목차

Abstract i
Table of Contents iii
List of Tables vii
List of Figures viii
Chapter 1. Research backgrounds and purpose 1
1.1. Research backgrounds 1
1.1.1. Cellular senescence 1
1.1.2. Intervertebral disc (IVD) degeneration (IVDD) 3
1.1.3. Association between IVDD and senescence 4
1.1.4. Challenges in systemic administration and multiple intradiscal injection 5
1.1.5. Cerebral ischemia-reperfusion (IR) injury in ischemic stroke 6
1.1.6. Association between cerebral IR injury and senescence 7
1.2. Purpose of research 8
Chapter 2. Experimental section 12
2.1. Cell culture 12
2.1.1. Isolation of AF and NP cells from IVDD patients 12
2.1.2. Isolation of AF+NP cells from healthy rat caudal discs 15
2.1.3. Rat brain cortex astrocytes culture 15
2.2. Preparation and characterization of PLGA nanoparticles 16
2.2.1. Synthesis of PLGA nanoparticles 16
2.2.2. Size and size distributions of PLGA nanoparticles 17
2.2.3. High-performance liquid chromatography (HPLC) analysis 18
2.2.4. Drug loading efficiency 18
2.2.5. In vitro drug release 20
2.2.6. Degradation of PLGA nanoparticles 21
2.3. In vitro assays 22
2.3.1. Effect of free ABT263 on disc cells 22
2.3.2. Effect of ABT263 released from PLGA-ABT on disc cells 23
2.3.3. OGD/R on astrocytes 23
2.3.4. Effect of concentration and treatment duration period of ABT263 on astrocytes 24
2.3.5. Immunocytochemistry 25
2.3.6. SA-β-gal staining of cells 26
2.3.7. Flow cytometry analysis for apoptosis 26
2.3.8. Enzyme-linked immunosorbent assay (ELISA) 27
2.3.9. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay 27
2.4. In vivo efficacy of PLGA-ABT in IVDD 28
2.4.1. Injury-induced IVDD rabbit models 28
2.4.2. Injury-induced IVDD rat models 29
2.4.3. In vivo retention of PLGA nanoparticles 30
2.4.4. In vivo toxicity of PLGA-ABT 31
2.4.5. In vivo efficacy of PLGA-ABT 32
2.4.6. SA-β-gal staining of disc tissues 33
2.4.7. Immunohistochemistry of the rat IVD tissues 34
2.4.8. qRT-PCR 35
2.4.9. TUNEL assay 37
2.4.10. MRI for rabbit 38
2.4.11. MRI for rat 38
2.4.12. MRI settings for rat 39
2.4.13. Safranin-O staining and hematoxylin and eosin (H&E) staining 39
2.4.14. Histological scoring 40
2.5. In vivo efficacy of ABT263 in cerebral IR injury 42
2.5.1. Transient MCAO rat models 42
2.5.2. In vivo efficacy of ABT263 on MCAO rat models 43
2.5.3. Immunohistochemistry of rat brain tissues 44
2.5.4. Measurement of infarct volume 45
2.5.5. Neurological function assessment 46
2.6. Statistical analysis 47
Chapter 3. Local delivery of ABT263 by PLGA-ABT for IVDD 48
3.1. Elimination of senescent disc cells by ABT263 in vitro 48
3.2. Preparation, characterization and in vitro efficacy of PLGA-ABT 58
3.3. In vivo retention of PLGA nanoparticle and toxicity of PLGA-ABT 62
3.4. Elimination of senescent cells by PLGA-ABT in vivo 64
3.5. Reduced expression level of SASP in vivo by PLGA-ABT 71
3.6. Inhibition of IVDD and restoration of IVD structure by PLGA-ABT in vivo 74
Chapter 4. ABT263 treatment for cerebral IR injury 82
4.1. Induction of senescence in rat cortex astrocytes by OGD/R 82
4.2. Selective elimination of senescent astrocytes by ABT263 in vitro 86
4.3. Elimination of senescent cells and attenuation of inflammation by ABT263 in cerebral IR-injured MCAO rats 90
4.4. Therapeutic effects of ABT263 in cerebral IR-injured MCAO rats 93
Chapter 5. Conclusion 95
References 97
Abstract in Korean 114