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研究生:謝克威
研究生(外文):Ko-Wei Sye
論文名稱:KIF3A和KIF5B的生理角色研究-以PC12細胞為研究模式系統
論文名稱(外文):Study of the physiological role of KIF3A and KIF5B–using PC12 cell as a model system
指導教授:林崇智林崇智引用關係高閬仙高閬仙引用關係
指導教授(外文):Chung-Chih LinLung-Sen Kao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:57
中文關鍵詞:驅動蛋白鈣離子鈣離子影像
外文關鍵詞:Kinesincalciumcalcium imagingPC12KIF3AKIF5B
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驅動蛋白構成了一個以微管為軌道進行運動的分子馬達超家族,其中有些可以在神經系統內調控胞器或是蛋白的極性運送造成神經元具有外型上或是功能上的差異性。因為神經生長因子(NGF)會誘導PC12細胞分化產生外表上類似神經元,而且細胞本體與神經纖維之間產生外形和功能的差異變化,因此KIF蛋白可能會去參與了這種由NGF所引起的PC12細胞分化的情形。本研究主要的目的是去瞭解是否KIF3和KIF5是否會去調控鈣庫或是囊泡的運送造成NGF對PC12細胞的鈣離子恆定和胞吐反應的影響。
過度表現shKIF5B會抑制NGF所引起的神經纖維生長。過度表現EGFP、DN-KIF3A-EGFP或是shKIF5B的NGF分化PC12細胞,以ATP刺激以鈣離子影像觀察神經纖維和細胞本體部份的細胞質游離鈣離子([Ca2+]i),發現在DN-KIF3A和shKIF3A的組別中,鈣離子的基礎值在神經纖維和細胞本體部份是較低的,但是此種降低[Ca2+]i的基礎值並不出現於非分化細胞(na��ve cells)。使用DsRed-Mito來觀察粒線體的分佈時,比較控制組與DN-KIF3A-EGFP的組別中分佈的結果,並沒有顯著的差異。此外只有TG((thapsigargin, ER鈣庫的抑制劑)而非CCCP(carbonyl cyanide m-chlorophenylhydrazone, 粒線體鈣庫的抑制劑)能消除shKIF3A對[Ca2+]i基礎值的影響以及神經纖維和細胞本體部份[Ca2+]i基礎值之差異,顯示KIF3A調控[Ca2+]i基礎值不是透過粒線體的鈣庫。由shKIF3A造成的[Ca2+]i基礎值降低,進一步造成了LDCV(Large dense core vesicle)的胞吐活性下降。在shKIF5B的組別中,亦造成類似shKIF3A降低NGF所分化的細胞中[Ca2+]i基礎值,儘管KIF5B knock down後分化的PC12細胞外型與na��ve細胞相似,這種降低現象是在NGF分化的PC12細胞所特有的。總結結果發現KIF3A和KIF5B在NGF處理的PC12細胞中鈣離子恆定扮演了一定的角色,但是KIFs是如何調控鈣離子恆定和胞吐反應還需要進一步的研究釐清。
Kinesins constitute a superfamily of microtubule-based motor proteins. Some of them regulate polarized transport of organelles and proteins in nerve system to result in differential morphology and functions in subcellular regions of neurons. Because nerve growth factor (NGF) induces na��ve PC12 cells to acquire neuronal-like differential morphology and functions among cell body and neurites. Therefore, KIFs seem to play a role in NGF-mediated differentiation of PC12 cells. This study is to understand whether KIF3 and KIF5, which possibly regulate transport calcium pools and vesicles, involves in the effects of NGF on calcium homeostasis and exocytotic activity of PC12 cells.
