JP2008131899A - Method for screening endoplasmic reticulum stress-related substance, and cell-evaluating chip for screening endoplasmic reticulum stress-related substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening an endoplasmic reticulum stress-related substance by which the substance related to the endoplasmic reticulum stress can readily and precisely be judged by utilizing an easily available cell line having a high reproduction rate, and to provide a cell-evaluating chip. <P>SOLUTION: The method for screening the endoplasmic reticulum stress-related substance includes culturing cells by changing the concentration of the endoplasmic reticulum stress-inducing substance stepwise, and by evaluating the influence of the test substance on the endoplasmic reticulum stress from the difference of the recovering concentration of the cell survival rate by the presence or absence of the test substance. In another embodiment, the method includes carrying out the cell culture by changing the concentration of the endoplasmic reticulum stress-inducing substance, and by evaluating the influence of the test substance from the difference of a BiP detection-starting concentration by the presence or absence of the test substance. Both the methods can be used in combination. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a screening method for screening a substance involved in endoplasmic reticulum stress, and further relates to a cell evaluation chip used for the screening.

  Oxidative stress is known as a biological stress related to the onset of diseases, and various functional substances that suppress this have been searched and studied. On the other hand, endoplasmic reticulum stress has recently been found as a new stress, and it has been suggested to be involved in various diseases as well as oxidative stress.

  Endoplasmic reticulum stress refers to a state in which unstable proteins in the course of biosynthesis in the endoplasmic reticulum fail to build a normal folding structure due to physical and chemical stimulation, and accumulate in the endoplasmic reticulum as abnormal proteins. . In the endoplasmic reticulum, unstable proteins in the course of biosynthesis are susceptible to physical and chemical stimuli, and these stimuli change into abnormal proteins with an abnormal folding structure. Proteins that are correctly folded in the endoplasmic reticulum are transported to the Golgi apparatus, while the abnormal protein that fails to fold is retained in the endoplasmic reticulum.

  In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, prion disease, and amyotrophic lateral sclerosis (ALS), abnormal proteins often form aggregates in nerve cells. It has been observed that the association with neurodegeneration has been strongly suggested. Recently, the relevance between endoplasmic reticulum stress and neuronal cell death has been pointed out one after another, and the role of endoplasmic reticulum stress in neurodegenerative diseases is attracting attention. In addition, endoplasmic reticulum stress is likely to occur in cells that actively synthesize proteins, such as pancreatic β cells that actively synthesize and secrete insulin, suggesting that it is also involved in the development of lifestyle-related diseases such as diabetes. Has been.

  Currently, oxidative stress is attracting a great deal of attention in the health-related market due to its wide involvement in aging and disease, but endoplasmic reticulum stress is likely to be a major factor comparable to this, and in the future, endoplasmic reticulum stress Interest is also expected to increase.

From such a situation, for example, research on the measurement method of endoplasmic reticulum stress and the search for substances that affect endoplasmic reticulum stress have been promoted in various directions (see, for example, Patent Literature 1 to Patent Literature 7). Useful information has been gained in research on endoplasmic reticulum stress.
JP 2001-066302 A JP 2003-212790 A JP 2004-309186 A Japanese Patent Laid-Open No. 2005-065652 JP 2005-204516 A JP 2005-245247 A JP 2006-034288 A

  By the way, many of the techniques described in each of the above-mentioned patent documents are such that the endoplasmic reticulum can be applied to special cells that have been genetically modified, or transgenic animals, specific pathological animals, specific cells lacking a specific gene, etc. The present invention relates to a technique in which a stress-inducing substance and a test substance are allowed to act, for example, to examine changes specific to apoptosis, and both require genetically modified cells and genetically modified animals.

  However, genetically modified cells and genetically modified animals are not easy to create, and there are many problems in making them general-purpose methods in terms of cost and the like. Moreover, although the method of measuring the expression level of an endoplasmic reticulum stress marker is proposed, there exists a problem that a complicated biochemical method is required. Furthermore, for example, Patent Document 6 proposes to search for substances that affect endoplasmic reticulum stress by simply examining apoptosis, but there are various variable factors, and the criteria for discrimination are not clear. Therefore, it is difficult to accurately select substances that affect endoplasmic reticulum stress.

