阅读说明：本技术 评价中性粒细胞的活性的方法 (Method for evaluating neutrophil activity ) 是由 数村公子 竹内康造 于 2019-06-10 设计创作，主要内容包括：本发明提供一种利用新的指标来评价中性粒细胞的活性的方法。本发明的方法是评价中性粒细胞的活性的方法,包括基于根据添加有中性粒细胞刺激剂的生物体试样的髓过氧化物酶活性或超氧化物产生活性的测定结果导出的指标,评价中性粒细胞的活性的工序,该指标是从添加中性粒细胞刺激剂的时刻起到髓过氧化物酶活性或超氧化物产生活性的峰向上升起的时刻为止的时间。(the present invention provides a method for evaluating the activity of neutrophils using a novel index. The method of the present invention is a method for evaluating the activity of neutrophils, comprising the step of evaluating the activity of neutrophils based on an index derived from the measurement result of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant is added, the index being the time from the time when the neutrophil stimulant is added to the time when the peak of the myeloperoxidase activity or superoxide generation activity rises upward.)

1. A method, characterized by:

The method is a method of evaluating the activity of neutrophils,

Comprising a step of evaluating the activity of neutrophils on the basis of an index derived from the measurement result of the myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant has been added,

The index is the time from the time when the neutrophil stimulant is added to the time when the peak of myeloperoxidase activity or superoxide generating activity rises upward.

2. The method of claim 1, wherein:

The step of evaluating includes a step of comparing the time with a reference value, and evaluating that the sensitivity of the neutrophil is decreased when the time is longer than the reference value.

3. The method of claim 1 or 2, wherein:

The biological sample is a sample containing whole blood.

4. The method of any one of claims 1 to 3, wherein:

The procedure for performing the evaluation further includes a step of evaluating the activity of neutrophils based on the peak area of myeloperoxidase activity or superoxide generating activity.

5. A method, characterized by:

The method is a method for collecting data for determining whether a subject is suffering from a disease accompanied by oxidative stress caused by excessive activation of neutrophils or is in a state accompanied by oxidative stress caused by excessive activation of neutrophils,

Comprising a step of deriving the data from the measurement results of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant has been added,

The data are the time from the time of addition of the neutrophil stimulator to the time of upward rise of the peak of myeloperoxidase activity or superoxide generation activity,

The biological sample is a biological sample obtained from the subject.

6. A method, characterized by:

The method is a method for collecting data for judging the therapeutic effect of a disease or condition associated with oxidative stress caused by excessive activation of neutrophils in a subject,

Comprising a step of deriving the data from the measurement results of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant has been added,

The data are the time from the time of addition of the neutrophil stimulator to the time of upward rise of the peak of myeloperoxidase activity or superoxide generation activity,

The biological sample is a biological sample obtained from the subject after treatment.

Technical Field

The present invention relates to a method for evaluating the activity of neutrophils.

background

Neutrophils exhibit chemotaxis for inflammatory cytokines, bacteria, and the like, and thus accumulate at inflammatory sites, have a phagocytic and bactericidal function, and the like, and play an important role in biological defense. As the mechanism of action of neutrophils to phagocytize and kill bacteria, generation of active oxygen (superoxide, hydrogen peroxide, etc.), generation of hypochlorous acid by Myeloperoxidase (MPO), and the like are known.

On the other hand, the generation of active oxygen due to excessive activation of neutrophils is pointed out to be a cause of oxidative stress, and causes cell or tissue disorders, leading to various diseases (see, for example, non-patent document 1). In recent years, it has been known that MPO released from neutrophils is also involved in lipid peroxidation, another cause of oxidative stress (for example, non-patent document 1).

As a method for evaluating the function of neutrophils, for example, patent document 1 discloses the following method: the method comprises a step of measuring a myeloperoxidase activity and a superoxide generating activity using the same sample containing whole blood, wherein at least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide generating activity is performed by detecting fluorescence by irradiating a sample vessel containing the sample with excitation light output from an excitation light source and detecting the released fluorescence by a fluorescence detector, and the excitation light source and the fluorescence detector are arranged on the same side with respect to an irradiation surface of the excitation light of the sample vessel, and the activity of neutrophils is evaluated based on the measured myeloperoxidase activity and superoxide generating activity.

