Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23750
DC FieldValueLanguage
dc.contributor許國堂zh_TW
dc.contributor陳春榮zh_TW
dc.contributor沈炯祺zh_TW
dc.contributor馬辛一zh_TW
dc.contributor.advisor蘇鴻麟zh_TW
dc.contributor.author張正一zh_TW
dc.contributor.authorChang, Cheng-Yien_US
dc.contributor.other中興大學zh_TW
dc.date2012zh_TW
dc.date.accessioned2014-06-06T07:21:06Z-
dc.date.available2014-06-06T07:21:06Z-
dc.identifierU0005-2007201110320100zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/23750-
dc.description.abstractGefitinib 是一種選擇性作用於上皮生長因子接受器的酪氨酸激酶抑制劑,目前仍嘗試於臨床上治療癌症病患,包括神經膠細胞瘤患者。然而目前對Gefitinib於神經膠細胞瘤所產生抑制癌細胞的詳細分子作用機轉仍不甚明瞭。Gefitinib可抑制神經膠細胞的生長,並誘使細胞產生細胞凋亡。於實驗中可發現Gefitinib經由內在細胞凋亡路徑誘使H4細胞死亡,其中作用的過程包括Bax蛋白於粒線體的位置改變,粒線體外膜的通透性改變,細胞色素C釋放至細胞質,caspase-3及caspase-9的活化。Gefitinib造成的神經膠細胞瘤凋亡的現象,可經由加入Bax siRNA及Bax蛋白通道抑制劑的方式抑制Bax的表現來緩和細胞凋亡,此現象說明Bax在此細胞凋亡過程扮演重要角色。Gefitinib造成Bad蛋白去磷酸化,特別是在Ser-112的位置上,此過程使Bad蛋白與Bcl-2及Bcl-xL結合的狀況增加。而Gefitinib造成Bad蛋白去磷酸化會伴隨著cAMP含量減少及protein kinase A的活性降低。Adenylyl cyclase 活化劑forskolin 可減緩Gefitinib造成的Bad蛋白去磷酸化,Bax蛋白於粒線體膜上位置的改變,caspase-3及caspase-9的活化,及細胞活性喪失等現象,而蛋白激酶A抑制劑H89則具有與forskolin相反的效果。而較無法解釋的是加入非選擇性蛋白磷酸酶抑制劑okadaic acid,可減緩Gefitinib所造成的影響,卻不改變Bad蛋白去磷酸化的程度。類似的情形在不同細胞株U87及T98G亦能觀察到,而U87本身具有較高的蛋白激酶A的活性。相對於H4細胞,U87對Gefitinib的反應較為遲緩且抗藥性較高。降低蛋白激酶A的活性可使H4,T98G及U87對Gefitinib毒性的敏感度增加,Bad於Serine-112位置去磷酸化程度,及caspase-3/caspase-9活化程度增加。本實驗發現gefitinib造成的神經膠細胞瘤凋亡的過程與Bad/Bax蛋白訊息傳導路徑有關。蛋白激酶A去活化在促使Bad蛋白引發細胞凋亡的過程中扮演著一定角色。zh_TW
dc.description.abstractGefitinib, a selectively epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is under clinical test and use in cancer patients, including glioma. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma remain largely uncharacterized. Gefitinib inhibited cell growth and induced apoptosis in human glioma cells. Gefitinib induced death of H4 cells with characteristics of intrinsic apoptotic pathway, including Bax mitochondrial translocation, mitochondrial outer membrane permeabilization, cytochrome c cytosolic release, and caspase-9/caspase-3 activation. The importance of Bax in mediating gefitinib-induced apoptosis was confirmed by the attenuation of apoptosis by Bax siRNA and Bax channel blocker. Gefitinib caused Bad dephosphorylation, particularly in serine-112, and increased its binding preference to Bcl-2 and Bcl-xL. The dephosphorylation of Bad in gefitinib-treated cells was accompanied by decreased intracellular cyclic AMP content and protein kinase A (PKA) activity. Adenylyl cyclase activator forskolin attenuated, but PKA inhibitor H89 augmented, gefitinib-induced Bad dephosphorylation, Bax mitochondrial translocation, caspase-9/caspase-3 activation, and viability loss. Intriguingly, a nonselective protein phosphatase inhibitor okadaic acid alleviated gefitinib-induced alterations, except Bad dephosphorylation. In parallel with the higher basal PKA activity, U87 cells showed a delayed and relatively resistant response to gefitinib treatment than H4 and T98G cells. The inactivation of PKA sensitized H4, T98G, and U87 cells towards gefitinib cytotoxicity, Bad dephosphorylation in serine-112, and caspase-9/caspase-3 activation. Our findings suggest the involvement of the Bad/Bax signaling pathway in gefitinib-induced glioma apoptosis. Furthermore, the inactivation of PKA was shown to play a role in triggering the proapoptotic function of Bad.en_US
