臺灣博碩士論文加值系統

神經退化性疾病隨著人類年齡層的提高而逐漸備受重視，相關的分子調控機制正逐步的被揭露。有越來越多的證據顯示，白血球的免疫功能在神經退化性疾病中扮演一個重要角色，然而此相關研究在台灣人中是相當稀少的。
本研究利用基因微陣列技術解析正常人、MCI與AD病人的血球基因表現的差異，並利用Q-PCR定量分析與驗證具差異表現的分子。期望發現在神經退化過程中所表現出的特殊分子以利於疾病的早期檢測。
分析基因微陣列晶片的結果顯示，正常人與AD病人在白血球中具有32個有表達差異的基因顯著地分別參與在ABC transporters、Ascorbate and aldarate metabolism、Gly/Ser/Thr metabolism、TGF-beta signaling pathway、ECM-receptor interaction pathway裡(T-test; p<0.05)。進一步分析MCI病人的基因表現顯示，具有8個有表達差異的基因分別參與在Purine metabolism、ABC transporters、Alkaloid biosynthesis、Ketone bodies metabolism pathway裡(ANOVA; p<0.05)。倍數差異的分析則顯示，有5個基因參與在Atrazine degradation、Cytokine-cytokine receptor interaction、Va/Leu/Ile biosynthesis pathway裡。Real-time PCR的結果則證實ACVR1B、GP1BA、GAMT、LAMB2、TNXB和ABCB1等五個基因具有顯著差異，而其中只有LAMB2與ABCB1的結果與基因微陣列晶片一致。總和以上結果，我們認為神經退化的過程可能與人體的代謝及訊息傳遞的過程有關，這個想法提供我們未來在進行神經性疾病的研究的一個新方向。

With the aging population, investigators have begun to put more focus on neurodegenerative disorders. The molecular bases underlying the pathogenesis of neurodegenerative diseases are gradually being discovered. There is increasing evidence showing that the immunological function of leukocytes play a central role in neurodegenerative disorders. However, there has relatively little research in the Taiwanese population.
The aim of this study was to investigate the differential expression of genes in peripheral leukocytes among normal, MCI and AD patients by DNA microarray approaches and validate the isolated gene by Q-PCR techniques, in order to develop a biomarker in neurodegenerative processing.
The microarray analysis result showed that there are 32 gene expression differences between NL and AD, significantly involved in ABC transporters, ascorbate and aldarate metabolism, Gly/Ser/Thr metabolism, the TGF-beta signaling pathway and the ECM-receptor interaction pathway (T-test; p<0.05). Further analyses with MCI data showed that there were 8 genes isolated that participate in purine metabolism, ABC transporters, alkaloid biosynthesis, and the ketone bodies metabolism pathway (ANOVA; p<0.05). Fold-change analysis showed that 5 isolated genes were involved in atrazine degradation, cytokine-cytokine receptor interaction and the Va/Leu/Ile biosynthesis pathway. Q-PCR result demonstrated ACVR1B, GP1BA, GAMT, LAMB2, TNXB and ABCB1 had significant differential expression, but only LAMB2 and ABCB1 were comparable with microarray data. In our opinion, neurodegenerative processing may correlate with metabolism and environmental information processing, such as signaling transduction. This result provides a direction for further psychiatric disorder research.

Abstract 3
中文摘要 4
Introduction 5
I. Alzheimer Disease Overview 5
1.1 History 5
1.2. Clinical Manifestation 6
1.3. Diagnosis 7
1.4. Prevalence 8
1.5. Causes 9
1.5.1. Pathology characteristic 9
1.5.2. Genetics characteristic 9
II. Immunology and neurology 11
2.1. Inflammation in CNS 12
2.2. Leukocytes 14
Purpose 19
Materials and methods 20
Patients 20
Blood sample and total RNA extraction: 20
cDNA synthesis from total RNA: 21
Gene chip: 21
Q-RT-PCR: 22
Results 23
I. Experimental design and gene chip analysis 23
1.2 Case selection and microarray experiment processing 23
1.3 Statistical analyse and in silico functional analysis 24
1.4 Fold-change analysis and in silico functional analysis 25
II. Quantification analysis 25
Discussion 27
I. Global alteration in NL and AD leukocytes gene expression. 27
1.1 The gene expression difference between AD leukocytes and brain. 27
1.2 The alterative gene expression in blood. 29
II. Global alteration in NL, MCI and AD leukocytes gene expression. 31
III. Quantification analysis 32
Appendix 35
Table 1. Samples with high quality RNA were selected to process microarray experiment. 35
Table 2. Selected samples were used in microarray experiment. 36
Table 3. The isolated genes with significant differential expression between normal and AD using GeneSpring software statistics analysis and functional analysis (T-test; P<0.05). 37
Table 4. The isolated genes with significant differential expression among normal, MCI and AD using GeneSpring software ANOVA analysis and functional analysis (P<0.05). 40
Table 5. The isolated genes with 2-fold change significant differential expression among normal, MCI and AD using GeneSpring software fold-change analysis and functional analysis (P<0.05). 41
Table 6. Primer sequences used in Q-PCR. 42
Table 7. The immunological factors of brain and peripheral blood on MCI and AD patients in the past report (until 2007). 44
Figure 1. Agarose gel electrophoresis of total RNA from each microarray sample stained with ethidium bromide. 45
Figure 2. The analysis opinion. 47
Figure 3. Primers specificity and efficiency. 48
Figure 4. Primers specificity and efficiency. 49
Figure 5. Gene expression Result by Q-PCR validation. 50
Figure 6. Serial variance expression genes isolated from ANOVA and 2-fold change analysis. 51
Figure 7. ABCB1 Result by Q-PCR validation (P=0.0274). 52
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