The Effects Of Lipofectamine In Streptozotocin-induced Diabetic Rats Transplanted NSCs Into The Subretinal Space

Objective To observe the expression of enhanced green fluorescent protein(EGFP) in NSCs, explore the feasibility of NSCs being gene target cells and EGFP being the tracer. To investigate the effects of Lipofectamine on NSCs transplanted into the subretinal space of rats with streptozotocin-induced diabetes. To learn the inflammation or the immunization after NSCs transplantation.Methods Diabetic rats were induced by injection of streptozotocin(STZ) intraperitondally. Four week,eight weeks and twelve weeks after the model being builded, the eyeballs were removed for making the retinal vascular network,PAS stained,HE stained and immunohistochemically stained.The NSCs were isolate from Wistar rat embryo brains, then cultured in serum free medium, and identified by immunocytochemisty. The NSCs were either transfected with pEGFP-N1 by Lipofectamine (as EGFP group) or uninfected (as control). The expression of EGFP in NSCs was detected by fluoresecent microscopy. Compare with the control, the cellular viability, the growth curves of the labeled cells were respectively analyzed. Transfection efficiencies were evaluated by flow cytometry. IN one experiment(A experiment), the NSCs were either transplanted into the subretinal space of rats. Toll-like receptor 4(TLR4) were measured by RT-PCR; the protein expression of TLR4 and activation of NF-κB were detected by Western-blot.IN another experiment (B xkperiment), The diabetic rats were randomly divided into three groups, NSCs group(A group),DMEM group(B group) and contract group(C group).To transplant NSCs into the subretinal space of rats of A group,DMEM into the subretinal space of rats of B group,no special deal with C group. Activation of NF-κB in retina were detected by Western blot.Results After the model being builded,the expression of VEGF are obviously up-regulated;there were patho-transforms in retinal vascular pericytes after four weeks;the formation of small capillary microaneuryms was occasionally recorded after eight weeks;the acellular capillaries occurred after twelve weeks.The NSCs were successfully cultured; The transfected NSCs expressed EGFP for a long-term. Similar morphological development and growth curves were found in 2 groups. And flow cytometry revealed that the highest transfection rate was up to 35.2%. IN A experiment,the expression of TLR4 and NF-κB had no differentiation between two groups.IN B experiment, compared with C group,western blotting showed up-regulation of NF-κB in A group and B group.Conclusions Diabetic rats model induced by injection of streptozotocin(STZ) was sucessful. The NSCs originated from Wistar rat embryo brains can be cultured in vitro under appropriate condition, and transfection of EGFP shows no significant effect on the proliferation of NSCs. Furthermore, transfection of NSCs mediated by Lipofectamine is effective and the reported gene has a long-term expression. That me thod can be further applied for the transplantation study of labeled cells. Lipofecta- mine has no effects on the NSCs subretinal transplantation in rats with diabetes. That method can be further applied for the transplantation study of NSCs. In diabetic rats ,the protein of NF-κB has a long-term expression after the NSCs subretinal transplantia- tion. This suggests that NSCs transplantiation induced the serious inflammation or the immunization.