WO2016010397A1 - Method for screening anti-inflammatory drug or anti-cancer drug - Google Patents

Abstract

The present invention relates to a method for screening an anti-inflammatory drug or anti-cancer drug, a composition for inflammation or cancer diagnosis, and a kit comprising the composition, and relates to a method capable of: detecting inflammation and cancer in vitro more specifically compared with an existing analysis method, using a cell membrane receptor of animal cells as a recognition component of inflammation and cancer; being cell-interactive; performing repetitive measurements on the same cells; and continuously observing a step in which inflammation and cancer are induced and inhibited.

Description

Anti-inflammatory or anticancer screening method

The present invention relates to a method for screening an anti-inflammatory or anticancer agent, a composition for diagnosing inflammation or cancer, and a kit including the composition.

Every year, a large number of animals are used worldwide for human experiments. Various kinds of animals such as rodents such as rats and guinea pigs, rabbits, dogs, cats, cows, pigs, and chimpanzees are used throughout the entire society, including medical and new drug development, as well as schools, military facilities, industrial facilities, cosmetics and daily necessities. Sacrificed for the experiment. Many animal experiments are conducted without ethical considerations, although there are alternative experiments for reasons such as simply satisfying intellectual curiosity, defending against legal proceedings, or reducing costs, and animal experiments on cosmetics are the most representative examples.

At present, the development of alternative experiments that do not use animals is now more quickly and accurately verifying the safety of cosmetics that were only possible with animal experiments. For example, the ocular mucosa test, which was repeatedly administered to a rabbit's eye, was replaced by a HET-CAM test using an egg and a test using an artificial model to evaluate blood vessel changes to determine the degree of stimulation. However, in the case of such a one-time limiting test method, there are alternative test methods, but there is still a problem in confirming the efficacy or toxicity obtained through repeated administration, and experiments using animals are still preceded. In addition, in the case of pharmaceutical products related to life and death, not functional products such as cosmetics, clinical trials using animals must be performed before clinical trials applied to humans. However, even if animal testing is preceded, problems such as immune rejection due to different immune systems between animals and humans are still emerging. Therefore, it is very urgent to develop alternative experimental methods that can solve such problems.

The concept of an animal cell-based biosensor that can detect analytes such as bacteria, toxins, environmental hormones, and harmful compounds by using human cells and animal cells instead of genes or antibodies, which have recently been used as core detectors of target substance analysis methods. This is emerging. A characteristic of biosensors using cells is that they can observe the physiological response of cells to stimuli such as heat, chemicals, shock, and infection from outside. These physiological responses have been reported to provide useful information such as cellular metabolism, cytotoxic responses to cytotoxicity, and bioavailability of drugs. In addition, because these features are circulated by metabolism, it is well suited to continuously observe the life reaction.

Therefore, animal cell-based biosensor-based technology has the advantage of being able to be extended not only to the food field but also to the environmental fields such as agriculture, defense, fisheries and livestock, and especially in the field of animal testing such as cosmetics and pharmaceuticals. As a tool for replacing animals (Asphahani and Zhang 2007).

However, the current trend of cell-based biosensors is obtained by detecting electrical signal changes or pH changes caused by changes in cell metabolism and morphology to external stimuli, or by developing and emitting chemicals and enzymes secreted into and out of cells. Techniques for detecting signals have been reported. Therefore, the cell-based assays developed to date are limited to the physical or chemical reactions of cells to external stimuli and thus have low specificity and very limited applications.

Inflammation is a defensive reaction in the body when a living tissue is damaged, causing redness, swelling, fever, and pain in one part of the body, such as trauma, burns, and bacterial invasion. Recently, many studies have reported that inflammatory reactions are closely linked to cancer, aging, and chronic inflammatory allergic diseases (asthma, rheumatoid arthritis, Alzheimer's disease). (Inflammation suppression), and methods of treating it are of great interest.

Existing inflammatory observation methods are used to confirm the expression level changes of inflammatory mediators and the activity of NF-κB, a transcriptional regulator that induces inflammatory responses.

As a method of confirming the change in the expression level of inflammatory mediators, it is possible to check the expression level of mRNA by PCR, the method by competition reaction with derivatives containing radioisotopes, chemical reaction or phosphor such as Fura2-AM. The method can be used to measure nitric oxide or cytoplasmic calcium concentration. In addition, the method of confirming the activity of NF-κB, a transcriptional regulator that induces an inflammatory response, can be tested for gene adhesion activity, activity according to the presence of NF-κB (in cytoplasm or nuclear membrane), and cytokine (cytokine). Immunoassay.

Except for the measurement of cytokines, the other methods can be observed only once by crushing cells or causing cell damage through surfactant and chemical reagent treatment. In addition, the method of measuring the concentration of NO discharged from the cell is low sensitivity, the method of measuring the calcium concentration inside the cell is not specific to the inflammatory response. Therefore, there is an urgent need for the development of alternative assays to observe more specific and continuous inflammatory responses.

In order to solve the conventional problems as described above, the present invention detects the inflammation caused by infection using the cell membrane receptor of the animal cells, to induce inflammation in vitro and can easily analyze the biosensing method and animal cells An object of the present invention is to provide an inflammation diagnosis system. In addition, it provides additional methods to induce mechanisms that suppress inflammatory responses in the body, cell-friendly immunoassay methods for measuring them semi-continuously, optical measuring devices for measuring them, and representative anti-inflammatory screening methods using the same. I would like to.

The present invention to achieve the above object,

(1) treating an inflammation or cancer inducing factor in a medium in which animal cells having a cell membrane receptor are cultured;

(2) after step (1), treating the medium with a detector-marker complex that specifically binds to the cell membrane receptor;

(3) acquiring an optical signal after the complex processing; And

(4) provides a cell-friendly immunoassay method characterized by measuring the inflammation or cancer through the obtained optical signal analysis.

The present invention also provides a method for screening an anti-inflammatory or anticancer agent.

In another aspect, the present invention provides a composition for diagnosing inflammation or cancer, comprising an animal cell having a cell membrane receptor and a detector-marker complex that specifically binds to the cell membrane receptor.

The present invention also provides an inflammation or cancer diagnostic kit comprising the diagnostic composition.

The present invention can detect infection and inflammation in vitro more specifically than conventional assays using cell membrane receptors of animal cells as a recognition component of infection and inflammation. In addition, since the cell-friendly measurement of infection and inflammation can be repeated on the same cell, by using it can be continuously observed the step of suppressing the inflammation induced.

In addition, the biosensing method using the animal cells of the present invention is not only applicable to the new drug screening system for treating inflammation as suggested in the present invention, but also to continuously measure repeated infection, inflammation, and anti-inflammatory. In this case, it is possible to replace clinical experiments using experimental animals.

In addition, by using a primary cell extracted from humans in place of the cell line used in the present invention, if the process of screening the drug can be applied to the field of personalized drug development according to the individual currently limited to the area of molecular diagnostics Do.

In addition, it can be widely used throughout the industry, such as water pollutant detection system, toxic substance detection system, virus detection system in livestock farms, biological process monitoring system, food production process monitoring system.

1 is a diagram showing the process of cell infection through bacterial invasion.

Figure 2 is a diagram showing a model for inducing and measuring in vitro the process of cell infection through bacterial invasion based on animal cells.

3 to 6 is a receptor for an infection present on the cell surface by infecting Shigella sonnei , a food-induced macrophage RAW264.7 cell line, in order to confirm the applicability of an animal cell-based induction model based on animal cells. Comparative analysis by measuring changes in protein level (FIG. 3) and gene level (FIG. 4) of TLR2 and changes in secretion amount of cytokines (TNF-α and IL-6), which are representative infection markers (FIG. 5, FIG. 6). It is.

7 is a model for continuously observing inflammation through the TLR regulatory mechanism.

8 to 11 is a graph showing the results of semi-continuously observed inflammation through the TLR1 regulatory mechanism.

12 to 13 shows a TLR immunoassay (FIG. 12) excluding a cell fixation process of crushing cells in order to observe inflammation continuously for the same cell (FIG. 12) and a TLR immunoassay including a cell fixation process (FIG. 13). ) Is a graph comparing.

14 to 16 is a cell-friendly optical measuring device (FIG. 14) for detecting an inflammatory signal and a processing method for analyzing the measured optical signal values (FIG. 15), and through which the cells are cultured without damage (FIG. Fig. 16 shows the results obtained.

17 to 18 are graphs showing the results of observing the cell-friendly inflammation continuous measurement model (FIG. 17) and two inductions of inflammation through the same cell (FIG. 18).

19 to 20 is a graph inducing an inflammatory response three times in the same cell, the response was measured by TLR immunoassay (Fig. 19) and cytokine assay (Fig. 20).

21 is a schematic diagram showing a schematic diagram (A, B) through which the inflammatory signal is transmitted through the NF-κB signaling process and an inflammation suppression strategy (C) using the corresponding mechanism.

22 to 24 are graphs showing the results of induction of anti-inflammatory treatment by treating CAF and sodium salicylate, an analgesic antipyretic acetaminophen, to prove the anti-inflammatory model shown in FIG. 21.

25 to 26 are graphs showing a method of semi-continuously measuring an anti-inflammatory response in an affinity with an animal cell (FIG. 25) and an experimental result thereof (FIG. 26).

27 to 30 is a graph showing a method for testing cytotoxicity and drug persistence through animal cells.

The cell's immune system is divided into innate and adaptive immune systems, each with a different function and role. Toll-like receptors (TLRs) are cell membrane receptors responsible for the innate immune system, which are activated immediately after infection and can quickly control the growth of infecting microorganisms, thus responsible for the early stage of infection until lymphocytes are responsible for infection. do. Recently, the innate immune system is very important in the defense mechanism of the human body, it is becoming increasingly clear that the fundamental function, and the innate immune system interaction and adaptive function of the innate immune system has become more important clinical significance.

