主題一： 探討人類血管收縮素轉換酶基因中-Alu-序列在神經細胞中對於血管收縮素轉換酶基因的功能調控 主題二： 探討護憶能對神經細胞的保護機制 - 建立天然藥物及健康食品對神經退化性疾病保護機制的分子分析平台

主題一： Functional regulation of Alu element of ACE gene in neuron cells 主題二： The neuroprotection of Hu Yi-Neng in neuron- molecular analysis platform of natural products and diet supplements for their protective effects on neurodegenerative diseases

蔡美蘭(Mei-Lan Tsai)

指導教授 ： 吳世忠

高雄醫學大學/健康科學院/醫學影像暨放射科學系碩士班/碩士(2012年)

摘要

主題一：
阿茲海默症為失智症中最常見的疾病，其病理特徵為神經纖維糾結、老年斑塊和神經細胞死亡。近年來，在阿茲海默症的遺傳因子的研究中指出，ACE 插入/刪除基因型的多形性和阿茲海默症之間具有高度的相關性。然而對於ACE基因第 16 個intron的插入序列—也就是所謂的 Alu 序列—是否具有生理功能，仍不清楚。因此我們建構含有ACE基因啟動子(promoter)片段的 p-ACEpro(f)-SEAP-Bas 載體。再分別將插入型序列及刪除型序列的片段接到 p-ACEpro(f)-SEAP-Bas 載體中啟動子序列的前面，以探討ACE基因intron 16中的Alu 序列是否有調控ACE啟動子轉錄的功能。以同步轉染和冷光酵素分析方法，分析轉染至神經母細胞瘤 SH-SY5Y 細胞中，各個建構載體的轉錄活性。結果顯示，含有插入型的正向 ACE 基因啟動子比含有刪除型的正向 ACE 基因啟動子，增加約 70 % 的轉錄活性。本研究是第一個發現，人類血管收縮素轉換酶基因第 16 個內含子的 Alu 序列，具有調控血管收縮素轉換酶啟動子轉錄活性的功能。本研究可以提供連結阿茲海默症病人中血管收縮素轉換酶的含量和血管收縮素轉換酶插入/刪除基因多型性與阿茲海默症之間的相關性。

主題二：
在工業發達與醫療進步的社會，人口老化而平均壽命也隨之增加，神經退化性疾病也有增加的趨勢，進而造成社會資源的負擔。雖有許多藥物開發使用於治療阿茲海默症，但是到目前為止，並沒有一個治療方法可以有效的治療或延緩阿茲海默症的症狀。而對於該疾病的致病機轉也仍有許多問題有待釐清。在藥物開發方面，有越來越多的研究指出天然藥物或營養補給品可以改善記憶、認知功能和防止神經退化。透過與祥和生物科技股份公司產學合作，提供一個複合配方的保健食品-護憶能，藉由探討其對於神經細胞的保護機制，建立一個藥物分子分析平台。我們分別使用 PrestoBlueTM 試劑和 DCFH-DA 染劑偵測過氧化氫刺激造成的細胞存活率下降和細胞內活性氧化物上升，檢測護憶能對過氧化氫誘導神經細胞受損的保護機制。結果顯示，護憶能可以減少過氧化氫誘導的細胞死亡和細胞內活性氧化物的含量，以及增加過氧化氫誘導的抗氧化基因-第一型血基質氧化酶的表現量。過氧化氫刺激會增加磷酸化 ERK1/2 和 Akt 的表現量。而預處理護憶能後予以過氧化氫刺激，則會使得過氧化氫誘導的磷酸化 ERK1/2 表現量增加，但是對於過氧化氫誘導的磷酸化 Akt 表現量則是沒有影響。這些結果指出，護憶能可能透過不同的訊息傳遞路徑，增加HO-1基因的表現對抗氧化壓力，達到保護神經細胞的效果。我們建立的藥物分子分析平台，未來可以用於檢測天然藥物或是營養保健食品對於神經退化性疾病的保護作用。

