臺灣博碩士論文加值系統

黃芩素是萃取自中草藥黃芩的主要類黃酮素，已經顯示具有抗癌的活性。Survivin蛋白具有促使癌細胞增生及腫瘤形成的作用。然而，黃芩素誘發人類癌細胞凋亡時，survivin蛋白的調控及功能仍不清楚。我們發現在不同人類膀胱癌細胞株包括TSGH8301、BFTC905、RT4、T24及HT1376皆高度表現survivin蛋白，並會集中表現在有絲分裂及細胞質分裂時期。當細胞處理40-80 �嵱 黃芩素，經24小時後，隨著處理濃度的提高，會顯著的增加膀胱癌細胞毒性、誘發細胞凋亡、G2/M時期增加及抑制細胞生長。同時，黃芩素會抑制cyclin B1、磷酸化cdc2 (Threonine-161)及survivin蛋白的表達。若前處理cdc2激酶的抑制劑，則會增加黃芩素所造成的細胞毒性。當我們利用基因轉殖survivin siRNA到人類膀胱癌細胞後，會抑制survivin蛋白表達，並活化γ-H2AX的表現，同時增加黃芩素在人類膀胱癌細胞毒性。此外，黃芩素會增加磷酸化p38 MAP激酶及Akt蛋白的表現量。以p38 MAP激酶抑制劑SB203580前處理膀胱癌細胞後，會減少黃芩素所造成的細胞毒性，並恢復survivin蛋白的表達量。相反的，以Akt抑制劑wortmannin前處理細胞，則會增加黃芩素所誘發的細胞毒性，並會抑制survivin蛋白的表達。綜合以上的結果，我們推測黃芩素誘發人類膀胱癌細胞死亡及細胞生長抑制，與降低survivin蛋白表達有關，並且survivin的表達分別可透過Akt及p38 MAP激酶路徑所正反調控。

Baicalein, a major flavonoid extracted from Scutellaria baicalensis, has been shown to exert anticancer activity. Survivin proteins are expressed in various cancer cells that promote cell proliferation and tumorgenesis. However, the role and regulation of survivin on the baicalein-induced apoptosis in human cancer cells remain unclear. Survivin proteins can expressed in variety of bladder cancer cells including TSGH8301, BFTC905, RT4, T24, and HT1376, which concentrated on the mitotic phase and cytokinesis. Baicalein (40-80 �嵱, 24 h) concentration-dependently increased the cytotoxicity, apoptosis, G2/M fractions, and growth inhibition in bladder cancer cells. The cyclin B1, phospho-cdc2 (threonine-161), and survivin proteins were decreased by baicalein. Pre-treatment with cdc2 inhibitors increased the baicalein-induced cell death. Transfection with survivin siRNA decreased the survivin expression but increased the γ-H2AX and the baicalein-induced cytotoxicity in bladder cancer cells. Moreover, baicalein increased phosphorylation of p38 MAP kinase and Akt in BFTC905 cells. A specific p38 MAP kinase inhibitor, SB203580, decreased the cytotoxicity and increased the survivin levels in the baicalein-treated cells. In addition, an Akt inhibitor, wortmannin, increased the cytotoxicity and decreased the survivin levels in the baicalein-exposed cells. Together, our results indicate that blockade of survivin expression mediate baicalein-induced cytotoxicity and growth inhibition, which is oppositely regulated by p38 MAP kinase and Akt in human bladder cancer cells.

目錄……………………………………………………………………….....Ⅰ
圖表目錄……………………………………………………………………Ⅳ
中英文對照表……………………………………………………........…. Ⅴ
中文摘要…………………………………………………………………....Ⅵ
英文摘要……………………………………………………………….…...Ⅶ
緒論………………………………………………………….……………......1
一、膀胱癌之簡介…………………………………………………………1
二、黃芩素之簡介………………………………………………………....2
三、survivin與細胞凋亡之關係…………………………………………..3
四、survivin與膀胱癌的關係……………………………………………..4
五、p38 MAP kinase的功能及表達……………………………………….4
六、Akt的功能及表達…………………………………………………….5
七、γ-H2AX的功能及表達……………………………………………...6
八、研究動機與目的………………………………………………............6
研究材料與方法……………………………………………………………7
一、化學藥品與抗體………………………………………………………7
二、細胞株…………………………………………………………............7
三、細胞培養………………………………………………………............8
四、細胞毒性分析…………………………………………………............9
五、細胞凋亡分析………………………………………………………....9

