JP2006075140A - Drug screening method - Google Patents

Abstract

Disclosed is a method for screening a drug capable of screening a substance that suppresses cell adhesion and / or cell migration of polymorphonuclear leukocytes at low cost and in a simple manner without using an animal for a test.
At least polymorphonuclear leukocytes, a test substance, and an activator of the polymorphonuclear leukocytes are added to an adhesive substrate coated on a solid support, and the polymorphism to the adhesion substrate is added. The cell adhesion ability of the polymorphonuclear leukocytes is evaluated by measuring the adhesion cells of the nuclear leukocytes. It is possible to screen for a substance that suppresses cell adhesion and / or cell migration of polymorphonuclear leukocytes at low cost, in a short time and easily without conducting animal experiments, and more efficiently develop pharmaceuticals. be able to.
[Selection figure] None

Description

  The present invention relates to a method for screening a drug effective for suppressing cell adhesion and / or cell migration.

  Polymorphonuclear leukocytes (hereinafter referred to as PMNs) are the cells that play the first line of defense against pathogenic bacteria and microorganisms, and adhere to cells through integrins, which are cell adhesion molecules, by various stimuli such as infection with pathogenic bacteria. It is known to cause. In addition, PMNs adhered through integrins are spread by reorganization of the intracellular actin skeleton, which is known to serve as a basis for strong adhesion to the substrate, cell migration (migration), and phagocytosis. Yes.

  Specifically, when pathogenic bacteria or microorganisms invade into the living body, PMNs circulating in the blood vessels sense and activate this, and (1) adhesion to the vascular endothelium, (2) outside the blood vessel It protects the living body through a series of processes such as leakage, (3) migration / accumulation at the site of inflammation, (4) phagocytosis of bacteria and microorganisms, and (5) sterilization. In this sterilization process, sterilization is performed by releasing various intragranular enzymes and active oxygen to bacteria engulfed by PMNs. In addition, the action of these intragranular enzymes and active oxygen is not specific to pathogenic bacteria, and is known to show damaging effects on host tissues.

  For this reason, the function of PMNs needs to be strictly controlled in time and space. However, disruption of these controls results in excessive accumulation of PMNs in the host tissue. PMNs attack even normal cells of the self to cause various tissue injuries and cause various inflammatory diseases, for example, autoimmune diseases such as rheumatoid arthritis and allergic diseases such as asthma. Furthermore, it also brings about an inflammatory reaction caused by cell adhesion of PMNs after transplantation in the body such as a medical device. About this inflammatory response, the host protein such as fibrinogen mainly coats the transplantation device, so that PMNs It is thought that adhesion is induced.

  In order to cope with the above excessive accumulation of PMNs, it is necessary to suppress cell adhesion and / or cell migration of PMNs, and discovery and development of substances exhibiting such inhibitory effect on PMNs are desired. .

On the other hand, as a method for screening medicinal substances, for example, Patent Document 1 discloses that atopic dermatitis is characterized in that a test substance is administered to an atopic dermatitis model animal and the effect of improving atopic dermatitis is assayed. A screening method for a substance for treating inflammation has been proposed. In this way, screening for medicinal substances is basically a method in which various substances are randomly administered to disease state model animals whose medicinal effects can be evaluated, their effects are examined, and substances that exert their medicinal effects are selected. It was.
JP 2004-41123 A

However, in screening methods based on animal experiments using disease model animals, in order to screen medicinal substances from countless substances,
There was a problem that considerable labor and a large amount of money were required.

  The present invention is intended to solve the above-described problems, and a method for screening a drug capable of screening a substance that suppresses cell adhesion and / or cell migration of polymorphonuclear leukocytes at low cost and easily without performing animal experiments. The purpose is to provide.

  As a result of intensive studies in view of the above problems, PMNs are prevented from cell adhesion and / or cell migration in blood vessels as a countermeasure against diseases such as autoimmune diseases caused by excessive function of polymorphonuclear leukocytes (PMNs). Or it is possible to use the medicine which suppresses. Therefore, using a plate in which a blood vessel wall is simulated, PMNs, a test substance, and an activator of PMNs are added to the plate, and then the number of cells or the amount of PMNs adhered to the plate It has been found that the effect of suppressing the PMN function of a drug can be evaluated inexpensively, easily and in a short time, and the present invention has been made.

