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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林萬寅 | |
dc.contributor.author | Yu-Jen Chen | en |
dc.contributor.author | 陳昱仁 | zh_TW |
dc.date.accessioned | 2021-06-12T18:30:47Z | - |
dc.date.available | 2009-08-28 | |
dc.date.copyright | 2007-08-28 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-08-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27969 | - |
dc.description.abstract | 腎上腺大腦白質退化症(X-linked adrenoleukodystrophy; X-ALD)是最常見的過氧化小體疾病,它導因於ABCD1基因的突變,而其所轉譯的ALDP蛋白質為包含腺苷三磷酸(ATP)結合區的過氧化小體膜蛋白。腎上腺大腦白質退化症的主要生化異常是在病人的組織、血液和纖維母細胞皆有極長鏈飽和脂肪酸(含有二十二個碳以上(≧C22:0)的飽和脂肪酸,主要為二十六個碳的飽和脂肪酸(C26:0))的過量堆積。ALDP 目前被認為是負責極長鏈脂肪酸運輸的蛋白質,但其致病的詳細分子機制和極長鏈脂肪酸堆積的生理效應至今仍然不清楚。為了研究極長鏈飽和脂肪酸堆積的可能生理效應,本論文以纖維母細胞和酵母菌為研究模式來分別探討其對細胞基因表現與脂肪酸代謝之影響。在正常體外培養之下,X-ALD 纖維母細胞內的C26:0 (佔總脂肪酸的0.02687%)是兩株正常纖維母細胞(分別佔總脂肪酸的0.0095%和0.01053%)的三倍左右。而經過1µg/ml C26:0的培養處理之後, X-ALD(佔總脂肪酸的0.052%) 和正常(佔總脂肪酸的0.021%)的纖維母細胞皆約為處理前的二倍左右。接著以微陣列晶片的方法來探討正常和X-ALD纖維母細胞經過極長鏈脂肪酸培養處理前後的基因表現差異發現,正常和X-ALD纖維母細胞以極長鏈脂肪酸培養處理前後並沒有明顯的基因表現差異。然而,以微陣列晶片與即時定量聚合酶鏈反應探討正常與X-ALD纖維母細胞間基因表現差異時,有6個基因呈現顯著的(大於兩倍)表現差異。以西方墨點法對其中兩個基因轉譯之蛋白質進行表現分析也證實, X-ALD較正常纖維母細胞的TFPI2蛋白質約有8-15倍的增加,DDEF2蛋白質約有1.5到3倍的降低。以1,2-dioleoylglycerol 對體外培養的X-ALD纖維母細胞處理之後發現,細胞中極長鏈脂肪酸的量降低了14~20%(其中C26:0降低了20%,約為正常與X-ALD纖維母細胞差異的三分之ㄧ),而其可能的機制為diacylglycerol 活化了ARF1 GAP的活性或是啟動了過氧化小體的分裂與增生,促進極長鏈脂肪酸的降解。綜合以上的結果顯示 ABCD1 基因的突變會導致纖維母細胞基因的差異性表達,且1,2-dioleoylglycerol 可降低體外培養的X-ALD 皮膚纖維母細胞之極長鏈脂肪酸的堆積。未來將持續研究探討1,2-dioleoylglycerol在不同劑量對減少極長鏈脂肪酸堆積的效能。
利用酵母菌模型,我們確認了FAT1基因的剔除會造成酵母菌體內極長鏈脂肪酸的增加。且在Fat1p蛋白質羧基端加入組胺酸標記,並不會顯著影響其在細胞內對極長鏈脂肪酸代謝的活性,但對於不同鏈長的脂肪酸仍會有不同程度的影響。利用大腸桿菌表現系統,我們已經成功地生產和純化重組的MBP-FAT1-6His融合蛋白質,並用以生產具特異性的anti-Fat1p抗體以偵測酵母菌蛋白質萃取物中的 Fat1p蛋白質。為了驗證純化的MBP-FAT1-6His融合蛋白質是否具有極長鏈乙醯基輔酶A合成酶(VLCS)的活性,我們以高效能液相層析(HPLC)與放射性同位素法(radioactive isotope)分析發現,並沒有顯著的極長鏈脂肪酸合成酶的活性。未來我們將持續探討MBP-FAT1-6His融合蛋白質失去極長鏈醯基輔酶A合成酶活性之原因,是否因為胺基端附加之麥芽糖結合蛋白(MBP)影響其活性或是在原核系統表達時缺乏適當之蛋白質轉譯後修飾所致. | zh_TW |
dc.description.abstract | X-linked adrenolukodystrophy (X-ALD) is impaired β-oxidation of very long chain fatty acids resulted from mutations of the ABCD1 gene which encodes a peroxisomal membrane protein (ALDP) with an ATP binding cassette. The principal biochemical characterization is abnormality of X-ALD which is the accumulation of saturated very long chain fatty acids (VLCFAs, ≧C22:0, mainly C26:0) in