Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20818
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dc.contributor.advisor陳健尉zh_TW
dc.contributor.advisorJeremy J.W. Chenen_US
dc.contributor.author佘慧芳zh_TW
dc.contributor.authorSher, Hui-Fangen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T07:14:36Z-
dc.date.available2014-06-06T07:14:36Z-
dc.identifier.urihttp://hdl.handle.net/11455/20818-
dc.description.abstractSalvia multiorrhizae Bunge,通常稱為丹參,是長久以來用於治療心血管疾病的傳統中草藥。丹參酮Ⅰ、丹參酮ⅡA和隱丹參酮是從丹參根部萃取出來主要的有效成份,前人的研究顯示具有抗發炎、抗氧化和細胞毒殺活性。在本研究中,想了解丹參酮Ⅰ、丹參酮ⅡA和隱丹參酮對於高侵犯性人類肺腺癌細胞株(CL1-5)的抑癌影響,並進而探討其作用機制。結果顯示丹參酮Ⅰ、丹參酮ⅡA及隱丹參酮在低濃度(10、10和5 µg/ml)時即明顯的抑制人類肺腺癌細胞生長,並隨時間增加而明顯增強其抑制能力。在與巨噬細胞共同培養後,肺癌細胞展現出更高侵犯潛力和分解細胞外基質能力,而丹參酮Ⅰ、丹參酮ⅡA及隱丹參酮能抑制所誘發的細胞遷徙能力和培養基中明膠酵素(gelatinase)的活性。以Matrigel為基礎的試管內侵犯能力分析結果顯示,丹參酮Ⅰ和丹參酮ⅡA能抑制macrophage-conditioned medium所誘發的肺癌細胞更高試管內侵犯能力。經即時定量聚合酶連鎖反應分析結果顯示,丹參酮Ⅰ及丹參酮ⅡA皆能抑制macrophage-conditioned medium所誘發之癌轉移相關基因IL-8 mRNA的表現量,並且隨著丹參酮Ⅰ及丹參酮ⅡA的濃度增高而減少。從丹參酮Ⅰ及丹參酮ⅡA能降低受IL-8啟動子調控的螢光酵素報導酵素活性,證實丹參酮Ⅰ及丹參酮ⅡA對於所誘發IL-8表現的抑制作用是在於轉錄作用階層。利用電泳移動偏移分析(EMSA),丹參酮Ⅰ明顯的降低macrophage-conditioned medium所誘發之轉錄因子NF-κB與AP-1與IL-8基因啟動子的結合活性,本研究結果證實丹參酮Ⅰ是直接抑制NF-κB及AP-1的結合活性且阻斷此訊息傳遞進而抑制CL1-5細胞中IL-8基因表現的增加。此外,經由細胞流速測定儀分析結果得知,在丹參酮ⅡA處理CL1-5肺癌細胞72小時後,凋亡的細胞數明顯地增加,而且隨著丹參酮ⅡA處理時間越長細胞產生凋亡的比例明顯增加,丹參酮ⅡA是藉由誘發肺腺癌細胞走向自然凋亡來抑制其生長與增殖。進一步,於癌細胞與巨噬細胞共同培養時加入這些丹參有效成份,利用癌轉移相關微陣列分析癌細胞對藥物反應的基因剖繪,以闡釋不同丹參有效成份各自的作用機轉。丹參酮Ⅰ、丹參酮ⅡA及隱丹參酮在非致死濃度下(10、10和5 µg/ml)抑制了一些癌轉移相關基因,包括PDGF-β、Shc、Shb、ephrin-A1、Rab8、MAPKK3和CD44等。並且從動物實驗發現,預先施打丹參酮Ⅰ確實能使腫瘤縮小。綜合以上這些實驗結果,證實丹參酮Ⅰ、丹參酮ⅡA和隱丹參酮具有抗癌及抗癌轉移能力的潛力,可能有助於發展癌症治療策略,或作為抗癌及輔佐化療用藥。zh_TW
dc.description.abstractSalvia miltiorrhizae Bunge, commonly known as “Danshen”, has long been used as a traditional Chinese herbal medicine for cardiovascular disorders. Tan- shinone Ⅰ, tanshinone ⅡA and cryptotanshinone are major active components isolated from the roots of Danshen, has been shown anti-inflammatory, anti- oxidative and cytotoxic activities. In this study, we investigated the anticancer effect and mechanisms of action of tanshinoneⅠ, tanshinone ⅡA and crypto- tanshinone in highly invasive human lung adenocarcinoma cell lines (CL1-5). Results showed that tanshinoneⅠ, tanshinone ⅡA and cryptotanshinone sig- nificantly inhibited CL1-5 cells growth in low concentration (10, 10 and 5µg/ml) and time dependent. After coculture with macrophages, lung cancer cell lines exhibited higher invasive potentials and matrix-degrading activities. TanshinoneⅠ, tanshinone ⅡA and cryptotanshinone inhibited macrophage-conditioned medium induced lung cancer cell migration activity and inhibited gelatinase activity in the media. Matrigel-based invasion assay showed that tanshinoneⅠand tanshinone ⅡA inhibited in vitro invasive activity of CL1-5 cells induced by macrophage- conditioned medium. TanshinoneⅠand tanshinone ⅡA inhibited macrophage- conditioned medium induced IL-8 mRNA levels in a dose-dependent manner as measured by real-time quantitative PCR. The inhibition of IL-8 induction by tan- shinoneⅠand tanshinone ⅡA is a transcriptional event, as shown by tanshinone Ⅰand tanshinone ⅡA significant reduction of IL-8 promoter-luciferase reporter activities. By electrophoretic mobility assay, tanshinoneⅠ significantly reduced the AP-1 and NF-κB binding activity induced by macrophage-conditioned medium. Altogether, our results demonstrate that tanshinoneⅠ inhibits IL-8 induction in CL1-5 cells; this inhibition is mediated by the attenuation of AP-1 and NF-κB binding activity and blocked signaling pathway. In addition, result from flow cyto- metry analysis revealed a time-dependent increase of apoptotic cells in 72 hours after treating CL1-5 cells with tanshinone ⅡA. Tanshinone ⅡA inhibits growth and proliferation of lung adenocarcinoma cells by inducing apoptosis. Further- more, put into these active components isolated from the roots of Danshen on cancer cells/macrophages cocultures, utilize metastasis-associated microarray to analysis the gene expression profiles of lung cancer CL1-5 cells that responding to differentially active components, can apply to explain the functional mecha- nisms of different components. Below sublethal concentrations of tanshinoneⅠ, tanshinone ⅡA and cryptotanshinone (10, 10 and 5 µg/ml), several metastasis- related genes were suppressed, including PDGF-β, Shc, Shb, ephrin-A1, Rab8, MAPKK3 and CD44. In addition, the results of animal experiment showed that tanshinoneⅠ can reduce the tumor volume. To sum up the above experimental results, we conclude that tanshinoneⅠ, tanshinone ⅡA and cryptotanshinone have anti-cancer and anti-metastasis potential, and may be helpful in treating cancer, or assist in chemotherapy.en_US
