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研究生:劉自強
研究生(外文):Tzu-Chiang Liu
論文名稱:神經元中間絲蛋白在PC-12細胞株過量表達之研究
論文名稱(外文):Overexpression of neuronal intermediate filament internexin in the PC-12 cell line
指導教授:錢宗良錢宗良引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:解剖學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:61
中文關鍵詞:中間絲蛋白神經元
外文關鍵詞:internexinPC12neurite outgrowthneuronPC-12
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摘要

神經元中間絲蛋白除了包括neurofilament triplet proteins (NF-L, NF-M, and NF-H),另外也包含peripherin以及internexin。在發育過程中,peripherin及internexin兩者會在週邊神經系統及中樞神經系統的神經細胞中表現。本研究主要目的之一是在探討internexin是否會增強神經纖維向外生長,因此我們將大鼠的internexin核酸序列末端接上綠色螢光蛋白質的核酸序列,再轉殖進入PC-12細胞株中表現。PC-12細胞株是由大鼠腎上腺髓質細胞瘤(adrenal pheochromocytoma)中分離建立起的神經細胞培養株,它可經由神經生長因子(nerve growth factor)的誘導,分化發育成為類似交感神經元的細胞。細胞經過藥物篩選之後,我們建立了五株會持續表現internexin末端接有綠色螢光(internexin-EGFP)的細胞株。本實驗中利用神經生長因子去誘導細胞分化,之後再利用西方點墨法及免疫染色去分析神經元中間絲蛋白之表現情形。另外,也利用反轉錄聚合酶連鎖反應(RT-PCR)分析不同時期分化中細胞的中間絲蛋白表現情形。

比較經過轉殖internexin-EGFP的細胞及沒有轉殖的PC-12控制組細胞,可以觀察到過量表達的internexin-EGFP蛋白質主要是位於細胞體及神經纖維的近端。我們也發現在利用神經生長因子誘導的早期,過量表達internexin-EGFP蛋白質會提升神經纖維向外生長的情形。另外,過量表達internexin-EGFP蛋白質也會增強NF-L及NF-M的表現情形。有趣的是,在利用神經生長因子誘導的晚期,有釵h經過internexin-EGFP轉殖的細胞會從培養皿中漂起。經由電子顯微鏡觀察,可以看到在經過internexin-EGFP轉殖的細胞中,有大量的神經元中間絲蛋白堆積,腫脹的粒腺體,以及內含有釵h高電子密度顆粒的神經纖維。這些觀察顯示在經過internexin-EGFP轉殖的細胞中,同時也正在進行神經退化及細胞死亡。

由本實驗的結果,可以推測internexin在神經細胞發育的過程中,對於神經纖維的向外生長,以及調控其他神經元中間絲蛋白的表現,扮演很重要的角色。另外,在接受神經生長因子誘導分化之後的細胞中,過量表達internexin-EGFP蛋白質的結果,很可能會引發細胞凋亡的機制。
Abstract

The neuronal intermediate filaments include not only the neurofilament triplet proteins but also peripherin and internexin. Both peripherin and internexin are expressed in the neurons of PNS and CNS during development. To determine whether neurite outgrowth is enhanced by internexin, the cDNA of rat internexin tagged with EGFP was transfected into a rat adrenal pheochromocytoma cell line PC-12 that responds to nerve growth factor (NGF) by induction of the neuronal phenotype. After selection, 5 stable cell lines were established. Selected stable clones were induced by NGF and examined for expression patterns of neuronal intermediate filaments by western blot and immunocytochemistry. Differentiating neurons were also collected after NGF induction for RT-PCR analyses.

Comparing the internexin-EGFP transfected stable cell line with non-transfected PC-12 cells, the overexpressed internexin-EGFPs were found mainly in cell bodies and the proximal part of neurites. We found that overexpression of internexin-EGFPs enhanced the neurite outgrowth of PC-12 cells at the early stages of NGF induction. It was also observed that the expression of NF-L and NF-M was up-regulated by the overexpression of internexin-EGFPs. Interestingly, internexin-EGFP transfected cells deattached from culture plates obviously at the late stages of NGF induction. Massive IF accumulations, swelling mitochondria, degenerating neurites with numerous electron-dense granules were observed ultrastructurally in the internexin-EGFP transfected cells. These observations may imply that cell death was undergoing in pINT-EGFP transfected cells.

From our study, it could be suggested that internexin may play an important role in the neurite outgrowth and regulate the expression of other neurofilaments during neuronal development. Apoptosis-like cell death could be also induced by the overexpression of internexin-EGFP in PC-12 cells after the NGF induction.
目 錄

壹、中英文摘要 -------------------------------------- 1

貳、緒論 ----------------------------------------------- 5

參、實驗材料與方法 -------------------------------- 8

肆、實驗結果 ---------------------------------------- 16

伍、討論 ---------------------------------------------- 23

陸、參考文獻 ---------------------------------------- 31

柒、圖表說明 ---------------------------------------- 36
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