氧化应激对人晶状体上皮细胞中SMP30蛋白表达变化的影响

发布时间：2018-03-16 23:16

本文选题：衰老标记蛋白30　切入点：人晶状体上皮细胞　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察氧化应激对人晶状体上皮细胞(human lens epithelial cells,HLECs)中衰老标记蛋白30(Senescence Marker Protein30,SMP30)表达变化的影响,为今后研究其在人晶状体上皮细胞及白内障发生发展过程中的作用奠定基础。方法:用不同浓度过氧化氢(0、100、200、300μM)刺激人晶状体上皮细胞24小时,建立不同浓度急性氧化应激模型,通过光镜、MTT检测分析细胞形态、状态,Western blot检测SMP30蛋白的表达情况。用不同浓度过氧化氢(0、25、50、75、100、125、150μM)刺激人晶状体上皮细胞2周,建立慢性氧化应激模型,诱导细胞衰老,通过光镜、MTT、β-半乳糖苷酶衰老细胞染色、细胞周期检测细胞氧化应激情况及评估细胞状态,用q-PCR、Western blot检测SMP30基因及蛋白的表达变化。结果:过氧化氢刺激HLECs24小时,随着过氧化氢浓度的增高,各处理组细胞在光镜下可见密度逐渐下降,变大变圆,300μM处理组细胞破碎,MTT结果提示细胞生存率逐渐降低,各浓度处理组与对照组相比差异均有统计学意义(P0.05),SMP30在100、200μM处理组的蛋白表达量均较对照组升高(P均0.05)。过氧化氢刺激HLECs14天后25μM处理组细胞形态与对照组无明显差异,50-125μM处理组细胞密度逐渐下降,细胞呈衰老状态,150μM处理组细胞碎裂。MTT结果提示25-50μM处理组较对照组相比生长曲线逐渐下移,75μM处理组仅维持细胞基本代谢活性,100-150μM处理组生长曲线则在早期大幅下降并维持。β-半乳糖苷酶衰老细胞染色显示随过氧化氢浓度升高,衰老阳性细胞增多,75-150μM处理组阳性细胞较对照组明显增多且90%,细胞周期则提示50-100μM处理组细胞增值阻滞于S和G2/M期,PI值逐渐升高。SMP30在25-100μM处理组与对照组相比mRNA表达均下降(P均0.01)。在25-50μM处理组SMP30蛋白的表达量与对照组相比无明显差异(P=0.695,P=0.126)。在75-100μM处理组SMP30蛋白的表达量均较对照组明显下降(P均0.01)。结论:SMP30蛋白在处于急性氧化应激状态下的人晶状体上皮细胞中表达上调。而在慢性氧化应激诱导衰老的早期阶段SMP30表达无明显变化,随着氧化应激增加和衰老加剧,SMP30的表达减少。SMP30参与人晶状体上皮细胞氧化应激损伤过程,可能与细胞衰老及白内障的发生发展有关。
[Abstract]:Objective: to observe the effect of oxidative stress on the expression of senescence Marker protein 30 SMP30 in human lens epithelial cells (HLECs). Methods: human lens epithelial cells were stimulated with different concentrations of hydrogen peroxide for 24 hours and acute oxidative stress models were established. The morphology of human lens epithelial cells was analyzed by light microscopy, and the expression of SMP30 protein was detected by Western blot. Human lens epithelial cells were stimulated with different concentrations of hydrogen peroxide for 2 weeks to establish a chronic oxidative stress model and induce cell senescence. The changes of SMP30 gene and protein expression were detected by light microscope MTT, 尾 -galactosidase staining, cell cycle detection of oxidative stress and evaluation of cell status. Results: hydrogen peroxide stimulated HLECs24 for hours. With the increase of hydrogen peroxide concentration, the density of cells in each treatment group decreased gradually under light microscope, and the MTT results showed that cell survival rate decreased gradually in the treatment group of 300 渭 M. Compared with the control group, the protein expression of P0.05SMP30 in the 100,200 渭 M treatment group was significantly higher than that in the control group (0.05 渭 M). There was no significant difference in cell morphology between the 25 渭 M group treated with hydrogen peroxide and the control group. The cell density decreased gradually in the treatment group. The results of cell fragmentation and MTT showed that the growth curve of 25-50 渭 M treatment group decreased gradually compared with that of the control group. The growth curve of 25-50 渭 M treatment group only maintained the basic metabolic activity of 75 渭 M treatment group was significantly decreased in the early stage. 尾 -galactosidase staining showed that with the increase of hydrogen peroxide concentration, The number of senescent positive cells increased significantly in 75-150 渭 M treatment group compared with the control group, and the cell cycle indicated that the cell proliferation of 50-100 渭 M treatment group was blocked at S and G _ 2 / M phase Pi value gradually increased. The mRNA expression of SMP30 in 25-100 渭 M treatment group was higher than that in the control group. The expression of SMP30 protein in 25-50 渭 M treatment group was not significantly different from that in control group. The expression of SMP30 protein in 75-100 渭 M treatment group was significantly lower than that in control group (P < 0.01). Conclusion the expression of SMP30 protein in the control group is in the state of acute oxidative stress. The expression of SMP30 did not change in the early stage of aging induced by chronic oxidative stress. With the increase of oxidative stress and the decrease of SMP30 expression in lens epithelial cells, SMP30 may be involved in the process of oxidative stress injury in lens epithelial cells, which may be related to cell aging and the development of cataract.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.1

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