Transplanted Bone Marrow Stromal Cells (BMSCs) Promote Functional Neurological Recovery And Relative Proteins After Transected Spinal Cord Injury

Objective:To investigate the therapeutic effects of transplanting allogeneic bone marrow stromal cells (BMSCs) via direct injection on spinal cord injury (SCI).Herein, we investigated whether BMSCs of multiple neurotrophic factor expression is associated with functional recovery and the part associated proteins of expression after spinal cord transaction injury.Method:BMSCs were obtained from Sprague-Dawley (SD) rat tibiae and femurs and the passage 3 cells were chosen to make immunocytochemical stain for CD44, CD29.The proliferation ability of MSCs was tested by MTT method.The expression of NTFs was detected in BMSCs by using RT-PCR and immunocytochemical stain.Rats'spinal cord were completely transected at T10 level and then detect the expression of part assosciated proteins at 1 day post operation (dpo),3dpo,14dpo,28dpo by Western Blot. BMSCs were injected into the injured spinal cord immediately. Twenty-four clean adult SD rats were randomly divided into tSCI operating groups,BMSC implantated groups,DMEM implatanted group. Then injected the cell suspension containing BMSC (2×106 cells/μl,10μl,) into the injury site of rat spinal cord in 6 sites separately in the experimental group.Before rats were killed, the Basso, Beattie and Bresnahan locomotor testing scale (BBB scores),CSEP,EPS was used to evaluate the rat's locomotor and sensory function of hindlimbs,The data were analyzed by One-way ANOVA and t Test.Results:The primary culture cells displayed a plastic adherent ability and appeared to be round and mononuclear 4 h after placement in the culture medium. The number of such cells and their intra-cellular particles obviously increased 24h later. Eight hours after culture, a few wing-like and flat cells showed their appearance. They were polygonal showing many process-like extensions but lacked a clear spatial view and appeared to be fibroblasts, with an oval-nucleus in the center and widely separated from each other. At about 96 h, most of the cells displayed wing-like appearance. A minority of them showed other morphology, but these gradually decreased with every passage of subculturing. About three passages later, the cells presented a uniform morphology showing swirling-or radiation-like growths. Immunocytochemistry showed 96.86% and 94.35%.BMSCs were positive for CD29 and CD44, both of which are known as markers for bone marrow mesenchymal stem cells, indicating that they comprised mostly of BMSC.In vitro, BMSCs could express multiple mRNA and protein for several neurotrophic factors, including TrkA, TrkB, TrkC, NGF, BDNF, IGF, EGF, VEGF, and GDNF.After the spinal cord injury, the expression trend of pyridoxal kinase protein and eukaryotic translation initiation factor-5A protein were first decreased and then increased to the normal level; the vimentin, tropomyosin and caspae-3 began to increase in the injured spinal cord at ldpo,and reached their peak value respectively at 14dpo,14dpo,3dpo,and then showed the trend of reduced to normal levels.After BMSC transplantation,the BBB score in BMSC group was 1.56 at 7 dpo, and increased markedly to 3.7 at 3wpo. This was followed by a more significant increase to 9.3 at 3mpo. Compared with the sham-operation group, the BBB score of animals in the group with only the cord transection and medium group and experimental group increased gradually from 1 to 12 WPO. There is no waveform of SEP could be recorded immediately in the all groups after injury, but after BMSC translation it's CSEP became from good to better and 12 weeks later was obviously improved. The latency of CSEP in BMSC group was reduced and its amplitude increased. No animal showed the abortion of CSEP waveform as a result of BMSC translation.However, throughout the experimental period, no SEP could be recorded in rats subjected to cord transection and medium group.A notable functional recovery was found concomitantly in BMSCs treated rats.Conclusion:The cultured BMSC in vitro have strong proliferative ability and high purity, which has the ability of secretion of multiple growth factor and can be treated as an ideal cell transplantation resource.After spinal cord injury,the proteins of metabolism,transcription and apoptosis showed a certain trend of change.BMSC transplantation into the spinal cord injury in rats significantly improved the restoration of motor and sensory function, and in 3 weeks and 12 weeks increased notablely. the recovey of BMSC transplantation into transected spinal cord injury may be associated with the secretion of various nutritional factors, and the over-expression or lack expression of the injury part protein.BMSC can be used as the most promising stem cell treatment for spinal cord injury and we provides the stem cells transplantation possibility of mechanism and theory for the clinical treatment.