  The present invention has been proposed in view of such a conventional situation, and it is a simple technique using a cell line that is easily available and has a high growth rate, but accurately identifies substances involved in endoplasmic reticulum stress. It is an object of the present invention to provide a screening method for an endoplasmic reticulum stress-related substance that can be performed, and further to provide a cell evaluation chip for screening an endoplasmic reticulum stress-related substance used therefor.

  In order to achieve the above-mentioned object, the screening method for a substance involved in endoplasmic reticulum stress according to the present invention comprises culturing cells while changing the concentration of an endoplasmic reticulum stress inducer in stages, and recovering cell viability by the presence or absence of a test substance. It is characterized in that the influence of the test substance on the endoplasmic reticulum stress is evaluated based on the difference in concentration. Alternatively, the concentration of the endoplasmic reticulum stress inducer is changed in stages, the cells are cultured, and the influence of the test substance on the endoplasmic reticulum stress is evaluated based on the difference in the BiP detection start concentration depending on the presence or absence of the test substance. It is characterized by. Furthermore, the concentration of the endoplasmic reticulum stress-inducing substance is changed in stages, and the cells are cultured. Based on the difference in the recovery concentration of the cell viability due to the presence or absence of the test substance and the difference in the BiP detection start concentration, It is characterized by evaluating the effect on ER stress.

  The inventors of the present invention have made various studies by searching for a method for accurately discriminating substances involved in endoplasmic reticulum stress. As a result, they found that when the concentration of the endoplasmic reticulum stress inducer exceeds a certain threshold, cell death is suppressed and cell viability is restored. That is, it has been found that as the concentration of the endoplasmic reticulum stress inducer acting on the cells is gradually increased, the cell viability decreases, but the cell viability is rapidly recovered at a certain concentration. It has been found that the concentration of the endoplasmic reticulum stress-inducing substance when the cell viability is restored shifts to a low concentration side or a high concentration side when substances involved in endoplasmic reticulum stress coexist.

  The first screening method of the present invention utilizes the above phenomenon. That is, when the concentration of the endoplasmic reticulum stress inducer is changed in stages and the cells are cultured, the cell viability recovery concentration (the endoplasmic reticulum stress inducer concentration at which the cell viability is restored) changes depending on the presence or absence of the test substance. The test substance is determined to be involved in endoplasmic reticulum stress.

  In addition, when an intracellular signal transduction system of endoplasmic reticulum stress protection mechanism possessed by cells was examined for BiP, which is one of the proteins that activate this mechanism, the concentration of endoplasmic reticulum stress inducer was also found. When a certain threshold value was exceeded, BiP was detected, and when a substance involved in endoplasmic reticulum stress coexists, it was found that the concentration at which this BiP was detected also shifted.

  The second screening method of the present invention utilizes such a phenomenon. That is, cells are cultured while changing the concentration of the endoplasmic reticulum stress inducer in stages, and when the BiP detection start concentration (the lowest endoplasmic reticulum stress inducer concentration at which BiP is detected) changes depending on the presence or absence of the test substance, The test substance is determined to be involved in endoplasmic reticulum stress.

  Furthermore, by discriminating based on the results of both of these, substances involved in endoplasmic reticulum stress are more accurately selected.

  If a substance that suppresses endoplasmic reticulum stress or, conversely, a substance that promotes endoplasmic reticulum stress can be selected accurately, it will be a major advance in researching endoplasmic reticulum stress. In the screening method of the present invention, genetically modified cells, genetically modified animals, etc. are not necessary, and screening can be performed using established cell lines widely used in the field of cell biology. In addition, a complicated biochemical method is unnecessary. Furthermore, unlike a technique for simply examining apoptosis or the like, it is difficult to be affected by cell culture accuracy and measurement accuracy, and can accurately grasp whether or not it affects endoplasmic reticulum stress.

  On the other hand, in the cell evaluation chip for screening an ER stress-inducing substance of the present invention, a first channel into which an ER stress inducer solution is introduced and a diluent for diluting the ER stress inducer solution are introduced. A second flow path is formed substantially in parallel, a plurality of branch flow paths are formed at predetermined intervals in the first flow path, cell culture cells are formed in the branch flow paths, and each branch flow is formed. A connecting flow path connecting the first flow path and the second flow path is formed at an upstream position of the path.

  In the cell evaluation chip having the above-described configuration, the ER stress inducer solution having a predetermined concentration is flowed through the first flow path, and the dilute liquid is flowed through the second flow path. A concentration of endoplasmic reticulum stress inducer solution is provided. Therefore, cell culturing performed by changing the concentration of the aforementioned endoplasmic reticulum stress inducer in stages is performed in a single cell evaluation chip, thereby realizing efficient screening.

