| Objective: The treatment of spinal cord injury(SCI) has been a big challenge for a long time. The main reason for the difficulty of SCI management is that the inability of neural cells to regenerate, thus the difficulty of the restoration of spinal cord function. At this present stage, cell replacement is the only possible method to solve this problem. Therefore, the discovery and investigation of neural stem cells(NSCs) bring new hope for the treatment of SCI. Unfortunately, researchers found that, with the absence of sufficient trophic support, NSCs that transplanted into the injured adult mammalian spinal cord showed insufficient ability of survival, migration and differentiation into neurons, and the restoration of damaged neural function was still very difficult. Based on above mentioned, gene transfection and/or the providing of exogenous neurotrophic factor(s) are necessary. Neurotrophin-3(NT-3) can promote the survival, migration and differentiation of NSCs into neurons, while tyrosine kinase C(TrkC) is the high affinity receptor of NT-3. Because general NSCs can not stably express enough amount of TrkC, the genetic modification of NSCs can perhaps promote the survival, migration and differentiation of NSCs into neurons witnin host injured spinal cord. In this research work, NSCs were transfected with TrkC gene, and then the situations of survival, migration and differentiation of trans-TrkC gene NSCs transplanted into injured spinal cord were observed. The therapeutic effect of trans-TrkC gene NSCs on the recovery of neural function after spinal cord injury was also investigated.Methods: In accordance with the synthesized primer of TrkC , and by using TrkC gene produced through PCR, TrkC-cDNA expressed plasmid was constructed with directional cloning, and DNA sequencing analysis was performed. And then pECFFP-C1-TrkC plasmid was constructed with the enzymedigestion and recombination of pEGFP-Cl and TrkC-cDNA plasmids and verified by enzyme digestion. Next, pEGFP-Cl-TrkC plasmid was transfected into in vitro NSCs with lipofectamine. Then fluorescent microscope, immunof luorescent staining, Western-Blot and MTT were uti 1 ized to observe and detect the TrkC gene expression and the change of proliferation viability of the transfected cells. Following to these work, rat spinal cord hemisection models were established with the "3-step roto-transsection method" set up by ourselves. The animals were randomly divided into six groups: normal control group, hemisection group, NSCs transplantation group, NSCs transplantation with the regional application of NT-3 group, trans-TrkC gene NSCs transplantation group and trans-TrkC gene NSCs transplantation with the regional application of NT-3 group. Nine days after the set up of animal models, cells transplantation into the injured spinal cord were performed. The BBB locomotor score were carried out respectively half, one and two months after cell transplantation, to observe the restoration of rats hindlimb motion function. And MEP and SEP were performed to detect the restoration of the upward and downward nerve conduction function of the injured spinal cord, also two months after cell transplantation. After the examination of MEP and SEP, animals were sacrificed, and double immunofluorescent staining was used to investigate the TrkC expression, survival, differentiation and migration of the transplanted cells within the host injured spinal cord.Results: The TrkC-cDNA expressing plasmid was successfully constructed, and DNA sequencing analysis proved that the obtained target gene has good consistency(99%) with the published TrkC sequence. The constructed pEGFP-Cl-TrkC plasmid satisfied the demand of this experiment, verified by enzyme digestion and electrophoresis. After the pEGFP-Cl-TrkC plasmid was transfected into NSCs, fluorescent microscope observation, immunofluorescent staining and Western-Blot indicated that transfected NSCs can efficiently and stably expressed the products of the target genes(GFP and TrkC). MTT showed apparent improvememnt of the proliferation viability of the transfected cells with the existence of... |
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