JP5757624B2 - Screening method for antiallergic factors - Google Patents

Description

  The present invention relates to a screening method for antiallergic factors, and more particularly to a screening method for antiallergic factors that screens substances that suppress the production of IgE antibodies produced by antibody-producing cells in vivo.

  In recent years, the number of allergic patients due to hay fever and house dust, which are urban diseases, has been rapidly increasing. In particular, hay fever is said to be 15 to 20% of the national population. Furthermore, it is said that this number will increase in the future, and it is getting worse. On the other hand, the symptoms of hay fever are diseases that have a great impact on life such as runny nose, cough, and itchy eyes, but they are not fatal, so many depend on their patience, Although various treatments have been tried in hospitals, there is no decisive treatment, and palliative treatment with steroidal anti-inflammatory drugs is being performed when symptoms are severe.

  This strong anti-inflammatory steroid-based anti-inflammatory agent has not only an anti-inflammatory action in the body but also a physiological action as a hormone because steroids are part of the hormone in the body, so there are also strong side effects. It has been pointed out. For this reason, it is important to use it under the strict supervision of a doctor, and many patients are concerned about the effects of such side effects. Functional foods do not aim to cure diseases like pharmaceuticals, but by incorporating them into daily eating habits, it is easy to prevent diseases and alleviate disease symptoms, so improve the quality of life for patients. There are great expectations as a means to make this possible.

  By the way, allergic diseases such as hay fever and atopic dermatitis are medically called type I allergy or immediate allergy, and many researchers are studying the onset mechanism. According to the knowledge clarified so far, the following is known. First, pollen, which is an allergen inhaled by respiration, binds to an IgE-type antibody as an immune response in the body. Furthermore, the combined product of pollen and IgE antibody binds to mast cells (mast cells). As a result, chemical substances such as histamine and leukotriene are released from the mast cells, and these substances cause inflammation of the nose, eyes and airways. In other words, IgE type antibodies are the trigger for allergies against pollen invasion. Therefore, a decrease in IgE antibodies in vivo is one method for suppressing allergic symptoms.

  In addition, the inventors of the present application have independently constructed a culture system using human peripheral blood lymphocyte cells that models the pharmacokinetics of human allergies, and have identified components exhibiting antiallergic effects from food components. Specifically, this technique is an allergy onset model detection system technique for culturing human blood cells that produce IgE antibody, which is an allergy trigger marker substance, and a test substance is added to the culture solution of blood cells. A decrease in IgE antibody production is measured and performed. Such a technique is extremely efficient because, unlike the conventional method of testing using a large amount of laboratory animals such as mice, hundreds of specimens can be tested simultaneously in about 10 days of culture. Furthermore, since a human cell is used, if an effective component in the searched factor is found, it is highly possible that the effect is exhibited in the living body, and the time required for searching can be shortened. In addition, by examining the interaction between the searched factor and the cell, it is possible to elucidate the action mechanism of the factor at the cellular level, and thus has many excellent advantages such as enabling scientific evaluation of food functions (non- Patent Document 1).

J. et al. Immunol. Methods 233 (2000) pp. 33-40 Issuer Elsevier

  However, since the allergic onset model detection system using cells described above uses blood cells related to immunity separated from the living body, blood cells are provided with the consent of healthy donors (providers) at each test. It was necessary to have you. As a result, there is a problem that the number of cells that can be used for the test is limited. In addition, there are problems such as individual differences among cell providers affecting the final test results, making it difficult to reproduce the data obtained as test results.

  Therefore, the present invention provides a screening method for an antiallergic factor that makes it possible to easily ensure the number of cells that can be used and to expect the reproducibility of the antiallergic effect of a test substance that is not affected by individual differences. The purpose is to provide.

