| Objective: In the previous research,after transplanting induced retinal pigment epithelium Stem Cells(iRPESCs)into the inferior Retinal cavity of retinal degeneration disease mice model,protective effects on the visual function of the mouse have been found.The mechanism might be paracrine.This study aims to observe the protective effect and decipher the mechanism of iRPESCs supernatant on light-damaged RPE cells,hoping to provide ideas for the cell-free therapy of RD diseases.Methods:1.To culture primary mouse retinal pigment epithelium(mRPE)cells,passaging and identifying cells.Immunofluorescence staining was used for mRPE identification.2.To establish the retinal degeneration cell model and identify the model through CCK-8 and flow cytometry:A self-made light damage chamber was used.mRPE cells were cultured under blue LED lamp for 6 hours,and then cultured for 24 hours for subsequent experiments,using CCK-8 assay and flow cytometry to exam the proliferation of light-damaged mRPE.3.Scrap mRPE cells off and transferred to the ultra-low adhesion petri dish to form spheroid cells.Immunofluorescence staining was used for iRPESCs identification.4.To evaluate the protective effect of iRPESCs supernatant on RD cells:Using Transwell to co-culture iRPESCs and light-damaged mRPE cells.(1)using Scratch test to evaluate migration of light-damaged mRPE cells;(2)using CCK-8 assay to test the proliferation of light-damaged mRPE cells;(3)using flow cytometry exam the apoptosis of light-damaged mRPE cells.Define experimental group as co-culture iRPESCs and light-damaged mRPE cells,iRPESCs treated with exosome blocker GW4869 co-culture with light-damaged mRPE cells as blockage group,light-damaged mRPE as the blank group.5.To decipher the mechanism of iRPESCs supernatant on retinal degeneration cells:(1)To extract the exosomes of iRPESCs,using LFQ analyze the protein contained in exosomes.(2)Western blot was performed to detect the relative expression levels of AKT,p AKT,PI3 K,Ppi3k proteins in the blockage group,experimental group and blank group for investigating the possible mechanism of iRPESCs supernatant on retinal degeneration cells.Result:1.Immunofluorescence staining suggests mRPE express RPE65 marker.2.the morphological observation and proliferation rate detection of mRPE cells after light exposure showed that the proliferation rate of mRPE cells decreased with the increase of light exposure time.3.Immunofluorescence staining suggests iRPESCs express Rho 、Opsin and Tubb3 marker.4.The protective effect of iRPESCs supernatant on light-damaged mRPE cells was compared between the experimental group and the blockage group.(1)Scratch test showed that the migration ability of the experimental group was significantly higher than that of the blockage group after 72 H co-culturing(P<0.01).(2)cck-8 assay showed that the proliferation ability of the experimental group was significantly higher than that of the blockage group after 48 H co-culturing(P<0.001).(3)Flow cytometry indicated that there was statistically significant difference in the mean apoptosis of light-damaged mRPE cells between the experimental group and the control group after 48 H and 72 H co-culturing(P<0.01).5.LFQ showed 24 proteins were differentially expressed in iRPESCs and mRPE exosomes,and many of them related to PI3K/AKT pathway.6.Western Blot analysis showed that p PI3K/PI3 K and pAKT/AKT were significantly different between the experimental group and the blockage group after 48 H co-culturing(P<0.01).Conclusion:1.The primary mRPE cells were successfully cultured and the light damaged model of mRPE was successfully established2.Using “sphere technology” successfully induced iRPESCs3.iRPESCs supernatant had the strongest protective effect on light-damaged cells when co-cultured for 48H4.In this model,iRPESCs supernatant possible play protective role though exosomes by activating PI3k/Akt pathways... |
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