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研究生:林姿廷
研究生(外文):Zih-Ting Lin
論文名稱:苦丁茶與空心蓮子草不同萃取物對初代免疫細 胞與自然殺手細胞免疫調節能力之影響及其與 前列腺癌細胞和乳腺癌細胞間交互作用
論文名稱(外文):Immunomodulatory effects of Ilex kudingcha and Alternanthera philoxeroides extracts in primary immune cells, natural killer cells and their interactions with PC-3 prostate cancer cells and MCF-7 breast cancer cells
指導教授:林金源林金源引用關係
口試委員:林璧鳳胡淼琳
口試日期:2014-07-09
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
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:2014
畢業學年度:102
語文別:中文
論文頁數:137
中文關鍵詞:空心蓮子草細胞凋亡細胞激素Fas 基因FasL 基因人類乳腺癌細胞 MCF-7人類前列腺癌細胞 PC-3苦丁茶發炎反應Th1/Th2 免疫平衡自然殺手細胞 NK92-MI促凋亡(Bax)/抗凋亡(Bcl2)基因
外文關鍵詞:Alternanthera philoxeroidesapoptosiscytokinesFas geneFasL genehuman breast adenocarcinoma MCF-7cellshuman prostate adenocarcinoma PC-3 cellsIlex kudingchainflammationTh1/Th2 immune balanceNatural killer NK92-MI cellspro- (Bax)/anti-a
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苦丁茶(Ilex kudingcha)又稱一葉茶,屬於傳統的飲品,文獻指出其具有降血糖及護肝之功能;空心蓮子草(Alternanthera philoxeroides (Mart.) Griseb)又稱革命草,原產於南美洲,具有清熱抗菌之效果,但是苦丁茶及空心蓮子草之免疫調節功能仍不清楚,因此本研究利用三種不同溶劑氯仿、甲醇及水分別萃取其成分,先利用小鼠脾臟細胞進行 Th1/Th2 免疫調節影響評估;篩選出兩種具有免疫調節潛力之萃取物後,再探討其對發炎及過敏反應之影響;並進一步利用自然殺手細胞之癌症免疫療法探討其對前列腺癌 PC-3 與乳腺癌 MCF-7 細胞株細胞凋亡之影響。
結果顯示六種萃取物中,苦丁茶氯仿萃取物(IKCE)使小鼠脾臟細胞免疫反應傾向 Th2 免疫平衡,而苦丁茶甲醇萃取物(IKME)則傾向促進 Th1 免疫平衡;因此以此兩種最具免疫調節潛力之萃取物探討其對小鼠腹腔巨噬細胞發炎之影響,結果發現單獨添加 IKCE 及 IKME 可輕微刺激巨噬細胞活性,推測可能具有提升先天性免疫之潛力。以脂多醣(LPS)刺激模擬發炎模式下,顯示 IKCE 及IKME 皆具有抗發炎潛力;在預防發炎模式下,IKCE 及 IKME 可顯著降低促發炎/抗發炎細胞激素之分泌比值,顯示兩者皆具有預防發炎之效果。另外,在抗過敏潛力評估方面,IKCE 及 IKME 皆具有顯著抑制小鼠肺臟肥大細胞去顆粒化之作用。
IKCE 及 IKME 已發現具有活化先天性免疫細胞之作用,因此進一步探討其在單獨添加或其與自然殺手細胞(NK92-MI)共同培養之條件培養液對前列腺癌細胞(PC-3)及乳腺癌細胞(MCF-7)之影響。結果發現 IKME 可抑制前列腺癌細胞生長;IKME 及其與自然殺手細胞 NK92-MI 共同培養之條件培養液皆可增加前列腺癌細胞促凋亡(Bax)及抗凋亡(Bcl2)基因表現之比值。另外,IKME 及其與自然殺手細胞 NK92-MI 共同培養之條件培養液,可促進前列腺癌細胞 Fas 基因之表現。
綜合本實驗結果顯示,IKME 可藉由單獨添加或透過調節自然殺手細胞免疫療法之作用方式,增加促凋亡 Bax 基因及 Fas 基因之表現,使前列腺癌細胞傾向促凋亡。
Ilex kudingcha, also known as a leaf tea, is a traditional beverage found to have hypoglycemic and hepato-protective functions. Alternanthera philoxeroides (Mart.) Griseb, also called alligator weed, originated in South America that exhibits heat-purging and antibacterial effects. However, immunomodulatory effects of Ilex kudingcha and Alternanthera philoxeroides remain unclear. To clarify the puzzle, Ilex kudingcha and Alternanthera philoxeroides were extracted using three different solvents, including chloroform, methanol and water, respectively. The extracts were first subjected to Th1/Th2 immunomodulatory study using mouse primary splenocytes in vitro. Two potential extracts were isolated to explore their anti-inflammatory and anti-allergic effects using mouse primary peritoneal macrophages and lung mast cells, respectively. Finally, potent extracts were further selected to treat human prostate adenocarcinoma PC-3 cells and breast adenocarcinoma MCF-7 cells using direct addition or natural killer cells’ immunotherapy.

