- 學位論文
褪黑激素（melatonin）對於大腦星狀神經膠質細胞在ionomycin作用下造成氧化壓力與粒線體膜電位變化所引起細胞凋亡之保護機制的探討
Melatonin inhibits oxidation-independent activation of mitochondrial permeability transition and apopotosis in rat brain astrocytes RBA-1
摘要
粒線體的功能除了是哺乳動物細胞能量產生中心外，有一些文獻也發現在細胞凋亡過程中粒線體同樣扮演著重要角色，鈣離子的?琠w也有關聯。粒線體本身是細胞最主要的活性氧分子(reactive oxygen species, ROS)之產生場所；活性氧分子是細胞進行有氧代謝過中所產生的副產物，包括超氧化物自由基(superoxide anion, .O2-)、過氧化氫(H2O2)與氫氧自由基(.OH)等。在神經細胞面臨損傷時會迅速活化或釋放一些免疫調節物，如有傷害性的一氧化氮(nitric oxide, NO)或活性氧分子，而進一步造成氧化神經毒性(oxidative neurotoxicity)。本論文使用雷射掃瞄共軛焦顯微鏡（confocal）觀察星狀膠質細胞(astrocyte)經過螢光染劑DCFH-DA 1μM（細胞內活性氧分子的測定，mROS）和TMRM 300 nM（粒線體膜電位的測定，△Ψm）雙染後；給於ionomycin 100 nM實驗結果顯示快速形成氧化壓力且粒線體膜電位二十分鐘後完全產生去極化現象，和活體細胞給於ionomycin作用三十分鐘內被PI（Propidium Iodid，鑑定細胞核是否發生濃縮）染上表示產生細胞凋亡現象。進一步證實ionomycin誘導早期粒線體膜電位去極化是否與MPT有關，細胞給於CsA（MPT inhibitor）結果顯示細胞三十分鐘內沒有死亡現象， ionomycin誘導氧化壓力減少百分之五十且粒線體膜電位未完全抑制；晚期細胞也呈現凋亡現象。接著使用抗氧化物質vitamin E來抑制氧化壓力的形成，造成細胞在沒有氧化壓力下，ionomycin促使鈣離子大量進入細胞內與粒線體之MPT的關連性。結果顯示粒線體的膜電位仍然緩慢去極化；表示鈣離子是主要扮演誘導細胞凋亡的重要角色，這當中也與MPT有關。進一步實驗證明褪黑激素(Melatonin)抗氧化劑保護星狀膠質細胞在給於ionomycin作用下，所誘導產生氧化壓力隨時間作用比較CsA有顯著減少及粒線體膜電位完全去極化的時間延後，且作用三十分鐘後沒染上PI染劑；由實驗證實褪黑激素(Melatonin)對星狀膠質細胞的細胞凋亡(apoptosis)現象有保護的作用。本論文進一步探討細胞在vitamin E合併使用melatonin後，給於ionomycin實驗結果證實粒線體膜電位去極化現象比單獨melatonin 或單獨vitamin E 或單獨CsA或vitamin E合併CsA，時間顯著延後至五十分鐘。當合併使用melatonin和CsA實驗結果看出粒線體膜電位去極化現象延後至七十分鐘。此現象可證實melatonin不僅可作為抗氧化物質，其作用在MPT pore 有關。由實驗結果比較性的總結：ionomycin < vitamin E < CsA < melatonin
並列摘要
Abstract Mitotochondrial function in mammalian is generally presented as the central pathway for energy metabolism, but several other aspects of mitochondrial function have been described, such as key roles in apoptosis, Reactive oxygen species production, thermogenesis and calcium signaling.Reactive oxygen species (ROS), such as superoxide anion(.O2-), hydrogen peroxide(H2O2)and hydroxyl radical(.OH), are by-products of aerobic metabolism in cells. Upon neuronal injury stimulation and release immune modulators such as the potentially damaging nitric oxide (NO) and reactive oxygen species. In this study, major technology used Lascer scanning confocal microscopy observation astrocytes cells line had double staning of ROS generation detected by DCFH-DA （1μM）and △Ψm depolarization detected by TMRM（300 nM）；After exposure ionomycin （100 nM），the study results indicated that ionomycin-induced mROS generation soon reached to a peak at where △Ψm depolarized completely (<20 min) and resulted in severe plasma membrane blebing and apoptotic condensation of nuclei and cell death (<30 min). To confirm ionomycin-triggered MPT as well as to examine whether the MPT involved in the early phase of ionomycin-induced △Ψm depolarization, cells were treated with a classic MPT inhibitor, CsA. No cell death was observed within 30 min of ionomycin exposure. Interestingly, inhibition of MPT significantly reduced ionomycin-induced mROS formation by about 50 % CsA did not completely inhibit ionomycin-induced apoptosis suggesting ionomycin-induced Ca2+ stress together with a MPT-independent mROS formation acted synergistically to cause mitochondrial dysfunction and later apoptotic cell death. To remove ionomycin-induced mROS formation for an “oxidation free-Ca2+ stress” and associated MPT during ionomycin exposure, cells were treated with a classic antioxidant vitamin E to mask ionomycin-induced ROS formation. Ionomycin-induced mROS generation was completely neutralized by vitamin E for an exclusive condition of “oxidation free-Ca2+ stress”. Nevertheless, “oxidation free-Ca2+ stress” induced a depolarization of △Ψm whose time course was slighter slower than that observed in the absence of vitamin E. These results thus suggest that Ca2+ plays a major role in ionomycin-induced apoptosis and “oxidation free-Ca2+ stress” is sufficient to trigger MPT and apoptosis. Melatonin significantly prevented ionomycin-induced depolarization of △Ψm in a manner different from that seen upon CsA treatment. At the end of recording (30 min), ionomycin-induced △Ψm declined to around 40 % of the control level. Its associated apoptotic death of cells was significantly prevented long after ionomycin exposure (>40 min) in the presence of melatonin. These results thus suggest, in addition to anti-mROS formation, melatonin may target furthermore on mechanisms that regulate Ca2+ stress-induced depolarization of △Ψm and Ca2+ stress-mediated MPT. In the presence of vitamin E, melatonin significantly prevented ionomycin-induced apoptosis. Cells survival rate was greatly improved up to near 100 % and to 70% at 30 min and 50 min, respectively, after ionomycin exposure. Melatonin completely prevented “oxidation free-Ca2+ stress”-induced depolarization of △Ψm and associated MPT. When effects of melatonin on “oxidation free-Ca2+ stress”-mediated △Ψm depolarization and MPT was compared to that provided by CsA, melatonin seems provides a better protection than CsA. With the protection provided by CsA in the presence of vitamin E, ionomycin-induced △Ψm depolarization and MPT, although prevented for longer than 26 min, declined to close 20% of the control level at 28 min after ionomycin exposure. effect of melatonin plus CsA on ionomycin-induced △Ψm depolarization and MPT. Interestingly, synergistic protections provided by CsA and melatonin prevented completely ionomycin-induced plasma membrane blebing and apoptotic death long after ionomycin exposure (>70 min). Ionomycin-induced depolarization of △Ψm and MPT was preserved to 80% of the control level even after 70 min of ionomycin exposure As a summary, degrees of protection provided by sole effect of vitamin E, CsA, or melatonin, as well as combination of these compounds during ionomycin exposure are listed below: ionomycin < vitamin E < CsA < melatonin < vitamin E + CsA < vitamin E + melatonin < melatonin + CsA.