香椿葉水萃取液對HL-60與 K-562 細胞株氧化性傷害之影響

越來越多的證據顯示，自由基在人類許多疾病的發生中扮演著十分重要的角色。現代醫學發現癌症的發生與自由基的侵襲有很直接的關係，因此，如何預防免於受到自由基的傷害是一項重要課題。白血病即俗稱的血癌，其傳統的治療方式有化學治療、放射線治療及骨髓移植，而目前以化學治療為主，但仍無法有效的治癒白血病，只能暫時的控制病情發展，且隨時有發病的危機存在。最終治癒的方法就只有骨髓移植，但骨髓配對的機率卻只有百萬分之ㄧ，因此對於如何能有效的輔助病況的發展為本實驗的動機。本研究先評估兩株人類白血病細胞株（HL-60與 K-562 cell line），受過氧化氫（hydrogen peroxide；以下簡稱H2O2）氧化性傷害之影響，建立H2O2作用之時間與劑量之模式，評估細胞受到氧化性傷害之情形。接著再探討HL-60與 K-562 cell line，在香椿葉水萃取液（Toona sinensis Roemor Concentrate；以下簡稱TS）作用下，TS造成HL-60與 K-562 cell line之50%細胞株致死劑量（lethal concentration of 50 %，以下簡稱LC50 ）之作用劑量及時間的條件。本實驗的設計，第一部份是以HL-60與 K-562 cell line當作實驗的材料，分別以不同濃度H2O2及不同時間作用HL-60與 K-562 cell line，評估HL-60與 K-562 cell line細胞存活率(cell viability)及細胞毒性(cytotoxicity)，求得LC50 之H2O2作用濃度與時間，作為後續H2O2引起氧化傷害之作用模式。結果顯示，H2O2造成HL-60 cell line LC50的條件為， 2.5 mM H2O2作用2小時。而H2O2造成K-562 cell line LC50的條件為，160 mM H2O2作用3小時。至於毒性效應方面，可觀察到HL-60 cell line在2.5 mM H2O2作用2小時的條件下，細胞乳酸去氫酶(lactate dehydrogenase，以下簡稱LDH)的釋出達47%。K-562 cell line在160 mM H2O2作用3小時的條件下，LDH的釋出達59%。因此HL-60 cell line採用2.5 mM H2O2作用2小時作為實驗之條件，而K-562 cell line採用160 mM H2O2作用3小時作為實驗之條件。在第二部份的實驗，HL-60 cell line使用0.27 mg/mL TS作用24小時作為實驗之條件，而K-562 cell line以25.5 mg/mL TS作用24小時，作為實驗之條件。第三部分的實驗採用丙酮醛（malondialdehyde；以下簡稱MDA）來評估脂質過氧化的程度與超氧陰離子歧化酶（superoxide dismutase；以下簡稱SOD）、麩胱甘肽過氧化酶(glutathione peroxidase；以下簡稱GPx)、麩胱甘肽還原酶(glutathione reducase；以下簡稱GRx)、麩胱甘肽(glutathione；以下簡稱GSH)等指標來評估抗氧化能力。實驗結果，可知當使用0.27 mg/mL TS作用24小時後，會造成HL-60 cell line的MDA上升，但無統計上的意義。若是將HL-60 cell line先以0.27 mg/mL TS作用24小時後，再給予2.5 mM H2O2作用3小時，會發現MDA也上升，但效果未達加成的效應。同樣的情形也表現在K-562 cell line。至於抗氧化能力的表現上，當使用0.27 mg/mL TS作用24小時後，再給予2.5 mM H2O2作用3小時，會造成HL-60 cell line的GSH下降，但無統計上的意義。若是將K-562 cell line先以25.5 mg/mL TS作用24小時後，再給予160 mM H2O2作用3小時，會造成K-562 cell line的GSH下降，且有統計上的意義。綜合本實驗的結果，香椿的加入並沒有加成效應的出現，因此本實驗的結果未達我們的預期目標。

關鍵字

抗氧化物；氧化性傷害； HL-60與 K-562 細胞株；香椿葉水萃取液

並列摘要

Many evidences demonstrated that free radicals play an important role in many human diseases. Several studies discovered that the occurrence of cancer is related with free radicals. Therefore, how to prevent the attack of free radical is an important topic. Traditional treatments of leukemia are chemotherapy and radiotherapy. Nowadays, chemotherapy is the first choice. However, the outcome of chemotherapy is limited. Bone marrow transplantion is a suitable therapy for leukemia, however, it is difficult to find a match pairs. This encouraged us to find out a suitable way for the treatment of leukemia. We used two human leukemia cell lines (HL-60 and K-562 cell lines) as the experimental materials in the first part of this study. These cell lines were treated by different concentrations of hydrogen peroxide (H2O2) in different time-courses to estimate the cell viability and cytotoxicity of HL-60 and K-562 cell. The second part, to estabilish the lethal concentration of 50% (LC50) of Toona sinensis Roemor Concentrate (TS), we adapted different concentration of TS to treat the two cell lines. The third part, we used malondialdehyde (MDA) to evaluate the lipid peroxidation and superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reducase (GRx), and glutathione (GSH) to evaluate the antioxidant activity. In result, we found that LC50 of HL-60 cell was incubated with H2O2 (2.5 mM) in 2 hours. The LC50 of K-562 cell was also incubated with H2O2 (160 mM) in 3 hours. The LC50 of HL-60 cell was incubated with TS (0.27 mg/mL) in 24 hours. The LC50 of K-562 cell was also incubated with TS (25.5 mg/mL) in 24 hours. In the level of MDA, the results showed that TS, in 0.27 mg/mL, could increase the level of MDA in HL-60 cell after treatment 24 hourse. There was no statistically significant (p>0.05). After HL-60 cell was incubated with TS (0.27 mg/mL), then, the HL-60 cell was also treated continuously with H2O2 (0.25 mM) in 2 hours. MDA was also elevated in this condition. In the level of GSH, K-562 cell was incubated with TS (25.5 mg/mL) in 24 hours caused the diminishing of GSH. There was no statistically significant (p>0.05). After K-562 cell was incubated with TS (25.5 mg/mL), then, the K-562 cell was also treated continuously with H2O2 (0.25 mM) in 3 hours. GSH was diminished in this condition. There is statistically significant (p<0.05).

並列關鍵字

anti -oxiadant ； oxiadtive damage ； K-562 cell line ； Toona sinensis Roemor Concentrate ； HL-60 cell line

參考文獻

1.Halliwell, B. G. J. Free Radicals in Biology and Medicine, Ed., Oxford University Press, Oxford, U.K.; 1990.

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