水稻 RE1M 與 RE31D 之染色體 DNA 之選殖與分析

The objectives of the current research are to clone the genomic clones of RE1M and RE31D from 10-day embryos of rice, to analyze these two genes, and to study the regulation of RE31D gene expression. The genomic clones of RE1M and RE31DG were isolated from genomic library of 5-day etiolated shoot of rice selected by plaque hybridization with the cDNA probe of RE1M and RE31D, and named RE1MG and RE31DG, respectively. The RE1MG is 1,853 bp long, and contains a 1,590-bp ORF, a 262-bp 5' untranslated region, and a 334-bp 3' untranslated region, but no intron. The molecular weight of deduced amino acid of RE1MG is 43.7 kDa with 401 amino acids and a pI of 11.34. The identity of RE1MG is not known at this moment. The RE31DG is 3,877 bp long, and contains a 1,775-bp ORF, a 1666-bp 5' untranslated region, a 138-bp 3' untranslated region, and two introns. The molecular weight of deduced amino acid of RE31DG is 60.5 kDa with 541 amino acids and a pI of 11.99. Either the nucleotide sequence or the deduced amino acid sequence of RE31DG is highly homologous with the plant globulin gene. Two chimeric constructs contained 5' promoter region of RE31DG (-367~47 and -890~47) and GUS coding region were transferred to 10-day embryos, mature embryos and calli of rice via gene gun mediated transformation. The results of GUS histochemical staining after 2 days of transformation show that the transient expression of GUS in both constructs were high in 10-day and mature embryos, but low in the calli. The promoter of RE31DG has embryo-specific characters.本研究之目的在於篩選水稻十天胚特有基因，RE1M 與 RE31D 之染色體組 DNA， 並分析RE31D 其啟動子調節。 本實驗以水稻5 天黃化苗之基因庫為材料，分別以RE1M 及RE31D 之cDNA為探針。篩選出RE1M 與RE31D 之染色體組 DNA，分別命名為RE1MG 及RE31DG。所選殖之RE1MG全長為1,853 bp具有1,590 bp之ORF，包含 5' 端上游啟動子有 262 bp， 3' 端非轉譯區共有 334 bp，沒有 intron。RE1MG 可轉譯出 401 個胺基酸，蛋白質分子量為 43.7 kDa，等電點為 11.34 。RE1MG 之 DNA 核酸序列，經由網路基因資料庫比對結果與其他發表的基因之相似性都很低，因此目前對其屬性仍未知。 RE31DG 全長有 3,877 bp，具有 1,775 bp 之 ORF，包含 5' 端上游啟動子 1,666 bp，3' 端下游區域 2,211 bp，有 2 個 intron。RE31DG 可轉譯出541 個胺基酸，蛋白質分子量為 60.5 kDa，等電點為11.99。將RE31DG 之 DNA 核酸列與推演出的胺基酸序列，經由網路基因資料庫比對之結果顯示與水稻的 globulin 基因有很高的相似性。 分別將 RE31DG 基因啟動子之 bp -368~47 (pBIA-4) 與 bp -890~47 (pBIA-9) 片段構築到 GUS 報導基因上，再利用基因槍轉移法分別傳送至台農67號水稻的十天胚、成熟胚與癒傷組織中。兩天後，經過 GUS 染色分析之結果顯示，此二段啟動子調節之 GUS 基因在十天胚與成熟胚的表現很明顯，在癒傷組織表現微弱，試驗結果顯示RE31DG 之啟動子具有胚獨特性之表現特性。目錄1 中文摘要3 英文摘要4 前言5 前人研究6 一、水稻簡介6 二、研究背景7 三、植物中儲藏性蛋白 (storage proteins) 之研究8 四、植物儲藏性蛋白－球蛋白 (globulin) 之研究11 五、水稻 glutelin 基因之研究12 材料與方法15 一、植物的種植與取胚15 二、質體中 cDNA 片斷的回收15 三、探針的合成16 四、單股 DNA 的抽取17 五、核酸定序18 六、序列分析19 七、質體的萃取方法19 八、染色體組 DNA 南方墨點分析20 九、染色體組基因庫的篩選20 十、噬菌體 Lambda DNA 的萃取方法22 十一、基因槍法轉殖23 十二、GUS 基因活性之分析23 十二、引子設計24 結果25 一、RE1M染色體基因組 DNA之篩選、核酸序列定序 與分析25 二、RE31D染色體基因組 DNA之篩選、核酸序列定序與分析26 三、RE31D基因啟動子之部份缺失構築與分析30 討論70 參考文獻7

