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目的观察转化生长因子-β(TGF-β)对人卵巢癌细胞系SK-OV-3活性氧簇(ROS)表达的影响,并探讨其可能机制。方法取对数生长期SK-OV-3细胞,随机分为两组,观察组用2 mL培养液+2μL无血清DMEM配制的5μg/mL TGF-β培养(最终浓度为5 ng/mL),对照组直接加2 mL培养液。流式细胞仪检测细胞内总的活性氧簇(ROS)产生及线粒体来源的ROS,实时荧光定量PCR检测细胞上皮细胞-间充质转化(EMT)变化各指标、线粒体呼吸链复合体Ⅲ组分mRNA。结果观察组及对照组总ROS产生量分别为3 159±42.92、1 062±24.41,两组比较,P<0.05。观察组及对照组细胞内线粒体来源ROS产生量分别为1 254±29.04、1 039±17.32,两组比较,P<0.05。观察组E-cadherin、N-cadherin、Vimentin、ZEB1分别是对照组的0.200 0、6.739 8、4.487 9、1.639 5倍,P均<0.05。观察组CYC1、TTC19、UQCRC1、UQCRC2、UQCR10、UQCRB、UQCRFS1、UQCRH、UQCRQ mRNA表达水平较对照组改变倍数分别为0.89、1.04、1.09、0.87、1.17、1.18、0.92、1.09、1.07倍,P均>0.05。观察组CYC1、TTC19、UQCRC1、UQCRC2、UQCRFS1蛋白水平较对照组改变倍数分别为1.01、0.97、1.07、0.99、1.03倍,P均>0.05。结论TGF-β诱导细胞内总的ROS表达增加,但过量ROS并非来源于线粒体,机制可能是通过调控细胞内氧化-抗氧化平衡系统的关键酶从而改变细胞内的ROS水平。
Objective To investigate the effect of transforming growth factor-β (TGF-β) on the expression of reactive oxygen species (ROS) in human ovarian cancer cell line SK-OV-3 and explore its possible mechanism. Methods SK-OV-3 cells in logarithmic growth phase were randomly divided into two groups. The observation group was cultured with 5 μg / mL TGF-β (final concentration was 5 ng / mL) in 2 mL culture medium and 2 μL serum-free DMEM. The control group directly added 2 mL of culture medium. The production of reactive oxygen species (ROS) and mitochondria-derived ROS were measured by flow cytometry. The changes of epithelial cell-mesenchymal transition (EMT) were detected by real-time fluorescence quantitative PCR. The mitochondrial respiratory chain complex III mRNA. Results The total ROS production in observation group and control group were respectively 3 159 ± 42.92 and 1 062 ± 24.41, P <0.05. The mitochondrial ROS production in observation group and control group were respectively 1 254 ± 29.04 and 1 039 ± 17.32, P <0.05. E-cadherin, N-cadherin, Vimentin and ZEB1 in the observation group were 0.200 0, 6.739 8, 4.487 9 and 1.669 respectively in the control group, all of which were less than 0.05. The changes of CYC1, TTC19, UQCRC1, UQCRC2, UQCR10, UQCRB, UQCRFS1, UQCRH and UQCRQ mRNA expression in the observation group were all 0.89,1.04,1.09,0.87,1.17,1.18,0.92,1.09,1.07 times, P > 0.05. The changes of CYC1, TTC19, UQCRC1, UQCRC2 and UQCRFS1 in the observation group were 1.01, 0.97, 1.07, 0.99 and 1.03 times more than those in the control group, respectively. CONCLUSION TGF-β induces an increase in total ROS expression, but excessive ROS does not originate from mitochondria. The mechanism may be through the regulation of the key enzyme in the intracellular oxidative-antioxidant balance system to alter intracellular ROS levels.