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研究生:許世典
研究生(外文):Shih-Tien Hsu
論文名稱:以基因晶片初探黃連對人體單核淋巴球免疫功能之影響
論文名稱(外文):Microarray analysis of immune genes regulation by Huang-Lian in human blood mononucleocytes
指導教授:陳光偉陳光偉引用關係
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:中西醫結合研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:80
中文關鍵詞:黃連基因晶片免疫
外文關鍵詞:Huang-Lianmicroarrayimmune
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目前醫學的發展趨勢,已由中醫、西醫兩系統分流並行,朝向中西醫結合的目標努力。以往中草藥中西結合研究,受限於舊有實驗方法,常就中藥中的單一成分探討機轉,易流於以偏概全。其次,實驗室操作的細胞、動物實驗結果,也常與中藥臨床所見脫節。如何結合現代醫學技術,重新審視中藥作用於人體的藥理機轉,成為現代中西醫結合的一大難題。
基因晶片,是近年來生命科學研究的一大利器。它可經由一次實驗,得到生物體所有基因表現量的數據。以基因晶片分析中醫複雜的證型變化或藥物組成,不但免除以偏概全的顧慮,更可以嘗試描繪出證型或藥物的基因圖譜,作為中西醫的交流與整合的橋樑。
本次實驗,藉由兩名健康成年男性服食黃連一週前後,抽取血液單核淋巴球中的RNA,利用Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array觀察黃連影響基因變化,探討黃連作用於人體的藥理機轉。黃連共同影響兩人的基因探針數目,表現上調達2倍的共879個,下調達2倍則為987個。經由分析黃連影響的基因,發現類Toll接受器、腫瘤壞死因子( TNF)、IL1、化學趨化激素、白血球Fc接受器的表現明顯下調,可能為黃連調整人體免疫功能、抑制人體發炎反應的機轉。對照濕熱下利、泄瀉、胃火牙痛、肝火脅痛、神昏譫語、癰腫瘡毒、耳目腫痛等黃連臨床療效,恰可由基因層次來驗證,提供一個中西醫結合研究的良好範例。
Background
Tranditional Chinese medicine (TCM) has been clinically extensively practiced in Chinese throughout thousands of years. Till now, the diagnostic and therapeutic concepts like disease patterns were not identical to those of modern western medicine. For further establishing a method to differ clinical disease patterns and understanding the mechanism of treatment, we try to elucidate the gene expression by microarray techonology.
In this study, we used microarray chip to compare with the changes in gene expression after treatment with Huang-Lian (the root of Coptis chinensis FRANCH) and differ the significant gene expression change about its immune function..

Material and methods
Examiners: 2 healthy males, no underling medical disorder
RNA-extraction: Fifty milliliters of peripheral blood was collected in EDTA-treated tubes. Mononucleocyte was isolated with Ficoll-paque Plus (Amersham biosciences). Then total RNA was extracted with the use of TRIzol reagent (Invitrogen Life Technologies). RNA quality was checked by UV spectrophotometer and Agilent 2100 bioanalyzer.
cRNA synthesis, labeling, hybridization with microaarray and expression profiling: We synthesized double strained cDNA from total RNA. Then Biotin-labeled cRNA was then synthesized from cDNA. cRNA was purified and fragmented into 30- to 200-base through the use of metal-induced hydrolysis. Then the biotin-labeled cRNA was hybridized onto Human genome U133 Plus 2.0 array( Affymetrix). Then the GeneChips were washed and stained in accordance with the intructions set forth in the Affymetrix GeneChip expression analysis manual. Finally, the array fluorescence singal was detected by Gene Array Scanner.
Drug treatment: After getting the pre-medication data, both the examiner should take Huang-Lian 6gm per day for 7 days, then collect their blood sample again and check the gene expression profile by microarray.
Statistical analysis: First, the mean background noise level is estimated and the intensity for each probe is adjusted to remove this. Next, probe-level data from the entire chipset are simultaneously normalized. Third, the normalized, backround-collected data is transformed to the log2 scale.

Results
Finally, we selected genes with arbitrary fold changes greater than 2 between post-treatment and before-treatment and both present in two cases for further discussion. The numbers of 2 fold up-regulated genes and down-regulated genes were 879 and 987.
In this pilot study, we try to elucidate the gene expression of the human peripheral blood mononucleocytes under the treatment by Huang-Lian and identify these differentially expressed genes with potential molecular targets for therapeutic options. Multiple group genes like Toll-like receptor, Fc receptor and chemokine production were all reduced and agreeable to the tranditional theropeutidal function of Huang-Lian. Further studies to confirm this results and find the detail pathway are needed.
目錄 i
圖目錄 ii
表目錄 iii
中文摘要 1
第一章 前言 1
第二章 文獻探討 3
第三章 材料與方法 22
第四章 結果 30
第五章 討論 63
第六章 結論 72
參考文獻 73
英文摘要 77
附件 79
謝辭 80
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