摘要
【目的】观察重组蛋白PTD-HSP27穿透视网膜视神经节细胞RGC-5的能力及其效率,测定重组蛋白PTD-HSP27对氯化钴(CoCl2)诱导的视网膜神经节细胞(RGC-5)细胞损伤的保护作用并初步探讨其机制。【方法】用FITC标记重组蛋白PTD-HSP27,荧光显微镜观察PTD-HSP27-FITC的穿透细胞膜的能力,用紫外分光光度计检测PTD-HSP27-FITC的穿膜效率,随后建立RGC-5细胞损伤模型。实验分为正常对照组、CoCl2损伤组、PTD-HSP27+CoCl2处理组,分别进行相应处理后,通过Annexin V-FITC/PI双染法检测各组细胞凋亡比例;Western Blotting检测各组细胞Bcl-2、Bax和Caspase-3等凋亡相关蛋白的表达水平。【结果】荧光显微镜下观察到重组蛋白PTD-HSP27-FITC能够有效通过细胞膜进入RGC-5细胞内,其进入细胞的效率为(47.29±2.33)%。CoCl2浓度依赖性地抑制RGC-5细胞存活率。比较正常对照组、CoCl2损伤组和PTD-HSP27+CoCl2处理组的检测结果示:PTD-HSP27能有效抑制CoCl2诱导的RGC-5细胞早期凋亡(P<0.01);PTD-HSP27能有效提高Bcl-2/Bax的表达比例,并抑制Caspase-3的激活(P<0.01)。【结论】重组蛋白PTD-HSP27能有效地穿过细胞膜进入RGC-5细胞内,且对CoCl2诱导的RGC-5细胞氧化应激损伤有保护作用,机制可能是通过调控Bcl-2/Bax的蛋白比例抑制细胞的早期凋亡。
【Objective】To detect the ability and the efficiency of recombinant PTD-HSP27 transport across RGC-5 cells and measure the role of recombinant PTD-HSP27 against oxidative stress damage induced by cobalt chloride on RGC-5 cells,and explored its potential mechanism tentatively.【Methods】RGC-5 cells were incubated with PTDHSP27 labeled with FITC,followed by the observation of fluorescence using fluorescence microscope.We also lysed the cells by radio immunoprecipitation assay and measure the transport efficiency of PTD-HSP27-FITC through ultraviolet spectrophotometer subsequently.We established the RGC-5 cell damage model.Cellular experiments were divided into three groups:normal control group,cobalt chloride damage group,PTD-HSP27+cobalt chloride treatment group.Those three groups of cells were experimented using Annexin V-FITC/PI staining kit:to detect apoptotic cell ratio;Western Blotting:to detect the expression level of apoptosis related protein,including Bcl-2,Bax and Caspase-3.【Results】The fluorescence of PTD-HSP27-FITC was visualized inside the RGC-5 cells using the fluorescence microscope,while the transport efficiency of PTD-HSP27-FITC was(47.29±2.33)%.Cobalt chloride inhibited the survival vitality of RGC-5 cells in a concentration-dependent manner.The comparison among normal control group,cobalt chloride damage group and PTD-HSP27+CoCl2 treatment group showed:PTD-HSP27 could effectively suppressed early apoptosis induced by co?balt chloride(P<0.01).Western Blotting results showed that PTD-HSP27 could effectively enhance the expression ratio of Bcl-2/Bax,and suppressed the activation of Caspase-3(P<0.01).【Conclusions】Recombinant protein PTD-HSP27 could protect RGC-5 cells against oxidative stress injury induced by cobalt chloride.Iit mainly regulated the expres?sion ratio of Bcl-2/Bax,suppressed early apoptosis and improved cell viability in RGC-5 cells.
作者
吴怡凝
陈梦婷
沙霄媛
刘莲
钟敬祥
WU Yi-Ning;CHEN Meng-Ting;SHA Xiao-Yuan;LIU Lian;ZHONG Jing-Xiang(Department of Ophthalmology,The First Affiliated Hospital of Jinan University,Guangzhou 510630,China;Department of Ophthalmology,Guangdong Women and Children Hospital,Guangzhou 511400,China)
出处
《中山大学学报（医学版）》
CAS
CSCD
北大核心
2019年第2期237-243,共7页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省中医药局科研项目(20161071)
广东省医学科研基金(A2017076)
关键词
视网膜神经节细胞
氧化损伤
热休克蛋白27
蛋白转导域
细胞凋亡
retinal ganglion cells
oxidative damage
heat shock protein 27
protein transduction domain
cell apoptosis
