Anti-inflammatory Substance Screening Model via Semi-continuous Monitoring of Mammalian Cell-surface Receptors
- 주제(키워드) Continuous monitoring , Mammalian Cell-surface Receptors , Toll-like Receptors , Anti-inflammatory Substance Screening , Mammalian Cell-based Biosensor
- 발행기관 고려대학교 대학원
- 지도교수 백세환
- 지도교수 안동준
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위구분 박사
- 학과 일반대학원 바이오마이크로시스템기술협동과정
- 원문페이지 265 p
- 실제URI http://www.dcollection.net/handler/korea/000000056898
- 본문언어 영어
- 제출원본 000045827215
초록/요약
The present dissertation study is a part of research that has been conducted with a view to developing biochips, which can replace therapeutic agents’ efficacy tests and toxicity tests using animals, as an ultimate goal. In particular, the purpose of this study is to devise anti-inflammatory agent screening system using animal cell receptors such as toll-like receptors (TLRs) as biomarkers. To this end, a method of measuring animal cells’ inflammatory responses was studied by repeatedly inducing them through external stimulations and measuring changes in the concentration of TLRs in semi-continuous mode. By utilizing this method, the cellular response pattern for the presence of anti-inflammatory candidate substances can be compared with the standard inflammatory and anti-inflammatory curves, enabling us to evaluate the accumulated efficacy and toxicity of test substances. To achieve the purpose of this study, the following milestones were set. First, we determined conditions for sensitive measurement of cellular responses to external bacterial infection through immunoassays of TLRs. Second, an in vivo-like cellular response pattern was presented by utilizing TLR regulation switching mechanism through which, upon stimulation, the cell-surface receptor concentration increased and then came back to the normal level while the same cell was under restoration. Third, an anti-inflammatory drug screening model was further developed, which was attained by evaluating anti-inflammatory effect of drug candidate against repeated stimulations for the same cells based on that of the standard drug obtained under the same conditions. Finally, we constructed a cell-based biosensor, using micro-porous membranes as solid matrix, for therapeutic substance screening, which was able to enhance the semi-continuous cellular responses of TLR expression. The detailed contents of the study for the individual milestones were presented below. First, for TLRs as biomarker indicating cellular response to bacterial infection, a new animal cell-based immunoassay method was developed. To establish optimal detection models for each bacterium, different animal cells were infected with different species of living food-poisoning bacteria and TLRs expressed as the cells’ inflammatory responses were then measured. For typical examples, the measurement of TLR1 on the A549 cells was optimal for Escherichia coli (E. coli) while the detection of TLR2 on RAW264.7 cells was more suitable for Shigella sonnei (S. sonnei). The TLR concentrations were determined by immunoassays using antibodies specific to each receptor. It was found that the analysis sensitivity was remarkably increased if the bacterium captured on the cell-surface was detected simultaneously with the analysis of TLR. Through such dual-immunoassays, S. sonnei could be detected in 5.1 h after infecting the animal cells with an extremely low concentration (about 3.2 CFU per well) of the bacterium. This test result corresponded to a 2.5 h faster detection than a typical result of the conventional immunoassays that simply rely on antigen-antibody bindings. In addition, immuno-magnetic enrichment providing an effect of concentrating bacterium to 50 times was introduced to ensure analytical specificity as well as high sensitivity showing the measurement at 3.4 h after inoculation under the same conditions as mentioned above. This result corresponded to shortening of the analysis time to a half compared to the conventional immunoassay. The effects that improved the analytical sensitivity could be attributable to the synergy of: 1) proliferation of living bacterium, 2) increased TLRs expression in response to the infection, and 3) autocrine signaling associated with cytokine expression. Furthermore, the immunoassay method for TLRs was improved enough for handling of the cells in biocompatible manner and then used to measure the cellular responses to repetitive bacterial stimulations based on TLR regulation switching mechanism. We employed a cell line, A549, as the host cell and a bacterium, Pseudomonas