纳米级石英与苯并芘联合作用对人支气管上皮细胞BEAS-2B的影响

发布时间：2018-03-06 03:31

本文选题：人支气管上皮细胞　切入点：纳米级石英　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[目的]研究纳米级石英与苯并芘联合作用对人支气管上皮细胞(BEAS-2B)的影响。 [方法]本实验选用人支气管上皮细胞为靶细胞,选用粒径为30nm的纳米级石英和3、4-苯并芘为研究对象,设置纳米级石英的浓度分别为25mg/L、50mg/L、100mg/L、300mg/L,3、4-苯并芘的浓度为5μmol/L,进行各物质的单独染毒和联合染毒。并设置正常达尔伯克必需基本培养基(Dulbecco's Minimum Essential Medium, DMEM)为空白对照组。细胞培养6、12、24、48、72小时后,用WST-1法进行细胞毒性试验。细胞培养6、12、24小时后用倒置相差显微镜对细胞形态生长情况进行观察；用流式细胞技术测定细胞内活性氧代谢产物(Reactive Oxygen Species, ROS)的表达情况；细胞培养6、24、48小时后用流式细胞技术测定细胞周期改变。结果应用SPSS17.0统计软件进行统计学处理。 [结果]1.细胞存活率的变化纳米级石英颗粒和苯并芘分别作用于人支气管上皮细胞后,随作用剂量增加、作用时间延长,细胞存活率呈下降趋势。纳米级石英和苯并芘联合作用组与纳米级石英单独作用组相比,细胞存活率下降更快。各实验组与对照组比较,细胞存活率差异均具有统计学意义(P0.05)。2.细胞形态学的变化空白对照组细胞形态学无明显变化。纳米级石英颗粒和苯并芘分别作用于人支气管上皮细胞后,随作用剂量增加、作用时间延长,细胞体积增大。当纳米级石英与苯并芘联合作用时,细胞形态学的变化明显增强。3.细胞内ROS的表达程度纳米级石英颗粒和苯并芘分别作用于人支气管上皮细胞后,随作用剂量增加、作用时间延长,与空白对照组比较,细胞内ROS表达的平均荧光强度均逐渐升高；当纳米级石英与苯并芘联合作用时,细胞内ROS表达的平均荧光强度明显升高(P0.01)。空白对照组细胞内ROS表达的平均荧光强度无明显变化(P0.05)。4.细胞周期的变化培养6小时后联合组细胞中DNA合成前期(G1期)细胞所占的比例为65.8±6.5%,与纳米级石英组和空白对照组相比无统计学差异(P0.05),随培养时间延长,联合组与纳米级石英组G1期细胞所占的比例逐渐下降,但联合组下降趋势更为明显(P0.01),空白对照组G1期细胞所占的比例无明显变化。培养6小时后联合组细胞中DNA合成期(S期)细胞所占的比例为21.9±2.1%,与纳米级石英组和空白对照组相比差异有统计学意义(P0.05),随培养时间延长,联合组、纳米级石英组S期细胞所占的比例均有上升,但联合组趋势更为明显(P0.01),空白对照组S期细胞所占的比例无明显变化(P0.05)。 [结论]受纳米级石英与苯并芘刺激后,人支气管上皮细胞丧失原有形态,细胞形态发生改变；细胞内活性氧代谢物过度表达：细胞周期由G1向S期过渡。其作用表现出时间-效应关系和剂量-效应关系,作用时间越长、作用剂量越大,其刺激作用越强。当二者联合作用时,对人支气管上皮细胞表现出更强的细胞毒性作用。
[Abstract]:[objective] to study the effect of combined action of nanometer quartz and benzopyrene on human bronchial epithelial cells (BEAS-2B). [methods] Human bronchial epithelial cells were selected as target cells, and 30nm nano-quartz and 3o 4-benzopyrene were selected as the research objects. We set the concentration of nanoscale quartz to 25 mg / L 50 mg / L 10 mg / L 10 mg / L 3 mg / L 3 mg / L 3 渭 mol / L benzopyrene 5 渭 mol / L for each substance separately and in combination, and set Dulbeccos Minimum Essential Media (DMEM), the basic medium required for normal Dalbek, as a blank control group. After cell culture, 612, 24, 4872 hours later, The cytotoxicity test was carried out by WST-1 method. The morphological growth of the cells was observed by inverted phase contrast microscope after 24 hours of cell culture, and the expression of reactive Oxygen species (ROS) was detected by flow cytometry. Cell cycle changes were measured by flow cytometry after 48 hours of cell culture. Results SPSS17.0 statistical software was used to analyze the changes of cell cycle. [results] 1. The changes of cell survival rate after treated with silica nanoparticles and benzopyrene, respectively, with the increase of the dose of action, the time of action was prolonged. The cell survival rate of the group treated with nano-quartz and benzopyrene decreased more rapidly than that of the group treated with nano-quartz alone, and the survival rate of each experimental group was higher than that of the control group. There was no significant change in cell morphology in blank control group. After treated with nanometer quartz particles and benzopyrene, the cell morphology increased with the increase of the dose. When the action time was prolonged, the cell volume increased, and when the nanoscale quartz was combined with benzopyrene, (3) the expression of ROS in cells was increased significantly. The effect of nano-quartz particles and benzopyrene on human bronchial epithelial cells increased with the increase of the dose, and the time of action was prolonged, compared with the blank control group. The average fluorescence intensity of ROS expression in cells increased gradually, and when combined with benzopyrene, The average fluorescence intensity of ROS expression in cells was significantly higher than that in control group (P 0.01). There was no significant change in the average fluorescence intensity of ROS expression in the blank control group. The cell cycle was changed after 6 hours of cell cycle change. After 6 hours of culture, the cells in the combined group were in the early stage of DNA synthesis and G1 phase. The proportion of cells was 65.8 卤6.5. There was no significant difference in the proportion of cells between the nano-quartz group and the blank control group (P 0.05), but with the increase of culture time, there was no significant difference between the two groups. The proportion of G1 phase cells in combination group and nanometer quartz group decreased gradually. However, the decreasing trend of the combined group was more obvious than that of the control group, but the percentage of G1 phase cells in the blank control group had no significant change. After 6 hours of culture, the proportion of the cells in the DNA synthesis phase of the combined group was 21.9 卤2.1, which was similar to that of the nano-quartz group and the blank group. The difference between the white control group and the control group was statistically significant (P 0.05). In the combined group, the percentage of S phase cells in the nano quartz group was increased, but the trend of the combined group was more obvious than that of the control group (P 0.01), but the percentage of S phase cells in the blank control group had no significant change. [conclusion] after being stimulated by nanometer quartz and benzopyrene, human bronchial epithelial cells lose their original morphology and change in morphology. Overexpression of Ros metabolites in cells: the cell cycle transitioned from G1 to S phase. The cell cycle showed a time-effect relationship and a dose-effect relationship. The longer the action time, the greater the dose. The stronger the stimulative effect is, the stronger the cytotoxic effect on human bronchial epithelial cells is when combined with them.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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