臺灣博碩士論文加值系統

位於人類粒線體第一蛋白質複合體的次單元 NDUFS7 (NADH dehydrogenase (ubiquinone) Fe-S protein 7)，是一個由細胞核基因所表現並且具有高度保留性的蛋白。NDUFS7 含有一個 [4Fe-4S] 的鐵硫中心 N2，其為第一複合體親水端上電子的最後接受者，同時也是 Quinone的還原者。在單純的模式生物以及病人檢體的研究中，NDUFS7 在第一蛋白質複合體的聚合和功能上扮演著重要的角色。NDUFS7 的突變目前已知與 Leigh 症候群 (Leigh syndrome)、躁鬱症有關。為了詳細探討 NDUFS7 在人類粒線體第一蛋白質複合體上的功能，本研究利用核糖核酸干擾技術 (RNAi) 降低 NDUFS7 在 T-REx293 細胞株的表現。結果顯示，NDUFS7 表現量的下降會導致細胞生長速度、三磷酸腺苷 (ATP) 產生量、耗氧量和第一蛋白質複合體活性的降低。這些證據指出，人類細胞中 NDUFS7 在第一蛋白質複合體的活性和能量的產生上扮演著重要的角色。

NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of the nuclear-encoded and highly conserved core subunits of mitochondrial complex I. This protein houses the tetranuclear iron-sulfur cluster N2, which is the terminal receptor of electrons and the reducer for quinone in complex I peripheral arm. NDUFS7 protein plays a critical role in complex I assembly and activity, according to the results of studies from simple organisms and human disease models. Mutations of NDUFS7 have been associated with Leigh Syndrome and bipolar disorder. To detailedly investigate the role of NDUFS7 in mitochondrial complex I of human cells, NDUFS7 was suppressed in human T-REx293 cells by RNA interference system in this study. In the NDUFS7 knockdown cells, the growth rate, ATP generation, oxygen consumption and complex I activity were reduced. These results provided the evidence of the essential role of NDUFS7 for complex I activity and energy production in human cells.

中文摘要................................................................................................................. i
Abstract .................................................................................................................. ii
Abbreviations ........................................................................................................ iv
Introduction ............................................................................................................ 1
Materials and Methods ......................................................................................... 11
1. Cell lines and culturing conditions ...................................................... 11
2. The sequence alignment and analysis of NDUFS7.............................. 11
3. Prediction of NDUFS7 sites possible involved in posttranslational modifications........................................................................................ 12
4. RNA extraction and reverse transcription ............................................ 13
5. Quantitative real-time PCR (Q-PCR) .................................................. 13
6. Growth curve ....................................................................................... 14
7. ATP determination ............................................................................... 14
8. Measurement of mitochondrial oxygen consumption .......................... 15
9. Assay of mitochondrial complex I activity .......................................... 16
Results .................................................................................................................. 18
1. The bioinformational analysis of NDUFS7 ......................................... 18
2. Reduced expression of NDUFS7 in T-REx293 cells ........................... 19
3. The growth rate of NDUFS7 knockdown cells was slower in glucose and galactose media ............................................................................. 19
4. Suppression of NDUFS7 expression in T-REx293 cells reduced the generation of ATP ................................................................................ 20
5. The oxygen consumption rate of NDUFS7 knockdown cells was decreased .............................................................................................. 21
6. The complex I activity was reduced in NDUFS7 knockdown cells .... 21
Discussion ............................................................................................................ 23
Tables ................................................................................................................... 30
Figures.................................................................................................................. 34
Reference ............................................................................................................. 42
Appendixes .......................................................................................................... 47

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