아주대학교

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TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………….i
TABLE OF CONTENTS……………………………………………………………….….. v
LIST OF FIGURES………………………………………..…………………………… viii
ABBREVIATIONS…………………………………………………………………………..x
I.INTRODUCTION 1
II. MATERIALS AND METHODS 7
A. Method 7
1. Epithelial cell culture and tTG activity assay 7
2. Intracellular reactive oxygen species (ROS) measurement 8
3. Immunoblot analysis 8
4. In vitro cross-linking reaction assay 9
5. Statistical analysis 9
B. Method 10
1. Materials 10
2. Cell culture 10
3. Real-Time polymerase chain reaction 10
4. Preparation of whole, cytosolic and nuclear extracts………………………………..11
5. Western blot analysis 12
6. Electrophoretic Mobility Shift Assay (EMSA) 13
7. Immunocytochemistry 13
8. Statistical analysis 14
C. Method 14
1. Cells culture and treatment with TDI 14
2. Real-time reverse transcriptase polymerase chain reaction……………………….... 15
3. Preparation of FITC-DBP conjugate and immunofluorescence staining 16
4.Flow cytometry analysis 16
5. Western blot analysis 17
6. Measurement of 1,25(OH)2D3 production and VEGF secretion 17
7. Statistical Analyses 18
III. RESULTS 19
A. Results 19
1. TDI-HSA conjugate increased tTG activity in A549 cells via ROS production 19
2. The cross linking between tTG and CK19 26
B. Results 29
1. TDI down-regulated FTL expression in A549 cells………………………………… 29
2. TDI down-regulated HO-1 expression in A549 cells ……………………..31
3. TDI inhibited Nrf2 translocation into nucleus ……………………………………….35
4. TDI inhibited the activation of MAP kinases ………………………………………..38
5. PPARγ agonists rescued the effect of TDI on the expression of HO-1/FTL …. …….41
C. Results 44
1. Suppresses of the uptake of FITC-VDBP into RLE-6TN cells by TDI……………. .44
2. Megalin mediated the endocytic uptake of FITC-VDBP into RLE-6TN cells……... 46
3. Downregulation of megalin mRNA and protein by TDI treatment in RLE-6TN……49
4. Decreased 1,25(OH)2D3 production by TDI ……………..51
5. Increased VEGF production and secretion through suppression of the
1,25(OH)2D3 production……………...…………………………………………………52
IV. DISCUSSION.................................................................................................................55
V. CONCLUSION................................................................................................................67
REFERENCES....................................................................................................................68
-국문요약-.............................................................................................................................85
















LIST OF FIGURES

Fig. 1. activation of tissue transglutaminase (tTG) with exposure to toluene
diisocyanate-human serum albumin (TDI-HSA) conjugate in A549 cells.. …21
Fig. 2. Measurement of intracellular reactive oxygen species (ROS) production
induced by toluene diisocyanate-human serum albumin (TDI-HSA) in A549 cells. 23
Fig. 3. Activation of tissue transglutaminase (tTG) by toluene diisocyanate-human
serum albumin (TDI-HSA) via reactive oxygen species (ROS) production in A549
cells. …..25
Fig. 4. Immunoblot analysis of bronchial epithelial cells using tissue transglutaminase (tTG)
and cytokeratin 19 (CK19). 28
Fig. 5. TDI downregulated ferritin expression in A549 cells. 31
Fig. 6. TDI downregulated the expression of several antioxidant proteins in A549 cells 34
Fig. 7. TDI inhibited the translocation of Nrf2. 37
Fig. 8. TDI inhibited activities of MAP kinases 40
Fig. 9. 15d-PGJ2 and rosiglitazone could rescue the effect of TDI on the expression of
HO-1/FTL. 43
Fig. 10. Time- and dose-dependent effect of toluene diisocyanate (TDI) on FITC-vitamin
D binding protein(VDBP) uptake in RLE-6TN cells……………………………….45
Fig. 11. comparison of FITC-vitamin D binding protein (VDBP) uptake…………….……48
Fig. 12. The effect of Toluene diisocyanate (TDI) on megalin expression………………...50
Fig. 13. The effect of Toluene diisocyanate (TDI) on 1,25(OH)2D3 production…………...52
Fig. 14. The effect of Toluene diisocyanate (TDI) and 1,25(OH)2D3 on VEGF production
and secretion……………………………………………………………………….54