臺灣博碩士論文加值系統

許多種類的活性氧化物種(ROS )，例如：氫氧自由基(.OH)和superoxide anion (O2-)會造成神經系統的病變，這可能是引發運動神經系統病變的一個重要原因。本篇論文是研究受神經支配的肌細胞所放出的NO，可以透過減少.OH對神經造成的傷害保護神經細胞。本實驗神經毒性的引發，是經由給予過氧化氫(H2O2)和亞鐵(Fe2+)以產生氫氧自由基所造成的，此時會使沒有任何肌細胞接觸的神經細胞(naive neuron)在約85分鐘時產生毒性而死亡，但是如果與一至三個肌細胞形成突觸的神經，其死亡時間則可延長至約180分鐘，若神經與一群肌細胞(如大於八個)接觸，則死亡時間更延長至約430分鐘。表示此保護作用與神經接觸的肌細胞有關，若以玻璃棒推一肌細胞與神經接觸亦具有類似的保護作用，但若是與fibroblast接觸，則沒有神經保護作用，所以當有氫氧自由基產生時，肌細胞會釋放出某種保護神經的物質。
使用一氧化氮合成酵素(NOS)的抑制劑NG-Nitro-L-arginine methylester (L-NAME)會拮抗肌細胞的保護作用，而solube guanylate cyclase(sGC)的抑制劑1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ)則不會影響肌細胞的保護作用。另外若使用一氧化氮的合成前趨物L-arginine以及低濃度的一氧化氮釋放劑sodium nitroprusside (SNP)都可以降低氫氧自由基所造成的naive神經毒性。但是cGMP的類似物Sp-cGMPS則不會影響氫氧自由基對naive神經細胞造成的傷害。因此一氧化氮(NO)可能為肌細胞釋放出的保護因子，且其保護作用與cGMP無關。神經滋養因子例如BDNF、 neurotrophin-3 (NT-3)以及GDNF也無法保護神經細胞對抗氫氧自由基(.OH)所造成的傷害。所以肌細胞可能不是釋放神經滋養因子來保護神經對抗H2O2的毒性。
若先在細胞內載入(loading)鈣離子(Ca2+)的螯合劑BAPTA，則可以明顯對抗氫氧自由基對神經細胞造成的毒性傷害，因此認為鈣離子是氫氧自由基造成神經毒性的一個重要物質。將Fura-2AM和DAF-2DA分別載入一天大的爪蟾細胞培養內一小時和三十分鐘，並且加入H2O2和Fe2+之後，可以在神經細胞和肌細胞都測量到鈣離子([Ca2+]i)和NO的增加，而且肌細胞內的NO增加也較神經細胞來的顯著。Fura-2AM在進入細胞後會被切割為Fura-2，此時可以與游離態的鈣離子作用，使用光學顯微鏡並在340nm及380nm二種波長的激發光，觀察細胞內鈣離子增加的情形。給予H2O2和Fe2+，神經與肌細胞的[Ca2+]i均會增加，而且需要胞外的鈣離子。此外DAF-2是脂溶性DAF-2衍生物，在進入細胞後會被胞內的脂解酵素切割成水溶性的DAF-2，此時DAF-2就可以與NO作用而形成另一種會產生螢光的化合物DAF-2T，並且不會逸出細胞外，此時使用共軛焦顯微鏡，在波長488nm的激發光下，DAF-2T就可以在515nm的散射波長下產生螢光。而且因為所形成的螢光化合物DAF-2T其結構是很穩定的不易再被分解，因此所產生的DAF-2T螢光是具累積性的，會隨著細胞內的NO與DAF-2作用而增加，我們發現給予H2O2和Fe2+，NO釋放增加，肌細胞之作用大於神經細胞。
利用電生理的方法，以whole-cell voltage-clamp的方式，在肌細胞記錄神經ACh (acetylcholine)自發性釋放引發的電流。如果在bath中先投與L-NAME之後，再加入H2O2和Fe2+則會大大增加神經細胞自發性釋放ACh的量。顯示肌細胞可以釋放NO防止H2O2和Fe2+在神經末梢引起的[Ca2+]i增加。因此推論肌細胞所釋放出的一氧化氮(NO)是經由一個和cGMP不相關的路徑，對抗氫氧自由基所造成的神經毒性，而提供神經細胞的保護作用，神經支配的肌細胞所釋放出的NO可能扮演了自由基的清除角色。

Reactive oxygen species (ROS) such as hydroxyl radical (.OH) and superoxide anion (O2-) generated in a variety of neuropathological processes may be important factors to contribute to the injury of motoneurons. Recent evidence suggests that nitric oxide (NO) is not only an important biological messenger but also has neuroprotective action. We here investigated the neuroprotective effects of endogenously released NO from myocyte against H2O2-mediated neurotoxicity. It is well known that .OH can be produced by ferrous iron (Fe2+) catalysis of the chemical breakdown of H2O2. Here we induced neurotoxicity by the bath application of H2O2 and Fe2+ in Xenopus 1-day-old nerve-muscle co-cultures. H2O2 plus Fe2+ caused the death of naive neuron about 85 min. However, myocyte contact delayed the neuronal death to 180 min, indicating that myocyte released neuroprotective factors upon the addition of H2O2 plus Fe2+. The neuroprotective action disappeared by acutely destroying the innervated myocyte by using a glass micropipette. Acute manipulation of a myocyte to contact with the growth cone of a naive neuron also exerts neuroprotective action. Neurons contacted by fibroblast die with 67 nin upon bath application of H2O2 plus Fe2+, indicating that fibroblast did not exert neuroprotective action against wxidative stress.
NG-Nitro-L-arginine methylester (L-NAME) inhibited the neuroprotective action of myocyte in both natural and manipulated synapses. ODQ, a soluble guanylate cyclase inhibitor, did not affect the neuroprotective effect of myocyte. L-arginine (1 mM) and low concentration of sodium nitroprusside (10 mM) but not Sp-cGMPS slightly antagonized the neurotoxic action of H2O2 in naive neurons. Chronic treatment with neurotrophic factors such as BDNF, NT-3 and GDNF did not exert neuroprotective action against oxidative injury in naive neurons.
Intracellular loading of BAPTA markedly inhibited the neurotoxic action of H2O2 in naive neurons, suggesting that Ca2+ is an important mediator leading to neuronal death. Intracellular Ca2+ and NO concentration were monitored by Fura-2 and DAF-2, respectively. Intracellular Ca2+ and NO levels increased in both neuron and myocyte upon the addition of H2O2 plus Fe2+. NO release is more prominent in innervated myocyte.
Myocyte was whole-cell voltage-clamped to record spontaneous ACh release from neuron and it was found that L-NAME pretreatment markedly increased ACh secretion in response to bath application of H2O2 plus Fe2+, indicating that neuronal Ca2+ elevation was enhanced by L-NAME under oxidative stress. Our results suggest that NO released from innervated myocyte protects neuron against neurotoxicity caused by H2O2 in a cGMP-independent pathway.

縮寫表‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧2
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧3
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧5
緒論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧8
實驗材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16
結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧27
討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧52
參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧58

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