JP2006184072A - Method for evaluating immunostimulating ability of test substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To collect a physiologically active substance suitable for measurement, that is, peripheral blood mononuclear cells (PBMC) and a test substance in vitro, which can be collected from a culture supernatant, and cytotoxic activity of PBMC And a method for evaluating the immunostimulatory ability of a test substance, which is simple and highly reliable, using a physiologically active substance that exhibits a high correlation with the PBMC and is less likely to vary depending on the PBMC used.
A method for evaluating the immunostimulatory ability of a test substance, comprising culturing the test substance together with peripheral blood mononuclear cells and detecting granulysin in the culture supernatant.
[Selection] Figure 4

Description

  The present invention relates to a simple and accurate method for evaluating the immunostimulatory ability of a test substance against mammals.

  In recent years, attention has been focused on healing and ameliorating diseases by enhancing immunity, and discovery of substances with immunostimulatory ability and immunostimulatory ability of substances not only in the pharmaceutical field but also in health foods and other fields. A lot of capital and effort has been invested in the evaluation. Conventionally, the presence or absence and degree of immunostimulatory ability of a test substance has generally been determined using cytotoxic activity as a marker.

  Cytotoxic activity was determined by what percentage of target cells were killed when mixed culture of peripheral blood mononuclear cells (PBMC) and target cells (tumor cells, etc.) and cell-cell contact. However, this method is cumbersome because the target cells need to be subcultured aseptically. In addition, since the determination is based on whether or not the target cell is injured, the value of the cytotoxic activity is likely to be influenced by the condition and vitality of the target cell. Fluctuation increases. In addition, the cytotoxic activity of PBMC obtained by separating from a subject is easily affected by storage conditions, time after blood collection, and the like, and it is essential to measure it on the day of blood collection or the next day at the latest. Furthermore, when blood with high cytotoxic activity is used, the value of cytotoxic activity reaches a plateau due to stimulation, and it tends to be difficult to compare immunostimulatory ability.

  Therefore, the method for measuring the cytotoxic activity is troublesome and has a problem that reliable data cannot be obtained unless a plurality of experiments are performed in order to obtain accurate data. Such a problem becomes a great obstacle especially when it is desired to screen many test substances. Therefore, an alternative method to measurement of cytotoxic activity has been desired.

  As a solution to this problem, a method of evaluating the immunostimulatory ability by measuring a physiologically active substance released from PBMC can be considered. According to this method, since it is only necessary to measure the physiologically active substance present in the supernatant, it is possible to collect a large amount of specimens in a lump by collecting the supernatant and storing it in a frozen state. Since it is not necessary to culture cells, it is convenient and convenient unlike the above method.

  Examples of physiologically active substances released from PBMC that are related to immunity include IFN-γ and IL-12.

Patent Document 1 discloses a method for confirming the immune state of a specimen provider by detecting extracellular granulysin in a body fluid specimen. The present invention shows that granulysin is recognized as a soluble protein outside the cell by being secreted extracellularly from NK cells and CTLs, and granulysin in plasma collected from cancer patients of different severity As a result of measuring the amount and comparing with the amount of granulysin in the plasma of healthy subjects, the amount of granulysin in plasma is almost the same as that of healthy subjects in cancer patients with low severity, whereas patients with high severity cancer (Immune status seems to be lower) was made by finding that the amount of granulysin in plasma was significantly reduced, and based on the behavior of granulysin in vivo .
International Publication Number WO 03/052417

  However, in order to accurately see the immunostimulatory ability using the physiologically active substance released by PBMC as an index, various things are required. That is, it is required that the correlation with the cytotoxic activity is large, that there is no large variation depending on the PBMC used, and that the immunostimulatory ability of small differences can be accurately evaluated. Even in Patent Document 1, the behavior of granulysin in vitro is not clarified.

  Therefore, a physiologically active substance suitable for evaluation, that is, when PBMC and a test substance are cultured in vitro, it can be collected from the supernatant (extracellular of PBMC) and has a high correlation with the cytotoxic activity of PBMC. There has been a demand for the discovery of a physiologically active substance having a relationship that is less likely to vary depending on the PBMC used, and a simple and reliable evaluation method using the physiologically active substance.