  The inventors of the present application have continued to examine in detail the mechanism of the allergy onset model detection system as an anti-allergy screening system using blood-derived primary cells. Using the provided blood cells, mixed cell culture of B lymphocytes producing IgE antibodies and T lymphocytes having an immunoregulatory effect was performed. In this mixed cell culture, it was found that a protein derived from the NOV gene was expressed on the cell membrane of T lymphocytes along with the production of IgE antibody of B lymphocytes. The NOV gene-derived protein is a cell activity information transmitter in the cell, and is considered to be involved in a cell proliferation signal and a signal related to cell adhesion (signal transmitter). However, its work is still under study and there are few reports. The inventors of the present application have found that the expression level of the NOV-derived protein changes with the antiallergic effect (antiallergic factor) of the test substance. The inventors of the present application have found that the expression of NOV gene is increased 5 times or more in the cell group of the allergy onset model detection system as compared with the cell group without the onset.

  That is, the inventors of the present application have found that when the expression level of NOV-derived protein on the cell membrane of T lymphocytes decreases, the production amount of IgE antibody, which is an allergic trigger substance, is suppressed accordingly. Furthermore, the inventors of the present application have found that this NOV-derived protein has a property of specifically binding to a test substance when an antiallergic substance is used as the test substance. In other words, it has been found that not only the method of examining the amount of IgE antibody but also the following screening may be performed to search for a factor that exerts an antiallergic effect by suppressing the production amount of IgE antibody.

  Therefore, a first aspect of the present invention is a screening method for an antiallergic factor for screening a substance that suppresses the production of IgE antibodies produced by antibody-producing cells in vivo, comprising a NOV-derived protein expressed in a cell line and Therefore, the method includes a determination step of screening a test substance that exhibits an antiallergic effect by suppressing the production of IgE antibody.

  Specifically, it is an assay method for searching for a component that suppresses the production of IgE antibody, and a test substance that binds to this NOV-derived protein is an antiallergic substance using the NOV-derived protein as a probe (search needle). It is a screening technique characterized by being. Specific examples of successful screening as antiallergic substances include, for example, a protein called glyceraldehyde 3-phosphate dehydrogenase derived from strawberries. The inventors of the present application have determined that protein antiallergic substances can be detected. Theoretically, since it only needs to bind to the NOV protein, it is basically determined that a substance having a molecular weight of 100 or more can be screened. In addition, although it is not protein by this inventor, it turns out that catechins currently said to be an antiallergic substance of tea can also be investigated by this screening system.

  The second aspect of the present invention is a method in which a substance having a high ability of directly binding to a reagent, NOV protein, is a relevant substance as a specific novel method for screening a substance having an antiallergic effect. That is, in the determination step, a high test substance is screened by comparing the binding affinity with the NOV-derived protein. The inventors specifically showed by experiments that the cell line is a human T lymphocytic leukemia cell line and the NOV-derived protein is a protein derived from the gene NOV.

  The third aspect of the present invention is that, as another new screening method, a test substance is added to a cell culture solution of T lymphocytes using T lymphocytes in which NOV protein is expressed on the cell membrane. In this case, if the expression level of NOV protein on the cell membrane decreases, the test substance is an antiallergic substance. That is, in the determination step, when the test substance is added to the culture solution of the cell line, a test substance that suppresses the expression level of the NOV-derived protein expressed by the cell line is screened. The inventors specifically showed by experiments that the cell line was the human T-cell leukemia cell line Molt-4. Since Molt-4 is a cell line with infinite growth ability, it can be subcultured and the culture environment can be kept constant. As a result, the number of cells that can be used can be easily and stably secured, and reproducibility of the antiallergic effect of the test substance that is not affected by individual differences can be expected.

  The test substances that can be used in the present invention are food materials, food compositions, chemical substances, and the like. In the case of water-soluble substances, these preparations are performed using water, physiological saline, culture medium, etc., and in the case of water-insoluble substances, they are dissolved by using a solvent such as ethanol, methanol, dimethyl sulfoxide or the like. That's fine.

