In vitro protective effect of recombinant prominin-1 combined with microRNA-29b on N-methyl-D-aspartateinduced excitotoxicity in retinal ganglion cells  

作　　者：Jun-Hua Li Guan-Shun Yu Yu-Da Wang Tian-Kun Li 

机构地区：[1]Eye Hospital and School of Ophthalmology and Optometry,Wenzhou Medical University,Wenzhou 325027,Zhejiang Province,China [2]Eye Hospital of Wenzhou Medical University Hangzhou Branch,Hangzhou 310020,Zhejiang Province,China [3]Tianjin Eye Hospital,Tianjin 300020,China

出　　处：《International Journal of Ophthalmology(English edition)》2023年第11期1746-1755,共10页国际眼科杂志（英文版）

基　　金：Supported by Zhejiang Provincial Natural Science Foundation of China(No.LY20H120003).

摘　　要：AIM:To determine the in vitro protective effect of recombinant prominin-1(Prominin-1)+microRNA-29b(P1M29)on N-methyl-D-aspartate(NMDA)-induced excitotoxicity in retinal ganglion cells(RGCs).METHODS:RGC-5 cells were cultured,and NMDAinduced excitotoxicity at the range of 100–800μmol/L was assessed using the MTT assay.NMDA(800μmol/L)was selected as the appropriate concentration for preparing the cell model.To evaluate the protective effect of P1M29 on the cell model,Prominin-1 was added at the concentration of 1–6 ng/mL for 48h,and the cell survival was investigated with/without microRNA-29b.After obtaining the appropriate concentration and time of P1M29 at 48h,real-time polymerase chain reaction(PCR)was utilized to detect the relative mRNA expression of vascular endothelial growth factor(VEGF)and transforming growth factor(TGF)-β2.Western blot detection was applied to measure the phosphorylation levels of protein kinase B(AKT)and extracellular regulated protein kinases(ERK)in RGC-5 cells after treatment with Prominin-1.Apoptosis study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of P1M29.Immunofluorescence analysis was used to analyze the expression levels of VEGF and TGF-β2.RESULTS:MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group(P<0.05).Real-time PCR data indicated that the expression levels of VEGF were significantly increased in both Prominin-1 and P1M29 groups compared NMDA and microRNA-29b group(P<0.05),while TGF-β2 were significantly decreased in both microRNA-29b and P1M29 groups compared NMDA and Prominin-1 group(P<0.05).Western blot results showed that both Prominin-1 and P1M29 groups significantly increased the phosphorylation levels of AKT and ERK compared to NMDA and microRNA-29b groups(P<0.05).Flow cytometry analysis revealed that P1M29 could prevent RGC-5 cell apoptosis in the early stage of apoptosis,while immunofluorescence results showed that P1M29 group had higher express

关 键 词：Prominin-1 microRNA-29b vascular endothelial growth factor transforming growth factor-β2 

分 类 号：R775[医药卫生—眼科]