自噬在年龄相关性白内障人晶状体上皮细胞氧化应激中的作用及机制研究

发布时间：2018-03-28 03:20

本文选题：自噬　切入点：年龄相关性白内障　出处：《浙江大学》2016年博士论文

【摘要】：年龄相关性白内障是一种中老年疾病,严重危害老年人视力的常见眼科疾病。氧化应激是白内障发病的主要因素之一。紫外线辐射、外源性氧化物质等均可以使晶状体上皮细胞(HLEs)产生活性氧片段(Reactive oxygen species, ROS),进一步使组织和细胞发生脂质过氧化以及DNA的损害,从而形成白内障。自噬是真核细胞的一种具有自我保护功能的生理现象,主要帮助细胞适应各种不良刺激,是细胞维持内环境的自稳并且实现自我更新的基本途径。上调自噬可通过激活磷酸化AKT通路,从而对HLEs的迁移和增殖具有明显的促进作用,下调自噬并抑制其功能。查阅相关文献,我们发现调控自噬水平对于HLEs的功能影响,特别在H2O2诱导的氧化应激中的作用未见相关报道。因此,本研究以HLEs-B3细胞株和人HLEs-B3自噬缺陷细胞株为实验对象,旨在探索自噬在人晶状体上皮细胞氧化应激中的保护作用及机理,为年龄相关性白内障的治疗提供一种新的治疗思路。本研究共分为三个部分,主要研究方法及结果如下。第一部分自噬水平与年龄相关性白内障程度的相关性。目的：研究自噬水平与年龄相关性白内障程度的相关性。方法：通过免疫荧光标记检测年龄相关性白内障病例的晶状体前囊膜上皮组织标本的自噬水平,分析其与白内障程度的相关性。步骤如下： (1)对右眼白内障手术病例,根据裂隙灯评判晶状体核性混浊的程度,将NC4级和NC1级白内障病例纳入统计； (2)收集NC4级和NC1级的晶状体上皮组织标本并固定；(3)对晶状体上皮组织标本进行LC3的免疫荧光标记并检测； (4)分析LC3荧光强度与白内障程度的相关性。结果：LC3荧光强度在NC4级白内障中较NC1级明显增强。结论：自噬水平与年龄相关性白内障程度呈一定的相关性。第二部分自噬在晶状体上皮细胞氧化应激中的作用。目的：比较HLEs-B3自噬缺陷细胞株和HLEs-B3细胞株,在外源性H202环境中细胞活力、氧化及抗氧化能力的差异。方法：通过HLEs-B3细胞转染PEP-KO和ATG7-’-质粒,构建PEP和A7细胞。然后将细胞分为六组,B3实验组和B3对照组；PEP实验组和PEP对照组;A7实验组和A7对照组。首先对比不同浓度的过氧化氢(H202)刺激对B3细胞株增殖活力的影响,确定合适的H202作用浓度；之后建立外源性H202相关的氧化应激模型,对B3、PEP、A7三种细胞进行细胞增殖、死亡、氧化及抗氧化能力等指标的检测：(1)通过CCK-8法检测H202对细胞增殖活力的影响；(2)通过流式细胞术Annexin V/7-AAD染色检测H202对细胞凋亡的影响； (3)通过2’7’-二氯荧光乙酰乙酸钠(DCFH-DA)荧光探针检测各组细胞内ROS荧光强度的变化; (4)运用酶生化法检测细胞内GSH、MDA的差异。结果：(1)CCK-8法显示300μM H2O2抑制细胞增殖活力,但A7组较其他组增殖增加; (2)Annexin V/7-AAD染色显示,300μM H2O2增加细胞死亡,但A7组较其他组,细胞死亡减少；(3)流式细胞仪检测显示,ROS荧光强度在A7组增加更明显； (4)酶生化法显示,H202刺激引起A7组GSH值下降更明显；而MDA值增加更明显。结论：在氧化应激环境下,如敲除自噬相关蛋白7,将降低细胞抗氧化能力,却能减少细胞死亡。第三部分自噬在晶状体上皮细胞氧化应激中的机制研究。目的：探讨自噬调控H202诱导的氧化应激的分子机制。方法：B3细胞株和HLEs-B3自噬缺陷细胞株在H202处理20 h后,采用Western blot检测各组Caspase-3、Caspase-8、 Caspase-9、Bcl-2、Bax的表达水平。结果：Western blot结果显示,H202刺激引起A7组Caspase-3、Caspase-9、Bax增加更明显,Bcl-2下降更明显,而Caspase-8无明显改变。结论：在氧化应激环境下,自噬与细胞凋亡呈相互抑制状态；自噬性细胞死亡可能在细胞死亡中占主要作用。
[Abstract]:Age related cataract is a common eye disease in senile diseases, serious harm to the elderly eyesight. Oxidative stress is one of the main factors of cataract. Ultraviolet radiation, exogenous substances can make the oxidation of lens epithelial cells (HLEs) to produce reactive oxygen species (Reactive oxygen segment species, ROS), and further make the organization cell lipid peroxidation and DNA damage, resulting in the formation of cataract. Autophagy is a physiological phenomenon in eukaryotic cells has the function of self-protection, to adapt to a variety of adverse stimulus help cells, is a basic way to maintain the internal environment of the cell homeostasis and achieve self-renewal. Autophagy can be activated by phosphorylation of AKT pathway. It has obvious effect on HLEs proliferation and migration, down-regulation of autophagy and inhibit its function. Access to relevant literature, we found that the regulation of autophagy in HL Effect of the function of Es, especially in the oxidative stress induced H2O2 has not been reported. Therefore, in this study, HLEs-B3 cells and HLEs-B3 cells autophagy defects as the experimental object, aims to explore the protective effect and autophagy in oxidative stress in human lens epithelial cells, which provides a new idea for the treatment of the treatment of age-related cataract. This study is divided into three parts, the main research methods and results are as follows. The first part of the correlation of autophagy and age-related cataract degree. Objective: To study the relationship between autophagy and the degree of age-related cataract. Methods: the level of autophagy in immunofluorescence detection of age-related cataract lens epithelial capsule tissue specimens, and analyze its