  According to the present invention, an unprecedented screening method for antiallergic factors can be obtained. In addition, it is possible to obtain a screening method for anti-allergic factors that makes it possible to easily secure the number of cells that can be used for screening and to expect the reproducibility of the anti-allergic effects of test substances that are not affected by individual differences. . Specifically, it becomes possible to search for a highly reproducible anti-allergic factor by cell lines, and furthermore, by examining the binding ability of NOV protein and a test substance biochemically, the anti-allergy can be extremely quickly and easily performed. It has become possible to screen substances.

It is the graph which showed the correlation with the expression level of a NOV origin protein expression cell, and IgE antibody production suppression effect. It is the graph which showed the IgE antibody production amount of the to-be-tested substance used in the case shown in FIG. It is the graph which compared the expression of NOV gene in a cell using a human cultured cell strain. It is the graph which compared the binding ability of NOV origin protein and an antiallergic factor.

  The present invention relates to a method for assaying a substance having antiallergic activity due to an IgE production inhibitory effect. More specifically, in the present invention, a NOV-derived protein that works in conjunction with suppression of the IgE production amount of antibody-producing cells binds to a test substance. Detect with. This makes it possible to realize an assay method in which a substance having a high binding affinity can be evaluated as an IgE production inhibitor, and thus an antiallergic substance. This is realized by immobilizing the NOV-derived protein as a probe on a BIACORE sensor chip CM5, which is a biomolecular interaction device, allowing a test substance to act on this chip and detecting its binding. Here, there is a human T-cell leukemia cell line Molt-4 that expresses NOV-derived protein on the cell membrane. This human T cell leukemia cell line Molt-4 can be cultured by using a basic synthetic medium such as RPMI 1640 medium containing about 10% of fetal bovine serum used as a growth factor in a general animal cell culture method. The culture medium is not limited to this, and a commercially available serum-free medium can be used as long as it is an animal cell culture medium.

  In the cultivation of Molt-4 cells, a test substance is added to a cell suspension adjusted to an appropriate cell density so as to have a final concentration of 5% or less in the culture solution. After culturing for several days, Molt-4 cells are collected, marked with a fluorescent dye using a specific antibody that binds to the NOV protein on the cell membrane, and the increase or decrease in the amount of fluorescence is quantified, and the amount of fluorescence decreases Is an antiallergic substance.

  In addition, although evaluation of the binding ability of NOV protein and a test sample uses the biomolecule interaction apparatus, it does not necessarily restrict to this. That is, there is a biochemical evaluation method such as an enzyme antibody method or a Western blotting method for evaluating the binding between proteins, which can be used.

  Examples of the present invention are illustrated below, but the present invention is not limited to the following examples.

[Separation of lymphocytes]
In order to obtain human peripheral lymphocytes, the peripheral blood of healthy subjects was separated by density gradient centrifugation. Peripheral blood is collected from a healthy person in a vacuum tube containing heparin, and 5 ml of peripheral blood is placed on a 4 ml blood separation agent (Ficoll; GE Healthcare) previously dispensed into a 15 ml centrifuge tube so as not to disturb the liquid level. The layers were layered and centrifuged at 400 × g for 30 minutes. The uppermost plasma layer was obtained and stored at -24 ° C. Next, the lymphocytes between the plasma layer and the Ficoll layer were collected, washed with a basic synthetic medium ERDF (Kyokuto Seiyaku), and centrifuged and washed three times under conditions of 400 × g and 5 min. The obtained lymphocytes were stored frozen at −85 ° C., and after thawing, the lymphocytes were washed with ERDF medium and used.

