利用基因晶片研究數種冬蟲夏草水萃液處理KG-1細胞之基因表現圖譜與標記基因

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研究生：	謝毓玲 Yu-Ling Hsieh
論文名稱：	利用基因晶片研究數種冬蟲夏草水萃液處理KG-1細胞之基因表現圖譜與標記基因 Using cDNA Microarray Technology to Analyze the Global Gene Expression Profiles and Select Marker Genes of KG-1 Cells Treated with Various Cordyceps sinensis Extracts.
指導教授：	葉世榮 S. R. Yeh 許志楧 Ian C. Hsu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2004
畢業學年度：	92
語文別：	中文
論文頁數：	77
中文關鍵詞：	基因晶片、中草藥、冬蟲夏草
外文關鍵詞：	microarray, Cordyceps sinensis
相關次數：	點閱：3 下載：0
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冬蟲夏草是一種菌蟲複合體的珍貴藥材，根據研究報告指出冬蟲夏草具有多種生物學上及藥理學上的作用，包括對肝、腎、心血管、內分泌系統等皆有活性，另有抗氧化、抗癌及免疫調節功能。【本草綱目拾遺】及【本草備要】兩書記載「夏草冬蟲乃感陰陽二氣而生，夏至一陰生，故靜而為草，冬至一陽生，故動而為蟲，以其得陰陽之氣全也，故能治諸虛百損。」『冬蟲夏草，甘平，保肺益腎，止血化痰，己勞嗽。四川嘉定府所產者最佳，雲南、貴州所產者次之。』古籍認為冬蟲夏草的子實體（草）與菌核（蟲）兩部位可能不同且具不同療效；另外產地似乎也是影響冬蟲夏草藥效的因素。
不同產地及不同部位的冬蟲夏草是否具有不同的生理及藥理功能？由於冬蟲夏草成分複雜，以一般傳統實驗方法無法對此中藥的功效及可能藥理機制進行全面性的了解。然而基因晶片可以一次觀察到數千個基因的表現，為一種可以全面性、大量的、且具平行處理能力的分子生物學研究新技術。因此，利用基因晶片研究冬蟲夏草影響模式細胞株的基因表現圖譜，可以全面性的評估不同產地或不同部位的冬蟲夏草之間功能性的異同。為了確認冬蟲夏草樣本的菌種以及評估樣本間品質的均一性，本研究進行了菌種基源鑑定、有機及無機化學圖譜鑑定等。本研究以急性骨髓性白血病細胞株－KG-1當作實驗的模式細胞株，利用冬蟲夏草對KG-1細胞株的生長效應做為生物指標篩選出適當的萃取液濃度，並依據基因晶片實驗結果挑選出最佳的實驗時間點。主要的基因晶片實驗首先經過統計學的迴圈設計，其結果也是經由嚴格的篩選以及統計學的運算，才篩選出初步的冬蟲夏草標記基因。此基因晶片的結果尚需經由Real-Time PCR的應證，才會成為確定的冬蟲夏草標記基因。利用本研究模式我們已經初步篩選出冬蟲夏草的標記基因，據此結果我們將能評估不同產地或不同部位的冬蟲夏草之間功能性的異同，並且進一步探討冬蟲夏草可能的藥理機制。

