臺灣博碩士論文加值系統

肺癌位居台灣惡性腫瘤中死亡原因的首位。儘管在治療上有多種臨床藥物的使用，大多數晚期肺癌患者皆會對治療藥物產生抗藥性，且在復發的案例中，因表皮生長因子接受器(Epidermal growth factor receptor, EGFR)突變(T790M)所造成的復發約佔一半，故找出治療抗藥性肺癌的方法為一當務之急。蜂膠素G (Propolin G)為台灣綠蜂膠萃取物中所分離出的活性物質，過去文獻指出其具有抗癌、抗細菌與抗發炎等功能。然而，蜂膠素G對於抗藥性肺癌的作用機制途徑尚未明瞭。故本研究欲探討台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞凋亡及細胞週期分子機制的影響。首先在細胞存活率的部分，分析蜂膠素G對各肺癌細胞之影響，其結果顯示四種非小細胞型肺癌細胞之存活率皆會隨蜂膠素G的濃度增加而受到抑制，尤以H1975細胞的抑制效果最顯著，其半抑制濃度(IC50)為6.80±0.38 μM。以流式細胞儀分析H1975細胞週期，發現到給予肺癌細胞H1975蜂膠素G 處理18小時後，會誘導其凋亡作用。而當利用凋亡蛋白抑制劑(Z-VAD-FMK)時，其能有效回復蜂膠素G造成的細胞凋亡。此外，蜂膠素G也會降低EGFR和信號傳導及轉錄激活蛋白(Signal transducer and activator of transcription 3, STAT3)的表現，且其所造成的EGFR降解為蛋白質穩定性的破壞，並非受轉錄調節影響。使用蛋白酶抑制劑(Carbobenzoxy-Leu-Leu-leucinal, MG132)時，蜂膠素G所造成的EGFR蛋白降解有回復的現象。接著探討18小時細胞凋亡作用前EGFR蛋白降解的原因，發現其中自噬相關蛋白(Microtubule-associated protein 1A/1B-light chain 3, LC3)的活化且使用自噬作用抑制劑(3-Methyladenine和Chloroquine)時，蜂膠素G造成的EGFR蛋白降解被有效回復。另外，使用活性氧化物質抑制劑(N-acetyl-L-cysteine)時，蜂膠素G所造成的EGFR蛋白降解也能有效回復且LC3II蛋白也有回復的現象。綜上所述，蜂膠素G可能藉由抑制EGFR蛋白相關訊號，進而誘導抗藥性人類非小細胞型肺癌細胞凋亡作用。

Lung cancer is the leading cause of malignant neoplasm death in Taiwan. Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by epidermal growth factor receptor (EGFR) T790M mutation in half of the relapses. Therefore, searching strategies for acquired mutated lung cancer treatment and/or prevention in urgency. Propolin G, a bioactive compound from Taiwanese propolis, has been demonstrated to possess anti-cancer, anti-bacterial, anti-inflammatory and so on. In this study, we focused on the anti-cancer mechanism of propolin G in acquired mutated lung cancer cells. The results revealed that the cell viability was inhibited by propolin G in a dose-dependent mode in a serial cell lung cancer lines. The acquired mutated lung cancer cells H1975 was more sensitive to propolin G at IC50 about 6.80±0.38 μM. Furthermore, apoptotic cells were accumulated after 18h of propolin G treatment. Pretreatment with caspase inhibitor, ZVAD-fmk, reversed propolin G-induced apoptosis. Molecularly, propolin G inhibited the expression of EGFR and signal transducer and activator of transcription 3 (STAT3) in a dose-dependent mode. Down-regulation of EGFR expression via propolin G was through EGFR protein stability disruption rather than transcriptional regulation. Pretreatment with proteasome inhibitor, MG132, reversed propolin G-induced EGFR degradation. Then we investigate the cause of EGFR degradation before 18h.We found that activation of LC3 and pretreatment with autophagy inhibitor, 3MA and CQ, reversed propolin G-induced EGFR degradation. In addition, treatment with reactive oxygen species inhibitor, NAC, reversed propolin G-induced EGFR degradation and LC3 activation. Collectively, inhibition acquired mutated lung cancer cells viability via propolin G might be through EGFR down-regulation following by apoptosis induction.