[In vitro allergy development model cell culture system]
In an ERDF medium containing 5% fetal bovine serum (FBS; Trace) and 10% human plasma, 2.5 × 10 6 cells / ml human peripheral blood lymphocytes removed in vitro and muramyl dipeptide (MDP) as an immunostimulant SIGMA) at 10 mg / ml, interleukin-2, -4, -6 (IL-2, -4, -6; R & D) at 10 ng / ml, and cedar pollen antigen (Cryj1; HAYASHIBARA) at 100 ng / ml In addition to the final concentration, an in vitro allergy model culture system was obtained. The main functions of the added immunostimulant are adjuvant activity that enhances the immune response to the antigen by MDP, promotion of T cell proliferation and differentiation by IL-2, induction of IgE class switch by IL-4, and IL-6 This is induction of B cell differentiation. 200 ml of human peripheral blood lymphocytes added with these immunostimulants and Cryj1 were dispensed into 96-well plates, and cultured in a 5% CO ₂ incubator at 37 ° C. for 10 days to induce IgE antibody production. The human plasma and human peripheral blood lymphocytes contained in this culture system were the same individuals.

[Correlation between NOV-derived protein expressing cells and IgE antibody production inhibitory effect]
By labeling the molecules present on the cell membrane with a fluorescent dye, the expression of NOV-derived protein on the cell membrane was confirmed using a flow cytometer capable of quantitative analysis. The test substance which has the effect which suppresses the production of IgE antibody in an in vitro allergy onset model cell culture system, ie, an antiallergic effect, was added and cultured for 10 days. Cells of the model culture system were labeled by the fluorescent antibody method using Goat anti NOV IgG (R & D, AF1640) and Donkey anti goat Ig's FITC (Santa Cruz Biotechnology, SC-3853). Then, the relative amount of NOV molecules on the cell membrane was measured. FIG. 1 is a graph showing the correlation between the expression level of NOV-derived protein-expressing cells and the IgE antibody production inhibitory effect. Compared with the control experiment of FIG. 1 (A), the addition of the antiallergic substance of FIG. 1 (B) shows that the expression level is reduced because the peak of the graph is shifted to the left. . FIG. 2 is a graph showing the IgE antibody production amount of the test substance used in the case shown in FIG. As shown in FIG. 2, it is shown that the expression of the NOV-derived protein was suppressed only in the cells of the culture system containing the test substance having an antiallergic effect.

[Identification of cells expressing NOV-derived protein on cell membrane]
There are currently no reports on cells expressing NOV-derived proteins. Therefore, by investigating the expression of the NOV gene in the cells using several types of cultured human cell lines, it was identified in which cells. The cell lines used were human B lymphocyte cell line SK729-1, human T lymphocyte cell line MOLT-4, and human B lymphocyte cell line DND39. After extracting RNA from these cells, cDNA was synthesized and real-time PCR was performed.

A) RNA extraction
Cultivate cultured cells 1 × 10 ⁷ cells using ISOGEN (Nippon Gene, 311-02501), add 0.2 ml of chloroform (Wako Pure Chemicals, 038-02606) to this, and shake vigorously for 15 seconds at room temperature. And allowed to stand for 5 minutes, and then centrifuged at 12K × g, 4 ° C. for 15 minutes. After centrifugation, the aqueous layer was collected, 0.5 ml of isopropanol (Wako Pure Chemicals, 168-21675) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, the supernatant was removed by centrifugation at 12K × g and 4 ° C. for 10 minutes, and 70% ethanol (Wako Pure Chemical Industries, Ltd., 057-00456) was added. Subsequently, the mixture was centrifuged again at 12K × g and 4 ° C. for 5 minutes, and the supernatant was discarded and air-dried for 5 minutes. To this, ddH ₂ O was added so that the RNA concentration was 5 μg / 9 μl to obtain an RNA sample.

