臺灣博碩士論文加值系統

在利用天然物篩選抗癌藥物的過程中，本實驗室發現由台灣杉心材萃取出的一種編號176的物質對於人類多種癌症細胞均具有細胞毒殺作用。在利用176處理Hep3B/T2細胞注入裸鼠的實驗中，發現致癌性很強的Hep3B/T2細胞其致癌性受到176的明顯抑制。我們也發現176會干擾細胞微細管聚合，引起細胞週期停滯於G2/M phase。細胞在176處理之後有細胞核微小化及DNA斷裂現象，判斷176所造成的細胞毒殺作用是透過誘發細胞凋亡。本論文研究重點即以人類肝癌細胞株HepG2/A2為受試模組，探討化合物176處理下所發生各種現象之關連，並試圖尋找176抑制癌細胞的機制。
實驗發現176 1g/ml處理24小時後對HepG2/A2細胞造成的毒殺作用為非可逆性。分析176處理後細胞週期及細胞週期蛋白之變化，發現176處理雖會造成細胞週期G2/M phase停滯及cyclin B1堆積，但此影響是可逆的，推測176 所造成的G2/M停滯為其影響細胞的過程，而非造成細胞死亡的主因。此外，以taxol處理HepG2/A2細胞作為比較，發現taxol處理造成之細胞毒殺及G2/M phase停滯現象都是不可逆的，但cyclin B1的堆積是可逆的。進一步分析176處理後HepG2/A2細胞MAPK(Mitogen Activated Protein Kinase)家族成員活化的情形，發現在176處理之後，細胞內JNK(c-Jun N-terminal Kinase)有被活化的跡象，與其他微細管干擾藥物如taxol處理之後的結果一致，說明176所造成的細胞程式凋亡可能是經由JNK的活化。我們並嘗試以microarray分析細胞週期中相關的基因，發現調控細胞分裂的基因cyclin B1、p55cdc、cdc25以及kap1在176處理之下有被活化的現象。
明顯地，176處理可以抑制人類肝癌細胞生長，而cyclin B1、p55cdc蛋白的誘發及JNK的活化可能是重要的調控機制。176毒殺肝癌細胞的詳細機轉及影響細胞週期相關蛋白表現量的真正意義有待深入了解，而對於176與其他微細管干擾藥物如taxol之間相似但又不完全相同的特性亦值得進一步探討。

Using natural products as a source for screening anticancer drugs, we found the cytotoxic compound 176 extracted from heartwood of Taiwania cryptomerioides Hayata. The tumorigenicity of Hep3B/T2 cells in nude mice was suppressed by treatment of compound 176. We found that compound 176 disrupted microtubule formation and caused cell arrest at G2/M phase. Detection of micronucleation and DNA fragmentation indicated that compound 176 causes cell death by inducing apoptosis. Using human hepatoma cell line HepG2/A2 as a model, we try to find out relationships between the effects caused by compound 176 treatment and the mechanism of its cytotoxicity to cancer cells.
It was found that the cytotoxic effect is non-reversible after 24hr compound 176 treatment. Cell cycle analysis showed that the cells arrested at G2/M is reversible. This suggests that G2/M arrest is a result of compound 176 treatment, not the cause of cell death. The protein level of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors were examined, The protein level of cyclin B1 increased after compound 176 treatment and is declined after drug removement. Activation of MAPK superfamily is also examined, compound 176 treatment induces c-Jun N-terminal Kinase (JNK) activation, which is related to apoptosis induction, on HepG2/A2 cells. Furthermore, using microarray, we had found several genes related with cell mitosis: cyclin B1, p55cdc, cdc25 and kap1 are expressed after 176 treatment.
The experiment has shown that compound 176 has strong cytoxicity to hepatoma cells. The elevation of cyclin B1, p55cdc protein level and activation of JNK might be important regulating signals, but the entire mechanism remains unclear. The similarity between compound 176 and other antimicrotubule agent, such as taxol, is also an interesting subject that requires further investigation, which may be able to provide more evidence of compound 176- caused cell death.
英文摘要.......................................................2
緒論...........................................................3
材料及方法.....................................................9
結果..........................................................17
討論..........................................................24
參考文獻......................................................29
圖表..........................................................33

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