| 摘要: | 本論文主要研究市面上常用且可能具有改善癌症之天然物對人類血癌（HL-60、U937、K562）等細胞株之影響，並探究其作用機轉。所試驗之天然物包括大陸產蜂膠（Propolis from China）、巴西產蜂膠（Propolis from Brazil）、台灣產赤松（Pinus densiflora）松針及台灣五葉松（Pinus morrisonicola）松針之抽提物（分別簡稱為RPS及FP）以及Pycnogenol（法國海濱松Pinus pinaster樹皮抽提物）。 實驗結果發現上述天然物皆具抑制人類血癌細胞增殖的效果，其中蜂膠、松針抽提物及Pycnogenol對此三類細胞之抑制作用呈劑量暨時間依存性關係。 大陸產蜂膠95﹪酒精粗抽提物對HL-60、U937及K562細胞之IC50（抑制細胞增殖達50﹪之濃度）分別為30.32、300.16、90.21 μg/ml，巴西產蜂膠酒精粗抽提物之IC50分別為150.48、350.35、180.09 μg/ml，赤松松針酒精抽提物（RPS）之IC50分別為166.07、93.96、177.29 μg/ml，台灣五葉松松針酒精抽提物（FP）之IC50則分別為209.01、100.18、199.07 μg/ml，而Pycnogenol之IC50分別為200.02、40.26、100.98 μg/ml。進一步以超臨界流體等不同方法抽提之各種蜂膠抽提物中，發現以大陸產蜂膠經超臨界流體萃取後所剩餘殘渣再以95﹪酒精抽提所得抽提物 (M-ReE) 對上述血癌細胞株有較好的抑制效果，IC50分別為16.80、24.30、6.20 μg/ml。 以細胞週期分析發現RPS及Pycnogenol能夠使HL-60之G0/G1期細胞族群比例顯著增加，同時伴隨有sub-G0/G1凋亡族群增加的現象。RPS（50～100 μg/ml）及Pycnogenol低劑量（50～125 μg/ml）處理下，會使HL-60細胞呈現NBT陽性反應、NSE陽性反應、並增加CD11b表現量，顯示其具有促進分化之能力。然而RPS及Pycnogenol對U937及K562並無促進分化之現象。高劑量（150～200 μg/ml） RPS及Pycnogenol對HL-60細胞均具毒殺作用，在分別以RPS、FP及Pycnogenol 個別的IC50處理下，三種血癌細胞均呈現細胞核斷裂與凋亡小體產生、增加phosphatidylserine翻轉至細胞膜外側，核DNA斷裂成180 bp梯度片段及Caspase 3被活化現象。此外，以Caspase 3抑制劑前處理一小時，可有效降低由RPS及Pycnogenol所誘發的Caspase 3活化及生長抑制作用。這些結果顯示松針抽提物及Pycnogenol抑制三種血癌細胞之生長與Caspase傳導之細胞凋亡路徑之誘發有關。 當RPS及Pycnogenol劑量高達250 μg/ml對正常人類Con A活化之單核白血球細胞 (PBMC)或人類其他正常細胞株並無顯著毒殺作用。因而據此推論RPS及Pycnogenol具有選擇性使血癌細胞凋亡及分化的能力，頗具未來發展為治療血癌疾病相關藥物之潛力，值得進一步研發。; We have investigated the effects of two pine needles extracts, RPS (Soxlet extract from Pinus densiflora) and FP (Soxle extract from Pinus morrisonicola), two ethanol extracts of raw propolis (obtained from China and Brazil), and Pycnogenol on human leukemia cells HL-60, U937 and K562. A dose-dependent decrease in cell proliferation was observed in all extracts investigated, but with different degrees of inhibition. The IC50 of RPS, FP, and Pycnogenol were 166.07, 209.01 and 200.02 μg/ml for HL-60; 93.96, 100.18 and 40.26 μg/ml for U937; and 177.29, 199.07 and 100.98 μg/ml for K562 cells, respectively. The IC50 of propolis from China were 30.32, 300.16 and 90.21 μg/ml for HL60, U937 and K562, respectively. The IC50 of propolis from Brazil were 150.48, 350.35 and 180.09 μg/ml for HL60, U937 and K562, respectively. In order to increase the amounts of flavonoids and to get rid of the wax-like materials and other undesired compounds, which may cause allergic reaction, raw propolis was extracted by various methods. Nine active propolis flavonoids were analyzed to indicate the amounts of active compounds in propolis. Most of the extracts exhibited a positive inhibitory effect. The inhibitory effects on the cell viability of leukemia cancer cells were also investigated. The amount of the total flavonoids in ReE extract of propolis from China were the highest one (92022 μg/g). The IC50 of ReE extract was the lowest one compared to the other propolis extracts. The IC50s were 16.80, 24.30 and 6.20 μg/ml for HL60, U937 and K562, respectively. These results indicated that the quantity of flavoids in ReE extract may be related to its significant growth-inhibiting effects. Further studies were performed to investigate the mechanisms involved in the growth-inhibiting effects on leukemia cells. The results from DAPI staining, subnuclei analysis, and DNA fragmentation assay indicated the induction of apoptosis in HL-60 cells by RPS and Pycnogenol . Cell cycle analysis revealed the significant increase of cell number in G0/G1 phase by RPS or Pycnogenol. Based on the changes of cell morphology, the increase of NBT reduction, NSE activity, and CD-11b-positive cells, we suggested that that RPS and Pycnogenol at a dose below 100 μg/ml has the ability to induce HL-60 to differentiation into monocytic lineage. The RPS or Pycnogenol induced apoptosis is mediated by activating caspase 3 activity. One hour pretreatment with of z-DEVD-fmk, a caspase 3 inhibitor, not only decreased the activity of caspase 3 but also reduced the percentage of apoptotic cells by RPS and Pycnogenol. |