dc.description.tableofcontents中文摘要..................................................................................................................i 英文摘要................................................................................................................iii 目錄........................................................................................................................v 表目錄...................................................................................................................vii 圖目錄..................................................................................................................viii 緒論.......................................................................................................................1 一、癌症形成之機轉...........................................................................................2 二、非小細胞肺癌...............................................................................................4 三、癌轉移..........................................................................................................6 四、血管新生......................................................................................................7 五、腫瘤相關巨噬細胞........................................................................................9 六、發炎反應與癌症..........................................................................................11 七、中草藥丹參.................................................................................................13 八、研究目的和策略..........................................................................................15 材料方法...............................................................................................................17 一、細胞株培養............................................................................................17 二、中草藥丹參有效成份............................................................................17 三、細胞存活能力分析................................................................................17 四、MTT分析法..........................................................................................17 五、癌細胞與單核球細胞共同培養系統.............................................................18 六、試管內細胞遷徙能力分析...........................................................................19 七、Gelatin zymography分析...........................................................................19 八、試管內侵犯能力分析...................................................................................19 九、總量核醣核酸的純化.............................................................................20 十、反轉錄反應與回收cDNA............................................................................20 十一、即時定量聚合酶連鎖反應.......................................................................21 十二、大腸桿菌勝任細胞的製備.......................................................................21 十三、大腸桿菌的轉形方法..............................................................................22 十四、少量質體DNA之製備............................................................................22 十五、短暫性轉染與螢光報導基因分析............................................................23 十六、細胞核萃取物的備製..............................................................................24 十七、EMSA (electromobility shift assay)........................................................25 十八、利用流式細胞儀來檢測CL1-5細胞凋亡的情況......................................27 十九、基因微陣列系統......................................................................................28 二十、雜交探針製備....................................................................................28 二十一、基因微陣列雜交法..........................................................................29 二十二、免疫化學之呈色偵測及顯色圖形之影像處理...................................29 二十三、資料分析..............................................................................................30 二十四、生物體內抗腫瘤影響(In vivo antitumor effect)分析..............................30 結果..............................................................................................................31 ~ 38 討論..............................................................................................................39 ~ 43 結論......................................................................................................................44 圖表..............................................................................................................45 ~ 69 參考文獻.......................................................................................................70 ~ 79 附錄..............................................................................................................80 ~ 88zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學院碩士在職專班zh_TW
dc.subjectDanshenen_US
dc.subject丹參zh_TW
dc.title中草藥丹參有效成分對人類肺腺癌細胞株抑癌的影響zh_TW
dc.titleThe anticarcinogenic effect of active components isolated from Chinese herbal medicine Danshen (Salvia miltiorrhiza Bunge) on human lung adenocarcinoma cellsen_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
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