Disclosure of Invention

Technical problem to be solved by the invention

Upon exposure to bacteria and fungi, neutrophils are absorbed into the neutrophils, forming phagocytes, in a manner encapsulated by the neutrophil plasma membrane. The phagosome then fuses with the particle and the particle contents are released into the phagosome. Active oxygen (superoxide, hydrogen peroxide) is produced by NADPH oxidase system formed on cell membranes (membranes of phagosomes), killing bacteria and fungi. In addition, the enzyme reaction of myeloperoxidase (EC number 1.11.2.2) contained in the contents of the granule is caused by hydrogen peroxide (H)₂O₂) And chloride ion (Cl)^-) Hypochlorous acid (HOCl) (or its halogen equivalent) is generated, killing bacteria and fungi.

in the method described in patent document 1, for example, after addition of a neutrophil stimulant, active oxygen and hypochlorous acid (or its halogen equivalent) are quantified, and the measured peak areas are calculated, thereby deriving the myeloperoxidase activity and the superoxide-producing activity. Then, the activity of neutrophils was evaluated based on the measured myeloperoxidase activity and superoxide generating activity.

On the other hand, the present inventors have found that, in a subject who is suffering from a disease such as arteriosclerosis or a condition in which neutrophil over-activation and is exposed to oxidative stress such as excessive fatigue, the time until the peak rises up is prolonged when myeloperoxidase activity and superoxide generation activity are measured by quantifying active oxygen and hypochlorous acid (or a halogen equivalent thereof) after addition of a neutrophil stimulant, based on the method described in patent document 1. The present invention is based on this novel finding, and an object thereof is to provide a method for evaluating the activity of neutrophils using a novel index.

Means for solving the problems

The present invention provides a method for evaluating the activity of neutrophils, comprising the step of evaluating the activity of neutrophils based on an index derived from the measurement result of the myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant is added, wherein the index is the time from the time when the neutrophil stimulant is added to the time when the peak of the myeloperoxidase activity or superoxide generation activity rises upward.

In the evaluation method of the present invention, the time from the time when the neutrophil stimulant is added to the time when the peak of the myeloperoxidase activity or superoxide generating activity rises upward (hereinafter also referred to as "index time") is used as an index, and thus the activity of neutrophils can be evaluated. The index time is prolonged in a subject suffering from a disease such as arteriosclerosis or a condition in which neutrophil is considered to be excessively activated and exposed to an oxidative stress such as excessive fatigue. This is considered to be due to the fact that neutrophils are over-activated, and as a result, the sensitivity to the following stimuli is reduced. Therefore, the evaluation method of the present invention can also be understood as a method of evaluating, for example, the sensitivity of neutrophils.

The step of evaluating may include a step of comparing the index time with a reference value, and evaluating that the sensitivity of the neutrophil is decreased when the index time is greater than the reference value.

The biological sample is preferably a sample containing whole blood. This is because the whole blood-containing sample does not require the isolation of neutrophils, and therefore, the operation is simple, and it is possible to obtain information that is closer to the dynamics in the living body including the interaction with various humoral factors and the like contained in the blood while avoiding the stress on neutrophils due to the isolation operation.

The step of evaluating may further include a step of evaluating the activity of neutrophils based on a peak area of myeloperoxidase activity or superoxide generation activity, in addition to the step of evaluating the activity of neutrophils based on the index time. In the case of subjects with an extended index time, a decrease in peak area is sometimes observed at the same time. Therefore, by performing the evaluation based on the peak area together, the activity of the neutrophil can be evaluated in a wide range. Further, exogenous factors such as "food" and the like, and the inflammation protective ability (antioxidant ability or ability to prevent oxidative stress) of antioxidant enzymes and the like possessed by the living body can also be evaluated together based on the peak area.