Among these, membrane receptors such as TLRs recognize signaling pathways that lead to the expression of various types of immune response genes, including inflammatory cytokines, by recognizing unique pathogen-associated molecular patterns (PAMPs) that only microorganisms possess. It is known to activate. Since this signaling pathway increases the expression level of cell membrane receptors, it is possible to indirectly confirm whether the inflammatory signaling pathway is activated through the expression level of the receptors, and when using this signaling pathway, the inflammatory response as in the cytokine measurement method. Screening for anti-inflammatory candidates that inhibit

In addition, since cell membrane receptors increase or decrease the amount expressed according to the intensity of stimulation by the regulatory mechanism in the body, when used as a measurement marker, it is possible to continuously observe the metabolism continuously occurring in the body. Therefore, the biosensor based on cytokine secretion or cell membrane receptor expression is not only able to measure the inflammatory response occurring in the body, but also to observe the anti-inflammatory mechanism in which inflammation is induced and suppressed because it can be continuously observed. It is possible to evaluate the performance of anti-inflammatory agents. In addition, the use of regulatory mechanisms in the body, it is possible to observe the continuous response of the animal cells through repeated inflammation induction and repeated anti-inflammatory treatment for it. Therefore, it is possible to conduct experiments to confirm efficacy or toxicity through repeated administration of anti-inflammatory agents, which is not possible in the one-time limited test method developed and in use.

Furthermore, studies have shown that cancer is inhibited when activating the innate immune system using agonist against TLR and that chronic inflammatory effects including Alzheimer's and rheumatoid arthritis are inhibited when antagonist is used to inhibit the innate immune system. Many have been reported. Therefore, there is a possibility of developing new types of anticancer drugs and anti-inflammatory drugs if we continue TLR research, which is a major factor in regulating the innate immune system activity.

In addition, by using the primary cells extracted from humans in place of the cell line used in the present invention, if the process of screening the drug can be applied to the field of personalized drug development according to the individual currently limited to the molecular diagnostic area Do.

Hereinafter, the present invention will be described in detail.

In order to enable the measurement of inflammation in vitro, an infectious agent is inoculated into a culture medium in which an animal cell is cultured, a step of treating an infected animal cell to an infected animal cell, and detection of a cell membrane receptor attached to a marker of the animal cell. Immunoassay method comprising the step of reacting with the sieve, and the process of recovering the cell membrane receptor through a detector attached with a marker in the same animal cell, a method for screening inflammation and cancer inhibitors, and Provided is an optical measuring device for measuring.

In more detail, the present invention,

(1) treating an inflammation or cancer inducing factor in a medium in which animal cells having a cell membrane receptor are cultured;

(2) after step (1), treating the medium with a detector-marker complex that specifically binds to the cell membrane receptor;

(3) acquiring an optical signal after the complex processing; And

(4) provides a cell-friendly immunoassay method characterized by measuring the inflammation or cancer through the obtained optical signal analysis.

The cell membrane receptor may be a TLR as an example. In order to continuously detect changes in TLR expression of living cells, a novel cellular immunoassay method that excludes chemical cell immobilization and cell membrane permeability that causes cellular damage has been devised. Needed. Typical cell-based immunoassay involves fixing cells to the bottom of the plate through formaldehyde and puncturing the outer membrane of the antibody through detergent (Tween20 or Triton X-100). The process of improving accessibility is included. These processes lead to cell death due to the characteristics of the chemical reagents used.

The animal cell may be adherent to a solid surface, for example, may be A549, a pulmonary epithelial cell line, as a cell line attached and growing on a plate surface. It is a cell that can be cultured without additional immobilization. In addition, the specific antibody to the cell membrane receptor TLR used in the immunoassay recognizes and binds a specific part of the TLR outside the cell membrane as a binding site, thereby inducing an immune response without damaging the cell membrane and increasing permeability. . Therefore, the present inventors can devise a method that excludes two processes leading to cell damage, and thus can provide a cell-friendly immunoassay method.

The cell-friendly immunoassay method of the present invention was intended to minimize shear stress caused by pipetting (culture medium supply device) by limiting the washing process necessary to supply the culture solution containing the antibody to three times. Cell culture medium containing 10% FBS was used as a buffer to facilitate nutrient supply to the cells during the immune response. At this time, the included FBS did not need an additional blocking agent because it simultaneously plays a role in reducing the non-specific signal in the immune response. However, the antibody concentration applied at this time should utilize twice the concentration of the conventional immunoassay.

The cell-friendly immunoassay method described above may be used as a method for observing inflammation and anti-inflammatory reactions in the same cell semi-continuously, and the description of the immunoassay method is the same as the anti-inflammatory or anticancer screening method of the present invention described below. Can be applied.

The immunoassay method does not immobilize the animal cells of step (1) through chemical treatment in order to minimize the damage of the cells, and then combines the detector with the labeled factor. Thereafter, washing may be performed through a culture solution containing FBS to stabilize cells. At this time, the labeling factor is not particularly limited, but may be an enzyme that generates an optical signal (hereinafter, also referred to as an “optical signal”) by a luminol emitter. That is, after the cleaning process, an optical signal may be obtained by adding a light emitter, for example, luminol. The light emitter is preferably added for a very short time of about 3 minutes or less.

The continuous detection of TLR expression in living cells required the design of new cellular immunoassays that eliminated chemical cell immobilization and increased membrane permeability, which cause cell damage. Typical cell-based immunoassay involves fixing cells to the bottom of the plate through formaldehyde and puncturing the outer membrane of the antibody through detergent (Tween20 or Triton X-100). The process of improving accessibility is included. These processes lead to cell death due to the characteristics of the chemical reagents used. A549, a pulmonary epithelial cell line introduced in the present invention, is a cell line that is attached to and grows on a plate surface, and is a cell that can be cultured without an additional immobilization process. In addition, the specific antibody to TLR used in the immunoassay recognizes and binds a specific part of the TLR existing outside the cell membrane as a binding site, thereby inducing an immune response without damaging the cell membrane and increasing permeability. Therefore, the present inventors have devised an immunoassay method that excludes two processes leading to cell damage. In order to confirm the applicability of the cell-friendly immunoassay, the present inventors conducted an experiment simultaneously with the existing immunoassay to compare the reactivity of TLRs. .

12 to 13 shows a TLR immunoassay (FIG. 12) excluding a cell fixation process of crushing cells in order to observe inflammation continuously for the same cell (FIG. 12) and a TLR immunoassay including a cell fixation process (FIG. 13). ) Is a comparison. As a result, TLR analysis was possible even when the cell fixation process was excluded, and it was confirmed that the ratio of the signal value to the background under the condition was higher than the conditions including the cell fixation process.

After the cell pulverized solution diluted 100-fold for 4 hours as shown in FIGS. 12 to 13, the cell immunoassay was performed (FIG. 12), and the signal-to- compared with the conventional immunoassay (FIG. 13). It was confirmed that the noise ratio was improved by 25%. This difference in results is due to the low background signal value, which is the formaldehyde reagent used to immobilize the cells. Formaldehyde reagents are chemical linkers that induce crosslinking of proteins with other proteins or DNA located around them, which modify the shape of some protein residues. Such modified proteins induce nonspecific binding with TLR antibodies, resulting in high background signal values. However, in the case of cell-friendly immunoassays, these reagents were excluded from the analysis process, resulting in a low background signal, resulting in a relatively high signal-to-noise ratio.

In addition, in order to measure TLRs without damaging the cells, it is also possible to introduce a technique for generating signals using photo-catalytic catalysis of horseradish peroxidase (HRP). The chromogenic immunoassay used in the above-described invention conditions promoted the action of oxidizing 3,3 ', 5,5'-tetramethylbenzidine (TMB), a substrate of reducing hydrogen peroxide, through HRP, an enzyme polymerized in a detection antibody. Use the principle of changing color. This principle is widely used as a method of confirming the presence of a target substance in an analytical sample. However, because the substrate is subjected to continuous oxidation regardless of the reaction over time, it is necessary to stop the enzyme reaction (treat a strong acid such as sulfuric acid) at an appropriate time (at the time when the reaction by the enzyme is saturated). This process serves to prevent the increase of the background signal that is accumulated. However, the reagents used to stop the reaction are highly acidic and cause cell damage.

14 to 16 is a cell-friendly optical measuring device (FIG. 14) for detecting an inflammatory signal and a processing method for analyzing the measured optical signal values (FIG. 15), and through which the cells are cultured without damage (FIG. 16).

That is, in the cell-friendly immunoassay method of the present invention, an optical measuring device can be used for analyzing the obtained optical signal, and the optical measuring device uses a telecentric lens on a cooled-CCD. By attaching it can be designed to adjust perspective and focus (FIG. 14). The above-described optical measuring device is constructed based on a cooled-CCD and a telecentric lens, but this does not limit the technical scope of the present invention.

 Since the cell image (FIG. 15 B-1) photographed by the optical measuring device varies in intensity of light emitted according to the degree of inflammation (FIG. 15 B-2 and B-3), the density of light through a commercially available program. The difference was converted into a numerical value and graphed (FIG. 15 B-4). In addition, through the microscopic cell observations, inflammatory immunoassay based on the luminescence signal detection method did not induce cell damage (Fig. 16 C-1, C-2, C-3). On the other hand, the color-based inflammatory immunoassay that was used previously induced cell damage and resulted in cell loss (Fig. 16 C-4). Such a phenomenon can be easily observed through a microscope as in FIG.

