Title Page
Abstract
초록
Contents
1. INTRODUCTION 14
2. MATERIALS AND METHODS 19
2.1. Culture and neural differentiation of human iPSCs 19
2.2. Production of lentivirus 20
2.3. Differentiation of astrocytes from NPCs 20
2.4. Telomerase inhibition 22
2.5. Senescence-associated β-galactosidase (SA-β-gal) staining 22
2.6. Immunocytochemistry (ICC) 23
2.7. Human cytokine array 24
2.8. Intracellular ROS measurement 24
2.9. Lipid droplet (LD) analysis 25
2.10. Measurement of mitochondrial membrane potential (MMP) 26
2.11. Analysis of mitochondrial morphology 26
2.12. Glutamate uptake assay 27
2.13. Synaptosome purification and in vitro engulfment assay 27
2.14. Telomere length measurement 27
2.15. RNA isolation, cDNA synthesis, and real-time PCR 28
2.16. Statistical analysis 28
3. RESULTS 31
3.1. The treatment of BIBR1532 induced morphological changes and telomere shortening in hPSC-derived astrocytes 31
3.2. Significant DNA damage and nuclear deformation occurred in BIBR astrocytes 38
3.3. BIBR astrocytes exhibited senescence-related phenotypes 41
3.4. BIBR astrocytes displayed defects in some astrocyte functions 47
3.5. BIBR1532 treatment induced reactivity in hPSC-derived astrocytes 54
4. DISCUSSION 56
5. REFERENCES 61
Table 1. PCR primer sequence 30
Fig. 1. Differentiation of hPSC-derived NPCs with BIBR1532 treatment 34
Fig. 2. Differentiation of BIBR astrocytes from hPSC-derived NPCs 35
Fig. 3. Morphological alterations in BIBR astrocytes 36
Fig. 4. Telomere shortening and reduced proliferation are induced in BIBR Astrocytes 37
Fig. 5. Treatment of BIBR1532 resulted in increased γH2AX foci 39
Fig. 6. Nuclear deformation accompanied by lamin-B1 reduction in BIBR astrocytes 40
Fig. 7. BIBR astrocytes exhibited increased SA-β-gal activity 44
Fig. 8. Comparison of the expression of SASP genes in control and BIBR astrocytes 45
Fig. 9. Examination of cytokine levels secreted in control and BIBR astrocytes 46
Fig. 10. Comparison of astrocyte function 50
Fig. 11. Examination of lipid droplet accumulation and lipid peroxidation 51
Fig. 12. Comparison of intracellular and mitochondrial ROS levels 52
Fig. 13. Examination of mitochondrial function and structure 53
Fig. 14. Expression of reactive genes in control and BIBR astrocytes 55