The results showed that Ilex kudingcha chloroform extract (IKCE) had potential to regulate Th1/Th2 immune responses toward Th2 immune balance and Ilex kudingcha methanol extract (IKME) toward Th1 immune balance among six different extracts.
Therefore, IKCE and IKME were screened to explore their anti-inflammatory effects using mouse peritoneal macrophages. IKCE and IKME treatments alone showed a mild stimulatory activity to macrophages, suggesting that both extracts might have potential to enhance innate immunity. IKCE and IKME treatments in the presence of lipopolysaccharides(LPS) were found to have anti-inflammatory potential. Furthermore, both IKCE and IKME had prophylactic effects against LPS-induced inflammation through inhibiting pro-/anti-inflammatory cytokine secretion ratios by peritoneal macrophages in a specified preventive experiment. In addition, IKCE and IKME inhibited the degranulation of mouse lung mast cells, suggesting that both extracts might have anti-allergic potential.

IKCE and IKME were suggested to have potential to enhance innate immunity; therefore IKCE and IKME were further selected to treat PC-3 and MCF-7 cells using direct addition or the conditioned medium of natural killer cells (NK92-MI) cultured with there potent extracts.The results showed that IKME inhibited the proliferation of human prostate adenocarcinoma PC-3 cells. Consequently, IKME and its conditioned medium of NK92-MI cells were further selected to treat PC-3 cells. Changes of mRNA expression levels of pro- (Bax)/anti-apoptotic(Bcl-2) genes in PC-3 cells were assayed using RT-qPCR. Results showed that expression ratios of Bax/Bcl-2 mRNA levels in PC-3 cells were significantly increased by IKME and its conditioned medium of NK92-MI cells. In addition, Fas gene expression levels in PC-3 cells were also increased by IKME and its conditioned medium of NK92-MI cells.

In conclusion, results from this study evidenced that IKME treatments might induce apoptosis in PC-3 cells through increasing pro-apoptotic Bax and Fas genes expression via
direct action or natural killer cells’ immunotherapy to achieve its anticancer effects.
摘要................................................................................................................................ I
Abstract ........................................................................................................................ III
目錄............................................................................................................................... V
表目錄........................................................................................................................... X
圖目錄........................................................................................................................ XII
縮寫對照表............................................................................................................... XIV
緒言................................................................................................................................ 1
第一章 文獻回顧.......................................................................................................... 2
第一節免疫反應........................................................................................................ 2
一、免疫系統 ...................................................................................................... 2
二、輔助型 T 細胞第 1 型與第 2 型之免疫平衡................................................ 8
三、促/抗發炎細胞激素 ..................................................................................... 10
第二節 發炎反應與癌症........................................................................................ 12
一、發炎反應與癌症 .......................................................................................... 12
二、細胞凋亡...................................................................................................... 13
第三節 苦丁茶及空心蓮子草................................................................................ 15
一、苦丁茶 ( Ilex kudingcha) ............................................................................. 15
二、空心蓮子草 (Alternanthera philoxeroides (Mart.) Griseb) ........................ 15
第四節 多酚類與萜類之免疫調節作用................................................................ 16
第五節 研究動機與目的........................................................................................ 18
第六節 論文架構與實驗設計................................................................................ 19
第二章 苦丁茶及空心蓮子草不同萃取物對初代脾臟細胞 Th1/Th2 免疫平衡之影
響.................................................................................................................................. 20
第一節 前言............................................................................................................ 20
第二節 材料與方法................................................................................................ 20
一、樣品製備 ...................................................................................................... 20
二、苦丁茶及空心蓮子草粉末樣品之水分及灰分含量測定 .......................... 23
三、苦丁茶與空心蓮子草不同萃取物之光譜掃描分析 .................................. 25
四、苦丁茶與空心蓮子草不同萃取物中總多酚類及總類黃酮含量測定 ...... 25
五、苦丁茶甲醇萃取物之高效能液態層析儀(HPLC)分析 ............................. 27
六、苦丁茶與空心蓮子草不同萃取物對 BALB/c 雌鼠脾臟細胞細胞之影響
.......................................................................................................................... 29
七、苦丁茶及空心蓮子草不同萃取物對 BALB/c 雌鼠脾臟細胞分泌細胞激素
之影響.............................................................................................................. 32
八、統計分析 ...................................................................................................... 35
第三節 結果............................................................................................................ 36