Therefore, the present invention has introduced a light-emitting technology that selects a different type of substrate and generates a signal without damaging the cells. Luminol is a light emitter that generates light through oxidation and is known to exhibit optimal reactivity at pH 8.5. Such Luminol emitters are known to inhibit poly (ADP-ribose) polymerase activity at 250-1000 uM concentration levels, but they are not involved in the expression of TLRs associated with inflammation. In addition, since the amount of luminol used in the luminescence reaction is a very small amount (60 pM), it is expected that it is less likely to cause cell damage and deformation.

In addition, hydrogen peroxide, a reducing agent known to cause oxidative stress, is exposed to cells for a very short time (within 3 minutes), and reacts very quickly with the substrate by HRP catalysis to convert to H ₂ O. The likelihood of inducing cell damage is very slim. In fact, after the reaction with the light-emitting reagent through the microscope and Janus green B method (mitochondrial staining method) as shown in Figure 16 as a result of measuring the cell activity (Fig. 16 C-1, C-2) It was confirmed that the same as.

Furthermore, in the present invention, rather than a multi-step procedure of enzyme-immunoassay using an enzyme in a detector that binds to cell membrane receptors such as Toll-like receptors (TLRs), the real-time cell membrane receptors are labeled through a label such as a phosphor and a quantum dot. Monitoring can be implemented to further reduce the time it takes to recognize an infection.

That is, the fluorescent substance and the quantum dots, which are markers, are added to the detector through a chemical method, and then an optimized amount is added before the inoculation of the infectious agent to the cultured animal cells. In the early inoculation, the detector and the marker rarely react with the cell membrane receptors of the animal cells, but the binding of the detector increases in proportion to the cell membrane receptor expressed on the cell surface over time after the inoculation of the infectious agent. Existing enzyme immunoassay was required to remove the unreacted detector by washing step by step. However, when applying a complex using a phosphor and a quantum dot, and excitation of the fluorescent substance and the quantum dot of the detector coupled to the cell membrane receptor by continuously irradiating light in a specific wavelength band without the process of washing / separation. This enables the construction of a system that can detect changes in cell membrane receptors in real time without the use of additional detectors by measuring the detected fluorescence signal. Therefore, the expression of toll-like receptors (TLRs), which are cell membrane receptors, can be observed in real time only by inoculation of an infectious agent, thereby changing the existing methods of complex food poisoning-causing bacteria and other methods based on animal cells. You can.

In the cell-friendly immunoassay method of the present invention, cell membrane receptors are toll-like receptors (TLRs), ion channel receptors, G-protein coupled receptors, and receptors. Guanylyl cyclase, Receptor tyrosine kinase (RTK), Cytokine receptor superfamily, Tyrosine phosphataase and Serine / Threonine protein kinase (Serine) / threonine protein kinases) is preferably at least one selected from the group consisting of, the toll-like receptors are Toll-like receptor 1 (TLR 1), Toll-like receptor 2 (TLR 2) and Toll-like receptor 4 (TLR It is preferable that it is at least one selected from 4). More preferably, the cell membrane receptors may be Toll-like receptors (TLRs).

In the cell-friendly immunoassay method of the present invention, the detector binds to the cell membrane receptor expressed on the surface of the animal cell, and the expression level of the cell membrane receptor can be confirmed by the labeling factor attached to the detector. From animal cells are infected to play a role in determining whether the inflammatory response occurs. In this case, the detector is preferably at least one selected from the group consisting of an antibody, a binding protein, a nucleic acid, an aptamer, and a peptide specifically bound to the cell membrane receptor. The detector may be more preferably an antibody.

In the cell-friendly immunoassay method of the present invention, the marker is attached to a detector and indicates whether the detector binds to a cell membrane receptor. The marker is a phosphor, a light emitter, an enzyme, a metal particle, a plastic particle, and a magnetic particle. It is preferably one or more selected from the group consisting of, more preferably an enzyme or a phosphor. Specifically, the phosphor includes FITC (fluorescein isothiocyanate (yellow green fluorescence), TRITC (tetramethylrhodamine isothiocyanate: red fluorescence), quantum dot (Quantum dot), and the like. Luminol), luciferin, and the like, and the enzymes include peroxidase (horseradish, horseradish), acid phosphatase, alkaline phosphatase, and glucose oxidase (glucose). oxidase), the metal particles include colloidal gold, the plastic particles include latex beads, and the like, and the magnetic particles include iron oxide nanoparticles, and the like. no.

In addition, the present invention

(1) treating an animal cell having a membrane receptor with an inflammation inducer or cancer inducer;

(2) after step (1), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor, and measuring the expression concentration of the cell membrane receptor;

(3) treating the animal cells with an anti-inflammatory or anticancer candidate after measuring the concentration; And

(4) after step (3), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor, and measuring the expression concentration of the cell membrane receptor,

It provides an anti-inflammatory or anti-cancer agent screening method, characterized in that the anti-inflammatory agent or anti-cancer agent is selected by comparing the expression concentration of the cell membrane receptors measured in step (2) and (4).

The screening method is preceded by the step of treating the animal cells with inflammation or cancer inducer, and then the anti-inflammatory agent or anti-cancer drug candidate.

That is, as an example, inflammation is induced by an inflammation inducer, and the concentration of the expressed membrane receptor is measured through a detector-labeled factor complex. Thereafter, sodium salicylate is treated as an anti-inflammatory candidate, and the concentration of the expressed membrane receptor is measured through a detector-labeled complex. Anti-inflammatory agents can be selected by comparing two concentrations of the cell membrane receptors obtained above.

In addition, the present invention

(1) treating an anti-inflammatory or anticancer candidate to an animal cell having a cell membrane receptor;

(2) after step (1), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor;

(3) treating the animal cell with an inflammation inducer or a cancer inducer after measuring the expression concentration of the cell membrane receptor after treating the detector-marker complex; And

(4) after step (3), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor, and measuring the expression concentration of the cell membrane receptor,

The anti-inflammatory or anti-cancer agent screening method is characterized in that the anti-inflammatory or anti-cancer agent is selected by comparing the expression concentrations of the cell membrane receptors measured in the above (3) and (4) steps.

The screening method is preceded by the step of treating the animal cell anti-inflammatory agent or anti-cancer drug candidate, and then proceeds to the step of treating the inflammation or cancer inducer.

That is, as an example, sodium salicylate, an anti-inflammatory candidate, is treated to animal cells, and a detector-labeled complex is treated to the animal cells to measure the concentration of the expressed membrane receptor. Thereafter, the inflammation inducer is treated with animal cells to induce inflammation, and then the detector-labeler complex is treated to measure the concentration of the expressed membrane receptor. Anti-inflammatory agents can be selected by comparing two concentrations of the cell membrane receptors obtained above.

As can be seen from the above description, by varying the timing of the anti-inflammatory agent or anti-cancer drug candidate treatment time, it is possible to confirm the degree of anti-inflammatory or anti-cancer activity different at the time of the candidate material treatment time.

As can be seen from the above description, by varying the timing of the anti-inflammatory agent or anti-cancer drug candidate treatment time, it is possible to confirm the degree of anti-inflammatory or anti-cancer activity different at the time of the candidate material treatment time.

In the anti-inflammatory or anticancer screening method of the present invention described above, the cell membrane receptor, the detector, and the marker may be equally applicable to the description of the cell-friendly immunoassay method of the present invention.

In addition, in the anti-inflammatory or anticancer screening method of the present invention described above, the animal cells may be cultured in a medium.

In addition, in the anti-inflammatory or anticancer screening method of the present invention described above, the expression concentration measurement of the cell membrane receptor may be performed by acquiring an optical signal and analyzing the obtained optical signal.

In other words, the immunoassay method using cell membrane receptors, which play a key role in the cell-friendly immunoassay method, uses specific biomaterials such as deoxyribonucleic acid (DNA) and antibodies, which are mainly used in the immunoassay method for the existing infectious agents. Rather, the innate immune response system was used directly as an infection sensor.

The anti-inflammatory or anti-cancer agent screening method of the present invention uses an immunoassay method of the present invention that detects infection from an infectious agent quickly through a detector having specificity and high affinity for a cell membrane receptor of an animal cell. Existing complex processes for determining efficacy can be simplified. In addition, since cell-friendly methods can be applied, the inflammatory process and anti-inflammatory process can be detected semi-continuously in the same cell, and the inflammatory response and anti-inflammatory effect can be analyzed, and even drug efficacy can be analyzed. That is, the screening method of the present invention is not a conventional one-time drug analysis method, and may also be capable of analyzing the toxicity of an anti-inflammatory agent or an anticancer agent.

The animal cell is a primary cell or cell line isolated from a tissue, and the primary cell or cell line is preferably an adherent cell or a floating cell. The adherent cells include epithelial cells, fibroblasts and endothelial cells, and the floating cells include T-cells, B-cells, dendritic cells, and monocytes. monocyte) and macrophage, but are not limited thereto.

The anti-inflammatory agent may use sodium salicylate and CAPE as an example of the present invention, but this is merely exemplary and does not limit the technical scope of the present invention. The anti-inflammatory agent is an anti-inflammatory agent, preferably a drug newly developed for anti-inflammatory purposes, an anti-inflammatory drug additionally expected, at least one selected from the group consisting of natural product extracts.

The inflammatory inducer may be a pathogenic microorganism in a form of inhibited activity as an example of the present invention, but this is merely exemplary and does not limit the technical scope of the present invention. Therefore, the induction factor is at least one selected from the group consisting of viable pathogenic microorganisms, pathogenic microorganisms inhibited by heat activity, pathogenic microorganisms inhibited by high frequency activity, and pathogen-associated molecular pattern obtained by separation and purification from pathogenic microorganisms. desirable.

