| Objectives1.To study the expression changes of neuroglobin(Ngb) after spinal cord injury (SCI) in rabbits.2.To isolate, culture and identify the rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro.3.To construct the Ngb recombinant lentvirus and infect BMSCs.4.To transplant the Ngb gene modified BMSCs to the injured site of rabbit spinal cord, then observe the viability of BMSCs, the changes of Ngb gene expressions, the excretion of Ngb protein and the therapeutic effect to rabbits after SCI.5.To investigate the protective mechanism of the Ngb gene modified BMSCs on SCI in rabbits.Methods1.The rabbit incomplete SCI model was established by an epidural balloon compression; the expressions of Ngb mRNA and protein in injured spinal cord tissue of rabbits after SCI at different time point were detected by Real-time PCR and Western blot.2.BMSCs were cultured by the density-centrifugation method combined with the wall-adhering method; the surface markers of BMSCs were detected by flow cytometer.3.The recombinant lentiviral vector carrying Ngb gene was constructed and transfected the 293T cells through liposome Lipofectamine 2000, and the lentiviral titer was detected by Real-time PCR; BMSCs were infected with Ngb recombined lentivirus, then the multiplicities of infection (MOI) was determined by expressions of fluorescence and the expressions of Ngb in the infected BMSCs were detected by Real-time PCR, Western blot and ELISA.4.The SCI rabbits were injected with NS, BMSCs, EGFP-BMSCs, LV-Ngb and Ngb-BMSCs. The hindlimb motor function of rabbits was evaluated according to the BBB scale, the expressions of green fluorescence in injured spinal cord tissue were observed by fluorescence microscope, and the expressions of Ngb in injured spinal cord tissue were detected by Real-time PCR and Western blot.5.The malondialdehyde (MDA) levels were detected by MDA test box; the changes of apoptosis index (AI) were detected by TdT-mediated UTP nick end labeling (TUNEL) assay.Results1.Ngb mRNA and protein were expressed in normal spinal cord tissue. At 6 hours after SCI, the expressions were significantly increased compared to the control group; the expressions gradually increased and reached the peak at 24 hours after SCI, then the expressions were still high at 72 hours after SCI, which were significantly increased compared to the control group (P <0.01); after that, the expressions gradually decreased, and at 7 days after SCI (the last observed time point), the expressions markedly decreased and closed to normal levels, which were not significantly difference compared to the control group (P > 0.05).2.BMSCs were successfully isolated, cultured, and amplified; the surface markers of BMSCs were detected by flow cytometer and found expressions of CD29 and CD90 were positive while that of CD34 and CD45 were negative, which coincided with the characteristics of BMSCs.3.The Ngb recombinant lentiviral vector was constructed. It was absolutely correct by enzyme digestion and sequencing identification, and it could transfect 293T cells and achieve expression. The lentiviral titer was 2×108 TU/ml; the best MOI that Ngb recombined lentivirus infected BMSCs was 100, and the expressions of Ngb mRNA and protein in infected BMSCs were stable and high. 4.Neurological improvements were significantly greater in the BMSCs, LV-Ngb EGFP/BMSCs, and Ngb/BMSCs groups compared to the NS group and that of the Ngb/BMSCs group was the most obvious. The expressions of green fluorescence in injured spinal cord tissue were observed in the EGFP/BMSCs, LV-Ngb and Ngb/BMSCs groups. The expressions of Ngb in injured spinal cord tissue were increased in the LV-Ngb and Ngb/BMSCs groups and that of the Ngb/BMSCs group was more obvious by the detection of Real-time PCR and Western blot.5.The levels of MDA and AI were significantly decreased in the BMSCs, EGFP/BMSCs, LV-Ngb, and Ngb/BMSCs groups compared to the NS group and that of the Ngb/BMSCs group was the most obvious.Conclusions1.The expression of Ngb was increased after SCI, and changed in a time-dependent manner. It was a kind of endogenous protective reaction mechanisms.2.BMSCs of high purity and homology were cultured by the density-centrifugation method combined with the wall-adhering method, and the biological characteristics remained stable following repeated passages.3.Ngb recombinant lentvirus were successfully structured. The Ngb gene was integrated with BMSCs and the expressions of Ngb were stable and high.4.After implantation of Ngb gene modified BMSCs to the injured site of spinal cord, BMSCs kept active and the expression of Ngb was significantly increased, which markedly promoted rabbit recovery of motor function after SCI and had protective effect on SCI..5.The possible nerve protective mechanisms of Ngb gene modified BMSCs on SCI were by antioxidant and anti-apoptosis.
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