热休克蛋白5调控内质网应激在视网膜色素上皮细胞保护中的作用

目的　探讨相对分子质量70 000的热休克蛋白5（heat shock 70 kDa protein 5，HSPA5）调控内质网应激在N-亚视黄基-N-视黄基乙醇胺（N-retinylidene-N-retinylethanolamine，A2E）联合蓝光诱导视网膜色素上皮（retinal pigment epithelium，RPE）细胞损伤对RPE细胞的保护作用。方法　建立A2E联合蓝光诱导RPE细胞损伤模型，显微镜下观察RPE细胞对A2E的摄取。在RPE细胞损伤后3 h、6 h、12 h、24 h和48 h，运用CCK-8法检测RPE细胞活力；Western blot检测RPE细胞内质网应激相关蛋白HSPA5和CHOP的表达情况。构建HSPA5 shRNA慢病毒载体抑制RPE细胞中HSPA5的表达，实验分3组，HSPA5干扰组转染了HSPA5干扰序列，阴性干扰组转染了阴性对照序列，野生型组为未转染的RPE细胞。检测在RPE细胞损伤时，干扰HSPA5对内质网应激相关凋亡分子CHOP、Caspase-12表达以及RPE细胞活力的影响。结果　细胞活力检测结果显示，A2E联合蓝光照射后3 h、6 h、12 h、24 h和48 h，RPE细胞活力分别是对照组的（80.9±6.2）%、（73.3±5.8）%、（70.6±5.4）%、（62.3±6.1）%和（51.4±4.5）%，与对照组相比差异均有统计学意义（均为P<0.05）。Western blot检测结果显示，HSPA5和CHOP蛋白表达在RPE细胞损伤后均有不同程度增高（P<0.05）。在A2E联合蓝光处理后12 h，HSPA5干扰组的CHOP和Caspase-12蛋白表达较对照组显著升高，并且RPE细胞活力明显下降（均为P<0.05）。结论　A2E联合蓝光可诱导RPE细胞发生损伤，损伤机制与内质网应激相关。HSPA5具有调控内质网应激，减轻A2E及蓝光损伤，促进RPE细胞存活的作用。

HSPA5 regulates endoplasmic reticulum stress for cell survival in retinal pigment epithelial cells

Objective　To investigate the protective role of heat shock 70 kDa protein 5 (HSPA5) in regulating endoplasmic reticulum (ER) stress for retinal pigment epithelium (RPE) cells survival after blue light and N-retinylidene-N-retinylethanolamine (A2E)-mediated damage.Methods　Experimental damage model of RPE cells was established by A2E and blue light treatment.Optical and confocal microscopy was applied to observe A2E intake of RPE cells.At 3 h，6 h，12 h，24 h and 48 h after RPE damage，cell viability was measured by a cell counting kit-8 (CCK-8) assay.The expression levels of ER stress related protein HSPA5 and C/EBP homologous protein (CHOP) were measured by western blot analysis.RNA interference of HSPA5 was performed by short hairpin RNA (shRNA).There were 3 groups.HSPA5-shRNA transfected group was exposed to LV-HSPA5-GFP-shRNA，negative transfected group was exposed to LV without carrying objective gene，and wild group was non-transfected RPE cells.Protein levels of CHOP and Caspase-12 as well as cell viability were evaluated after silencing of HSPA5 in blue light-mediated damage of A2E-laden RPE cells.Results　The cell viabilities were (80.9±6.2)%，(73.3±5.8)%，(70.6±5.4)%，(62.3±6.1)% and (51.4±4.5)% at 3 h，6 h，12 h，24 h and 48 h after A2E and blue light exposure that of negative transfected group，with statistical difference (all P<0.05).The expression levels of HSPA5 and CHOP in A2E-treated RPE cells significantly increased at different time points post-illumination (P<0.05).At 12 h after A2E and blue light exposure，HSPA5 interference group revealed a significant up-regulation of CHOP and caspase-12 expression，as well as an evident decrease in RPE cells viability compared with wild group (P<0.05).Conclusion　A2E-ladened RPE cells suffer damage after blue light exposure，and ER stress participates in it.HSPA5 regulates ER stress to alleviate A2E and blue light damage for RPE cells survival.