- 學位論文
蟛蜞菊功能性粗萃物與主成份抑制乳癌細胞增生之作用研究
Experimental antibreast cancer potential of functional extract and components of Wedelia chinensis
摘要
蟛蜞菊(Wedelia chinensis)，俗稱黃花蜜菜，是青草茶的原料之一，國內學者已經證實可以抑制前列腺癌(Prostate Cancer)，但對於乳癌(Breast cancer)方面的效用尚不清楚， 所以本研究著手研究探討蟛蜞菊對乳癌細胞的影響。而本研究選用的乳癌細胞株分別為MDA-MB-231(estrogen-negative；雌激素受體陰性 )和MCF-7 (estrogen-positive；雌激素受體陽性)。 首先我們先研究，黃花蜜菜的乙酸乙酯層粗萃物(EAW)是否可以抑制乳癌細胞的增生，經由細胞毒性分析(MTT assay)、細胞存活率分析(Trypan blue dye exclusion assay)、細胞聚落形成分析(Colony formation assay)，發現可以有效抑制乳癌細胞的增生，再來經由非固著依賴性生長分析(Anchorage-independent transformation assay)觀察到可以降低MDA-MB-231細胞惡化的特性，接著用HPLC分析，發現蟛蜞菊主成份含有蟛蜞菊內酯(Wedelolactone；WL)和去甲基蟛蜞菊內酯(Demethylwedelolactone；DWL)。所以接著研究這兩種蟛蜞菊主成份(WL and DWL)對抑制乳癌細胞增生的影響，結果發現，WL和DWL跟EAW一樣可以抑制乳癌細胞的增生，和降低MDA-MB-231細胞惡化的特性。另外經由細胞凋亡測定和細胞螢光染色測定(Annexin V/PI和DAPI stain)發現DWL會造成乳癌細胞凋亡，接著用西方墨點法(Western blot)發現DWL造成的細胞凋亡(apoptosis)，會同時影響內生性凋亡路徑(Death-receptor-dependent pathway)和外生性凋亡路徑(Death-receptor-independent pathway)；而WL這個藥物經由細胞週期測定和西方墨點法(Cell cycle assay和Western blot)發現隨著時間的增加和劑量的提高會降低細胞週期調控的G0/G1 phase蛋白的表現。綜合以上實驗，初步發現蟛蜞菊其中的主成份DWL會使MDA-MB-231細胞走向細胞凋亡；WL會使MDA-MB-231細胞停滯在G0/G1期，但是更詳細的分子機制，仍待進一步釐清。
並列摘要
Wedelia chinensis, is one component of Herb Juice. Scholar in Taiwan have already proved that can restrain prostate cancer; however, as for its effect of breast cancer, people are not familiar with that. In this way, we focused on the influence of Wedelia chinesis on breast cancer cells. Moreover, we chose MDA-MB-231(estrogen-negative) and MCF-7(estrogen-positive) as breast tumor cell lines. First of all, we studied whether EAW can restrain the proliferation of cancer cells or not. Through MTT assay , Trypan blue assay , Colony formation assay, this research found that EAW can restrain the proliferation of cancer cells. Secondly, we discovered that EAW can restrain the cell deterioration of MDA-MB-231 through anchorage-independent transformation assay. Then, found that there are WL and DWL by HPLC analysis, in Wedelia chinensis. Furthermore, this research discussed the influence of WL and DWL to restrain breast cancer cells from proliferation and we also found that WL and DWL can restrain breast cancer cells from proliferation and restrain the cell deterioration of MDA-ME-231 cells at the same time. Moreover, this research discovered that the apoptosis on dose dependent and time course by DWL would influent the death-receptor-dependent pathway and death-receptor-independent pathway. Through the experiments above, this research found that DWL would cause the apoptosis of MDA-MB-231 cells. In addition by cell cycle assay and western blot analysis, WL lower the expression of G0/G1 phase related cell cycle regulated protein, which is related to the cell cycle as dose dependent and time course. In conclusion, this research found DWL induce apoptosis, WL cell cycle arrest on G0/G1 phase. Nevertheless, the detailed molecular mechanisms need to be identitied in the future.