patient’s tissues, plasma and skin fibroblasts. ALDP is a putative transporter of very long chain fatty acids, but the detailed molecular mechanism of the disease and physiological effects of the accumulated VLCFAs are still not clear. To investigate possible physiological effects of the accumulated VLCFAs, fibroblast cell models were used to examine gene expression profiles before and after hexacosanoic acid (C26:0, 1µg/ml) challenge. Before hexacosanoic acid challenge, the intracellular level of C26:0 in X-ALD fibroblasts (0.02687% of total fatty acids) was about 3 folds to that of two fibroblast cell lines derived from normal controls (0.0095% & 0.01053% of total fatty acids). After hexacosanoic acid challenge, about 2-fold increase of C26:0 level was determined in X-ALD (0.052% of total fatty acids) compared to that of normal fibroblasts (0.021% of total fatty acids). Gene expression profiles were also compared between normal and X-ALD fibroblasts before and after VLCFA challenge using a microarray strategy. There was no significant difference in gene expression profiles before and after VLCFA challenge as analyzed by cDNA microarray of approximately 8,000 unique genes. Nevertheless, there were 6 differentially expressed genes of >2-fold differences between cultured X-ALD and normal skin fibroblasts by both cDNA microarray and real-time PCR. It was estimated an 8-15-fold increase of TFPI2 protein and a 1.5-3-fold decrease of DDEF2 protein expression in X-ALD compared to that in normal fibroblasts using Western blotting. After treatment of 1,2-dioleoylglycerol (1,2-DOG), the accumulation of VLCFAs was reduced by 14~20% (the 20% reduction of the intracellular C26:0 level in X-ALD fibroblasts after 1,2-DOG treatment was approximately one-third of the difference between normal and X-ALD skin fibroblasts before treatment). The mechanism of this reduction may possibly be due to the ability of phosphatidic acid (PA) and diacylglycerol (DAG) to initiate peroxisome division and proliferation resulting in the increased activity of VLCFAs β-oxidation or to activate the activity of ARF1 GAP involved in peroxisomal biogenesis. The above results suggest that mutations in the ABCD1 gene resulted in differential gene expression, and 1,2-DOG is useful for the reduction of VLCFAs in X-ALD skin fibroblasts model in vitro.
