臺灣博碩士論文加值系統

壬基酚（Nonylphenol；NP）為界面活性劑經由微生物分解後的代謝產物，屬於外因性內分泌干擾物質（endocrine disrupting chemicals；EDC），會干擾正常的內分泌表現以及造成生物生理活性的改變。由於NP具有生物累積性可經由食物鏈的傳遞進而累積人體當中並對健康造成影響。為建立一個以基因體訊號為基礎的NP監測系統，本研究使用抑制性扣減雜交法（suppressive subtractive hybridization；SSH）獲得NP處理後具有差異性表現之基因，進行多齒新米蝦（Neocaridina denticulata）作為檢測NP之指標生物的評估。本研究以0.5 mg/L 的NP處理多齒新米蝦一天及七天，經扣減後，NP處理一天得到19個正向扣減的基因和46個負向扣減的基因；NP浸泡七天得到33個正向扣減的基因和34個負向扣減的基因；為探討NP對於生殖的影響，以抱卵的多齒新米蝦及未抱卵的多齒新米蝦進行扣減得到105個正向扣減的基因。
挑選出23個與NP處理的相關基因及7個抱卵相關的基因進行差異性的確認，共有19個基因表現具有差異性，在NP相關基因測量結果顯示NP會促進未知基因NP1-18及壓力相關基因rNP1-10表現量上升；而防禦相關基因NP1-2、轉錄因子NP1-7、發育相關基因NP1-9、代謝相關基因rNP1-8及未知基因NP1-17、rNP1-16、rNP1-17、rNP1-19、rNP1-20、rNP1-21、rNP1-22及rNP1-27表現量則下降；在抱卵相關基因方面，則有5個基因確實會因抱卵而表現量增加。測量差異性表現基因於0.001、0.01及0.5 mg/L之NP處理樣本的靈敏度，結果顯示，14個NP相關的基因中，4個基因於三個濃度NP處理後的表現均有顯著差異，包括壓力相關基因rNP1-10及未知基因rNP1-17、rNP1-20及rNP1-27；代謝相關基因rNP1-8及未知基因rNP1-22二個基因於0.001 和 0.5 mg/L的NP處理時表現有差異；發育相關基因NP1-9則於0.01 和 0.5 mg/L的NP處理時表現有差異；其餘7個NP調控基因只在0.5 mg/L的NP處理組有差異。此外，5個生殖相關基因在不同濃度NP處理的表現皆無差異。統整所有基因在不同濃度NP處理後的表現情形，初步建立一個偵測的NP模式組。

Nonylphenol is a metabolic product from the microbial degradation of detergents. Because of disrupting function of normal endocrine and physiological activity, NP is regarded as an endocrine disrupting chemical（EDC）. NP exerts its effect through the trophic chain and bioaccumulation in human. In this study, genomics signals were used to assess toxicity of NP in Neocaridina denticulata. We identified differentially expression genes of Neocaridina denticulata exposed to NP by suppression subtractive hybridization（SSH）. Neocaridina denticulata treated with 0.5 mg/L NP for 1 day, 19 expressed sequence tags （ESTs） were obtained in forward subtracted library and 46 ESTs in reverse subtracted library. When exposed in 0.5 mg/L NP for 7 days, we obtained 33 ESTs in forward subtracted library and 34 ESTs in reverse subtracted library.
14 ESTs recognized up-regulated or down-regulated genes in NP-related gene library and gestation-related library have been reported to be associated with immunity（NP1-2）, stress（rNP1-10）, development（NP1-9）, metabolism（rNP1-8）, translation factor（NP1-7）,and unknown（NP1-17, rNP1-16, rNP1-17, rNP1-19, rNP1-20, rNP1-21, rNP1-22,and NP1-27）. Evaluating the sensitivity of each gene for NP, a change of 4 genes, including rNP1-10、 rNP1-17、rNP1-20、rNP1-27, was detected after treatment with 0.001, 0.01, and 0.5 mg/L for one day. A change of gene NP1-9 was detected after treatment with 0.01 and 0.5 mg/L of NP. The level of the other genes expression was different after treatment with 0.5 mg/L of NP. It suggests that differentially expression genes pattern can be used as a model for detecting toxicity of NP.