Cordyceps, a highly valued traditional Chinese medicine, comprising the caterpillar larvae invaded by the parasitic fungus Cordyceps sinensis. Previous studies have demonstrated that Cordyceps exhibits a broad variety of biological and pharmacological effects towards immune, cardiovascular, hepatic, and renal systems. It has been noted in a famous Chinese medical literature Ben-Cao-Gang-Mu-Shi-Yi (Supplements to Compendium of Materia Medica) written by Zhao Xue-Min in 1765 that the growing courses of fruiting body and larvae’s body are influenced by Yin and Yang, respectively. Thus, it is believed that Cordyceps tonifies Yin and Yang only when both the fruiting body and larvae’s body are consumed altogether. Cordyceps is found in Tibet, Qinghai, Si-chuan and Yun-nan provinces of China, where the altitude is between 3500 to 5000 meters. However, relatively little information is available about the biological and pharmacological resemblance between fruiting body and larvae’s body of Cordyceps as well as Cordyceps of different geographical origin.
The constituents of Cordyceps are very complex. It is difficult to comprehensively analyze the possible pharmacological functions using traditional experimental techniques. However, the advent of DNA microarray technology allows researchers to monitor the expression levels of thousands of genes simultaneously. Thus, we would like to apply microarray technology to explore whether it is possible to distinguish the differences between aboveground and underground parts of Cordyceps as well as Cordyceps samples of distinct geological origins. In order to assure the authentication of Cordyceps sinensis, DNA sequence analysis of 18S rRNA gene was performed. In addition, HPLC and ICPMS analysis were performed to reveal the chemical fingerprints of various Cordyceps samples. A human acute myelogenous leukemia cell line KG-1 was used as the experimental cell model. Biological experiment and microarray experiment were performed to determine proper treating period as well as Cordyceps extract concentration. Statistics has played an important role on the experimental design and analysis of microarray data. A set of “Cordyceps marker genes” was identified as significantly influenced by Cordyceps extracts based on statistical methods. These preliminary results will be confirmed by Real-Time PCR assay. According to these results, we will be able to further evaluate the functional distinctions between different parts of Cordyceps and Cordyceps of diverse geological origins. Furthermore, possible pharmacological mechanisms underlying the gene expression data could also be examined in the future.

中文摘要……………………………………………………………………....…I
英文摘要……………………………………………………………………...…II
誌謝......................................................................................................................III
目錄......................................................................................................................IV
圖表目錄.............................................................................................................IX
第一章 序論
1.1 研究動機........................................................................................................1
1.2 研究目的........................................................................................................2
1.3 研究流程........................................................................................................2
第二章 原理
2.1 基因晶片………………………………………………………….......….....3
2.1.1 基因晶片種類…………………………………………………................ 3
2.1.2 基因晶片原理………………………………………………..…...............4
2.1.3 基本實驗流程………………………………………………….................5
2.2 基因晶片應用於中草藥之研究…………………………………...........…11
2.3 冬蟲夏草……………………………………………………........………...12
2.3.1 生活史………………………………………………………................…12
2.3.2 藥效機制……………………………...……………………...............…..14
2.3.3 菌種鑑定………………………………………………............................14
2.4  KG-1 細胞株…………………………………….…………..…........…...15
第三章 實驗步驟與設計
3.1 實驗設計………………………………………......……………………....17
3.2 冬蟲夏草樣品品質管制............................................................................. 18
3.2.1菌種基源分析-18S rRNA序列比對.........................................................18
3.2.2無機化合物化學圖譜鑑定-感應偶合電漿質譜儀...................................18
3.2.3  有機化合物化學圖譜鑑定-HPLC..........................................................20
3.3 生物活性指標篩選......................................................................................20
3.3.1 冬蟲夏草水萃液之備製...........................................................................20
3.3.2 細胞增殖曲線...........................................................................................21
3.3.3  Mock實驗...............................................................................................21
3.3.4 時間點決定實驗.......................................................................................22
3.4 主要晶片實驗與迴圈設計..........................................................................22
3.5 資料處理與統計分析..................................................................................24
3.5.1 以廻圈設計估計基因表現差異...............................................................24
3.5.2  F-檢定......................................................................................................28

