The Treatment Of Acute Spinal Cord Injury By Combined Transplantation Of Activated Schwann Cells And Human Umbilical Cord Blood Mesenchymal Stem Cells

objective To observe the capability of Human Umbilical Cord Blood mesenchymal Stem Cells(HUCBMSCs) to differentiate into neurogenic cells in vitro and vivo under the impact of Activated Schwann cells’(ASCs) secreting sorts of neuronotrophins. The influence of microenvironment after spinal cord injury(SCI) on differentiation of HUCBMSCs were also evaluated. Finally, we discuss whether these two types of cells grafted into the injured site of spinal cord, have the abity of promoting axonal regeneration and functional recovery after SCI.Method Firstly, bilateral saphenous nerves of four weeks’ old Wister rats were ligated. One week later, far end of saphenous nerves were isolated and cultured by way of dual-enzyme digestion method combined with mechanical separation. HUCBMSCs were cultured through isolating HCMNCs by Ficoll’s way and then Plastic-adherent culture. Firstly, two methods were used for HUCBMSCs culture: all-trans retinoic acid+bFGF+EGF (control group),1/2DMEM+ASCs culture(medium after ASCs cultured2days, experimental group). Immunohistochemistry, Western-Bolt semi-quantitative and Real-time PCR (NF200、 GFAP、MBP) quantitative analysis were used to evaluate neurogenic differentiation of HUCBMSCs. Then, SCI model in Wistar rat (Female,8weeks’ old) was created at T10with NYU Impactor-Ⅱmachine with10g×50mm weight drop.80SCI rats were classified into4groups randomly and evenly:blank control group(DMEM), ASCs transplantation group, HUCBMSCs transplantation group and co-transplantation group of ASCs and HUCBMSCs.1,2,3,4weeks after operation,2animals of each group were sacrificed, and injured spinal cord tissue were used for Western-Bolt and Real-time PCR analysis(NF200、GFAP、MBP). BBB score was carried out at the3rd day and at weekly after intervals injury.12weeks later,8rats in each group were chosen out to carry out10%BDA anterograde tracing mark. Then,2weeks later, the animals were sacrificed, the spinal cord tissue with injured lesion was taken out and fast cryostat sections(5μm) was made followed by BDA developing, HE and immunochemistry(NF200,MBP,GFAP) staining. The pictures were processed by microimage analysis software (Image pro plus5.0), the IOD value of positive response area was counted to compare among four groups.Result ASCs and HUCBMSCs can be steadily cultured at least4and6passages separately after isolation and purification in vitro. The results of immunohistochemistry staining, such as NF200、GFAP and MBP, confirm that HUCBMSCs can differentiate into neurogenic cells under the effect of ASCs and RA+bFGF+EGF. The statistical analysis of Western-Bolt and RT-PCR(NF200、 GFAP、MBP) indicate that there exists differences between ASCs and RA+bFGF+EGF group. From the4th week after injury, BBB score in co-graft group was significantly higher than that in other3groups (p<0.05). The rats of co-graft group recoverd to the largest extent. BDA anterograde tracing mark showed that there were more regenerated nerve fibers pass through the injured site in co-graft group than that in other3groups, while little in control group(p<0.05). HE staining showed that the injured cavity was the smallest one in cell co-graft group when compared with other3groups.Conclusion HUCBMSCs can proliferate and differentiate into neurogenic cells under the effect of numerous neurotrophic factors in vivo and in vitro. The animal experiment results indicate that the co-transplantation of HUCBMSCs and ASCs is more effective than single cell in promoting axonal regeneration and functional recovery from rat’s spinal cord injury. HUCBMSCs differentiates into neuron and oligodendrocytein, which are beneficial to regeneration axonal extension and the link between synapses by filling the tissue gap and making up the spinal cavity, in lesion area. The co-transplantation fully takes advantage of ASCs, which have a higher activity than Schwann cells, to promote HUCBMSCs differentiate into neurons, enhance axonal regeneration and functional recovery.