aeruginosa (P. aeruginosa) in the lysate form, as stimulant. When stimulated, the selected cell-surface receptor, TLR1, on the host cells recognized specific pathogen-associated molecular pattern (PAMP) of the infectious agent, which eventually activated nuclear factor-κB (NF-κB) pathways through a series of processes. The resultant, up-regulated TLR concentration was measured through the detection of light signal generated by the enzyme label chemically associated with the specific antibody bound to the cell-surface receptor. The TLR concentration increased in the state of stimulation was down-regulated and come back to the normal level when the cell culture medium was replaced with the standard medium containing animal serum. Major factors that affected the cell-surface receptors up- and down-regulations were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and time interval for each regulation. Under the optimal conditions of these variables, the TLR regulation switching mechanism could be repeated three times for the same cells immobilized on the microtiter plate surfaces. The effect of antibody remaining after immunoassay or enzyme substrate (e.g., hydrogen peroxide) was minimized in each repetitive stage. Such analytical technique for the TLR-based cellular response could be highlighted by the fact that accumulated responses to repetitive stimulations were observed with the human-derived cells, which may be essential for therapeutic drug development. Using the change in TLR concentration expressed by the bacterial stimulation, a drug screening model was proposed based on the technique of semi-continuous measurement of the cellular response to test substance. The pattern of response to the substance was obtained through the same method and compared with the standard inflammatory and anti-inflammatory curves. The inflammatory curve was derived by measuring the TLR1 concentrations for the stimulated cells and normal cells, respectively, and then by plotting the ratio of the two concentration values against the cell culture time. Likewise, the anti-inflammatory curve was obtained using the same process except for the pretreatment of the stimulated cells with sodium salicylate known to suppress the activity of NF-κB. Based on the patterns of the two standard curves, the anti-inflammatory effects of CAPE, which was a honeycomb extract as a candidate substance for anti-inflammation, and acetaminophen, known to be a simple antipyretic drug as the negative control, were able to be distinguished. Furthermore, the drug screening model developed through the analysis of cellular responses to repeated stimulations could also determine the persistence of efficacy and accumulation of toxicity for tested substance. A cell-based biosensor was finally constructed by using polyester membrane as solid matrix for cell fixation so that the drug screening model using semi-continuous patterns of the animal cells’ inflammatory and anti-inflammatory responses can be simply reproduced. The membrane disc surfaces were treated to be hydrophobic and then used to immobilize the A549 cells on the limited area from the center. The membrane discs with the immobilized cells were triplicately stacked within a plastic frame to increase the cell concentration per unit volume. This configuration of cell fixation mimicked the in vivo conditions where cells were grown in three-dimensional structures and, thus, ensured the cells to secure spaces for accommodation when they proliferated over time. Comparing to the microtiter plate-based cell culture system, the repetition number of measuring cellular response to the stimulation-restoration cycle was indeed extended from three times to four times. In addition, the micro-porous membranes as solid matrix also enabled us to exchange the aqueous medium in flow-through mode, contributing a remarkable reduction in the immunoassay time along with the high cell concentration. Since the anti-inflammatory drug screening model presented in this study semi-continuously measured inflammatory or anti-inflammatory responses for the same cells, it could provide experimental data for the persistence of drug efficacy and accumulation of toxicity for tested substance. Such pre-clinical information cannot be obtained from the animal cell-based one-off screening systems previously developed to date. Therefore, the novel biosensing concept based on semi-continuous cellular response patterns could be more appropriate to replace animal experiments for testing new drugs than the conventional screening system.