  The method for screening a drug according to claim 1 of the present invention is a method for screening a drug effective for suppressing cell adhesion and / or cell migration of polymorphonuclear leukocytes, wherein the adhesion is coated on a solid support. At least polymorphonuclear leukocytes, a test substance, and an activator of the polymorphonuclear leukocytes are added to the sex substrate, and the number of adherent cells or the amount of adherent cells of the polymorphonuclear leukocytes to the adhesion substrate is measured. Thus, the cell adhesion ability of the polymorphonuclear leukocytes is evaluated.

  The drug screening method according to claim 2 of the present invention is characterized in that, in claim 1, the adhesive substrate is fibrinogen.

  Further, the drug screening method according to claim 3 of the present invention is characterized in that, in claim 1 or 2, the activator is phorbol myristic acid acetate.

  The drug screening method according to claim 4 of the present invention is characterized in that, in claim 1 or 2, the activator is phorbol myristate acetate and magnesium ion.

  Furthermore, the drug screening method according to claim 5 of the present invention is characterized in that in any one of claims 1 to 4, the number of adherent cells is measured using a phase contrast microscope.

  The drug screening method according to claim 6 of the present invention is the method for screening a drug according to any one of claims 1 to 4, wherein the adherent cells are stained with an aqueous solution containing crystal violet, and the amount of the adherent cells is measured using a spectrophotometer. It is characterized by measuring.

  According to the drug screening method of claim 1 of the present invention, a substance that suppresses cell adhesion and / or cell migration of polymorphonuclear leukocytes can be screened inexpensively, in a short time, and easily without performing animal experiments. It is possible to develop a medicine more efficiently. In addition, to screen for drugs caused by cell adhesion of polymorphonuclear leukocytes such as rheumatoid arthritis, asthma, allergic diseases, inflammatory skin diseases, atopic skin diseases, transplant rejection diseases, transplant rejection diseases, etc. Useful.

  According to the method for screening a drug according to claim 2 of the present invention, since cell adhesion ability is evaluated using fibrinogen which is present in a large amount in plasma and can be easily adjusted, other expensive adhesive substrates (for example, Compared with fibronectin, collagen, ICAM-1, etc.), it is possible to perform screening of many kinds of specimens at low cost.

  According to the drug screening method of claim 3 of the present invention, cell adhesion can be promoted by directly activating integrin expressed in polymorphonuclear leukocytes by phorbol myristic acetate.

  According to the drug screening method of claim 4 of the present invention, phorbol myristic acid acetate and magnesium ions can activate integrin expressed in polymorphonuclear leukocytes and promote cell adhesion.

  According to the method for screening a drug described in claim 5 of the present invention, it can be measured with a general-purpose laboratory instrument at a low cost in a short time.

  According to the drug screening method of the sixth aspect of the present invention, it can be measured with a general-purpose experimental instrument at a low cost in a short time.

  Hereinafter, the present invention will be described in more detail. The method for screening a drug of the present invention is a method for screening a drug effective for suppressing cell adhesion and / or cell migration of polymorphonuclear leukocytes, wherein at least an adhesive substrate coated on a solid support is applied to at least By adding polymorphonuclear leukocytes, a test substance, and an activator of the polymorphonuclear leukocytes, and measuring the adherent cells of the polymorphonuclear leukocytes to the adhesion substrate, the cells of the polymorphonuclear leukocytes It is characterized by evaluating adhesive ability.

  The cell adhesion referred to in the present invention means a state in which a cell is attached to a substrate and spreads flatly after being attached to the substrate to cause spreading. In addition, in order for cells to extend, reorganization of the intracellular actin skeleton system is necessary.

  The adhesive substrate used in the present invention refers to various substrates that bind to β2-integrin (CD11b / CD18), which is one of cell adhesion molecules expressed on the cell surface of polymorphonuclear leukocytes (PMNs). Include. Specific examples include fibrinogen which is an extracellular matrix, ICAM-1 expressed on vascular endothelial cells, or iC3b which is a degradation product of complement C3.

  The support used in the present invention includes a culture plate suitable for immobilizing an adhesive substrate.