In the present invention, the quantitative analysis described by the concentration analysis of the cell membrane receptor may use a statistical analysis method (one-way analysis of variance followed by Turkey's post hoc multiple range tests), and the distribution range of the effective P-value value. Can be distinguished accordingly. For example, *** very highly significant (P <0.001), ** highly significant (P <0.01), * significant (P <0.05), No significance (ns) (P> 0.05).

In another aspect, the present invention provides a composition for diagnosing inflammation or cancer, comprising an animal cell having a cell membrane receptor and a detector-marker complex that specifically binds to the cell membrane receptor. The inflammation or cancer diagnostic composition may be prepared based on the cell-friendly immunoassay method of the present invention, or an anti-inflammatory or anti-cancer screening method.

The present invention also provides an inflammation or cancer diagnostic kit comprising the diagnostic composition. The inflammation or cancer diagnosis kit may be attached with a well and a marker containing an animal cell and detecting a cell membrane receptor of the animal cell. That is, the present invention provides a kit for diagnosing infection and observing inflammation and anti-inflammatory reactions using animal cells. In this case, the kit may further include a capture recognition component for recognizing the infectious agent.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

1 to 2 are examples, a process of infecting cells through bacterial invasion (FIG. 1) and a model of inducing and measuring such infection process in vitro based on animal cells (FIG. 2). When the pathogen invades (Fig. 1 A-1), NF-κB, which is a transcription factor that induces an inflammatory response, is activated through specific binding of the cell membrane receptor TLR with the pathogen PAMP (Fig. 1 A-2) (Fig. 1 A-1). A-3). Activated NF-κB induces inflammatory response while inducing TLR expression (Fig. 1 A-4) or secrete cytokine, an inflammatory inducer (Fig. 1 A-5), and re-recognizes the inflammatory response continuously. Induces or induces an inflammatory response of surrounding cells (FIGS. 1A-6). The method of inducing and measuring such an infection process in vitro is as follows (FIG. 2).

Infectious pathogens are added to animal cells (infectants) immobilized in a limited space and cultured (FIG. 2 B-1). The injected infectious agent is specifically recognized through the TLR present on the animal cell membrane surface, and as an infection signal thereto, the expression level of the TLR is increased (Fig. 2 B-2). The concentration of TLR expressed in the cell membrane was measured by sequentially reacting a specific antibody against TLR with a polymerized secondary antibody (FIG. 2 B-3), and comparing the change in expression level of TLR through comparison with normal cells. Check it.

As shown in FIG. 1, when infecting an animal cell by inoculating an infectious agent in the medium, Toll-like receptors (TLRs), which are one of cell membrane receptors exposed to a certain amount of animal cell surface, are exposed to specific pathogen-associated molecular patterns ( Pathogen-associated molecular patterns (PAMPs) sites are recognized, resulting in signaling that alerts the cell to external invasion.

Transcription factors (NF-κB) activated by intracellular signaling increase the expression of cell membrane receptors, such as toll-like receptors (TLRs) in animal cells, to counteract further stimulation of infectious agents. The increased cell membrane receptors, i.e., toll-like receptors (TLRs) as an example, deliver stronger warning signals of infection into cells, thereby accelerating the induction of inflammatory responses. As the infection time progresses, the amount of Toll-like receptors (TLRs), which cause inflammatory responses to the infection, is continuously expressed in the extracellular membrane. Such cellular reactions occur in proportion to the intensity of the source and the infection time. .

Activated transcription factor (NF-κB) is also a pro-inflammatory cytokines of interleukin-1 and tumor necrosis factor-α through transcription of inflammation-related genes. , TNF-α), Interferon-γ, etc. secrete extracellularly, which stimulate other cells to induce additional inflammatory responses, leading to additional expression of Toll-like receptors (TLRs) in other cells. do.

Therefore, in the present invention, in order to induce the above-mentioned infection process in vitro, animal cells were used as infectious agents as shown in FIG. The model selects toll-like receptors whose expression changes through infection as cell membrane receptors, and to measure the selected cell membrane receptors, antibodies specific for toll-like receptors and secondary antibodies attached to enzymes, and substrates for the enzymes. Were reacted sequentially.

3 to 6 is a receptor for an infection present on the cell surface by infecting Shigella sonnei , a food-induced macrophage RAW264.7 cell line, in order to confirm the applicability of an animal cell-based induction model based on animal cells. Comparative analysis by measuring changes in protein level (FIG. 3) and gene level (FIG. 4) of TLR2, and changes in secretion amount of cytokines (TNF-α and IL-6), which are representative infection markers (FIG. 5, FIG. 6). It is. As a result, the expression level of TLR was increased at both the protein level and the gene level according to the concentration of the infectious agent and the reaction time (Fig. 3 and Fig. 4), and a stronger signal was obtained when using a live infectious agent (solid line graph of Fig. 3). ). When analyzing two types of cytokines, the same results as TLRs were obtained (FIGS. 5 and 6). This proved the applicability of the IVD model.

As a result, when the infection was induced by treating S. sonnei , a bacterium that causes phage poisoning bacteria, to the mouse-derived macrophages, as shown in FIG. 3, the expression level of TLR2 was observed to be changed. The level of TLR2 expressed with the time of inoculation of bacteria and with the population of inoculated bacteria increased, indicating that the inflammatory signal is continuously increased by the transcriptional regulator NF-κB. In addition, when inoculated with live S. sonnei , the expression of TLR2 was further increased because animal cells and bacteria were simultaneously cultured in the cell medium.

The change in the expression level of the protein in the animal cell can be proved through the change in the expression level of the mRNA, which is a transcription gene for the protein. As shown in FIG. 4, the expression level of the mRNA is increased in proportion to the concentration of the inoculated bacteria. It was. In addition, as shown in Figures 5 and 6, even when measuring the amount of pro-inflammatory cytokine, a reaction product of NF-κB, a transcriptional regulator activated through an infection signal, the concentration and activity of the inoculated bacteria and the time of inoculation reaction It was confirmed that the secretion amount of the cytokine according to the change. This proved that the animal cell-based in vitro experimental model established by the inventor induces infection similarly in the body and can be observed by TLR immunoassay.

7 is a model for continuously observing inflammation through the TLR regulatory mechanism. As mentioned in FIGS. 1 to 6, when pathogens invade the body, signals are transmitted into cells by TLRs (FIG. 7A-1), and cytokines are activated by transcriptional regulators activated through such signaling. It is secreted (Fig. 7 A-2) or the expression level of TLR is increased (Fig. 7 A-3). This process is called up-regulation. After the infectious agents are removed, TLRs overexpressed through the up-regulation process are regulated to normal levels through the down-regulation process. Β-arrestin binds preferentially to inhibit signaling of overexpressed TLRs (FIG. 7 B-1). TLR bound to β-arrestin undergoes endocytosis, which is introduced into the cell by the AP2-clathrin structure (FIG. 7 B-2). TLR constructs that enter the cell are combined with ubiquitin, a signal marker that induces degradation (FIG. 7 B-3), and is finally degraded by lysosome, a proteolytic organelle (FIG. 7 B-4). .

Membrane receptors, such as toll-like receptors, are usually present at minimal concentrations on the cell membrane surface to continuously monitor for infection by pathogens. As shown in the up-regulation of FIG. 7, when Toll-like infection occurs, each of the toll-like receptors present at the minimum concentration level transmits an infection signal to the body through specific binding with the pathogen (FIG. 7A-1). ). The signal transmitted to the body activates NF-κB to induce an inflammatory response in cells and secrete cytokines to induce an inflammatory response of surrounding cells (FIG. 7 A-2). In addition, by increasing the concentration of the receptor on the cell membrane surface (Fig. 7 A-3) (up-regulation) in preparation for further infection. After the infection by the pathogen is eliminated, a down-regulation of the elevated receptor levels occurs to a normal level. This mechanism prevents the overexpressing toll-like receptors from causing persistent inflammatory reactions. To do this. The down-regulation process begins with the attachment of β-arrestin, which interferes with the signaling of toll-like receptors (Fig. 7 B-1), and the desensitized toll-like receptor surrounded by the AP2-clathrin structure and into the cytoplasm. Inducing endocytosis is induced (FIG. 7 B-2). The structure introduced into the cytoplasm is combined with ubiquitin, which is a signal marker for inducing proteolysis (Fig. 7 B-3), and is degraded by lysosome, a proteolytic organelle (Fig. 7 B-4). ). Therefore, the present inventors hypothesized that the continuous measurement of cell membrane concentrations of toll-like receptors, which are closely related to the induction of inflammatory responses, would be able to continuously observe the inflammatory responses in the body. Inflammation continuous observation model was established through TLR control mechanism.

Pseudomonas , a pneumonia-inducing bacterium, as an infectious agent for implementing the inflammatory continuous observation model shown in FIG. aeruginosa The bacterial disruption solution, A549, a human-derived lung cell line, was selected as the subject of infection, and the inflammatory response was induced in vitro, and the response of the cells was observed through changes in TLRs (FIGS. 8 to 11). After 4 hours of infection as shown in FIG. 8, the concentration of TLRs increased through the infection process was reduced to normal levels after the infection was removed and the recovery solution was supplied at the same time. It was confirmed that (Fig. 8). This phenomenon was confirmed that the process is induced regardless of the concentration of the infected source, the higher the concentration of the source was confirmed that the recovery rate is lower. Among them, the highest recovery rate was obtained under the condition of 100-fold dilution of bacteria, and the recovery rate reached 80% (Fig. 9). In addition, such cell reactivity was not induced in one-off but was commonly induced in two or three times. This experimental result is an important evidence that can support the hypothesis established in FIG. However, since the experimental results shown in FIGS. 8 to 11 are limited semi-continuous measurements obtained by crushing cells in order to prepare several bundles of cells and observe TLRs according to their respective time periods, There is a need for techniques to measure infection, recovery and the concentration of TLRs without cell damage.