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LIST OF FIGURES
ABBREVIATIONS
CHAPTER
1. INTRODUCTION
1.1. Organization of the Dissertation
1.2. Objective
1.3. Specific Goals
1.4. Experimental Design
1.4.1. Cell-surface Receptor-based Sensing of Bacterial Stimulation
1.4.2. Semi-Continuous Monitoring of Repetitive Stimulations
1.4.3. In vitro Inhibition of Inflammatory Response
1.4.4. Anti-inflammatory Substance Screening Model
1.4.5. Polyester Membrane-based Anti-inflammatory Drug Screening System
1.5. References
2. RESEARCH BACKGROUND
2.1. Definition of Animal Testing
2.2. Cases where Animal Testing is Utilized
2.2.1. Pure Researches
2.2.2. Applied Researches
2.3. Problems in Animal Testing and Related Regulations
2.4. Necessity of Alternatives to Animal Testing
2.5. Current State of Alternatives to Animal Testing
2.5.1. Bacterial Model for Carcinogen Assays
2.5.2. Fungal Model for Mammalian Drug Metabolism
2.5.3. Lower Animal Models: Caenorhabditis elegans Disease Models
2.5.4. Lower Animal Models: Zebrafish Disease Models
2.5.5. Cell Culture and Tissue Engineering Models
2.6. Limitation and Prospect of Alternatives to Animal Testing
2.7. Reference
3. NOVEL SENSING MODELS FOR BACTERIAL CONTAMINATION BASED ON TOLL-LIKE RECEPTORS EXPRESSION
3.1. Introduction
3.2. Materials and Methods
3.2.1. Materials
3.2.2. Preparations of bacterial and mammalian cells
3.2.3. Optimization of TLRs expression-based assays
3.2.4. Performance characterization of TLR-based assays
3.2.5. Development of specific bacterial detection protocol
3.3. Results and Discussion
3.3.1. Analytical concept
3.3.2. Propagation of TLRs expression by infection
3.3.3. Development of TLR-based bacterial detection system
3.4. References
4. SEMI-CONTINUOUS MONITORING OF CELLULAR RESPONSE USING TOLL-LIKE RECEPTOR REGULATION SWITCHING
4.1. Introduction
4.2. Materials and Methods
4.2.1. Materials
4.2.2. Preparation of bacterial lysate and mammalian cell
4.2.3. Conventional analytical procedures for TLR and for cell densities
4.2.4. Optimization of TLR up- and down-regulation conditions
4.2.5. Analysis for TLR regulation switching without cellular damage
4.3. Results and Discussion
4.3.1. Analytical Model based on TLR Regulation Switching
4.3.2. Determination of Optimal TLR Regulation Conditions
4.3.3. Repetitive Immuno-analysis of Cellular Response to Stimulation
4.4. References
5. REPETITIVE CELLULAR RESPONSE PATTERN ANALYSIS REGARDING EFFICACY AND TOXICITY OF ANTI-INFLAMMATORY SUBSTANCES
5.1. Introduction
5.2. Materials and Methods
5.2.1. Materials
5.2.2. Preparation of Bacterial Lysate and Mammalian Cells
5.2.3. Analytical Procedures for TLR Induced by Bacterial Stimulation
5.2.4. Suppression of Cell Receptor Upregulation Using a Chemical Inhibitor
5.2.5. Semi-continuous Biosensing Model for Anti-inflammation
5.3. Results and Discussion
5.3.1. Establishing an Experimental Model for Inflammation Inhibition Monitoring
5.3.2. Simulation of Anti-inflammatory Substance Screening
5.3.3. Characterization for Potential Cytotoxicity and Duration of Inhibition
5.4. Reference
6. BIOSENSING OF REPETITIVE BACTERIAL STIMULATIONS FOR THE SAME MAMMALIAN CELLS IMMOBILIZED ON POLYESTER MEMBRANE
6.1. Introduction
6.2. Materials
6.2.1. Preparation of Bacterial Lysate and Mammalian Cell
6.2.2. Analytical Procedures for TLR Induced by Bacterial Stimulation
6.2.3. Optimization of Cell-based, Flow-Through Immunoassay Kit
6.2.4. Semi-continuous Biosensing Model for Anti-inflammation
6.3. Results and Discussion
6.3.1. Diffusion-facilitated, Cell-based Immunoassay Concept
6.3.2. Cell Immobilization on Polyester Membrane and Its Characterization
6.3.3. Construction of High-performance Cell-based Biosensor System
6.3.4. Characterization of Cellular Responses to Repetitive Stimulations for the Same Cells
6.4. References
7. CONCLUSIONS
APPENDIX A
APPENDIX B
APPENDIX C
