| Abstract: | 本研究提議運用DNA微陣列(cDNA晶片)基因表達的分析方法，去探討為何不同的中草藥方劑可以治癒同樣疾病之分子機轉，以建立「同病異治」的理論基礎。在此運用中藥草醫治國人主要之肝癌為模式，使用多種方劑的煎煮液，各以不同稀釋濃度處理後，測定其生長被抑制的程度，以判定這些方劑對不同肝癌細胞株之療效(即鑑定「同病異治」療效)。對於可被方劑抑制之肝癌細胞株，其mRNA被逐一萃取出來做人類基因之微陣列基因表達分析，並歸類(Profiling)因用藥所引發基因表達的異同。如此中醫中「辨證論治」在分子層次之依據也就建立了。第一年的目標包括製作人類基因cDNA晶片，及測試多種方劑抑制肝癌細胞株生長的效果，倘若時間許可也會逐一對於可被有效方劑抑制之肝癌細胞株中，萃取其mRNA從事cDNA晶片之核酸雜交融合反應，以測得初步基因表達的類型。經過一年的努力，我們完成以下的工作：(1)完成6,400點人類基因晶片的製作，可以適時執行Gene expression profiling實驗。(2)完成3種方劑(龍膽瀉肝湯、丹梔逍遙散、Genistein)對4種肝癌細胞株(Chang Liver、Hep3B.1-7、HepG2、HA22T)及1株胎兒肝細胞株(WRL68)生長抑制的效果，並發現在低濃度時，這些方劑對肝癌細胞株的生長有促進作用，而在高濃度時才有抑制效果。至於Genistein則無抗肝癌效果。(3)Hep3B.1-7肝癌細胞株於龍膽瀉肝湯的有效(抑制)濃度處理後，其mRNA被用來與基因晶片做雜交融合反應，並得到滿意結果。以上的成果在我們客觀的評估下，認為達成當初計畫的年度目標。
This research project intended to use the global gene expression profiling method to assess gene expression patterns in a variety of hepatocellular carcinoma cell lines induced by many traditional Chinese herbal drugs that have been suggested to be anticancer. The results could then be correlated to the drug efficacies of suppressing the growth of the cancer cell lines, so that similar gene expression patterns induced by the different drugs can be analyzed and served as the basis of the molecular model for the 「同病異治」 theory. The goal for the first year was to produce cDNA microarrays, test the growth inhibitory effects of the collected Chinese herbal drugs on liver cancer cell lines, and if time allowed microarray hybridization was to be performed with mRNA from those cancer cells treated with effective doses of the drugs. After one year's works, we accomplished the following items: (1) A cDNA-chip containing approximately 6,400 human genes/ESTs was produced that was used immediately for gene expression experiments. (2) Three herbal drugs (龍膽瀉肝湯, 丹梔逍遙散, and soya Genistein) have been tested for the effectiveness of inhibiting the growth of 4 hepatocellular carcinoma cell lines (Chang Liver, Hep3B.1-7, HepG2, and HA22T) and 1 fetal liver cell line (WRL68). We found that, at the lower concentrations, the formal two drugs stimulated the growth of the cells, but became inhibitory when the high concentrations of the herbal extracts were applied. Genistein, however, did not inhibit the growth of any liver cancer cells. (3) Since time allowed us to work further, mRNA from Hep3B.1-7 cells, which had been previously treated with 2,000μg/ml of 龍膽瀉肝湯, were isolated to hybridized with the cDNA-chips that yielded a satisfactory result. Based on the data we obtained in this year, we believe that the goals of the grant proposal have been achieved. |