第四章 結果
Part I. 冬蟲夏草的品質管制............................................................................31
4.1 冬蟲夏草菌種分析-18S rRNA序列比對結果...........................................31
4.2無機化合物化學圖譜鑑定-感應偶合電漿質譜儀結果..............................31
4.3有機化合物化學圖譜鑑定-HPLC圖譜比較結果.......................................33
4.3.1 青海冬蟲夏草不同部位水萃液之HPLC圖譜.......................................33
4.3.2  西藏冬蟲夏草不同部位水萃液之HPLC圖譜......................................34
Part II. 冬蟲夏草的生物活性指標...................................................................36
4.4 冬蟲夏草水萃液處理之細胞增殖曲線......................................................36
4.4.1 青海菌核（QW）水萃液對KG-1細胞之增殖抑制率..........................36
4.4.2 青海子座（QFB）水萃液對KG-1細胞之增殖抑制率..........................37
4.4.3 青海全蟲（QCS）水萃液對KG-1細胞之增殖抑制率.........................37
4.4.4 西藏菌核（TW）水萃液對KG-1細胞之增殖抑制率............................38
4.4.5 西藏子座（TFB）水萃液對KG-1細胞之增殖抑制率...........................39
4.4.6 西藏全蟲（TCS）水萃液對KG-1細胞之增殖抑制率...........................39
4.4.7 不同產地及部位蟲草水萃液之細胞增殖抑制率...................................40
4.5 決定時間點實驗..........................................................................................41
4.5.1  Mock實驗...............................................................................................41
4.5.2  表現差異基因之挑選.............................................................................42
4.5.3  叢集分析.................................................................................................43
Part III. 篩選冬蟲夏草的標記基因.................................................................44
4.6 主要基因微陣列實驗..................................................................................44
4.6.1 細胞增殖抑制率.......................................................................................44
4.6.2 樣本RNA 品質鑑定結果........................................................................45
4.6.3 各片晶片實驗之原始圖檔.......................................................................46
4.6.4 以外加指示控制基因評估各片晶片實驗...............................................46
4.6.5 選點標準與歸一化處理...........................................................................48
4.6.6 矩陣運算後之結果...................................................................................52
4.7 可靠基因群與冬蟲夏草標記基因群之選取..............................................53
4.7.1 選取可靠基因群.......................................................................................53
4.7.2 選取冬蟲夏草標記基因群.......................................................................53
4.8 叢集分析結果..............................................................................................58
第五章 討論
5.1 品質管制……………………………………………...…………………...59
5.2 生物活性指標..............................................................................................60
5.3 篩選冬蟲夏草標記基因..............................................................................63
5.3.1 實驗設計與實驗品質控管.......................................................................63
5.3.2 冬蟲夏草樣本間之差異程度...................................................................65
5.4 冬蟲夏草數種功能相關基因群..................................................................67
5.4.1 免疫/發炎反應相關基因群......................................................................68
5.4.1.1  CD69基因表現上調............................................................................68
5.4.1.2  IL1-β基因表現下調............................................................................68
5.4.1.3  STAT1基因表現上調，STAT5A基因表現下調...............................69
5.4.2  與抗氧化作用相關基因群.....................................................................69
5.4.3  與細胞內醣類代謝相關基因群.............................................................70
5.4.4  與細胞遷移/附著相關基因群................................................................70
5.5 展望..............................................................................................................71
參考文獻…….………………………………………………………………....72

