儿茶素二聚体抑制氧化应激诱导PC-12细胞损伤机制的实验研究

发布时间：2018-07-29 09:45

【摘要】：背景:众所周知,天然食物来源的抗氧化物,尤其是抗氧化植物化学物质,被认为是无害的天然抗氧化剂,其对一系列的慢性健康问题,如衰老、心血管疾病和癌症等,都具有明显的预防治疗作用。近年来,来源于食物的天然抗氧化物引起了广大研究人员的兴趣,尤其是那些可减缓衰老和降低年龄相关性退行性疾病风险的抗氧化物越来越受到关注。许多抗氧化物,比如酚类化合物(例如多酚类、类黄酮类和单宁类)、类胡萝卜素、有机酸和多糖等,已被发现存在于很多天然植物资源中,并且已经被广泛应用于食物和药物之中。CD提取自紫色丝膜菌(家族:丝膜菌科),具有显著的抗氧化效果,对氧化应激诱导PC-12细胞损伤具有较强的保护作用。CD在抗氧化应激损伤中具有的优势,使其可能为年龄相关性退行性疾病的治疗提供了一个新的解决方案或思路。目的:探讨丝膜菌提取物CD对H_2O_2诱导PC-12细胞氧化应激损伤的保护作用与分子机制。明确CD对神经元性PC-12细胞内ROS变化的影响,探究线粒体来源的ROS信号在H_2O_2诱导的PC-12细胞凋亡过程当中的作用,从而进一步探讨研究CD可能的神经细胞保护作用机制。方法:1.在96孔板中用不同浓度的H_2O_2(50,100,150,200,250,300μmol/L)处理PC-12细胞,分别作用2h、4h、6 h、12 h、24 h和48 h后,加入10μL/孔的MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,中文化学名为3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,商品名:噻唑蓝)孵育细胞,去除培养液,加入150μL/孔的二甲基亚砜(Dimethyl Sulfoxide,DMSO)溶解,应用酶标仪检测吸光度来检测细胞活性;2.H_2O_2(150μmol/L)处理PC-12细胞2 h后,去除培养液,加入含维生素C(Vitamin C,Vc)(150μmol/L)或CD(4μg/m L)的培养液处理PC-12细胞12 h小时后,应用MTT试验检测细胞活性;3.H_2O_2(150μmol/L)处理PC-12细胞2 h后,去除培养液,加入含Vc(150μmol/L)或CD(4μg/m L)的培养液处理PC-12细胞12 h小时后,使用DCFH-DA(二氯二氢荧光素-乙酰乙酸酯)孵育后应用荧光显微镜观察细胞内ROS分布水平,应用流式细胞术检测细胞内ROS变化程度;4.H_2O_2(150μmol/L)处理PC-12细胞2 h后,去除培养液,加入含Vc(150μmol/L)或CD(4μg/m L)的培养液处理PC-12细胞12 h小时后,应用DNA染料荧光染料Hoechst 33342染细胞核,在荧光显微镜下观察不同分组的细胞核的变化;5.H_2O_2(150μmol/L)处理PC-12细胞2 h后,去除培养液,加入含Vc(150μmol/L)或CD(4μg/m L)的培养液处理PC-12细胞12 h小时后,JC-1染色后,应用荧光显微镜观察细胞膜电势变化水平,应用流式细胞术检测线粒体膜电势变化程度;6.H_2O_2(150μmol/L)处理PC-12细胞2 h后,去除培养液,加入含Vc(150μmol/L)或CD(4μg/m L)的培养液处理PC-12细胞12 h小时后,应用Western blot(蛋白质印迹法)检测线粒体介导凋亡相关蛋白PARP-1(多聚二磷酸腺苷-核糖聚合酶的裂解片段1,poly ADP-ribose polymeras-1),Bax(Bcl2-Associated X protein),Bcl-2(B淋巴细胞瘤-2基因,B-cell lymphoma-2),caspase-3(含半胱氨酸的天冬氨酸蛋白水解酶3,cysteinyl aspartate specific proteinase-3)和caspase-9(含半胱氨酸的天冬氨酸蛋白水解酶9,cysteinyl aspartate specific proteinase-9)蛋白在PC-12细胞中的表达。结果:1.成功建立了H_2O_2诱导PC-12细胞氧化应激损伤模型;2.H_2O_2抑制PC-12细胞活性并诱导细胞凋亡,联合应用Vc或CD后,减低了H_2O_2对PC-12细胞的毒性效应;3.H_2O_2使PC-12细胞ROS水平增加,具有浓度和时间依赖性,联合应用Vc或CD后,PC-12细胞的ROS水平明显降低;4.CD能够抑制H_2O_2诱导PC-12细胞凋亡,减少H_2O_2所致的胞内ROS升高,降低由线粒体功能障碍导致的凋亡;5.H_2O_2降低PC-12细胞的线粒体膜电势,导致线粒体功能障碍,最终导致细胞凋亡,联合应用Vc或CD后,抑制PC-12细胞的线粒体膜电势下降;6.H_2O_2上调PC12细胞凋亡蛋白PARP-1、cleaved caspase-3、caspase-9和Bax的表达,联合应用Vc或CD降低H_2O_2诱导的PARP-1、cleaved caspase-3、caspase-9和Bax的表达水平;7.H_2O_2下调PC12细胞抗凋亡蛋白Bcl-2的表达,联合应用Vc或CD上调PC-12细胞内Bcl-2的表达水平,增强了细胞活力,降低了细胞凋亡。
[Abstract]:Background: it is well known that natural food sources of antioxidants, especially antioxidant phytochemicals, are considered as harmless natural antioxidants, which have obvious preventive treatment for a series of chronic health problems, such as aging, cardiovascular disease and cancer. In recent years, natural antioxidants derived from food have been caused by the natural antioxidants. Many antioxidants, especially those that reduce the risk of aging and age related degenerative diseases, have attracted more and more attention. Many antioxidants, such as phenols, flavonoids and tannins, carotenoids, organic acids and polysaccharides, have been found in many natural plants. .CD has been widely used in food and medicine, and has been widely used in food and medicine. It has a significant antioxidant effect. It has a strong protective effect on oxidative stress induced PC-12 cell damage and.CD has some advantages in antioxidant stress injury, which may be an age related degenerative disease. The treatment of the disease provides a new solution or idea. Objective: To explore the protective effect and molecular mechanism of CD on oxidative stress injury induced by H_2O_2 in PC-12 cells. The effect of CD on the changes of ROS in neuronal PC-12 cells, and to explore the ROS letter number derived from mitochondria in the process of PC-12 cell apoptosis induced by H_2O_2 To further explore the possible mechanism