- 學位論文
神經醯胺誘導神經膠瘤細胞粒線體功能損傷及細胞凋亡
Induced Mitochondrial Dysfunction and Apoptosis of Rat Glioma Cells by C6 Ceramide
摘要
神經醯胺(ceramide)是磷脂質的二級訊號傳遞因子，在細胞生長過程與刺激反應中扮演必要的調節者。此外也被證實為造成細胞氧化壓力的來源之ㄧ，不但參與發展中的神經細胞凋亡，更被證實和神經退化性疾病的生成有關。有文獻指出，ceramide可能經由在粒線體膜上形成通道(channel)，造成細胞色素(cytochrome)釋放至細胞質，誘導細胞凋亡的發生。本論文主旨為探討C6 ceramide造成大鼠神經膠瘤細胞(C6 glioma cells)之粒線體功能缺損進而造成細胞色素c (cytochrome c)釋出導致細胞凋亡之研究。從實驗結果得知，在25 ?嵱 C6 ceramide的作用後會降低神經膠瘤細胞的存活率。以25 ?嵱 C6 ceramide培養24小時後，細胞存活率降至對照組的30.8% ± 2.2% (n=3, p＜0.001)，並且經H2DCFDA染色檢測後發現細胞內活性氧自由基(reactive oxygen species, ROS)的產生於六小時C6 ceramide處理後增加為對照組的209.4% ± 23.3% (n=3, p＜0.01)，粒線體膜電位(mitochondria membrane potentials, Δ??)在12小時處理後則下降為29.9% ± 2.1% (n=3, p＜0.001)。粒線體ATP含量隨刺激時間增加而遞減，在18小時減少為4.4% ± 2.8% (n=3, p＜0.001)。此外，C6 ceramide的處理也會造成粒線體DNA的斷損突變(mitochondrial DNA rearragement)，於六小時C6 ceramide處理後，其斷損突變情形增加。當以Annexin V-FITC/ Propidium iodide (PI)染色分析偵測細胞死亡情形，發現以25 ?嵱 C6 ceramide培養24小時後，與對照組比較細胞凋亡現象增加了34.7%。當我們以帶有螢光基團的C6 ceramide (NBD-C6 ceramide)與細胞進行培養，利用雷射共軛焦顯微鏡觀察，發現NBD-C6 ceramide與粒線體的螢光影像圖位置相同並重疊在一起，顯示C6 ceramide進入細胞後會在粒線體上形成堆積。以西方墨點法分析細胞凋亡蛋白質變化，結果顯示經25 ?嵱 C6 ceramide處理後，與對照組比較cytochrome c自粒線體釋出到細胞質的情形增加為212.5%，促使細胞走向凋亡，我們添加粒線體膜通透性改變孔(MPT pore) 的抑制劑環孢黴素A (cyclosporin A)，看到因C6 ceramide刺激所導致的cytochrome c釋出被部分抑制，與對照組比較減少為115.2%，另外，我們以C6 ceramide的活性抑制物C6 dihydroceramide (DH C6)處理後，發現因25 ?嵱 C6 ceramide刺激所導致的cytochrome c釋出由269.8% ± 28.1%降低為114.0% ± 6.6%，我們推測C6 ceramide除了可能經由傳統的mitochondrial-dependent cell apoptosis 路徑造成細胞死亡，另外，有可能經由C6 ceramide堆積在粒線體上形成cytochrome c釋出的通道(leakage)。當我們以C6 ceramide處理純化後的功能性粒線體，發現C6 ceramide可以直接與粒線體作用，逕自使粒線體的呼吸率下降、cytochrome c釋出。由此我們推測，C6 ceramide經由堆積在大鼠神經膠瘤細胞之粒線體上，引發大量ROS產生造成氧化性傷害，粒線體Δ?擏幭僈P功能缺損，並在粒線體外膜上形成孔洞，誘導儲存於膜間隙的cytochrome c釋出，促使細胞走向凋亡。C6 ceramide進入細胞後在粒線體上形成堆積，可能是造成粒線體功能缺失與細胞凋亡的主因。
並列摘要
Ceramide has been proven an origin of oxidative stress, not only participate in neuron programed cell death, but involve in variety of neurological disorders such as epilepsy, Alzheimer’s and Parkinson’s diseases, and cerebral ischemia. Recent evidence have demonstrated that ceramide-induced apoptosis might be mediated by ceramide-forming mitochondrial membrane channel and cytochrome c release into cytoplasm. In this study, we proposed that ceramide-induced C6 glioma cells apoptosis via mitochondrial dysfunction, forming mitochondrial membrane leakage and induce cytochrome release. We investigated the cytoeffects of C6 ceramide on glioma cells damages including mitochondrial DNA instability, mitochondrial dysfunction and cytochrome c release. We performed several experiments including cell viability by dye exclusion assay, cell apoptosis by Annexin V/PI flow cytometry analysis, ROS generation and mitochondria membrane potentials (Δ??) by flow cytometry, ATP generation by ATP-luciferin assay, mitochondrial DNA rearragement by long-PCR analysis and cytochrome c release by western blotting. In order to identify the cell accumulation of C6 ceramide and ceramide localization, we detected the ceramide marker NBD-C6 ceramide by confocal microscope. A declined cellular viability was found to be 30.8% ± 2.2% in 25 ?嵱 C6 ceramide-treated cells (p＜0.001). A 2.1 folded-increase of ROS generation at 6 h (p＜0.01) and 70% reduction of mitochondria membrane potentials at 12 h (p＜0.001) were found. In addition, we found the ATP content was declined to 4.4% ± 2.8% (p＜0.001) at 18 h and showed a time-dependent reduction. After 25 ?嵱 C6 ceramide treatment, the apoptotic cells were increased 34.7% and raised apoptotic fraction was also detected. We also found NBD-C6 ceramide colocalized with mitochondria. In addition, we also found a 2.1 folded-increase of cytochrome c releasing from mitochondria into cytoplasm followed 25 ?嵱 C6 ceramide treatment. Moreover, the use of cyclosporin A (MPT pore inhibitor, CsA) was found to partially block of cytochrome c release to 115.2% in the ceramide-treated cells. On the other hand, using C6 dihydroceramide (DHC6) as biological inactivator of C6 ceramide was also declined the release of cytochrome c from 269.8% ± 28.1% to 114.0% ± 6.6%. Those evidences indicated that not only C6 ceramide mediated the traditional mitochondrial-dependent cell apoptosis pathway, but accumulation of ceramide in the mitochondria possibly contributing to mitochondrial dysfunction and forming mitochondrial membrane leakage for cytochrome c release. Further more, we isolated the functional mitochondria from glioma cells, the reduced oxygen consumption rates and the induction of cytochrome c release were also revealed in C6 ceramide-treated mitochondria. According to our results, we suggested that ceramide-induced cell apoptosis might be mediated by intramitochondrial ceramide induced mitochondrial malfunction.