The Experiments On The In Vitro Purification Of Human Fetal Olfactory Ensheathing Cells And Transplantation

Objective.To obtain the ideal methods of drawing the materials from OB and puring OECS; To select optimal chance to cure SCI with cell transplantation by comparing three different transplantation occasion after SCI; Try to find out whether united transplanation of OECS and ESC possess synergistic effects to promote axonal regeneration after rats suffer spine cord transection . Methods: We made a contrast of the following procedures: three methods to draw the materials from OB;three methods to pure the OECS; three kinds of petri dish which process differently; three different density to inoculate OECS. The optimal method of harvesting seeds cells could be found. The experiments of transplantation would be carried out using purified cells above-mentioned: We divided 20 adult female Sprague-Dawley rats into three groups,to make hemisection hole model of spinal cord .The animals of the first group transplanted OECS as soon as they suffered injury .While the animals of the second and the third group transplanted equivalent cells 2 weeks and 4 weeks respectively after they were operated .All of animals would be put to death 1 month later. We evaluated their nervous function in various occasion after they suffered injury .Meanwhile we calculated the quantities of red nucleus great cells of midbrain to evaluate regenerated extent of spinal cord axons indirectly. We used HOECS of three months embryonic age which were cultured for 14 days in vitro primarily and cervicothoracic ESC freshly gained from fetal rats of 14 days embryonic age as grafts to cure hemisection cavity of rats spinal cord. We would judge good and bad between two intervention studys and combined two agents above-mentioned by these methods as follow: To proceed animals praxiology evaluation regularly; To make pathological observation about axonal regeneration where spinal cord was transected and transplantation was performed; To confirm whether grafts were survival andThe experiments on the in vitro purification of human Fetal olfactory ensheathing cells and transplantation_____Abstractwere integrated with host tissue by immunology detection; To retrogradely trace corticospinal tract and rubrospinal tract using HRP-TMB, counting motoneuron which was tagged ?In our experiment, 36 adult SD rats were fabricated as spinal cord hemisection cavity model, which were randomly divided into four groups: The first group that was pluged gelatin foam and D/F12 medium into focal lesion was taken as matched control. The second group were transplanted with OECS and the third with ESC. We performed associated transplantation with both OECS and ESC in the fourth group animals. All animals were put to death and took out of spinal cord 4 weeks after transplantation. The regeneration and the remyelination of axis-cylinder in focal lesion were detected by pathological and immunohistochemical methods. A part of transplanted OECS in our experiment were labelled with nuclear marker Hoechst(H33342).Result: We had screened out the best method of drawing the materials from OB, which was crushing directly. The OECS purity could be achieved to 94.66% by immunoabsorption .Coating the petri dish is profitable to pure the cells. Meanwhile the appropriate cells density inoculated is important for eventual purity of culture. The differently occasional transplantation of OECS would creat different effects as follow: The results of the group transplanted 2W after SCI and the group transplanted 4W after SCI were superior to the one of the first group which were transplanted immediately after SCI..There were significant difference between the three groups (P<0.05). When we observed animals histomorphological change of associated transplantation experiment, We found that transplanted OECS integrated well with host tissue.Regenerated axis-cylinder were found in focal lesion. Either OECS or regenerated axons expressed MBP and NGFR.In the same way, ESC was found to integrate well with host tissue. The grafts were filling the cavity leaved by spinal injury. They formed tight junction with host...