Protective Effect Of CNTF Gene-Modified BMSCs On Retinal Ganglion Cells Of Optic Nerve Crush Model In The Rat

Objective To investigate the protective effect of ciliary neurotrophic factor(CNTF) gene-modified bone marrow-derived mesenchymal stem cells(BMSCs) on retinal ganglion cells(RGCs) of optic nerve crush(ONC) in a rat model.Methods1. Construction of CNTF-BMSCs:Rat secreted-CNTF gene cDNA was synthesized and subcloned into a lentiviral vector plasmid pHIV-dTomato to construct recombinant vector CNTF-dTomato. CNTF-dTomato/pHIV-dTomato plasmid were co-transfected into293T cell line with packaging plasmid psPAX2and enveloping plasmid pMD.2G to produce recombinant lentivirus CNTF-lenti and control-lenti. Rat BMSCs were infected with CNTF-lenti/control-lenti to produce CNTF-BMSCs and control-BMSCs. Expression of dTomato and efficiency of infection was evaluated under the fluorescence microscope. Uninfected BMSCs(pure-BMSCs) were served as the blank control. CNTF protein levels in the supernate was detected by enzyme-linked immunosorbent assay(ELISA) and compared among the pure-BMSCs group, control-BMSCs group and CNTF-BMSCs group. Adipogenic and osteogenic differentiation of CNTF-BMSCs were induced using adipogenic/osteogenic-inducible medium and identified using oil-red O staining and alizarin red staining.2. Establishment of rat ONC model and animal groups:Sixty adult male SD rats were divided into blank control group(group A), operating control group(group B), PBS treating group(group C), pure-BMSCs treating group(group D), control-BMSCs treating group(group E) and CNTF-BMSCs treating group(group F).and10rats in every group. Both eyes of every rat in group B,C,D,E,F were applied to establish ONC model. Except of group A and B,10μL of corresponding treatment liquid was instantly injected into the vitreous of injured eyes.3. Effects of CNTF-BMSCs on the ONC model:Animals were sacrificed at the14th day after injury, paraffin sections and hematoxylin-eosin(HE) staining were performed to assess the morphological change of the injured retinas, and total number of cells in the ganglion cell layer was counted, Immunofluorescence staining was used to localize CNTF expression. Western blot was used to detect activited Caspase-3and Brn3a expression in the retina, and the obtained images were scanned to calculate the relative expression levels of different proteins in Image J software.Results1. CNTF-BMSCs was successfully constructed: The results of DNA sequencing showed that CNTF-dTomato plasmid was successfully constructed. The infection rate of CNTF-lenti was approximately95%. ELISA showed that in the post-infected day1.2,3and7, the CNTF protein level in the supernate was significantly higher in the CNTF-BMSCs group than those in the pure-BMSCs group and control-BMSCs group (P all=0.000). In the CNTF-BMSCs group, the CNTF protein level in the supernate was significantly increased in infected2,3,7days compared with1day(P=0.013,0.004,0.042). The adipogenic-induced cells showed the red staining to oil red O, and osteogenic-induced cells presented the orange staining to alizarin red.2. The ONC rat model was successfully established:HE staining showed the severe vacuolation and disorganization of RGCs in group B and C, however group D, E and F showed less vacuolar changes of RGCs.3. Protective effects of CNTF-BMSCs on ONC model:The number of cells in the ganglion cell layer in group D,E and F were lower than that in group A, and higher than other groups(P all<0.05). Immunofluorescence showed that there was no CNTF expression in group A, a large number of CNTF expression was detected in retinal ganglion cell layer(GCL) of group F, and other groups showed a small amount of CNTF expression in GCL. The relative expression of activated Caspase-3in group F was lower than that in other groups(P all=0.000):The relative expression of Brn3a in group F was lower than that in group A, while higher than other groups(P all<0.05).Conclusions BMSC line with stable overexpression of CNTF was successfully established by lentiviral vectors. By administered intraocularly, the recombinant cell CNTF-BMSCs can provide a significant protective effect on RGCs in the rat ONC model.