8 to 11 are the results of semi-continuously observed inflammation through the TLR1 regulatory mechanism. The human-derived cell line, A549 lung infection mainly were utilizing the lysate of bacterial pneumonia caused by the bacteria Pseudomonas aeruginosa infectious agents. After 4 hours of infection, the infectious agent was removed and observed over 24 hours with the recovery solution, and it was confirmed that the concentration of TLR decreased to the normal level (FIG. 8). This phenomenon showed a recovery rate of nearly 80% when infected with 100-fold diluted bacteria (Fig. 9). In addition, it was possible to observe up to three repeated infections under optimized conditions (Figs. 10 and 11). However, these experimental results are limited semi-continuous observations measured by crushing several bundles of cells over time.

TLR immunoassay based on cell-friendly luminescence was performed, and a detector equipped with a telecentric lens was mounted on a cooled digital camera as shown in FIG. 14 to measure the signal value. In optical measuring equipment including a lens, signal measurement sensitivity is changed according to a reference focus. However, since animal cells are attached to and grow on the surface, it is very limited to focus on the surface of each cell in the system where the surface of the cell must be measured at the top. Therefore, in order to solve this problem, a cell well plate made of a black plastic material, in which the bottom of the surface on which the cells are cultured is a highly permeable glass material and the light is not reflected, is introduced.

The focus of the detector is on the bottom, so that the reflected light is concentrated on the edge of the plate. As a result, as shown in FIG. 15, a circular-type signal pattern in which optical signals reflected to the bottom of the glass material are concentrated on the surface portion of the well edge may be obtained. The image measured by the luminescence detector (B-1 in Fig. 15) is converted into a digital signal through a Java-based Image J program (Figs. 15B-2 and B-3), and the converted signal values are plotted graphically. (FIG. 15 B-4). As a result, the cells infected with the bacterial grind fluid as shown in B-2 and B-3 of FIG. 15 were observed to exhibit the light signal by TLRs with increased expression levels. It was confirmed that only TLR was measured.

As mentioned above, the cell-friendly immunoassay method of the present invention, an optical signal acquisition and analysis of the application of the inflammation measurement technology through the experiment to induce inflammation in the same cell and to measure it.

17 to 18 are cell-friendly inflammatory semi-continuous measurement model for the same cell (FIG. 17) and the results of observing two times of inflammation induction (FIG. 18). Cell-friendly semi-continuous measurement of cell fixation step (Fig. 17 A-1), TLR concentration measurement step of the initial cell (Fig. 17 A-2), cell preparation step (Fig. 17 A-3), inflammation induction step (Fig. 17 A-4), TLR expression expressed through induction of inflammation (Fig. 17 A-5), and recovery (Fig. 17 A-6). Except for the cell immobilization step, the process is circulated sequentially to induce repeated inflammation. As a result, it was observed that inflammation was induced and recovered twice in the same cell (FIG. 18).

The results of the experiment were based on the immunoassay of the TLRs of the experimental group treated with the infectious agent and the control group not treated with the infectious agent. Converted to and plotted in a linear graph (Figs. 18 and 19). This process was applied to the pneumonia in vitro model (model infected with P. aeruginosa , a pneumococcal agent in A549, a pulmonary epithelial cell) shown in FIGS. As a result, as shown in Figure 18, the inflammation signal is increased compared to the background signal in the step of causing inflammation through infection, the signal value is reduced to near the normal value in the step of removing and recovering the source of infection by replacing the culture medium I could confirm it.

19 to 20 induce an inflammatory response three times in the same cell, the response was measured by TLR immunoassay (Fig. 19) and cytokine assay (Fig. 20). The TLR immunoassay showed a pattern in which the ratio of signal value to background increased regularly during three inflammatory response measurements (FIG. 19). In contrast, in the case of the cytokine TNF-α, the signal value was constantly reduced (Fig. 20, upper curve), and IL-6 was observed in a pattern in which the signal value was kept constant (Fig. 20, Bottom curve).

In addition, as shown in FIG. 19, it could be demonstrated that the semi-continuous inflammation measurement is reproduced even in two or three reinfection conditions rather than one time. This results in the same result as the limited inflammatory semicontinuous observation through the TLR control mechanism shown in FIGS. 8 to 11, demonstrating that the immunoassay method proposed in the present invention can measure inflammation continuously without damage on the same cell. .

In addition to the cell-friendly immunoassay method of the present invention, there is a method for measuring cytokines secreted through inflammation as mentioned in A-5 of FIG. 1. . The cytokine measurement method is a method for obtaining an immunoassay by obtaining a cell culture solution for each process in FIG. 17, and is a very simple technique for observing an inflammatory response without cell damage. However, cytokines are secreted at very low concentrations (less than 10 pg / mL) in human-derived cells, which is very limited for inflammatory reactions. Unlike this, in the case of rat-derived cells as shown in FIG. 20, a measurable level of cytokine is secreted, but the tendency is different depending on the type of cytokine. Semi-continuous measurement of cytokine inflammation in rat-derived cells revealed that the amount of TNF-α secreted decreases with repeated cycles, while IL-6 is constantly secreted. As described above, although the experimental method for measuring cytokines is simple, the measurement results vary depending on the analyte (type of cytokines) and there is a problem that it cannot be applied to human-derived cells.

Thus, there are two ways to measure inflammation continuously. The first model measures changes in the expression level of TLR in the human-derived cell line A549, and the second model measures cytokines (IL-6) in the mouse-derived cell line RAW264.7. The first model uses human-derived cell lines, which makes it very suitable for new drug screening applications (such as drug efficacy and sustainability and toxicity testing). On the other hand, the second model, which utilizes a mouse-derived cell line, is very easy to analyze, making it more suitable for environmental, harmful bacteria and virus monitoring.

In addition, the present invention, using a cell-friendly immunoassay method of the present invention that can measure the inflammation or cancer semi-continuously screening method for selecting an anti-inflammatory agent or anti-cancer agent that suppresses the inflammatory response and carcinogenesis from anti-inflammatory agent or anti-cancer drug candidates Can provide.

21 is a schematic diagram (A, B) through which the inflammatory signal is transmitted through the NF-κB signaling process and an inflammation suppression strategy (C) using the corresponding mechanism. As mentioned in FIGS. 1 to 6, invasion of the pathogen activates the transcriptional regulator NF-κB and, as a result, secretes TLR expression (A-1) and cytokines (A-2). In contrast, inflammation is induced by stimulation other than infection as follows (B). Through external stimulation (B-1), bradykinin, an inflammation-inducing substance present in the body, is synthesized (B-2). The synthesized bradykinin is recognized by the bradykinin receptor and stimulates NF-κB (B-3). Stimulated NF-κB expresses the bradykinin receptor, which induces inflammation continuously (B-4), while synthesizing another pro-inflammatory agent, prostaglandin (B-5), which promotes inflammation through vasodilation. Cause (B-6). Therefore, the inventors inhibited the activity of NF-κB, which plays a pivotal role in the induction of inflammation (C-1), thereby inhibiting the expression of TLR and cytokines and bradykinin receptors (C-2), thereby preventing anti-inflammatory A strategy of inducing (C-3) was devised. As shown in FIG. 21, the innate immune response through TLR recognition begins when the TLR recognizes an invasion of an infectious agent. TLRs specifically bind to pathogen-associated molecular patterns (PAMPs) of infectious agents to deliver foreign invasion signals into cells, and these signals activate the transcriptional regulator NF-κB. Activated NF-κB expresses cytokine, a signaling agent between cells, spreads infection to adjacent cells and increases the number of inflammatory receptors such as TLR. Induce as much as possible (infection process). As such, NF-κB is a regulator responsible for the expression of cytokine and inflammation-related protein receptors, which are known to be activated by external stimuli (e.g. burns, pain, etc.) that cause inflammation rather than infection. have. When cells are exposed to external stimuli that cause inflammation, including infection, the kinin-kallikrein system synthesizes bradykinin (inflammatory markers that induce vasodilation) in the body, which is recognized by the inflammation-related receptor bradykinin receptors. The process of doing this happens. The binding between the bradykinin and the receptor activates NF-κB, and the activated NF-κB pathway increases the population of the bradykinin receptor, while inducing a mechanism for synthesizing another inflammatory marker, prostaglandin (inflammation) process). As such, the infection and inflammatory processes occurring in the body are induced through the NF-κB pathway, of which NF-κB activity plays a pivotal role in the mechanism of inflammation. Therefore, the present inventors control the inflammatory response through the inhibition of NF-κB activity as shown in FIG. 21C through the theoretical background that NF-κB activity significantly influences the expression of inflammation-related proteins and related receptors. We propose a technique for screening materials.

As an example, to demonstrate the performance of the above-mentioned techniques for screening anti-inflammatory agents, reagents that inhibit the activity of NF-κB in A549 cells (pulmonary epithelial cells) that caused inflammation through infection (e.g., CAPE and sodium salicylate ) To build up an animal model that inhibits the inflammatory response, and the experiment was performed to predict whether the inflammation was inhibited (inhibition of NF-κB activity) by changing the expression level of TLR. Such a cell-based inflammatory measurement system can be used as a system for screening anti-inflammatory candidates, and can be a base technology of experimental models that can replace animal experiments.