In the yeast model, we confirmed that deletion of the FAT1 gene resulted in the accumulation of VLCFAs in vivo. The addition of His tag at the carboxyl terminus of Fat1p, though not significantly affecting cellular VLCFA metabolism, does show different influences on cellular VLCFA accumulation of different carbon chain lengths. We had successfully expressed and purified recombinant Fat1p and used it to produce anti-Fat1p antibody with high purity for use in the detection of Fat1p in yeast protein extract. In order to verify whether purified Fat1p has VLCS activity in vitro, MBP-FAT1-6His fusion protein expressed in prokaryotic over-expression system was subjected to activity evaluation. There was no apparent fatty acyl-CoA synthetase activity for purified MBP-FAT1-6His using both C18:0 and C26:0 as substrates by both HPLC and radioactive isotope based analyses. The cause for the lack of VLCS activity for MBP-FAT1-6His will be further investigated. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:30:47Z (GMT). No. of bitstreams: 1 ntu-96-D91223022-1.pdf: 2631582 bytes, checksum: fd7a2bd340bb98a32d1c258b6fe9d18b (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 謝誌 I
摘要 II Abstract V Contents VIII Figure contents XI Table content XII Chapter 1. Introduction 1 Chapter 2. Materials and Methods 13 2-1. Cell culture for RNA preparation 14 2-2. Cell culture for saturated VLCFAs analysis 15 2-3. Cell was treated with 1,2-dioleoylgercerol for saturated VLCFAs analysis 15 2-4. VLCFAs analysis for skin fibroblast 15 2-5. Quiagen RNeasy mini kit for isolation of total RNA from human skin fibroblast 16 2-6. Reverse transcription Labeling 17 2-7. Microarray hybridization and washing 17 2-8. Microarray scanning and image analysis 18 2-9. Real-time PCR (qRT-PCR) 18 2-10. Western blot analysis for fibroblast 19 2.11. Yeast culture for fatty acid analysis 20 2-12. Fatty Acid Analysis for yeast 21 2-13. Cloning of His-tagged FAT1 (FAT1-6His) for E. coli expression system 23 2-14. Purification of FAT1-4His 24 2-15. Purification of MBP-FAT1-6His 25 2-16. Production and purification of antibody against FAT1-4His 25 2-17. Western blot analysis for yeast extract protein 26 2-18. MALDI-TOF analysis 27 2-19. Expression of Fat1p in yeast 27 2-20. Activity assay of MBP-VLCS by HPLC 28 2-21. Assay of Fatty Acyl-CoA Synthetase Activity by radioactive isotope method 28 Chapter 3. Study of Differential Gene Expression For X-Linked Adrenoluekodystophy Using Fibroblasts as a model 30 3-1. Accumulation of C26:0 in both X-ALD and normal skin fibroblasts by hexacosanoic acid treatment 30 3-2. Gene Expression Profiling Using Microarray Analyses 30 3-3. Verification of Differential Gene Expression by Real-Time PCR (qRT-PCR) and Western Blotting 31 3-4. 1,2-dioleoylglycerol (1,2-DOG) treatment decreases the accumulation of VLCFAs in fibroblasts 33 3-5. Discussion 35 3-6. Conclusion 39 Chapter 4. The study of the effect of very long chain acyl-CoA synthetase on the accumulation of cellular VLCFAs using a yeast model 52 4-1. VLCFA levels in yeasts 52 4-2. Heterologous over-expression of the His-tagged yeast FAT1p in E. coli and purification using Ni2+ resin 54 4-3. The expression and purification of MBP-FAT1-6His protein using E. coli system 55 4-4. The purification of anti-Fat1p antibody using affinity chromatography 55 4-5. The expression levels of Fat1p in yeast 56 4-6. Activity assay of MBP-FAT1-His in vitro by HPLC and radioactive isotope method 56 4-7. Discussion 58 4-8. Conclusion 61 Chapter 5. References 79 | |
dc.language.iso | en | |
dc.title | 以纖維母細胞和酵母菌為研究模式探討腎上腺大腦白質退化症之生化特性 | zh_TW |
dc.title | Biochemical characterization of X-linked adrenoleukodystrophy using a fibroblast cell model and a yeast model | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 盧志峰,王進賢 | |
dc.contributor.oralexamcommittee | 張煥宗,羅禮強,李憶菁 | |
dc.subject.keyword | 腎上腺大腦白質退化症,極長鏈乙醯基輔酶,A合成酶,極長鏈飽和脂肪酸,基因表現,微陣列晶片,即時定量聚合酶,鏈反應,融合蛋白質, | zh_TW |
dc.subject.keyword | X-linked adrenolukodystrophy,Very long chain acyl-CoA synthetase,Very long chain fatty acid,Gene expression profil,, Microarray,Real-time PCR,MBP-FAT1- 6His fusion protein, | en |
dc.relation.page | 82 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-08-02 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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