The Neuroprotective Mechanism by Sesame Antioxidants

中文摘要 腦血管病變目前佔臺灣死亡率第二，即使腦中風病變輕微，也使病患及家屬經濟上及精神上產生極大的負擔。中風主要的病因是因血管栓塞造成腦組織缺血, 使局部神經元因缺血而缺氧以至死亡。目前我們已經了解部份中樞神經損傷死亡的病理機轉是腦缺血/缺氧後，神經元的能源供應系統受到干擾，並且產生自由基及一氧化氮導致細胞二度的傷害。最近幾年的研究更發現氧化產生的自由基也是重要的細胞死亡原因，也發現了許多抗氧化基因及反應機制。芝麻自古用於營養滋補具多種療效，由於我們對芝麻種子的蛋白質基因有一系列的研究成果，對其成分已經很瞭解，但對其抗氧化物的保護細胞各種作用仍然未明，因此我們將純化芝麻的各種抗氧化物成分，來探討其保護作用。 近年來的研究神經膠細胞包括星狀膠細胞及微膠細胞是神經系統不可缺少的非神經元細胞，對腦有多種功能，並能釋放多種細胞間素（介白質）負責修補受傷害的神經元及其軸突，幫助組織的復原，這種反應目前稱為神經免疫反應，但其反應經由複雜的細胞內訊息傳遞機制調控，而腦缺血及再灌流的過程亦會刺激星狀膠細胞及微膠細胞產生發炎反應，釋出一氧化氮及前列腺素並產生氧化自由基，反而對神經元造成傷害，目前尚不完全了解這些系統的生化調控，因此本實驗分別利用離體的實驗研究不同種類的神經膠細胞的死亡機制。 微膠細胞是一種類似巨噬細胞，存在於腦中具有保護與修補中樞神經系統功能之細胞，並且在中樞神經系統疾病中對病理機制與神經免疫反應，扮演了重要角色。活化的微膠細胞能釋放自由基物質，而此自由基之釋放可以藉由微膠細胞受到LPS (lipopolysacchariges) 的刺激後，由一連串之訊息傳遞系統所引起之反應造成。 芝麻油含有木質酚，例如芝麻素與芝麻醇素；芝麻油因為含有高劑量的抗氧化物質例如芝麻素，可以讓麻油具有更長之保存時間，因此我們將測試芝麻抗氧化物質對微膠細胞在氧化逆境的反應表現。由於PC12細胞是廣泛被使用的神經模式細胞，因此我們也將使用此PC12細胞，進一部確認芝麻素與芝麻醇素對神經細胞的保護作用；我們將研究在過氧化氫或缺氧缺葡萄糖等逆境下芝麻素與芝麻醇素對PC12細胞與BV-2微膠細胞之作用 MAPKs (mitogen-activated protein kinases) 已經知道是參與調控細胞生存與死亡之蛋白酵素，細胞在缺氧缺葡萄糖逆境下造成之MAPK已知有ERK (extracellular signal-regulated protein kinase)、JNK (c-Jun N-terminal protein kinase)、p38MAPK等蛋白酵素參與其中，因此我們將測試芝麻素與芝麻醇素對這些蛋白激酶之調控作用。當完成了細胞實驗，進一部我們也將進行動物實驗，以探討芝麻抗氧化物對活體實驗是否具有保護神經細胞作用，以印證離體實驗的結果，並希望未來能應用在臨床上。ABSTRACT In Taiwan, death caused by stroke is the second highest among all the causes. Ischemic brain injury produced by stroke is a major cause of human neurological disability. Even miner stroke caused paralysis of the patient and resulted in both finacial and mental stress of the family. The disease is due to neuronal cell death by focal hypoxic insult of the brain tissue. It is known that the insult rapidly decreased energy level of neuron and induced neuronal cell death. Recently, reactive oxygen species (ROS) and nitric oxide (NO) are known important factors for cell injury and antioxidants may protect cells from this attack. Sesame has been use for its nourishment and medicinal effects since ancient time. We have reported and sequenced all major proteins in sesame seeds. However, we know very little about the protective effect of each ingredient. Therefore, we will purify and study the antioxidant ingredients in sesame seed and study its effect in microglia cells. Recent studies revealed that astrocytes and microglia are important cells in the brain and can be activated by pathological condition to induce neuroimmune responses by releasing ROS and cytokines. The cytokine is then mediated through receptors to stimulated growth and repairing the damage neuron. Thus, the neuroimmune responses of glial cells are very important factor for the recovery of a patient suffering from stroke. Nevertheless, over reaction of microglia and astrocytes may release oxidative moleculars such as superoxides and nitric oxide that may cause a second damage of the neuron. Therefore, we want to examine cell damage and cell death mechanisms in brain ischemia and to suggest therapeutic approaches directed at specific injury mechanisms. Microglia, a resident macrophage-like population of brain cells, have been proposed to play a role in host defense and tissue repair in the CNS (central nervous system). However, microglia have also been proposed to play a pathogenetic role in immunologically mediated CNS diseases. Nitric oxide (NO), cytokine, and reactive oxygen species (ROS) released from activated microglia and other glial cells may