| Background and Objective: Spinal cord injury(SCI) is a common injury during traffic, mineral working and sports accident, with high rate of disability and mortality. In recent years, the treatment of SCI has been concentrated on the transplantation of neural stem cells. However, some studies demonstrated that the transplanted suspension-cultured embryonic neural stem cells, could only differentiate into astrocytes in the injured spinal cord microenviroment, but the neuronal-restricted precursor cells(NRP) and the glial-restricted precursor cells(GRP) could keep survival and differentiate. The adherent cultured neural precursor cells are composed mainly of NRP and GRP. It is not clear that which type of cells will be suitable for the transplantation treatment of SCI. The present study compared the features of neural precursor cells under suspension culture and adherent culture for the transplantation treatment of SCI.SCI contains primary injury and secondary injury. Primary injury is inconvertible. So the treatment of SCI is mainly focused on the secondary injury. Some studies had shown that lipid peroxidation mediated by oxygen free radical played a major role on the secondary spinal cord injury. The extent of lipid peroxidation also affects the fate of engrafted cells in great part. According to the theory of traditional Chinese medicine, the paraplegia caused by SCI belongs to the field of body laziness and paralysis. Its treatment is mainly depended on activating blood circulation, removing blood stasis, deoppilating du channel, dredging meridian, tonifying pneuma,invigorating kidney and replenishing essence. The kidney in charge of bones produces essence depending on the incessant supplying of spleen and stomach, which is the foundation of acquiring nutriments and the source of metaplasia between pneuma and blood. Renal injury can make essence lost. Blood and essence may transform each other. Nourishing piyin remedy has the function of tonifying pneuma, activating blood, invigorating kidney, replenishing essence, engorging spleen and stomach, and so on. It can protect neurocyte by enhancing antioxygenic activity of the mitochondria, accommodating phospholipid metabolism, inhibiting responsive gliosis and inflammatory reaction. The present study investigated the protective role and mechanism of serum nourishing piyin remedy on NRP/GRP lipid peroxidation injury.The mild SCI model(2.5g×10cm) produced by Allen or NYU method was extensively applied for the SCI research. The extent of tissue damage and pathological changes was mild due to the minor contusion of spinal cord. While moderate SCI model(5g×10cm) showed heavier morphological and pathological changes, it afforded poorer microenviroment and peroxidation injury for the cells. The present study evaluated the treatment of moderate SCI with the transplantation of NRP/GRP cells protected with nourishing piyin remedy.Part 1 Investigation of growth characteristics of fetal spinal cord derived neural precursor cells with suspension and adherent cultureObjective: To compare the growth characteristics of fetal spinal cord derived neural precursor cells in both suspension and adherent culture, so as to select the optimal candidate population of cells for treatment of spinal cord injury.Method: Cells were isolated from fetal spinal cord tissue of E13.5 SD rats, and cultured with both suspension and adherent method. Morphological changes during cell growth was observed, and cellular growth curve was drawn. 72 hours after culture, BrdU-labelled proliferating rate and celluar expression of Nestin, PSA-NCAM, and A2B5 was detected with either immunofluenrescence or FACS method. Some of the cultured cells wereinduced to differentiate by fetal bovine serum.Results: Cells cultured with suspension method showed typical morphology of neurospheres and robust growth, while adherently cultured cells showed bipolar morphology typically and sporadic growth. BrdU- labelled proliferating rate of cells cultured with suspension method was 28.50%±4.67% compared to 9.11%±1.82% with adherent method(P<0.05). Celluar immunofluenrescence showed positive for Nestin, PSA-NCAM and A2B5 in both groups, however, ratios of each were not uniform. The adherent culture were composed mainly of NCAM and A2B5 positive cells(>80%). Cells in both groups, after induction invitro, were all able to differentiate into mature neurocytes—NF-200,GFAP, Gal-C positive cells.Conclusion: Proliferating activity of cells with suspension culture is superior than those with adherent culture. Cell population with suspension culture are basically composed of neural stem cells and progenitor cells in early stage, while adherent culture are chiefly formed by the NRP/GRP(>80%). Cells cultured with both method can differentiate into neuron, astrocyte and oligodentrocyte after induction by fetal bovine serum.Part 2 The protection of lineage-restricted neural precursors with nourishing piyin remedy against lipid peroxidation injuryObjective: