| [Objective]:To investigate the effects of co-transplantation of bone marrow mesenchymal stem cells(BMSCs) and olfactory ensheathing cells(OECs) on theⅧand IX layers neural survival, glial scar and nerve regeneration in rats subjected to spinal cord transaction.[Methods]:Rat femurs derived BMSCs were isolated invitro and the passage3 cells were chosen to make immunocytochemistry stain for CD44; OECs were isolated and cultured from new born SD rat's olfactory bulb and were identified by p75 immunostaining. Twenty-five clean grade and healthy male SD rats were randomly divided into sham operation group (CG group), surgical control group (SG group), simple BMSC transplantation group(BMSCs), simple OEC transplantation group (OECs) and co-transplation of BMSC and OEC group (BMSCs+OECs). The BMSCs were labeled by Dil and OECs were labeled by Hoechst33342, BMSCs or OECs were transplanted into the rostral, scar and caudal site of the transected cord of rats in the chronic phase (14 days post operation) by using the stereotaxic apparatus.6 months later, the spinal cord was obtained and prepared for paraffin section. Hematoxylin stain were performed to explore theⅧand IX layers neural survival and Immunohistochemistry was used to detect the glial scar and host neural regeneration, further count the number and area of survival neuron, count the number of GFAP and APC-positive cells in glial scar and analyze their immunostaining OD values. And immunohistochemistry were used to detect the regeneration of host's PKCγ,5-HT and CGRP-positive neve fibers. SPSS11.5 software package were used for statistical analysis.[Results]:1:Positive staining of CD44 and p75 was detected respectively in BMSCs and OECs by immunocytochemistry.2:The effect of cell transplantation for host's neural survival:The quantity and area ofⅧ,Ⅸneurons in CG group were decreased significantly when compared with that of sham group (p<0.05).①To compared the quantity and area ofⅧneurons with every group, there is no significant changes in BMSCs group and CG group (p>0.05), the quantity in OECs group exceed than CG group(p<0.05), and that in BMSCs+OECs group exceed than BMSCs group or OECs group(p<0.05).①To compared the quantity of IX neurons with every group, there is no significant changes in BMSCs or OECs group and CG group (p>0.05). There was increased significantly in BMSC+OECs group compared with that of BMSCs group(p<0.05). There was no detective changes of BMSC+OEC group compared with OECs group.③To compared the area of IX neurons with every group, there is no significant changes in BMSCs or OECs group and CG group (p>0.05). The area of IX neurons in OEC group increased significantly compared with CG group(p<0.05). There was increased significantly in BMS C+OECs group compared with that of simple BMSCs or OECs group(p<0.05).3:The effect of cell transplantation for astrocyte and oligodendrocyte in host's scar tissue.3.1:The effect of cell transplantation for astrocyte in host's scar tissue. Afer rats SCT, the quantity of GFAP positive cells and its OD values increased significantly compared with SG group(p<0.05).①To compared the quantity of GFAP positive cells with every group:Its quantity in BMSC or OEC group decreased significantly compared with CG group(p<0.05). Compared with the CG group, at the CST site, BMSC or OEC group detected no significantly change(p>0.05), at the gray mater and posterior horn in BMSC or OEC group the quantity of GFAP positive cells decreased significantly compared with CG group(p<0.05). The quantity of GFAP positive cells in BMSC+OECs group decreased significantly compared with BMSC or OEC group(p<0.05). At the CST site in rostral scar, the GFAP positive cells'number increased significantly when compared with BMSC group(p<0.05), while have no significantly change compared with OECs group(p>0.05). At the gray matter in rostral scar and posterior horn in caudal scar, there were decreased significantly compared with simple BMSC or OEC transplantation group(p<0.05).②To compared the OD values of GFAP'immunostaining with every group:Its quantity in BMSC or OEC group decreased significantly compared with CG group(p<0.05). At CST site in rostral scar and posterior horn in caudal scar in BMSC group decreased significantly compared with CG group(p<0.05), while at gray matter in rostral scar detected no significantly change compared with CG group(p>0.05). At the CST site in OEC group rostral scar there is no significantly change compared with CG group(p>0.05), while the gray matter in rostral scar and posterior horn in caudal scar decreased significantly compared with CG group(p<0.05); The OD values in BMSC+OEC decreased significantly compared with BMSC or OEC simple group(p<0.05), At the gray matter in rostral scar and posterior horn in caudal scar in BMSC+OEC group decreased significantly compared with BMSC or OEC group(p<0.05). At the CST site in rostral scar there is no significantly change compared with BMSC or OEC group(p>0.05).3.2:The effect of cell transplantation for olygodendrocyte in host's scar tissue. Afer rats SCT, the quantity of APC positive cells and its OD values decreased significantly compared with SG group(p<0.05).