According to the evaluation method of the present invention, it is possible to distinguish subjects who are in a state in which neutrophil is considered to be over-activated and thus exposed to oxidative stress, such as, for example, suffering from diseases such as arteriosclerosis, and excessive fatigue. Accordingly, the present invention also provides a method for collecting data for determining whether a subject is suffering from a disease associated with oxidative stress due to excessive activation of neutrophils or is in a state associated with oxidative stress due to excessive activation of neutrophils, the method comprising a step of deriving the data from a measurement result of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant is added, the data being a time period from a time point at which the neutrophil stimulant is added to a time point at which a peak of the myeloperoxidase activity or superoxide generation activity rises upward, the biological sample being a biological sample obtained from the subject.

According to another finding discovered by the present inventors, in a subject who is in a condition where it is considered that neutrophils are excessively activated and are exposed to an oxidative stress state, such as, for example, suffering from a disease such as arteriosclerosis, or excessive fatigue, the index time is shortened after the condition is improved, as compared with that in the case of the condition. Accordingly, the present invention also provides a method for collecting data for determining the therapeutic effect of a disease or condition associated with oxidative stress caused by excessive activation of neutrophils in a subject, comprising the step of deriving the data from the measurement result of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant has been added, the data being the time from the time when the neutrophil stimulant has been added to the time when the peak of the myeloperoxidase activity or superoxide generation activity rises upward, the biological sample being a biological sample obtained from the subject after treatment. Effects of the invention

The present invention can provide a method for evaluating the activity of neutrophils using a novel index. The evaluation method of the present invention can evaluate the sensitivity of neutrophils by using the index time as an index. According to the present invention, it is also possible to determine, for example, whether or not a subject suffers from a disease accompanied by oxidative stress due to excessive activation of neutrophils, whether or not the subject is in a state accompanied by oxidative stress due to excessive activation of neutrophils, and to determine the therapeutic effect of the disease or state accompanied by oxidative stress due to excessive activation of neutrophils.

Drawings

FIG. 1 is a graph showing the results of measurement of myeloperoxidase activity and superoxide generating activity in test example 1 using a biological sample containing whole blood collected from a patient suffering from lower limb arteriosclerosis obliterans and undergoing a peripheral vascular intervention.

FIG. 2 is a graph showing the results of analysis of the correlation among the peak area of superoxide generating activity before treatment, the peak area of myeloperoxidase activity, and the index time of superoxide generating activity in test example 1.

FIG. 3 is a graph showing the results of measuring myeloperoxidase activity and superoxide generating activity in test example 2 using a biological sample containing whole blood collected from a healthy person.

FIG. 4 is a graph showing the results of measuring myeloperoxidase activity and superoxide generating activity using a biological sample containing whole blood collected from a healthy person and deriving the index time in test example 3.

FIG. 5 is a graph showing the results of measuring myeloperoxidase activity and superoxide generation activity and deriving index time in test example 3 using a biological sample containing whole blood collected from a patient with lower limb arteriosclerosis obliterans (before treatment).

FIG. 6 is a graph showing the results of measuring myeloperoxidase activity and superoxide generating activity and deriving index time in test example 3 using a biological sample containing whole blood collected from a patient with lower limb arteriosclerosis obliterans (1 month after the intervention).

FIG. 7 is a graph showing the results of measuring myeloperoxidase activity and superoxide generating activity using a biological sample containing whole blood collected from a patient with a brain disease and deriving an index time in test example 3.

FIG. 8 is a graph showing the results of analysis of the correlation between the MPO activity in plasma and the MPO activity upon stimulation (MPO activity measured by addition of a neutrophil stimulator) using the biological sample of test example 1 in a reference example.

Detailed Description

The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

The method for evaluating the activity of neutrophils (also referred to as "evaluation method") according to the present embodiment includes a step of evaluating the activity of neutrophils (also referred to as "evaluation step") based on an index derived from the measurement result of the myeloperoxidase activity or superoxide generating activity of a biological sample to which a neutrophil stimulant is added, and as the index, a time from the time when the neutrophil stimulant is added to the time when the peak of the myeloperoxidase activity or superoxide generating activity rises upward is used.

