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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉逸軒(I-Hsuan Liu) | |
dc.contributor.author | Yen-Ju Chan | en |
dc.contributor.author | 詹雁茹 | zh_TW |
dc.date.accessioned | 2021-07-11T14:44:17Z | - |
dc.date.available | 2021-10-14 | |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78162 | - |
dc.description.abstract | 在斑馬魚胚胎發育前期,其卵黃中膽固醇主要以游離膽固醇形式存在,先前的研究結果暗示斑馬魚胚胎吸收卵黃中膽固醇的機制可能與哺乳類動物腸道吸收膽固醇機制相類似。除此之外,另有實驗證實於哺乳類腸道中主要參與膽固醇吸收的基因也同時也在斑馬魚卵黃囊膜中表現,如:Mttp及Npc1l1基因。而在哺乳類腸道吸收膽固醇的機制中,第二型固醇酰基轉移酶 (Sterol O-acyltransferase,Soat2)為另一相當重要的功能性蛋白。Saot2是細胞內能將長鏈脂肪酸與膽固醇進行酯化形成膽固醇酯的酵素,使膽固醇以膽固醇酯的形式透過淋巴管進入循環系統被運送。過去我們利用in situ hybridization實驗方法證實Soat2同樣的會在斑馬魚胚胎發育時表現,因此推測於斑馬魚胚胎發育過程中,Soat2對卵黃中膽固醇的利用也扮演相當重要的角色。
為了觀察斑馬魚胚胎發育時利用卵黃中膽固醇時Soat2所扮演的角色,我們利用轉錄激活因子樣效應因子核酸酶(transcription activator-like effector nucleases, TALEN)技術建立Soat2基因剔除之斑馬。在所有帶有異常Soat2基因序列的斑馬魚子代中,篩選出於基因第四號外顯子中有八個去氧核醣核苷酸被剔除的基因序列型態。在體外的細胞實驗,我們利用HEK293細胞株過量表現正常或異常Soat2 蛋白功能並且測量細胞整體膽固醇酯含量做為細胞進行膽固醇酯化的能力判斷,結果證實表現正常Soat2基因功能的細胞組表現顯著較高的膽固醇酯含量,由此證實我們透過TALEN技術得到的soat2基因序列異常確實會造成Soat2失去正常基因功能。進一步觀察soat2基因異常時對於胚胎產生的影響,包含胚胎死亡率、胚胎卵黃面積大小變化以及胚胎整體膽固醇酯含量變化,結果顯示當Soat2發生異常時,並不會對胚胎存活率和發育造成嚴重影響,但卵黃從胚胎出生後第24小時就顯著較大,而膽固醇酯則在第72小時偵測為顯著較低。 為了更明確了解soat2基因功能與卵黃面積大小之間的關聯,我們將帶有不同soat2 基因型態的胚胎做區分,卻發現在第三世代的胚胎卵黃大小並不因帶有不同的soat2基因型而有差異,針對此特別的現象,我們進一步利用soat2特定的morpholino oligomer繼續進行實驗。利用morpholino暫時性抑制野生型斑馬魚soat2基因功能同樣會造成胚胎卵黃較大的現象,但是當作用於soat2基因異常的子代時則看不見差異,根據先前的研究結果暗示此現象可能為某特定基因被剔除時,特定機制或基因將被啟動以彌補胚胎發育時所需要的特定基因功能表現。另外,為了更加確認Soat2基因功能和卵黃面積大小與膽固醇酯之間的關係,額外在soat2剔除子代的卵黃中給與正常soat2 RNA片段,希望藉此能回復胚胎因失去Soat2功能所產生的異常型態。然而此實驗結果顯示即使於卵黃中給予soat2 RNA片段仍無法回復卵黃面積較大及膽固醇酯含量較低的現象。為了證實在Soat2異常的斑馬魚中有彌補性機制被啟動,針對與膽固醇代謝相關的基因表現進行觀察,結果發現在胚胎第24、48小時時羥甲基戊二酸單醯輔酶A (hydroxymethylglutaryl-CoA, HMG-CoA)大量表現,但在第96小時則顯著下降,除此之外,soat1於胚胎發育前期表現量叫野生型較高,而卵磷脂胆固醇酰基转移酶(lecithincholesterolacyltransferase,LCAT)則在第96小時大量表現。 綜合上述,我們證實Soat2為胚胎利用卵黃中物質時所需的媒介之一,此基因功能異常時將會影響卵黃物質的吸收利用並影響卵黃大小。除此之外,Soat2也影響胚胎中膽固醇酯的含量,當Soat2異常時會導致胚胎發育於第72小時時膽固醇酯顯著下降。特別的是在這些帶有soat2基因序列異常的斑馬魚中,可能由於無法正常利用卵黃中膽固醇,在胚胎發育第24、48小時時透過大量自行合成膽固醇以彌補膽固醇不足,而到第96小時則開始透過Abca1和Lcat基因吸收卵中膽固醇,因此使膽固醇自行合成作用下降。 | zh_TW |
dc.description.abstract | Free cholesterol is the major form of yolk cholesterol in early stage of zebrafish embryogenesis. Accumulating evidences suggested that the mechanism for zebrafish embryo to absorb cholesterol and lipid from yolk is similar to that in mammalian intestines where the Soat2 plays a role in cholesterol trafficking. Sterol O-transferase2 (Soat2) is an intracellular enzyme that can transfer long-chain fatty acyl-CoA to cholesterol to form cholesteryl ester (CE), the major form of cholesterol with the delivery of lipoproteins. We previously demonstrate that soat2 was expressed in the zebrafish yolk sac membrane and Soat2 depleted embryos showed abnormal yolk consumption. From the evidences, we hypothesized that Soat2 plays a role in the mechanism of yolk cholesterol trafficking during zebrafish embryogenesis.