  As a result of intensive studies to solve the above problems, the present inventors have found that when PBMC and a test substance are mixed and cultured, there is a high correlation between the amount of granulysin present in the culture supernatant and the cytotoxic activity. And the amount of granulysin in the supernatant was found to have little variation between PBMCs even when experiments were conducted using PBMCs collected from the blood of multiple people, and the present invention was completed. did.

  That is, the present invention is a method for evaluating the immunostimulatory ability of a test substance, comprising culturing the test substance together with peripheral blood mononuclear cells (PBMC) and detecting granulysin in the culture supernatant.

  When the test substance is cultured with PBMC, if the test substance has immunostimulatory ability, PBMC is activated and granulysin production is enhanced. However, when the test substance does not have immunostimulatory capacity, the production of granulysin is not enhanced. Part of the produced granulysin is secreted from PBMC and present in the culture supernatant, and the amount of granulysin detected from the culture supernatant shows a high correlation with the cytotoxic activity. Therefore, by detecting granulysin in the culture supernatant, it is possible to evaluate whether or not the test substance has immunostimulatory ability and its degree.

According to this method, unlike the cytotoxic activity measurement method, it is not necessary to aseptically subculture target cells (generally, K562, which is a tumor cell), and it is not necessary to keep the state of the target cells constant. It is. In addition, since it is necessary to measure the cytotoxic activity within several hours (generally after about 2 to 4 hours) after reacting PBMC and K562, it is not possible to redo or store the measurement specimen. Even a small number of samples must be measured within the same day, but in the method according to the present invention, the supernatant can be collected and stored frozen, and can be measured later. . In particular, when measuring a plurality of test substances, it is efficient because a sample is stored and measured in a lump.
In addition, even when using PBMCs from different individuals, the values do not fluctuate greatly, so highly reliable data can be obtained without performing multiple experiments, and the immunostimulatory ability of the test substance is compared. It is also convenient.

  If ELISA is used as a means for detecting granulysin, measurement is performed with reference to a standard substance, so that there is little fluctuation in value, and a large amount of samples can be processed at once. In addition, since there is no need to use a radioisotope, there is no problem that it must be measured at a facility where use of a radioisotope is permitted, and there is no problem such as an influence on the environment or the health of the measurer. Furthermore, the measurement is simple and does not require special and expensive measuring equipment.

  If screening of immunostimulatory substances is performed using the evaluation method according to the present invention, substances having immunostimulatory ability can be efficiently discovered from a large number of test substances.

  According to the evaluation method according to the present invention, it is only necessary to culture the test substance together with PBMC and measure granulysin in the culture supernatant. Therefore, unlike the measurement of cytotoxic activity, it is not necessary to culture the target cells. In addition, since the supernatant can be stored and measured later, there are few time restrictions and the stored samples can be measured together. Furthermore, even if different PBMCs are used, there is no significant difference in the measurement results, so highly reliable data can be obtained without performing multiple experiments, which is very effective for screening a large number of test substances. It is.

  Peripheral blood mononuclear cells (PBMCs) generally contain monocytes and lymphocytes (natural killer (NK) cells, T lymphocytes, B lymphocytes). Peripheral blood is collected with heparin and centrifuged using Ficoll. It can be obtained by separating or the like. In the present invention, it refers to one containing at least either natural killer cells (NK cells) or cytotoxic T lymphocytes (CTL).

  Granulysin is one of a series of molecules with cytotoxic activity released by natural killer cells (NK cells) and cytotoxic T lymphocytes (CTL) to cause cell death in the target cells. And antibacterial activity has been reported.

  Hereinafter, the present invention will be described in more detail with reference to examples. The test substances and measurement methods used in the examples are as follows. IFN-γ was used as a substance to be compared with granulysin.

