臺灣博碩士論文加值系統

Dexamethasone已經知道可以抑制癌細胞的侵襲性，但是其中的機制還不清楚。本研究探討人類惡性神經膠質瘤細胞中dexamethasone抑制細胞侵襲性的機制。人類惡性神經膠質瘤細胞具有內生性MMP-2的活性，且其活性與細胞的惡性度有關。p38 MAPK的抑制劑SB203580與p42/44 ERK的抑制劑PD98059都可以降低人類惡性神經膠質瘤細胞U87MG中MMP-2的活性。Dexamethasone可以誘發U87MG細胞中MKP-1的表現，所誘發的MKP-1隨著dexamethasone濃度的增加而增加，並隨著時間的增加而持續性表現。轉錄作用的抑制劑
actinomycin D與轉譯作用的抑制劑cyclohexamide都可以阻斷U87MG細胞中dexamethasone所誘發MKP-1的表現，顯示dexamethasone所誘發MKP-1的表現是經由新蛋白的合成。RU486 (糖皮質激素受體拮抗劑)與triptolide (MKP-1阻斷劑)都可以降低U87MG細胞中dexamethasone所誘發的MKP-1，RU486可以逆轉dexamethasone所抑制的細胞侵襲性。NO donor sodium nitroprusside (SNP)可以逆轉U87MG細胞中dexamethasone所抑制MMP-2的活性。Triptolide可以逆轉U87MG細胞dexamethasone所抑制iNOS的活性。綜合以上結果可以得知，在U87MG細胞中，dexamethasone透過誘發MKP-1而抑制iNOS的表現及NO的釋出，進而抑制MMP-2的活性與細胞侵襲性。

Dexamethasone has been shown to inhibit tumor invasion, however,
the underneath mechanisms have not been elucidated. In the present study, we investigated the mechanism of which dexamethasone regulated the invasion in human malignant glioma cells. We found that human malignant glioma cells constitutively expressed MMP-2, which is well correlated with their malignancies. Treatment of glioma cells with SB203580 (a p38 MAPK inhibitor) or PD98059 (a p42/44 ERK inhibitor) reduced MMP-2 activity. Incubation of human malignant glioma cells with dexamethasone resulted in a dose- and time-dependent induction of a dual specific MAPK phosphatase, MAPK phosphatase-1(MKP-1). Treatment of glioma cells with actinomycin D (a transcription inhibitor) or cyclohexamide (a translation inhibitor) before the addition of dexamethasone decreased MKP-1 protein level, suggesting dexamethasone-induced MKP-1 expression required de novo protein synthesis. Pretreatment of cells with RU486 (a glucocorticoid receptor antagonist) or triptolide (a pharmacological MKP-1 blocker) reversed the inhibition of MMP-2 activity and cell invasion by dexamethasone. Treatment of glioma cells with NO donor, sodium nitroprusside (SNP), reversed the inhibition of MMP-2 activity by dexamethasone. In addition, pretreatment of triptolide reversed dexamethasone-reduced inducible nitric oxide synthase (iNOS) expression. Taken together, our data suggest that dexamethasone may inhibit tumor cell invasion and MMP-2 activity through MKP-1 induction, and that NO is the positive regulator of MMP-2 in human malignant glioma cells.

壹、緒論..........................................1
一、惡性腦瘤.......................................1
二、基質金屬蛋白酶 (MMP)............................1
三、Dexamethasone.................................4
四、MAP kinase phosphatase-1......................5
五、NO............................................5
貳、研究材料與方法..................................7
一、研究材料.......................................7
(一)、實驗細胞株...................................7
(二)、藥品試劑.....................................7
(三)、常用儀器....................................10
(四)、常用溶液....................................11
二、研究方法......................................14
(一)、神經膠質瘤細胞的培養..........................14
(二)、細胞蛋白質製備(preparation of cell lysate)...14
(三)、蛋白質定量法(protein assay)..................15
(四)、西方點墨法(Western blotting).................15
(五)、Gelatin zymography..........................15
(六)、細胞存活率分析方法(MTT assay).................16
(七)、siRNA transfection..........................16
(八)、細胞侵襲性實驗(Invasion assay)................17
参、實驗結果與分析..................................18
一、在不同惡性度的神經膠質瘤細胞中MMP-2的活性..........18
二、Dexamethasone誘發U87MG細胞中MKP-1的表現..........18
三、在U87MG細胞中MMP-2的表現透過MKP-1................19
四、NO參與U87MG細胞中MMP-2的活性.....................20
肆、結論與討論......................................21
伍、參考文獻........................................24
實驗結果圖表........................................35

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