摘要目的 观察蓝光诱导人视网膜色素上皮(RPE)细胞外泌体对核苷酸结合寡聚化结构样受体蛋白(NLRP3)炎性体相关因子表达的影响.方法 人RPE细胞株贴壁细胞分为实验组和对照组.实验组细胞以蓝光照射6 h建立光损伤模型;对照组细胞常规培养.分级低温超速离心法获得两组细胞外泌体,透射电子显微镜观察其形态;蛋白免疫印迹法(Western blot)检测两组细胞外泌体表面CD63及白细胞介素(IL)-1β、IL-18、半胱氨酸天冬氨酸蛋白酶(caspase-1)蛋白相对表达水平.将两组细胞外泌体作用于正常RPE细胞,据此分为蓝光诱导细胞外泌体+实验组、正常细胞外泌体+对照组.培养24 h后,Western blot检测两组细胞外泌体中IL-1β、IL-18、caspase-1蛋白表达;荧光实时定量聚合酶链反应检测两组细胞中NLRP3 mRNA表达.结果 实验组细胞发生损伤失去原有形态;外泌体均呈双凹托盘状,直径50~200 nm;实验组细胞外泌体中IL-1β(t=18.04)、IL-18(t=12.55)、caspase-1(t=14.70)蛋白表达量明显高于对照组,差异有统计学意义(P<0.001).蓝光诱导细胞外泌体+实验组细胞外泌体中IL-1β(t=18.59)、IL-18(t=23.95)、caspase-1(t=35.27)蛋白表达量明显高于正常细胞外泌体+对照组,差异均有统计学意义(P<0.001);蓝光诱导细胞外泌体+实验组、正常细胞外泌体+对照组细胞内NLRP3 mRNA相对表达量分别为1.000±0.069、0.200±0.010,两者比较,差异有统计学意义(t=12.20,P<0.001).结论 蓝光诱导RPE细胞外泌体可使RPE细胞内NLRP3炎性体相关细胞因子IL-1β、IL-18、caspase-1蛋白和NLRP3 mRNA表达上调.

abstractsObjective To observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3). Methods Cultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Results Blue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04),IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P<0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P<0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P<0.001). Conclusion The expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.