葛根素对培养的晶状体上皮细胞氧化损伤的保护作用

doi:10.3321/j.issn:0412-4081.2008.02.015

CORC > 北京大学 > 生命科学学院

	葛根素对培养的晶状体上皮细胞氧化损伤的保护作用; The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells
	郝丽娜 ; 何守志 ; 罗秀梅 ; 马清敏 ; 毛绮妍 ; 凌亦凌
刊名	中华眼科杂志
	2008
关键词	晶体上皮细胞过氧亚硝基酸葛根素细胞,培养的 Lens,crystalline Epithelial cells Peroxynitrous acid Puerarin Cells,cultured
DOI	10.3321/j.issn:0412-4081.2008.02.015
英文摘要	目的探讨过氧亚硝基阴离子(ONOO-)对培养的兔晶状体上皮细胞(LEC)造成的影响以及葛根素(Pur)的对抗作用.方法为实验研究.原代培养兔LEC,将其第3或第4代用作实验.实验分为:(1)对照组:加入无热源生理盐水200 μl.(2)ONOO-组:加入ONOO-200 μl使之终浓度为0.5 mmol/L.(3)Pur组:同时加入5 μg/ml ONOO- 和10μg/ml的Pur.各组加入试剂总容积为200 μl,继续培养24 h.收集细胞分别用免疫荧光技术检测ONOO-的标记物硝基酪氨酸(NT)在LEC中的抗原表达变化;免疫印迹法检测晶状体组织中NT蛋白表达变化;光学显微镜观察细胞形态;基因组DNA电泳、流式细胞仪和Fas/FasL免疫组织化学染色检测细胞凋亡.采用单因素方差分析和q检验对数据进行分析.结果实验6～24 h期间,对照组LEC呈现绿色荧光;ONOO-组:LEC颜色逐渐由黄绿色转变为橘黄色荧光;Pur组LEC颜色逐渐由淡绿色转变为淡黄色和淡黄绿色荧光;对照组NT蛋白质表达极微弱;ONOO-组:实验各期均有NT蛋白质表达,且呈由弱到强的趋势(A值为77.22±2.44,145.00±3.94,235.78±5.97).Pur组实验6 h表达微弱(A值为72.78±2.64),12 h表达增强(A值为84.94±3.01),但至24 h表达强度减弱(A值为74.44±3.00);计算机图像分析表明,实验6、12及24 h,3组NT蛋白质平均表达水平比较,差异有统计学意义.对照组细胞形态和基因组DNA电泳正常,流式细胞仪检查可见极轻微凋亡,但细胞膜和细胞浆均未见Fas/Fas L的表达;ONOO-组出现了明显的细胞形态改变,基因组DNA电泳可见典型的"梯形条带",随时间延长细胞凋亡逐渐加重(q=78.12,82.76,69.98,P＜0.01)并可见Fas/Fas L的表达;而Pur组细胞形态、基因组DNA电泳以及流式细胞仪检查大致正常,未见Fas/Fas L的表达.结论葛根素可减轻ONOO-所致培养的LEC凋亡.Fas/Fas L信号转导途径可能影响并加强了ONOO-介导的LEC的凋亡过程.; Objective To investigate the peroxynitrite damage to the lens epithelial cells(LEC)and the prevention of this damage by puerarin in vitro. Methods T his paper was experimental study.Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment.The experiment groups included:(1)Control group:Heat-pathogen free saline(NS)200 μl was added to the medium;(2)ONOO- group:ONOO- 200 μl was added to obtain the terminal concentration at 0.5 mmo/L;(3)Puerarin group:5 μg/ml ONOO- and 10 μg/ml puerarin were added simultaneously.Then,the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine(NT),a symbol of the ONOO-,was tested with immunofloureseine technique.The expression of NT protein was examined with Western blot method.The cell morphology was observed with light microscope.Cell apoptosis was examined via DNA ladder. Flow cytometry and Fas/FasL immunohistochemical staining.These datas were analyzed by one-way-ANOVA and q test. Results During the 6 to 24 hours of experiment period,green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow,then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine(NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group(A=77.22±2.44,145.00 ±3.94, 235.78 ±5.97).At 6 hours,a slight expression of NT could be seen in the control group(A=72.78 ± 2.54),this increased at 12 hours(A=89.94±3.01)and decreased at 24 hours (A=74.44 ±3.00). With computer photo-analysis,there were significant differences between the control,STZ and puerarin groups at different period during the experiment(q=78.12,82.76,69.98,P＜0.01).In the control group, cell morphology and gene DNA ladder were normal,minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical"ladder bands"as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. Conclusions The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin.Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.; 中文核心期刊要目总览(PKU); 中国科技核心期刊(ISTIC); 中国科学引文数据库(CSCD); 0; 2; 163-169; 44
语种	中文
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/265291]
专题	生命科学学院
推荐引用方式 GB/T 7714	郝丽娜,何守志,罗秀梅,等. 葛根素对培养的晶状体上皮细胞氧化损伤的保护作用, The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells[J]. 中华眼科杂志,2008.
APA	郝丽娜,何守志,罗秀梅,马清敏,毛绮妍,&凌亦凌.(2008).葛根素对培养的晶状体上皮细胞氧化损伤的保护作用.中华眼科杂志.
MLA	郝丽娜,et al."葛根素对培养的晶状体上皮细胞氧化损伤的保护作用".中华眼科杂志 (2008).