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粒線體DNA匱乏C6神經膠瘤細胞對於神經醯胺誘發細胞凋亡之抗性研究

Resistance to Ceramide-induced Apoptosis in the Mitochondrial DNA-depleted C6 Glioma Cells

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[[abstract]]粒線體在細胞凋亡發生的訊號傳遞途徑中扮演著一個重要調節者的角色。粒線體DNA (mtDNA)匱乏的細胞株對於TRAIL (TNF-related apoptosis-inducing ligand)所誘發的細胞凋亡感受性極低。在本實驗中我們利用溴化乙醯(ethidium bromide, EtBr)及二脫氧胞苷(zalcitabine, 2''-3''-dideoxycytidine, ddC)處理C6神經膠瘤細胞(C6 glioma cell)後,以同步定量聚合酶連鎖反應來偵測mtDNA套數,確認得到不同程度mtDNA匱乏的細胞,將所有藥劑移除72小時之後再次測量mtDNA套數的回復情形。各組細胞中mtDNA套數約為:control組, 100%; 1μM EtBr組, 52%; 25 μM EtBr組, 13%; 5 μM ddC組, 25%; re-1 μM EtBr組, 92%; re-25 μM EtBr組, 52%; re-5 μM ddC組, 51%。細胞的生長速率以25 μM EtBr組生長速率最低,細胞存活率以25 μM EtBr組細胞存活率最低。粒線體膜電位(mitochondrial membrane potential, △Ψ) 的測量結果顯示添加EtBr均導致△Ψ下降。實驗結果發現mtDNA套數的下降在本實驗中與粒線體ATP的形成能力無絕對關係。觀察粒線體電子傳遞鏈酵素群蛋白質(ND6、Core 2及F1α)的表現量後,結果顯示mtDNA套數的下降直接影響mtDNA調控的蛋白質ND6,但在藥劑移除之後表現量大幅高於控制組,顯示有抑制回饋現象。在確認了mtDNA套數的下降所造成的細胞生理的影響之後,使用共軛焦顯微鏡偵測細胞色素C (cyt c)的轉位作用及以流式細胞儀計算細胞凋亡的發生率。實驗結果顯示只有25 μM EtBr組具有明顯的cyt c轉位作用及較高的細胞凋亡發生率。若以25 μM ceramide誘發細胞凋亡,發現部分mtDNA套數下降的細胞對其細胞凋亡的誘發敏感性較小。而先使用3 μM cyclosporin A (CsA) 處理30 分鐘再以25 μM ceramide誘發細胞凋亡,各組細胞皆有細胞凋亡發生率下降的現象,顯示其細胞凋亡確實是由ceramide所誘發。實驗證實mtDNA套數確實會影響細胞生理功能,並且對於ceramide所誘發的細胞凋亡具有抗性。

[[abstract]]Mitochondria is one of the master regulators in apoptosis signaling pathway. Mitochondrial DNA (mtDNA)-depleted cell line has lower sensitivity to apoptosis which inducing by TRAIL (TNF-related apoptosis-inducing ligand). In this study, we created C6 glioma cells with mtDNA depletion by treatment with ethidium bromide (EtBr) and zalcitabine (ddC) for 72 hours. The mtDNA copy numbers were determined from cells treated with EtBr and ddC followed removing incubated reagent 72 hours. MtDNA copy numbers were determined by with real-time polymerase chain reaction (real-time PCR) and listed as followed list: control: 100%, 1μM EtBr: 52%, 25 μM EtBr: 13%, 5 μM ddC: 25%, re-1 μM EtBr: 92%, re-25 μM EtBr: 52%, re- 5 μM ddC: 51%. We counted cell number for cell growth rate. The cell viabilities were also analyzed by MTS assay. Mitochondrial membrane potential (△Ψ) analyzed by JC-1 stainning indicated that EtBr groups had lowest △Ψ. Contents of cellular ATP generation were determined represented that ditacted lower mtDNA copy number was not direct proportion to ATP generation. Estimated mitochondrial electron transport chain (ETC) proteins (ND6, Core 2 and F1α) expression with Western blotting, the result indicated that cells harboring lower mtDNA copy number had lower mtDNA encoded protein, but mtDNA copy number restores and more higher than control cell after all drugs were removed. To confirme the influence of mtDNA depleting to cells, we observed the Cytochrome c (cyt c) translocation with confocol microscopy and calculated apoptotic proportion with flow cytometery. The result showed only the 25 μM EtBr treatment group has conspicuous effect of cyt c translocation and higher apoptotic ratio. The cell apoptosis induced with 25 μM ceramide showed that mtDNA-depleted cells were not susceptible for ceramide treatment. Moreover, cells incubated with 3 μM cyclosporine A (MPT pore inhibitor, CsA) for 30 min before 25 μM ceramide treatment showed lower apoptosis proportion occurred in all groups, this data indicated the cell apoptosis was induced by ceramide. According to our results, we suggest that decreased mtDNA copy number affected cell physiology and resisted to ceramide-inducing apoptosis.