中文摘要.........................................................ii
英文摘要.........................................................iv
致謝...............................................................v
縮寫表.............................................................vii
目錄...........................................................viii
第一章、文獻回顧.................................................1
一、肺癌..........................................................1
(一)概論..........................................................1
(二)臨床治療方法......................................................1
(三) 抗藥性肺癌機轉-表皮生長因子突變(EGFR mutation)...................3
二、表皮生長因子接受器(EGFR)........................................4
(一)概論.........................................................4
(二)訊息傳遞路徑...............................................4
(三)肺癌中的角色..............................................5
三、細胞凋亡........................................................7
(一)概論.......................................................7
(二)內在路徑和外在路徑............................................7
(三)與自噬作用的相關性.............................................9
(四)與活性氧化物質(ROS)的相關性...................................10
四、台灣蜂膠.........................................................11
(一)蜂膠之來源與分類..........................................11
(二)台灣蜂膠的特性...............................................12
(三)台灣蜂膠的生理功能..............................................13
1.抗腫瘤生長...................................................13
2.抗菌能力.......................................................13
3.誘導細胞凋亡作用.................................................14
第二章、研究目的與設計..............................................15
一、研究目的...................................................15
二、實驗設計.....................................................15
三、實驗流程圖..........................................................16
第三章、材料與方法.................................................17
一、實驗材料.............................................................17
(一)台灣蜂膠萃取物蜂膠素G...........................................17
(二)細胞株.......................................................17
(三)培養基..........................................................17
(四)化學藥品..........................................................17
(五)抗體..........................................................19
二、實驗儀器......................................................20
三、實驗耗材.......................................................21
四、常用溶液配置.........................................................22
五、實驗方法.............................................................25
(一)細胞培養(Cell culture) ................................................25
(二)細胞存活試驗(MTT assay) ..................................25
(三)細胞週期分析試驗(Flow cytometry) ...........................26
(四)西方墨點法(Western blotting assay) .................................27
(五)細胞集落形成試驗(Colony formation assay) ......................29
(六)mRNA表現量測定(mRNA expression assay) ...........................29
第四章、統計分析...................................................32
第五章、實驗結果................................................33
一、台灣蜂膠萃取物對抗藥性肺腺癌細胞株H1975存活率之影響...................33
二、蜂膠素G對四種非小細胞型肺腺癌細胞株存活率之影響.........................33
三、蜂膠素G對抗藥性肺腺癌細胞株H1975集落形成之影響....................34
四、蜂膠素G對抗藥性肺腺癌細胞株H1975週期之影響.........................34
五、蜂膠素G對抗藥性肺腺癌細胞株H1975細胞凋亡之影響.....................35
六、蜂膠素G對抗藥性肺腺癌細胞株H1975蛋白及mRNA表現之影響................36
七、蜂膠素G對抗藥性肺腺癌細胞株H1975之LC3II蛋白表現影響.....................38
八、自嗜作用抑制劑 (3-Methyladenine and Chloroquine)對於抗藥性肺腺癌細胞株H1975蛋白表現之影響.....................................................38
九、活性氧化物質抑制劑(NAC)對於抗藥性肺腺癌細胞株H1975蛋白表現之影響...........39
十、蛋白酶體抑制劑(MG132)對於抗藥性肺腺癌細胞株H1975蛋白表現之影響.............40
第六章、討論......................................................42
一、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975生長之影響....................42
二、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975細胞週期之影響............42
三、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975細胞凋亡作用之探討....43
四、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975之EGFR訊息傳遞路徑探討..........44
五、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975自噬作用之探討............45
六、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975中活性氧化物質角色之探討.........47
七、台灣蜂膠萃取物蜂膠素G對抗藥性肺癌細胞H1975之EGFR降解的其它路徑探討........49
第七章、圖表.................................................50
第八章、參考文獻..............................................70
第九章、附圖...................................................77
第十章、附表....................................................82
 

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