Regulation Of Math5on Retinal M(u|¨)ller-derived Stem Cells Differentiating Into Retinal Ganglion Cells

Chapter1Primary culture and purification of rat retinal Muller cellsObjective:To culture the rat retinal Muller cells in vitro and improve the purification method.Methods:Retina of SD rat at postnatal10-21days were dissociated from the eye balls and cultured in DMEM containing10%fetal bovine serum(FBS) after enzymatic digestion. After changing the culture medium6-8days later, the retinal cells were divided into two groups, of which cells of group A were passaged by complete pancreatic enzyme digestion method when cells of group B were passaged by repeated incomplete pancreatic enzyme digestion method.The expression of GS protein was measured by immunocytochemical staining. The purity of retinla Miiller cells was examined by fluorescence-activated cell sorter(FACS). The expression of the specific markers mRNA of retinal Miiller cells and other retinal cells was measured by RT-PCR. And statistical analysis was used to test the difference between the two groups.Results:FACS demonstrated that the purity of Muller cells of group A(cells were passaged by complete pancreatic enzyme digestion method) was75.41%when purity of group B(cells were passaged by repeated incomplete pancreatic enzyme digestion method) was98.01%. RT-PCR analysis indicated that the specific markers of Miiller cells(GS、 Vimentin、Clusterin、Carbonic anhydrase) mRNA was at a higher level in group B; and the specific marker of other retinal cells mRNA was expressed at a low level in group A when there was almost no expression in group B. Under the fluorescence microscope, about83.20%±5.16%of the cells were GS positive in group A, and about98.50%±1.08%of the cells were GS positive in group B which is significantly higher than group A(P<0.005).Conclusion:The repeated incomplete pancreatic enzyme digestion method is an efficient and practical method to purify retinal Miiller cells.Chapter2Cultivation and identification of retinal stem cellsObjective:To culture and identify the retinal stem cells.Methods:High-purity retinal Muller cells were cultured in dedifferentiation medium(containing N2supplement, B27supplement,20ng/ml EGF and10ng/ml bFGF) in vitro, and observed under phase-contrast microscopy. The expression of Pax6and Nestin(specific markers of RSCs) protein was measured by immunocytochemical staining and western blot. The expression of Pax6and Nestin mRNA was measured by RT-PCR. And cell proliferation was identified by Edu staining. Results:Cells formed cell spheres after culturing in dedifferentiation medium for3-7days, and cells retained the power of proliferation. Immunocytochemical staining analysis demonstrated that about92.94%±6.48%of the cells were Pax6positive,85.96%±6.04%of the cells were Nestin positive, and Edu staining was positive in most of the cell spheres. RT-PCR and western blot analysis both showed that there was expression of retinal stem cells specific transcripts in cell spheres but not in Muller cells.Conclusion:Retinal stem cells were successfully cultivated in the dedifferentiation medium. Retinal Muller cells were an accessible source of retinal stem cells.Chapter3Regulation of Math5on retinal stem cells dedifferentiating into retinal ganglion cellsObjective:To investigate the regulation of Math5on retinal stem cells dedifferentiating into ganglion cells.Methods:Retinal stem cells were cultured in vitro and transfected by constructed PGC-FU-Math5-GFP lentivirus with PGC-FU-GFP vector and non-transfection as contrast. Cells were observed under phase-contrast microscopy daily. And the expression of Brn3b(specific marker of retinal ganglion cells) was measured by immunocytochemical analysis. Results:After transfection for7days, immunocytochemical analysis demonstrated that retinal stem cells could differentiate into Brn3b positive retinal ganglion cells. The percentage of retinal ganglion cells was50.40±8.22%in group A(Math5lentivirus transfected),13.3±13.25%in group B(empty vector transfected), and9.10±3.21%in group C(non-transfected). The differences between group A and group B or C were statistically significant(P<0.001).Conclusion:Math5can up-regulate retinal ganglion cells differentiation. It can promote retinal stem cells to differentiate into retinal ganglion cells.