臺灣博碩士論文加值系統

在台灣男性中，鼻咽癌在十大癌症中排名第十位，其致死率也是第十位。而高度的淋巴或遠端轉移常是鼻咽癌病患低存活率的主要原因之一。目前對於調控著鼻咽癌遠端轉移的分子層面還不明確，因此，本研究的目的即是尋找並確認鼻咽癌細胞具高度侵犯能力的基因。研究方法是取NPC-BM1與NPC-BM2兩株鼻咽癌細胞株，首先以Matrigel invasion assay的方法來篩選出具高度侵犯能力的子細胞株。經由Matrigel invasion assay及 in vitro wound healing assays的結果，確定其侵犯及移行能力在高子代細胞中有上升的驅勢。經過cDNA microarray的分析後，發現在子代中有7個基因的表現量有2倍上升的情形，21個基因的表現量下降的現像。經由RT-PCR及western blot的分析後，發現14-3-3sigma、annexin II、及gp96在子代的表現量皆較低。經由進一步的使用siRNA抑制此三個基因的表現後，發現14-3-3sigma對於細胞的侵犯及移行能力均有增加的現象; annexin II對於細胞的移行能力有增加的現象;而gp96對於細胞的侵犯及移行能力亦均有增加的現象。實驗結果証明14-3-3sigma、annexin II、及gp96在鼻咽癌細胞的侵犯及/或移行能力扮演著負向調控的角色。

In Taiwan, nasopharyngeal carcinoma (NPC) is the 10th leading cancer in male. However, since high invasive and metastatic potential, the survival rate of NPC in late stage is relative low among all cancers. The molecular factor that contributes to NPC invasion is obscure. In this study, we intend to identify the genetic alterations associated with cellular invasion in NPC. Using Matrigel and Transwell selection method, we have established four generations of invasion subline cells from two NPC parental cell lines (BM1 and BM2), which provide materials for further screening of potential invasive-associated genes. Matrigel invasion and in vitro wound healing assays demonstrated these subline cells possess higher invasion and migration ability, suggesting the successfulness of the subline establishment. cDNA microarray analysis was used to screen potential invasive genes, and total of 7 genes were found over-expressed and 21 under-expressed in invasion subclones, which maybe associate with invasion mechanism. In which, three candidate genes were further verified by RT-PCR and western blot: 14-3-3sigma, annexin II, and GP96. Results show that 14-3-3sigma, annexin II, and gp96 were down-expressed with the ascending generations of invasion subclones. After knock down the expressions of 14-3-3sigma, annexin II, and gp96 by siRNA technique, the cell invasion and/or migration ability were augmented, whereas no significant effect on cell growth or colony formation. Those data demonstrate that 14-3-3sigma, annexin II, and gp96 play negative regulatory roles in the invasion and migration of NPC cells.

Background and Significance 1
NPC pathology 1
NPC staging and prognosis 2
Genes associated with the invasion/metastasis of NPC 3
Experimental design and Study Aim 5
Materials and Methods 6
Cell & cell culture 6
Establishment of highly invasive subclones using Matrigel invasion model 7
RNA extraction and cDNA Microarray analysis 7
RT-PCR (Reverse transcriptase-polymerase chain reaction) analysis 8
Protein extraction 9
Immunoblot analysis 9
siRNA plasmids transfection 10
Matrigel invasion assay 11
Would healing migration assay 11
Cell growth assay 12
Colony formation assay 12
Results 13
Establishment of highly invasive nasopharyngeal carcinoma subclones 13
Characterization of the migration and invasive ability in NPC subclone cells 14
Screening potential invasive genes in NPC cells by microarray analysis 14
Expression of three candidate invasive genes (14-3-3, annexin II, and gp96) in NPC invasive subclones 15
Effective inhibition of specific gene expression by siRNA 16
14-3-3 plays a negative regulatory role in NPC cell invasion and migration 17
Annexin II play a negative regulatory role in NPC cell migration 17
Gp96 plays a negative regulatory role in NPC cell invasion and migration 18
Discussion 19
Reference 23

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