The evaluation method of the present embodiment may further include a step of adding a neutrophil stimulant to the biological sample (also referred to as an "addition step"), a step of measuring myeloperoxidase activity or superoxide generating activity using the biological sample to which the neutrophil stimulant is added (also referred to as a "measurement step"), and a step of deriving the index (index time) from the measurement result of the myeloperoxidase activity or superoxide generating activity (also referred to as a "derivation step"). However, the evaluation method of the present embodiment does not necessarily have to include the addition step, the measurement step, and the derivation step, and may be configured to perform only the evaluation step using an index time derived separately.

The biological sample may be a sample collected from a living body and containing neutrophils. Neutrophils are contained in, for example, blood, saliva, gingival sulcus exudate. The biological sample may be, for example, a sample containing a stock solution of a sample collected from a living body (for example, blood (whole blood), saliva, gingival crevicular fluid), or a sample containing a treatment solution obtained by treating a sample collected from a living body and concentrating or separating neutrophils. The treatment solution obtained by concentrating or separating neutrophils may be prepared by a conventional method such as a method of concentrating or separating neutrophils using a flow cytometer.

the biological sample may be a sample stock solution or a treatment solution stock solution collected from a living body, or may be a sample obtained by diluting a sample or a treatment solution collected from a living body with a physiological saline solution, a buffer solution, or the like. In the dilution, the dilution ratio may be appropriately set as long as the myeloperoxidase activity or superoxide generating activity can be detected. For example, when whole blood is diluted, the dilution is preferably 10 to 750 times, more preferably 50 to 500 times, still more preferably 100 to 400 times, yet more preferably 200 to 300 times, and particularly preferably 220 to 280 times.

The neutrophil stimulator may be any substance that activates the function (e.g., chemotaxis and phagocytosis) of neutrophils. Examples of the neutrophil stimulators include formylmethionyl leucylphenylalanine (fMLP: neutrophil chemotactic peptide), phorbol-12-myristate-13-acetate (PMA), and modified zymosan (OZ). These can be used alone in 1 or more than 2 kinds combined use.

Myeloperoxidase activity or superoxide generating activity can be measured, for example, by detecting fluorescence, luminescence, or absorbance over time using a fluorescence indicator, a luminescence (e.g., chemiluminescence) indicator, or an absorbance indicator. The indicator may be a commercially available indicator or a commercially available assay kit.

The myeloperoxidase activity can be measured, for example, using a fluorescence indicator, a luminescence indicator, or a light absorption indicator that reacts with hypochlorous acid (or its halogen equivalent) produced by myeloperoxidase. Examples of such an indicator include Aminophenyl fluorescein (APF), taurine/TNB (see J.Clin.Invest., Vol.70, pp.598-607,1982), 8-amino-5-chloro-7-phenylpyrido [3,4-d ] pyridazine-1, 4- (2H,3H) dione (L-012: see Anal biochem., Vol.271(1), pp.53-58,1999).

The reaction of APF with HOCl can be detected by fluorescence (e.g., excitation wavelength 490nm, fluorescence wavelength 515 nm). The reaction of taurine/TNB with HOCl can be detected by absorption (e.g., wavelength 412 nm). The reaction of L-012 with HOCl is less specific, but can be detected by chemiluminescence (e.g., wavelength 455 nm).

the superoxide generating activity can be measured, for example, using a fluorescent indicator, a luminescent indicator, or an absorbing indicator that reacts with superoxide. Examples of such an indicator include 2-methyl-6-phenyl-3, 7-dihydroimidazo [1,2-a ] pyrazin-3-one (CLA), 2-methyl-6- (4-methoxyphenyl) -3, 7-dihydroimidazo [1,2-a ] pyrazin-3-one (MCLA), 2-methyl-6-p-Methoxyphenylethynylimidazopyrazinone (MPEC), indocyanine type imidazopyrazinone compounds (NIR-CLA), 2- [2,4,5, 7-tetrafluoro-6- (2-nitro-4, 5-dimethoxyphenylsulfonyloxy) -3-oxo-3H-xanthen-9-yl ] benzoic acid (BES-So).