  According to the present invention, it is possible to accurately discriminate a substance involved in endoplasmic reticulum stress while being a simple technique using a cell line that is easily available and has a high proliferation rate. In addition, there is not one type of method for reducing endoplasmic reticulum stress, but it can include induction of endoplasmic reticulum chaperones, attenuation of translation, and degradation of abnormal proteins. To make these three types function efficiently, reduce stress. Sometimes it is better to increase it. In the present invention, it is possible to perform evaluation by setting a wide concentration range, and it is possible to perform wide evaluation on increase and decrease of stress. Therefore, by utilizing the present invention, it is possible to dramatically develop research on endoplasmic reticulum stress.

  Hereinafter, a screening method for an endoplasmic reticulum stress-involved substance to which the present invention is applied and a cell evaluation chip for endoplasmic reticulum stress-involved substance screening will be described in detail with reference to the drawings.

  Endoplasmic reticulum stress means that unstable proteins in the course of biosynthesis in the endoplasmic reticulum are susceptible to physical and chemical stimuli, and that stimulation causes accumulation of proteins with an abnormally folded structure in the endoplasmic reticulum. . In response to these endoplasmic reticulum stresses, the cells basically undergo ER-specific stress response mechanisms (crisis management) called UPR (Unfolded protein response) and ERAD (Endoplasmic reticulum-associated degradation), resulting in abnormal protein accumulation. Try to avoid bankruptcy.

  The ERAD is a system in which abnormal proteins in the endoplasmic reticulum are dragged to the cytoplasm side by an unexplained molecular mechanism involving ubiquitination and degraded by the proteasome. UPR, on the other hand, induces a molecular chaperone localized in the endoplasmic reticulum at the transcription level to prevent transcription of abnormal proteins, and translation suppresses protein synthesis at the translation level to reduce the endoplasmic reticulum load. It is divided into a suppression mechanism. For example, mammalian UPR is skillfully controlled by at least three types of endoplasmic reticulum transmembrane proteins, IRE1, PERK, and ATF6, which are expressed with BiP expression. For example, PERK suppresses translation and suppresses the generation of abnormal proteins. ATF6 induces transcription and enhances the folding ability in the endoplasmic reticulum. IRE1 activates ERAD-related factors and enhances degradation capacity.

  BiP binds to the sensor regions of IRE1, PERK, and ATF6 when not stressed, and dissociates when stressed to multimerize IRE1 and PERK, and ATF6 is activated with a monomer. That is, BiP functions as an endoplasmic reticulum stress sensor. It is known that activated IRE1 shifts to the apoptotic pathway, PERK shifts to translational repression, and ATF6 shifts to transcriptional repression.

  Although the detailed mechanism of the screening method of the present invention is unknown, it can be said that the above-described stress response mechanism is used, and it serves as a recovery of cell viability based on the stress response mechanism or as an endoplasmic reticulum stress sensor. By analyzing BiP, it is determined whether or not the test substance is involved in endoplasmic reticulum stress.

  First, a first embodiment of the screening method of the present invention will be described. The screening method of the first embodiment utilizes recovery of cell viability based on a stress response mechanism, and cultivates cells by changing the concentration of an endoplasmic reticulum stress inducer in stages, and the presence or absence of a test substance. The change of the cell viability recovery concentration (the endoplasmic reticulum stress inducer concentration at which the cell viability is restored) due to is observed. Then, the effect of the test substance on endoplasmic reticulum stress-induced apoptosis is evaluated using the pattern as an index. Specifically, when the concentration of the endoplasmic reticulum stress inducer that restores cell viability when the test substance is added or not is changed (shifted), the test substance is involved in the endoplasmic reticulum stress. Judge that. When the change is not observed, it is determined that the test substance is not involved in endoplasmic reticulum stress.

  FIG. 1 schematically shows the relationship between the endoplasmic reticulum stress inducer concentration and cell viability. As the endoplasmic reticulum stress inducer concentration increases, the cell viability gradually decreases, but at a certain concentration, the cell viability is rapidly recovered. Although the details are not clear about the reason, it is presumed that the above-mentioned stress response mechanism is involved. Here, for example, the cell viability recovery concentration when no test substance is added is C1. When a similar experiment is performed in a system to which a test substance is added, the cell viability recovery concentration may shift to a low concentration side and become C2, as indicated by a one-dot chain line in the figure. In such a case, it is presumed that the test substance had some influence on the endoplasmic reticulum stress. For example, the shift to the low concentration side is considered to suggest an action of suppressing endoplasmic reticulum stress-induced apoptosis in a test substance or an action of recovering endoplasmic reticulum stress.

