臺灣博碩士論文加值系統

中文摘要 :
以MTT還原能力方法自27種中藥之91個粗萃物概略篩選出保護神經元細胞免於glutamate及 A 毒性之保護成分，初步發現人參水層及五種北茵層粗萃物對於glutamate 所引起之毒性具有保護潛力，因此針對此二種中藥進行保護機制之探討。結果顯示人參水層及北茵陳分離層28-30、28-39具有使glutamate處理之大白鼠大腦皮質神經元細胞MTT還原能力增加，但在高濃度下對神經細胞則具有毒性。 以2’,7’-dichlorofluorescein diacetate水解試驗來測量glutamate所引起神經元細胞高反應性氧化物 (Reactive oxygen species，ROS) 的產生，發現分別以2 M及5 M glutamate處理30分鐘，神經元細胞ROS含量分別增加為對照組的1.4和2.1倍。若以50 g/ml及100 g/ml之北茵陳分離層28-30、28-39先行處理30分鐘，再以2或5 M之glutamate 處理30分鐘，發現兩種北茵陳分離層皆可對glutamate造成細胞內ROS的增加具有抑制效果，至於人參水層對glutamate造成之細胞內ROS累積沒有明顯影響。以5 M glutamate處理神經元細胞，細胞內鈣離子濃度比值迅速增加至對照組的2.1倍，預先以100 g/ml之人參水層、北茵陳分離層28-30或28-39處理30分鐘，發現皆可抑制細胞內鈣離子濃度。細胞內鈣離子增加會活化nNOS進而產生NO，最後導致cGMP量的增加。因此探討中藥粗萃物是否影響細胞內cGMP含量，發現人參水層和北茵陳分離層28-30可以抑制細胞內cGMP濃度的增加，而北茵陳分離層及28-39對glutamate所引發細胞內cGMP的濃度增加無明顯影響。
另外以1至40 M 之 A處理神經瘤細胞 (Neuroblastoma) Neuro 2A 24小時，發現MTT還原能力隨 A 濃度增加而下降，計算細胞數目則當低濃度 A處理時，細胞數目並未改變，高濃度則使細胞數目減少。分別以Lactate dehydrogenase之釋出及Trypan blue染色法測量，結果顯示A1至40 M並未造成細胞死亡，以BrdU incorporation試驗發現細胞增生的抑制與A25-35呈現濃度效應，顯示高濃度 A 對 Neuro 2A 造成之作用可能是抑制 Neuro 2A 細胞的增生。為釐清Neuro 2A細胞增殖與何種傳遞系統有關，分別以SB 203580 (p38抑制劑)、PD 98059 (ERK活化之抑制劑)、LY 294002 (PI3K抑制劑)及Genistein (tyrosine kinase抑制劑) 處理細胞，結果暗示ERK、PI3K及tyrosine phosphorylatiuon與 Neuro 2A細胞之增生有密切關連。分析A對幾種kinase的影響發現 : (1) A 降低了PI3K與tyrosine phosphorylated蛋白的結合；(2) A 不影響ERK的活化；(3) A 增加或減少了蛋白的tyrosine phosphorylation； (4) A短暫增加JNK的活化。以北茵陳粗萃物之各分離層處理2小時，再以1 M A 處理24小時，發現水層粗萃物，分離層4、9-9、9-20、9-21C、16-14、16-18、16-20-16、28、28-19、28-20-4、28-21C、28-24、28-39具有使MTT還原能力增加之效果，其它分離層及純化合物則無保護效果。CD光譜顯示具保護作用的分離層9-21C和16-20-16會影響A25-35的聚合，不具保護作用的分離層16-1-2則不影響A25-35的聚合。結果顯示這兩種分離層之保護效果乃在影響A25-35之聚合狀態而達成。

英文摘要：
For searching the neuroprotective agents against glutamate-induced neurotoxicity, ninety-one fractions of crude extracts from twenty-seven kinds of Chinese herbal medicine were screened by using cortical neurons as model system. The cell viability was determined by MTT reduction assay. The preliminary results showed that the water extraction of Panax ginseng C.A. Meyer and five fractions of water extraction of Origanum vulgare L. were the most effective to reduce the neurotoxicity induced by glutamate. The mechanisms of neuroprotective effect of these fractions against glutamate-induced neurotoxicity were therefore investigated. The glutamate-induced production of reactive oxygen species (ROS) in cortical neurons was determined by 2’,7’-dichlorofluorescein diacetate assay. Treatment of cortical neurons with 5 M of glutamate for 30 min increased the ROS level to be 2.1 fold of that in control cells. Pretreatment of fractions 28-30 or 28-39 of Origanum vulgare L. significantly decreased the glutamate-induced increase of ROS level in cortical neurons. Water extraction of Panax ginseng C.A. Meyer, however, did not affect the ROS level induced by glutamate. Treatment of cortical neurons with 5 M of glutamate elevated the intracellular concentration of calcium to be 2.1 fold of that in control cells. Fractions 28-30 or 28-39 of Origanum vulgare L. or water extraction of Panax ginseng C.A. Meyer blocked the glutamate-induced increase of intracellular concentration of calcium. The increase of calcium concentration activates the activity of nNOS and leads to the production of NO, thereby elevating the intracellular level of cGMP. Water extraction of Panax ginseng C.A. Meyer and fraction 28-30 of Origanum vulgare L. significantly decreased the glutamate-induced increase of intracellular concentration of cGMP in cortical neurons. Fractions 28-39 of Origanum vulgare L., however, did not show any effect on the intracellular concentration of cGMP induced by glutamate.
In search of the neuroprotective reagents against A-induced neurotoxicity, the same crude extracts were screened on Neuro 2A cells. A was toxic to Neuro 2A cells as determined by MTT reduction assay. The cell viability, however, was not affected by the treatment of Aas determined by LDH release and trypan blue exclusion. The proliferation of Neuro 2 A cells was found to be blocked by A as determined by cell number counting and the incorporation of BrdU. Inhibitor studies suggested that the signaling pathway of ERK, PI3K and tyrosine phosphorylation may be involved in the proliferation of Neuro 2A cells. Therefore, tyrosine phosphorylation of proteins, activation of ERK, JNK, and the association of PI3K with tyrosine phosphorylated proteins were elucidated. Results showed that : (1) A decreased the association of PI3K with tyrosine phosphorylated proteins, (2) A did not affect ERK activation, (3) A increased or decreased the tyrosine phosphorylation of proteins, (4) A25-35 increased transitorily the JNK activation.
Among the ninety-one fractions, thirteen fractions of water extraction of Origanum vulgare L. were potential to decrease the neurotoxicity induced by A as determined by MTT reduction. The mechanism of neuroprotective effect of these fractions against A-induced neurotoxicity was investigated. CD spectra showed that fraction OV 9-21C and 16-20-16 of Origanum vulgare L interfered the A25-35 aggregation, and fraction 16-1-2 of Origanum vulgare L. did not change A25-35 aggregation. Thus, the neuroprotective effect of Origanum vulgare L. may be mediated by altering the A25-35 aggregation.

目 錄
圖表目錄---------------------------------------------Ⅰ
縮寫表-------------------------------------------------Ⅳ
中文摘要----------------------------------------------Ⅵ
英文摘要----------------------------------------------Ⅷ
緒論---------------------------------------------------- 1
研究目的-----------------------------------------------11
實驗材料-----------------------------------------------14
實驗方法-----------------------------------------------20
實驗結果-----------------------------------------------28
討論-----------------------------------------------------38
結論-----------------------------------------------------48
參考文獻-----------------------------------------------49
圖表-----------------------------------------------------57

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