The reaction of CLA with superoxide can be detected by chemiluminescence (e.g., 380nm maximum luminescence wavelength). The reaction of MCLA with superoxide can be detected by chemiluminescence (e.g., maximum luminescence wavelength 465 nm). The reaction of MPEC with superoxide can be detected using chemiluminescence (e.g., maximum luminescence wavelength of 430 nm). The reaction of NIR-CLA with superoxide can be detected by chemiluminescence (e.g., 800nm maximum luminescence). The reaction of BES-So with superoxide can be detected by fluorescence (e.g., excitation wavelength 505nm, fluorescence wavelength 544 nm).

The evaluation using the evaluation method of the present embodiment can be achieved by measuring one of the myeloperoxidase activity and the superoxide generating activity, but both the myeloperoxidase activity and the superoxide generating activity may be measured on the same biological sample, for example, based on the method described in patent document 1.

The detection of fluorescence, luminescence, or absorbance can be performed using a known apparatus such as a fluorescence photometer, a luminescence measuring apparatus, or an absorbance photometer. In addition, detection of fluorescence when the biological sample contains whole blood is preferably performed based on the method described in patent document 1.

The index time is the time from the time when the neutrophil stimulant is added to the time when the peak of the myeloperoxidase activity or superoxide generating activity rises upward. A method of deriving the index time will be described with reference to fig. 3. FIG. 3 is a graph showing the results of measuring myeloperoxidase activity and superoxide-producing activity using a biological sample containing whole blood collected from a healthy person. In the graph of fig. 3, the horizontal axis represents elapsed time (measurement time), and the right vertical axis and the left vertical axis represent myeloperoxidase activity (fluorescence emission amount (measurement value)) and superoxide generation activity (chemiluminescence amount (measurement value)), respectively.

FIG. 3 is a graph collectively showing the results of blood collection performed every morning and every day for 3 consecutive days for the same healthy person, and the measurement of myeloperoxidase activity and superoxide-producing activity for each biological sample (sample containing whole blood); and about 1 month later, blood was collected again, and the myeloperoxidase activity and superoxide-producing activity were measured with respect to the obtained biological sample (sample containing whole blood). Among them, the healthy people complain that fatigue was not eliminated and was extremely fatigued only before the day 1, and were in a state that it was considered that neutrophils were over-activated and faced an oxidative stress state.

In fig. 3, arrow a indicates the timing of addition of the neutrophil stimulant. Arrows B and C indicate the time when the peak of the superoxide generating activity rises upward in the measurement using the biological sample on day 2 and day 1, respectively. Similarly, arrow D and arrow E indicate the time when the peak of myeloperoxidase activity rises upward in the measurement using the biological sample on day 2 and day 1, respectively. For example, the time from the time indicated by the arrow A to the time indicated by the arrow C is an index time derived from the superoxide generating activity measured in the biological sample on day 1. For example, the time from the time indicated by the arrow a to the time indicated by the arrow B is an index time derived from the superoxide generating activity measured in the biological sample on day 2. In the example of fig. 3, the index time on day 1, at which the subject (healthy person) complains of extreme fatigue, shows a larger value than the index time on day 2. In addition, as can be understood from FIG. 3, the evaluation using the evaluation method of the present embodiment can be achieved by measuring only one of the myeloperoxidase activity and the superoxide generating activity.

