rhEPO对光损伤诱导的RPE细胞凋亡的抑制

目的研究重组人促红细胞生成素(rhEPO)在人视网膜色素上皮(RPE)细胞光化学损伤中的保护作用及其作用机制,为年龄相关性黄斑变性等的药物治疗和预防提供理论依据。方法取成人ARPE-19细胞株传代培养的2~5代细胞建立光损伤模型,光照12h后再培养24h终止培养,采用AnnexinV-FITC/PI流式双染法检测不同浓度的rhEPO干预治疗前后RPE细胞凋亡的变化;采用酶联免疫吸附实验(enzyme linked immunosorbant assay,ELISA)及免疫组织化学法检测不同浓度的rhEPO干预治疗前后RPE细胞caspase-3及Bcl-2表达的变化;并添加AG490(Jak2激酶抑制剂),探讨人rhEPO对人RPE细胞光化学损伤的保护性作用机制。结果各rhEPO组均可明显减少光化学损伤诱导的人RPE细胞的凋亡,呈浓度依赖性,以40IU·ml-1EPO组结果最明显,为(4.93±1.45)·ml-1;在40IU·ml-1EPO组caspase-3浓度最低,为(0.125±0.029)μg·L-1;在40IU·ml-1EPO组Bcl-2表达最高(168.21±3.87);在加入AG490组,人RPE细胞凋亡增高为(11.29±2.11)·ml-1;caspase-3浓度增高为(0.362±0.042)μg.L-1;Bcl-2表达降低。rhEPO抑制凋亡作用均被阻止。结论rhEPO可抑制光化学损伤诱导的人RPE细胞的凋亡,抑制caspase-3的浓度,上调Bcl-2的表达,对人RPE细胞的光化学损伤有保护治疗作用;其保护作用机制主要通过EPO与受体结合后激活Jak2激酶途径完成的。

Inhibition of light-induced apoptosis of RPE cells by recombinant human erythropoietin

Aim This study aimed to assess the protection of recombinant human erythropoietin (rhEPO) in light-induced injuries in human retinal pigment epithelial(RPE)cells by researching the inhibition of rhEPO for apoptosis in human RPE cells by light-induced injuries.Methods Cultured human RPE cells were exposed to light of 8 w (2 000±500) lux for 12hours,then the culture were stopped at 24 hours after 12hours light stimulation. The effect of inhibiting apoptosis of rhEPO was detected by AnnexinV-flunorescein isothiocyanate/Propidium iodium labeling and flow cytometry. The enzyme linked immunosorbant assay(ELISA)and immunocytochemical staining were used to assess the expressions of caspase-3 and Bcl-2 treated by different doses of rhEPO in light-induced injury on human RPE cells and research the protective mechanism of rhEPO by adding AG490(the special inhibitor of Jak2).Results There was a obviously increased effects on inhibiting apoptosis in every rhEPO group, which was the most conspicuous in 40 IU·ml-1 rhEPO group,and the value was (4.93±1.45)·ml-1. The decrease of expression of caspase-3 was most obvious in 40 IU·ml-1 rhEPO group, and the value was (0.125±0.029) μg·L-1. The increase of expression of Bcl-2 was the most obvious in 40 IU·ml-1 rhEPO group and the value was 168.21±3.87. But these effects on inhibiting apoptosis in rhEPO group were restrained by adding AG490, the value of apoptosis was (11.29±2.11)·ml-1 and the density of caspase-3 increased to (0.362±0.042) μg·L-1,the expression of Bcl-2 dropped.Conclusion It is suggested that rhEPO can inhibit the apoptosis of human RPE cells in the light-induced injuries and inhibit the expression of caspase-3 and up-regulate the expression of Bcl-2, so rhEPO can protect the light-induced injuries for human RPE cells. Its protective mechanism is accomplished principally by the pathway of combining EPO with EPOR ,then the combination activates Jak2.