双氢杨梅树皮素诱导人视网膜母细胞瘤细胞氧化及凋亡的研究
发布时间：2018-03-12 18:25
本文选题：双氢杨梅树皮素 切入点：人视网膜母细胞瘤细胞 出处：《广西医科大学》2011年硕士论文 论文类型：学位论文
【摘要】：目的:通过检测双氢杨梅树皮素(APS)对体外培养的人视网膜母细胞瘤细胞株(HXO-RB44)增殖的影响,观察细胞凋亡、线粒体跨膜电位、细胞内活性氧(ROS)的变化,探讨APS诱导人视网膜母细胞瘤(RB)细胞凋亡的氧化作用机制。 方法:取对数生长期的人HXO-RB44进行实验。1.观察APS干预下细胞的生长曲线:设7个不同药物浓度(50.00、33.33、22.22、14.84、9.90、6.60、4.40)μg/ml和空白对照组,分别作用0、24、48、72h后,绘制生长曲线图。2.观察APS对细胞增殖的抑制作用:7个浓度的APS干预RB细胞24h后,观察其形态学变化, MTT法、软琼脂克隆形成法观察RB细胞的体外抑制作用。3.AO/EB染色观察细胞凋亡形态学变化:选取3个不同浓度(50.00、14.84、4.40)μg/ml的APS干预RB细胞24h后,染色,荧光显微镜观察,并拍照。4.流式细胞仪检测细胞凋亡:设3个药物浓度组(50.00、14.84、4.40)μg/ml和空白对照组,作用24h后,用流式细胞仪检测细胞凋亡情况。5.流式细胞仪检测线粒体跨膜电位及ROS的变化:选取对数生长期细胞培养,设3个药物浓度组和空白对照组培养24h后,染色,流式细胞仪检测线粒体跨膜电位及ROS的变化。 结果:1.APS干预后,RB细胞的生长受到明显抑制,随着药物浓度的增加呈剂量依赖性;作用24h时抑制作用最明显。2.通过倒置显微镜可观察到RB细胞出现形态学的变化。细胞的IC50为14.71μg/ml;药物浓度为9.9μg/ml以上时单细胞克隆形成抑制率为100㳠,药物浓度为4.4μg/ml,抑制率为52.2㳠。3.AO/EB荧光染色,药物作用后出现了明显的凋亡细胞,表现为细胞皱缩、核染色质浓缩、核碎裂等凋亡特征性的形态学改变。4.APS对细胞凋亡有明显的诱导作用。药物作用24h后的凋亡率分别为88.13%、58.46%、14.83%,各浓度组凋亡率与空白对照组(4.82%)比较,差异均有显著性(P0.05),各浓度组细胞凋亡率之间差异也均有显著性(P0.05)。5.APS能降低RB细胞线粒体跨膜电位。APS处理24h后不同浓度组(50.00、14.84、4.40、0)μg/ml的平均荧光强度分别为20.70、55.63、88.57和146.53,药物组与空白对照组比较差别有统计学意义( P均 0. 01),三个浓度之间差异有统计学意义( P 0. 01)。6.APS能促进RB细胞内ROS的产生。APS处理24h后不同浓度组(50.00、14.84、4.40、0)μg/ml的平均荧光强度分别为54.87、43.03、36.80和20.93,药物组与空白对照组比较差别有统计学意义( P均 0. 01),三个浓度之间差异有统计学意义( P 0. 01)。 结论:1.APS对RB细胞的体外增殖有明显的抑制作用。2.APS可诱导体外培养的RB细胞凋亡。3.APS诱导RB细胞凋亡与线粒体内跨膜电位的下降、细胞内ROS产生增加有关。
[Abstract]:Aim: to investigate the effects of dihydromyricetin (APS) on the proliferation of human retinoblastoma cell line HXO-RB44 in vitro, and to observe the changes of apoptosis, mitochondrial transmembrane potential and intracellular reactive oxygen species (Ros). To investigate the oxidative mechanism of APS induced apoptosis of human retinoblastoma (RBB) cells. Methods: human HXO-RB44 in logarithmic growth period was taken for experiment. 1. Observe the growth curve of human HXO-RB44 in logarithmic growth phase. Observe the growth curve of cells treated with APS: set up 7 different concentrations of 5 0.0033X 33.33C 22.2222 14.84 渭 g / ml and control group 6.604.40) 渭 g / ml and control group, respectively, after 72 hours of treatment, the cells were exposed to 0.24 渭 g / ml and 0.24 渭 g / ml, respectively. To observe the inhibitory effect of APS on the proliferation of RB cells: 7 concentrations of APS were used to observe the morphological changes of RB cells after 24 hours of intervention, and MTT method was used to observe the effects of APS on the proliferation of RB cells. The inhibitory effect of RB cells in vitro was observed by soft Agar Clone formation method. 3. AO- / EB staining was used to observe the morphological changes of apoptosis. Three different concentrations of APS (50.004 ~ 14.84 渭 g / ml) were selected to interfere with RB cells for 24 h, then stained and observed by fluorescence microscope. Cell apoptosis was detected by flow cytometry: three drug concentration groups were divided into three groups (50.0044.40) 渭 g / ml and blank control group (control group). After 24 hours of treatment, the cell apoptosis was detected by flow cytometry. Cell apoptosis was detected by flow cytometry .5.The changes of mitochondrial transmembrane potential and ROS were detected by flow cytometry. The cells were cultured in logarithmic growth phase and cultured for 24 hours in three drug concentration groups and blank control group. The changes of mitochondrial transmembrane potential and ROS were detected by flow cytometry. Results 1. The growth of RB cells was significantly inhibited after intervention with APS in a dose-dependent manner with the increase of drug concentration. Morphological changes of RB cells were observed by inverted microscope. The IC50 of RB cells was 14.71 渭 g / ml, and the inhibition rate of single cell clone formation was 100 渭 g / ml when drug concentration was more than 9.9 渭 g / ml. The drug concentration was 4.4 渭 g / ml and the inhibition rate was 52.2? 3. AO- / EB fluorescence staining showed that apoptotic cells appeared after drug treatment, which showed cell shrinkage and nuclear chromatin concentration. The apoptotic characteristic morphologic changes such as nuclear fragmentation. 4. APS could induce apoptosis obviously. The apoptotic rate of APS was 88.13 ~ 58.46% and 14.83% respectively after 24 hours of treatment. The apoptotic rate of each concentration group was compared with that of the blank control group (4.82%). There were significant differences in apoptosis rate between different concentration groups. P0.05N. 5. APS could decrease mitochondrial transmembrane potential of RB cells. APS treatment for 24 hours, the average fluorescence intensity of different concentration groups was 20.7055.63 88.57 渭 g / ml and 146.53 渭 g / ml, respectively. There was significant difference between the white control group and the control group (P = 0.01, P = 0.01, P < 0.01). 6. APS could promote the production of ROS in RB cells. After 24 hours of treatment with APS, the average fluorescence intensity of the three concentration groups was 50.0014. 844.40%) 渭 g / ml, respectively. The difference between the drug group and the blank control group was statistically significant (P all 0.01), and the difference between the three concentrations was statistically significant (P 0.01). Conclusion: 1. APS can inhibit the proliferation of RB cells in vitro. 2. APS can induce RB cell apoptosis in vitro. 3. APS induced RB cell apoptosis is related to the decrease of mitochondrial transmembrane potential and the increase of intracellular ROS production.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.7
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本文编号：1602743
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