Title Page
Contents
Abstract 10
I. INTRODUCTION 13
1. Role of macrophages and macrophage polarization in inflammatory disease 13
2. Quantum dots nanoparticle 14
3. The cellular uptake mechanism of nanoparticles 15
II. MATERIALS AND METHODS 19
1. Materials 19
2. Cell culture 19
3. Treatment of QDs on RAW 264.7 cells 19
4. Preparation and characterization of QD complex 20
5. Cell transfection 20
6. Cell viability 21
7. RNA preparation, reverse transcription-PCR (RT-PCR) and real-time RT-PCR 22
8. Western blotting 22
9. Flow cytometry 23
10. Immunofluorescence staining 23
11. Inhibition of endocytosis 24
12. Fluorescence microscopy 24
13. Statistical analysis 25
III. RESULTS 32
1. A murine macrophage cell line, RAW 264.7 cells, has low transfection efficiency 32
2. Characterization and effects of three types of QDs on RAW 264.7 cells 42
3. The fabrication and characterization of QD complex 51
4. QD complex at 80 pmol concentration are more efficient for internalization in RAW 264.7 cells 57
5. QD complex is suitable for the efficient delivery of small size nucleotides in RAW 264.7 cells 62
6. The uptake mechanism of small RNAs-QD complex depends on not only Clathrin- but also Caveolin-mediated endocytic pathway 69
7. Small RNAs-QD complex successfully escape from the endosomes in RAW 264.7 cells 74
8. QD complex/miR-10a efficiently inhibit M1 and promote M2 polarization in RAW 264.7 cells 78
9. QD complex/miR-10a inhibit LPS-induced activation of NF-кB (p65), mitogen-activated protein kinase (MAPK), and AKT signaling pathways 81
IV. DISCUSSION 84
V. CONCLUSION 87
REFERENCES 89
ABSTRACT IN KOREAN 100
Table 1. List of oligonucleotides 26
Table 2. Primer sequences for RT-PCR and real-time RT-PCR 27
Table 3. List of primary antibodies 28
Table 4. List of secondary antibodies 30
Table 5. The function of pharmacological inhibitors for endocytosis 31
Figure 1. M1 and M2 phenotypes of macrophages 18
Figure 2. Evaluation of the transfection efficiency of plasmid DNA in... 37
Figure 3. Evaluation of the transfection efficiency of small RNAs in... 41
Figure 4. The characterization of three types of QDs with different... 44
Figure 5. Amine-QDs were poorly internalized and have a toxicity 46
Figure 6. Carboxyl-QDs were internalized with high efficiency and... 48
Figure 7. PEG-QDs were internalized with high efficiency and have... 50
Figure 8. A schematic diagram of the fabrication of QD complex 53
Figure 9. The characteristics of modified QDs 56
Figure 10. Evaluation of the delivery efficiency of GFP plasmid DNA-... 59
Figure 11. Evaluation of the delivery efficiency of small RNAs-QD... 61
Figure 12. Evaluation of the transfection efficiency of pDNA-QD complex 65
Figure 13. Evaluation of the transfection efficiency of small RNAs-QD complex 68
Figure 14. Cellular uptake mechanisms of QDs and QD complex 73
Figure 15. Subcellular localization of small RNAs-QD complex 77
Figure 16. Effect of QD complex/miR-10a to modulate macrophage polarity 80
Figure 17. Effect of QD complex/miR-10a on the phosphorylation of... 83
Figure 18. Cartoon illustrating the concept that summarized the effect... 88