In vitro Modeling of Noise-Induced Hearing Loss using in vivo Data of Gene Expression Changes or Direct Noise Stimuli

목차 (Table of Contents)

• I. INTRODUCTION 1
• II. STUDY 1: Identification of genetic factors associated with NIHL 8
• 1. Materials and methods 8
• 1-1. Animal models 8
• 1-2. Noise exposure 8
• 1-3. Auditory brainstem response (ABR) 8
• 1-4. Hematoxylin and eosin staining and immunohistochemistry 9
• 1-5. RNA sequencing 10
• 1-6. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) 11
• 1-7. Antibodies and reagents 13
• 1-8. Statistical analysis 13
• 2. Results 14
• 2-1. NIHL Mouse Model for PTS 14
• 2-2. Morphological analysis of the cochlear tissues of NIHL animal models 16
• 2-3. Transcriptomic profiles of RNA sequencing through hierarchical clustering, KEGG pathway and GSEA analysis 19
• 2-4. IPA analysis 22
• 2-5. Inflammation, cell death and mitochondrial oxidative stress markers within the cochlear tissue of NIHL mice 26
• 2-6. TNFSF10 expression within the cochlear tissue 28
• 2-7. Comparison of gene expression patterns related to the metabolic pathway and ROS mechanisms in the NIHL animal model 30
• III. STUDY 2: In vitro modeling of NIHL using chemical drug in auditory hair cells 32
• 1. Materials and methods 32
• 1-1. HEI-OC1 Cell Culture and drug treatments 32
• 1-2. Immunohistochemistry 32
• 1-3. qRT-PCR 32
• 1-4. Western blot analysis 32
• 1-5. Mitochondrial DNA Copy-Number Analysis 33
• 1-6. Measurement of ATP Content 33
• 1-7. MitoTracker staining 34
• 2. Results 35
• 2-1. Drug-induced NIHL mimetic in vitro model 35
• 2-2. Protein expression in the apoptosis, necroptosis, and ER stress pathways of the drug-induced NIHL mimetic in vitro model 37
• 2-3. Effects of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) to the HEI-OC1 for increasing mitochondrial biogenesis 39
• 2-4. Effects of BCH on the TG-induced NIHL mimetic HEI-OC1 cells 41
• 2-5. Effects of BCH Administration on an in vivo NIHL Model 43
• IV. STUDY 3: In vitro modeling of NIHL using noisy sound 45
• 1. Materials and methods 45
• 1-1. Applying sound stimulation to the HEI-OC1 cells 45
• 1-2. Cell viability 45
• 1-3. Immunohistochemistry 45
• 1-4. qRT-PCR 46
• 1-5. Mitochondrial ROS detection 46
• 1-6. TUNEL staining 46
• 2. Results 47
• 2-1. Protocol for noise stimulation and cell viability in a NIHL mimetic model 47
• 2-2. Inflammation, cell death, and various markers expressed in HEI-OC1 cells following noise stimulation 49
• 2-3. Mitochondrial ROS production in noise-stimulated HEI-OC1 cells 52
• 2-4. Apoptosis in HEI-OC1 cells and hair cell loss in cochlear explants induced by noise stimulation 54
• V. DISCUSSION 56
• VI. CONCLUSION 66
• VII. REFERENCES 67
• 국문요약 78