The activator of polymorphonuclear leukocytes used in the present invention is a substance that activates β2 integrin by stimulating PMNs and induces cell adhesion of PMNs via β2 integrin. Specific examples include phorbol myristate acetate (phorbol 12-myristate 13-acetate, PMA), Mg ^{2+ and the} like. It is known that the activation of integrin is induced by directly activating protein kinase C (PKC) with PMA (Fagerholm S, et al, J Biol Chem 277 (3), 1728-1738, 2002). ). It is also known that Mg ²⁺ significantly increases the substrate binding ability by binding to the Mg ²⁺ / Ca ²⁺ binding site in the extracellular region of the CD11b chain (Yan SR, et al, J Inflamm 45 (4), 297-311, 1995). Further, Ca ²⁺ is not directly involved in integrin substrate binding, and is thought to contribute to maintaining the structure of the activated integrin molecule.

  In the present invention, the cell adhesion ability of PMNs is evaluated by measuring the adherent cells of PMNs to the adhesion substrate. Examples of methods for measuring and evaluating the adherent cells include the following two methods.

1. Morphological evaluation Test substances, Mg ²⁺ , and PMNs are added to an adhesive substrate coated on a solid support to induce cell adhesion. Thereafter, the morphological change of the cells is observed with a general-purpose experimental instrument such as a phase-contrast microscope (for example, magnification: 200-600 times), and stretched cells that are flattened by adhering to the adhesive substrate The number of cells and the number of unattached cells are measured, and the spreading rate is calculated, and then the spreading evaluation is performed.

2. Crystal Violet Staining Method A test substance, Mg ²⁺ , and PMNs are added to an adhesive substrate coated on a solid support to induce cell adhesion. Thereafter, the cells are washed with HEPES buffer to remove non-adherent cells, and crystal violet staining is added to the plate on which the adherent cells remain to stain cell nuclei. After staining, the staining solution is removed, and excess staining solution is removed by washing with HEPES buffer. Next, after adding an elution buffer, the mixture is stirred to elute crystal violet, the absorbance at a wavelength of 540 nm is measured, the relative adhesion rate (%) is calculated, and then cell adhesion is evaluated.

  The drug screening method of the present invention is a method that uses a general-purpose laboratory instrument, does not perform animal experiments, and inexpensively, in a short time and easily, a substance that suppresses cell adhesion and / or cell migration of polymorphonuclear leukocytes. It is possible to screen. Furthermore, the active ingredient can be analyzed in detail using the characteristics that can be screened in a short time and simply. In other words, after purifying a substance having an active ingredient, by applying the obtained fractions to the screening method of the present invention and narrowing down candidate compounds, the active ingredient can be identified and drug development can be performed more efficiently. it can.

  The drug screening method of the present invention screens drugs for diseases associated with tissue damage caused by PMNs, such as rheumatoid arthritis, asthma, allergic diseases, inflammatory skin diseases, atopic skin diseases, transplant rejection diseases, and the like. Useful for.

  Furthermore, it is known that extracellular matrix molecules (for example, fibronectin) involved in adhesion between cells and extracellular matrix are also involved in cancer cell adhesion and cell migration in the process of cancer metastasis. Therefore, polymorphonuclear leukocytes in the screening method of the present invention may be used in place of cancer cells for the development of drugs that act as cancer metastasis inhibitors.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited thereto.

Evaluation of anti-inflammatory effect of drug by PMNs cell adhesion ability test: This evaluation method evaluates the anti-inflammatory effect by measuring the adhesion ability of PMNs to the surface on which the fibrinogen coat is applied. Specifically, PMNs are suspended in a solution containing a test substance, and adhesion is induced by stimulation with Mg ²⁺ which is an activator of PMNs and phorbol myristate acetate (phorbol 12-myristate 13-acetate, PMA). Then, morphological evaluation by photography and colorimetric quantitative evaluation by crystal violet staining / staining liquid extraction of cells were performed. The work procedure is described below.

(1) Preparation of fibrinogen-coated plate Fibrinogen was dissolved in HEPES buffer (5 mM HEPES / 150 mM NaCl / 5 mM glucose), and this fibrinogen solution (1 mg / mL) was dispensed into a 96-well plastic microplate in 300 μL portions at room temperature. After standing for 1 hour or longer, the solution was discarded and washed 3 times with HEPES buffer to prepare a fibrinogen-coated plate.