The soat2-knockout zebrafish (F0) was generated by using transcription activator-like effector nuclease (TALEN) system and further produced F1 generation by outcrossing F0 with AB wild-type zebrafish. Among all F1 zebrafish, only one mutant allele type was found with an 8-nucleotide deletion at exon4 of soat2 genomic DNA sequence. To confirm the effect of mutant sequence, HEK293 cells were used to examine zebrafish Soat2 enzymatic activity. Cells overexpressing mutant Soat2 showed significantly lower CE levels compared to those overexpressing normal Soat2, indicating the success of loss-of-function by Soat2 mutation. To test the importance of Soat2 in zebrafish embryogenesis, we characterized the phenotype of soat2-knockout descendants. Results suggested that the mortality and gross morphology of mutant F2 descendants were not significantly different from AB wild-type embryos, but with significantly larger yolk size and lower CE levels at 72 hour-post-fertilization (hpf) compared to AB wild-type zebrafish. Surprisingly, the yolk sizes of the F3 descendants were no significant difference among siblings with different soat2 genotypes. To characterize the phenomenon, soat2-knockdown AB wild-type morphants were evaluated which showed significantly larger yolk sizes. The soat2-knockdown in Soat2 mutation descendants were further carried out to test whether a genomic compensation mechanism is processed in Soat2 mutation descendants to compensate Soat2 loss-of-function. Soat2-knockdown in Soat2 mutation zebrafish showed no difference of yolk sizes from control Soat2 mutation zebrafish, indicating some factors were inherited from maternal zebrafish or a compensatory mechanism is activated to rescue the phenotype from Soat2 loss-of-function. To further understand the interactions between soat2 genotypes and phenotypes of yolk sizes and CE levels, the wild-type soat2 RNA was injected into yolk area of Soat2 mutation descendants at 3hpf and then analyzed the yolk sizes and CE levels of the embryos. Results suggested that wild-type soat2 RNA failed to rescue the phenotypes of Soat2 mutation descendants, indicating the phenotypes were affected by some factors inherited from parents. To verify the speculation of a compensatory mechanism activated in the soat2-knockout zebrafish, gene expression of several genes relevant to cholesterol metabolism were detected. Results showed that HMG-CoA was overexpressed in embryos at 24, 48 hpf. The expression of soat1was higher at earlier stage of embryogenesis, while lcat was overexpressed in embryos with mutant Soat2 at 96 hpf. In conclusion, Soat2 plays a role in yolk cholesterol trafficking during zebrafish embryogenesis which could affect the yolk sizes and CE levels of zembryos. Among the soat2-knockout zebrafish, compensatory mechanisms were activated to compensate Soat2 loss-of-function. The overexpression of HMG-CoA was detected in soat2-knockout descendants at 24, 48 hpf. The overexpression of Lcat was detected at 96 hpf. The results indicated that zebrafish with mutant Soat2 may increase the de novo synthesis at 24, 48 hpf, while rescue the yolk cholesterol absorption by Abca1 and Lcat at 96 hpf to compensate for Soat2 loss-of-function. