臺灣博碩士論文加值系統

指導教授推薦書…………………………………………………………..
論文口試委員審定書……………………………………………………..
誌謝……………………………………………………………………iii
中文摘要………………………………………………………………iv
Abstract…………………………………………………………………v
目錄……………………………………………………………………vi
圖目錄………………………………………………………………ix
表目錄………………………………………………………………x
第一章 緒論 1
1.1 前言 1
1.2 ADAR 1
1.3 ADAR與肌肉生成 (myogenesis) 的關聯 2
1.4 Flnb 3
1.5 Igfbp7 4
1.6 實驗動機 5
1.7 實驗設計 6
1.7.1確認Flnb與Igfbp7是否會受到RNA編輯 6
1.7.2探討RNA編輯是否調控Flnb與Igfbp7 RNA及蛋白的表現 6
1.7.3探討Flnb與Igfbp7編輯前後在肌肉生成中的角色 7
1.7.4釐清RNA編輯如何透過調控Flnb與Igfbp7影響肌肉生成 7
第二章 實驗方法與材料 8
2.1 細胞培養、轉染試驗與樣本收取 8
2.2 蛋白質製備 9
2.3 收集細胞外液 9
2.4 西方墨點法 (Western blot；WB) 9
2.4.1 SDS-PAGE 9
2.4.2 轉印(transfer) 10
2.4.3 Blocking及抗體 (antibody) 辨識 10
2.5 RNA萃取及反轉錄聚合酶鏈鎖反應 (RT-PCR) 11
2.5.1 RNA萃取 11
2.5.2 RT-PCR 11
2.6建構Flnb與Igfbp7表現載體 11
2.7聚合酶鏈鎖反應 (Polymerase Chain Reaction；PCR) 13
2.8建構的pZback質體的確認 13
2.8.1 pZback ligation 13
2.8.2質體轉型 13
2.8.3質體純化 14
2.9即時聚合酶鏈鎖反應 (Quantitative-PCR；Q-PCR) 14
2.10胜肽競爭 (Peptide competition) 14
2.11免疫沈澱法 (Immunoprecipitation；IP) 15
2.12 Coomassive Blue 15
2.13 Silver staining 15
第三章 實驗結果 17
3.1細胞分化前後的Flnb蛋白表現沒有差異 17
3.2編輯後的Flnb蛋白在分化後的肌肉細胞中表現量上升 17
3.3建構編輯前後的Flnb與Igfbp7表現載體 17
3.4 Igfbp7蛋白的分泌功能與RNA編輯的關聯 18
3.5以銀染分析RNA編輯對Igfbp7與其他蛋白交互作用的影響 18
3.6以銀染分析RNA編輯對Flnb與其他蛋白的交互作用的影響及對Filamin家族同源與異源二聚體的影響 20
3.7探討編輯事件對於Filamin家族調控肌動蛋白聚合的影響 20
第四章 討論 22
4.1探討受到RNA編輯的Flnb對細胞肌肉生成的影響 22
4.2探討受到RNA編輯的Igfbp7在細胞內外對肌肉生成的影響 23
參考文獻 24
圖表 27
附錄 38


圖表目錄
圖表 27
Figure 1. 在分化後的細胞中，Flnb受到RNA編輯的程度增加 27
Figure 2. 以Flnb-Q2R抗體專一性辨識Flnb-QR在分化後的表現 28
Figure 3. Flnb表現載體轉染進細胞後的RNA level 及蛋白表現 30
Figure 4. 比較不同的Igfbp7表現載體在細胞內的蛋白表現 31
Figure 5. 比較編輯前後Igfbp7在細胞內外蛋白表現 34
Figure 6. 分析RNA編輯對Igfbp7在細胞內外組成的影響 35
Figure 7. 分析RNA編輯對Flnb在細胞內外組成的影響 36
Figure 8. 分析RNA編輯對Flnb穩定細胞骨架結構特性的影響 37
附錄 38
Table 1. 聚合酶鏈連鎖反應引子序列與載體修飾引子序列 38
Table 2. 實驗試劑成分與配方 40
Table 3. OVA-peptide之組成、peptide purification kit 抗體純化流程圖及純化抗體命名 41

Athanasiadis, A., Rich, A., and Maas, S. (2004). Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome. PLoS biology 2, e391.
Chen, C.X., Cho, D.S., Wang, Q., Lai, F., Carter, K.C., and Nishikura, K. (2000). A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains. Rna 6, 755-767.
Desterro, J.M., Keegan, L.P., Lafarga, M., Berciano, M.T., O'Connell, M., and Carmo-Fonseca, M. (2003). Dynamic association of RNA-editing enzymes with the nucleolus. Journal of cell science 116, 1805-1818.
Garrett, S.C., and Rosenthal, J.J. (2012). A role for A-to-I RNA editing in temperature adaptation. Physiology 27, 362-369.
Hsieh, C.L., Liu, H., Huang, Y., Kang, L., Chen, H.W., Chen, Y.T., Wee, Y.R., Chen, S.J., and Tan, B.C. (2014). ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions. Cell death and differentiation 21, 707-719.
Patterson, J.B., and Samuel, C.E. (1995). Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase. Molecular and cellular biology 15, 5376-5388.
Poulsen, H., Nilsson, J., Damgaard, C.K., Egebjerg, J., and Kjems, J. (2001). CRM1 mediates the export of ADAR1 through a nuclear export signal within the Z-DNA binding domain. Molecular and cellular biology 21, 7862-7871.
Price, M.G., Caprette, D.R., and Gomer, R.H. (1994). Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils. Cell motility and the cytoskeleton 27, 248-261.
Sansam, C.L., Wells, K.S., and Emeson, R.B. (2003). Modulation of RNA editing by functional nucleolar sequestration of ADAR2. Proceedings of the National Academy of Sciences of the United States of America 100, 14018-14023.
Sheen, V.L., Feng, Y., Graham, D., Takafuta, T., Shapiro, S.S., and Walsh, C.A. (2002). Filamin A and Filamin B are co-expressed within neurons during periods of neuronal migration and can physically interact. Human molecular genetics 11, 2845-2854.
Soltow, Q.A., Zeanah, E.H., Lira, V.A., and Criswell, D.S. (2013). Cessation of cyclic stretch induces atrophy of C2C12 myotubes. Biochemical and biophysical research communications 434, 316-321.
van der Flier, A., Kuikman, I., Kramer, D., Geerts, D., Kreft, M., Takafuta, T., Shapiro, S.S., and Sonnenberg, A. (2002). Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits. The Journal of cell biology 156, 361-376.
Verhagen, H.J., de Leeuw, D.C., Roemer, M.G., Denkers, F., Pouwels, W., Rutten, A., Celie, P.H., Ossenkoppele, G.J., Schuurhuis, G.J., and Smit, L. (2014). IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival. Cell death &; disease 5, e1300.
Xie, Z., Xu, W., Davie, E.W., and Chung, D.W. (1998). Molecular cloning of human ABPL, an actin-binding protein homologue. Biochemical and biophysical research communications 251, 914-919.
Xu, W., Xie, Z., Chung, D.W., and Davie, E.W. (1998). A novel human actin-binding protein homologue that binds to platelet glycoprotein Ibalpha. Blood 92, 1268-1276.
Yang, Y., Zhou, X., and Jin, Y. (2013). ADAR-mediated RNA editing in non-coding RNA sequences. Science China Life sciences 56, 944-952.