(2) Adhesion ability test 1: morphological evaluation A test substance, Mg ²⁺ (final concentration 0.6 mM), and PMNs (final concentration 3 × 10 ⁶ cells) were prepared on the fibrinogen-coated plate prepared as described in (1) above. / ML) and made up with HEPES buffer to a final volume of 100 μL. Next, after stirring, preincubate for 10 minutes at 37 ° C., and dilute PMA to 50 μg / mL with HEPES buffer so that this PMA is added to a fibrinogen-coated plate to which PMNs and the like are added to a final concentration of 1 μg / mL. Added. The cells were further incubated at 37 ° C. for 20 minutes to induce cell adhesion. Microscopic images of the cells with a phase contrast microscope were taken with a digital camera, and the number of stretched cells that were flattened after adhering to fibrinogen and the number of cells that were not adhered were measured from the photographs to evaluate the stretch. In addition, the extension rate (Spreading) (%) was computed as the extension rate as the number of extended PMNs / total number of PMNs × 100 (%). The number of cells was counted for each of the randomly photographed images, and the average value ± standard deviation was indicated.

(3) Adhesive ability test 2: Crystal violet staining method As shown in (2) above, after induction of adhesion with PMA, the cells were washed three times with 100 μL of HEPES buffer to remove non-adherent cells. To the plate on which the adherent cells remained, 100 μL of a 0.4 wt / v% aqueous solution of crystal violet was added to stain the cell nucleus for 30 minutes. After staining, the staining solution was removed, and the excess staining solution was removed by washing with HEPES buffer. After adding 100 μL of the eluate, the mixture was stirred to elute crystal violet, and the absorbance at a wavelength of 540 nm was measured. For the evaluation of cell adhesion, relative adhesion rate (%) was calculated with the cell adhesion (positive control) when 0.6 mM Mg ²⁺ and 1 μg / mL PMA was added to the fibrinogen coated plate as 100%. .

(4) Effect of extracellular pH The extension rate and relative adhesion rate of PMNs were examined when the pH of the HEPES buffer was changed to 7.2, 6.9, 6.6, 6.3, and 6.0, respectively. It was. In addition, as a test substance, the HEPES buffer solution of each pH was used, and the elongation ratio and the relative adhesion ratio were calculated by the same method as in the adhesion test 1 and 2 described above except for the pH.

  The results are shown in FIGS. FIG. 1 shows that in the morphological evaluation, the pH of the HEPES buffer was changed to (a) pH 7.2, (b) pH 6.9, (c) pH 6.6, (d) pH 6.3, (e) pH 6.0. The microscope image of the cell when changing is shown. In the figure, black cells indicate cells that have adhered and flattened to fibrinogen, while white cells indicate non-flat cells. FIG. 2 is a graph showing the influence of extracellular pH on extension by calculating the extension rate (%) by counting the number of PMNs extended and the total number of PMNs from the photograph of FIG. The test was performed 4 times and a standard error bar was attached. FIG. 3 is a graph showing the effect of extracellular pH on cell adhesion. As shown in FIGS. 1 to 3, cell adhesion and decrease in extension after adhesion were observed as the pH decreased. In addition, when comparing the morphological evaluation of FIG. 2 and the crystal violet staining method of FIG. 3, the relative adhesion rate obtained by the crystal violet staining method is higher than the extension rate obtained from the morphological evaluation. There was little influence. This is probably because PMNs cells do not reach flatness but there are PMNs adhered to fibrinogen.

Cell adhesion ability test of polymorphonuclear leukocytes using watercress extract as test substance:
Watercress powder was extracted with hot water, the extract was powdered by freeze-drying, and then a methanol extract extracted with 100% methanol was recovered. Thereafter, methanol contained in the methanol extract was removed by evaporation. The extract from which methanol was removed was dissolved (100 mg equivalent / mL) in HEPES buffer (pH 7.2) and used as a test substance. Further, the stretch rate (%) was calculated by the same method as the morphological evaluation method described in Example 1. The test was performed 4 times and a standard error bar was attached. The results are shown in FIG. Further, the relative adhesion rate (%) was calculated by the same method as the crystal violet staining method described in Example 1. The test was performed three times and a standard error bar was attached. The results are shown in FIG.

