감사의 글
국문개요
Contents
Ⅰ. Introduction 15
Ⅱ. Reagents 20
1. Cell lines 20
2. Materials and Reagents 20
3. Instruments 22
Ⅲ. Methods 24
1. Purification of VC 24
2. Hemagglutination and sugar specificity test 25
3. SDS-PAGE 25
4. Cell culture 26
5. MTT assay 26
6. Cell growth determination 27
7. VCA-induced apoptosis 27
7-1 Nuclear staining 27
7-2 DNA fragmentation 28
7-3 Flow cytometric analysis 28
8. Protein preparation and Western blotting analysis 29
9. Telomearse assay 29
10. Total RNA preparation and PCR amplification 31
10-1 Total RNA isolation 31
10-2 Reverse transcriptase-polymerase chain reaction 31
11. Statistical analysis 33
Ⅳ. Results 35
1. Purification of VC 35
2. Hemagglutination and sugar specificity test 35
3. SDS-PAGE 35
4. VCA induces cell death in cells via apoptosis 37
4-1 Cytotoxic activities 37
4-2 Morphological changes of apoptotic nuclei 41
4-3 Flow cytometric analysis of VCA-treated cells 47
5. Correlation between p21 expression and VCA-induced apoptosis 53
6. VCA-induced apoptosis through activation of caspase-3 proteases in HL-60, SK-Hep-1 and Hep 3B cells 55
7. Caspase-3 inhibitor prevented apoptosis induced by VCA in HL-60 cells 59
8. VCA induced apoptosis through activation of Bax and inhibition of Bcl-2 in SK-Hep-1 and Hep 3B cells 61
9. VCA inhibited telomerase activity in SK-Hep-1 and Hep 3B cells 63
10. Effect of VCA on TNF-α gene expression 65
Ⅴ. Discussion 69
Ⅵ. Conclusion 74
Ⅶ. References 76
Abstract 85
Table 1. Primer sequences for cytokine cDNA amplification 34
Fig. 1. SDS-PAGE patterns of lectins (A) in the absence or (B) in the presence of reducing agent 36
Fig. 2. Dose-and time-dependent viability of HL-60 cells treated with VCA 38
Fig. 3. Dose-dependent viability of B16BL6 cells treated with VCA 39
Fig. 4. Dose-and time-dependent viability of SK-Hep-1 and Hep 3B cells treated with VCA 40
Fig. 5. Morphological changes of HL-60 and B16BL6 by treatment of VCA 42
Fig. 6. Morphological changes of (A) SK-Hep-1 and (B) Hep 3B by treatment of VCA 43
Fig. 7. DNA fragmentation induced by VCA in HL-60 cells 44
Fig. 8. DNA fragmentation induced by VCA in B16BL6 cells 45
Fig. 9. DNA fragmentation induced by VCA in SK-Hep-1 and Hep 3B cells 46
Fig. 10. Flow cytometry analysis of HL-60 cells in the absence of VCA 48
Fig. 11. Flow cytometry analysis of HL-60 cells in the presence of VCA 49
Fig. 12. Flow cytometry analysis of B16BL6 cells (A) in the absence or (B) in the presence of VCA (50 ng/mL) 50
Fig. 13. Flow cytometry analysis of SK-Hep-1 cells (A) in the absence or (B) in the presence of VCA (10 ng/mL) 51
Fig. 14. Flow cytometry analysis of Hep 3B cells (A) in the absence or (B) in the presence of VCA (10 ng/mL) 52
Fig. 15. Effect of VCA on the expression level of p53 and p21 in SKHep-1 and Hep 3B cells 54
Fig. 16. Cleavage of PARP (A) and activation of caspase-3 proteases (B) in HL-60 cells induced by VCA 57
Fig. 17. Cleavage of PARP (A) and activation of caspase-3 proteases (B) in SK-Hep-1 and Hep 3B cells induced by VCA 58
Fig. 18. (A) Inhibition of VCA-induced apoptosis, (B) Cleavage of PARP, and (C) activation of caspase-3 proteases in HL-60 cells by a caspase-3 specific inhibitor, z-DEVD-FMK 60
Fig. 19. Effect of VCA on the expression level of Bcl-2 and Bax in SK-Hep-1 and Hep 3B cells 62
Fig. 20. Inhibitory effect of VCA on the telomerase activity in SKHep-1 and Hep 3B cells 64
Fig. 21. Dose-dependent viability of RAW 264.7 cells treated with VCA 66
Fig. 22. Relative abundance of TNF-α mRNA in RAW 264.7 cells after in vitro exposure to LPS or VCA 67
Fig. 23. Effects of VCA on TNF-α gene expression 68