NF-κB is a transcriptional regulator consisting of subunit proteins such as P60 protein that binds to DNA, P50 protein that allows passage through the nuclear membrane, and Iκβα that binds to and inhibits the activity of the two preceding proteins.

22 to 24 are the results of induction of anti-inflammatory treatment by treating NF-κB inhibitors CAPE and sodium salicylate and analgesic antipyretic acetaminophen to prove the anti-inflammatory model shown in FIG. CAPE used in the experiment is known to inhibit NF-κB that enters the nuclear membrane and bind to DNA, and sodium salicylate inhibits the activity of NF-κB by inhibiting the activity of an enzyme that activates NF-κB. come. This phenomenon was also observed through the TLR-based inflammation measurement method (Figs. 23 and 24), in particular, the degree of inhibition was different depending on the time point of sodium salicylate treatment (Fig. 23B-1). , B-2). In addition, acetaminophen, which is known to not induce anti-inflammatory, did not show any difference when compared with the inflammatory signal (FIG. 24).

As shown in FIG. 22, NF-κB activity in the cytoplasm is inhibited by Iκβα. However, when TLR and PAMP are combined by infection, Iκβα kinase is activated, thereby removing Iκβα. NF-κB activated by the eliminated Iκβα passes through the nuclear membrane and binds to the DNA present inside the nuclear membrane to produce inflammation-related proteins. CAPE used in the experiment is an anti-inflammatory candidate substance that is known to interfere with the transcription of NF-κB that enters the nuclear membrane by binding to P65 protein. Another reagent, sodium salicylate, is a salicylate-based reagent that inhibits the activity of IκB kinase and maintains the binding of Iκβα, including aspirin, which is widely known as an anti-inflammatory agent. In addition, acetaminophen (an analgesic antipyretic agent) with no anti-inflammatory effect was selected as a negative control to demonstrate that the TLR-based inflammatory assay responds to the extent of NF-κB activity.

As shown in FIG. 23, after treatment with sodium salicylate diluted by concentration and P. aeruginosa , which is a pneumococcal bacterium, was treated with A549, which is a pulmonary epithelial cell, the amount of TLR expression was analyzed. It was confirmed that the amount of TLR expressed according to the inhibition. In particular, when the anti-inflammatory agent was first treated before infection (FIG. 23 B-2), the expression of TLR was more strongly inhibited than when the anti-inflammatory agent was simultaneously treated with the infection (Fig. 23 B-1). This means that the anti-inflammatory effect is different depending on when the anti-inflammatory agent is treated. If such a phenomenon is used, it is possible to determine when to take the anti-inflammatory agent properly. The anti-inflammatory response was measured using the expression level of TLR was observed even under the condition of CAPE treatment separately concentration (Fig. 24 C-1) as shown in FIG. In particular, nearly 95% of TLR expression inhibitory effect (anti-inflammatory effect) was observed under CAPE treatment at 180 uM. In contrast, when treated with analgesics (FIG. 24 C-2), it was confirmed that TLR is constantly expressed regardless of the concentration of the reagent to be treated, and experimental results indicate that the expression of TLR is limited to the activity of NF-κB. Could prove that.

In order to repeatedly implement the anti-inflammatory reactions shown in FIGS. 22 to 24 in the same cell without being limited to one time, an experiment was carried out to measure inflammation in a semi-continuous manner.

25 to 26 is a method for semi-continuously measuring the anti-inflammatory response to the cell-friendly in animal cells (Fig. 25) and the experimental results (Fig. 26). Cell-friendly anti-inflammatory semi-continuous measuring method is the same as the method described in Figures 17 to 18 cell fixation (Fig. 25 A-1) and TLR concentration measurement of the initial cells (Fig. 25 A-2), cell preparation step (Fig. 25 A-3) and anti-inflammatory treatment, inflammation induction (Fig. 25 A-4), expression suppressed TLR concentration measurement (Fig. 25 A-5) and recovery (Fig. 25 A-6). When the anti-inflammatory agent was not treated, the ratio of signal values to the background when inflammation was induced or recovered was higher than that when the anti-inflammatory agent was treated, and these signal values accumulated in the second cycle to form a steep rise curve (FIG. 26). B-1). On the contrary, when the anti-inflammatory agent was treated, a gentle upward curve was formed, and as in the result of FIG. 26, the gentle slope of the curve was different according to the time point of the anti-inflammatory agent treatment (FIGS. 26B-2 and B-3. ).

Semi-continuous measurement of cell-friendly anti-inflammatory response is as shown in the schematic diagram of FIG. 25, the cell fixation step (A-1), the TLR concentration measurement step (A-2), the cell preparation step (A-3), and the inflammation induction step (A-3) of the initial cells. -4), TLR concentrations expressed through inflammation induction (A-5), and recovery (A-6) is divided into steps, except for the cell immobilization step is circulated sequentially to repeatedly induce inflammation and inhibition and The recovery process is in progress. In addition, sodium salicylate, an inflammation inhibitor, was treated in a cell preparation step or an inflammation induction step, and as shown in FIG. As a result, as shown in Fig. 26, when the anti-inflammatory treatment was induced, the anti-inflammatory treatment showed stronger anti-inflammatory response than when the anti-inflammatory treatment and inflammation induction were performed at the same time. Such a phenomenon was observed up to 2 times in the same cell. This proved that when the system designed by the inventor was used, the phenomenon of inducing and inhibiting inflammation in the same cell was observed semi-continuously, and an example of selecting an anti-inflammatory agent as an application thereof was further proposed.

In order to confirm the applicability and applicability of the technique for measuring inflammation in a semi-continuous manner to the same cell, an experiment was conducted to confirm the continuity and toxicity of the anti-inflammatory agent when repeatedly treated. Most anti-inflammatory screening systems currently developed are one-time tests that verify the performance of many types of drug candidates with high-throughput. These test systems only check whether anti-inflammatory activity is limited to one-time, so when the reagent candidate group is treated, additional cytotoxicity experiments or animal-based tests are performed to confirm the duration of drug efficacy or cell toxicity. Clinical trials must be complemented. However, in the case of the semi-continuous inflammation measuring system based on the animal cell membrane receptor of the present invention, the re-infection of the same cells is induced and the condition is monitored semi-continuously so that the duration of the drug efficacy after treatment with the anti-inflammatory agent or the anti-inflammatory agent treatment Applicable for testing primary cytotoxicity and cumulative cytotoxicity following repeated anti-inflammatory treatments. Therefore, in order to prove the applicability of cell membrane receptor-based anti-inflammatory screening system, we selected an inflammatory standard model that induces re-infection and recovery three times, and conducted experiments in which three anti-inflammatory agents were treated in various models.

27 to 30 is a method for testing cytotoxicity and drug persistence through animal cells. The cytotoxicity of the inhibitor can be confirmed by the difference in signal values against the background of normal cultured cells and cells damaged by the toxicity of the inhibitor (FIG. 27), and the appropriate level of inhibitor concentration can be selected through the cytotoxicity test. It was. This reactivity was introduced into three drug sustainability test to confirm that the drug is continued without toxicity accumulated for 80 hours (Fig. 28, 29). In addition, the results of experiments with different treatment times confirmed that this pattern was not applied after 20 hours (FIG. 30).

As shown in FIG. 27, three times of reinfection and recovery without induction of anti-inflammatory agents (CAPE) were induced, the infection signal value was about 3 compared to the background signal, and the recovery value was about 2. In contrast, when anti-inflammatory agents (90 uM CAPE and 50 uM salicylate) were treated before the induction of inflammation, signal values of about 2 during the induction of inflammation and about 1.5 during the recovery were shown. As shown in FIGS. 22 to 24, when the anti-inflammatory treatment did not increase or decrease the TLR signal value, it was confirmed that the anti-inflammatory condition is not toxic to the cells. In contrast, when treated with a high concentration of anti-inflammatory agent as shown in Figure 28 (180 uM CAPE), it was confirmed that the damage of the cells is not induced to increase the ratio of the signal value against the background from the inflammatory process. This is due to the rise of nonspecific background signals and the loss of immobilized cells due to cell damage. Since the two cell characteristics are an objective indicator of cell damage, the cytoreceptor-based anti-inflammatory screening system was able to test not only the efficacy of the anti-inflammatory agent but also the toxicity of the drug. The constant ratio of inflammatory signal to background signal during three treatments of 90 uM CAPE and 50 mM sodium salicylate means that the reagents are anti-inflammatory without toxicity to the cells at the indicated concentrations. In addition, in the case of acetaminophen as shown in Figure 29, it was confirmed that there is no toxicity as well as anti-inflammatory action even at 10 mM concentration.

In addition, to determine the persistence and resistance of drug efficacy, anti-inflammatory treatment was performed at different times to compare the anti-inflammatory properties under each condition. As shown in FIG. 30, when the anti-inflammatory agent was treated only in the first and second circulation reactions by dividing the three inflammatory reactions according to each cycle, the ratio of the signal value to the background was different in the inflammatory induction process except the third circulation reaction. 2 was represented and 1.5 was shown in the recovery process. In the third cycle of the anti-inflammatory treatment, the ratio of 2.5 in inflammation and 2 in recovery was found. Similarly, when anti-inflammatory agents were treated in cycles 1 and 3, the rate of signal values close to the normal infection level was observed only in the second infection. Especially, in the second recovery, the ratio of the inflammatory signal value to the background signal close to 2, which is the same as the pattern recovered after the normal infection, was shown because the TLR expressed at the high level remained. In addition, since the ratio of a constant signal value appeared during the three repeated reactions as described above, the concentration of the anti-inflammatory agent used in the experiment proves that there is no toxicity even if repeated use. This phenomenon cannot be attempted in the existing reaction of one-time anti-inflammatory reactions in the same cell, which means that the drug persistence test and toxicity test, which can be performed only in experimental animals, can be replaced by a cell receptor-based system. . Therefore, when the system is utilized, the screening test to confirm the efficacy of the drug at the same time as well as the continuity and toxicity test of the drug and iterative dosing test to repeatedly check these tests will be able to proceed at once. Such a cell receptor-based complex test method is expected to replace the existing test methods using laboratory animals.