dc.description.tableofcontents中文摘要..................................................i Abstract.................................................ii 目 錄..................................................iii 圖目次...................................................vi 壹、前言..................................................1 一、促上皮細胞生長因子傳遞路徑與神經膠細胞瘤..............1 二、以上皮細胞生長因子接受器為標的治療腫瘤................2 三、B-細胞淋巴瘤基因2(Bcl-2 )相關蛋白造成神經膠細胞瘤細胞凋亡........................................................2 四、以抑制促上皮生長因子接受器為標的的方式治療腫瘤所造成的抗藥性狀況..................................................3 1.自分泌(autocrine)或旁分泌(paracrine)....................3 2.促上皮生長因子接受器產生突變............................3 3.傳導路徑下游分子持續活化................................3 4.替代路徑活化............................................4 五、Gefitinib於神經膠細胞瘤患者使用狀況及其面臨的困境.....4 六、研究動機..............................................5 貳、研究材料與方法........................................6 一、試驗藥物及細胞株培養..................................6 二、細胞蛋白質之萃取 (Collection of cell lysate)..........6 三、細胞凋亡及活性評估....................................6 1.Trypan blue dye exclusion, MTS assay, 及 LDH diagnostic kit.......................................................6 2.流式細胞儀(flow cytometry, FACE scan)...................7 3.DNA fragmentation assay.................................7 4.Caspase activity assay..................................7 四、神經細胞瘤細胞轉染....................................8 1.小干擾RNA(small interfering RNA, siRNA)轉染.............8 2.Mcl-1及Akt質體轉染:為使神經膠細胞瘤過度表現.............8 五、細胞核質蛋白萃取 (Nuclear/ Cytosol protein extraction)...............................................8 六、免疫墨點分析及免疫沉澱分析(Immnuoblot, immunoprecipitation)......................................9 1.西方墨點分析(Western blot)..............................9 2.免疫沉澱分析(immnuoprecipitation)......................10 七、粒線體膜電位測量.....................................10 八、Cyclic AMP(cAMP)含量檢測.............................10 九、Protein kinase A (PKA)活性測量.......................11 十、統計分析.............................................11 參、實驗結果.............................................12 一、Gefitinib對人類膠細胞瘤細胞具有生長抑制(cytostatic)及細胞毒殺(cytotoxic)的效果..................................12 二、Gefitinib能誘使人類神經膠細胞瘤細胞產生細胞凋亡......12 三、Gefitinib誘使神經膠細胞瘤產生細胞凋亡過程中粒線體扮演的角色.....................................................13 四、Gefitinib對上皮細胞生長因子接受器下游細胞訊息傳導所造成的改變及此改變對細胞所造成的影響.........................13 1. Gefitinib 抑制上皮生長因子/上皮生長因子接受器的細胞傳遞訊號.......................................................13 2.JAK/STAT pathway.......................................14 3.MAPK pathway...........................................14 4.PI3K/AKT pathway .......................................15 5.cAMP/PKA pathway .......................................15 五、Bcl-2 家族在gefitinib 所造成的細胞凋亡過程中所扮演的角色及與EGFR下游訊號改變的相互關係...........................15 1. 經gefitinib處理誘發的細胞凋亡現象中Bcl-2家族及與細胞凋亡相關的蛋白質的表現狀況...................................15 2. Bax蛋白與經gefitinib處理誘發的細胞凋亡現象有關........16 六、Gefitinib減少Bad磷酸化的程度並引使神經膠細胞瘤細胞產生細胞凋亡...................................................16 七、Gefitinib與PKA的相互關係.............................17 1. Gefitinib會抑制PKA的活性..............................17 2. PKA與蛋白磷酸酶(protein phosphatases)與gefitinib誘發細胞凋亡的過程有關...........................................17 3. 不同細胞株細胞凋亡反應的差異及此差異與PKA活性的相關性.17 4. H89提高gefitinib對細胞的毒性反應......................18 肆、討論與結論...........................................19 伍、附表與圖表...........................................23 陸、參考文獻.............................................63zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.subjectGefitiniben_US
dc.subjectGefitinibzh_TW
dc.subjectglioma cellen_US
dc.subjectapoptosisen_US
dc.subject神經膠細胞瘤zh_TW
dc.subject細胞凋亡zh_TW
dc.titleGefitinib誘發神經膠細胞瘤細胞凋亡機制探討zh_TW
dc.titleMolecular mechanisms of gefitinib-induced apoptosis in experimental glioma cellsen_US
dc.typeThesis and Dissertationzh_TW
item.grantfulltextnone-
item.openairetypeThesis and Dissertation-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.cerifentitytypePublications-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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