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Docetaxel에 의한 p65의 핵내 이동 억제에 관한 연구

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Author(s)
이정희
Issued Date
2007
Keyword
Docetaxel|p65|이동억제|핵내이동|유전자 발현
Abstract
주목나무에서 추출한 항암제인 docetaxel이 큰 포식세포에서 lipopolysaccharide (LPS)에 의한 iNOS 유전자의 발현에 미치는 영향에 대해 연구하였다. 본 연구를 통하여 docetaxel (0.1 mM)을 8시간 전처리하면 LPS에 의해 유도되는 inducible nitric oxide synthase (iNOS) 유전자의 발현을 억제함을 확인하였다. Docetaxel은 큰 포식세포에서 일산화질소의 생성을 유의성 있게 억제하였으며, Western blot과 reverse transcriptase-polymerase chain reaction (RT-PCR)을 통하여 iNOS 유전자 발현을 억제시킴으로써 일산화질소의 생성을 억제함을 확인하였다. iNOS에 대한 면역형광염색실험결과, iNOS의 발현이 docetaxel에 의해 억제됨을 재확인하였다. Electrophoretic mobility shift assay를 수행한 결과 docetaxel이 nuclear factor-kB/Rel (NF-kB/Rel)의 DNA 결합 활성을 억제함을 확인하였다. 또한 NF-kB/Rel를 구성하는 중요한 단백질인 p65에 대한 면역형광염색을 통하여 docetaxel이 p65의 핵내 이동을 억제함을 확인하였다. 이러한 결과들로부터 docetaxel이 NF-kB/Rel 의 활성을 억제함으로써 iNOS 유전자의 발현을 억제함을 확인하였다.|In this study, docetaxel, an antitumor agent derived from yew tree, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. In this report we described that the pretreatment of macrophages with docetaxel (0.1 mM) for 8 h inhibited LPS-induced iNOS gene expression. Pretreatment of RAW 264.7 cells with docetaxel significantly inhibited LPS-stimulated nitric oxide (NO) production. Western immunoblot of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression in RAW 264.7 cells. Immunocytochemical staining of iNOS further confirmed that pretreatment of macrophages with docetaxel inhibited macrophage activation. Electrophoretic mobility shift assay showed that docetaxel inhibited NF-kB/Rel DNA binding. Immunocytochemical staining of p65, a component of NF-kB/Rel, showed that docetaxel inhibited nuclear translocation of p65 protein. Collectively, these series of experiments indicate that docetaxel inhibits iNOS gene expression by blocking NF-kB/Rel activation.
Alternative Title
Inhibition of p65 nuclear translocation by docetaxel
Alternative Author(s)
Lee, Jung-Hee
Affiliation
조선대학교 대학원
Department
일반대학원 의학과
Advisor
임진수
Awarded Date
2007-08
Table Of Contents
Ⅰ. INTRODUCTION = 1
Ⅱ. MATERIALS AND METHODS = 5
A. Materials = 5
B. Cell culture = 5
C. Isolation of peritoneal macrophages = 5
D. Nitrite quantitation = 6
E. Western immunoblot analysis = 6
F. RT-PCR = 7
G. Transient transfection of RAW 264.7 cells = 7
H. Electrophoretic mobility shift assay (EMSA) = 9
I. Statistical analysis = 10
Ⅲ. RESULTS = 12
A. Effect of docetaxel on nitrite production in macrophages = 12
B. Effect of docetaxel on iNOS gene expression in macrophages = 17
C. Inhibition of NF-kB/Rel in response to docetaxel in LPS-stimulated RAW 264.7 cells = 23
D. Inhibition of NF-kB/Rel nuclear translocation by docetaxel in LPS-stimulated RAW 264.7 cells = 27
Ⅳ. DISCUSSION = 34
Ⅴ. REFERENCES = 37
영문초록 = 41
Degree
Doctor
Publisher
조선대학교 대학원
Citation
이정희. (2007). Docetaxel에 의한 p65의 핵내 이동 억제에 관한 연구.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/6814
http://chosun.dcollection.net/common/orgView/200000234297
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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