一、苦丁茶及空心蓮子草不同萃取物之萃取率 .............................................. 36
二、苦丁茶及空心蓮子草粉末樣品之水分及灰分分析 .................................. 36
三、苦丁茶及空心蓮子草不同萃取物之光譜掃描 .......................................... 36
四、苦丁茶及空心蓮子草不同萃取物之總多酚及類黃酮成分分析 .............. 37
五、以高效液態層析測定苦丁茶甲醇萃取物組成分分析 .............................. 41
六、苦丁茶及空心蓮子草不同萃取物對 BALB/c 雌鼠初代脾臟細胞生長之影
響...................................................................................................................... 46
七、苦丁茶及空心蓮子草不同萃取物對 BALB/c 雌鼠初代脾臟細胞分泌細胞激素之影響...................................................................................................... 49
第四節 討論............................................................................................................ 57
第五節 結論............................................................................................................ 58
第三章
苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對發炎及過敏反應之影響...... 59
第一節 前言............................................................................................................ 59
第二節材料與方法.................................................................................................. 59
一、實驗架構 ...................................................................................................... 59
二、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對 BALB/c 雌鼠初代巨噬細胞促發炎與抗發炎細胞激素分泌之影響.............................................................. 60
1.模式 A:單獨添加樣品對初代巨噬細胞細胞激素分泌之影響 .................. 60
2.模式 B:模擬在發炎模式下樣品與 LPS 共同存在對初代巨噬細胞細胞激
素分泌之影響.................................................................................................. 61
3.模式 C:模擬預防模式下樣品對初代巨噬細胞細胞激素分泌之影響 ...... 61
4.模式 D:模擬治療模式下樣品對初代巨噬細胞細胞激素分泌之影響 ...... 61
三、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對 BALB/c 雌鼠肥大細
β-hexosaminidase 釋放之影響........................................................................ 64
四、統計分析 ...................................................................................................... 68
第三節結果.............................................................................................................. 69
一、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對 BALB/c 雌鼠初代巨噬細胞促發炎與抗發炎細胞激素分泌之影響.............................................................. 69
二、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對 BALB/c 雌鼠肥大細胞去顆粒化之影響.......................................................................................................... 81
第四節 討論............................................................................................................ 83
第五節 結論............................................................................................................ 84
第四章 苦丁茶氯仿及苦丁茶甲醇萃取物對人類乳癌細胞與前列腺癌細胞生長之
影響.............................................................................................................................. 85
第一節 前言............................................................................................................ 85
第二節材料與方法.................................................................................................. 85
一、實驗設計 ...................................................................................................... 85
二、實驗樣品配製 .............................................................................................. 86
三、自然殺手細胞 NK92-MI 細胞株之培養 .................................................... 86
四、苦丁茶氯仿萃取物與苦丁茶甲醇萃取物對自然殺手細胞 NK92-MI 細胞
株細胞毒性評估.............................................................................................. 87
五、苦丁茶氯仿萃取物與苦丁茶甲醇萃取物對自然殺手細胞 NK92-MI 細胞
株細胞激素分泌之影響.................................................................................. 88
六、條件培養液之收集 ...................................................................................... 91
七、人類前列腺癌 PC-3 細胞株與人類乳癌 MCF-7 細胞株之培養 .............. 91
八、苦丁茶氯仿萃取物和苦丁茶甲醇萃取物及條件培養液對人類前列腺癌
PC-3 細胞株與人類乳癌 MCF-7 細胞株之影響 .......................................... 93
九、苦丁茶甲醇萃取物和其與 NK92-MI 細胞之條件培養液對人類前列腺癌
細胞促/抗凋亡及 Fas 基因 mRNA 表現之影響 ........................................... 95
十、統計分析 .................................................................................................... 102
第三節結果............................................................................................................ 103
一、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對自然殺手 NK92-MI 細胞生長
之影響............................................................................................................ 103
二、苦丁茶氯仿萃取物與苦丁茶甲醇萃取物、PMA 及 DEX 對自然殺手細胞
NK92-MI 細胞株細胞激素分泌之影響....................................................... 105
三、苦丁茶氯仿萃取物及苦丁茶甲醇萃取物對人類前列腺癌細胞與乳腺癌細
胞生長之影響................................................................................................ 108
四、苦丁茶甲醇萃取物對人類前列腺癌細胞促凋亡與抗凋亡基因 mRNA 表
現之影響........................................................................................................ 117
五、苦丁茶甲醇萃取物和其與自然殺手 NK92-MI 細胞之條件培養液對人類
前列腺癌細胞 Fas 基因 mRNA 表現之影響 .............................................. 120
六、苦丁茶甲醇萃取物對自然殺手細胞 NK92-MI 細胞株 FasL 基因 mRNA
表現之影響.................................................................................................... 123
第四節 討論.......................................................................................................... 125
第五節結論............................................................................................................ 126
第五章 總討論與總結.............................................................................................. 127
第六章 參考文獻...................................................................................................... 129
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