  When the relationship between the endoplasmic reticulum stress inducer concentration and the cell viability is examined, minute peaks and valleys may be observed due to various variation factors or measurement errors in cell culture. In such a case, as an index for determining whether or not the cell viability recovery concentration is reached, a difference in the viability before and after the cell viability is recovered can be mentioned. For example, in FIG. 1, when the survival rate L1 in the valley before the cell survival rate is recovered and the survival rate in the mountain after the cell survival rate is recovered is L2, the difference between the survival rate L1 and the survival rate L2 ( If L2-L1) is 50% or more of the survival rate L2, it may be determined that the cell survival rate is restored.

  In order to perform the screening of the first embodiment described above, it is necessary to culture cells in which the concentration of the endoplasmic reticulum stress-inducing substance is changed in stages depending on whether the test substance is present or not. At this time, any cell can be used as the cell to be used, but a cell line that is readily available, stable, and has a high growth rate is preferable. Specific examples include human leukemic T cell lines (Jurkat cells), melanoma cells, PC12 cells, colon cancer cells, and the like.

  Moreover, the endoplasmic reticulum stress inducer to be used is also arbitrary. For example, in addition to tunicamycin, thapsigargin (Tg) which decreases Ca in the endoplasmic reticulum, Brefeldin A (BFA) which stops transport between the endoplasmic reticulum and the Golgi apparatus, 2-deoxyglucose (2-DG) which causes depletion of glucose, reduction Although DTT etc. which are agents can be mentioned, tunicamycin is the most typical endoplasmic reticulum stress inducer. The concentration range of the endoplasmic reticulum stress inducer and the step of concentration when changing the concentration stepwise can be arbitrarily set. For example, when tunicamycin is used, the concentration range is about 0 to 2.5 μM, The concentration may be changed in steps of about 0.2 μM.

  Cell viability can be calculated, for example, by a cell proliferation test similar to the MTT assay. In addition, for example, apoptosis or necrosis can be determined by staining cultured cells with a predetermined dye and observing the cells with a fluorescence microscope or the like. For example, the trade name Hoechst 33342 passes through the nuclear membrane, is taken up by DNA and emits blue fluorescence, and thus can be used to detect living cells and early apoptosis. Propidium iodide (PI) does not permeate the nuclear membrane, is taken up by the DNA of dead cells and emits red fluorescence, and therefore can be used for detection of late apoptosis and necrosis.

  The above is the first embodiment of the screening method of the present invention. In addition to the restoration of the cell viability, it is determined whether or not the test substance is involved in endoplasmic reticulum stress by the aforementioned BiP analysis. Is possible. This is the second embodiment of the screening method of the present invention.

  As described above, it is speculated that BiP works as an endoplasmic reticulum stress sensor. Therefore, it is considered that the state of endoplasmic reticulum stress can still be grasped by analyzing this BiP. As a result of repeated experiments based on such knowledge, the present inventors have found that when the endoplasmic reticulum stress inducer concentration increases, BiP is detected when a predetermined concentration (BiP detection start concentration) is reached, When the test substance is involved in endoplasmic reticulum stress, the inventors have found that the BiP detection start concentration shifts.

  The present embodiment uses this to determine whether or not the test substance is involved in endoplasmic reticulum stress. That is, the BiP detection start concentration is monitored, and when the BiP detection start concentration changes (shifts) between when the test substance is added and when the test substance is not added, as in the first embodiment, Is considered to be involved in ER stress. When the change is not observed, it is determined that the test substance is not involved in endoplasmic reticulum stress.

  In the second embodiment as well, in the screening, it is the same that the cells cultured with the concentration of the endoplasmic reticulum stress-inducing substance changed in stages are used for cases where the test substance is present and not present. The same applies to cells and endoplasmic reticulum stress inducers used. BiP analysis can be performed by electrophoresis or the like.

