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研究生:張宴偢
研究生(外文):Yen-Chiu Chang
論文名稱:內質網與高基氏體蛋白質是細胞架構的重要部份
論文名稱(外文):Characterization of endoplasmic reticulum and Golgi apparatus proteins as part of cell architecture defined by in situ subcellular fractionation
指導教授:蔡有光
指導教授(外文):Yeou-Guang Tsay
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:59
中文關鍵詞:原位生物化學萃取細胞架構細胞骨架核基質質譜分析蛋白質體學內質網高基氏體
外文關鍵詞:in situ subcellular fractionationcell architecturecytoskeletonnuclear matrixmass spectrometryproteomicsendoplasmic reticulumGolgi apparatus
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細胞生物學家於廿世紀時發現了細胞骨架(cytoskeletons),其廣義的定義包括了,核骨架、核纖層和細胞外基質,其形成貫穿於細胞核、細胞質、細胞外的一體化網絡結構。然而,現今的細胞骨架相關研究仍局限於探討其本身的解剖學、功能以及結構。在第二章中,我們利用modified in situ subcellular fractionation的萃取方式,收集到五管針對子宮頸癌HeLa細胞進行原位生物化學萃取後的萃取分管。接著,我們透過膠體電泳分析及質譜分析技術將這五管萃取分管作進一步的分析。其中,由於細胞殘骸可以抵抗嚴苛的萃取條件並且保留了大部分的細胞形態,因此,我們認為它是一個真核細胞的基本結構組織,並將它定義為細胞架構 (cell architecture)。在這個子宮頸癌細胞架構中,我們成功鑑定到590個蛋白質,也在進一步透過Gene Ontology database分析之後,發現的其中還包含著18% 和8% 的蛋白質過去被認為與內質網和高爾基氏體相關。同樣地,我們也針對經其他四個步驟萃取後的萃取分管作分析,並發現在這四管萃取分管中所鑑定到的蛋白質,與細胞架構萃取分管中的蛋白質特徵截然不同,因此我們更加確信了此架構蛋白質體的獨特性質。透過Gene Ontology databases和Ingenuity Pathway Analyses databases等生物資訊平台的分析,我們也鑑定到這群細胞架構蛋白質可能參與在蛋白質轉運、磷脂質合成和膜成塑形功能中。此外,我們也藉由免疫化學分析來進一步驗證部分具有代表性的內質網和高爾基體蛋白的確存在於細胞架構中。 例如,寡糖基轉移酶 48 kDa次單位蛋白質(DDOST)和易位子相關蛋白(TRAP-α)即為細胞架構中的內質網蛋白質。
Cell biologists discovered cytoskeleton in the 20th century. By extension, the term "cytoskeleton" includes nuclear matrix, nucleoli, and extracellular matrix, forming an integrated network that connects the nucleus, cytoplasm, and extracellular matrix. Nowadays, the cytoskeleton-related research is still limited to its own anatomy, function, and structure. In Chapter II, we have collected five subcellular fractions using modified in situ subcellular fractionation. These five fractions were subjected to electrophoresis and mass spectrometry analyses. Since the remaining fraction is resistant to stringent extraction and preserves most of cell morphology, we propose to term such residual structure as cell architecture, the basic structural organization of a cell. We found that HeLa cell architecture has ~590 proteins, and among which 18% and 8% are indeed associated with the keywords endoplasmic reticulum or Golgi, respectively, based on Gene Ontology database. We have studied the protein groups removed with a four-step extraction procedure, and found that these groups have features quite distinct from the residual fraction, underscoring the unique properties of the cell architecture proteome. With information in Gene Ontology and Ingenuity Pathway Analyses databases, we identify the possible roles of this part of cell architecture in protein transport, phospholipid biosynthesis and membrane shaping. We also employed immunochemical analyses to demonstrate several ER and Golgi proteins are bona fide cell architecture proteins. For example, dolichyl-diphospho-oligosaccharide-protein glycosyltransferase 48 kDa (DDOST) subunit and the alpha-subunit of the translocon-associated protein (TRAP-α) are in the ER part of the cell architecture.
<TABLE OF CONTENTS>

致謝 I
中文摘要 III
ABSTRACT V
TABLE OF CONTENTS VII
LIST OF FIGURES IX
CHAPTER I Literature Review 1
1.1 Introduction of endoplasmic reticulum and Golgi apparatus 2
1.2 Proteomics ressearch of endoplasmic reticulum and Golgi apparatus 5
1.3 Define the sturdy cell architecture by in situ subcellular fractionation 8
CHAPTER II Subproteomic analyses based on in situ subcellular fractionation identified HeLa cell architecture 13
SUMMARY 14
INTRODUCTION 15
MATERIALS AND METHODS 18
RESULTS 25
DISCUSSION 46
REFERENCES 50
SUPPLEMENT 54

<LIST OF FIGURES>

CHAPTER I
Figure 1. Illustration of the procedure of in situ subcellular fractionation. 10

CHAPTER II
Figure 2. SDS-PAGE analyses of proteins present in five subcellular fractions obtained from HeLa cells.27
Figure 3. Functional characterization of HeLa cell architecture proteome based on the information in Gene Ontology database.28
Figure 4. Functional characterization of proteomes from Triton X-100, DNase, RNase and high-salt fractions of HeLa cells based on the information of Gene Ontology database.31
Figure 5. Distribution of endoplasmic reticulum proteins in five subcellular fractions.34
Figure 6. Distribution of Golgi apparatus proteins in five subcellular fractions.35
Figure 7. Western blot analysis of untreated on treated HeLa cells with antibodies against endoplasmic reticulum protein, TRAP-α.39
Figure 8. Western blot analysis of untreated on treated HeLa cells with antibodies against endoplasmic reticulum protein, DDOST.40
Figure 9. Western blot analysis of untreated on treated HeLa cells with antibodies against endoplasmic reticulum protein, ERLIN1.41
Figure 10. Western blot analysis of untreated on treated HeLa cells with antibodies against Golgi apparatus protein, GOLPH4.42
Figure 11. Western blot analysis of untreated on treated HeLa cells with antibodies against Golgi apparatus protein, GALNT2.43
Figure 12. Immunofluorescence microscopic analysis of untreated on treated HeLa cells with antibodies against ERLIN1, DDOST and PDI (endoplasmic reticulum proteins).44
Figure 13. Immunofluorescence microscopic analysis of untreated on treated HeLa cells with antibodies against GALNT2 (Golgi apparatus proteins).45

SUPPLEMENT
Supplemental Figure 1. Phase-contrast microscopy of untreated HeLa cells (A) and HeLa cells after Triton X-100 (B), DNase I (C), RNase A (D), 2M NaCl (E) treatment.54
Supplemental Figure 2. Top 5 canonical pathways derived from Ingenuity Pathway Analysis (IPA) of endoplasmic reticulum-associated cell architecture proteins.55
Supplemental Figure 3. Top 5 canonical pathways derived from Ingenuity Pathway Analysis (IPA) of Golgi-associated cell architecture proteins.56
Supplemental Figure 4. Western blot analysis of untreated on treated HeLa cells with antibodies against Golgi apparatus protein, TGN46.57
Supplemental Figure 5. Western blot analysis of untreated on treated HeLa cells with antibodies against Golgi apparatus protein, β-COP.58
Supplemental Figure 6. Immunofluorescence microscopic analysis of untreated on treated HeLa cells with antibodies against β-COP (Golgi apparatus proteins).59
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