在許多癌症中(例如皮膚癌)，上皮生長因子受體（EGFR, Epidermal
Growth Factor Receptor）及Cyclin D1 通常有過度的蛋白質表現、異常增生及突變情形。利用MTT assay，我們證實了松杉靈芝甲醇萃取物能在時間與劑量的調控下抑制A431 的生長。因此接下來欲探討松杉靈芝甲醇萃取物對於EGFR 過份表現的人類表皮癌細胞株A431 的抗癌作用。我們證實了松杉靈芝甲醇萃取物在時間與劑量的調控下抑制A431 的癌細胞生長。其次藉由西方墨點法偵測一系列下游訊息的蛋白質表現探討詳細分子機轉。我們證明了松杉靈芝甲醇萃取物在時間與劑量的條件下負調控EGFR、cyclin D1 與 CDK4 的蛋白質表現量並且降低了A431 癌細胞株的增生。在降解途徑方面，我們也發現了藉由加入蛋白酶抑制劑 MG132，松杉靈芝甲醇萃取物負調控了cyclin D1 的情況會回升，進而推論 cyclin D1 會走ubiquitin-dependent 的蛋白酶降解途徑。進一步我們又發現，松杉靈芝甲醇萃取物可能會提高AKT 及GSK3β的去活化作用。藉由這些實驗，我們可以推論松杉靈芝甲醇萃取物造成EGFR 蛋白質過表現的人類表皮癌細胞株A431 細胞週期停滯可能是經由PI3K/AKT 途徑進而造成cyclin D1 蛋白降解。此外，松杉靈芝甲醇萃取物不僅造成p21 的蛋白質表現增加而抑制了細胞週期中G1 期的cyclin D1 並且降低CDK4 的蛋白質表現。綜合以上之研究推測造成cyclin D1 減少的原因可能是由於蛋白酶調控的降解途徑以及CDK 抑制劑( p21)的表現提高，最終造成松杉靈芝甲醇萃取物抑制 EGFR 蛋白過表現的人類表皮癌細胞株。

The Epidermal Growth Factor Receptor (EGFR) and cyclin D1 are frequently amplified, overexpressed, or mutated in many cancers, including skin. We have previously demonstrated that the locally cultivated Ganoderma tsugae (G. tsugae, Lingzhi) extract possessing anti-cancer and anti-angiogenic properties in vitro and in vivo. The aim of the present work was to investigate the role of G. tsugae extracts in anti-cancer properties of EGFR-overexpressing human epidermoid carcinoma A431 cells. Using MTT assay, we demonstrated that G. tsugae extracts could inhibit the growth of A431 cells in a dose- and time-dependent manner. Western blotting analysis was used to investigate the mechanism of these effects. We demonstrated here a dose- and time-dependent down-regulation of expression of EGFR, cyclin D1 and CDK4 by G. tsugae extracts those correlate with the decrease in the proliferation of A431 cells. We also found that G. tsugae extracts-induced down-regulation of cyclin D1 was reversed by proteasome inhibitor, MG132, suggesting the role of ubiquitin-dependent proteasomal pathway. Furthermore, G. tsugae extracts treatment could cause the de-phosphorylation of constitutively active AKT and GSK3-beta. These finding suggest that G. tsugae extracts induces cell cycle arrest through proteasomal degration of cyclin D1 in EGFR-overexpressing human epidermoid carcinoma A431 cells might via the phosphatidylinositol 3-kinase/Akt-dependent pathway. Additionally, G. tsugae extracts could dramatically induce the expression of p21 while significantly inhibit the expression of the G1 phase cell cycle regulatory gene such as cyclin D1 and could suppress the expression of CDK4 protein. Taken together, our results suggest that proteasome-mediated down-regulation of cyclin D1 and up-regulation of CDK inhibitor might contribute to the antiproliferative effect of G. tsugae extracts against EGFR-overexpressing human epidermoid carcinoma.