Biosensing using cell membrane receptors, which play a key role in the inflammation-inducing system of the cells, does not use specific biomaterials such as deoxyribonucleic acid (DNA), antibodies, etc., which are mainly used in biosensing methods for infectious agents. It is characteristic that the innate immune response system was used directly as an infection sensor. In this way, complex procedures such as culturing a large number of infectious agents can be eliminated only by inoculation of the corresponding infectious agent, so that the process can be shortened and simplified.

The present invention provides a biosensing method that detects infection from an infectious agent quickly through a detector having a high affinity specific to a cell membrane receptor of an animal cell, thereby simplifying existing processes for measuring infection and inflammatory responses. This allows semi-continuous detection of inflammatory and anti-inflammatory processes in the same cell.

In other words, rather than measuring the physical or chemical changes of animal cells to pathogens, cell membrane receptors such as Toll-like receptors (TLRs), which play a key role in the cell's inflammation system, are introduced and directly detected for infectious agents. While improving specificity and sensitivity, it is possible to indirectly derive experimental results obtained only through animal experimentation in vitro.

Furthermore, it is possible to reinforce the miniaturized measuring equipment by introducing IT technology, and thus use the biosensing method of the present invention in the field outside the laboratory.

The present invention will be described in more detail with reference to the following examples. However, these embodiments are merely exemplary and do not limit the technical scope of the present invention.

Example  1. Cell Culture and Attachment

Fetal Bovine Serum (FBS) (10% (v / v), final concentration) and penicillin-streptomycin solution (Penicillin-streptomycin, 1% (v / v), final concentration) mixed in RPMI1640 medium Using a medium, 96-well cell culture plate so that human lung epithelial cell line A-549 (ATCC CCL-185) can be maintained in the number of 6 x 10 ⁴ cells per well. 200µl each was injected into the cells), followed by incubation for 24 hours under the condition that the temperature was maintained at 37 ° C and carbon dioxide (CO ₂ ) 5%. After confirming that A-549 was fixed on the well surface through culture (100X), the medium was removed by an inhaler to remove the penicillin-streptomycin solution, that is, antibiotics, and then DPBS (+ / +) was added to 200 Wash with addition of μl. After washing, the antibiotics were removed by inhaler again, and then RPMI1640 medium serum-free medium was added to 200 μl to give starvation. It is preferable that the animal cell used in the biosensing method is in a cell starvation state. This is because the animal cell line is cultured on a cell culture substrate such as a 96-well microtiter plate and the like, and the animal cell can react sensitively to external stimuli caused by an infectious agent through a state of attachment and starvation. Play a role. When utilizing a mouse macrophage RAW264.7 (KCLB 40071), it was used in place of Dulbecco's Modified Eagle's Medium (DMEM).

Example  2. Through infectious agents Inflammation

Using a medium mixed with RPMI1640 medium and Dulbecco's Modified Eagle's Medium (DMEM) with FBS (10% (v / v), final concentration) and penicillin-streptomycin solution (1% (v / v), final concentration), Inject 200 µl of each A-549 and RAW264.7 into a 96-well cell culture plate to maintain the number of 6 x 10 ⁴ cells per well, and keep the temperature at 37 ° C and carbon dioxide (CO ₂ ) at 5%. The cells were attached to the surface and incubated for 24 hours. After incubation, the penicillin-streptomycin solution, i.e., the medium was removed with an inhaler for antibiotic removal, followed by washing with 200 μl of DPBS (+ / +), followed by removal of the remaining antibiotics with the inhaler, and serum-free for each cell. 200 μl of the medium was added to starvation for 1 hour.

Live food poisoning bacteria ( Sigella) for infection sonnei ) and high-frequency pulverized bacterial lysate ( Pseudomonas aeruginosa ) was diluted to the concentrations indicated in the serum-free medium used for each animal cell, and 200 µl of each well was inoculated to perform infection for the indicated time under the same conditions as described above. Wells with no addition of infectious agents were used as controls. At the end of each infection time, medium and Escherichia coli were removed through an inhaler, and washed once with Dulbecco's Phosphate Buffered Saline / modified with Calcium and Magnesium (DPBS (+ / +)) in 200 μl.

Example  3. Cell based Enzyme development  Assay (if cell damage is induced)

Infected cells were treated with 100 μl of 4% formalin solution for 30 minutes at room temperature to fix the infected cells in the wells. 200 μl of casein protein-phosphate buffer solution (Casein-PBS) dissolved in Dulbecco's Phosphate Buffered Saline / modified without Calcium and Magnesium (DPBS (-/-)) at a concentration of 0.5% (w / v). The reaction was performed at 100% humidity to react for 1 hour at 37 ^° C.

Next, TLR rabbit polyclonal antibody that specifically binds to TLR was used at a rate of 1/300 using Casein-PBS (Casein-PBS-Tw) containing 0.1% (v / v) tween 20. After diluting and dispensing 100 μl each reaction for 1 hour under the same conditions, discard the reaction solution and washed three times by 200 μl three times using phosphate buffer solution (PBST) containing 0.1% tween 20. Removed. Next, HRP conjugated goat anti-rabbit IgG, a secondary antibody, was also diluted to a ratio of 1/5000 using Casein-PBS (Casein-PBS-Tw) containing 0.1% (v / v) tween 20, and then 100 μl each. After dispensing and reacting for 1 hour under the same conditions, the resultant was washed three times with PBST in the same manner. 50mM acetate, 1% 3, 3 ', 5,5'-tetramethylbenzidine (TMB) (w / v) and 3% hydrogen peroxide (v / v) in a ratio of 1000: 10: 1 A solution was prepared, and 200 µl of the color substrate solution was observed to observe the color reaction for 15 minutes, and then 50 µl of 2M sulfuric acid was dispensed to stop the reaction. The finally developed signal was detected by ELISA plate reader (Versamax; Molecular Device, USA) at 450 nm wavelength. The detected result is graphically graphed through the MS excel program as shown in FIG. 3.

Example  4. Molecular Biology Marker analysis

To confirm the expression level of TLR in molecular biology, infected cells were washed three times with 200 µl each using Dulbecco's Phosphate Buffered Saline / modified without Calcium and Magnesium (DPBS (-/-)), followed by RNA using TRIzol reagent from Invitrogen. Was extracted. Abcam TaqMan reverse transcriptase was reacted with the extracted RNA to synthesize complementary DNA (cDNA). The reverse transduction process was induced by Bio-Rad's MJ Mini Thermal Cycler. The induction conditions are 10 minutes cooling at 25 ° C., 1 hour at 37 ° C. and 1 hour extended at 42 ° C., and finally 5 minutes at 95 ° C. for inactivation. Synthesized cDNA was quantified by NanoDrop from Thermo Fisher Scientific.

Two gene sequences (5-TTGCTCCTGCGAACTCCTAT-3 and 5-AGCCTGGTGACATTCCAAGA-3) for TLRs were designed for real-time polymerase chain reaction, respectively. The indicator was inserted through. Real-time polymerase chain reaction was performed using Applied Biosystems' ABI 7500 Real-Time PCR System, and the conditions of use were as follows. Procedure 1: 50 ° C for 2 minutes and 95 ° C for 10 minutes, Process 2: 95 ° C for 15 seconds and 60 ° C for 1 minute. Procedures 1 and 2 were repeated 40 times. In addition, two gene sequences (5-TGTGTCCGTCGTGGATCTGA-3 and 5-CCTGCTTCACCACCTTCTTGAT-3) were additionally prepared to use glyceraldehyde 3-phosphate dehydrogenase (mGAPDH) as a control, and the threshold TLR mRNA ratio was increased. It was quantified by the cycle method. The result was plotted through the MS excel program as shown in FIG.

Example  5. Inflammatory oil  Secretion of cytokines

A cytokine immunoassay kit sold by R & D system was used to measure TNF-α and IL-6 secreted from infected cells. Inactivated S. sonnei bacteria and bacteria inactivated by treatment with antibacterial proclin300 were diluted with DMEM medium and treated with mouse macrophage RAW264.7 for a predetermined time as described in Example 2 to induce an inflammatory response. To measure the cytokines secreted through bacterial infection, the culture medium was diluted (1/5 TNF-α and 1/2 IL-6) through the dilution buffer provided in the assay kit to fix the antibody against the cytokine. The reaction was carried out for 2 hours at room temperature. After the reaction, washing was performed five times through the washing buffer provided in the kit, and 100 μl of the polymerized detection antibody was reacted at room temperature for 1 hour. After washing once more, the color reaction is induced and measured through the color substrate as specified in Example 3. The measured results are graphically plotted as shown in FIGS. 5 and 6.

