Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36922
標題: 增加丹參癒合組織中丹參酮含量以及丹參酮 I 抑制非小細胞肺癌作用之研究
Study on increasing the yield of tanshinones for the calli of Salvia miltiorrhiza Bunge and the anticancer effects of tanshinone I in none-small-cell-lung cancer cell
作者: 李鎮宇
Lee, Chen-Yue
關鍵字: Denshan;丹參;Tanshinones;Lung Cancer;丹參酮;肺癌
出版社: 農藝學系所
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摘要: 
丹參為唇形科之多年生草本植物,許多文獻記載其具有心血管之療效,為重要之傳統中草藥。本論文的主要目標是有系統的研究丹參有效成份之藥物基因體學:利用農桿菌轉殖、有效成份誘導、主動誘變以及生物晶片等系統進行丹參有效成分之研究,以及了解丹參酮抑制肺癌生長與轉移之可能機制。
本論文發現利用丹參癒合組織培養以植物生長調節劑2, 4-D或zeatin誘導隱丹參酮,丹參酮I及丹參酮IIA之含量有明顯之差異,另亦探討丹參植株再生與農桿菌轉殖之最適條件。並從中發展主動誘變轉殖系有效成份之篩選平台,其篩選系統是利用植物生長調節劑2, 4-D及細胞本身顏色之變化進行轉殖系之篩選;1435個轉殖細胞系經過非成份誘導培養之細胞外觀顏色篩選後,進行HPLC成分分析以及南方墨點法鑑定,篩選獲得T1-T6個主動誘變細胞系,其有效成份(丹參酮I、丹參酮IIA與隱丹參酮)相較於非轉殖丹參細胞系於非誘導之情況下,其細胞成份產量則可以增加5-100倍。
本論文進行抑癌藥物平台之開發與丹參有效成份抑制肺癌細胞CL1-5藥理機制研究,且完成丹參酮成份抑制人類肺癌細胞CL1-5之動物試驗,並發現丹參酮I抑制肺癌細胞CL1-5之可能機制。以MTT、細胞群聚能力分析、細胞遷徙能力、Gelatin zymography分析和試管內侵犯能力等不同試驗分析,發現丹參酮I在CL1-5百分之十致死濃度內可以抑制試管內人類肺腺癌細胞之生長、轉移以及膠原蛋白酶之表現;經IL-8啟動子報導基因與EMSA分析結果發現丹參酮I可抑制轉錄因子AP-1 and NF-κB與IL-8啟動子之結合造成IL-8 mRNA表現減少;利用人類微陣列晶片分析發現丹參酮I可以抑制癌細胞增殖與轉移相關之訊號傳遞,其中包含PDGF-β, Shc, Shb, ephrin-A1, Rab8, MAPKK3, CD4 and Rac 1等路徑。
研究也發現,利用Real-time PCR以及cDNA microarray的方法,發現丹參酮I能夠抑制由marcophage-conditioned medium所誘導Rac1的表現與活性,另外也發現丹參酮I能夠抑制CL1-5肺癌細胞的移動和形態改變。丹參酮I可以抑制由marcophage-conditioned medium所誘導Rac1下游與肌動蛋白相關的α-actinin與β-catenin蛋白質以及cofilin的去磷酸化作用。
由上述之結果顯示丹參有效成份未來將具有增產之可能性,而丹參酮I具有抑制肺癌生長與轉移之能力,未來將有可能成為肺癌治療之輔助藥物。

Salvia miltiorrhiza Bunge, generally known as Danshen, the perennial herb is a traditional Chinese herbal medicine with multiple functions. However, its systematic study on pharmacogenomics has not been well established. In this study, by using an omnibus strategy, we investigate Agrobacteria transgenic, ingredients inducible, T-DNA activation tagging and microarray system, as well as the anti-cancer and anti-invasive mechanisms in non-small-cell lung cancer (NSCLC).
We have set up a zeatin/2-4D inducible system of Danshen active ingredients, and confirmed by HPLC. Furthermore, the regeneration and Agrobacterium-transgenic systems were established by a BA regenerative and selective medium. The results of indicated that there are more than six independently transgenic cell lines with red color from 1435 transgenic lines while grown in non-inducible medium. ATM lines T1-T6 showed significant increases in the yields of tanshinone-I (up to 43-fold), tanshinone-IIA (up to 26-fold) and cryptotanshinone (up to 104-fold) compared with those of the nontransgenic lines on 2,4-D medium. Interestingly, the yield of cryptotanshinone from line T4 on 2,4-D medium was two times higher than that of the nontransgenic lines on trans-zeatin riboside medium.
For the pharmacologic study of tanshinones, we found that the active ingredients including tanshinoneⅠand tanshinoneⅡA can significantly inhibit cell growth, migration, invasion, gelatinase activity, and IL-8 transcriptional regulation of human lung adenocarcinoma cell CL1-5 with highly invasive capability. Interestingly, only TanshinoneⅠ could significantly reduce AP-1 and NF-κB binding activity of CL1-5 cells measured by EMSA, as well as the tumorigenesis of mice. However, tanshinoneⅡA revealed a much stronger pro-apoptotic activity than TanshinoneⅠ measured by flow cytometry analysis. To further interpret the mechanisms of Danshen on anti-metastasis and anti-angiogenesis, human cDNA microarray was applied to this study. The results showed that tanshinoneⅠcan suppress several metastasis-related genes including PDGF-β, Shc, Shb, ephrin-A1, Rab8, MAPKK3, CD4 and Rac 1.
Furthermore, tanshinone I significantly block increased the expression of focal adhesion proteins α-actinin and β-catenin, and upregulated phosphorylation of cofilin through the inhibition of Rac1 expression and activity. The in vivo antimetastatic effects of tanshinone I were evaluated in SCID mice following a tumor xenograft implantation of CL1-5 cells. Tanshinone I (0.3 mg per kg daily) inhibited lung metastasis and cofilin dephosphorylation in CL1-5-bearing mice.
These data suggested that Danshen has the potential for increasing the yields of tanshinons. On the other hand, tanshinone I may have the potential for anti-cancer and anti-metastasis, and may be helpful in treating the cancer, as a medicine or an assistant chemotherapy in lung cancer.
URI: http://hdl.handle.net/11455/36922
其他識別: U0005-3107200812092200
Appears in Collections:農藝學系

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