participate in the neurodegenerative process. The inducible isoform, iNOS, is rapidly transcribed and expressed in microglia and astrocytes after stimulation with bacterial lipopolysaccharide (LPS). LPS binding to the Toll-like receptor 4 (TLR4) activates a complex array of intracellular signaling pathways involving tyrosine kinases, mitogen-activated protein (MAP) kinases, and NF-kB mediated gene expression. Sesame oil contains significant amounts of lignans, such as sesamin (a typical lignan with b-b linkage) and sesamolin (a compound with a phenyl-group with an acetal oxygen bridge). It has been known for a long time that sesame oil is highly resistant to oxidative deterioration compared to other edible oils due to its superior antioxidative activities. Therefore, we were interested in examining the effect of sesame antioxidants, sesamin and sesamolin, on the activation of BV-2 cells under LPS-stimulation and oxidative stress. Since PC12 cells are widely known as neuronal model cells, these results might have wide implications concerning the protective effect of the sesame antioxidants, sesamin and sesamolin, on the CNS. Therefore, in the present study we evaluated the effect of sesamin and sesamolin on preventing hypoxic or H2O2-stressed death of PC12 and BV-2 microglial cells by analyzing LDH release and viability of PC12 and BV-2 microglial cells. In many cell types, numerous extracellular stimuli regulate growth, differentiation and apoptosis through activation of protein kinase cascades. Members of the mitogen activated protein kinases (MAPKs) family have been implicated in regulation of cell survival and cell death under numerous conditions. Hypoxia causes the activation of several MAPKs: extracellular signal-regulated protein kinases (Erk1/2), c-jun NH2-terminal kinase (JNK) and p38 MAP kinase signaling pathways. Therefore, we studied whether the protective effect of sesame antioxidants was mediated through the inhibition of MAP kinase and apoptosis pathways in hypoxic PC12 and BV-2 microglial cells. After studying the protective mechanisms of sesame antioxidants in PC12 and microglia cells following hypoxia, we will test in vivo for the protective effects of sesamin and sesamolin. The evidence of antioxidants suggest their roles for neuroprotection. Since the expression of the iNOS mRNA and NO production are induced by ATP and LPS, dose-dependently in cultured rat microglia. Therefore, we were interested to study the effect of antioxidants on the immune response in BV-2 microglial, neuronal and PC12 cells under hypoxic, or LPS- stimulations. Therefore, our specific aims are: 1. To purify and identify the antioxidant ingredients in sesame seed. 2. The effect of sesame ingredients on hypoxic, or LPS-stimulated BV-2 microglia on ROS, NO production. 3. Study the mechanism of these antioxidative effects on hypoxia-stimulated BV-2 microglial, neuronal and PC12 cells. 4. To test the neuroprotective effects of sesamin and sesamolin in vivo.Chapter 1. Effect of Sesame Antioxidants on LPS-induced NO Production by BV-2 Microglial Cells. Chapter 2. Protective Effects of Sesamin and Sesamolin on BV-2 Microglia Cells under Hypoxia. Chapter 3. Protective Effects of Sesamin and Sesamolin on Hypoxic Neuronal Cells, PC12 Cells, and in vivo test

芝麻素及芝麻醇素用於預防中風及保護神經退化之用途

本發明係關於芝麻素(sesamin)與芝麻醇素(sesamolin)用於製造供預防中風及保護神經退化之醫藥品的用途，及關於包含芝麻素及/或芝麻醇素之醫藥組合物