  10 kinds of animal lactic acid bacteria (lactobacillus lactis) isolated from animal fermented dairy products and intestinal tracts and 3 kinds of plant lactic acid bacteria (lactobacillus pentosus) isolated from pickles were used as test substances (hereinafter these 13 kinds). Lactic acid bacteria are referred to as A, B, C, D, E, F, G, H, I, J, K, L, and M, respectively). Lactic acid bacteria were used as heat-killed cells at a concentration of 5 μg / ml.

The detection of granulysin was performed using the ELISA method. The procedure is shown below.
Granulaicin sandwich ELISA method
A mouse anti-human granulysin monoclonal antibody (clone RB1) diluted to 5 μg / ml was added to the solution using 100 mM sodium carbonate buffered ficoll solution, and this was dispensed into wells of a 96-well microplate. Immobilization is carried out by contact overnight at ° C. The antibody-immobilized plate is washed with a washing solution (0.1% Tween20 / PBS) and then blocked with a blocking solution (10% fetal bovine serum / washing solution). After washing the antibody-immobilized plate with the washing solution, the standard product diluted with the blocking solution (the plasmid used for immunization when obtaining the monoclonal antibody was transfected into COS-7 cells, The culture supernatant of the eye is used as a standard product of 1,000,000 unit / ml) or 50 μl of each sample is added and reacted. Next, a biotinylated anti-granulinicin monoclonal antibody (clone RC8) biotinylated by a conventional method is reacted, washed with PBST (0.1% Tween20 / PBS), and then diluted with PBST to contain HRP (Horseradish Peroxidase) -labeled streptavidin. React. After washing with PBST, a TMB enzyme substrate for detection is added to the plate, reacted, and then measured with a plate reader.

The cytotoxic activity was measured using the following method. K562 was used as a tumor cell (target cell).
Cytotoxic activity measurement method
K562 target cells are labeled with the fluorescent dye DiO and reacted with PBMC for 2 hours. A cell membrane-impermeable nucleic acid-binding fluorescent dye PI is added to the culture solution. Injured K562 target cells are double-stained with DiO and PI, whereas non-injured K562 target cells are single-stained with DiO, and are detected using a flow cytometer. .

“Examination of reaction time”
Human peripheral blood was blood-collected with heparin, and PBMCs were separated by Ficoll-pack density centrifugation. The separated PBMC was adjusted to a final concentration of 2 × 10 6 cells / ml using RPMI 1640 medium containing 10% fetal bovine serum. Lactic acid bacteria A at a concentration of 5 μg / ml was added to this and cultured in a 5 ml lymphocyte culture tube. The culture was performed under conditions of 5% carbon dioxide gas and 37 ° C. The supernatant was collected every 3 hours and the amount of granulysin in the supernatant was measured by the granulysin sandwich ELISA method. The results are shown in FIG.

  An increase in the amount of granulysin in the PBMC supernatant was observed from about 6 hours after stimulation with lactic acid bacteria. After 15 hours, there is a clear difference between the amount of granulysin in the PBMC supernatant measured without addition of lactic acid bacteria (no stimulation), and it is clear that lactic acid bacteria A has the ability to promote granulysin secretion. became. The amount of granulysin in the supernatant reached a plateau in 18 hours. From the results of Example 1, the culture time of PBMC and lactic acid bacteria was determined to be 21 hours.

"Examination of granulysin production promoting ability by lactic acid bacteria A to I"
Culturing PBMC with lactic acid bacteria for 21 hours in the same manner as in Example 1, then centrifuging, allowing PBMC to settle in the lower layer of the test tube, recovering the culture supernatant, and purifying the supernatant by granulinicin sandwich ELISA. The amount of granulysin in the medium was measured. The results are shown in FIG.

  When lactic acid bacteria A to I were added, the amount of granulysin in the culture supernatant was increased as compared with the control (the amount of granulysin in the culture supernatant when no lactic acid bacterium was added). From the results of the Examples, it was found that lactic acid bacteria A to I have the ability to enhance granulysin production and the degree thereof.

