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.

[Abstract ] Objective C reactive protein (CRP), an in-flammatory maker, increased significantly among diabetes mellitus and other metabolic diseases.Meanwhile, adiponectin plays a vital role in anti-atherogenic, anti-inflammatory and anti-diabetic poten-tials.Further, it decreased in diabetes mellitus.To investigate the effects of C-reactive protein in the expression of high-molecular-weight adiponectin ( HMWA) and adiponectin multimerization. Methods The fully differentiated 3T3-L1 cells were respectively treated with 50μg/mL CRP for 0 h、6 h、12 h and 24 h , and different doses of CRP with 0μg/mL、5μg/mL、25μg/mL、50μg/mL for 24 h.The expres-sion of HMWA was further detected by Western blot.Additionally,
the mRNA expressions of adiponectin assembly related genes ( Ero1-L、DsbA-L、ERp44 ) were detected by Real time PCR after 50μg/mL CRP treatment for 24 h. Results After 24 h treatment, 25μg/mL CRP and 50μg/mL CRP resulted in a substantial reduction ( [70 ±7]%vs [44 ±7]%, P<0.05) while 5μg/mL CRP revealed no change.With the dose of 50μg/mL CRP treated, the ex-pression of HWMA were both inhibited after the 12 h and 24 h CRP treatment ([71 ±6]%vs [48 ±11]%, P<0.05), but for the 6 h CRP treatment group, HWMA remained unchanged.Additionally, CRP inhibited Ero1-L(86 ±10)%and DsbA-L(72 ±6)%gene expression and upregulated the expression of ERp44(141 ±23)%. Conclusion CRP decreases HMWA expression in a dose and time-dependent manner and inhibits the multimerization of adiponectin, thus weaken the benefits of adiponectin in diabetes.