圖表目錄

圖2.1 cDNA微陣列系統，利用空心針將cDNA點印至表面處理過之玻璃載具上..............3
圖2.2 Affymetrix公司以光蝕刻法製造之寡核苷酸基因晶片...............................................3
圖2.3 中心法則（Central Dogma），敘述 DNA → RNA → 蛋白質的流程，幾乎是所有生物體內生命現象運作的基本機制...............................................................................5
圖2.4 基因微陣列系統基本實驗流程圖，主要包含（a）玻片備製及探針基因點印（Printing）、（b）雜合反應（Hybridization）、（c）玻片影像掃描（Scanning）、（d）影像數據分析（Analys等四部分....................................................................................6
圖2.5 點印用之狹縫針..............................................................................................................7
圖2.6 微陣列點印系統之針座，一次可以同時使用48根針進行點印工作..........................7
圖2.7 基因微陣列點印系統之自動化機械手臂，每一批可以點印出含有超過三萬點之玻共137片...........................................................................................................................7
圖2.8 UV -Crosslinking增強DNA探針與玻片的黏合..............................................................7
圖2.9 非直接標記法原理，以寡腺苷鏈進行第一次雜合反應，再以3 DNA擷取序列的互補序列進行第二次雜合反應...........................................................................................9
圖2.10 Axon 4000B 掃描系統..................................................................................................9
圖2.11 Cy-3、Cy-5螢光染劑之激發光譜...............................................................................10
圖2.12 以GenePix 5.0進行圈點的動作................................................................................10
圖2.13 冬蟲夏草外觀..............................................................................................................13
圖2.14 冬蟲夏草生活史，幼蟲在土壤中避冬感染蟲草真菌大量繁殖稱「冬蟲」，夏初，蟲草菌繼續成長在蟲體頭部長出棍棒狀子實體，人稱「夏草」.........................13
圖2.15 KG-1細胞於顯微鏡下（1000X）.............................................................................16
圖2.16 樹突狀細胞分化圖.....................................................................................................16
圖3.1 實驗流程圖...................................................................................................................18
圖3.2 冬蟲夏草之A.子座（子實體）B.菌核（蟲）...........................................................21
圖3.3 時間點決定實驗設計圖，control為不處理冬蟲夏草，QW為處理青海菌核水萃液之樣本，數字為處理之天數.......................................................................................22
圖3.4 樣本之設計，由產地分為青海與西藏再依部位分為子實體、菌核、全蟲三部分，加上內部控制組與控制組共八個樣本.......................................................................23
圖3.5 主要實驗迴圈設計設計圖，紅色字為樣本代號而藍色數字為片子編號...............23
圖4.1 18S rRNA序列比對結果...............................................................................................31
圖4.2 冬蟲夏草與其菌絲體產品叢集分析後之樣品關連性................................................33
圖4.3 青海冬蟲夏草不同部位水萃液之HPLC圖譜...........................................................34
圖4.4 西藏冬蟲夏草不同部位水萃液之HPLC圖譜...........................................................35
圖 4.5 以不同濃度之青海菌核冬蟲夏草水萃液處理KG-1細胞之細胞增殖抑制率.......36
圖 4.6以不同濃度之青海子座冬蟲夏草水萃液處理KG-1細胞之增殖抑制率................37
圖 4.7以不同濃度之青海全蟲冬蟲夏草水萃液處理KG-1細胞之增殖抑制率.................38
圖 4.8 以不同濃度之西藏菌核冬蟲夏草水萃液處理KG-1細胞之增殖抑制率..............38
圖 4.9 以不同濃度之西藏子座冬蟲夏草水萃液處理KG-1細胞之細胞殘存率..............39
圖 4.10 以不同濃度之西藏全蟲冬蟲夏草水萃液處理KG-1細胞之增殖抑制率............40
圖4.11 不同產地與部位冬蟲夏草處理KG-1細胞之增殖抑制率折線圖.........................40
圖4.12 Mock實驗之MA圖，A值小於2，表示系統誤差甚小而系統靈敏度高...........42
圖4.13 時間點實驗叢集分析結果，第二天與四、八天基因表現圖譜有明顯的不同... 44
圖4.14 細胞殘存率比較圖....................................................................................................45
圖4.15處理蟲草水萃液四天細胞抽出之RNA之電泳圖與5S、18S及28S rRNA含量圖................................................................................................................................46
圖4.16為原始影像檔.............................................................................................................46
圖4.17 良好之指示控制基因在M-A圖分佈情形.................................................................48
圖4.18 不良之指示控制基因在M-A圖分佈情形..............................................................48
圖4.19數據分析之流程.........................................................................................................48
圖4.20為16片玻片之M-A圖，由圖可知皆為良好之實驗結果.....................................49
圖4.21 增加批次因子之σ分佈圖，A為一個γ所算出之σ分佈圖，B為五個γ所算出之σ分佈圖.................................................................................................................51
圖4.22 各樣品之基因表現差異，控制組內誤差λ3-λ4之分佈遠小於其他樣本對控制組之分佈.........................................................................................................................53
圖4.2 叢集分析結果...............................................................................................................58
圖5.1 為以青海冬蟲夏草水萃液20μl/ml處理細胞之結果.................................................62
圖5.2冬蟲夏草標記基因進行叢集分析之後這七個樣本的基因表現相關性.....................67

表4.1 冬蟲夏草與其菌絲體產品之微量無機物比例表.......................................................32
表4.21 各時間點顯著差異基因數..........................................................................................43
表4.3 冬蟲夏草標記基因群....................................................................................................54

                                

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