Title Page
Contents
국문초록 10
ABSTRACT 12
I. Introduction 14
1. Mast cells in health and disease 14
2. Cereblon 23
II. Objectives 27
III. Materials and Methods 28
A. Experimental Animals 28
B. Animal Sampling 28
C. Histological Analysis for Toluidine Blue Staining 29
D. Bone Marrow Extraction and Bone Marrow-Derived Mast Cell Culture 29
E. Toluidine Blue Staining of BMMCs 32
F. Immunoblotting (Western Blot) 32
G. Immunocytochemistry 35
H. Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) 36
I. ELISA 38
J. Statistical Analyses 38
IV. Results 39
A. Diabetic animals had increased body weight but decreased heart weight and femur and tibia lengths 39
B. Diabetic hearts had increased numbers of mast cells 41
C. The wildtype and diabetic bone marrow-derived mast cells showed phenotypic differences throughout passages 43
D. The diabetic BMMC group showed lower levels of CRBN 48
E. Tryptase concentration was higher in the supernatant of immature BMMCs 53
F. mRNA levels showed no significant difference between the two groups 56
G. Diabetic BMMCs contained higher levels of CRBN in the cell culture supernatant 58
H. cJun as the common denominator between mast cell proteins and cereblon 60
I. The diabetic BMMC showed an increased protein expression in the inflammatory cytokine TNFα 63
J. CRBN is expressed in cardiac tissues of diabetic mice 65
V. Discussion 67
VI. Conclusion 71
References 72
Appendix 75
Table 1. List of Western Blot Primary Antibodies 34
Table 2. List of primer sequences used in RT-PCR 37
Figure 1. Mast cell development and heterogeneity. 16
Figure 2. Mechanisms of mast cell activation. 17
Figure 3. Comparison of various BMMC protocols. 22
Figure 4. Biological regulation and role of CRBN in health and disease. 24
Figure 5. Diagram of bone marrow extraction for bone marrow-derived mast cell (BMMC) culture and maintenance. 31
Figure 6. Phenotypic comparison of experimental animals. 40
Figure 7. Diabetic hearts had higher mast cell densities. 42
Figure 8. Observable phenotypic differences of BMMCs throughout passages. 45
Figure 9. Positive trend in cell viability and average cell sizes throughout passages. 46
Figure 10. Diabetic BMMC groups showed delayed maturation compared to the wildtype BMMC group. 47
Figure 11. CRBN levels were lower in the diabetic BMMCs at full maturation. 50
Figure 12. CRBN levels were lower in BMMCs at passage 5. 52
Figure 13. Tryptase concentration was higher in the supernatant of immature diabetic BMMCs. 55
Figure 14. mRNA levels showed no significant difference between the two groups. 57
Figure 15. Diabetic BMMCs had higher levels of CRBN in the culture supernatant. 59
Figure 16. JNK and cJun expressions were lower in the diabetic BMMCs. 61
Figure 17. mRNA expression of cJun and JNK1 showed minimal differences between the two groups. 62
Figure 18. Diabetic BMMCs had higher protein levels of the inflammatory cytokine TNFα. 64
Figure 19. Cardiac tissue protein expression. 66
Figure 20. Localization of CRBN expression in mast cells. 76