石蓮花熱乙醇萃取物對血癌細胞株毒殺及免疫活化功能之探討

Study on the effects of hot ethanol-extract of Graptopetalum paraguayense E. Walther on apoptosis of leukemia cells and on activation of immuno-functions

蔡旻芬(Min-Fen Tsai)

指導教授 ： 張基隆

高雄醫學大學/醫學院/生物化學研究所/碩士(2007年)

摘要

石蓮花為多年生草本植物，屬於景天科 ( Crassulaceae ) 石蓮花屬 ( Graptopetalum )。民間常做為抗癌食物，然而其藥理、細胞毒性、細胞生理作用及抗癌機制目前仍尚未清楚。本研究主要探討石蓮花對血癌細胞株包括 U937, Jurkat和 Raji 細胞株之毒殺機制並進行其與人類週邊血液單核細胞 ( Peripheral blood mononuclear cells；PBMC ) 及動物免疫功能相互作用之研究。首先以 trypan blue exclusion 方法測定石蓮花熱乙醇萃取物 ( P-HE ) 對細胞毒殺能力，結果顯示隨著石蓮花熱乙醇萃取物 ( P-HE ) 濃度增加，對細胞毒殺能力越顯著，之後以 flow cytometry分析 subG1 期及 DNA 進行 agarose gel 電泳，分析 DNA 片斷化的情形。皆可觀察出隨著 P-HE 濃度增加，細胞凋亡情形越明顯且同時偵測出粒線體膜電位的下降情形，及以 western blotting 分析發現 cytochrome c、凋亡蛋白酵素 caspase 3 及 caspase 9 增加，而抗凋亡蛋白 Bcl-2 和 Bcl-XL 表現量下降，但對於 PBMC 並無影響。
另ㄧ方面，在 P-HE 對免疫功能之探討，利用 flow cytometry偵測 T 細胞和 B 細胞表面抗原，由結果發現，可刺激 Th 細胞和B 細胞的增殖表現，以及 U937 經 P-HE 刺激後能增強巨噬細胞吞噬能力，使其細胞內大量表現活性氧化物 ( reactive oxygen species；ROS )，以 RT-PCR 偵測 TNF-α、IFN-γ、IL-1、IL-6 mRNA 表現量增加，TGF-β 及 IL-10 表現量降低。在動物實驗中，以 ( 0.57mg/kg/day ) P-HE 餵食 Balb/c 小鼠八週，利用 ELISA法偵測小鼠血清中，TNF-α 和 IFN-γ 表現量同時增加。由以上結果顯示，石蓮花熱乙醇萃取物 ( P-HE ) 具有可提升動物體內及細胞免疫因子釋出量。

關鍵字

石蓮花 ； 血癌細胞珠 ； 細胞凋亡 ； 人類週邊血液單核細胞 ； 免疫活性

並列摘要

Graptopetalum paraguayense is a perennial herbaceous plant that belongs to Crassulaceae subject and Graptopetalum genus. Crassulaceae subject has been served as an edible vegetable for protecting against cancer for a long time. However, the efficacy and mechanism of Graptopetalum paraguayense on leukemia are still unclear. In this study, we explored the cytotoxic activity and underline mechanisms of Graptopetalum paraguayense on leukemia cell lines including U937, Jurkat and Raji cells. Trypan blue exclusion method showed the hot ethanolic extract of Graptopetalum paraguayense ( P-HE ) induced cell cytotoxicity of U937, Jurkat and Raji cells in a dose-dependent manner. In addition, flow cytometry analysis showed that P-HE induced a cell accumulation of subG1 phase, and gel electrophoresis demonstrated nuclear DNA fragmentation induced by P-HE. Furthermore, mitochondria membrane potential (ΔΨm) as well as cytochrome c release was reduced; the caspase3/9 activity was increased and anti-apoptosis protein Bcl-2 and Bcl-XL were decreased in leukemia cell lines. In contrast, those effects of P-HE were not on PBMC.
P-HE also stimulate T cell and B cell proliferated, meanwhile, the cell macrophage phagocytosis ability of U937 was increased as detected the intracellular reactive oxygen species（ROS）formation. The RT-PCR results showed TNF-α、IFN-γ、IL-1、IL-6 mRNA expressions were increased and TGF-β、IL-10 mRNA were decreased . Serum TNF-α and IFN-γ levels of Balb/c mice were increased as feed (0.57mg/kg/day) P-HE for 8 weeks.
So far, our results indicate there may be some components of Graptopetalum paraguayense E. Walther, which are effective on the immuno-functions and are relative to antioxidant activation resulting in enhancement of immunity in vivo, and those are worthy to be further studied.

並列關鍵字

Graptopetalum paraguayense E. Walther ； leukemia cell lines ； apoptosis ； PBMC ； activation of immuno-functions

參考文獻

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