Title Page
Contents
국문초록 10
ABSTRACT 12
I. Introduction 14
1. Cardiac fibrosis 14
2. Cereblon (CRBN) 15
3. Transient receptor potenial Ankyrin 1 (TRPA1) 16
II. Objectives 21
III. Materials and Methods 22
1. Animal tissue lysate 22
2. Cell culture 22
3. Plasmid construction 22
4. Plasmid DNA cloning and extraction 23
5. Plasmid DNA transfection 23
6. Co-immunoprecipitation 24
7. siRNA transfection and adenovirus infection 24
8. Cell surface expression assay 25
9. Western Blot 26
10. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) 28
11. Drug treatment 31
12. Immunocytochemistry (ICC) 31
13. Ca2+ influx measurement[이미지참조] 32
14. Analysis 34
IV. Results 35
1. CRBN deficiency increases TRPA1 protein levels 35
2. Presence of CRBN decreases TRPA1 protein levels 37
3. CRBN and TRPA1 directly interact with each other 40
4. CRBN reduces functional TRPA1 42
5. TRPA1 potentially plays a role in cardiac fibrosis 45
6. TRPA1 deficiency decreases fibrotic markers in TGF-β1-induced cardiac fibrosis in MCF 50
7. Inhibition of TRPA1 by CRBN attenuate cardiac fibrosis induced by TGF-β1 in MCF 52
8. Inhibition of TRPA1 by CRBN alleviates fibrosis through TGFβ1-ERK1/2-SMAD2 pathway 54
V. Discussion 56
VI. Conclusion 63
References 65
Table 1. List of antibodies used in Western blot and immunostaining experiments. 27
Table 2. List of primer sequences used in RT-PCR 30
Table 3. Components of Ca2+ buffer used in Ca2+ influx measurement (1 L)[이미지참조] 33
Figure 1. Mammalian TRP ion channel family tree. 17
Figure 2. Structure of TRPA1 channel. 18
Figure 3. CRBN-TRPA1 as possible target for heart failure treatments. 21
Figure 4. CRBN deficiency increases TRPA1 levels. 36
Figure 5. CRBN decreases TRPA1 levels. 38
Figure 6. CRBN directly interacts with TRPA1. 41
Figure 7. CRBN decreases functional TRPA1. 43
Figure 8. TRPA1 in heart failure. 46
Figure 9. Inducing cardiac fibrosis with MCF in vitro. 48
Figure 10. TRPA1 is increased in TGF-β1-induced cardiac fibrosis. 49
Figure 11. Knockdown of TRPA1 decreases fibrotic markers in MCF. 51
Figure 12. Overexpression of CRBN decreases TRPA1 and fibrotic marker levels in TGF-β1-induced cardiac fibrosis. 53
Figure 13. Overexpression of CRBN decreases the phosphorylation of SMAD2 and ERK1/2. 55
Figure 14. Proposed mechanism on the role of CRBN and TRPA1 in cardiac fibrosis. 64