①To compared the quantity of APC positive cells with every group:BMSC or OECs group's cells number exceed than CG group(p<0.05), At the CST site in rostral scar in BMSC group increased significantly compared with CG group(p<0.05), while there is no significantly change when compared OECs group with CG group(p>0.05). At the gray matter in rostral scar and posterior horn in caudal scar there was increased significantly when compared BMSC or OEC group with CG group(p<0.05). The quantity of APC positive cells in BMSC+OEC group increased significantly compared with BMSC goup(p<0.05). There was no siginificantly change in rostral scar CST site when compared with BMSC group(p>0.05). There was increased significantly in rostral scar gray matter and caudal scar posterior horn when compared with BMSC goup(p<0.05). The quantity of APC positive cells in BMSC+OEC group have no significantly change compared with OEC group(p>0.05). While at CST site and gray matter in rostral scar and posterior horn in caudal matter there was increased significantly when compared with OEC group(p<0.05).①To compared the OD values of APC immunostaining with every group:BMSC or OEC group decreased significantly compared with CG group(p<0.05). At CST site in rostral scar have no significantly change when compared BMSC or OEC group with CG group(p>0.05). At gray matter in rostral scar and posterior horn in caudal scar in BMSC or OEC group increased significantly compared with CG group(p<0.05). The OD values in BMSC+OEC group increased significantly compared with BMSC group(p<0.05). At the CST site and gray matter in rostral scar increased also significantly compared with BMSC group(p<0.05). While there was no detective change when compared BMSC+OEC group with BMSC group at caudal scar posterior horn(p>0.05). The OD values of APC immunostaining in BMSC+OEC group increased significantly compared with OEC group as well as at the CST site and gray matter in rostral scar and posterior horn in caudal scar(p<0.05).4:The effect of cell transplantation for nerve regeneration in host's scar tissue.①The regeneration of PKCγpositive nerve fibers:Afer SCT, the PKCγpositive neve fibers in all animals have no significantly regeneration, all of them is not grow into scar.②The regeneration of 5-HT positive nerve fibers:The spinal cord 5-HT positive fibers in all group grow into scar. It is grow into scar 532.60±145.74μm in CG group, 1698.00±880.81μm in BMSC group,659.80±271.54μm in OECs group, 1492.00±337.52μm in BMSCs+OECs group. The regeneration longth of 5-HT positive fibers in BMSCs or OECs groups increased significantly compared with CG group(p<0.05). The regeneration longth of 5-HT positive fibers in BMSCs+OECs group increased significantly compared with OECs group, while there is no significantly change when compared with BMSCs group(p>0.05).③The regeneration of PKCγpositive nerve fibers:After SCT, the CGRP positive nerve fibers in CG group and BMSC group have no regeneration detected, both of them not grow into scar tissue. While there is 1962.40±441.14μm apart from rostral scar in CG group,313.60±109.79μm apart from rostral scar in BMSC group. The CGRP positive nerve fibers in OEC group host's spinal cord grow into scar tissue, and the regeveration longth was 198.00±85.26μm. There was also regeneration detected in BMSC+OEC group, while its regeneration longth was 531.80±162.62μm. To compared the longth of CGRP positive cells with every group. There was increased significantly when compared cell transplantation groups with CG group (p<0.05), While the CGRP positive nerve fibers regeneration longth in BMSC+OEC group exceed than BMSC or OEC group(p<0.05). [Conclusion]:1:The neurons inⅧandⅨlayers of host were atrophied or even died after spinal cord transection.2:The simple BMSC transplantation can't promote host'sⅧ,Ⅸneuron survive, also can't inhibit its degeneration,; The simple OEC tansplantation can promote host's spinalⅧnotⅨneuron's survival, OECs can inhibit host's spinal neuron degeneration. The effect of co-transplantation of BMSCs and OECs exceed than BMSCs simple transplantation in promoting host'sⅧand IX neurons survival,and also exceed than OECs simple transplantation in promoting host'sⅧnot IX neurons survival. By repressing host's neurons degeneration, co-transplantation have a notable effect when compare with BMSCs or OECs simple transplantation.3:After SCT, the glial scar were conformed, the GFAP immunoreactive was increased. The nerve fibers in spinal cord arised demyelination. APC immunoreactive was decreased. BMSCs and OECs simple-or co-transplantation can improve the microenviroment in glial scar by promoting oligodendrocytes survival and surpressing the conformation of glial scar. The effect of co-transplantation are more notable.4:After rat SCT, the host's PKCγor CGRP positive nerve fibers can't regeneration into scar, yet 5-HT positive neurite can regeneration and grow into scar. Neither simple BMSCs or OECs transplantation nor co-transplantation can promote PKCγpositive nerve fibers regeneration. Simple BMSCs transplantation and co-transplantation of BMSCs and OECs but not OECs simple transplantation can promote 5-HT positive nerve fibers regeneration. BMSCs or OECs simple transplantation and co-transplantation can promote CGRP positive nerve fibers regeneration, OECs group's CGRP positive neurite can grow into scar. The co-transplantation have a more notable effect in promoting regeneration. |
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