臺灣博碩士論文加值系統

缺血性腦中風已知會造成腦組織受損，嚴重時可能致命。腦組織缺血時，會產生過多的活性氧物質(ROS)，而腦組織的抗氧化能力又偏低，因此缺血或再灌流(復血)產生的氧化壓力一直是腦細胞及組織受損的主要原因之一。如何有效提升缺血腦細胞之抗氧化能力，對腦中風的治療或預防上均極其重要。星芒狀細胞在腦組織中數目最多，且具有較高之抗氧化能力。又乙醛脫氫酶3A1(ALDH3A1)，在眼角膜中被指出具有抗氧化之功能。本論文因此針對ALDH3A1在缺血(GOSD)星芒狀細胞中是否扮演抗氧化及保護性的角色角色及中藥水粹物ZC008是否能提升此細胞中ALDH3A1之活性做評估。實驗中利用台盼藍染染色法、西方墨點法、Zymography Assay、DCF、OPA、GR及TBARS試驗法，評估體外缺血 (GOSD) 6小時對星芒狀細胞的存活、ALDH3A1蛋白及活性表現、胞內及胞外ROS含量、GSH含量、NADPH含量及丙二醛(MDA)含量的影響。另將二甲氨基苯甲醛(DEAB)或ZC008外加於GOSD星芒狀細胞中，做同上之分析。研究結果顯示，神經、微膠及星芒狀細胞經GOSD(6小時)處理後，星芒狀細胞的存活率最高，細胞內與細胞外的ROS最少，ALDH3A1的蛋白與活性反應最顯著，顯示星芒狀細胞在面對缺血壓力(GOSD)時，的確有較佳之抗氧化能力。為了釐清ALDH3A1是否及如何調控GOSD星芒狀細胞的抗氧化能力，實驗中將ALDH3A1抑制劑(DEAB)外加入GOSD星芒狀細胞中，發現在GOSD星芒狀細胞中ALDH3A1能抑制胞內ROS但提升細胞外ROS的含量，及提高細胞存活率的功能。由於在眼角膜中ALDH3A1能藉由提升NADPH及GSH來清除ROS，並降低脂質酸化的程度，論文中因此亦針對這些因子的濃度變化做分析，結果發現，GOSD活化的ALDH3A1對GOSD星芒狀細胞內ROS的抑制與NADPH或GSH無關，對脂質酸敗之程度亦不具調控性。已知ZC008具有抗氧化、抗發炎及抗中風的潛能，論文中因此進一步評估ZC008是否能透過提升缺血星芒狀細胞活性，提升抗氧化能力進而保護細胞。結果發現，ZC008可進一步提升GOSD星芒狀細胞中ALDH3A1之蛋白與活性表現，進而提升GOSD星芒狀細胞之存活率。雖然外加ZC008可提升GOSD細胞內ROS及抑制胞外ROS的含量，但DEAB測試的結果發現ZC008提升之ALDH3A1明顯具有降低胞內及胞外ROS含量的功效。雖然ZC008可顯著提升GOSD星芒狀細胞中NADPH及GSH的含量，但僅有GSH的提升和ALDH3A1有關，表示ZC008提升之ALDH3A1可藉由提升GSH之含量降低細胞之氧化壓力。另外，亦驗證GOSD對星芒狀細胞脂質酸敗的抑制作用不受ZC008及ALDH3A1之調控。整體而言，本論文首次在星芒狀細胞中中發現 GOSD及ZC008可藉由提升ALDH3A1的蛋白表現及活性，進而強化細胞之抗氧化能力對抗缺血導致的氧化壓力。ZC008活化的ALDH3A1，可能透過提升GSH來抑制GOSD星芒狀細胞內、外之ROS濃度。此研究除初步釐清ZC008之抗氧化機轉外，更進一步確認其在腦中風治療上的潛在價值。