  Screening based on the change in the cell viability recovery concentration depending on the presence or absence of the aforementioned test substance (screening in the first embodiment), and screening based on the change in the BiP detection start concentration depending on the presence or absence of the test substance (second embodiment) Screening) may be performed alone or in combination. By using these two screenings together, more accurate evaluation becomes possible. For example, if the cell viability recovery concentration shifts to a lower concentration side by addition of the test substance and the BiP detection start concentration also shifts to the lower concentration side, the test substance suppresses endoplasmic reticulum stress-induced apoptosis with considerable probability. It is possible to predict that it has an action or an action to restore endoplasmic reticulum stress.

  Next, a cell evaluation chip capable of easily and collectively culturing cells in which the concentration of an endoplasmic reticulum stress inducer is changed in stages will be described.

  FIG. 2 shows an example of a cell evaluation chip to which the present invention is applied. This cell evaluation chip has a first channel 1 into which an endoplasmic reticulum stress inducer solution is introduced on a substrate and a second channel 2 into which a diluent for diluting the endoplasmic reticulum stress inducer solution is introduced. It is formed in parallel (substantially parallel, and a slight angle deviation is allowed), and the branch flow paths 3a to 3f and the cell culture cells 4a to 4f, as well as the first flow path and the second flow path. The connecting flow paths 5a to 5f that connect these flow paths are formed.

  Here, a material such as polydimethylsiloxane (PDMS) can be used for the substrate, and chips of various shapes can be formed by optical processing or the like. A second substrate is stacked on the substrate on which each of the above-described flow paths and cell culture cells is formed to form a sealed structure, and a glass substrate is preferable as the second substrate. By using a glass substrate as the substrate to be stacked, the state of the cell culture cell can be confirmed through the glass substrate, and the cell culture cell can be directly subjected to spectroscopic analysis or the like. The PDMS and glass are both characterized by low toxicity to cells and withstand long-time infiltration.

  The branch flow paths 3a to 3f are branched from the first flow path 1 in the orthogonal direction, and are provided in the first flow path 1 at substantially equal intervals. For example, circular cell culture cells 4a to 4f are formed in the middle of each of the branch channels 3a to 3f, and cell culture is performed in the cell culture cells 4a to 4f. In the cell culture cells 4a to 4f, discharge channels 6a to 6f are formed as downstream portions of the branch channels 3a to 3f, and excess solution and the like are discharged from here. Moreover, a cultured cell can be introduce | transduced into each cell culture cell 4a-4f from these discharge flow paths 6a-6f.

  In the first flow channel 1, a connecting flow that branches from the second flow channel and connects the first flow channel 1 and the second flow channel 2 is located upstream of the branch flow channels 3 a to 3 f. Paths 5a to 5f are formed. The endoplasmic reticulum stress inducer solution introduced into the first channel 1 is sequentially diluted with a diluent (buffer) flowing into the first channel 1 through the connection channels 5a to 5f, and the first flow It mixes in the downstream parts 1a-1f of the path | route 1, and is inject | poured into each cell culture cell 4a-4f. Therefore, endoplasmic reticulum stress inducer solutions having different concentrations are injected into the cell culture cells 4a to 4f. For example, the ER stress inducer solution with the highest concentration is injected into the cell culture cell 4a, and the ER stress inducer solution with the lowest concentration is injected into the cell culture cell 4f. The concentration of the endoplasmic reticulum stress inducer solution injected into each of the cell culture cells 4a to 4f can be arbitrarily changed, for example, by changing the thickness (cross-sectional area) of the connection channels 5a to 5f. .

  When screening a substance to be detected using the above-described cell evaluation chip, first, an equal amount of cells are introduced and fixed in the cell culture cells 4a to 4f of the cell evaluation chip. The cells to be introduced are, for example, human leukemic T cell lines (Jurkat cells), melanoma cells, PC12 cells, colon cancer cells and the like. The living cells are adsorbed in the cell culture cells 4a to 4f and are naturally fixed.

  Next, an endoplasmic reticulum stress inducer solution having a predetermined concentration is injected from the inlet 10 of the first channel 1, and a diluent (buffer) is injected from the inlet 20 of the second channel 2. As a result, endoplasmic reticulum stress inducer solutions having different concentrations in stages are injected into the cell culture cells 4a to 4f. After the injection of the endoplasmic reticulum stress inducer solution is completed, the cells are cultured in the cell culture cells 4a to 4f under predetermined culture conditions.

