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研究生:陳建村
研究生(外文):Chen Chien-Tsun
論文名稱:粒線體DNA4,977bp斷損突變對神經膠融合細胞在doxorubicin誘導下進行細胞凋亡之影響
論文名稱(外文):Studies on Doxorubicin-Induced Apoptosis in Glial Cybrids Harboring 4,977 bp-Deleted Mitochondrial DNA
指導教授:魏耀揮魏耀揮引用關係
指導教授(外文):Wei Yau-Huei
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:73
中文關鍵詞:粒線體細胞凋亡
外文關鍵詞:mitochondriaapoptosis
相關次數:
  • 被引用被引用:0
  • 點閱點閱:125
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  • 下載下載:28
  • 收藏至我的研究室書目清單書目收藏:0
粒線體在動物細胞的生與死之間扮演著相當重要的角色,它ㄧ方面透過內膜上的氧化磷酸化系統產生大量的ATP,供給細胞執行各種生理作用所需的能量;另一方面則透過接受外來的刺激訊號而改變膜的結構,釋放出死亡訊息分子,進而調控著細胞的死亡。目前已知許多的粒線體疾病都和粒線體DNA的突變有很密切的相關性,這些粒線體疾病大多都是神經肌肉病變或是神經退化性的疾病,例如慢性進行性外眼肌麻痺症候群(chronic progressive external ophthalmoplegia,CPEO)的患者有很高的比例會有粒線體DNA 4,977 bp大幅斷損突變。本實驗室先前的研究發現,當人類骨髓瘤細胞帶有這樣的粒線體DNA大幅斷損突變時,會使得這些細胞在紫外線照射下更容易進入細胞凋亡。而我的實驗目的便是探討粒線體DNA 4,977 bp大幅斷損突變對神經膠細胞發生細胞凋亡的影響,以了解粒線體DNA斷損突變是否造成細胞功能缺陷與不正常的凋亡,而與神經肌肉組織退化及粒線體疾病的發生有關。
在本研究中我使用doxorubicin作為誘發帶有粒線體DNA 4,977 bp斷損突變神經膠融合細胞(glial cybrid)進入細胞凋亡的刺激物,以探討斷損突變對融合細胞發生細胞凋亡的影響。Doxorubicin為臨床上常用之抗癌藥物,其引發細胞凋亡的機制乃透過活化CD95L/CD95死亡受體路徑,以及造成細胞內產生大量活性氧分子(reactive oxygen species, ROS)等方式使細胞死亡。利用trypan blue exclusion方法評估細胞存活率,我發現在0.6 microM doxorubicin處理48小時後,突變型細胞的存活率較野生型細胞來得低(31.7 % vs. 59.7 %)。在caspase 3活性分析中則發現,突變型細胞的活性在doxorubicin處理36小時之後約為野生型細胞的2倍,處理48小時之後則為野生型的1.5倍。此外透過西方墨點法我可觀察到caspase 3與caspase 9活化及cytochrome c自粒線體釋放的情形,這證實了融合細胞的死亡是經由粒線體調控的細胞凋亡路徑造成的結果。最有趣的是,藉著流式細胞計數儀測量細胞內活性氧分子含量,我發現在未經doxorubicin處理時,突變型細胞內ROS含量是野生型細胞的1.4倍,而在0.6 microM doxorubicin處理2小時之後,可以看到突變型細胞有大量的ROS產生,野生型細胞增加的情形則不是那麼顯著,這樣的現象可能與突變細胞較易發生細胞凋亡有關。從上述觀察到的結果可以發現突變型細胞確實較野生型細胞更容易在doxorubicin刺激後發生細胞凋亡。另一方面,我也觀察到了這些融合細胞死亡受體調節路徑中FADD (Fas-associated death domain)的磷酸化、caspase 8活化以及Bax蛋白轉位至粒線體的情形,顯示死亡受體路徑也共同參與神經膠細胞的死亡,但野生型細胞與突變型細胞FADD的磷酸化與Bax的轉位並沒有顯著差異。
本研究結果顯示粒線體DNA 4,977 bp斷損突變可能會造成粒線體結構與功能上的異常,使得突變型細胞平時就有較高的氧化壓力,粒線體在doxorubicin刺激下也較容易產生ROS,而使得細胞更容易發生凋亡。本研究也進一步證明粒線體DNA上的4,977 bp斷損突變影響細胞凋亡的行為,與CPEO等粒線體疾病的發生有密切的關係。
Mitochondria play important roles in life and death of mammalian cells. They serve not only as the major energy generator, but also a key regulator of apoptosis. It is well known that there is a wide spectrum of mutations and deletions in mitochondrial DNA (mtDNA), and these genetic defects can cause an array of mitochondrial diseases, mostly neuromuscular disorders such as CPEO (chronic progressive external ophthalmoplegia) syndrome. In one of our previous studies, we demonstrated that the human cells with mtDNA large scale deletions are more susceptible to UV-induced apoptosis. I have been interested in knowing whether similar effects of mtDNA deletions can be observed in glial cells, which are important supporting cells in neuronal tissues.
I used doxorubicin as an apoptotic agent in human glial cybrids harboring CPEO-associated 4,977 bp deletion of mtDNA (termed mutant cybrids). Doxorubicin is an anti-cancer drug which can trigger apoptosis via the CD95L/CD95 pathway and reactive oxygen species (ROS) over-production in cells. By trypan blue exclusion assay, I observed lower cell viability in mutant cybrids compared with wild-type cybrids (31.7 % vs. 59.7 %) after treatment with 0.6 microM doxorubicin for 48 hours. In caspase 3 activity assay, I found that the activity of the mutant cybrid was 2 times higher than that of wild-type cybrid after treated with 0.6 microM doxorubicin for 36 hours, and was 1.5 times higher after 48 hours. By Western blot analysis, I observed activation of caspase 3 and caspase 9 and the release of cytochrome c from mitochondria upon 0.6 microM doxorubicin treatment, indicating that mitochondria was involved in doxorubicin-induced glial cybrid death. Most interestingly, by using flow cytometry, I found that the basal ROS level in the mutant cybrid was 1.4 times higher than that in the wild-type cybrid. Moreover, the ROS over-production was more pronounced in mutant cybrids after treatment with doxorubicin for 2 hours. These observations together indicate that glial cybrids with 4,977 bp-deleted mtDNA were more susceptible to apoptosis compared with cybrids harboring wild-type mtDNA. On the other hand, I monitored FADD (Fas- associated death domain) phosphorylation, caspase 8 activation and translocation of Bax to mitochondria by Western blot in these cybrids under doxorubicin treatment. The results showed that death receptor-mediated apoptosis was also involved. However, the patterns of the above events showed no significant differences between wild-type and mutant cybrids.
The 4,977 bp deletion of mtDNA may cause alterations in mitochondrial structure and function in mutant cybrids, and thereby resulted in higher oxidative stress and more ROS production under doxorubicin stimulation. This could be the reason why the mutant cybrids were more susceptible to apoptosis by doxorubicin treatment. The present studies provide evidence to support the notion that mtDNA deletion and susceptibility to apoptosis may play important roles in the pathogenesis of mitochondrial diseases such as CPEO syndrome.
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