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:44:17Z (GMT). No. of bitstreams: 1 ntu-105-R03626004-1.pdf: 2628713 bytes, checksum: 0ed5f41adfc0465a80ee42add25cfcdf (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii ABSTRACT iv CONTENTS vii LIST OF FIGURES xi LIST OF TABLES xiii Chapter 1 Introduction 1 1.1 Importance of Cholesterol and cholesterol in the yolk 1 1.2 General cholesterol trafficking mechanism 7 1.3 Cholesterol esterification in the yolk sac membrane 9 1.4 Zebrafish as an excellent research model 10 1.5 Editing DNA sequence for functionally studying a gene 11 1.5.1 TALEN 11 1.5.2 CRISPR-Cas9 12 1.5.3 DNA repaired pathway 12 1.6 The yolk syncytial layer in early zebrafish embryogenesis 16 1.7 Lipid and cholesterol trafficking during zebrafish embryogenesis 17 Chapter 2 Specific aim 19 Chapter 3 Materials and Methods 20 3.1 Zebrafish 20 3.2 Total genomic DNA extraction 20 3.3 RNA extraction and reverse-transcription polymerase chain reaction 21 3.4 Real-time PCR analysis 21 3.5 Whole-mount in situ hybridization 22 3.6 Real-time Polymerase chain reaction (PCR) for soat2 targeted sequence 23 3.7 Amplification of specific DNA fragments 23 3.8 Transfect pSoat2-IRES2-EGFP to human embryonic kidney cells (HEK293 cells) and in vitro test zebrafish Soat2 enzymatic activity 24 3.9 Generate soat2-knockdown zebrafish by morpholino 25 3.10 Quantitate total cholesterol, free cholesterol and cholesteryl ester 26 3.11 Zebrafish yolk size determination 27 3.12 Statistical analysis 27 Chapter 4 Results 30 4.1 Expression pattern of zebrafish soat2 by whole-mount in situ hybridization 30 4.2 Generate soat2 knockout zebrafish descendants 30 4.3 in vitro test Soat2 enzymatic activity in HEK293 cells 31 4.4 Survival rate and yolk sizes of Soat2 mutation embryos 32 4.5 in vivo test zebrafish Soat2 enzymatic activity 32 4.6 The interaction of yolk sizes and soat2 genotypes 33 4.7 Generation of soat2 knockdown zebrafish by morpholino 33 4.8 Phenotypes of yolk sizes and cholesteryl ester levels of yolk-specific soat2-knockdown zebrafish 34 4.9 Rescue Soat2 loss-of-function zebrafish by wild-type soat2 RNA 35 4.10 Activated compensatory mechanism in soat2-knockout zebrafish 35 Chapter 5 Discussion 53 5.1 The mechanism with Soat2 for trafficking yolk cholesterol in zebrafish embryos may execute since 24hpf 53 5.2 Zebrafish Soat2 induced cholesteryl ester accumulation in embryo proper rather than in yolk. 54 5.3 A genetic compensatory mechanism is activated in soat2-knockout zebrafish. 55 5.4 Gaps between measuring yolk sizes and phenotypes of Soat2 loss-of-function 57 5.5 Both knocking-out and knocking-down technologies are essential for functional study of a gene. 57 5.6 The mechanism of yolk cholesterol trafficking 58 Chapter 6 Conclusions 60 Chapter 7 Reference 63 | |
dc.language.iso | en | |
dc.title | 第二型固醇酰基轉移酶 (Soat2) 在斑馬魚胚胎發育時期對卵黃中膽固醇之輸送和利用所扮演的角色 | zh_TW |
dc.title | Sterol O-acyltrasnferase 2 contributes to yolk cholesterol trafficking during zebrafish embryogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳洵一,丁詩同,陳靜宜,管永恕 | |
dc.subject.keyword | 斑馬魚,卵黃囊膜,第二型固醇?基轉移?,膽固醇酯,膽固醇, | zh_TW |
dc.subject.keyword | zebrafish,soat2,yolk sac membrane,cholesteryl ester,yolk cholesterol, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201601922 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-05 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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