Establishment and Characterization of an Immunoregulatory Microglial Cell Line

다국어 초록 (Multilingual Abstract)

Microglia play a central role in neuroinflammation during Alzheimer's disease (AD). However, their dysregulation underpins disease pathology. Dehydrogenase/reductase 9 (DHRS9), previously implicated in a regulatory marker of stable macrophages, remained unexplored in microglial function. This study investigates the impact of DHRS9 overexpression in human microglial HMO6 cells. DHRS9 promoted an anti-inflammatory microglial phenotype, characterized by the downregulation of M1 markers (CD86, iNOS), upregulation of M2 markers (CD206, ARG1), and enhanced expression of anti-inflammatory cytokines (IL-10, TGF-β), along with reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β). DHRS9 overexpression also enhanced Aβ phagocytosis and clearance, with correlation to the induction of the TREM2 phagocytic receptor. DHRS9-overexpressing microglia also ameliorated Aβ-induced neurotoxicity in SH-SY5Y cells. Mechanistically, DHRS9 activated the retinoic acid pathway in microglia cells. Collectively, our findings identify DHRS9 as a key regulator of microglial polarization and function, bridging metabolic signaling with immunomodulation and Aβ clearance. This positions DHRS9 as a promising therapeutic target for modulating microglial function in AD.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 1.1. Neurodegenerative diseases 1
• 1.2. Microglia – Macrophages of the central nervous system (CNS) 2
• 1.3. Microglia as a therapeutic target for neurodegenerative disease 3
• 1.4. Dehydrogenase/reductase 9 (DHRS9). 6
• 1.5. Research Objectives 7
• 2. MATERIALS AND METHODS 9
• 2.1. Cell culture 9
• 2.2. Gene cloning and plasmid construction 9
• 2.3. Cell transfection 10
• 2.4. DHRS9 gene knockdown 11
• 2.5. Beta-amyloid preparation 11
• 2.6. Thioflavin T assay 11
• 2.7. Microglia cell treatment 12
• 2.8. Phagocytosis assay 12
• 2.9. RNA preparation and real-time quantitative PCR 12
• 2.10. Reactive oxygen species (ROS) measurement 13
• 2.11. Neuronal cell treatment 13
• 2.12. Western blot analysis 14
• 2.13. Flow cytometry 15
• 2.14. Enzyme-linked immunosorbent assay (ELISA) 16
• 2.15. Statistical analysis 16
• 3. RESULTS 19
• 3.1. Construction and verification of EGFP and DHRS9 plasmids 19
• 3.2. Optimization of transfection conditions for HMO6 cells 24
• 3.3. Validation of DHRS9 overexpression in HMO6 cells after transfection 28
• 3.4. Microglial phenotypic shift in HMO6 cells following DHRS 9 overexpression 31
• 3.5. DHRS9 regulates microglia function through the retinoic acid pathway 35
• 3.6. DHRS9 overexpression decreases LPS-induced inflammation and oxidative stress in microglia 39
• 3.7. DHRS9 overexpression decreases Aβ-induced inflammation and ROS production in microglia 46
• 3.8. DHRS9 enhanced the phagocytosis of Aβ in HMO6 cells 56
• 3.9. DHRS9-overexpressing HMO6 reduced Aβ-induced neurotoxicity in SH-SY5Y 61
• 4. DISCUSSION 64
• 5. REFERENCE 72