Experimental Study Of Hyperbaric Oxygen Combined With Human Umbilical Cord Mesenchymal Stem Cell Transplantation In The Treatment Of Spinal Cord Injury In Rats

Part 1.Isolation and Identification of HUCMSCsObjective:To isolate and characterize mesenchymal stern cells derived from human umbilical cord Wharton’s Jelly.Methods:MSCs were derived from human umbilical cord Wharton’s jelly using a consecutive enzyme digestion method of both collagenase and trypsin,FACS analysis was done to access the phenotype of HUCMSCs. Moreover, the HUCMSCs were induced to differentiate into adipocytes and Chondrogenic, Osteogenic.Results:HUCMSCs could be efficiently derived from human umbilical cord Wharton’s jelly. Primary cells could attach around 24-48h after plating. Around 5-7 days the first passage could be made. Primary cells could be expanded within two-three weeks in 107 to 108 range. Human umbilical cord Wharton’s jelly MSCs could be efficiently induced to differentiate into adipocytes and Chondrogenic, Osteogenic. Phenotype of HUCMSCs was stable even after prolonged ex vivo cell culture of 20-30 passages. Conclusion:We described the isolation, maintenance,ex vivo expansion and characterization of HUCMSCs derived from human umbilical cord Wharton’s jelly.These HUCMSCs are multi-potent, extremely stable in ex vivo culture,and can be efficiently induced These HUCMSCs hold great promise for future cell based therapy.Part 2 Transdifferentiation of human umbilical cord mesenchymal stromal cells into neural stem cellsOBJECTIVE:To explore differentiation of human umbilical cord-derived mesenchymal stem cells into neural stem cells induced by the human epidermal growth factor and Human basic fibroblast growth factor-2.METHODS:The human umbilical cord-derived mesenchymal stem cells were induced by basic fibroblast growth factor and epidermal growth factor in vitro. The cell morphology was observed by invert microscope. The differentiation status was detected by immunofluorescence. The Nestin expression in mRNA level before and after induction was detected by real-time PCR.RESULTS:The neural stem cell balls were observed after induction. And the Nestin was detected by immunofluorescence and western blot. Nestin could further differentiate to the neuronal markproteins neuron-specific enolase, microtubule-associated protein 2 protein and glial fibrillary acidic protein.CONCLUSION:From this study show that basic fibroblast growth factor and epidermal growth factor can induce human umbilical cord-derived mesenchymal stem cells to differentiate to neural stem cells.Part 3 Hyperbaric oxygen combined with human umbilical cord Mesenchymal Stem Cells transplantation for rat spinal cord injuryOBJECTIVE:To investigate the effect of HUCMSCs transplantation plus hyperbaric oxygen (HBO) on repair of rat SCI.METHODS:Eighty female rats were divided randomly into four groups:sham group; saline transplantation group; HUCMSCs transplantation; HUCMSCs transplantation+HBO group. At 1,2,3,4weeks post-injury, A behavioral test (BBB)was performed to measure functional recovery of the hind limb postoperatively.Four weeks post-transplantation, Frozen spinal cord sections were immunohistochemically stained for observating the survival and differentiation of HUCMSCs. Myoleperoxidage(MPO) activity and ELISA was measured to study inflammation after spinal cord injury.RESULTS:The average BBB scores in the HUCMSCs transplantation+HBO group were significantly better than in the other groups from the 2nd week post-SCI. By the 4th week, The surviving amount of transplanted cells was also signifieantly greater than in the HUCMSCs group.CONCLUSION:HBO combined with HUCMSCs transplantation has synergistic effects and can improve functional recovery after SCI. This may be explained by the important role of HBO in promoting the survial of transplanted HUCMSCs.Part 4 The effects of hyperbaric oxygen on macrophage polarization after rat spinal cord injuryObjective:The present study was performed to examine the effects of hyperbaric oxygen therapy (HBOT) after spinal cord injury (SCI) with a special focus on its effect on macrophage activation and cell apoptosis.Methods:T10 clip compressive injury animal model was established. Rats were divided into three major groups:(A) sham operation; (B) SCI+ normobaric air (NBA; 21% oxygen at 1 ATA) and (C) SCI+ hyperbaric air (100% oxygen at 2.8 ATA). The macrophage activation, cell apoptosis, tissue protection and functional recovery was detected in three groups.Results:HBOT was associated with marked changes in the SCI environment, with significant increases in the numbers and levels of M2 phenotype:(arginase-1-positive), and decreased numbers of M1 phenotype (iNOS-positive). This was associated with simultaneous attenuation of MPO activity, inhibition of cell apoptosis (TUNEL-positive cells, expression of caspase-3 and Bax) and downregulation of proinflammatory factors, such as IL-1β, TNF-α and IL-17. Luxol fast blue (LFB) and GAP-43 staining showed that the levels of spared myelination and spared axons in the HBOT group were significantly higher than those in the NBA group. These changes were associated with improved functional recovery.Conclusions:Our results suggest that HBOT can promote the activation of M2 macrophages and effectively reduce the development of SCI-associated inflammation, which may further inhibit cell apoptosis, promote the neuroprotection and contribute to functional recovery after SCI in rats.