"Examination of the ability of lactic acid bacteria A to I to promote cytotoxic activity"
In the same manner as in Example 2, after stimulating PBMC with lactic acid bacteria for 21 hours, K562 target cells labeled with the fluorescent dye DiO were reacted with PBMC for 2 hours, and the cytotoxic activity was measured. The results are shown in FIG. The cytotoxic activity of PBMC was enhanced by stimulation with lactic acid bacteria. From these results, it was found that lactic acid bacteria A to I have the ability to enhance cytotoxic activity.

"Correlation between the amount of granulysin in the supernatant and the cytotoxic activity upon stimulation with lactic acid bacteria"
FIG. 4 shows the correlation between the amount of granulysin in the PBMC supernatant and the cytotoxic activity. As shown in the figure, a strong correlation was observed between the amount of granulysin in the supernatant upon stimulation with lactic acid bacteria and the cytotoxic activity. The correlation coefficient was 0.8862.

  FIG. 5 shows the granulysin ratio (prepared from the results of Example 2) and the cytotoxic activity ratio (created from the results of Example 3) in the supernatant when the control is 1. As shown in FIG. 5, according to the method for measuring the amount of granulysin in the supernatant, it is possible to clearly grasp the immunostimulatory ability with a small difference compared to the cytotoxic activity. On the other hand, the amount of granulysin in the supernatant did not fluctuate extremely for each test substance, so that it was not necessary to carry out dilution or the like, and the measurement was simple.

“Examination of individual differences in granuleicin production by PBMC (1)”
For the same test substance, an experiment was performed using PBMC separated from the blood of three persons (each person is represented as SGHMKY), and the amount of granulysin in the culture supernatant was compared. The results are shown in Table 1 and FIG.

  The amount of granulysin in the PBMC supernatant was measured using PBMCs isolated from different individuals, and there was no difference between individuals, and there were no individuals that showed extremely high values or almost no granulysin was detected. . For each lactic acid bacterium, when the individual showing the maximum value was compared with the individual showing the minimum value, the maximum value did not exceed twice the minimum value. Furthermore, regardless of which individual PBMC was used, the amount of granulysin in the supernatant showed a high value in the order of G, A, E, B, F, D, I, C, H (however, only HM, D and F are the same value).

"Examination of individual differences in PBMC granulysin production (2)"
In order to further examine the individual differences in the amount of granulysin in the PBMC supernatant, using PBMC separated from the blood of 4 subjects different from Example 4 (each represented as KT, KA, SK, SY) Experiments with five types of lactic acid bacteria were performed, and the amount of granulysin in the culture supernatant was compared. The results are shown in Table 2 and FIG.

  Similar to Example 4, also in Example 5, granulysin in the PBMC supernatant showed no extremely high individual or hardly detected individual, and the difference between individuals was small. For each lactic acid bacterium, when the individual showing the maximum value was compared with the individual showing the minimum value, the maximum value did not exceed three times the minimum value. Furthermore, regardless of which individual PBMC was used, the amount of granulysin in the supernatant showed a high value in the order of G, K, and J. Regarding L and M, only one person showed a high value in the order of M and L, and the other three people showed a high value in the order of L and M.

  From Example 4 and Example 5, it can be seen that the amount of granulysin in the PBMC supernatant has little variation among individuals, and it is possible to obtain highly reliable data without repeating the experiment using a plurality of specimens. It was.

  From the results of the above Examples, it is clear that there is a high correlation between the amount of granulysin in the PBMC supernatant and the cytotoxic activity of PBMC when PBMC is cultured with the test substance. It was found that the immunostimulatory ability of the test substance can be evaluated by measuring the amount of granulysin in the supernatant instead of measuring the injury activity. In addition, it becomes clear that the measurement of the amount of granulysin in the PBMC supernatant is simple, and that individual differences due to the PBMC used are small, and the method according to the present invention is a method for evaluating the immunostimulatory ability of a test substance It was found that the method is particularly suitable as a method for screening a substance having immunostimulatory ability from a large number of test substances.

“Comparative Example 1”
Measurement of the amount of IFN-γ in the PBMC supernatant upon stimulation with lactic acid bacteria After stimulation of PBMC with lactic acid bacteria for 21 hours in the same manner as in Example 1, the PBMC culture supernatant was recovered and measured by ELISA using a commercially available ELISA kit. The amount of IFN-γ in the supernatant was measured by the method. The results are shown in Table 3 and FIG.

