The pilot study on rapamycin retarding the differentiation of RPE cells in vitro / 中华实验眼科杂志

ABSTRACT

Background Retinal pigment epithelial (RPE) cell transplantation is a novel approach to the treatment of hereditary retinal diseases, however, human-derived RPE cell line occurs de-differentiation during in vitro cell culture.Studies showed that early abnormal activation of Akt/mammalian target of rapamycin (mTOR) signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of mTOR pathway will be helpful for the retard of de-differentiation of RPE cells.Objective This study aimed to investigate whether rapamycin can suppress the activation of mTOR pathway and promote differentiation of ARPE19 cells.Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group.DMSO or rapamycin with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamycin-treated group, respectively.The cells of each group were collected 24 hours and 48 hours after cultured.The expression of zonula occludens-1 (ZO-1) in the cells was examined by immunofluorescence.The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot, respectively.The detected results were compared between the two groups.Results ZO-1 was expressed in both group,but the fluorescence intensity was evidently enhanced in the rapamycin-treated group.The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97％ , 29.71％ and 13.00％ in the rapamycin treated group compared with the control group 24 hours after cultured (P=0.04,0.04,0.04) , and the expression levels of RPE65, LRAT, rLBP1, BEST1 , keratin18 and MERKT mRNA elevated by 174.00％ , 88.00％ , 56.18％ ,193.81％ ,10.83％ and 35.02％ in the rapamycin-treated group in comparison with the control group 48 hours after cultured (P =0.00,0.04,0.01,0.04,0.04,0.03).In addition, the expressions of p-mTOR, p-P70S6 and p-S6 protein were weaker in the rapamyein-treated group than those in the control group both 24 hours and 48 hours after cultured.Twenty four hours after cultured,the expression level of ZO-1 protein raised by 40％ in the rapamycin-treated group compared with the control group (P =0.01);while 48 hours after cultured,the expression levels of ZO-1 ,MERKT, catenin and LRAT proteins elevated by 36.00％ ,57.37％, 13.68％ and 41.07％ in the rapamycintreated group in comparison with the control group (P=0.01,0.00,0.04,0.04).Conclusions Rapamycin can suppress the activation of mTOR signaling pathway and up-regulate the expressions of RPE specific genes in ARPE19 cells.Inhibition of mTOR pathway might be an effective way for culturing RPE cells in vitro.