청각 세포간 신호전달과 관련된 Cisplatin의 이독성에 대한 은행엽 추출물의 예방효과
Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication
- 주제(키워드) "Gap Junction , Connexin , Hearing loss , Apoptosis , Gingko biloba extracts , Cisplatin"
- 발행기관 아주대학교
- 지도교수 정연훈
- 발행년도 2013
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016585
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. HEI-OC1 auditory cells were cultured and treated with cisplatin (50µM) and EGb (300µg/ml) for 24 h, and then analyzed by immunocytochemistry (Annexin V/ propidium iodide) and Western blots. The function of GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1–10µM) and EGb (50–400µg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP- ribose polymerase (PARP) bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of Cx 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 kHz and 32 kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of important mechanisms of cisplatin-induced ototoxicity.
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ABSTRACT i
TABLE OF CONTENTS ii
LIST OF FIGURES iii
ABBREVIATION iv
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIAL AND METHODS 7
A. In vitro experiments – apoptosis of auditory cells 7
1. Auditory cell culture 7
2. Cell viability assay 7
3. 4'6-Diamidino-2-phenylindole (DAPI) staining 8
4. Western blot analysis of caspase-3, poly-ADP-ribose polymerase (PARP), Cx26, and Cx43 8
5. Quantitative real time-polymerase chain reaction (qRT-PCR) of Cx26, Cx30, Cx31, and Cx43 9
6. Immunocytochemistry of Cx26, Cx30, Cx31, and Cx43 10
7. Scrape-loading dye transfer (SLDT) assay 10
B. Exvivo experiments - invitro organ of Corti culture 12
C. In vivo experiments 13
1. Auditory Brainstem Response (ABR) Test 13
2. Phalloidin staining 14
3. Scanning electron microscope (SEM) 14
Ⅲ. RESULTS 16
A. In vitro experiments – apoptosis of auditory cells 16
1. Protective effects of EGB 761 on cisplatin-induced death in HEI-OC1 cells 16
2. Protective effects of EGB 761 on cisplatin-induced apoptosis 18
3. Protective effects of EGb 761 on the inhibition of GJIC by cisplatin 20
B. Ex vivo experiments - in vitro oC culture 26
C. In vivo experiments 28
1. Shift of ABR thresholds 28
2. Cochlear morphology - phalloidin staining and SEM 30
Ⅳ. DISCUSSION 32
Ⅴ. CONCLUSION 39
REFERENCES 40
국문요약 56
