Transplantation Of Mesenchymal Stem Cells For Early-stage Retinopathy In Diabetic Rats

Objective:The research firstly use fBM-MSC in the treatment of early-stage retinopathy in DM animal model.We have compared curative effects in the early-stage retinopathy in diabetic rats between two types of mesenchymal stem cells transplanted via two pathways separately. The object of this study was to find safe and effective donor cell type and transplantion method, and provide basic theories and experiments’criterions for clinical treatment applied to DR.Methods:hUC-MSC and fBM-MSC have been isolated,cultured and identified in vitro in serum-free sterile medium. The passage5was transfected with HIV lentivector containing the cDNA encoding green fluorescence protein (GFP) for transplantation. Two types of P5MSC were transplanted intravitreally and intravenously separately into4-week diabetic rat models induced by STZ and normal rats. Blood glucose, weight, FFA and other general situation of rats in each group were measured pre-transplant and post-transplant1w,4w,8w. Changes of inner retinal and outer nuclear layer thickness were detected by HE staining. The migration, survival, and integration of donor cells transplanted were observed by immunofluorescence microscopy, in addition of muller cell hyperplasia.The expressions of proteins of GFAP and Rhodopsin were compared among different groups by semi-quantitative Western-Blotting. Full-field F-ERG recorded changes of retinal function in each group. Statistical analysis was used to draw a conclusion.Results:1. hUC-MSC and fBM-MSC can be easily isolated and cultured separately from human umbilical cord and fetal bone marrow, with high purity, uniform shape and typical MSC morphology.They both express MSC-specific antigen markers, have strong amplification ability and maintain poorly differentiated status and pluripotency in vitro;2.No hypoglycemic effect has been observed over the entire current period after hUC-BMC and fBM-BMC were transplanted intravitreally and intravenously separately;3.hUC-MSC and fBM-MSC show similar biological characteristics when transplanted in the same way.After intravitreal transplantation, two types of cultured MSC can migrate and home in the retina of4-week diabetic rats,and there was a slight increase in the total count of cells in the4-week group, with a decrease at the8th week. Donor cells were mostly present along with NFL and GCL, only a few cells migrated into as far as OPL during8weeks. Doner cells transplanted either intravenously or in normal control rats didn’t show obvious migration. There was no evidence of tumor formation or other untoward pathology in all groups throughout the observation period;4.Retinal structure, function and expression levels of specific proteins have already changed around4weeks in a rat diabetic model;5. Intravitreal and intravenous injections are both safe, convenient and effective way for transplantation, and suitable for clinical application. Local, but not systemic transplantation is more conducive for donor cells migrating and homing in the diseased retina. In our study, protective effects on the structure, function and protein expression of the retina have all been observed in four experiments groups during8weeks after transplantation,and it was obvious at about the4th week. When efficacy were compared,it was found that hUC-MSC was better than fBM-MSC, intravitreal injection was significantly better than intravenous injection.Conclusion:1. hUC-MSC and fBM-MSC can be easily isolated and cultured, they both have strong amplification ability and maintain poorly differentiated status and pluripotency in vitro;2.hUC-MSC and fBM-MSC can sense and adapt to the microenvironment of early-stage retinopathy in DM, when transplanted intravitreally they can migrate and home in the diseased retina;3. Curative effects were positive when either of two types of MSC transplanted via either of two pathways in the early-stage retinopathy in diabetic rats.Efficacy was obvious at about the4th week,when compared,it was found that hUC-MSC was better than fBM-MSC, transplantation by intravitreal injection was significantly better than intravenous injection;4.hUC-MSC may have broad prospects for application in the therapeutic areas of retinal degenerative diseases such as DR.