The Study On Neural Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Promoted By Human Schwann Cells

ObjectiveTo observe the influence of human Schwann Cells (hSCs) on the survival and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in Wistar rats with spinal cord injury (SCI); To describe the expression of the time-dependent characteristic of the Glial fibrillary acidic protein (GFAP), Myelin basic protein (MBP) and High molecular weight neurofilament (NF-H), and to explore the possible mechanism in the process of the neural differentiation of hUC-MSCs promoted by hSCs.MethodThe sciatic nerves of the healthy patients who need the treatment of amputation due to trauma were removed under sterile conditions, then, the nerves were cultured by way of dual-enzyme digestion method combined with mechanical separation, the cells were purified by the method of low concentrations of trypsin digestion combined with differential adhesion. The hSCs of P4were identified with S-100antibody, and the hSCs of P5were used in this study. The hUC-MSCs were provided by Tianjin Concord Stem Cell Gene Engineering Company, the cells were identified by flow cytometry and found to comply with the standards of stem cells, the hUC-MSCs used in this study were also P5. Eighty8-week-old female Wistar rats were subjected to T10SCI by Impactor Model-Ⅱ type device and divided into four groups randomly and evenly:DMEM control group (A), hSCs transplantation group (B), hUC-MSCs transplantation group (C), co-transplantation group of hSCs and hUC-MSCs (D). The2nd and4th week after transplantation,2rats of each group were drawn by cardiac perfusion, then the spinal cord tissues with injured lesion were taken out (the length=1.5cm), the removed spinal cord tissues were made into paraffin sections for immunofluoresence staining (GFAP, MBP and NF-H) while the implanted cells were labeled by MAB1281. The1st,2nd,3rd and4th week after transplantation, the spinal cord tissues with injured lesion of each group were taken out by the same way, and the expression of GFAP, MBP and NF-H were detected by Western-Blot and Real-Time PCR technology. The data were finally analyzed by the software of SPSS16.0.ResultsOn the3rd day of primary culture, the hSCs can climbed out from the edge of the tissue block, a large number of growing cells can be observed on the9th day. The cells can be passaged after culturing for2weeks, it can be steadily cultured at least5passages in vitro.Immunofluorescence staining showed that the MAB1281positive cells can not be seen in group A, while other groups were positive, and the MBP and NF-H positive cells can be seen in group D, while other groups were negative.The detection by Western-Blot, Real-Time PCR technology showed that the three neural markers of group A were gradually increased with time, and the expression of GFAP was significantly increased at the2nd week following transplantation while other two kinds of markers were significantly increased at the3rd week (The GFAP band gray-value ratio of2nd week to1st week was1.34:1, while PCR ratio was (2.86±0.33):1, the difference was statistically significant (P<0.05); The MBP band gray-value ratio of3rd week to1st week was3,43:1, while PCR ratio was (6.47±0.62):1; The NF-H were4.12:1and (6.78±0.77):1respectively, the difference was statistically significant (P<0.05). Compared with the group A of the corresponding time point, both the expression of MBP and NF-H were higher in group B, C and D, while the expression of GFAP were lower, more precisely, the expression of MBP, NF-H were the highest in group D (The MBP and NF-H band gray-value ratio of3rd week to1st week between Group D and Group A both was2.11:1), the difference was statistically significant (P<0.05); However, there was no significant change of the GFAP expression at2nd,3rd and4th week in group D (The band gray ratio of each week were1:1.12:1.13:1.14)(P>0.05).ConclusionhUC-MSCs can survival for at least4weeks in SCI Wistar rats with the effect of hSCs; hSCs can promote hUC-MSCs to differentiate into neuron and oligodendrocyte; after the interaction of hSCs and hUC-MSCs, the formation of glial scar can be inhibited; the expression of GFAP, MBP and NF-H showed a certain amount of time-dependent characteristic.