The timing at which the peak of myeloperoxidase activity or superoxide generating activity rises upward can be determined, for example, as follows: after a neutrophil stimulant was added to a biological sample, measurement was continued for a sufficient time until a peak of myeloperoxidase activity or superoxide generating activity appeared, and then a graph of the measured values with respect to the measurement time was plotted, from which the time (vicinity of foot of peak) at which the slope of the measured values was changed with respect to the background level was determined.

for example, when the myeloperoxidase activity is measured by quantifying hypochlorous acid (or its halogen equivalent) produced by the myeloperoxidase, the index time is usually 290 to 470 seconds. The index time shifts to a larger value (e.g., more than 500 seconds) because the sensitivity of neutrophils to a stimulus is reduced in a subject who is suffering from a disease such as arteriosclerosis and a condition in which neutrophils are considered to be over-activated and exposed to an oxidative stress such as excessive fatigue.

For example, in the case of measuring the superoxide-producing activity by quantifying superoxide dismutase, the index time is usually 65 to 150 seconds. The index time shifts to a larger value (e.g., more than 200 seconds) because the sensitivity of neutrophils to a stimulus is reduced in a subject who is suffering from a disease such as arteriosclerosis and a condition in which neutrophils are considered to be over-activated and exposed to an oxidative stress such as excessive fatigue.

the activity of neutrophils can be assessed based on the derived index time. As described above, in the case where the index time shows a larger value, it indicates that the sensitivity of the neutrophil to the stimulus is decreased. For example, when the derived index time exceeds a predetermined reference value, it is possible to evaluate that the sensitivity (sensitivity to stimulation) of neutrophils is decreased. Similarly, when the derived index time is a predetermined reference value, it can be evaluated that the sensitivity of the neutrophil (sensitivity to a stimulus) is normal.

The reference value may be set as appropriate according to the purpose. For example, the reference value may be derived by taking an average or the like from the index time measured for a large number of human samples. The reference value may be set to a plurality of values according to, for example, sex, age, and the like. Further, the measured value in the health state may be used as a reference value for a specific individual. In the method of collecting data for determining the therapeutic effect of a disease or condition associated with oxidative stress due to excessive activation of neutrophils, the index time before therapy may be used as a reference value.

In the evaluation method of the present embodiment, the evaluation step may include a step of evaluating the activity of neutrophils on the basis of a peak area of myeloperoxidase activity or superoxide generation activity, in addition to the step of evaluating the activity of neutrophils on the basis of the index time. By performing the evaluation based on the peak area together, the activity of the neutrophil can be evaluated in a wide range. Further, exogenous factors such as "food" and the like, and the inflammation protective ability (antioxidant ability or ability to prevent oxidative stress) of antioxidant enzymes and the like possessed by the living body can also be evaluated together based on the peak area.

The evaluation method of the present embodiment can be used, for example, in a method (first data collection method) for collecting data for determining whether or not a subject is suffering from a disease associated with oxidative stress due to excessive activation of neutrophils or is in a state associated with oxidative stress due to excessive activation of neutrophils, a method (second data collection method) for collecting data for determining a therapeutic effect on a disease or a state associated with oxidative stress due to excessive activation of neutrophils in a subject, and the like.

The first data collection method of the present embodiment is a method including a step of deriving an index time from a measurement result of myeloperoxidase activity or superoxide generation activity of a biological sample to which a neutrophil stimulant is added, and collecting the derived index time as the data. The biological sample is a biological sample obtained from a subject.

Examples of the diseases accompanied by oxidative stress caused by excessive activation of neutrophils include arteriosclerosis such as lower limb arteriosclerosis obliterans, cancer, inflammatory diseases, hyperlipidemia, diabetes, alzheimer's disease, parkinson's disease, alcohol dependence, and the like. The state associated with oxidative stress due to excessive activation of neutrophils includes, for example, excessive fatigue, smoking after strenuous exercise, aging, and the like.

the second data collection method of the present embodiment is a method including a step of deriving an index time from the measurement result of myeloperoxidase activity or superoxide generating activity of a biological sample to which a neutrophil stimulant is added, and collecting the derived index time as the data. The biological sample is a biological sample obtained from a subject after treatment.

The second data collection method may further include a step of deriving the index time from a biological sample obtained from the subject before the treatment, and further collect the derived index time (before the treatment) as the data.

The first data collection method and the second data collection method according to the present embodiment are specifically described above.