Effect Of Transplantation Of Embryo Neural Stem Cells Modified By VEGF In Acute Spinal Cord Injury In Rats

PARTâ… Isolation, cultivation and identification of NSCs and gene transfectionEXP.â… Isolation, cultivation and identification of NSCs from brain of Wistar rat embryoObjective: To study the methods by which neural stem cells from brain of Wistar rat embryo are isolated and cultivated. To provide the source of neural stem cells for the study of base and application on neural stem cells. Methods: Brain tissue is acquired from Wistar rat embryo. Neural stem cells were cultured in serum-free medium. Cells were identified by immunochemistry. Results: Succeeding in isolating and cultivating neural stem cells which can be passed continuously and proliferated into neurons and astrocytes. Conclusions: There are neural stem cells which have the capacity for multipotentiality in brain of Wistar rat embryo, and they could be the resource of NSCs in research.EXP.â…¡The research of transfection of Green fluorescent protein to neural stem cell through lipofectamineObjective 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. Methods The NSCs were isolated from Wistar rat embryo brains. 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. Results 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 15.2%. Conclusions The 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 method can be further applied for the transplantation study of labeled cells.EXP.â…¢The research of transfection of vascular endothelial growth factor and green fluorescent protein to neural stem cell through lipofectamineObjective To observe the expression of EGFP, the effect and expression of VEGF in NSCs after co-transfection of vascular endothelial growth factor and green fluorescent protein. Methods The NSCs were isolated from Wistar rat embryo brains. The NSCs were either transfected with VEGF and pEGFP-N1 by Lipofectamine (as VEGF-EGFP group) or uninfected (as control). To observe the expression of EGFP, growth of sypnase after derivation and the expression of VEGF by RT-PCR. Results The expression of EGFP in NSCs was also detected by fluoresecent microscopy in VEGF-EGFP group. Compare with the NSCs group and EGFP-NSCs group, the number of synapse increased significantly(P<0.05). The expression of VEGF increased significantly than control group in RT-PCR(P<0.05). Conclusions VEGF may promote the growth of sypnase, and the VEGF can express in mRNA level after co-transfection. PARTâ…¡Effect of transplantation of modified neural stem cells in acute spinal cord injuryEXP.â… Making and evaluating a rat model of acute spinal cord injuryObjective: To make a model of acute spinal cord injury in rats, which can be used in studying the change of NF200 in spinal cord and rule about the behavioral. Method: All the 36 adult Wistar rats were made as SCI models by improved Allen's weight drop methods. The rats were randomly divided into two groups: the control group, the SCI group. The change were evaluated by NF200 detects, axon count and the slope test at 2 weeks after SCI respectively. Results: Compared with the control group, there was great difference of morphology in the SCI group and the axon count and the slope test were descended obviously in the SCI group(P<0.05). Conclusions: Spinal cord injury model in rats by this method was reproducible. It is relatively little that the injury is resulted from the operation on the rats being made during making model. Following the SCI there were some changes on rat behavioral and injury. The pathophysiological changes in the rats with SCI are similar to that in the patients with SCI. The model could be used in research.EXP.â…¡Effect of transplantation of neural stem cells modified by VEGF in acute spinal cord injuryObjective: To study the effect of the treatment with neural stem cells modified by VEGF on acute spinal cord injury. Methods: All the 64 adult Wistar rats were made as SCI models by improved Allen's weight drop methods. They were randomly devided into 4 groups:control group, NSCs group, EGFP-NSCs group and VEGF-EGFP-NSCs group. Each group was transplanted with different implant. To observe the condition of NSCs by immunofluorescence and recovery of motor function by the slope test, evaluate the expression of VEGF by Western blot and the situation of aptosis by TUNEL after transplantation for 2 weeks. Results: After transplantation for 2 weeks, there were cells expressed EGFP and NSE, VEGF expression in VEGF-EGFP-NSCs group increased significantly than other groups in Western blot(P<0.05); apoptotic cells decreased significantly than other groups in TUNEL(P<0.05); motor function score increased significantly than other groups(P<0.05). Conclusions: The NSCs transplanted can survive and differentiation in the spinal cord, and VEGF may alleviate apoptosis and premote nerve restoration.