中文摘要 1
英文摘要 3
前言 4
文獻回顧 7
一、外因性內分泌干擾物質 7
（一）外因性內分泌干擾物質簡介 7
（二）壬基酚（Nonylphenol, NP） 8
1. 壬基酚簡介 8
2. 壬基酚影響生物的相關研究 9
（1）脊椎動物 9
（2）無脊椎動物 10
（3）哺乳動物 11
3. 壬基酚的流佈 11
二、污染物檢測 13
（一）物化檢測法 13
（二）生物檢測法 14
1. 指標生物 14
2. 生物指標 17
三、差異性表現基因體研究方法 20
（一）差異型表現反轉錄聚合酵素鏈鎖反應（Differential display
RT-PCR；DD RT-PCR） 20
（二）基因陣列（gene array） 21
（三）扣減法 22
實驗目的及實驗策略 25
材料方法 26
一、實驗用蝦 26
二、多齒新米蝦浸泡處理 26
（一）壬基酚配製 26
（二）浸泡處理 26
三、萃取蝦組織的mRNA 27
（一）蝦組織的製備 27
（二）Total RNA的萃取 27
（三）RNA瓊酯膠體電泳 28
（四）mRNA的純化 29
四、抑制性扣減雜交(suppression subtractive hybridization；SSH) 30
（一）第一股cDNA的合成 31
（二）第二股cDNA的合成 31
（三）限制酵素Rsa I的作用 32
（四）接合子（adaptor）連接至檢測組（tester）的cDNA片 32
（五）第一次雜合反應 33
（六）第二次雜合反應 34
（七）核酸聚合酵素連鎖反應（polymerase chain reaction；PCR） 34
（八）β- actin檢驗扣減效率 36
五、扣減cDNA基因庫(subtracted cDNA library)的建立 37
（一）聚合酵素鏈鎖反應（PCR）之產物的轉殖 37
（二）細胞轉形（transformation） 38
（三）轉形細胞的篩選 38
1. 聚合酵素鏈鎖反應（PCR） 38
2. 核酸點漬法（DNA dot blot） 39
（1）陽性轉形株的核酸轉漬於硝化纖維膜上 39
（2）探針（probe）的製備 40
（3）DNA雜合反應（DNA hybridization） 41
（4）雜合反應的檢測 ( Detection ) 41
（四）限制酵素切割圖譜的分析 42
1. 少量質體的製備 42
2. 限制酶切割 43
六、扣減後cDNA的序列於資料庫的比對與結果分析 43
（一）序列重組 43
（二）比對分析 44
七、扣減後cDNA的再次確認 44
八、基因靈敏度測試 46
結果 48
ㄧ、不同濃度壬基酚（NP）浸泡多齒新米蝦不同天數後之原酚氧化酵素（proPO）基因的表現 48
二、抑制性扣減雜交法之扣減樣本 49
（一）NP相關基因的扣減樣本 49
（二）抱卵相關基因的扣減樣本 50
三、抑制性扣減雜交法之扣減基因庫 50
（一）NP相關基因的扣減基因庫 50
（二）抱卵相關基因的扣減基因庫 51
四、轉型株基因的定序與序列比對 52
（一）NP處理之基因庫的相關基因 52
（二）抱卵基因庫之相關基因 53
五、基因庫中差異表現基因（differential expression genes）的確認 53
（一）NP處理之差異表現基因 53
（二）抱卵之差異表現基因 55
六、基因靈敏度測試 55
討論 57
一、扣減雜交所得之差異性表現基因的應用 57
二、多齒新米蝦之基因表現做為生物指標的可行性 58
三、扣減法作為差異性基因篩選的問題 60
四、扣減所得之差異性表現基因的生理意義 61
（一）血清素（Hemocyanin） 61
（二）cathepsin L 62
（三）磷酸海藻糖合成酶（Trehalose-6-phosphate synthase 1） 63
（四）未知功能基因 64
結論 65
參考文獻 66
圖表 79
表一、本研究所使用的引子 79
表二、壬基酚浸泡處理一天組之扣減基因庫的篩選資料 80
表三、抱卵相關基因扣減後基因庫的篩選資料 81
表四、壬基酚處理一天後之正向扣減基因庫比對結果 82
表五、壬基酚處理一天後之反向扣減基因庫比對結果 84
表六、壬基酚浸泡一天後之正反扣所得基因的功能分類整理表 87
表七、抱卵相關基因片段基因庫比對結果 89
表八、第一天正扣cDNA片段檢測所用之引子 90
表九、第一天反扣cDNA片段檢測所用之引子 91
表十、抱卵相關基因檢測所用之引子 93
表十一、壬基酚正負調控基因之靈敏度檢測表 94
表十二、NP處理七天後之正向扣減基因庫比對結果 95
表十三、NP處理七天後之反向扣減基因庫比對結果 98
表十四、第七天正負扣減基因庫的篩選資料 100
圖一、烷基苯酚聚乙氧基醇類分解路徑 101
圖二、抑制性扣減雜交法中所用之接合子及引子 102
圖三、抑制性扣減雜交法流程圖 103
圖四、實驗架構 104
圖五、pGEM-T Vector 105
圖六、多齒新米蝦分浸泡於不同濃度壬基酚後的不同天數之proPO基因
表現 106
圖七、多齒新米蝦分浸泡於不同濃度壬基酚後的不同天數之PO活性 107
圖八、雙股cDNA的確認 108
圖九、抑制性扣減雜交法之正、反向雜合產物經兩次PCR後的確認 109
圖十、抑制性扣減雜交法之正、負向扣減產物的扣減效率檢測 110
圖十一、抱卵相關基因扣減效率檢測 111
圖十二、第一天反扣所得基因庫經PCR放大之結果 112
圖十三、第一天正扣所得基因庫經PCR放大之結果 113
圖十四、第七天反扣所得基因庫經PCR放大之結果 114
圖十五、第七天正扣所得基因庫經PCR放大之結果 115
圖十六、第一天及第七正反向扣減基因庫進行核酸點漬法篩選 116
圖十七、壬基酚處理一天之正扣基因片段表現的確認 118
圖十八、壬基酚處理一天之反扣基因片段表現的確認 119
圖十九、抱卵相關基因表現的確認 121
圖二十、壬基酚正調控基因之靈敏度測試 122
圖二十一、壬基酚負調控基因之靈敏度測試 123
附錄 125
實驗藥品之成分與配製 125

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