  As shown in FIG. 4, the product without the watercress extract (concentration 0 mg equivalent / mL) showed an extension of about 78%, whereas the product with the watercress extract added (concentration 100 mg equivalent / mL). ) Only about 10% showed extension. On the other hand, as shown in FIG. 5, those without added watercress extract (concentration 0 mg equivalent / mL) were those with PMNs cells adhering to about 100% fibrinogen and added watercress extract (concentration 100 mg equivalent / mL). mL) PMNs cells adhered to about 80% fibrinogen. From this result, it was confirmed that the watercress extract component significantly suppressed the extension of polymorphonuclear leukocytes. Moreover, since cell adhesion shows the state which the cell has adhered to the substrate and has produced the extension, it is thought that the watercress extract suppresses only extension and does not suppress adhesion. In addition, watercress is conventionally said to be effective for asthma, and in the method of the present invention, since the watercress extract suppresses cell spreading of polymorphonuclear leukocytes, the method of the present invention is effective for asthma and the like. It is considered useful for screening for drugs effective for allergic diseases.

Cell adhesion ability test of polymorphonuclear leukocytes using cytochalasin B which is a polymerization inhibitor of actin as a test substance:
First, the extension ratio (%) was calculated by the same method as the morphological evaluation method described in Example 1. The test was performed 4 times and a standard error bar was attached. The results are shown in FIG. Next, the relative adhesion rate (%) was calculated by the same method as the crystal violet staining method described in Example 1. The test was performed three times and a standard error bar was attached. The results are shown in FIG.

  As shown in FIG. 6, the stretch ratios when cytochalasin B was not added (concentration 0 μM) and when cytochalasin B was added (concentration 2 μM) were about 77% and about 21%, respectively. On the other hand, as shown in FIG. 7, the relative adhesion rate (%) when cytochalasin B was not added (concentration 0 μM) and when cytochalasin B was added (concentration 2 μM) was about 109% and about 118%, respectively. there were. Thus, cell adhesion refers to a state in which cells are attached to the substrate and cause extension, and thus it is considered that cytochalasin B only suppresses extension and does not suppress attachment. In addition, since the extension after cell adhesion requires reorganization of the intracellular actin skeleton, the extension was suppressed by the inhibition of actin polymerization by cytochalasin B, which is known as an actin polymerization inhibitor. This has been observed by the method of the present invention. Therefore, the method of the present invention can evaluate the function inhibitory effect of polymorphonuclear leukocytes of a test substance (drug etc.), and can screen for a drug effective for suppressing not only cell adhesion but also cell migration.

In the morphological evaluation of Example 1 of the present invention, the pH of the HEPES buffer was changed to (a) pH 7.2, (b) pH 6.9, (c) pH 6.6, (d) pH 6.3, (e) pH 6 The microscopic image of the cell when changed to 0.0 is shown. It is a graph which shows the influence of extracellular pH with respect to the cell extension of Example 1 of this invention. It is a graph which shows the influence of extracellular pH with respect to the cell adhesion of Example 1 of this invention. It is a graph which shows the result of the cell extension by the watercress extract of Example 2 of this invention. It is a graph which shows the result of the cell adhesion by the watercress extract of Example 2 of this invention. It is a graph which shows the result of the cell extension by cytochalasin B of Example 3 of this invention. It is a graph which shows the result of the cell adhesion by cytochalasin B of Example 3 of this invention.

Claims (6)

  A method for screening a drug effective for suppressing cell adhesion and / or cell migration of polymorphonuclear leukocytes, comprising an adhesive substrate coated on a solid support, at least polymorphonuclear leukocytes, a test substance, The polymorphonuclear leukocyte activator is added, and the cell adhesion ability of the polymorphonuclear leukocyte is evaluated by measuring the number of adherent cells or the amount of adherent cells of the polymorphonuclear leukocyte to the adhesion substrate. A method for screening a drug characterized by comprising:   The method for screening a drug according to claim 1, wherein the adhesive substrate is fibrinogen.   The method for screening a drug according to claim 1 or 2, wherein the activator is phorbol myristic acetate.   The method for screening a drug according to claim 1 or 2, wherein the activator is phorbol myristic acid acetate and magnesium ion.   The method for screening a drug according to any one of claims 1 to 4, wherein the number of adherent cells is measured using a phase contrast microscope. The method for screening a drug according to any one of claims 1 to 4, wherein the adherent cells are stained with an aqueous solution containing crystal violet, and the amount of the adherent cells is measured using a spectrophotometer.