correlation with the degree of cataract. The steps are as follows: (1) cases of right eye cataract surgery, according to the Evaluation of slit lamp lens nuclear opacity level, NC4 level and NC1 level of cataract cases included in the statistics; (2) collection of NC4 grade and NC1 grade of lens epithelial tissue specimens and fixed; (3) LC3 immunofluorescence labeling and detection of lens epithelial tissue; (4) the correlation analysis of the fluorescence intensity of LC3 with the degree of cataract. Results: the fluorescence intensity of LC3 was significantly enhanced than grade NC1 in NC4 cataract. Conclusion: the correlation between autophagy and age-related cataract. The second part of autophagy in lens epithelial cells in oxidative stress by autophagy defects. Objective: To compare HLEs-B3 and HLEs-B3 cell lines, cell viability in the exogenous H202 environment, the difference between oxidation and antioxidant ability. Methods: the HLEs-B3 cells transfected with PEP-KO and ATG7- '- plasmid construction of PEP and A7 cells. Then the cells were divided into six groups, B3 Experimental group and B3 control group; PEP group and PEP control group; A7 group and A7 control group. Firstly, comparison of different concentrations of hydrogen peroxide (H202) stimulation effect on B3 cell proliferation activity, determine the appropriate concentration of H202; after the establishment of the oxidative stress model, the effect of exogenous H202 on B3 and PEP A7, three kinds of cell death detection, cell proliferation, oxidative and antioxidative indexes such as: (1) detected by H202 CCK-8 method on cell proliferation activity; (2) by flow cytometry Annexin V/7-AAD staining was used to detect the effect of H202 on cell apoptosis; (3) the 2 '7' - two acetyl chloride sodium acetate (DCFH-DA) fluorescence probe to detect the intracellular ROS fluorescence intensity; (4) using enzyme biochemical method to detect GSH, MDA difference. Results: (1) 300 M CCK-8 assay showed that H2O2 inhibited cell proliferation activity, but increase the proliferation of A7 group than in the other group Plus; (2) Annexin V/7-AAD staining showed that 300 M H2O2 increased cell death, but the A7 group compared with other groups, to reduce cell death; (3) flow cytometry showed that the fluorescence intensity of ROS increased more significantly in the A7 group; (4) showed the enzyme biochemical method, by A7 group GSH decreased more obviously stimulated H202; while the MDA value increased more significantly. Conclusion: under oxidative stress, such as knockdown of autophagy related protein 7, will reduce the antioxidant ability of cells, but it can reduce cell death mechanism. The third part is the research of autophagy in oxidative stress in lens epithelial cells. Objective: to explore the molecular mechanism for oxidative stress and autophagy regulation H202 induced. Methods: B3 cell line and HLEs-B3 cell line H202 in autophagy defects after 20 h treatment, using Western blot to detect the Caspase-3, Caspase-8, Caspase-9, Bcl-2, the expression level of Bax. Results: Western blot results showed that A7 caused Cas H202 stimulation group Pase-3, Caspase-9 and Bax increased more significantly, Bcl-2 decreased more significantly, while Caspase-8 did not change significantly. Conclusion: autophagy and apoptosis are mutually inhibited under oxidative stress, and autophagic cell death may play a leading role in cell death.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R776.1

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