| Objective:The purpose of this study was to certify the ability of Muller cells for producing retina progenitor cells in vitro.Methods:Muller cells were isolated from rat retina,and proliferating cells were expanded in serum-containing medium.The third or fourth passage of cells were identified by RT-PCR and Immunocytochemistry analysis.For dedifferentiation, the cultured cells were transferred to the sphere-culture medium composed of DMEM/F-12 supplemented with 1×N2,bFGF and EGF for 3-5days. At last, Cells in this stages were identified by immunocytochemical analysis and RT-PCR.Results:Approximately 95.31±2.68%and 93.04±4.15%of cells in the culture were Muller cells as revealed by expressing glutamate-aspartate transporters (GLAST) and glutamine synthetase (GS) immunoreactivities. RT-PCR analysis also revealed that the culture was enriched for Miiller cells and not contaminated with other retinal cells.After 3-5 days cultuerd in the the sphere-culture medium, the Miiller cells became round and differentiate to neurospheres.95.07±1.35%of cells in the neurosphere were positively reacted for Nestin,10.34±3.26% for GFAP and 90.26±4.12%for BrdU.Conclusion:New born SD rat Muller cells can generate clonal neurospheres,which consist of proliferating and multipotent cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina. Objective:The purpose of this study was to certify the ability of Muller cells for producing neural stem cells in vitro and to evaluate the possibility of transferring math5 gene to the stem cells which produces by Miiller cells via electroporation.Methods:Firstly we created the PEGFP-N1-Math5 plasmid. Secondly the neurospheres were transfected either by electroporation or by lipofection using the PEGFP-N1-Math5 plasmid.At last,the cells were analyzed 24h,48 h,3d,4d,1w and 2w after transfection and detect the effectiveness of transfection by fluorescence microscopy.Results:The recombinant transformers were digested with EcoR I and BamH I. The fragment was about 450 bp, indicating that Math5 fragment had been inserted into the vector. Moreover, The result of DNA sequencing also confirmed that the recombinant plasmid of Math5 was constructed correctly.The PEGFP-N1-Math5 plasmid were transfected into the neurospheres.The transfection efficiency was superior with electroporation (15.54±2.43%) as compared to lipofection (0.13±0.17%) at forty-eight hours after transfection.Conclusion:Electrotransfection is a method which can induce stem cells express gene effectively.This study may lay a fundation for further study on stem cells and gene therapy for neural regeneration in retina. Objective:The purpose of this study was to certify the ability of math5 on the differentiation of retinal Muller cells into retinal ganglion cells.Method:The neurospheres dedifferentiated from Miiller cells were divided into three groups:(A) neurospheres transfected by PEGFP-N1-Math5; (B) neurospheres transfected by PEGFP-N1; and (C) neurospheres without transfection.Then they were transferred to the culture medium, which was composed of Brain-derived neurotrophic factor (BDNF; 1 ng/ml),Retinoic acid (RA; 1μM) and 1% Fetal bovine serum at 37℃in 5%CO2. After 7 days,cells were fixed using cold 4% paraformaldehyde for immunocytochemical analysis.Result:After plating, cells in the spheres migrated outwards and began to differentiate. It was found that the percentage of RGCs in group transfected by PEGFP-N1-Math5(61.10±1.93%) was higher than that of the group transfected by PEGFP-N1 and the group without transfection.They were statistically significant(P<0.01).Conclusion:Math5 can promote the differentiation of retinal Miiller cells into retinal ganglion cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina.
|
PDF Full Text Request