六、粒線體膜電位分析……………………………………………………10
七、細胞生長分析…………………………………………………............10
八、細胞週期分析…………………………………………………............10
九、西方墨點法……………………………………………………………11
十、間接免疫螢光分析……………………………………………………12
十一、共軛焦螢光顯微鏡觀察……………………………………………13
十二、基因轉殖技術………………………………………………………14
十三、統計與數據分析…………………………………………………….14
結果…………………………………………………………………………..15
ㄧ、survivin 在不同人類膀胱癌細胞株的表達及分佈…………………15
二、黃芩素(baicalein)誘發膀胱癌細胞死亡、生長抑制及細胞週期停止
…………………………………………………………..……...............15
三、黃芩素抑制cyclin B1及磷酸化cdc2蛋白的表達…………………16
四、黃芩素會誘發粒線體膜電位的改變及細胞凋亡…………...............16
五、黃芩素抑制survivin蛋白但誘發γ-H2AX蛋白的表達……………17
六、阻斷survivin會誘發γ-H2AX表達及細胞死亡……………………18
七、p38 MAP激酶參與調控黃芩素抑制survivin蛋白的表達…………...18
八、Akt參與黃芩素抑制survivin蛋白的表達…………………………..19
討論…………………………………………………………………..............20
一、黃芩素可能藉由抑制survivin蛋白的表達而使細胞週期停止及造成
細胞凋亡…………………………………………………………...…20
二、Cyclin B1及磷酸化cdc2蛋白參與黃芩素誘發細胞週期停止及細胞
凋亡…………………………………………………………………...21
三、黃芩素誘發p38 MAP激酶的活化參與抑制survivin蛋白的表達..21
四、Akt參與調控黃芩素抑制survivin蛋白的表達…………………….22
五、總結……………………………………………………………………23
參考文獻……………………………………………………………………24
圖一、不同種類的類黃酮素之化學結構……………………………………30
圖二、不同種類的類黃酮素對膀胱癌細胞之存活率的影響……………….31
圖三、黃芩素對膀胱癌細胞之細胞生長的影響…………………………….32
圖四、黃芩素對膀胱癌細胞週期的影響…………………………………….33
圖五、黃芩素對膀胱癌細胞中cyclin B1及cdc2蛋白表達的影響…………..34
圖六、cdc2的抑制劑對黃芩素誘發膀胱癌細胞毒性的影響…………….....35
圖七、黃芩素對膀胱癌細胞粒線體膜電位的影響…………………………36
圖八、黃芩素對膀胱癌細胞凋亡的影響…………………………………….37
圖九、人類膀胱癌細胞中survivin蛋白的表達及分佈…………………….38
圖十、黃芩素抑制膀胱癌細胞中survivin表達但誘發γ-H2AX蛋白表達..39
圖十一、轉殖survivin siRNA抑制膀胱癌細胞中survivin蛋白表達並誘發
γ-H2AX表現……………………………………………………...40
圖十二、survivin siRNA增加黃芩素所抑制的膀胱癌細胞存活率…………41
圖十三、黃芩素對膀胱癌細胞中p38 MAP激酶之表現…………………….42
圖十四、抑制p38 MAP激酶對黃芩素抑制膀胱癌細胞生長的影響………43
圖十五、抑制p38 MAP激酶對黃芩素在膀胱癌細胞中survivin、Bcl-2及
XIAP蛋白表達之影響…………………………………………….44
圖十六、黃芩素對膀胱癌細胞中磷酸化AKT蛋白的影響…………………45
圖十七、Akt抑制劑(wortmannin)對黃芩素在膀胱癌細胞之存活率的影響……………………………………………………………….....46
圖十八、Akt抑制劑(wortmannin)對黃芩素在膀胱癌細胞中survivin蛋白
之表達情形………………………………………………………...47
圖十九、黃芩素抑制人類膀胱癌細胞中survivin表達的分子機制圖…….48

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