非瑟酮对人晶状体上皮细胞增殖和凋亡的影响

发布时间：2018-03-22 18:26

本文选题：非瑟酮　切入点：人晶状体上皮细胞　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：研究非瑟酮(fisetin, Fis)在生理状态下及氧化应激状态下对人晶状体上皮细胞(human lens epithelial cells, HLECs)增殖和凋亡的影响。方法：(1)实验准备：将冻存的人晶状体上皮细胞株(SRA01/04)复苏后,置于含10%胎牛血清的低糖DMEM培养基(葡萄糖终浓度5.56mmol/L)中培养,传代,收集对数生长期细胞进行以下实验。(2)分组及干预：用0.25%胰酶消化收集同一代贴壁细胞,制成细胞悬液计数后,随机分为空白对照组(不加干预)；Fis组(分别加入5μg/ml、10μg/ml、20μg/ml浓度的Fis)；H2O2组(加入300μmol/L过氧化氢(Hydrogen peroxide, H2O2)); Fis+H2O2组(先加入5μg/ml、10μg/ml、20μg/mlFis,1小时后加入300μmol/L H2O2共同培养)。(3)形态学观察：培养12h和24h后倒置显微镜观察以上各组细胞的形态学改变,随机照相(×100倍,×200倍),对比各组细胞形态学变化。(4)Fis对HLECs增殖的观察：采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium, MTT)比色法分别检测Fis在有或无H2O2作用12小时和24小时后对HLECs增殖的影响。(5)Fis对H202诱导的HLECs凋亡的观察：采用异硫氰酸(FITC-Annexin-v, Annexin-V)和碘化丙锭(Propidium Iodide, PI)标记的双染色免疫荧光法,流式细胞术(flow cytometric analysis, FCM)检测Fis在与H202共同作用24小时后HLECs凋亡率的变化。结果：(1)与空白对照组比较,加入5～20μg/mlFis培养12小时和24小时,对HLECs的增殖无明显影响(P0.05),细胞形态无明显变化。(2)与空白对照组比较,H2O2组较多细胞出现细胞密度降低以及典型的细胞形态学改变,细胞增殖能力明显降低,凋亡率明显增加,差异有显著统计学意义(P0.01)。加入5-20μg/mlFis预处理组,H2O2诱导的典型形态改变的细胞减少,细胞增殖能力明显改善,并且随时间和Fis浓度的增加作用更明显(P0.05)；Fis作用24h时,细胞凋亡率逐渐降低,与H2O2组比较,差异有统计学意义(P0.05),并且随Fis浓度增加作用更明显(P0.05)。结论：(1)在生理条件下,在一定浓度和作用时间范围内,非瑟酮对HLECs的增殖无明显影响。(2)在氧化应激条件下,在一定浓度和作用时间范围内,非瑟酮能明显改善HLECs的增殖能力,且呈剂量-效应与时间-效应关系。(3)在氧化应激条件下,在一定浓度范围内,非瑟酮能明显抑制HLECs凋亡,且呈剂量-效应关系。
[Abstract]:Aim: to study the effects of Fisetin (Fis) on proliferation and apoptosis of human lens epithelial cells (HLECs) in physiological and oxidative stress. Methods: the cryopreserved human lens epithelial cells (LECs) were resuscitated after cryopreserved human lens epithelial cells (HLECs) were resuscitated. Cultured in 10% fetal bovine serum low-glucose DMEM medium (glucose final concentration 5.56 mmol / L), subcultured, collected logarithmic growth phase cells for the following experiment. 2. Intervention: digesting and collecting the same generation of adherent cells with 0.25% trypsin. After making cell suspension count, They were randomly divided into two groups: control group (Fis group without intervention) (adding 5 渭 g / ml 10 渭 g / ml 10 渭 g / ml ~ (20) 渭 g/ml) to H _ 2O _ 2 group (adding #number0# 渭 mol/L hydrogen peroxide, H _ 2O _ 2), Fis H2O2 group (5 渭 g 路ml ~ (10) 渭 g 路ml ~ (10) 渭 g 路ml ~ (20) 渭 g 路ml ~ (-1)) and then adding 300 渭 mol/L H2O2 for 1 hour. Morphological observation: 12 h and 24 h later, the morphology of Fis H2O2 group was observed. The morphological changes of the above groups were observed by inverted microscope. Random radiographs (脳 100 times, 脳 200 times) were used to compare the morphological changes of cells in each group. The proliferation of HLECs was observed by Fis thiazolyl tetrazolium (MTT) colorimetric method. The proliferation of HLECs was detected by Fis after 12 hours and 24 hours of H2O2 treatment, respectively. Observation of HLECs apoptosis induced by H202 by FITC-Annexin-V (Annexin-V) and Propidium Iodide (Pi) labeled by FITC-Annexin-V (Pi). Flow cytometric analysis (FCM) was used to detect the changes of HLECs apoptosis rate in Fis treated with H202 for 24 hours. Results compared with control group, Fis was cultured with 5 渭 g/mlFis for 12 hours and 24 hours. Compared with the control group, more cells in H 2O 2 group had decreased cell density and typical cell morphological changes, and the cell proliferation ability was significantly decreased, and the apoptosis rate was significantly increased compared with the control group. The difference was statistically significant (P 0.01). In the 5-20 渭 g/mlFis pretreatment group, the cells with typical morphological changes induced by H _ 2O _ 2 decreased, the cell proliferation ability was improved significantly, and the effect was more obvious with the increase of the concentration of Fis and the concentration of P0.05Fis for 24 h. The apoptosis rate decreased gradually, and the difference was statistically significant compared with that of H2O2 group, and the effect was more obvious with the increase of Fis concentration. Conclusion: under physiological conditions, in a certain concentration and within a certain time range, the cell apoptosis rate was significantly increased with the increase of Fis concentration. There was no significant effect of fenetherone on the proliferation of HLECs. (2) under oxidative stress, under certain concentration and action time range, fenetherone could obviously improve the proliferative ability of HLECs, and showed a dose-effect and time-effect relationship under oxidative stress. In a certain concentration range, fenetherone could significantly inhibit HLECs apoptosis, and showed a dose-effect relationship.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R776.1

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