrophil function test system according to claim 1, wherein the active oxygen producing ability of the neutrophil is detected by chemiluminescence. 2. The neutrophil function test system according to claim 1, wherein the active oxygen producing ability of the neutrophil is detected by colorimetry. 2. The hydrogel exhibits a thermoreversible sol-gel transition phenomenon in which the hydrogel gels at a low temperature and gels at a high temperature, and the gel is substantially insoluble at a temperature higher than the sol-gel transition temperature. The neutrophil function testing system according to any one of ~ 3. The neutrophil function testing system according to any one of claims 1 to 4, wherein the hydrogel contains a neutrophil chemotactic factor. A neutrophil function test method using the neutrophil function test system according to any one of claims 1 to 5 as an indicator of chemiluminescence amount or absorbance as an active oxygen production ability to be detected. After measuring the active oxygen producing ability of neutrophils that have entered the hydrogel at a temperature higher than the sol-gel transition temperature using the neutrophil function testing system according to claim 4, the sol-gel A method for examining neutrophil function, comprising dissolving a hydrogel at a temperature lower than a transition temperature and measuring the number of cells invading the hydrogel. The neutrophil function testing method according to claim 7, wherein the number of cells is measured by measuring ATP activity.