of the neuroprotective effect of CD. Methods: 1. in 96 Kong Banzhong, PC-12 cells were treated with different concentrations of H_2O_2 (50100150200250300 mu mol/L), and 2H, 4h, 6 h, 12 h, 24 h and 48 h were added to the 10 micron L/ aperture. Omide, the Chinese chemical was named 3- (4,5- two methyl thiazole -2) -2,5- two phenyl tetrazolium bromide, the commodity name: thiazolium incubated cells, the culture solution was removed, the two methyl sulfoxide (Dimethyl Sulfoxide, DMSO) was dissolved in the 150 mu L/ hole, and the activity of the cell was detected by the enzyme labeling instrument. The 2.H_2O_2 (150 mu mol/L) treated 2 PC-12 cells and removed the culture. After adding vitamin C (Vitamin C, Vc) (150 mu mol/L) or CD (4 g/m L) to treat PC-12 cells for 12 h hours, MTT test was used to detect cell activity. After 3.H_2O_2 (150 mu) treated 2 cells, the culture solution containing 150 mu (150 mu) or 4 micron (4 mu) was used to treat 12 hours. After incubation with two chloro two fluorescein acetoacetate (two chlorofluorescein acetoacetate), the intracellular ROS distribution was observed by fluorescence microscopy, and the changes in intracellular ROS were detected by flow cytometry. After 2 h of PC-12 cells were treated with 4.H_2O_2 (150 mu mol/L), the culture solution was removed and the incubating liquid containing Vc (150 u mol/L) or CD (4 mu g/m L) was used to treat 12 hours of PC-12 cells. NA dyestuff dye Hoechst 33342 dyed the nucleus and observed the changes of nuclei in different groups under the fluorescence microscope; after 5.H_2O_2 (150 mol/L) treatment of PC-12 cells 2 h, the culture solution was removed, the medium containing Vc (150 mu) or CD (4 mu g/m L) was added to the PC-12 cells for 12 hours. After dyeing, the cell membrane was observed by fluorescence microscope. The level of potential change was measured by flow cytometry. After 6.H_2O_2 (150 mu mol/L) treatment of PC-12 cells 2 h, the culture solution was removed, and PC-12 cells were treated with Vc (150 mu mol/L) or CD (4 u g/m L) to treat PC-12 cells for 12 h hours. -1 (lysate fragment 1, poly ADP-ribose polymeras-1), Bax (Bcl2-Associated X protein), Bcl-2 (B lymphocyte tumor -2 gene, B-cell), and cysteine (cysteine containing aspartate protein hydrolase 3) and cysteine The expression of aspartate protein hydrolase 9, cysteinyl aspartate specific proteinase-9) protein in PC-12 cells. Results: 1. the H_2O_2 induced oxidative stress damage model of PC-12 cells was successfully established, 2.H_2O_2 inhibited the activity of PC-12 cells and induced apoptosis, and the toxic effect of H_2O_2 on the cells was reduced after the joint application of Vc or CD; 3. H_2O_2 increased the level of ROS in PC-12 cells and had a concentration and time dependence. After the combination of Vc or CD, the ROS level of PC-12 cells decreased significantly. 4.CD could inhibit H_2O_2 induced apoptosis of PC-12 cells, reduce intracellular ROS increase caused by H_2O_2 and reduce apoptosis induced by mitochondrial dysfunction, and reduce mitochondrial membrane electricity. Potential, leading to mitochondrial dysfunction, and eventually leading to apoptosis, combined with Vc or CD, to inhibit the decrease of mitochondrial membrane potential in PC-12 cells; 6.H_2O_2 up regulation of the expression of PC12 cell apoptotic protein PARP-1, cleaved Caspase-3, caspase-9 and Bax The expression of anti apoptotic protein Bcl-2 in PC12 cells was down regulated by 7.H_2O_2. The expression of Bcl-2 in PC-12 cells was up-regulated by Vc or CD, and cell viability was enhanced and apoptosis was reduced.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151

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