  After cell culture, cell viability is measured by spectroscopic analysis or the like in the cell culture cells 4a to 4f. Or you may take out the cultured cell from each cell culture cell 4a-4f, and may measure a cell viability. The relationship between the endoplasmic reticulum stress inducer concentration measured here and the cell viability becomes the reference pattern, and the concentration at which the cell viability recovers becomes the reference endoplasmic reticulum stress inducer concentration.

  Next, the test substance is introduced and the cells are cultured, and the procedure in this case is almost the same. The only difference is that the endoplasmic reticulum stress inducer solution injected from the inlet 10 of the first channel 1 and the diluent (buffer) injected from the inlet 20 of the second channel 2 have the same concentration. Is to mix the test substance. By mixing the test substance of the same concentration (ideally the same concentration, ideally a slight deviation is allowed) into the endoplasmic reticulum stress inducer solution and diluent (buffer), The test substance concentrations in the endoplasmic reticulum stress inducer solution injected into the cell culture cells 4a to 4f are all the same. Moreover, the endoplasmic reticulum stress inducer concentration in the endoplasmic reticulum stress inducer solution injected into each cell culture cell 4a-4f changes in steps.

  Even when the test substance is introduced, the cells are cultured in the cell culture cells 4a to 4f under the same culture conditions. After cell culture, cell viability is measured by spectroscopic analysis or the like in the cell culture cells 4a to 4f. Or the cell cultured from each cell culture cell 4a-4f is taken out, and a cell viability is measured. The pattern obtained thereby is compared with the reference pattern, and when the concentration at which the cell viability is restored is shifted from the reference endoplasmic reticulum stress inducer concentration, the test substance is a substance that affects the endoplasmic reticulum stress. judge. Conversely, if the concentration at which the cell viability is restored is the same as the reference endoplasmic reticulum stress inducer concentration, the test substance is determined to be a substance that does not affect endoplasmic reticulum stress.

  In addition, about the cell culture | cultivated in each cell culture cell 4a-4f, you may evaluate a test substance by extracting and analyzing BiP, and the endoplasmic reticulum stress inducer which recovers the cell viability The test substance may be evaluated based on both the concentration and the BiP detection start concentration.

  Hereinafter, specific examples of the present invention will be described based on experimental results.

(1) Cell treatment (1-1) Cell culture The human leukemic T cell line Jurkat used from RIKEN Cell Bank. Jurkat cells were used in RPMI 1640 medium (GIBCO) containing 10% inactivated fetal bovine serum (FETAL BOVINE SERUM: FBS) (GIBCO) in a 150 mm petri dish (Corning) at 37 ° C. and 5% CO ₂ . Cultured. Passage was performed once every 3 days by adding 1/10 volume of the cell suspension to RPMI 1640 medium containing 10% inactivated FBS. For cell storage, cells that reached approximately 70% confluence were collected by centrifugation (1,000 rpm, 5 minutes) and suspended in a 1 ml cell banker (Nippon Zenyaku Kogyo Co., Ltd.) (1.5 × 10 ⁶ cells). / Ml) was added to a storage tube (IWAKI). Then, it put into the bicell and left still at -80 degreeC for 1 day, and preserve | saved in liquid nitrogen. Since cells lost their morphological changes when they were subcultured, stock cells stored once a month were thawed again and subcultured again. In addition, since cells immediately after thawing may have abnormal growth due to freezing stress, cells that have been passaged three or more times were used in the experiments.

(1-2) Tunicamycin (Tm) treatment The Tm medium was prepared as follows. Tm (WAKO) was dissolved in DMSO, diluted to an appropriate concentration with RPMI 1640 medium, applied to 0.22 μm New Terradisk (KURABO), and filter sterilized. Jurkat cells were transferred to a 15 ml centrifuge tube (IWAKI) after approximately 70% confluence, and centrifuged (1,000 rpm, 5 minutes). The number of cells was counted using a hemocytometer, adjusted to a cell concentration of 2 × 10 ⁵ cells / ml, and cultured in Tm medium under conditions of 37 ° C. and 5% CO _{2 for} an appropriate time.

(1-3) Simultaneous treatment of Tm and linoleic acid After linoleic acid was dissolved in DMSO, the linoleic acid was diluted to an appropriate concentration with RPMI 1640 medium, subjected to 0.22 μm New Terradisk (KURABO), and filter sterilized. Then, it added to Tm culture medium so that final concentration might be set to 25 micromol. Jurkat cells were transferred to a 15 ml centrifuge tube (IWAKI) after approximately 70% confluence, and centrifuged (1,000 rpm, 5 minutes). The number of cells was counted using a hemocytometer, adjusted to a cell concentration of 2 × 10 ⁵ cells / ml, and cultured in a Tm medium containing linoleic acid at 37 ° C. and 5% CO _{2 for} an appropriate time.