Cerebral ischemia can lead to severe brain damage or even death of the patients. Ischemia/reperfusion (I/R)-induced reactive oxygen species (ROS) and brain''s low antioxidant activity all contribute to the increased oxidative stress in ischemic brain that consequently can lead to severe brain injury. How to down regulate the oxidative stress in ischemic brain has continuously been a hot issue in the control of cerebral ischemia. It is known that brain astrocytes have the highest anti-oxidative activity compared to other brain cell types and ALDH3A1 (aldehyde dehydrogenase 3A1 ) in mammalian corneal epithelium plays critical and multifaceted roles in protecting the corneal from oxidative stress. The primary goals of this thesis study were to know whether ALDH3A1 also plays similar roles in protecting astrocytes from ischemia-increased oxidative stress and could the Chinese herb extract from Graptopetalum paraguayense (GP) leafs, ZC008, up-regulate ALDH3A1 activity to promote the protection of ischemic astrocytes against oxidative stress. In the study, the trypan blue dye exclusion assay, Western blotting, zymography assay, DCF (dichlorofluorescein) assay, OPA (o-phthaldialdehyde) assay, GR (glutathione reductase) assay and TBARS (TBA-reactive substances) assay, were respectively used to determine the survival, protein expression and activity of ALDH3A1, intracellular and extracellular level of ROS, the amount of intracellular NADPH, GSH and MDA of the ischemic astrocytes (under the glucose, oxygen and serum deprivation or GOSD condition) , in the absence or presence of DEAB (diethylaminobenzaldehyde; ALDH3A1 inhibitor) or ZC008. The results showed that under the GOSD (6 h) stress, astrocytes have the highest survival rate and ALDH3A1 activity and lowest amount of intracellular and extracellular ROS, compared to microglia and neurons, indicating GOSD-treated astrocytes indeed have the highest anti-oxidative potential among the three brain cell types. When DEAB was added into the GOSD-treated astrocytes, the protein expression level and activity of ALDH3A1and the survival rate of astrocytes were all significantly decreased whereas the intracellular ROS was increased, indicating GOSD-triggered ALDH3A1 is critical for the protection of astrocytes against GOSD-induced oxidative stress. It is known that ALDH3A1 can elevate NADPH and GSH to down regulate ROS level and oxidative rancidity of fat to protect corneal epithelium from oxidative stress. Whether ALDH3A1can act the same in GOSD-treated astrocytes was also investigated. The results showed that intracellular levels of NADPH, GSH and MDA (malondialdehyde; the product of oxidative rancidity of fat) were all significantly decreased in GOSD-treated astrocytes, indicating GOSD-increased oxidative stress is likely due to the reduction of NADPH and GSH but did not cause any oxidative rancidity of fat. DEAB blocking assay further demonstrated that GOSD-triggered ALDH3A1 did not play a part in up-regulating the amount of NADPH and GSH nor in oxidative rancidity of fat in GOSD astrocytes, suggesting that ALDH3A1-decreased intracellular ROS was NADPH and GSH independent. Results from our early study have demonstrated that ZC008 can protect brain from ischemic injury. The underlying mechanisms responsible for ZC008-mediated brain protein however, remain still unknown. The impacts of ZC008 upon ALDH3A1 and the protection of GOSD-treated astrocytes against oxidative stress were therefore examined. The results showed that ZC008 (at medium dose) can further increase the protein expression and activity of ALDH3A1, intracellular ROS (not cytotoxic), NADPH, GSH and the survival rate of GOSD astrocytes but significantly decreased the extracellular ROS level and showed no influence on MDA. ZC008-activated ALDH3A1 appeared to play critical roles in up-regulating GSH (not NADPH) and down regulating intracellular and extracellular ROS to protect astrocytes from GOSD-induced oxidative stress. Neither ZC008 nor ALDH3A1 involved in the oxidative rancidity of fat in GOSD-treated astrocytes. In overall, the role of ALDH3A1 in self- and ZC008-mediated protection of astrocytes against ischemia (GOSD)-induced oxidative stress has been clarified for the first time in an in vitro ischemic model. ZC008-activated ALDH3A1 can effectively down regulate the intracellular and extracellular ROS, in a GSH-dependent manner, to protect astrocytes against GOSD-induced oxidative stress. Through the study, the therapeutic value of ZC008 in the control of cerebral ischemia can be further confirmed mechanistically.

