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研究生:雷雅婷
研究生(外文):Ya-Ting Lei
論文名稱:建立細胞微陣列晶片系統與探討Syndecan-2誘發絲狀偽足與層足之訊息傳導途徑
論文名稱(外文):Establishment of cell array system and investigation of the downstream signaling of Syndecan-2 in filopodia and lamellipodia formation
指導教授:薛一蘋
指導教授(外文):Yi-Ping Hsueh
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:110
中文關鍵詞:細胞微陣列晶片系統Syndecan-2絲狀偽足層足
外文關鍵詞:cell arraySyndecan-2filopodialamellipodia
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結合生物晶片與細胞轉染的概念,Cell array (細胞微陣列晶片) 利用逆向轉染 (又稱為固相轉染) 的方法將欲研究的基因送入哺乳類動物的細胞株裡,可應用於大量的基因篩選。我們在不同的細胞株內,包括COS,HEK293T,rat pheochromocytoma PC12與neuroblsatoma Neuro-2A,分別建立了適當的逆向轉染條件。此外我們亦利用這些條件在pheochromocytoma PC12中檢視實驗室現有的基因對於細胞形態的影響。當大量表現CINAP、syndecan-2或syndecan-3時,可誘發細胞形態改變。反之,大量表現CASK與Tbr-1則無明顯影響。綜合此部分實驗結果可知我們所建立的cell array條件已可應用於大量基因篩選,此外,CINAP,syndecan-2和syndecan-3在細胞形態上扮演了重要的角色。
Syndecan家族是細胞表面主要的heparan sulfate proteoglycans。Syndecan-2是此家族的成員之一。若在不同細胞株內大量表現全長的syndecan-2時,可誘發filopodia與lamellipodia的表徵。我們利用共轉染syndecan-2與Rac1T17N (Rac1 dominant negative form) 到細胞內並觀察其表徵,證實syndecan-2誘發表徵的過程中除了需要Cdc42以外,仍須Rac1的參與。為進一步瞭解還有哪些訊息傳導路徑參與syndecan-2誘發表徵的途徑中,在經轉染syndecan-2的細胞培養液中,我們添加不同的kinase抑制物,其中PKA的抑制物 – KT5720與H89不論濃度高低,皆可抑制syndecan-2誘發的表徵;此外,在養殖HEK293T的細胞培養液中添加forskolin也可誘發部分syndecan-2的表徵,故推測cAMP/ PKA參與了syndecan-2誘發表徵的途徑。我們亦使用cAMP與PKA activity assay去偵測大量表現syndecan-2細胞內cAMP 濃度與相對PKA活性,雖不見cAMP濃度有顯著差異,但PKA活性有些微提升的趨勢。最後我們依照syndecan-2蛋白結構的區域構築了不同缺失的mutants,其中syndecan-2 C-terminal的C1與V區域在syndecan-2誘發表徵中扮演重要角色,此外,大量表現不具有cytoplasmic domain 的syndecan-2 mutant中,不見細胞內PKA活性提升的現象。綜合上述結果,可知syndecan-2藉由C1與V區域,活化了PKA路徑來誘發filopodia與lamellipodia的表徵。
Cell microarray assays have been established by reverse transfection, a modified transfection method also known as solid-phase transfection, which is a systematic and high-throughput approach for studying the function of genes on a genome-wide scale. Cell microarray was therefore adapted for different cell lines, such as COS, HEK293T, rat pheochromocytoma PC12, and neuroblsatoma Neuro-2A cells. To verify the system for screening, we examine the morphology of different cells, which were transfected with defined cDNAs. Over-expression of syndecan-2, syndecan-3 or CINAP promoted filopodia formation of PC-12 cells. In contrast, over-expression of CASK or Tbr-1 had no effect on cell morphology of PC-12 cells. The results not only support that the system established is ready for genome-wide screening but also indicate that syndecan-2, syndecan-3 and CINAP play a role in cell morphology.
Syndecan-2 is one of the members of syndecan family, a major heparan sulfate proteoglycan on plasma membrane (HSPG). Syndecans mediate cell-cell and cell-matrix adhesion thereby controlling cell movement and morphology. Syndecan-2 induced not only filopodia but also lamellipodia. The effect of syndecan-2 on cell morphology was reproducible in different cell lines. Rac1 dominant negative mutant blocked syndecan-2 induced phenotype, supporting that Rac1 is necessary for syndecan-2 induced phenotype. In addition to Rac1, several evidences support that PKA is the downstream signaling molecule of syndecan-2. First of all, PKA inhibitors – KT5720 and H89 – prevented the formation of filopodia and lamellipodia. Secondary, addition of forskolin in HEK293T cells also induced filopodia formation. Finally, PKA activity was slightly increased in syndecan-2 over-expressed cells. The cytoplasmic domain of syndecan-2 responsible for filopodia and lamellipodia was mapped by the C-terminal deletion mutants of syndecan-2. The C1 and V regions of syndecan-2 were found to be important for filopodia formation, and they are also responsible for the enhancement of PKA activity. Our results support that via the C1 and V region, syndecan-2 activates PKA pathway and induces filopodia and lamellipodia formation.
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