To investigate the protection of lineage-restricted neural precursor cells with nourishing Piyin remedy preventing lipid peroxidation injury.Method: Lipid peroxidation model induced by H2O2 was established, and divided as normal group, damage group, intervented group with blank or remedy serum, and damage group intervented with blank or remedy serum aforehand. Then, compared celluar morphology, drew celluar survival curve, detect the content of MDA and the archaeus of SOD, and observed the fluorescence and apoptotic bodies following fluorescein staining with hoechst33258.Results: H2O2 cut down the celluar survival rate and speed up the apoptosis notably, meanwhile, enhanced the content of MDA and decreased the archaeus of SOD in cells. After intervention with serum nourishing piyin remedy, the celluar survival rate raised at the same condition. It could also prevent the augment of MDA and the decrease of SOD, and alleviate the apoptosis.Conclusion: The serum nourishing piyin remedy could protect the lineage-restricted neural precursors against lipid peroxidation by preventing the augment of MDA and the decrease of SOD, and improving the survival rate and existent status of these cells.Part 3 The treatment of spinal cord injury with the transplantation of lineage-restricted neural precursors protected with nourishing piyin remedy into ratsObjective: To investigate the therapeutic effectiveness of lineage- restricted neural precursors(NRP/GRP) transplanted into rat spinal cord after moderate contusion, and the protective effectiveness of serum nourishing piyin remedy on the survival, migration and differentiation of transplanted cells in vivo.Method: Sixty adult female SD rats were randomly divided into six groups as operated control group(Group A), contused control group(Group B), culture medium control group(Group C), NRP/GRP engrafted group(Group D), and NRP/GRP engrafted group combined with blank serum or nourishing piyin remedy serum (Group E, F). Injury models were established by self-designed electronic impact device with the impacting force of 5g×10cm. BBB scale and cortical somatosensory evoked potential (CSEP) was observed. Survival, migration and differentiation of engrafted NRP/GRP labelled with BrdU were also detected at 4 days, 2 weeks and 5 weeks after transplantation.Results: BBB scores of Group F increased significantly (P<0.05) at 2 weeks post-engraftment, and those of Group D, E were statistically higher at 4 weeks post-engraftment (P<0.05). CSEP wave of Group F showed slight recovery at 5 weeks post-engraftment. Survival, migration and differentiation of engrafted cells in Group D, E, F were illustrated at 4 days, 2 weeks, and 5 weeks after transplantation respectively, among which Group F was the best. At the last time-point, relative integrity of injured region was shown in Group D and E, meanwhile, further integrity, most of the lesion cavity filled up, was demonstrated in Group F.Conclusion: The lineage-restricted neural precursors can survive, migrate and differentiate into mature phenotypes—neuron, astrocyte and oligoden- trocyte, after immediate transplantation into moderately contused rat spinal cord. The serum nourishing piyin remedy can benefit the survival, migration, and differentiation of transplanted NRP/GRP by ameliorating the microenvironment of injured spinal cord. Transplantation with NRP/GRP could facilitate recovery and reorganization of nerve fibre in moderate SCI models, and reveal synergistic effect when used combined with serum nourishing piyin remedy.From above, we concluded that:1. The suspension cultured cells were composed of neural stem cells and neural progenitor cells, while the adherent cultured cells were mainly of NRP/GRP (>80%). Both of the cells cultured with different method could differentiate into neuron, astrocyte and oligodentrocyte induced by fetal bovine serum.2. The serum nourishing piyin remedy provided some protective role for the NRP/GRP to prevent lipid peroxidation injury, through preventing the excessive decrease of SOD, attenuating the overproduction of MDA, and consequently, improving survival rate and existent status of the NRP/GRP.3. The NRP/GRP could retain survival, migrate and differentiate into mature phenotypes—neuron, astrocyte and oligodentrocyte, aftertransplantation into the moderate contused spinal cord, thus, facilitate the recovery and regeneration of nerve fibre.4. The serum nourishing piyin remedy could improve the SCI microenvironment, decrease the lipid peroxidation injury, benefit the survival, migration and differentiation of transplanted NRP/GRP, and demonstrate synergistic effect when used in combination.5. How to improve the proliferation ability of NRP/GRP, and how to enhance the antioxygenic activity of nourishing piyin remedy, still need to be further investigated. There are many prospective aspects for the transplantation of NRP/GRP combined with tissue engineering scaffold and gene transfection technique. |
PDF Full Text Request