臺灣博碩士論文加值系統

紫色禿馬勃 (Calvatia lilacina)屬於馬勃菌科，也是常見的真菌食用菇類，最初的生長地是在中國與台灣，紫色禿馬勃更是一種中藥材，有消腫止血、利喉解毒等功用。
本研究探討紫色禿馬勃蛋白質萃取液造成人類大腸癌SW480細胞的內質網壓力與存活率分析，當內質網中的壓力訊號達到最高點時，可以促使鈣離子快速從內質網中釋放，會引起ER stress的反應，增加調控CHOP、ATF-4以及GRP78的表現，並且誘導細胞凋亡。
研究結果顯示，紫色禿馬勃蛋白質萃取液100 µg/ml藥物處理6小時，結果發現其細胞質內的細胞空泡隨之增加；更進一步藉由細胞週期的分析發現，紫色禿馬勃蛋白質萃取液也會造成SW480細胞的SubG1細胞群和小片段DNA斷裂(oligonucleosomal DNA fragmentation)、內質網壓力的指標GRP78表現增加，另外也會造成PERK蛋白下游的eIF2α蛋白磷酸化，進而促使CHOP轉錄因子的活化細胞凋亡調控蛋白Bax的表現量增加、細胞內鈣離子上升。用紫色禿馬勃蛋白萃取液處理細胞後，發現未處理組及處理組的細胞凋亡分別是3.0%及45.8%，但若在加入紫色禿馬勃蛋白質萃取液前先加入不同功能的抑制劑：Salubrinal、SP600125、BAPTA、N-乙醯半胱胺酸 (N-acetylcysteine，NAC)，細胞凋亡率分別下降為22.7%、18.7%、19.7%、19.2%。
由以上結果闡明了紫色禿馬勃蛋白萃取液造成SW480細胞內質網壓力訊息與引發細胞凋亡之間的相互關係。

目錄
中文摘要 ………………………………………………..………………i
英文摘要 ……………………………………………..………………..iii
目錄 ....………………….……………………………………………….v
第一章 緒論 ……………………………………………………............1
第二章 文獻探討……………………………….…………….................2
第一節 大腸直腸癌 (Colorectal cancer) ..............................................2
第二節 紫色禿馬勃(Calvatia lilacina，CL) ....................................2
第三節 內質網壓力(Endoplasmic Reticulum stress，ER stress) …3
第四節 內質網壓力(ER stress)與凋亡(apoptosis)之關係 .............4
第五節 Mitogen-activated protein kinase (MAPK) pathway與凋亡(apoptosis) 之關係 .............................................................6
第六節 Mitogen-activated protein kinase (MAPK) pathway與ER stress之關係 …………………..………..............................6
第七節 鈣離子(Ca2+)與ER stress……………………......................7
第八節 鈣離子(Ca2+)與apoptosis之關係 ………………................7
第九節 GSH耗空與內質網壓力(ER stress)之關係 …...................8
第三章 實驗目的 ..................................................................................10
第四章 研究方法 ..................................................................................11
第一節 材料 …………………..……………….……….................11
一、 菇類萃取與蛋白質定量 ...................................................11
二、 細胞株部分 .......................................................................11
三、 細胞培養部分 ...................................................................12
四、 細胞存活率偵測與細胞週期部分 ………………...........12
五、 Western blotting .................................................................13
六、 儀器項目 …...................................................……………14
七、 鈣離子及内質網壓力 .......................................................14
八、 其他 …...........................................................………........15
第二節 方法 ..........................................................................................16
一、紫色禿馬勃(CL)蛋白質的萃取法 ......................................16
二、人類大腸癌細胞(SW480)培養 ...........................................17
三、應用流式細胞儀分析細胞週期 ..........................................18
四、應用ER Tracker dye觀察細胞內質網壓力的空泡產生 ….18
五、利用西方墨點法分析細胞內蛋白表現 …...........................19
六、應用流式細胞儀偵測鈣離子變化 ......................................21
七、統計方法 ..............................................................................22
第五章 實驗結果 …..............................................................................24
第一節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480細胞週期的影響…………..………………………………....23
第二節 紫色禿馬勃萃取液加熱與加入Proteinase K觀察對SW480細胞細胞週期的影響 ...........................……….24
第三節 紫色禿馬勃蛋白萃取液造成大腸直腸癌細胞SW480細胞型態之影響 ….……………………………………...24
第四節 紫色禿馬勃蛋白萃取液造成大腸直腸癌細胞SW480、HT-29及DLD-1細胞型態與ER stress相關性之影響 ...25
第五節 紫色禿馬勃萃取液對SW480、HT-29、DLD-1細胞存活率之影響 ……………………………………………….26
第六節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的有關內質網壓力的相關蛋白p-eIF2之影響 ..................26
第七節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的有關內質網壓力的相關蛋白ATF-3之影響 .....................26
第八節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的有關內質網壓力的相關蛋白ATF-4之影響 ….................27
第九節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的有關內質網壓力的相關蛋白CHOP/GADD 153之影響 ..27
第十節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的有關內質網壓力的相關蛋白GRP78之影響 .....................28
第十一節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480內Ca2+之影響 ……….........................................................28
第十二節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480的caspase 4、9活性的影響 …............................................29
第十三節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480內Bax蛋白及其量化圖的變化 ….............................................29
第十四節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480內p-ERK蛋白及其量化圖的變化 ….................................30
第十五節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480內p-38蛋白及其量化圖的變化 ……….............................30
第十六節 紫色禿馬勃蛋白萃取液對大腸直腸癌細胞SW480內p-JNK蛋白及其量化圖的變化 ……..............................31
第十七節 使用JNK抑制劑(SP600125)、ROS抑制劑、鈣離子抑制劑(BAPTA)來觀察紫色禿馬勃萃取液對SW480細胞內CHOP蛋白變化................................................................31
第十八節 紫色禿馬勃蛋白萃取液使大腸直腸癌細胞SW480細胞凋亡主要路徑的探討 .....................................................32
第十九節 紫色禿馬勃蛋白萃取液造成大腸直腸癌細胞SW480細胞凋亡的相關路徑 …………………..……...................33
第六章 討論 ..........................................................................................34
參考文獻 ................................................................................................38
圖表 ........................................................................................................43

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