Example  6. TLR Control  Limited inflammation through Semicontinuous observation

In order to observe the inflammation semi-continuously by using the TLR control mechanism shown in Figure 7, preferentially by dispensing 200 μL of medium without FBS to the cell culture well cultured in several bundles of animal cell lines as in Example 1 Cell starvation was induced for 2 hours under conditions maintained at 37 ° C. and carbon dioxide (CO ₂ ). In some cell line bundles induced cell starvation, the cells were immobilized as in Example 3, and then a color-immune response to TLR was performed. The rest of the cell line was maintained at a temperature of 37 ^° C. and carbon dioxide (CO ₂ ) by 200 μl of serum-free medium diluted with infectious agents by removing the medium after the preparation period in which the cells were starved. Infected for 4 hours under conditions. In some infected cell line bundles, the cells were immobilized as in Example 3, and then a color-immunized reaction was performed. The remaining cell line bundle was washed three times through a medium containing 10% FBS after infection, and the wells were washed with 200 μl of 10% FBS-containing medium at 37 ° C. and carbon dioxide (CO _2). ) Recovery reaction was induced for 24 hours under the condition of 5%. Each cell line bundle was subjected to colorimetric reaction as in Example 3 in 6-hour units as shown in FIG. As a control, a bundle of cell lines that did not induce infection was prepared, and the ratio of the inflammatory signal value to the background signal value was calculated and plotted in FIG. 9. The maximum recovery rate is the ratio of the expression level of TLR decreased during the recovery process to the expression level of TLR increased during the infection process. The semi-continuous observation of such limited inflammation was repeated twice and three times, and the results are graphically plotted in FIGS. 10 and 11.

Example  7. Cell-friendly enzyme luminescence immunoassay

For cell-friendly enzyme-immunoimmunoassay, infected cells were washed three times with 200 μl each using Dulbecco's Phosphate Buffered Saline / modified without Calcium and Magnesium (DPBS (-/-)), followed by TLR binding specifically to TLR. Rabbit polyclonal antibody was diluted at a rate of 1/100 using a medium containing 10% FBS (RPI1640 for A549, DMEM for RAW264.7), and 100 μl were dispensed under the same conditions. After reacting for 1 hour, the reaction solution was discarded and unreacted antibody was removed by washing three times with 200 μl using the same medium. Next, HRP conjugated goat anti-rabbit IgG, a secondary antibody, was also diluted at a rate of 1/2500 using a medium containing 5% FBS, and then reacted for 1 hour under the same conditions. Washed twice.

A solution for generating a luminescence signal was prepared by mixing a solution of Supersignal West Femto sold by Thermo Fisher Scientific in a ratio of 1: 1, and the inventors at a point in time after 3 minutes by dispensing 200 μl of the light emitting substrate solution. Measured using an optical measuring device based on a cooled CCD camera (Fig. 14). After luminescence measurement was completed, the remaining substrate was removed by washing three times with 200 μl using a medium containing 10% FBS.

Example  8. Cell-Friendly Inflammation on the Same Cell Semi-continuous measurement

In order to measure cell-friendly inflammation semi-continuously in the same cell (A549) as shown in Figure 7 (A), an inflammatory response was induced as in Example 2, and cells for TLR as shown in Example 7 Friendly enzyme luminescence immunoassay was performed. To measure initially expressed TLR, after cell adhesion process (before cell preparation period), cell-friendly enzyme luminescence immunoassay was performed, and after enzyme preparation, inflammatory response, and recovery, Luminescent immunoassay was performed. Sequential enzyme luminescence immunoassay was performed on cells that did not react with the infectious agent as a control.

The cycle was performed up to two or three times and is graphically illustrated in FIGS. 18 and 19. In addition, the same procedure was applied to RAW264.7 cells, and the pattern of cytokines secreted was measured and analyzed as in Example 5 and illustrated in FIG. 20.

Example  9. NF through -κB inhibitors Anti-inflammatory reaction  Induction and observation

In order to simulate the anti-inflammatory mechanism in which inflammation is induced and inhibited in vitro, the anti-inflammatory response was induced by treating an inflammatory response-induced cell with an NF-κB inhibitor as in Example 2, and measured. Sodium salicylate and CAPE were selected as NK-κB inhibitors, and sodium salicylate was diluted in serum-free medium to prepare 500 mM standard solution, and CAPE was diluted in DMSO to prepare 180 mM standard solution.

As shown in Example 2, cells fixed on the cell culture well surface for 24 hours were washed with 200 µl of DPBS (+ / +), and the remaining antibiotics were removed by inhaler again. When the inhibitor is pretreated, 200 μl of the inhibitor diluted by concentration is added to the serum-free medium and treated for 2 hours. After pretreatment of the inhibitor, the bacterial lysate was reacted in the same manner as in Example 2, and signal values were measured by colorimetric immunoassay shown in Example 3. In addition, the negative control group did not induce inflammation, and the positive control group was selected for the case of induction of inflammation without treatment with an inhibitor, and the experiments were conducted simultaneously under the same conditions. The results are shown in bar graphs in FIGS. 23B-1 and 24C-1. In addition, the same experiment was performed by treating acetaminophen but not NF-κB inhibitor, and the results are shown in (C-2) of FIG. 24.

In the case of simultaneous treatment of inhibitors, the serum-free medium containing no inhibitors is treated for 2 hours before inducing inflammation, followed by bacterial crushing fluids that induce inflammation and inhibitors that inhibit inflammation. It was mixed and treated. The result is shown in FIG.

Example  10. Semi-continuous on the same cell Anti-inflammatory reaction  Induction and measurement

In order to repeatedly observe the anti-inflammatory action as shown in FIGS. 22 to 24 on the same cell, the cell-friendly luminescence immunoassay shown in Example 7 was applied.

Semi-continuous measurement of anti-inflammatory activity on the same cell is the same as the method shown in Figure 17, after the cell attachment process (before the cell preparation period), preferentially cell-friendly enzyme luminescence immunoassay to measure the initially expressed TLR Enzyme luminescence immunoassay was performed sequentially after cell preparation (or inhibitor treatment), after inflammatory reaction, and after recovery. As mentioned above, the inhibitor was pretreated in the cell preparation process, and its concentration was 50 mM for sodium salicylate and 90 µM for CAPE. Sequential enzyme luminescence immunoassay was performed on the cells that did not react the infectious agents with the negative control group, and the cells treated with the inhibitor and the reactants with the positive control group. The results obtained are plotted as a linear graph over time in terms of ratio of signal values to background. Two anti-inflammatory semi-continuous measurements are shown in FIG. 26 and three anti-inflammatory semi-continuous measurements are shown in FIGS. 27 to 30.

In addition, the inhibitor was treated according to the predetermined number of cycles in order to confirm the drug sustainability according to the number of inhibitor treatment times, the results are shown in FIG.

Claims (13)

1. (1) treating an inflammation or cancer inducing factor in a medium in which animal cells having a cell membrane receptor are cultured;

   (2) after step (1), treating the medium with a detector-marker complex that specifically binds to the cell membrane receptor;

   (3) acquiring an optical signal after the complex processing; And

   (4) a cell-friendly immunoassay method characterized by measuring inflammation or cancer through the obtained optical signal analysis.
2. The method according to claim 1,

   The marker is a cell-friendly immunoassay method, characterized in that the enzyme or a phosphor that generates an optical signal by the Luminol (Luminol) emitter.
3. The method according to claim 1,

   The optical signal analysis is a cell-friendly immunoassay method characterized in that using a detector equipped with a telecentric lens in the cooling digital camera.
4. The method according to claim 1,

   The animal cell has a cell-friendly immunoassay characterized in that the adhesion to the solid surface.
5. The method according to claim 1,

   Cell-friendly immunoassay method characterized in that it does not include the process of chemical cell immobilization and cell membrane permeability.
6. The method according to claim 1,

   The cell membrane receptors include toll-like receptors (TLRs), ion channel receptors, G-protein coupled receptors, and receptor guanylyl cyclases. ), Receptor tyrosine kinase (RTK), cytokine receptor superfamily, Tyrosine phosphatases and serine / threonine protein kinases Cell-friendly immunoassay characterized in that above.
7. (1) treating an animal cell having a membrane receptor with an inflammation inducer or cancer inducer;

   (2) after the step (1), the animal cell is treated with a detector-marker complex that specifically binds to the cell membrane receptor, and the expression concentration of the cell membrane receptor is measured;

   (3) treating the animal cells with an anti-inflammatory or anticancer candidate after measuring the concentration; And

   (4) after the step (3), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor, and measuring the expression concentration of the cell membrane receptor,

   The anti-inflammatory agent or anti-cancer agent screening method, characterized in that the anti-inflammatory agent or anti-cancer agent is selected by comparing the expression concentration of the cell membrane receptor measured in step (2) and (4).
8. (1) treating an anti-inflammatory or anticancer candidate to an animal cell having a cell membrane receptor;

   (2) after step (1), treating the animal cell with a detector-labeled complex which specifically binds to the cell membrane receptor;

   (3) treating the animal cell with an inflammation inducer or a cancer inducer after measuring the expression concentration of the cell membrane receptor after treating the detector-marker complex; And

   (4) after step (3), treating the animal cell with a detector-marker complex that specifically binds to the cell membrane receptor, and measuring the expression concentration of the cell membrane receptor,

   The anti-inflammatory agent or anti-cancer agent screening method, characterized in that the anti-inflammatory agent or anti-cancer agent is selected by comparing the expression concentration of the cell membrane receptors measured in step (3) and (4).
9. The method according to any one of claims 7 to 8,

   The detector is at least one selected from antibodies, binding proteins, nucleic acids, aptamers and peptides, anti-inflammatory or anticancer screening method.
10. The method according to any one of claims 7 to 8,

    The marker is at least one selected from phosphors, light emitters, enzymes, metal particles, plastic particles and magnetic particles, anti-inflammatory or anticancer screening method.
11. The method according to any one of claims 7 to 8,

    A method for screening an anti-inflammatory or anticancer agent, characterized in that it is semi-continuously screenable.
12. Composition for diagnosing inflammation or cancer comprising an animal cell having a cell membrane receptor and a detector-marker complex that specifically binds to the cell membrane receptor.
13. Inflammation or cancer diagnostic kit comprising the diagnostic composition of claim 12.

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