  The amount of IFN-γ in the PBMC supernatant varied greatly depending on the test substance. That is, it is either not detected at all or shows a high value, and since the difference in values is large, it was necessary to perform measurement after diluting.

“Comparative Example 2”
FIG. 9 shows the correlation between the amount of IFN-γ in the supernatant and the cytotoxic activity in the PBMC supernatant. The correlation between the amount of IFN-γ in the supernatant upon stimulation with lactic acid bacteria and the cytotoxic activity was low, and the correlation coefficient was 0.5009.

“Comparative Example 3”
Examination of individual differences in IFN-γ production by PBMC An experiment was conducted using PBMCs separated from the blood of five people (each person is represented as KA ka HMKYSK), and IFN in the PBMC supernatant. -The amount of γ was compared. The results are shown in Table 4 and FIG.

  Individual differences in the amount of IFN-γ in the PBMC supernatant vary greatly. There are individuals with extremely high values (KA, HM, KY) and individuals with almost no detection (ka, SK). It was. In addition, even when compared between individuals with high IFN-γ responsiveness (or between individuals with low IFN-γ responsiveness), the measured values sometimes differed by 7 times or more, and the type of lactic acid bacteria showing the highest value also differed between individuals.

  From the results of Comparative Examples 1 to 3, when PBMC was cultured together with the test substance, no clear correlation was found between the amount of IFN-γ in the PBMC supernatant and the cytotoxic activity of PBMC. The method of measuring the amount of IFN-γ in the supernatant cannot be used in place of the measurement of the damaging activity, and the amount of IFN-γ in the PBMC supernatant varies greatly depending on the PBMC used. It was found that the substance was not suitable for evaluating the immunostimulatory ability of the substance.

"Conclusion"
From the results of Examples, it was found that the immunostimulatory ability of a test substance can be evaluated by detecting granulysin in the supernatant instead of the cytotoxic activity measurement method. Further, in the method according to the present invention, it is not necessary to culture K562, and since the supernatant can be stored frozen and measured at a later date, the measurement is much simpler than the measurement of cytotoxic activity. The convenience was high.
From the results of Comparative Examples, granulysin is particularly suitable for evaluation of immunostimulatory ability among physiologically active substances that can be collected from the culture supernatant when PBMC and a test substance are cultured in vitro. I understood that.

  The method according to the present invention can be used for screening of drugs that utilize immunostimulatory ability (for example, therapeutic drugs for cancer and infectious diseases).

The line graph which shows the time-dependent change of the amount of granulysin in a PBMC supernatant. The bar graph which shows the amount of granulysin in PBMC supernatant at the time of culture | cultivating with various lactic acid bacteria. The bar graph which shows the cytotoxic activity of PBMC when it culture | cultivates with various lactic acid bacteria. The graph which shows the correlation of the amount of granulysin in PBMC supernatant, and cytotoxic activity. The bar graph which shows the granulysin ratio (left) and cytotoxic activity ratio (right) in a PBMC supernatant when a control is set to 1. The bar graph which shows the amount of granulysin in the PBMC supernatant of each individual | organism | solid (S.G., H.M., K.Y.). A bar graph showing the amount of granulysin in the PBMC supernatant of each individual (K.T., K.A., S.K., S.Y.). The bar graph which shows the amount of IFN-γ in the PBMC supernatant when cultured with various lactic acid bacteria. The chart which shows the correlation of the amount of IFN-γ in PBMC supernatant and cytotoxic activity. The bar graph which shows the amount of IFN-γ in the PBMC supernatant of each individual (K.A., k.a., H.M., K.Y., S.K.).

Claims (3)

  A method for evaluating the immunostimulatory ability of a test substance, comprising culturing the test substance together with peripheral blood mononuclear cells and detecting granulysin in the culture supernatant.   The evaluation method according to claim 1, wherein ELISA is used as a means for detecting granulysin.   A screening method for an immunostimulatory substance using the evaluation method according to claim 1 or 2.

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