關鍵字

阿茲海默症 ； 血管收縮素轉換酶神經保護效果

並列摘要

主題一：
Alzheimer's disease (AD) is the most common form of degenerated dementia with the pathological findings of neurofibrillary tangle, senile plaque, and neuronal loss. For more than a decade, ACE insertion/deletion (I/D) polymorphism has been implicated to be associated with AD. However, whether the insertion sequence, Alu element, in the intron 16 of human ACE gene plays functional role remains to be defined. To examine the regulation of the Alu element on the ACE promoter activity, the pACEpro(f)-SEAP-Bas vector was constructed by fusing human ACE promoter region into pSEAP-Basic vector, andthe I- and D-form fragment in the intron 16 were inserted before the ACE promoter sequence of the pACEpro(f)-SEAP-Bas vector. Transient transfection and luciferase reporter assay were applied to assay the transcriptional activity of each constructed reporter in SH-SY5Y cells. Our results show that the transcriptional activity of the reporter vector with I form fragment increases ~70% than of the vector with D form fragment. The present study first indicates that the Alu sequence in human ACE intron 16 shows regulatory function on the ACE promoter activity. Our findings mayprovide evidence to bridge the gap between the protein level of ACE associated with AD patients and the ACE polymorphism (I/D) associated with AD.

主題二：
The growing aged population in industrialized countries has led to an increased prevalence of late-onset neurodegenerative disorders, which imposes an enormous financial and social burden on health-care systems and society as a whole. Though a number of drugs have been approved for the treatment of AD, there is currently no effective treatment to halt the progression or prevent the onset of AD. More and more studies suggest that natural products or diet supplements may potentially hinder neurodegeneration, and improve memory and cognitive function. By industry-university cooperation, we aimed to study a comprehensive capture of diet supplements- Hu-Yi-Neng provided by HSIANG HO Biotechnology Company Limited and to estabolish a molecular analysis platform for examining the protective effects of natural products and diet supplements on neurodegenerative diseases. H2O2-induced neuronal toxicity was characterized in SH-SY5Y human neuroblastoma cells by the decrease of cell viability using PrestoBlue™ assay and by the increase of intracellular reactive oxygen species (ROS) level using DCFH-DA (2’, 7’-dichlorodihydrofluorescin diacetate) assays. The increased cell damage and intracellular ROS accumulation induced by H2O2 were reversed by treatment of Hu-Yi-Neng. We found that the mRNA expression of HO-1 gene was increased in SY5Y exposing to H2O2. Interestingly, the expression induced by H2O2 was further enhanced when the cells pretreated with Hu-Yi-Neng. We also observed that phosphorylation of ERK1/2 and Akt were increased by H2O2 stimulation. When cell pretreated with Hu-Yi-Neng, the phosphorylation of ERK1/2 by H2O2-induced was higher than only H2O2-induced, but the phosphorylation of Akt by H2O2 -induced was no effect. The data suggest that neuroprotective effect of Hu-Yi-Neng though against H2O2-induced cell damage or enhance H2O2-induced HO-1 gene expression may be due to different pathway. The molecular analysis platform of H2O2-induced cell damage could be use to study the neuroprotection of natural products or diet supplements on neurodegenerative diseases in the future.

並列關鍵字

Alzheimer's disease ； Angiotensin converting enzyme ； Neuroprotective effect

參考文獻

1.Martin, J.B., Molecular basis of the neurodegenerative disorders. N Engl J Med, 1999. 340(25): p. 1970-80.

2.Davie, C.A., A review of Parkinson's disease. Br Med Bull, 2008. 86: p. 109-27.

3.Coppede, F., An overview of DNA repair in amyotrophic lateral sclerosis. ScientificWorldJournal, 2011. 11: p. 1679-91.

4.Bano, D., et al., Neurodegenerative processes in Huntington's disease. Cell Death Dis, 2011. 2: p. e228.

5.Prince, M., R. Bryce, and C. Ferri, The benefits of early diagnosis and intervention, in World Alzheimer Report.