本實驗目的為探討大黃素誘導小鼠血癌細胞株(WEHL-3)產生 細胞凋亡的機制，由上述種種實驗結果可得到以下幾個結論：
(一)、大黃素能夠抑制血癌細胞株的存活率，這表示大黃素對於癌細 胞具有毒殺作用。
(二)、利用 DNA Fragmentation、Comet assay 和 DAPI staining 可觀察
到DNA 斷裂形發生，而得知 DNA 損傷的程度。
(三)、利用流式細胞儀偵測分析，血癌細胞株經 Emodin 作用之後，
皆會釋放出大量的鈣離子以及活性氧化物質(ROS)，而導致粒線體膜 通透性改變、膜電位下降。
(四)、利用流式細胞儀也可測得 Caspase-3、-8、-9 活性皆有上升的 表現。
(五)、藉由西方墨點法探討了當粒線體膜電位改變時，一些與細胞凋 亡有關的蛋白表現量以及觀察和內質網壓力產生有關的蛋白變化情 形，此外，免疫螢光染色的實驗利用共軛焦顯微鏡觀察細胞凋亡的 情形。由種種實驗結果可推論Emodin 誘導老鼠血癌細胞株(WEHI-3) 產生細胞凋亡可能和Mitochondria-dependent apoptosis pathway、ER stress pathway 兩者路徑皆有關。
(六) 大黃素影響體內免疫機轉及細胞吞噬之抗癌作用探討，藉由 in Cytochrome c ↑ Apaf-1 ↑
Bcl-xL ↓
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Emodin inhibits murine leukemia WEHI-3 cells in vitro and enhances macrophage phagocytosis in vivo
Yuan-Chang Chang Major professor: Tung-Yuan Lai
Graduate institute of Chinease Medical science, China Medical University
Abstract
Emodin is extracted from Rheum palmatum L. that had been used as folk medicine in Chinese population. Emodin have been shown to induce cell death and apoptosis in many human cancer cell lines, such as lung, cervical, melanoma, esophagus and liver cancers. However, there is no available information to address Emodin induced apoptosis in WEHI-3 cells. We examined the effects of Emodin on murine leukemia WEHI-3 cells and enhance macrophage phagocytosis in vivo. Therefore, the results from flow cytomertic analysis indicated that Emodin arrest cell cycle in G0/G1 and induced apoptosis in examined cell line.
We also used DAPI stain and DNA gel electrophoresis to confirm Emodin induced apoptosis in WEHI-3 cell line. Flow cytometric also used for analysis the levels of reactive oxygen species, Ca2+ and mitochondrial membrane potential (MMP) in WEHI-3 cell lines. The results showed Emodin promoted ROS and Ca2+ production and decreased the MMP level. Western blotting show that Emodin treatment gradually decreased the levels of anti-apoptotic protein (Bcl-2), but increased the levels of the pro-apoptotic protein (Bax). Emodin promoted the release of cytochrome c from mitochondria based on the changes of
In this study, we examined the in vivo effects of Emodin on leukemia WEHI-3 cells and on macrophage phagocytosis. The weights of the livers and spleens were decreased in the Emodin-treated groups compared to the control groups. The Emodin treatment increased the percentage of CD11b and CD19 marker cells in WEHI-3-injected mice and decreased the percentage of Mac-3 indicating that the differentiation of the precursor of macrophage and B cells was inhibited. The Emodin treatment promoted the activity of macrophage phagocytosis in the peripheral blood mononuclear cells (PBMC) and peritoneal cells. In conclusion, Emodin can inhibit WEHI-3 cells in vitro and promote macrophage phagocytosis in vivo.
keywords：Emodin, Rheum palmatum, WEHI-3, apoptosis, phagocytosis