不同分化程度之人類肝癌細胞株中MnSOD表現調控之探討

Study on the Regulation of MnSOD Expression in Human Hepatoma Cell Lines at Different Differentiated Stage

張照瑋(Chao-Wei Chang)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/碩士(2001年)

摘要

自由基中的活性氧分子(reactive oxygen species, ROS)乃是細胞正常代謝過程中所產生的高活性代謝物。而活性氧分子又與許多細胞生理過程息息相關，包括經由訊息傳遞路徑促使細胞增生、分化、癌化甚至於凋亡(apoptosis)。然而，活性氧分子也能經由直接與生物分子作用導致細胞傷害。在本實驗室之前的研究結果指出，分化程度較高的肝癌細胞株(Hep G2、Hep 3B)，其內生性抗氧化酵素的表現，包括catalase (CAT)、manganese superoxide dismutase (MnSOD)、glutathione reductase (GRx)的活性，明顯的較分化程度較低的肝癌細胞株(SK-Hep-1, HA22T/VGH)高出許多。而為了解釋不同分化程度肝癌細胞株之抗氧化能力，則以50 μΜ H2O2 作為外在氧化壓力，發現只有分化程度較低肝癌細胞株的MnSOD活性有被誘導增加之情形。顯示出分化程度較低之肝癌細胞株比分化程度較高之肝癌細胞株具有較佳之氧化壓力耐受性。因此，本論文以不同分化程度的肝癌細胞株為細胞模式，探討不同分化程度肝癌細胞株的內在氧化壓力及其調控方式。結果發現，分化程度較高肝癌細胞株的內在氧化壓力確實比分化程度較低肝癌細胞株來得高。利用流式細胞儀的技術，經由以carboxy-2’,7’-dichlorofluorescein diacetate (carboxy-DCFDA)檢測知分化程度不同肝癌細胞的細胞膜對螢光染劑之通透性無明顯差異；而以2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)，dihydroethidium (HEt)和dihydrorhodamine 123(DHR123)分別檢測細胞內H2O2、. O2-和粒線體H2O2的含量，發現分化程度較高肝癌細胞株在細胞內H2O2，. O2-和粒線體之H2O2之相對比率都比分化程度較低肝癌細胞株來得高，其平均值分別差了5.70倍(p<0.01)、2.18倍(p<0.01)及2.35倍(p<0.01)。由上述結果推測，高活性的抗氧酵素表現是為了用來平衡較高的內在氧化壓力。而之前文獻中指出，MnSOD基因前5’untranslation region 具有NF-κB、AP-1及SP-1等對活性氧分子敏感的轉錄因子結合位，故在不同分化程度肝癌細胞株MnSOD的調控方面，則利用electrophoretic mobility shift assay (EMSA)的方法去探討這些對活性氧分子敏感的轉錄因子活化的情形。結果顯示，以50 μΜ H2O2處理不同分化程度肝癌細胞時，發現NF-κB及AP-1皆只有些微的活化，平均倍數小於1.5倍；而分化程度較低的肝癌細胞則有明顯的Sp-1活化的情形，其平均倍數均大於5倍，故推測不同分化程度肝癌細胞在處於氧化壓力時，Sp-1在MnSOD表現的調控上扮演相當重要的角色，而其詳細的作用機制則需再進一步的探討。

關鍵字

錳超氧岐化酵素

並列摘要

Free radicals, especially the reactive oxygen species (ROS) are highly reactive metabolites that are generated during normal cell metabolism. The ROS has been shown to correlate with various biological processes such as differentiation, proliferation, carcinogenesis and apoptosis through the signaling pathway. However, the ROS can cause cellular damages by direct interaction with bio-molecules. Our previous results showed that the well-differentiated hepatoma (Hep 3B, Hep G2) exhibited higher expression profile of antioxidant enzymes, including catalase (CAT), manganese superoxide dismutase (MnSOD) and glutathione reductase (GRx) than poor-differentiated hepatoma (SK-Hep-1 and HA22T). The activity of MnSOD, but not other antioxidant enzymes, could be induced in poorly-differentiated HCC cells after treatment with 50 μM H2O2. These results suggested that poorly-differentiated HCC cells might have better antioxidative ability than well-differentiated ones. Therefore, in this report, we will elucidate the endogenous oxidative stress and different regulatory events between poorly and well-differentiated HCC cells. By flow cytometry, our results demonstrated the relative ratio of intracellular H2O2、. O2- and mitochodrial H2O2 were about 5.7 folds (p＜0.01)、2.18 folds(p<0.01) and 2.35 folds(p<0.01) for well-differentiated over poorly-differentiated cells, respectively. Furthermore, electrophoretic mobility shift assay (EMSA) was used to elucidate the participation of ROS-sensitive transcription factors. After treatment with 50 μM H2O2, the results showed that poorly and well-differentiated HCC cells exhibited only minor differences for both the activities of NF-κB and AP-1, the average fold less than 1.5. However, the poorly-differentiated HCC cells were vice versa for Sp-1. Their average folds were more than 5. Thereby, we suggested that Sp-1 might play important roles of MnSOD expression in HCC cells under oxidative stress. Nevertheless, it warrants further investigation.

並列關鍵字

MnSOD

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