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아스피린 불내성 천식환자에서 류코트리엔 제 1 수용체의 유전적 다형성과 IL-4에 의해 유도되는 류코트리엔 제 1 수용체의 전사조절기작에서 류코트리엔 조절제의 영향에 관한 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 박해심 | - |
| dc.contributor.author | 오정미 | - |
| dc.date.accessioned | 2019-10-21T06:46:01Z | - |
| dc.date.available | 2019-10-21T06:46:01Z | - |
| dc.date.issued | 2006-08 | - |
| dc.identifier.other | 1545 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/16374 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의학과,2006. 8 | - |
| dc.description.tableofcontents | ABSTRACT ⅰ TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF TABLES vii Ⅰ. INTRODUCTION 1 Ⅱ. MATERIAL AND METHODS 8 A. Subjects 8 B. Identification of genetic polymorphism in CysLTR1 gene 8 C. Plasmid construction for promoter activity and transfection 10 D. Nuclear extracts preparation and electrophoretic mobility shift assay 11 1. Nuclear extracts preparation 11 2. Electrophretic mobility shift assay (EMSA) 12 3. Sequence mutation analysis 13 E. CysLTR1 promoter deletion and luciferase reporter assay 14 F. Phamacological treatments and reporter gene assay 15 G. CysLTR1 expression by RT-PCR, Realtime-PCR and flowcytometry 16 1. RT-PCR analysis for CysLTR1 mRNA 17 2. Realtime-PCR analysis for CysLTR1 mRNA 17 3. CysLTR1 expression by flowcytometry 18 H. Statistical analysis 19 Ⅲ. RESULTS 20 A. Identification of three genetic polymorphisms in CysLTR1 gene 20 B. Genetic association study of CysLTR1 genetic polymorphism 20 1. Clinical characteristics of the study subjects 20 2. Genotype distribution of CysLTR1 3 SNPs 23 3. Association analysis of AIA associated quantitative phenotypes 25 4. Effect of 3 SNPs on transcriptional activity 28 5. Identification of unknown nuclear protein binding by EMSA 28 C. Characterization of CysLTR1 promoter in A549 cells 31 D. Transcriptional regulation of CysLTR1 gene by IL-4 33 1. Expression of CysLTR1 33 2. Effect of genetic polymorphism on the transcription regulation by IL-4 33 E. Effect of leukotriene modifier on the transcription regulation by IL-4 36 F. Effect of dexamethasone on CysLTR1 mRNA expression in A549 cells 38 Ⅳ. DISCUSSION 41 Ⅴ. CONCLUSION 49 REFERENCE 50 국문요약 64 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 아스피린 불내성 천식환자에서 류코트리엔 제 1 수용체의 유전적 다형성과 IL-4에 의해 유도되는 류코트리엔 제 1 수용체의 전사조절기작에서 류코트리엔 조절제의 영향에 관한 연구 | - |
| dc.title.alternative | Jung Mi Oh | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Jung Mi Oh | - |
| dc.contributor.department | 일반대학원 의학과 | - |
| dc.date.awarded | 2006. 8 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 565545 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001545 | - |
| dc.subject.keyword | AIA | - |
| dc.subject.keyword | ATA | - |
| dc.subject.keyword | CysLTs | - |
| dc.subject.keyword | CysLTR1 | - |
| dc.subject.keyword | SNP | - |
| dc.subject.keyword | MK-571 | - |
| dc.subject.keyword | Dexamethason | - |
| dc.subject.keyword | IL-4 | - |
| dc.description.alternativeAbstract | Background: In recent, intensive studies have attempted to elucidate the genetic background of aspirin intolerant asthma (AIA), but genetic factors associated with its pathogenesis are still not completely understood. Cysteinyl leukotrienes (CysLTs) play important roles in eosinophilic airway inflammation of AIA via CysLTR1-mediated mechanism. Leukotriene modifier MK-571, a selective CysLTR1 antagonist, is widely used in the management of asthma. We selected Cysteinyl leukotruene receptor 1 (CysLTR1) among leukotriene ？ related genes and screened for genetic variation in the 5？ upstream region of CysLTR1 gene. This study was designed to investigate whether MK-571 possesses inhibitory effect on transcriptional induction of CysLTR1 induced by IL-4 or not. Materials and methods: Genetic variation in 5？ upstream region of CysLTR1 gene were screened in 43 normal healthy controls by direct sequencing and then genetic association study of CysLTR1 genetic polymorphisms were performed in 105 aspirin intolerant asthma (AIA) patients, 111 aspirin tolerant asthma (ATA) patients, and 125 normal healthy controls (NC) . Promoter activities according to the genetic polymorphism were examined by luciferase reporter assay. Transcriptional regulation of endogeneous CysLTR1 gene induced by IL-4 was investigated using lung carcinoma cell line, A549 cells, by real time PCR and flowcytometry. Effect of MK-571 on the transcriptional induction of CysLTR1 by IL-4 was examined by luciferase reporter assay. Results: In the screening of 5？ upstream region of CysLTR1 gene in 43 normal healthy controls, three single nucleotide polymorphisms (SNPs) including C-634T, A-475C, and A-336G were identified. Though a genetic association studies of 3SNPs in 105 aspirin -intolerant asthma (AIA) patients, 111 aspirin tolerant asthma (ATA) patients, and 125 normal healthy controls (NCs), significant association of 3 SNPs with AIA were found. There were significant differences in allele frequencies of the 3 SNPs within male subjects; the common 3 SNP haplotypes, ht1 [C-A-A], was associated with decreased disease risk (p=0.031 for AIA vs. NC; p=0.022 for AIA vs. ATA), and the ht2 [T-C-G] haplotype with increased disease risk (p=0.031 for AIA vs. NC; p=0.031 for AIA vs. ATA). Moreover, 3 SNPs can modulate CysLTR1 gene expression; promoter activity was enhanced with ht2 [T-C-G] construct in comparison of ht1 [C-A-A] construct in jurkat cells, U937 cells, and A549 cells. Endogeneous level of CysLTR1 gene was remarkably increased by IL-4 in A549 cells. Promoter activity was also significantly increased with the ht2 [T-C-G] construct in comparison with ht1[C-A-A] construct in IL-4 primed A549 cells (p=0.008), which was significantly inhibited by pre-treatment with MK-571(p=0.004), not with dexamethasone in which ht1 [C-A-A] construct promoter activity was not significantly inhibited by dexamethasone. Conclusion: These results suggest that genetic variants of CysLTR1, ht2 [T-C-G], can up-regulate CysLTR1 expression and significantly induced CysLTR1 expression by IL-4. MK-571 may be more beneficial to control asthmatic symptoms, especially in patients carrying variant genotype of the CysLTR1 promoter. | - |
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