B) Synthesis of cDNA The ThermoScript ^™ RT-PCR System (Invitrogen, 11146-016) was used to synthesize cDNA from the isolated RNA. Random Hexamer (1 μl), RNA (9 μl), and 10 mM dNTP Mix (2 μl) were mixed and incubated at 65 ° C. for 5 minutes. Thereafter, 4 μl of 5 × cDNA Synthsis Buffer, 1 μl of 0.1M DTT, 1 μl of RNaseOUT ^™ (40 U / μl), 1 μl of DEPC-treated water and 1 μl of ThermoScript ^™ RT (15 units / μl) were added and mixed. Then, it was made to react at 25 degreeC for 10 minutes, 50 degreeC for 50 minutes, and 85 degreeC for 5 minutes, and synthesize | combined cDNA. Thereafter, 1 μl of RNase H was added and incubated at 37 ° C. for 20 minutes to decompose the remaining RNA.

C) Real-time PCR (NOV)
Using cDNA as a template, mix 1 μl of cDNA, 1 μl of Forward Primer, 1 μl of Reverse Primer, 12.5 μl of iQ ^™ SYBR (registered trademark) Green Supermix (BIORAD, 170-8882), 9.5 μl of DEPC-treated water, and mix NOV gene Real-time PCR was performed. In addition, β-actin was used as an internal standard. The PCR conditions at this time were to activate the enzyme at 95 ° C. for 3 minutes, and carried out 40 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The primers used have the following sequences.

NOV
Forward primer: 5'-TGGTCCCTCATAGCCCTCAG-3 '(SEQ ID NO: 1)
Reverse primer: 5'-CCCTGCTAATATGCTGTTTCTGC-3 '(SEQ ID NO: 2)

β-actin
Forward primer: 5'-GACTTCGAGCAAGAGATG-3 '(SEQ ID NO: 3)
Reverse primer: 5'-GCCAGACAGCACTGTGTT-3 '(SEQ ID NO: 4)

  FIG. 3 is a graph comparing the expression of NOV genes in cells using human cell lines. As a result, as shown in FIG. 3, it was found that many NOV genes were expressed in the MOLT-4 cell line. Thus, NOV-derived protein was expressed in human T lymphocyte cell lines. There is a correlation between the suppression of IgE antibody production, which is a method for detecting the antiallergic effect, and the decrease in the expression level of NOV-derived protein on the T lymphocyte cell membrane used in the present invention. By investigating the expression level of NOV-derived protein using 4 cell lines, a novel anti-allergic factor search method could be established.

[Evaluation of binding ability of NOV-derived protein and antiallergic factor]
In order to evaluate the binding ability between the NOV-derived protein and the anti-allergic factor, the binding between molecules was measured using a biomolecular interaction device BIACORE capable of kinetically measuring. In other words, NOV-derived protein (R & D, 1640-NV) is immobilized on BIACORE sensor chip CM5 (BIACORE, BR-1000-12), and 120 substances that have anti-allergic effects and 120 substances that do not have anti-allergic effects are identified. Flow through the channel for 2 seconds and graphed the binding affinity with the NOV-derived protein. FIG. 4 is a graph comparing the binding ability of a NOV-derived protein and an antiallergic factor. As shown in FIG. 4, the anti-allergic factor has recorded a change in a graph showing a high binding ability compared to the control experiment. As a result, it was found that NOV-derived proteins and antiallergic substances have high binding affinity, and conversely, those that are not antiallergic substances have extremely low binding affinity. It was found that a novel anti-allergic factor can be searched by measuring the binding ability to the test substance using the NOV protein itself for the anti-allergic factor search.

Claims (2)

A screening method for an antiallergic factor for screening a substance that suppresses production of IgE antibodies produced by antibody-producing cells in vivo,
With a binding affinity between NO V protein expressed in cell lines, anti-allergic factor of screening comprising determining step of screening a test substance to exert anti-allergic effects by inhibiting the production of the IgE antibody Method. In the determination step, the binding affinity of the NO V protein is to screen for a test substance is higher than the binding affinity with those that are not the NOV protein and anti-allergic agent, anti-allergic agent according to claim 1, wherein Screening method.