謝誌-----------------------------------I
中文摘要-------------------------------II
英文摘要-------------------------------III 
目錄-----------------------------------V
表目錄---------------------------------VIII
圖目錄---------------------------------IX
附錄目錄-------------------------------X
縮寫表---------------------------------XI

第一章、	前言-實驗緣起及目的---------1
第二章、	序論------------------------2 
第三章、	第一節、細胞週期------------2
壹、細胞週期概論---------------------2
貳、細胞週期之調控-------------------3
叁、細胞週期蛋白---------------------5
第二節、表皮生長因子-----------------6
壹、表皮生長因子受體概論（EGFR）-----6
貳、表皮生長因子受體與癌症的關係-----7
叁、針對表皮生長因子受體的治療方法---8         
第三節、靈芝-------------------------9
壹、靈芝概論-------------------------9
貳、靈芝的主要成份與功效-------------10
参、靈芝與癌症的關係-----------------11    
肆、松杉靈芝-------------------------12
研究方向-----------------------------13
第三章、材料與方法-------------------14
第一節、實驗材料及相關儀器-----------14
壹、細胞株---------------------------14
貳、抗體-----------------------------14
叁、化學藥品與試劑-------------------14
肆、主要儀器及器材-------------------15
第二節、實驗方法---------------------16
壹、松杉靈芝萃取物之製備-------------16
貳、細胞存活率分析-------------------16
叁、西方墨點法-----------------------17
第四章、實驗結果---------------------18
壹、松杉靈芝甲醇萃取物影響人類表皮癌細胞A431之細胞形態---18
貳、松杉靈芝甲醇萃取物抑制人類表皮癌細胞A431之生長-------18
叁、松杉靈芝甲醇萃取物影響人類表皮癌細胞A431的細胞週期之相關蛋白之表現-----19
一、 松杉靈芝甲醇萃取物抑制人類表皮癌細胞A431的cyclin D1及CDK4的表現量-----19
二、松杉靈芝甲醇萃取物增加人類表皮癌細胞A431的CDKi （p21）的表現-----------20
肆、松杉靈芝甲醇萃取物對人類表皮癌細胞A431的cyclin D1蛋白分解途徑之探討-----20
伍、松杉靈芝甲醇萃取物抑制人類表皮癌細胞A431的EGFR蛋白之表現---------------21
陸、松杉靈芝甲醇萃取物抑制人類表皮癌細胞A431的MAPK訊息傳遞途徑-------------22
第五章、討論----------------23
第六章、結論----------------26
參考文獻--------------------27
表--------------------------41
圖--------------------------45
附錄------------------------55
表目錄----------------------IV
表1、細胞株基本資料---------41
表2、西方墨點法使用之抗體---42
表 3、化學藥品及試劑--------43
圖目錄----------------------V
圖1、細胞週期的調控機轉-----4
圖2、表皮生長因子受體蛋白所調控之相關訊息傳遞路徑-----7
圖3、松杉靈芝甲醇萃取物對於人類表皮癌A431細胞株外觀型態之影響---------45
圖4、松杉靈芝甲醇萃取物對於人類表皮癌細胞A431細胞生長之影響---------46
圖5、松杉靈芝甲醇萃取物對於人類表皮癌A431細胞株之細胞週期相關蛋白之影響-----------47
圖6、松杉靈芝甲醇萃取物對於人類表皮癌A431細胞株cyclin D1蛋白分解途徑之影響-------48
圖7、松杉靈芝甲醇萃取物對於人類表皮癌A431細胞株之p-GSK3-beta蛋白表現量之影響---49
圖8、松杉靈芝甲醇萃取物抑制人類表皮癌A431細胞株p-Akt蛋白表現量-----------------50
圖9、松杉靈芝甲醇萃取物對於人類表皮癌A431細胞株之EGFR蛋白表現量之影響-----------51
圖10、松杉靈芝甲醇萃取物抑制人類表皮癌A431細胞株之EGFR下游MAPK ( Erk )蛋白質表現----------52
圖11、松杉靈芝甲醇萃取物抑制人類表皮癌A431細胞株之EGFR下游MAPK ( p38 )蛋白質表現----------53
圖12、松杉靈芝甲醇萃取物抑制cyclin D1之可能途徑示意圖---54
附錄目錄-----------------VI
附錄1、細胞培養使用之緩衝液-----------55
附錄2、西方墨點法使用之試劑溶液-------56
附錄3、Cyclin D1的降解途徑------------58

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