Overexpression of shKIF5B instead of shKIF3A or DN-KIF3A-EGFP reduced NGF-induced neurite outgrowth. NGF-differentiated PC12 cells overexpressing either EGFP as control, or DN-KIF3A or shKIF5B were stimulated by ATP to evoke [Ca2+]i increase, and changes of [Ca2+]i in cell body and neurites of these cells were visualized by calcium imaging. DN-KIF3A and shKIF3A reduced resting [Ca2+]i in cell bodies and neurites of NGF-differentiated PC12 cells, but difference of resting calcium among cell bodies and neurites is not affected, i.e. higher resting [Ca2+]i in neurite than in cell body. To test whether mitochondria may be re-distributed by DN-KIF3A to affect calcium homeostasis in PC12 cells, subcellular distribution of mitochondria was visualized by DsRed-Mito. There is no significant difference between control and DN-KIF3A-overexpressing cells. Moreover, only TG (thapsigargin, ER calcium pump inhibitor) instead of CCCP (carbonyl cyanide m-chlorophenylhydrazone, inhibitor to deplete mitochondrial calcium pool) reduces differential resting [Ca2+]i among neuritis and cell bodies and the effects of DN-KIF3A on resting [Ca2+]i, and support that KIF3A regulates resting [Ca2+]i is not mediated by mitochondria calcium pools. Such reduction of resting [Ca2+]i by shKIF3A results in decreased LDCVs (Large dense core vesicle) exocytosis. Similar to shKIF3A, shKif5B reduces resting calcium level only in NGF-treated cells instead of na��ve cells, even morphology of NGF-treated and na��ve KIF5B knock down cells are similar. In conclusion, KIF3A and KIF5B play a role in calcium homeostasis only in NGF-treated PC12 cells. But the mechanism in how KIFs regulate calcium homeostasis and exocytosis needs to be further studied.
Abstract 5
中文摘要 7
中英對照表 8
縮寫表 9
1 序論 10
1.1功能性神經連接對於正確神經傳導的重要性 10
1.2 NGF對PC12細胞的分化影響 11
1.2.1 NGF的訊息傳導對PC12細胞外型的影響 11
1.2.2 NGF與PC12細胞中囊泡的關係 11
1.2.3NGF與PC12細胞內鈣離子恆定的關係 12
1.3 驅動蛋白 (Kinesins) 13
1.3.1驅動蛋白概覽 13
1.3.2驅動蛋白與細胞外型的關係 14
1.3.3驅動蛋白與離子通道的關係 14
1.3.4驅動蛋白與囊泡運送的關係 15
1.4 研究目的 15
2 材料與方法 17
2.1 細胞培養 17
2.2 質體DNA 18
2.3 細胞轉染 (Transfection) 18
2.4 慢病毒(lentivirus)的製備 19
2.5 鈣離子影像 19
2.6 全反射螢光顯微鏡 (TIRFM) 20
2.7 Total RNA萃取及RT-PCR 21
2.8統計 21
2.9藥品配方 22
2.10使用藥品清單 22
3 結果 23
3.1 KIF3A和KIF5B在NGF處理後PC12細胞的表現 23
3.3 KIF3A 對分化的PC12細胞在 [Ca2+]i的影響 25
3.4 抑制KIF3A或KIF5B的表現對PC12細胞在 [Ca2+]i的影響 27
3.5 KIF3A對LDCVs胞吐作用的影響 30
4 討論 31
4.1 KIF3A和KIF5B影響PC12細胞鈣離子恆定的可能機制 31
4.2 KIF3A和KIF5B在囊泡分泌上扮演的角色 32
5參考文獻 34
6 圖表 40
圖一 KIF3A和KIF5B在PC12細胞內的表現 40
圖二 比較PC12細胞在NGF處理後,型態上的差異 41
圖三 KIF3A 對分化的PC12細胞在神經纖維數量和長度上的影響 42
圖四 DN-KIF3A-EGFP的分佈 43
圖五 shKIF3A慢病毒對於PC12細胞內KIF3A和KIF5B表現的抑制 44
圖七 DN-KIF3A-EGFP對分化的PC12細胞 [Ca2+]i反應的影響 46
圖八 DN-KIF3A-EGFP對於PC12細胞內粒線體分佈的影響 47
圖九 加入不同的鈣庫抑制劑對於KIF3A knock down的PC12細胞中[Ca2+]i的影響 49
圖十 KIF5B knock down對PC12細胞 [Ca2+]i反應的影響 50
7附錄 52
附錄一 KIF3A 對分化的PC12細胞在神經纖維數量和長度上的影響 52
附錄二 本研究中所用的PCR引子 53
附錄三 DsRed Mito與DN-KIF3A的質體圖譜 54
附錄四 細胞本體、神經纖維末端以及整隻細胞的定義 55
附錄五 製備shRNA 慢病毒質體圖譜及shRNA序列 56
附錄六 慢病毒製備與感染簡易流程圖 57
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