壹、前言………………………………………………………………………………………………………………….1
一、腦中風…………………………………………………………………………1
二、缺血性中風的傷害機轉………………………………………………………1
三、星芒狀細胞……………………………………………………………………2
四、缺血對星芒狀細胞造成的傷害………………………………………………2
五、星芒狀細胞對缺血腦組織的保護……………………………………………2
六、活性氧化物質(ROS)之種類及生成………………………………………….3
七、缺血在星芒狀細胞中造成的氧化壓力及傷害………………………………3
八、缺血星芒狀細胞的抗氧化能力………………………………………………4
九、乙醛脫氫酶(ALDH)…………………………………………………………..5
十、ALDH3A1在不同組織中之功能…………………………………………….5
十一、ALDH3A1的抗氧化機轉………………………………………………….6
十二、脂質過氧化或酸敗(rancidity)……………………………………………...6
十三、ALDH3A1和缺血性中風的相關性……………………………………….7
十四、ZC008的抗氧化活性………………………………………………………7
十五、研究目標……………………………………………………………………8
貳、實驗材料與方法………………………………………………………………………………………………9
一、實驗材料………………………………………………………………………9
二、動物來源及繁殖………………………………………………………………9
三、初代星芒狀細胞及微膠細胞之培養………………………………………..10
四、神經細胞之培養……………………………………………………………..10
五、體外缺血處理(GOSD，glucose- oxygen- serum- deprivation)……………..11
六、藥物處理……………………………………………………………………..11
七、細胞存活率之分析（Trypan Blue dye Exclusion Assay）…………………11
八、蛋白質萃取與定量…………………………………………………………..12
九、蛋白質表現量分析（西方墨點法，Western blotting）…………………...12
十、ALDH3A1活性測式（Zymography Assay）………………………………13
十一、ROS分析（DCFH assay）……………………………………………….13
十二、GSH測驗 (OPA assay)…………………………………………………...14
十三、NADPH測驗 (Gluathione reductase assay)……………………………...15
十四、脂質酸敗測試（TBARS assay）………………………………………...15
十五、統計分析…………………………………………………………………..15
參、結果………………………………………………………………………………………………………………..16
一、在GOSD壓力下，星芒狀細胞具有較高之存活率……………………….16
二、在GOSD壓力下，星芒狀細胞具有較佳清除ROS的能力，釋放的ROS最少……………………………………………………………………………….16
三、在GOSD壓力下，星芒狀細胞的ALDH3A1蛋白表現及活性均顯著提升…………………………………………………………………………………..16
四、ALDH3A1具有抑制胞內ROS聚集但促進ROS外排的功能，進而保護星芒狀細胞在GOSD壓力下之存活。………………………………………….17
五、ALDH3A1對GOSD星芒狀細胞胞內ROS的抑制和NADPH或GSH無關…………………………………………………………………………………..18
六、GOSD提升之ALDH3A1和星芒狀細胞中脂質酸敗的被抑制無關……..18
七、ZC008可進一步提升GOSD星芒狀細胞中ALDH3A1之活性及蛋白表現…………………………………………………………………………………..18
八、ZC008可藉由提升ALDH3A1保護GOSD星芒狀細胞之存活………….19
九、ZC008可藉由提升ALDH3A1抑制GOSD星芒狀細胞ROS之釋放…...19
十、ZC008提升之ALDH3A1可藉由提升GOSD星芒狀細胞中GSH之含量來降低細胞之氧化壓力…………………………………………………………..20
十一、ZC008及ALDH3A1不參與調控GOSD對星芒狀細胞中脂質酸敗的抑制…………………………………………………………………………………..20
肆、討論………………………………………………………………………………………………………………..21
一、GOSD星芒狀細胞具有較高的ALDH3A1活性反應，藉此可提升其抗氧化能力……………………………………………………………………………..21
二、首次發現ZC008可提升GOSD星芒狀細胞中ALDH3A1的活性保護細胞之存活……………………………………………………………………………..21
三、ALDH3A1如何抑制GOSD星芒狀細胞胞內之ROS目前尚不清楚……22
四、ALDH3A1可能透過提升ROS（H2O2）的外滲性增加GOSD星芒狀細胞胞外ROS的含量，進而對周圍其它腦細胞造成氧化性傷害…………………22
五、ALDH3A1對胞內脂質的氧化酸敗不具調控性…………………………...23
六、ZC008可藉由ALDH3A1提升GOSD星芒狀細胞內之GSH，並抑制胞內及胞外ROS的含量進而保護細胞之存活………………………………………24
七、ZC008提升的胞內ROS，可能對GOSD星芒狀細胞不具傷害性………24
八、總結…………………………………………………………………………..25
伍、中文參考文獻…………………………………………………………………..42
陸、英文參考文獻…………………………………………………………………………………………………42

中文參考文獻
王偉迪(2010) 石蓮花粉劑對缺血腦組織保護機轉之探討 國立國立中興大學碩士論文

行政院衛生署(2015)中華民國一零三年國人死因統計結果
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