Cell-penetrating Ability and Protective Effect of Recombinant PTD-HSP27 on RGC Against Hypoxia-induced Damage / 中山大学学报(医学科学版)

ABSTRACT

@#【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 PTD- HSP27 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 cobalt 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 expression ratio of Bcl-2/Bax，suppressed early apoptosis and improved cell viability in RGC-5 cells.