(2) Measurement of cell viability using cell proliferation test Cell viability was produced by a cell proliferation test similar to the MTT assay (Cell titer 96 Aqueous (Promega)). This is because the tetrazolium compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt) is contained in cells. It is utilized that it is converted into formazan having absorption at 490 nm by dehydrogenase. The structures of MTS and formazan are shown in Chemical Formula 1.

The concentration was adjusted by adding BPA to RPMI 1640 medium containing 10% inactivated FBS, and added to a 96-well plate (IWAKI). 2 × 10 ⁴ cells were added per well. 24 and 48 hours after the start of the treatment, 20 μl of a mixed solution of MTS / PMS (phenazine method) (MTS solution: PMS solution = 20: 1) was added to each well and cultured for 2 hours (37 ° C., 5% CO 2). ₂ ). After culturing, the absorbance at 490 nm was measured with a microplate reader (BIO RAD, model 450) (control wavelength: 655 nm). Based on the absorbance of untreated cells, the relative cell viability was calculated according to the following formula. In the formula, X represents the value of the medium alone. The cell survival rate after Tm treatment (24 hours treatment) is shown in FIG. 3, and the cell survival rate after Tm and linoleic acid simultaneous treatment (24 hours treatment) is shown in FIG.
Cell viability = (experimental value−X / control value−X) × 100

  First, as shown in FIG. 3, when the cells were exposed to Tm, the survival rate gradually decreased, and recovery of the survival rate was observed at around Tm 1.7 μM. This was not time dependent but concentration dependent. On the other hand, when linoleic acid as the test substance was added, the recovery point of the survival rate shifted to the low concentration side (around Tm 0.8 μM). Therefore, the linoleic acid can be determined to be a substance involved in endoplasmic reticulum stress.

(3) Western blotting (3-1) Sample preparation Jurkat cells were treated with various concentrations of Tm or Tm and linoleic acid, cultured for an appropriate time, and then samples (2 × 10 ⁶ cells) were collected. Cells collected in 50 μl of Lysis buffer (Table 1) were lysed, and 50 μl of 2 × SDS-PAGE sample buffer (Table 2) was added. The solution was uniformly dissolved by sonication with ice cooling for about 20 to 30 seconds. The mixture was heat-treated at 98 ° C. for 5 minutes, ice-cooled for 5 minutes, and centrifuged (12,000 rpm, 5 minutes), and subjected to 10% SDS-PAGE.

(3-2) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
A 40% acrylamide (Daiichi Kagaku) solution (Table 5) was added so that the concentration gel (Table 3) was 4% and the separation gel (Table 4) was 10%. As the electrophoresis buffer, an SDS-PAGE buffer (Table 6) was used. As the molecular weight marker, a prestained protein marker (Nacalai Tesque) was used.

(3-3) Blotting of target protein on PVDF membrane Polyvinylidene difluoride (PVDF) membrane of the same size as the SDS-PAGE gel is immersed in 100% methanol for 20 seconds, and about 30 minutes in solution B (Table 8). Shake. Two sheets of absorbent paper (ATTO) having the same size as the gel were immersed in solution A (Table 7), one in solution B, and three in solution C (Table 9). Moreover, the gel after electrophoresis was shaken in B liquid for 30 minutes. As a blotting apparatus, a horizon blot (ATTO) was used. Two sheets of absorbent paper immersed in the liquid A were stacked on the lower electrode wetted with the liquid A, and one sheet of absorbent paper immersed in the liquid B was stacked thereon. A PVDF membrane was further stacked on top, and the gel was placed so that bubbles could not enter over several ml of solution B. Thereafter, a small amount of liquid C was applied from above, and three sheets of absorbent paper immersed in liquid C were stacked in the same manner and pressed firmly from above to bring the gel, PVDF membrane, and absorbent paper into close contact. Finally, the upper electrode was lowered, and current was applied at 100 mA (about ² mA per 1 cm ^{2 of} gel area) for 35 minutes to blot the target protein on the PVDF membrane.

