臺灣博碩士論文加值系統

Gas7涉及神經分化與骨骼生成的過程，然而，對於此兩種發育形成的機制尚不明瞭，為了了解神經突觸形成的機制，本篇論文以Gas7為餌用來研究與之共同參與、影響突觸生成的蛋白。由免疫沉澱及MALTI-TOF MS方法，我們找到了在PC12細胞中與Gas7作用、且已知亦是神經突觸生成必需的中間絲蛋白 - peripherin。pull down分析及間接免疫染色螢光顯微鏡的觀察中發現，Gas7與peripherin共位的情況傾向於發生在一段特殊的時期，伴隨著Gas7表現 (如過量表現或NGF誘導分化) 的PC12細胞；更進一步的是，兩者因NGF誘導而表現的時間相近。他們共同表現的位置發生在細胞本體而比較不像是延展中的突觸，且蛋白皆可快速的在NGF處理下3~12小時累積。而蛋白穩定度的分析顯示，在未分化的PC12細胞中，Gas7持續處於被分解的狀態，卻可快速累積在NGF誘導的情況。基於以上的結果，我們推論Gas7與peripherin也許可以共同調節且協同作用以控制神經突處的生成。

Gas7 is involved in neurite outgrowth and bone morphogenesis. However, the underlying mechanism for the development of each phenomenon is not clear. To assess the mechanism of neurite outgrowth, Gas7 was used as a bait to search interacting proteins which might represent components of the complex that drives neurite outgrowth. By use of immnoprecipitation and MALTI-TOF MS, peripherin, a component of interfilament known to be required for neurite outgrowth, was identified as a Gas7 interacter in PC12 cells, a pre-neuronal cell. Pull-down analysis and indirect immunofluorescence microscopy indicate that the complex of Gas7 and peripherin was detected preferentially in a specific period of time, concomitantly occurred with Gas7 expression, following cell differentiation induced by NGF. Furthermore, the two proteins were temporally co-expressed in PC12 cells following the NGF-treatment. They specifically co-localized at cytoplasm of the cell body, but less likely in the region of out-reaching neurites. Surprisingly, both proteins were rapidly accumulated in cells within 3~12 hrs by NGF. Analysis on protein stability suggests that Gas7 is constantly degraded in non-differentiating cells, whereas it is rapidly accumulated in NGF-induced differentiating cell conditions. Based on these results, we proposed that Gas7 and peripherin might be co-regulated and act in concert in the control of neurite outgrowth.

目 錄
指導教授推薦書
口試委員會審定書
授權書………………………………………………………………… iii
誌謝……………………………………………………… ………… iv
中文摘要………………………………………………………………… v
英文摘要……………………………………………………………… vi
目錄…………………………………………………………………… vii
圖表附錄 ix
壹. 緒 論……………………………………………………… ……… 1
一.何謂生長休止基因 …………………… … … … 1
二.已知的生長基因種類………………………………… …… 2
三.Gas7的生化性質與生理功能…………………… 5
四.本論文之研究目的 9
貳. 材料與方法…………………… 10
一.質體構築 ( plasmid construction )……… … …10
二.反轉錄聚合酶連鎖反應RT-PCR ( reverse transcription
polymerase chain reaction ) 11
三.細胞培養、藥物處理及質體轉染 12
四.細胞萃取液的製備 13
五.製備級免疫沉澱法 (preparative protein immunoprecipitation) 13
六.His釣餌 (His pull down) 14
七.聚丙烯醯胺膠體電泳 (SDS-PAGE) 15
八.西方轉漬法 (Western blotting) 15
九.銀離子染色 (Silver Stain) 16
十.間接免疫染色 (indirect immunofluorescence) 17
叁. 結 果…………………………………………………………… 18
一. 與Gas7作用相關蛋白- peripherin………… 18
1. PC12細胞分化後與Gas7作用的蛋白 ─ peripherin 18
2. 部分Gas7與peripherin在分化中PC12細胞內分佈一致 20
3. 新發現類似peripherin的mRNA 20
二. Gas7穩定性探討 21
肆. 討 論……………………… 23
一. 與Gas7作用相關蛋白- peripherin … …… 23
二. Gas7穩定性探討 ………………………………… 26
圖表附錄……………………………………… …… 27
參考文獻………………………………………………………… … 40

圖表附錄
Figure 1 NGF induced neuritogenesis in PC12 cell model. 27
Figure 2A Immunoprecipitate of Gas7 and associated proteins in PC12 cells with NGF treatment or not. 28
Figure 2B Determination by MALDI-TOF MS that the 83 kDa band is Growth-arrest-specific protein 7 (GAS-7) 29
Figure 2C Determination by MALDI-TOF MS that the 57 kDa band is Peripherin 30
Figure 3 His-pull down experiments confirm the interactive relationship of Gas7 and peripherin in NGF-treated PC12 cell. 31
Figure 4 Colocalization of Gas7 and peripherin proteins in PC12 cells by indirect immunofluorescence microscopy. 33
Figure 5A Colocalization of Gas7 and peripherin proteins in PC12 cells by indirect immunofluorescence confocal microscopy. 34
Figure 5B Three dimensional merge of confocal microscopy. 35
Figure 6 Novel form of peripherin transcript. 36
Figure 7 Induction of Gas7 and peripherin mRNA and protein in PC12 cells by NGF. 37
Figure 8 Similar degradation rate of Gas7 protein in NGF treated and untreated PC12 cells. 38
Figure 9 Gas7 is ubiquitinated in PC12 cells. 39

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