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研究生:盧信芳
論文名稱:台灣五葉松松針免疫活性之探討
論文名稱(外文):Evaluation of immune activity of Pinus morrisonicola hay
指導教授:許祥純許祥純引用關係
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:台灣五葉松免疫評估細胞激素反轉錄競爭定量聚合酶鏈反應
相關次數:
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松樹的民間療法已施行多年,有許多研究報告指出多種松樹具有調節免疫系統的功能,但過去對於台灣本土松樹生理活性的探討卻為數不多。因此本研究將針對台灣五葉松,分別以體內及體外試驗為模式,利用反轉錄競爭定量聚合酶鏈反應,定量老鼠體內免疫系統中細胞激素,間白素二、四、十、十八及γ-干擾素的基因表現。
對Th 1細胞分泌之細胞激素調節情形在體內試驗部分,餵食中濃度(19.5 mg/day/mouse)及高濃度(39 mg/day/mouse)松針萃取物時,較能刺激IL-2及IFN-γ的產生;在沒有conA的刺激情況下, IL-2以高濃度餵食四週時,較能提高其表現量,IFN-γ方面,只有在餵食中、高濃度八週時可明顯提高基因之表現。對Th 2細胞分泌之細胞激素調節情形方面,以餵食低濃度(1.95 mg/day/mouse)松針萃取液四週時,較能提高IL-4及IL-10的表現。在IFN-γ/ IL-4 的比值方面,以中、高濃度松針萃取物餵食小鼠可增加IFN-γ/ IL-4 的比值,降低了IL-4 的表現;而餵食低濃度時, 比值則會降低。而IL-18表現量在以中、高濃度餵食時,可明顯增加其表現量。而體外實驗結果則趨向在中、低濃度(0.0975 mg/ml、0.0195 mg/ml)較能刺激IL-2及IL-4的分泌,在高濃度(1.95 mg/ml)時雖會抑制脾臟細胞增殖及造成細胞的死亡,但卻可刺激脾臟細胞中IFN-γ的表現。綜合實驗結果得知,不同濃度的松針萃取液可刺激不同之Th1及Th2細胞激素之基因表現;在中、高濃度時有利於細胞性的免疫反應,低濃度時則傾向於體液性之免疫反應的發生。
目錄
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖索引 XII
表索引 XIII
壹、前言 1
貳、文獻整理 3
第一節、免疫系統之簡介 3
一、免疫系統之介紹及分類 3
(一) 淋巴細胞 3
(二) 吞噬細胞 4
二、T淋巴細胞與細胞激素 4
第二節、五葉松之簡介及文獻回顧 12
第三節、反轉錄競爭性定量 17
叁、材料與方法 20
第一節、試驗材料 20
一、萃取物之製備 20
第二節、體內試驗 20
一、餵食劑量 20
二、實驗動物 21
三、餵養方式 21
四、脾臟細胞之收集及培養 21
第三節、體外試驗 22
一、實驗動物 22
二、脾臟細胞之收集及培養 22
第四節、脾臟細胞之細胞激素基因表現檢測 23
一、RT-qcPCR之操作步驟 23
1、細胞RNA之萃取 24
2、逆轉錄反應 25
3、IL-10、IL-18 standard及competitor的製作 25
3.1、IL-10、IL-18 standard引子對之設計 25
3.2、聚合酶連鎖反應 26
3.3、IL-10及IL-18 standard之選殖 29
3.3.1、接合作用 30
3.3.2、轉形作用 31
3.3.3、篩選 31
3.4、質體之純化 32
3.5、IL-10及IL-18 standard之定序 33
3.6、IL-10及IL-18 competitor之選殖 33
二、利用qc-PCR定量餵食不同濃度松針萃取後小鼠體
內細胞激素之基因表現 35
1、標準曲線之建立 35
2、樣品之檢測 36
3、統計分析 36
肆、結果 37
一、體重變化 37
二、松針萃取液每日平均攝取量 37
三、IL-10和IL-18標準品選殖定序結果 37
四、小鼠體內細胞激素基因之表現之定量結果 38
1、標準曲線之建立 38
2、體內試驗 39
2.1、IL-2 mRNA之表現 39
2.2、IFN-γmRNA之表現 40
2.3、IL-4 mRNA之表現 40
2.4、IFN-γ/ IL-4比值 41
2.5、IL-10 mRNA之表現 42
2.6、IL-18 mRNA之表現 42
3、體外試驗 43
3.1、IL-2 mRNA之表現 43
3.2、IFN-γmRNA之表現 44
3.3、IL-4 mRNA之表現 44
3.4、IFN-γ/ IL-4 比值 45
3.5、IL-10 mRNA之表現 45
伍、討論 64
陸、結論 72
柒、參考文獻 73
圖索引
圖一、Th1及Th2細胞分化之過程 7
圖二、IL-10 competitor設計之原理 34
圖三、BALB/c小鼠餵食不同濃度松針萃取液四週之體重變
化情形 46
圖四、BALB/c小鼠餵食不同濃度松針萃取液八週之體重變
化情形 47
圖五、IL-10之選殖標準品序列與GenBank小鼠序列經BestFit比
之結果 49
圖六、IL-18之選殖標準品序列與GenBank小鼠序列經BestFit比對之結果 50
圖七、IL-10之qc-PCR產物電泳圖 51
圖八、IL-10標準曲線及回歸方程式 52
表索引
表一、IL-10和IL-18標準品及競爭子引子對之序列 27
表二、IL-2、IL-4和IFN-γ標準品及競爭子引子對之序列 28
表三、BALB/c小鼠之三種濃度松針萃取液每日平均攝取量 48
表四、餵食不同濃度松針萃取液四週及八週後脾臟中IL-2 mRNA的表現量 53
表五、餵食不同濃度松針萃取液四週及八週後脾臟中IFN-γ mRNA的表現量 54
表六、餵食不同濃度松針萃取液四週及八週後脾臟中IL-4 mRNA的表現量 55
表七、餵食不同濃度松針萃取液四週及八週後脾臟中IFN-γ/ IL-4 mRNA之比值 56
表八、餵食不同濃度松針萃取液四週及八週後脾臟中IL-10 mRNA的表現量 57
表九、餵食不同濃度松針萃取液四週及八週後脾臟中IL-18 mRNA的表現量 58
表十、不同濃度松針萃取液直接刺激ICR小鼠脾臟細胞IL-2 mRNA之表現量 59
表十一、不同濃度松針萃取液直接刺激ICR小鼠脾臟細胞IFN-γ mRNA之表現量 60
表十二、不同濃度松針萃取液直接刺激ICR小鼠脾臟細胞IL-4 mRNA之表現量 61
表十三、不同濃度松針萃取液直接刺激ICR小鼠脾臟細胞後IFN-γ/ IL-4 mRNA之比值 62
表十四、不同濃度松針萃取液直接刺激ICR小鼠脾臟細胞IL-10 mRNA之表現量 63
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