(3-4) Activation of BiP The PVDF membrane after blotting is immersed in 20 ml of blocking buffer (5% (w / v) skim milk in TBST (Table 10)) for 1 hour, and washed 3 times for 5 minutes each with TBST solution. Thereafter, the protein on the PVDF membrane and the primary antibody of BiP (Funakoshi, Anti-BiP, (H-129), Human (Rabbit)) were reacted overnight at 4 ° C. After the reaction, the plate was washed with TBST solution three times for 5 minutes, and the protein on the PVDF membrane and the secondary antibody (anti-rabbit IgG (SIGMA)) were reacted at room temperature for 2 hours. After the reaction, the plate was washed with TBST solution three times for 5 minutes, immersed in AP buffer (Table 12) for 5 minutes, and then immersed in a developing solvent (Table 13) for staining. After staining, the plate was washed 3 times for 5 minutes each with a TBS solution (Table 11). The detection result of BiP expression after Tm treatment (6 hour treatment) is shown in FIG. 5, and the detection result of BiP expression after Tm and linoleic acid simultaneous treatment (6 hour treatment) is shown in FIG.

  5 and 6, the BiP expression point (BiP detection start concentration) is shifted from 0.3 μM to 0.1 μM on the low concentration side by the addition of linoleic acid. Therefore, it can be judged from this that the linoleic acid is a substance involved in endoplasmic reticulum stress.

It is a figure which shows typically the relationship between an endoplasmic reticulum stress inducer density | concentration and cell viability. It is a schematic plan view which shows an example of a cell evaluation chip | tip. It is a figure which shows the cell survival rate after processing for 24 hours by Tm. It is a figure which shows the cell viability after carrying out simultaneous treatment with Tm and linoleic acid for 24 hours. It is a photograph which shows the detection result of BiP expression after processing for 6 hours by Tm. It is a photograph which shows the detection result of BiP expression after 6-hour simultaneous treatment with Tm and linoleic acid.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st flow path, 2nd flow path, 3a-3f branch flow path, 4a-4f cell culture cell, 5a-5f connection flow path, 6a-6f discharge flow path

Claims (9)

  Culturing the cells with different concentrations of the endoplasmic reticulum stress inducing substance, and evaluating the effect of the test substance on the endoplasmic reticulum stress based on the difference in the recovery concentration of cell viability depending on the presence or absence of the test substance A screening method for a characteristic endoplasmic reticulum stress-related substance.   Culturing the cells at different concentrations of the endoplasmic reticulum stress inducer, and evaluating the influence of the test substance on the endoplasmic reticulum stress based on the difference in the BiP detection start concentration depending on the presence or absence of the test substance A screening method for a substance involved in endoplasmic reticulum stress.   Culturing the cells by changing the concentration of the endoplasmic reticulum stress inducing substance in stages, to the endoplasmic reticulum stress of the test substance based on the difference in the cell viability recovery concentration depending on the presence or absence of the test substance and the difference in the BiP detection start concentration A screening method for a substance involved in endoplasmic reticulum stress, characterized by evaluating the effect of ER.   The screening method for a substance involved in endoplasmic reticulum stress according to any one of claims 1 to 3, wherein the cell is a cell line.   The screening for a substance involved in endoplasmic reticulum stress according to claim 4, wherein the cell is at least one selected from human leukemic T cell line (Jurkat cell), melanoma cell, PC12 cell, and colon cancer cell. Method.   6. The screening method for a substance involved in endoplasmic reticulum stress according to claim 1, wherein the endoplasmic reticulum stress inducer is tunicamycin. A first flow path into which the endoplasmic reticulum stress inducer solution is introduced and a second flow path into which a diluent for diluting the endoplasmic reticulum stress inducer solution is introduced are formed substantially in parallel.
A plurality of branch channels are formed at predetermined intervals in the first channel, and cell culture cells are formed in the branch channels,
A cell evaluation chip for screening an endoplasmic reticulum stress-related substance, wherein a connecting flow path connecting the first flow path and the second flow path is formed at an upstream position of each branch flow path.   8. A cell evaluation chip for screening an endoplasmic reticulum stress-related substance according to claim 7, wherein a discharge channel is formed in each cell culture cell, and cultured cells are introduced into the cell culture cell from these discharge channels. .   A test substance is dissolved in substantially the same concentration in an endoplasmic reticulum stress inducer solution introduced into the first channel and a diluent introduced into the second channel, and is introduced into the cell culture cell. The cell evaluation chip for screening an endoplasmic reticulum stress participation substance according to claim 7 or 8.

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